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A Textbook of
Industrial Microbiology
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 Wulf Crueger
Anneliese Crueger

BIOTECHNOLOGY:
A Textbook
of Industrial Microbiology
SECOND EDITION

Editor of the English edition:

Thomas D. Brock

Sinauer Associates, Inc. + Sunderland, MA 01375

Dr. W. Crueger
Technical Microbiology Center
Dr. A. Crueger
Biochemical Process Development
Bayer AG
Friederich-Ebert Strasse 217
5600 Wuppertal 1
Federal Republic of Germany

Library of Congress Cataloging-in-Publication Data

Crueger, Wulf. ,
[Lehrbuch der angewandten Mikrobiologie. English]
Biotechnology : a textbook of industrial microbiology / Wulf
Crueger, Anneliese Crueger : editor of the English edition. Thomas
D. Brock. -- 2nd ed.
en
Translation of: Lehrbuch der angewandten Mikrobiologie.
Includes bibliographical references.
ISBN 0-87893-131-7
1. Industrial microbiology. I. Crueger, Anneliese. IJ. Brock,
Thomas. III. Title.
QR53.C7813 1990
660 ’ .62--dc20
89-26191
CIP

All rights reserved.

Copyright © 1989 R. Oldenbourg Verlag GmbH, Munchen.

English translation copyright © 1990 by Science Tech Publishers.
Printed in the United States of America.

Original translation by Caroline Haessly. Editorial supervision and new

translation by Thomas D. Brock. Production supervision by Katherine M. Brock
(Science Tech Publishers, Madison, Wisconsin).

Address orders and correspondence to

Sinauer Associates, Inc., Sunderland, MA 01375, USA

Bias
53s 22 el
 Foreword

Biotechnology deals with the use of living organisms or their products in large-scale
industrial processes. It is an old field that has been rejuvenated in recent years because
of the development of genetic engineering techniques. At present, biotechnology is
in an amazing growth phase whose end is nowhere in sight. Industrial microbiology,
a central part of biotechnology, matured as a science in the antibiotic era, and the
large-scale manufacture of microbial products became a multibillion dollar industry.
Genetic engineering has now made possible the directed construction of microorga-
nisms that will do almost anything, and new products are being announced almost
daily. Not only are new classes of substances being sought for human therapy, but
cost-effective processes are being developed for major organic chemicals. Yet the mere
engineering of a microbe is not enough. Large-scale, economically viable production
must be attained. The industrial microbiologist knows that there are vast difficulties
in the transfer of a laboratory process to the production plant. There are, however,
some well-established principles of industrial microbiology, and it is the purpose of
this textbook to enunciate them.
Although there are a number of advanced textbooks and research monographs
dealing with industrial microbiology, there has been no up-to-date book suitable for
use in universities and colleges. The first edition of this book has been used widely
in university classes, not only in North America but throughout the world. The book
obviously met a real need and I am pleased that a new edition can now be published.
This second edition is based on a new German-language edition published in 1989.
In the years since the first edition appeared, a number of important advances in
industrial microbiology have taken place; all of these advances are described in some
detail in this book. Among the new material is, of course, an extensive update of the
material on genetic engineering, including the use of recombinant DNA (rDNA) tech-
niques for strain selection and for the production of pharmaceutically useful proteins,
enzymes, and amino acids. A whole series of new rDNA products, whose development
makes use of the principles laid out in this book, are appearing on the market. Other
major new developments include the use of immobilized enzymes and cells for large-
scale processes. Biochemical reactors employing new membrane technology are dis-
cussed, as are membrane-based biosensors used in medicine and laboratory analysis.
Some of the older industrial processes for the production of organic acids, alcohols,
aldehydes, and flavor and aroma ingredients have been greatly improved in recent
years, and these new developments are covered. The purification of industrial me-
tabolites is almost a special branch of biotechnology, and new or improved chro-
matographic and extraction methods are presented.

Vv
vi / FOREWORD
As in the first edition, this book has been carefully edited to make it accessible to
the English-speaking student. I have translated the new material myself, taking pains
to ensure that the vocabulary is appropriate for an undergraduate student. In order
to bring out this new edition as rapidly as possible, the translation was made directly
from the German manuscript.
Students will certainly find this text useful. Research scientists and technicians al-
ready employed in industrial microbiology will also benefit from this practical over-
view of the field. In addition to industrial microbiologists, others who may find this
book valuable include food scientists, environmental scientists, chemical engineers,
and organic chemists.
The authors and I are pleased that we are able to offer this modern, attractive,
accessible book, which should be of great value to both students and practicing re-
searchers.
November 1989
Thomas D. Brock
Department of Bacteriology
University of Wisconsin
Madison, Wisconsin, USA
 Contents

Introduction
Screening for new metabolites
2.1 General
2.2 Primary and secondary metabolites
2.3 Strains used in screening
2.4 Test systems
Strain development
3.1 General
3.2 Mutation
3.3 Selection of mutants
3.4 Recombination
3.5 Regulation
3.6 Gene technology
3.7 Use of genetic methods
Substrates for industrial fermentation
4.1 Substrates used as carbon sources
4.2 Substrates used as nitrogen sources
Methods of fermentation
5.1 Introduction
5.2 Growth kinetics of microorganisms
5.3 Fermenter systems
5.4 Stirring and mixing
5.5 Gas exchange and mass transfer
5.6 Scale up
5.7 Sterilization of gases and nutrient solutions
5.8 Fermentation processes
5.9 Instrumentation
5.10 Use of computers
6 Product recovery
6.1 Introduction
6.2 Unit operations in product recovery
6.3 Yield

Vii
viii /CONTENTS
7 Organic feedstocks produced by fermentation 124
Zed General 124
122 Ethanol 124
7.3 Acetone/butanol fermentation 129
7.4 Glycerol 131
8 Organic acids 134
8.1 Introduction 134
8.2 Citric acid 134
8.3 Gluconic acid, gluconolactone, and glucose oxidase 142
8.4 Acetic acid 143
8.5 Lactic acid 147
8.6 Kojic acid 148
8.7 Itaconic acid 148
9 Amino acids 150
9.1 Introduction 150
932 Commercial uses of amino acids 151
9.3 Methods of production 153
9.4 Strains for amino acid production 156
9.5 Process control 157
9.6 Product recovery 158
95% Production of individual amino acids 158
9.8 L-Glutamic acid 158
9.9 L-Lysine 164
9.10 L-Tryptophan 169
10 Nucleosides, nucleotides, and related compounds 175
10.1 Introduction 175
10.2 Structure and biosynthesis 176
10.3 Regulation 178
10.4 Production 178
11 Enzymes 189
1 Introduction 189
PTZ Amylases 191
11.3 Glucose isomerases 198
11.4 L-Asparaginases 203
Ts Proteases 203
11.6 Rennin 205
by Pectinases 206
11.8 Lipases 207
11:9 Penicillin acylases 208
11.10 Lactases 210
en Stabilization of enzymes and cells 210
12 Vitamins 219
hae Introduction 219
1222 Vitamin B,, 219
 CONTENTS / ix

12.3 Riboflavin 222

12.4 $-Carotene 226
13 Antibiotics 229
13.1 Introduction 229
13.2 6-Lactam antibiotics 233
13.3. Amino acid and peptide antibiotics 243
13.4 Carbohydrate antibiotics 249
13.5 Macrocyclic lactone antibiotics : 256
13.6 Tetracyclines and anthracyclines 262
13.7 Nucleoside antibiotics 267
13.8 Aromatic antibiotics 268
13.9 Other commercially produced antibiotics 270
14 Ergot alkaloids 274
14.1 Occurrence and significance 274
14.2 Developmental cycle of Claviceps a
14.3 Structure 279
14.4 Biosynthesis 277
14.5 Production of ergot alkaloids 279
14.6 Regulation of alkaloid production in cultures 281
14.7 Strain development 283
15 Microbial transformations 286
15.1 Introduction 286
15.2 Types of bioconversion reactions 286
15.3 Procedures for biotransformation 290
15.4 Applications of bioconversions 291
15.5 Transformation of steroids and sterols 292
15.6 Transformation of nonsteroid compounds 298
15.7 Transformation of antibiotics 300
15.8 Transformation of pesticides 301
16 Single-cell protein (SCP) 306
16.1 Introduction 306
16.2 Production of single-cell protein from alkanes 307
16.3 Bacteria which utilize methane 309
16.4 Methanol fermentations 309
16.5 Single-cell protein from wood 314
16.6 Single-cell protein from carbohydrates 315
16.7 Single-cell protein from sewage 315
17 Newer approaches to sewage treatment 317
17.1 Introduction 317
17.2 Starter cultures for treatment processes 318
17.3 Aerobic sewage treatment—airlift process 319
17.4 Aeration with pure oxygen 320
17.5 Methane production S21
x / CONTENTS
18 Leaching 326
18.1 Introduction 326
18.2 Organisms for leaching 326
18.3 Chemistry of microbial leaching 327
18.4 Commercial processes 327
19 Extracellular polysaccharides 330
20 Other fermentation processes and future prospects 335
Index 343
 Introduction

Biotechnology is the use of microbiology, bio- velopments in the production of wine, vinegar,
chemistry, and engineering in an integrated fash- beer, and sake, and with the traditional fungal
ion with the goal of using microorganisms and fermentations used in Asia and Africa for the
cell and tissue cultures (or their parts) to man- production of food. An experimental approach to
ufacture useful products. Biotechnology can be the production of microbial metabolites only be-
divided into two categories which are sometimes gan at the beginning of the 20th century. Up until
called ”traditional biotechnology” and ”new bio- the time of World War II, the main microbial
technology”. The major products of the tradi- products that had developed from this experi-
tional biotechnology industry are food and flavor mental approach were enzymes such as pro-
ingredients, industrial alcohol, antibiotics, and teases, amylases, and invertase.
citric acid. These products amount, on a world- A major breakthrough in biochemical and mi-
wide basis, to about 300 billion dollars annually. crobial engineering occurred after World War II
The new biotechnology, which involves the use as a result of the large-scale production of the
of the newer techniques of genetic engineering first antibiotic, penicillin. In order to produce this
and cell fusion to produce organisms capable of antibiotic economically, important engineering
making useful products,. provides at present developments had to be made, including the de-
products with a total value of less than a billion velopment of techniques for large-scale sterili-
dollars. In the future, however, it is predicted that zation, aeration, and growth of microorganisms.
the new biotechnology will account for a much In addition, genetic methods for microbial strain
larger fraction of the total biotechnology indus- improvement were perfected.
try. From World War II up until about 1960, the
Industrial microbiology, the major founda- major new biotechnology products were anti-
tion of biotechnology, arose out of empirical de- biotics. Through intense efforts of the pharma-
2 / CHAPTER 1 / INTRODUCTION

ceutical industry, numerous new antibiotics were Table 1.1 Patent applications in 1984 for three
countries with major biotechnology industries.
discovered and of these around 20 were put into
commercial production. In addition, in this early Country Molecular Fermentation
biology patents patents
post-World-War-II period, processes were de- (including
veloped for the chemical transformation of ste- enzymes)
roids, and the culture of animal cells for the pro- USA 100 160
duction of virus vaccines was perfected. Japan 90 700
In the period from 1960 through 1975, new Federal Republic of
Germany 12 55
microbial processes for the production of amino
acids and 5'-nucleosides as flavor enhancers
were developed, primarily in Japan. In addition,
the major “highlight” of the period since 1975,
numerous processes for enzyme production for
in actuality other products are economically more
industrial, analytic, and medical purposes were
important. For instance, the production of
perfected. During this same period, successful
ethanol by immobilized cells has become a major
techniques for the immobilization of enzymes
process. The enzyme glucose isomerase has be-
and cells were developed. During this time a fur-
come a 27 million dollar industry and is used to
ther development was the use of continuous fer-
produce high-fructose syrup which itself has a
mentation for the production of single-cell pro-
value of 2.5 billion dollars. Aspartame, a major
tein from yeast and bacteria for use as human
artificial sweetener, is produced microbially.
and animal food. Single-cell protein processes
Many new antibiotics have been introduced.
were developed using microorganisms capable of
Cheap fats are being increased in value by en-
using petroleum-based starting materials such as
zymatic esterification, the enzymes being micro-
gas oil, alkanes, and methanol. In this same pe-
bial products. The biodegradation of persistent
riod, microbial biopolymers such as xanthan and
chemicals using specially developed microbial
dextran, used as food additives, were also de-
strains as starter cultures is being field-tested.
veloped into commericial processes. Somewhat
To place in perspective research activities in
distinct processes that were advanced during this
traditional biotechnology and the ”new” bio-
period were the use of microorganisms for ter-
technologies (using genetic engineering, etc.),
tiary oil recovery (an aspect of geomicrobiology)
Table 1.1 provides a comparison of the number
and the perfection of techniques for anaerobic
of patent applications in the whole field of bio-
cultivation of microorganisms, derived out of
technology for three major industrial countries,
studies on the sewage treatment process.
U.S.A., Japan, and the Federal Republic of Ger-
Since 1975 biotechnology has entered some
many. As can be seen, traditional biotechnolo-
important new phases. First was the develop-
gies still dominate, especially in Japan.
ment of the hybridoma technique for the pro-
duction of monoclonal antibodies, of interest pri-
marily in the medical diagnosis field. Soon after REFERENCES
was the production of human proteins using ge-
Demain, A.L. and N.A. Solomon. 1986. Manual of In-
netically engineered Escherichia coli. The first dustrial Microbiology and Biotechnology. American
product, human insulin was introduced in 1982, Society for Microbiology, Washington, D.C.
followed soon by Factor VIII, human growth hor- Moo-Young, M. (editor). 1985. Comprehensive Biotech-
nology. The Principles, Applications and Regulations
mone, interferons, and urokinase. At present, a
of Biotechnology in Industry, Agriculture, and Medi-
vast array of human proteins are in the devel- cine. Volume 1, The Principles of Biotechnology: Sci-
opment stage. entific Fundamentals; Volume 2, The Principles of Bio-
technology: Engineering Considerations; Volume 3,
Although the production of human proteins
The Practice of Biotechnology: Current Commodity
by engineered bacteria is generally recognized as Products; Volume 4: The Practice of Biotechnology:
 REFERENCES / 3

Specialty Products and Service Activities. Pergamon 4, Microbial Products II; Volume 5, Food and Feed
Press, Oxford. Production with Microorganisms; Volume 6a, Biotrans-
Rehm, H.J. and Reed, G. (editors). 1981-1988. Biotech- formations; Volume 6b, Special Microbial Processes;
nology. A Comprehensive Treatise. Volume 1, Microbial Volume 7a, Enzyme Technology; Volume 7b, Gene
Fundamentals; Volume 2, Fundamentals of Biochem- Technology; Volume 8, Microbial Degradations. VCH
ical Engineering; Volume 3, Biomass, Microorganisms Publishers, Weinheim, Germany and Deerfield Beach,
for Special Applications, Microbial Products I; Volume Florida.
 Screening
for new
metabolites

pected and the method is widely used in the

2.1 GENERAL
antibiotic industry.
The biochemical capabilities of microorganisms 5. Gene cloning (Section 3.6), in which genes
are vast, and a wide variety of new or unusual may be transferred between unrelated strains
compounds may be produced by various micro- which are producers of known substances. Al-
bial isolates. One of the main tasks of the in- ternatively, transfer may be to nonproducers
dustrial microbiologist is to develop procedures which contain ”silent” genes, leading to the
for obtaining new microbial metabolites. There generation of modified or even new sub-
are five distinct approaches: stances. This will undoubtedly be the method
of choice in the future.
1. Screening for the production of new metab-
olites with new isolates and/or new test In order to be successful, screening must be
methods. This is the only way to obtain com- an interdisciplinary activity, combining the ac-
pletely new classes of substances. tivities of microbiology, chemistry, biochemistry,
2. Chemical modification of known microbial engineering, and bibliographics. Microbiology is
substances. involved in the isolation and identification of mi-
3. Biotransformation (Chapter 15), which re- croorganisms, strain preservation, testing for bi-
sults in change in a chemical molecule by ological activity, and fermentation practice. Bio-
means of a microbial or enzymatic reaction. chemistry provides the analytical procedures
4. Interspecific protoplast fusion (Section 3.4), needed as well as the approaches to purification
which is a means of recombining genetic in- of the biologically interesting molecules whereas
formation from rather closely related producer synthesis of substrates and inhibitors falls under
strains. New or hybrid substances are ex- the purview of the chemist. The bibliographic
 2.2 PRIMARY AND SECONDARY METABOLITES / 5

specialist searches the literature as well as the a group of closely related structures; one
various computerized data bases. The engineer's strain of Streptomyces, for example, produces
activity focuses on the development of technical 32 different anthracyclines.
equipment needed for the successful process. * Some organisms form a variety of different
Screening can never be considered a routine classes of substances as secondary metabo-
activity, since the methods must always be lites.
adapted to the newest techniques and knowl- . The regulation of the biosynthesis of secon-
edge. The goal is always to detect and identify dary metabolites differs significantly from
new substances of commercial interest and to that of the primary metabolites.
separate them in the quickest possible way from
the numerous easily detected substances that are There are several hypotheses about the role
of no commercial interest. Some of the most in- of secondary metabolites, of which Hans Zah-
telligent screening methods that are currently ner’s, illustrated in Figure 2.1, is the most elegant.
used are the products of Japanese scientific ac- Besides the five phases of the cell’s own metab-
tivity, particularly the group of Omura. For in- olism (intermediary metabolism, regulation,
stance, 42 completely new compounds were transport, differentiation and morphogenesis),
found by Omura (1986) using systems that de- secondary metabolism is considered a “playing
tected microbially produced substances with field” for the evolution of further biochemical
antibacterial, antimycoplasmal, antianaerobe, development, which can proceed without dam-
antifungal, antiparasite, and antitumor activity. aging primary metabolism. Genetic changes
Substances were also found that acted as her- leading to the modification of secondary metab-
bicides and as inhibitors of penicillin, elastase, olites would be expected not to have any major
and adenosine deaminase. effect on normal cell function. If a genetic change
Further overviews of the newer screening leads to the formation of a compound that in
methods are given by Vandamme (1984), Box some way is beneficial, then this genetic change
(1985), Verrall (1985), Cheetham (1987), and would be fixed in the cell’s genome, perhaps be-
Elander (1987). coming essential. In this way the former secon-
dary metabolite would be converted into a pri-
2.2 PRIMARY AND SECONDARY mary metabolite.
METABOLITES
Bu'Lock borrowed the term “secondary metab-
olite” from plant physiology in 1961 and applied Vv es
it to microbiology. While primary metabolites GE PO
are essential for life and reproduction of cells,
and primary metabolism functions similarly in
all microorganisms, the following is true for sec-
ondary metabolites:
e Every secondary metabolite is formed by only
a few organisms.
e Secondary metabolites are seemingly not es-
sential for growth and reproduction.
+ Their formation is extremely dependent on Figure 2.1 The five levels of primary metabolism with
environmental conditions. the “playing field” of secondary metabolism (Zahner,
e Some secondary metabolites are produced as 1979).
6 / CHAPTER 2 / SCREENING FOR NEW METABOLITES

altitudes, cold habitats, sea water, deep sea, de-

Screening for new metabolites
serts, geysers, and petroleum fields are being ex-
There are no universal screening methods. The amined. Depending on the inoculum source and
success of a screening program depends upon the enrichment procedure, specific groups of orga-
selection of appropriate tests as well as appro- nisms may be isolated. Table 2.1 presents some
priate microorganisms to be tested. The capacity examples of the kinds of organisms that can be
of an industrial screening group for isolation of isolated with various enrichment methods. The
microorganisms and thorough testing is around isolation of strains can be carried out with the
1000-2000 strains per year. following scheme: 1) The soil or water sample is
Today, most screening programs focus on suspended in a definite amount of sterile water
chemotherapeutically useful products for the fol- to which Tween has been added as an emulsi-
lowing areas: activity against antibiotic-resistant fying agent. The sample is vigorously agitated.
strains, tumors, and viruses, as well as a search 2) The supernatant is diluted 107—1079, 3) Sam-
for enzyme inhibitors and pharmacologically ac- ples from this dilution series are plated on var-
tive substances (hormones, etc.). Better starter ious culture media and then incubated. 4) Single
cultures for the food industry as well as micro- colonies from the plates are picked and purified
organisms that are capable of degrading hazard- by restreaking. 5) The pure strains are main-
ous and persistent chemicals are also sought. tained as agar cultures in test tubes.
Of the 10,000 antibiotically active com- The screening procedure can often be
pounds known in the late 1980’s, 67% are pro-
speeded up by testing the initial isolates directly
duced by microorganisms (67% by actinomy- for biological activity. Some examples of proce-
cetes, 9% by other bacteria, and 15% by fungi). dures that can be carried out directly on agar
Additionally, about 2000 other biologically ac-
plates are given in Table 2.2. The soil or water
tive secondary metabolites are known, as well as
samples are diluted directly onto the test plates
a large number of enzymes.
and only those colonies showing activity are iso-
lated.
2.3 STRAINS USED IN SCREENING The variability of metabolites produced by
individual genera is somewhat limited except in
The success of a screening program depends on
the streptomycetes. For instance, except for in-
both the kinds of organisms used and the meth-
dustrial enzymes, Bacillus strains almost exclu-
ods for detection of activity. Currently, the choice
of strain has a 30-40% influence on the outcome, sively produce peptide antibiotics.
In one extensive study, 20,000 Actinoplanes
the test procedure a 60-70% influence.
A gram of soil contains between 10°-108 bac- strains were isolated, of which 13,000 were
teria, 10*-10° actinomycete spores, and 107-104 screened for the formation of antibiotics. Within
fungal spores. Less than 1% of the world’s mi-
croorganisms have been intensively studied. Table 2.1 Enrichment of microorganisms by selection
Above all, the approximately 100,000 known of appropriate culture conditions
fungi have been poorly studied, so that a vast Enrichment methods Type of isolate
number of new natural products can be expected Extreme pH values (pH 2-4) Acidophiles
from this group in the future. Low temperatures (4-15°C) Psychrotrophs
In the isolation of new metabolic products, High temperatures (42-100°C) Thermophiles
High NaCl concentrations Nocardia, halophiles
researchers try to isolate strains from extreme or N, atmosphere Anaerobes
unusual environments in the hope that such Chitin as growth substrate Lysobacter
strains may be capable of producing new metab- Wood bark, roots Myxobacteria
Pollen grains Actinoplanes
olites. For instance, microorganisms from high
'
 24 1EST SYSTEMS /°7
Table 2.2 Test systems for screening of metabolites _
meso-diaminopimelic acid, a component of the
Product sought Test system cell wall of bacteria, but did not inhibit the in-
Antibiotics Agar plates with strains of test corporation of leucine, an indicator of protein
microorganisms?. Inhibition synthesis. In the third phase, substances whose
zones as indicator of activity molecular weights were greater than 1000 were
G-Lactamase-resistant Agar plates with test
antibiotics microorganisms to which 6- eliminated by use of membranes, because larger
lactamase has been added molecules often elicit undesirable side effects
Proteases Agar plates with casein, when used therapeutically. With these three pro-
selection of colonies which
produce clear zones on the cedures, culture filtrates of 10,000 strains (fungi,
turbid plates bacteria and actinomycetes) were screened. A
Amylases Agar plates with starch, new antibiotic, azureomycin, was discovered,
selection of colonies after
staining with iodine and six known antibiotics were also reisolated.
Lipases Agar plates with oil emulsion, The antibiotic penicillin is a B-lactam, and
selection of colonies after one mode of resistance is through the production
precipitation of free fatty
acids with Ca?* of B-lactamase, an enzyme which splits the 6-
Phosphatases Agar plates with lactam ring. Inhibitors of 6-lactamase might thus
phenolphthalein-diphosphate prove useful in permitting penicillin therapy
and pH indicator, selection
based on color change against resistant organisms. For the screening of
NAD Agar with microorganism microbial 6-lactamase inhibitors, supernatants of
auxotrophic for NAD the cultures were placed on agar plates contain-
*For example, Staphylococcus aureus, Proteus vulgaris, Can- ing penicillin or cephalosporin and one of the 6-
dida albicans, Penicillium avellaneum, bacteria resistant to lactamase-producing microorganisms. Thus it
or hypersensitive to aminoglycoside, macrolides, or 6-lac-
tam antibiotics. could be determined during the first screening
whether different 6-lactamases could be inhib-
ited.
a ten year span, 41 new antibiotics were isolated Nozaki et al. (1987) used a screening proce-
and characterized from these strains. These anti- dure with #-lactam-hypersensitive mutants to
biotics turned out to be almost all either acetyl- isolate the antibiotic lactivicin, an antibiotic
malonyl or amino acid derivatives. which combines with the penicillin-binding site
of gram-negative and gram-positive bacteria
2.4 TEST SYSTEMS even though it lacks a 6-lactam ring.
Fleck and Strauss (1975) used molecular bi-
The success of a screening procedure is quite de- ology tests to discover an antitumor metabolite.
pendent on the development of “intelligent” Continuous fermentation can provide a
tests with which known or undesirable anti- method to isolate from mixed cultures strains
biotics can be eliminated and compounds with with improved properties. By raising the tem-
the required properties can be recognized. For perature or the alcohol concentration, microor-
instance, in one screening program (Omura et al., ganisms can be selected that are either thermo-
1979), a procedure was set up to discover inhib- philic or alcohol tolerant. Similar tests can be
itors of cell wall biosynthesis, because all known used to isolate strains that produce temperature-
antibiotics with this mode of action have low stable extracellular enzymes.
toxicity. In the first phase, screening was done
for metabolites which inhibited Bacillus subtilis,
but did not inhibit Acholeplasma laidlawti (which REFERENCES
lacks a cell wall). In the second phase, substances Box, S.J. 1985. Approaches to the isolation of an uniden-
were sought which inhibited the synthesis of tified microbial product. pp. 32-51. In Verrall, M.S.
8 / CHAPTER 2 / SCREENING FOR NEW METABOLITES

(editor), Discovery and Isolation of Microbial Products. lactam antibiotic to penicillin-binding proteins. Nature
Ellis Horwood Publishers, Chichester, U.K. 325; 179-180.
Cheetham, P.S.J. 1987. Screening for novel biocatalysts. Omura, S. 1986. Philosophy of new drug discovery. Mi-
Enzyme and Microbial Technology. 9: 194—213. crobiological Reviews 50: 259-279.
Elander, R.P. 1987. Microbial screening, selection and Omura, S., H. Tanaka, R. Oiwa, T. Nagai, Y. Koyana, and
strain improvement. pp. 217-251. In: Bu'Lock, J. and Y. Takahashi. 1979. Studies on bacterial cell wall in-
B. Kristiansen (editors). Basic Biotechnology. Academic hibitors, vol. VI, Screening method for the specific in-
Press, London. hibitors of peptidoglycan synthesis. J. Antibiotics
Fleck, W. and D. Strauss. 1975. Leukaemomycin, an anti- 32:978-984.
biotic with antitumor activity, vol. 1, Screening, fer- Vandamme, E.J. 1984. Antibiotic search and production:
mentation, and biological activity. Z. Allg. Mikrobiol. an overview. pp. 3-31. In Vandamme, E.J. (editor).
15:495-503. Biotechnology in industrial antibiotics. Marcel Dekker,
Lancini, C. 1980. Screening for new antibiotics. Lecture. New York.
Int. School of General Genetics: Microbial Breeding II, Verall, M.S. (editor). 1985. Discovery and isolation of mi-
June 3-13, 1980. Erice/Italy. crobial products. Ellis Horwood, Chichester.
Nozaki, Y., N. Katayama, H. Ono, S. Tsubotani, S. Harada, Zahner, H. 1979. What are secondary metabolites? Folia
O. Okazaki, and Y. Nakao. 1987. Binding of a non-6- Microbiol. 24:435-443.
 Strain
development

frequently the end result of complex, highly reg-

3.1 GENERAL
ulated biosynthetic processes, a variety of
With the exception of the food industry, only a changes in the genome may be necessary to per-
few commercial fermentation processes use wild mit the selection of high-yielding strains.
strains isolated directly from nature. Mutants or For a cost-effective process, strains with im-
recombinants which are specifically adapted to proved fermentation properties may also be
the fermentation process are used in the pro- needed. Depending on the system, it may be de-
duction of antibiotics, enzymes, amino acids, and sirable to isolate strains which require shorter fer-
other substances. The objective of a genetic strain mentation times, which do not produce undesir-
improvement program depends on the process. able pigments, which have reduced oxygen
In general, the major motivation for industrial needs, with lower viscosity of the culture so that
strain development is economic, since the me- oxygenation is less of a problem, which exhibit
tabolite concentrations produced by wild strains decreased foaming during fermentation, which
are usually too low for economical processes. are able to metabolize inexpensive substrates,
Through an extensive strain development pro- with tolerance to high concentrations of carbon
gram (which may require several years), yield or nitrogen sources, or with resistance to bacte-
increases up to 100 times or more can usually be riophage.
attained. The success of these programs depends Wild strains frequently produce a mixture of
greatly on the substance to be examined. For in- chemically closely related substances. Mutants
stance, the yield from products involving the ac- which synthesize one component as the main
tivity of one or a few genes, such as enzymes, product are preferable, since they make possible
can be increased simply by raising the gene dose. a simplified process for product recovery.
However, with secondary metabolites, which are Changes in the genotype of microorganisms can
10 / CHAPTER 3 / STRAIN DEVELOPMENT

lead to the biosynthesis of new metabolites. development program each method should com-
Thus, mutants which synthesize modified anti- plement the other. In the past, procedures for
biotics may be selected. achieving genetic recombination in industrially
One of the most significant approaches to important production strains hardly existed, so
strain improvement can be anticipated from the that the spectacular successes of strain devel-
use of recombinant DNA techniques. Bringing opment in industry are basically due to the ex-
together in one organism genes from several or- tensive application of mutation and selection. Ta-
ganisms has the potential for not only increasing ble 3.1 shows that the success of this method has
yields but also for producing entirely new sub- been impressive. However, current data on the
stances. Perhaps of even greater significance is production levels of industrial high-performance
the use of recombinant DNA techniques for the
mutants are rarely published, so that the data in
production by microbes of nonmicrobial prod-
Table 3.1 may not show the full extent of de-
ucts, such as insulin, somatostatin, human
velopment.
growth hormone, virus vaccines, and interferon.
Although yields of penicillin in the early days
However, one difficulty in applying the
newer genetic techniques to improvement of ex- of industrial production were less than 100
isting processes is that the organisms of widest units/ml, today the penicillin yield is around
use in industry are unfortunately not the orga- 85,000 units/ml (approx. 50 g/1). Because of the
nisms for which the greatest amount of basic ge- low yield per weight of substrate used (weight
netic information is available. There is an ap- of penicillin produced per weight of glucose used
parent gap between basic research and industrial is around 0.12), continued increases can be ex-
application. Biosynthesis and regulation, along pected in the future. The success of these purely
with the genetic fundamentals of industrially im- empirical strain development programs depends
portant microorganisms, must be understood be- on an optimal use of mutagenesis procedures in
fore the combination of empirical procedures cur- combination with an effective system for select-
rently used can be replaced by appropriate new ing high-yielding strains.
approaches. In the last ten years in several in-
dustrial firms, steps to bridge the gap between
basic knowledge and industrial application have Spontaneous and induced mutations
been made, and we can anticipate a marked in- Mutations occur in vivo spontaneously or after
crease in the effectiveness of strain improvement induction with mutagenic agents. Mutations can
programs. Protoplast fusion, site-directed muta- also be induced in vitro by the use of genetic
genesis, or recombinant DNA methods are ex- engineering techniques. The rate of spontaneous
amples of the use of newer technologies which mutation depends on the growth conditions of
have been especially useful in the production of
the organism and is between 107" and 10-5 per
primary metabolites such as amino acids, but are
generation and per gene; usually the mutation
also finding increasing use in strain development
programs for antibiotics.
In the present chapter we present the fun- Table 3.1 Increase in antibiotic production through
mutation and selection between 1943-1961
damental genetic approaches to strain improve-
ment and show how these approaches are used Antibiotic Productivity at Productivity of
in practice. time of discovery high yield mutants
units/ml units /ml
Penicillin 20 (1943) 8000 (1955)
3.2 MUTATION Streptomycin 50 (1945) 5000 (1955)
Erythromycin 100 (1955) 2000 (1961)
Introduction Chlortetra- 200 (1948) 4000 (1959)
cycline
Changes in the genotype are caused by mutation Oxytetracycline 400 (1950) 6000 (1959)
and genetic recombination. In a balanced strain (Alikhanian, 1962)
 3.2 MUTATION / 11

rate is between 107 and 10, All mutant types Although genome mutations are important in
are found among spontaneous mutations, al- plant genetics, mutations used in microbial strain
though deletions are relatively frequent. The improvement usually are point mutations; chro-
causes of spontaneous mutations which are thus mosome mutations also occur (e.g. deletions, du-
far understood include integration and excision plications) but are of minor significance.
of transposons, along with errors in the func-
tioning of enzymes such as DNA polymerases,
Repair mechanisms
recombination enzymes, and DNA repair en-
zymes. Because of the low frequency of spon- Mutant formation is a complex process. Premu-
taneous mutations, it is not cost-effective to iso- tational lesions of DNA occur spontaneously or
late such mutants for industrial strain through the action of mutagenic agents; only part
development. The mutation frequency (propor- of these result in stable mutants during subse-
tion of mutants in the population) can be signif- quent replication. The following structural
icantly increased by using mutagenic agents changes occur in DNA:
(mutagens): it may increase to 1075-10 for the + Pyrimidine dimers, in which two adjacent py-
isolation of improved secondary metabolite pro- rimidines on a DNA strand are coupled by
ducers or even up to 10°7-10" for the isolation additional covalent bonds and thus lose their
of auxotrophic mutants. ability to pair.
Spontaneous and induced mutants arise as a e Chemical changes of single bases, such as al-
result of structural changes in the genome: kylation or deamination, thus causing
changes in the pairing properties of the DNA.
«+ Genome mutation may cause changes in the ¢ Crosslinks between the complementary DNA
number of chromosomes. strands, which prevent their separation in
e Chromosome mutation may change the or- replication.
der of the genes within the chromosome, e.g. ¢ Intercalation of mutagenic agents into the
by deficiency, deletion, inversion, duplica- DNA, causing frameshift mutations.
tion, or translocation. e Single-strand breaks.
¢ Gene or point mutations may result from ¢ Double-strand breaks.
changes in the base sequence in a gene.
These premutational structural changes can
lead to mutations directly by causing pairing er-
Most information is available about point rors in replication or indirectly by error-prone
mutations. Through base substitution, a base pair repair in the next following round of DNA rep-
in the wild type allele may be replaced by an- lication.
other base in the mutant allele. Several kinds of Repair systems play a significant role in the
changes are recognized. A transition is an ex- mutation process. As a result of repair, poten-
change of a purine with another purine or a py- tially lethal changes in the DNA may be elimi-
rimidine with another pyrimidine. A transver- nated. If the repair systems function in an error-
sion refers to the substitution of a pyrimidine free manner, potentially mutagenic lesions are
with a purine or vice versa. One characteristic of eliminated before they can be converted into final
point mutants is that they can revert. Another mutations. A number of repair systems have thus
category includes those called frameshift mu- far been discerned in microorganisms, particu-
tations, which result when one nucleotide or larly bacteria. In the following, these repair
more is inserted or deleted, thus altering the mechanisms are discussed briefly.
reading frame in the following transcription and
translation processes, and leading to a changed Photoreactivation Short-wavelength ultraviolet
amino acid sequence in the resulting protein. irradiation (254 nm) affects DNA in a number of
12 / CHAPTER 3 / STRAIN DEVELOPMENT

elebekeabeleE
ways, but a well-established action is the for- GYyVA RSIK CE FASAG
mation of thymine dimers, a state in which two
adjacent thymine molecules are chemically
joined, so that replication of the DNA cannot UV Irradiation |

occur. When such an ultraviolet-irradiated pop-

ulation is subsequently exposed to visible light
of a wavelength of 300 to 450 nm, the survival
shake ahha
rate increases and the frequency of mutation de-
UV Endonuclease |
creases. This is due to the activation of a pho-

before.
hebe
toreactivating enzyme (photolyase) which splits GA T=C
A G
thymine dimers. In the dark, this enzyme bonds.
with thymine dimers; in the presence of light the
5’-Endonuclease >
enzyme splits the dimers into monomer pyrim-
idines. Up to 80% of the thymine dimers existing Nae

hepti &
GA Q G
in the genome can be photoreactivated. UV-in-
duced DNA crosslinks can also be photoreacti- --.[SPJOH P Pale

vated. This repair system functions in an error- DNA Polymerase |

free manner and thus does not allow mutations
to occur. Gon Are TERE ING)

_.habe} ron fete ==

Excision repair In contrast to photoreactivation,
which is possible with single-stranded DNA, the Polynucleotide
complementary strand is required for excision re- ligase

~bebebebebele-
pair. In a dark reaction, damages to the DNA,
such as ultraviolet-induced pyrimidine dimers,
alkylation, or deamination, are recognized by
specific DNA endonucleases. Different repair
Figure 3.1 Repair of DNA containing pyrimidine dimers
paths are involved according to each DNA lesion. by nucleotide excision
Excision repair can be partially prevented by in-
hibitors such as caffeine, acriflavin and 8-meth-
oxypsoralen. of the pyrimidine dimer and 3’ hydroxyl and 5’
The mechanism of excision repair for ultra- phosphoryl] termini are produced. With the help
violet induced lesions is different than that for of a 5’ exonuclease and DNA Polymerase I, a 6-
lesions induced by alkylation or deamination. In to 7-nucleotide-long oligonucleotide is cut out
one mechanism of repair of ultraviolet lesions, a along with the dimer and the resulting gap is
nucleotide-excision repair mechanism is oper- expanded to approximately 30 nucleotides. The
able. In effect, defective nucleotides are cut out missing nucleotides are filled by DNA polym-
and replaced, according to the mechanism illus- erase I (polA) starting from the 3’ end and are
trated in Figure 3.1. For this mechanism to op- connected by a polynucleotide ligase (lig). This
erate, the normal DNA replication process is not repair mechanism is almost error-free and mu-
required. tation is thus avoided.
In some bacteria, an ATP-dependent endonu- Another mechanism for repair of ultraviolet
clease has been demonstrated in ultraviolet-ex- damage is termed recA-dependent repair. This
posed cells. This endonuclease is controlled by second repair path requires DNA replication and
three genes, uvrA, uvrB and uvrC. The endonu- seems to be mutagenic since large gaps of up to
clease splits a phosphodiester bond on the 5’ side 1500 nucleotides are cut out. For this repair
 3.2 MUTATION / 13

mechanism to function recA, recB, recC, lexA, DNA strand. The sequence following is similar
uvrD and polC genes are needed. This type of to nucleotide excision repair, but it has not yet
repair is a form of the so-called SOS response been determined whether identical enzymes are
(see later). involved. Uracil DNA glycosylase and hypoxan-
Excision repair of alkylated and deaminated thine DNA glycosylase, both involved in the re-
DNA operates in a different manner. In this re- pair of deaminated DNA, have been found in
pair path, modified bases are recognized and cut various bacteria.
out. It is therefore also knownvas base excision
repair (Figure 3.2). In alkylated DNA of Micro- Postreplicative recombination repair If a DNA
coccus luteus it has been shown that the 3-meth- strand contains a lesion which hinders base pair-
yladenine formed is recognized by an N-glyco- ing, a gap in the daughter strand of approxi-
sylase, which splits the N,C'1 bond between the mately 1000 nucleotides is formed during rep-
base and deoxyribose, so that a purine-free site lication. According to Figure 3.3, which is
is formed in the DNA. 7-Methylguanine is tol- partially hypothetical, these gaps are filled with
erated by the cells so that formation of this al- material from the parent strands through recom-
kylated base is not mutagenic. The lesion is rec- bination processes (by the action of the recA gene
ognized by a special AP endonuclease (AP = product). The repair of the parent strands occurs
apurinic or apyrimidinic site), which splits the through repair replication with the daughter
strands as a matrix by means of DNA Polymerase
I. Several replication and recombination stages
are involved in this repair process.
A
T ae ee
Nerd Ne a

ahah ålalis Egpte PÅBE = SS

SE ar oN PS ae ea
Alkylation | | Deamination

eit cag la ee cl fa aa ERR

N-Glycosylase Uracil(HX)-DNA-
Glycosylase
FA U (HX)
ZG TAS C G
__trbebebebete-
AP-Endonuclease
*G-T A’ C G
KOHL KK
-PRYPLPL PPPYR--
Mæn
Exonuclease PS

AG TTA ke G

aa Ron pJP--
DNA-Polymerase |
XCM yA C A G
OHK Do i
_bebebel the ee
DNA Ligase —— eu 7

2G EAM TG YSN aN ÆRE err

--./RPR KR ÅRARIER
Figure 3.3 Model of post-replicative repair. a. DNA dou-
Figure 3.2 Base excision as a mechanism for the repair ble strand with premutative lesions (__/\_); b. Gaps in
of alkylated DNA (I) or deaminated DNA (II) with uracil daughter strands which have resulted during replication;
(U) or hypoxanthine (HX). *G = 7-methylguanine; “A = c. and d. Hypothetical exchange of parent strand DNA
3-methyladenine and replication repair (Smith, 1978)
14 / CHAPTER 3 / STRAIN DEVELOPMENT

“SOS” Repair Photoreactivation, excision re- induced condition, the product of the lexA gene
pair, and postreplicative recombination generally binds to the identical operator sequence of sev-
operate as error-free mechanisms. Error-prone— eral unlinked genes: recA, umuDC, and uvrA). Af-
and therefore mutation-inducing—methods also ter initiation by a regulatory signal, several SOS
exist, of which the best known is the SOS-repair functions are derepressed (for example, DNA ex-
system of E. coli. Overlapping gaps, such as the onuclease V), and DNA replication can restart at
ones which can result in errors in both comple- the chromosomal origin. The repair is performed
mentary strands in the replication of DNA, by a DNA polymerase which differs from the
would be lethal for the cell, but are filled by the constitutive DNA polymerases I, II and III in that
SOS repair despite the absence of DNA template it continues to carry out DNA synthesis in spite
(Figure 3.4). Thus the chemical structure of DNA of the lesion in the template, which takes place
is reconstructed, but the heredity information is mainly when ”false” bases are incorporated. It
defective; as a result, SOS repair very likely re- is assumed that a new polymerase is not induced
sults in mutations. In contrast to the constitutive but rather a factor is formed which lowers the
repair systems thus far described, the SOS repair proof-reading of one or more DNA polymerases.
activity is inducible, being repressed in untreated
wild type cells. Exposure to ultraviolet radiation Adaptive repair In Escherichia coli, increased re-
or the action of other mutagens which damage sistance to the mutagenic and lethal effects of
DNA or cause an inhibition of replication (such high doses of alkylating agents has been found
as treatment with the antibiotic mitomycin C) to occur after lengthy treatment with sublethal
induce the mechanism of SOS repair. In the un- concentrations of such agents. This effect, which

A B

Figure 3.4 SOS repair in Escherichia

Aa 3. Ba coli (based on Witkin, 1976)
A. Excision repair: a. DNA with py-
rimidine dimers (__/\__). b. Excision.
c. Repair replication stops at the sec-
sg ond dimer. Exonuclease activity con-
Ab. : z tinues. d. SOS repair is induced. It po-
ÅD SGD Bb lymerizes the DNA above and

|
beyond the dimer and causes a mu-
tation through erroneous incorpora-
tion (x). e. The second dimer is re-
a paired through nucleotide excision so
Ac 2
that both DNA strands carry the er-
ror.
B. Post-replication Repair: a. DNA
with pyrimidine dimers; b. During
DNA replication, gaps result in the
Ad daughter strands, which overlap and
cannot be closed by recombination re-
pair. c. SOS repair is induced, as in
Ad; d. The second gap is closed by
recombination. In the following rep-
lication, a daughter strand produces
overlapping gaps again. See Bb; e.
The second gap is also closed through
Be SOS polymerase
 3.2 MUTATION / 15

cannot be elicited by ultraviolet radiation, can bé ultraviolet radiation (UV). The wavelengths ef-
traced to a further inducible repair system. The fective for mutagenesis are between 200-300 nm
number of alkylated bases, particularly O*-meth- with an optimum at 254 nm, which is the ab-
ylguanine, in the genome is reduced by this sorption maximum of DNA. The most important
adaptive repair mechanism which works almost products of UV action are dimers (thymine-thy-
without error. In this way, the synthesis of an mine, thymine-cytosine and cytosine-cytosine;
O%-methylguanine DNA methyl transferase is in- Figure 3.5) formed between adjacent pyrimidines
duced. In uninduced E. coli between 13-60 mol- or between pyrimidines of complementary
ecules of this methyl transferase are present strands, which results in crosslinks. Ultraviolet
whereas induced cells contain more than 3000 radiation mainly induces transitions of GC — AT;
molecules. Another enzyme, 3-methyladenine transversions, frameshift mutations and dele-
DNA glycosylase II, is involved in the break- tions are also found.
down of 3-methyl adenine, 3-methyl guanine, 7- During the repair of ultraviolet lesions, up to
methyl adenine, and 7-methyl guanine. In the 1000 pyrimidine dimers can be repaired, and
induced cell the glycosylase content is increased with the exception of adaptive repair all repair
by a factor of 20. The mechanism of the induction systems are involved. To increase the frequency
of these enzymes is not understood. The capacity of mutation, the error-free mechanisms of pho-
of this repair system is limited however. O%- toreactivation and excision repair must be pre-
methylguanine accumulates when larger muta- vented by carrying out all manipulations under
gen doses are used, and the mutation frequency long-wavelength visible light (> 600 nm) and/
is directly proportional to the O%-methylguanine or through the use of caffeine or similar inhibitors
concentration. The enzymatic mechanism in- of repair. The SOS repair system is primarily re-
volved in the elimination of the compound is not sponsible for the production of mutations.
yet understood.
Long-wavelength ultraviolet radiation Radiation
Reaction mechanisms of mutagens at wavelengths of 300-400 nm has less lethal and
mutagenic effects than short-wavelength UV.
Many mutagens induce more than one type of However, if the exposure of cells or bacterio-
potentially mutagenic lesion. Thus, they fre- phages to long-wavelength UV is carried out in
quently cause mutation directly as a result of the presence of various dyes which interact with
pairing errors and indirectly as a result of errors DNA, greater death rates and increased mutation
during the repair process. In the following, the frequency result. Especially effective activators of
most commonly used mutagens are listed, to- long-wavelength UV are the psoralen deriva-
gether with their molecular reaction mecha- tives. 8-Methoxypsoralen (Figure 3.6, Structure
nisms. Detailed descriptions can be found in ref-
erences cited at the end of this chapter.

tere
Mutagenesis through radiation HN SN
Both ultraviolet radiation and ionizing radiation
are used in mutagenesis studies. The mecha-
nisms of mutagenesis are quite different for each
type of radiation, however. R R
Short-wavelength ultraviolet One of the more Figure 3.5 Thymine-cytosine-cyclobutane dimer, the
effective mutagenic agents is short-wavelength photoproduct formed as a result of ultraviolet radiation
16 / CHAPTER 3 / STRAIN DEVELOPMENT

sorption of a second photon causes the coupling

of the pyrimidine-psoralen monoadduct with an
additional pyrimidine. Biadduct formation be-
tween complementary strands of nucleic acid re-
OMe
sults in crosslinks. These lesions cannot be pho-
toreactivated, although they are eliminated
B CH;-0-S02-CH; through nucleotide excision repair in conjunction
oO CH,— CH,- 0 -SO,-CH, with the mutation-causing SOS repair system.
D CH, CH, 0-S0,- 0 -CH,—CH,
Ionizing radiation Jonizing radiation includes X-
rays, y-rays, and 6-rays, which act by causing
BOR GORE
20 "CbGS=0H-C0H-3C0R, ionization of the medium through which they
pass. These rays are usually used for mutagenesis
only if other mutagens cannot be used (e.g. for
E Ab GERNeale
cell material impenetrable to ultraviolet rays).
O=N—-N—
CHz Single- and double-strand breaks occur with a
G CH.N, significantly higher probability than with all
other mutagens. Ninety percent of the single-
strand breaks are repaired by nucleotide excision;
4 Os sp HED
the recA-dependent repair mechanism is also in-
O=N—N-CHh, volved. Double-strand breaks result in major
structural changes, such as translocation, inver-
| ClCH Ore -S eH CHE
sion or similar chromosome mutations. Therefore
ultraviolet radiation or chemical agents are nor-
i
ai
Br mally preferable for mutagenesis in industrial
strain development.
07 ™N
H
Mutagenesis with chemical agents
| SS A variety of chemicals are known which are mu-
K Dee 2 yds tagenic, and these may be classified into three
groups according to their modes of action:
Beer Bort.
+ Mutagens which affect nonreplicating DNA
Figure 3.6 Structure of different mutagens ¢ Base analogs, which are incorporated into
A. 8-Methoxypsoralen; B. Methylmethanesulfonate
(MMS); C. Ethylmethanesulfonate (EMS); D. Diethylsul-
replicating DNA due to their structural sim-
fate (DES); E. Diepoxybutane (DEB); F. N-Methyl-N’-ni- ilarity with one of the naturally occurring
tro-N-nitrosoguanidine (NTG); G. Diazomethane; H. N- bases.
Methyl-N-nitrosourea (NMU); I. Di-(2-chlorethyl)-sulfide
(mustard gas); J. 5-Bromouracil (BU); K. Acridine orange
e Frameshift mutagens, which enter into DNA
(AO) during replication or repair and through this
intercalation cause insertion or deletion of
one or a few nucleotide pairs.
A) intercalates between the base pairs of double-
stranded DNA and after the absorption of long- Chemicals which affect nonreplicating DNA A
wavelength UV, an adduct is formed between the number of chemicals are known which cause di-
8-methoxypsoralen and a pyrimidine base. Ab- rect damage to nonreplicating DNA. Nitrous
 3.2 MUTATION / 17

acid (HNO,) deaminates adenine to hypoxan- ization and pairs then with adenine, so that
thine and cytosine to uracil. Through the through hydroxylamine action GC—AT transi-
changed pairing properties of the deamination tions are caused.
products (hypoxanthine pairs with cytosine, ur- Another group of chemicals affecting non-
acil with adenine) AT — GC and/or GC — AT replicating DNA are the alkylating agents. Ex-
transitions occur. In addition, nitrite induces cept for ultraviolet radiation, alkylating agents
crosslinks between the complementary strands. are the most potent mutagenic system for prac-
Figure 3.7 shows the establishment of the mu- tical application. Compounds frequently used in-
tation after two generations. Excision and recom- clude ethyl methanesulfonate (EMS), methyl
bination repair are involved in the elimination of methanesulfonate (MMS), diethylsulfate (DES),
deamination products. Besides point mutations, diepoxybutane (DEB), N-methyl-N’-nitro-N-ni-
deletions occur relatively frequently with nitrous trosoguanidine (NTG), N-methyl-N-nitroso-urea
acid treatment. and mustard gas (see structures B-I, Figure 3.6).
Hydroxylamine (NH,OH) reacts with pyrim- Transitions, transversions, deletions and frame-
idines, but only the reaction with cytosine is mu- shift mutations occur as a result of the action of
tagenic, whereby the amino group is replaced alkylating agents.
with a hydroxylamino group. The hydroxyl- Mutagenesis with alkylating agents occurs
amine derivative from cytosine shows tautomer- via various pathways. These compounds cause
the formation of a whole spectrum of alkylated
bases in DNA, along with phosphotriester, pu-
NH» OH rine-free sites and single-strand breaks. Although
N= | N HNO, ou 7-alkylguanine is in all cases the most common
alkylation product, it does not result in muta-
by Sl Sn &
H H tions. O%-alkylguanine and O*-alkylthymine are
the most important premutational lesions, and as
a result of pairing errors, mainly AT — GC tran-
sitions are elicited (direct mutagenesis). A second
process which also results in mutation is the in-
oud = i}
duction of error-prone SOS repair (see above)
when relatively high doses of mutagen are used.
It has been suggested that the occurrence of

Il 0, 1 LY so Ål mutations in Escherichia coli is dosage-dependent

Haar i aay in relation to its repair system: At a low level of

alkylation of DNA the constitutive error-free sys-
a ER Me en tems perform the repair and mutations seldom

Kleen ree peo occur. At higher mutagenic doses, on the other

| | || | hand, the performance of the constitutive repair

systems is not sufficient and the adaptive repair
Va oe oad es enzymes are induced. At even higher doses, the

TL ! asolaser Aylmer et enzymes involved in SOS repair are also in-

(EL | Siaae
0—
— IE |
duced. Between the error-free adaptive repair
and the error-prone SOS system there is com-
Transition GC AT Transition AT —= GC
petition for the repair of DNA lesions. The fre-
quency of mutation is critically dependent upon
Figure 3.7 Mutation caused by nitrous acid which of these repair systems is working.
18 /CHAPTER 3 / STRAIN DEVELOPMENT

The use of N-methyl-N'-nitro-N-nitroso- Base analogs are of minor importance for

guanidine (NTG) in a mutation program is dif- practical application, because for industrially im-
ficult because of its carcinogenic effects, but it is portant strains which are poorly understood ge-
one of the most effective chemical mutagens. A netically it can be rather costly to set up the op-
large proportion of mutants is found under op- timal conditions for mutagenesis. As an example
timal conditions with a low killing rate. In Strep- of the difficulties, the incorporation of BU in
tomyces coelicolor, for instance, 8-10% of the sur- DNA takes place only if the organism is growing
vivors are found to be auxotrophs, and in under thymine deficiency. Thus, thymine auxo-
Escherichia coli up to 50% of the surviving pop- trophs are frequently used; however, use of such
ulation consists of mutants. Ninety percent of the auxotrophs can lead to complications in strain
mutations induced by NTG are GC — AT tran development, since in many cases auxotrophic
sitions; to a small extent deletions and frameshift mutation results in reduced production of the de-
mutations are also found as a result of the dele- sired substance.
tion of GC pairs.
The exact molecular reaction mechanism of Frameshift mutagens Frameshift mutagens in-
NTG is not yet understood. The compound is tercalate into the DNA molecule and cause errors
easily decomposed in vivo, and in acidic solu- which result in an alteration of the reading frame,
tions nitrous acid is formed. Although nitrous resulting in the formation of faulty protein or no
acid is a mutagen, it is not effective in the pH protein at all. The most commonly used frame-
range where NTG is active (pH 6—9); diazometh- shift mutagens are the acridine dyes, such as ac-
ane (Figure 3.6, Structure G), a strongly meth-
ridine orange (structure in Figure 3.6 K), pro-
ylating agent, is formed under alkaline condi- flavine and acriflavine. The induction of
tions. insertions or deletions is dependent on the ability
Besides the alkylation of nonreplicating of an acridine dye to become inserted between
DNA, the main point of action of NTG is at the two neighboring bases of a DNA strand. The size
of the acridine molecule, 3.4 A, is exactly equiv-
replication point of DNA, through a change in
alent to that of a mononucleotide. Intercalation
DNA polymerase III during DNA replication. In
occurs most likely in DNA segments with iden-
this process, there is incorrect duplication in a
tical base pairs in areas of strand irregularities,
short segment of the DNA until the defective
such as at the end of a chromosome, near the
polymerase is replaced by an intact molecule.
replication fork, or at a site undergoing recom-
This explains the observation that NTG muta-
bination. A proposed model for the mode of ac-
tions frequently occur in gene clusters.
tion is illustrated in Figure 3.9.
Although acridines are useful is research,
Base analogs Because of their structural simi- they are not very suitable for a routine isolation
larity, base analogs such as 5-bromouracil (BU, of mutants in strain development: although they
Figure 3.6 J) or 2-aminopurine (AP) are incor- are strong mutagens for bacteriophages T2 and
porated into replicating DNA instead of the cor- T4, they have little or no mutagenic effect in bac-
responding bases thymine and adenine. The an- teria.
alogs tautomerize more frequently than the
natural bases: BU in keto form pairs with ad-
enine, whereas BU in enol form pairs with guan- Further methods of mutagenesis
ine. If the keto form of BU is incorporated, there Strains used industrially are usually less well
is a AT — GC transition caused by tautomeri- characterized genetically than the organisms
zation; if the incorporation takes place in the enol commonly used in basic research and there are
forma GC — AT transition is caused (Figure 3.8). practically no applicable processes other than
 3.2 MUTATION / 19

SA A A
y,ae Use Oe ee
—A— se BBU == LO EET
Vax (ANE | vige
———S\——— Tautomerization == O=>
5] i oe
——BU.— Tautomerization BU,

AR Seerne
Figure 3.8 Mutation via action of 5-
bromouracil (BU). A. BU is incorpo- —C— \
rated in keto form (BU,); Tautomeri- —BU.— > - fh —
zation during replication causes an

ile cong Gath

Tautomerization Sf
AT — GC-transition. B. BU is incor-
N
se EAS
porated in enol form (BU.); Tauto-
—6—
merization during replication causes — Oo =—BU-—
a GC — AT-transition

mutagenesis with radiation or chemical agents. transversions, depending on the mutator gene.
For the sake of completeness, several interesting Three such mutator genes have been demon-
possibilities are mentioned here which have thus strated in Escherichia coli. Because of the high
far found only limited application. rate of mutation, the handling of such mutator
strains in production may present difficulties.
Mutator genes In Escherichia coli the frequency
of mutation can be increased by a factor of 100 IS-Elements, transposons, and bacteriophage Mu.
through the introduction of a mutator gene. The The occurrence of mutations through integration
cause of this effect is an error-prone DNA po- into DNA of prophages or IS-elements and tran-
lymerase which frequently makes mistakes copy- sposons is well known in Escherichia coli and
ing a template, resulting in either transitions or Salmonella typhimurium. IS-elements are DNA
sequences of variable length (800-1400 base
pairs) which can be incorporated in different sites
CGCAGCTTTTACCGAT
GCGTCGAAAATGGCTA
of the genome and released again. Integration
4 and excision take place in recA-independent re-
tand a
combination. This applies also to transposons
CGCAGCTTTTACCGAT CGCAGCTTTTACCGAT (genetic elements containing flanking [S-ele-
GCGTCG fac GGCTA GCGITCGA ATGGCTA
ments in inverse orientation, often with anti-
| Degradation
hain |
ct biotic-resistance genes) and the temperate bac-
CGCAGCTTTTAC Cc CGCAG TTACCGAT teriophage Mu.
aC)
GCGTCG AAATG G GCGTCGAATGGCTA
| I Mispairing These elements destroy the function of the
Resynthesis y gene at the site of their integration. The incor-
CGCAGCTTT
!

A7 Cc COCAG
() MAACE GAT poration of [S1 also has a polar effect on the genes
GCG T CGAAA pA G GC GTC GAATGG Cal
! 4 distal with respect to the promotor. The genes
Ligase Ligase
are barely or not at all transcribed, probably due
to the blocking of mRNA synthesis. [S2 bears a
Insertion of A Deletion of AA
promotor, which, when incorporated in the ap-
Figure 3.9 Possible mechanism for the production of propriate orientation, results in the constitutive
frameshift mutations expression of genes located downstream. More-
20 / CHAPTER 3 / STRAIN DEVELOPMENT

over, IS-elements cause chromosome abbera-

tions. In particular IS1 causes deletions, whereas
IS2 causes duplications. Transposon mutagenesis
is discussed in a later section.

Comutation and sequential mutagenesis

Nitrosoguanidine (NTG), which causes multiple
mutations at the replication point through its ef-
fect on DNA polymerase III, can be used to in-
duce mutations in certain sites of the genome by
means of a process called comutation or sequen-
tial mutagenesis. Mutants
survivors
million
per

Comutation When a mutation is induced in a

specific locus, a large number of further muta- Normalized replication time
tions, so-called comutations, may be found in
closely linked genes. In E. coli 40% of comuta- Figure 3.10 Sequential mutagenesis of Escherichia coli
TAU-bar (Cerda-Olmedo et al., 1968). Samples of a syn-
tions are concentrated in a region of about 50,000
chronized culture (25°C) were mutagenized at 5 min. in-
base pairs (about 1/60 of the genome). In Strep- tervals with nitrosoguanidine (0.1 mg/ml; pH 5.5) and
tomyces coelicolor the comutation region is about assayed for revertants to arg*, pro* and his*.
twice as large. By using a selective marker, such
as the reversion of an auxotrophic mutant, clones
maximum is characteristic of the position of a
can be isolated after NTG mutagenesis which
specific marker on the genetic map. Sequential
carry mutations in neighboring genes of the se-
mutagenesis has already been used in several or-
lective marker. In some cases this mutagenesis
ganisms. By this means, genetic maps can be
in a specific operon can be used in strain devel-
drawn up; moreover, specific genes can be mu-
opment, provided the genes controlling produc-
tated as desired, provided the time of replication
tion have been mapped.
is known.
Sequential mutagenesis The replication of cir-
cular chromosomes is a sequential process. In Directed mutagenesis
Escherichia coli, the replication point moves from
a fixed starting point O (origin) to an end point The methods of mutagenesis which have been
T (terminus) in a bidirectional fashion. In an ex- discussed up to this point are completely undi-
ponentially growing, nonsynchronized bacterial rected. The development of gene technology has
culture, the replication of individual chromo- led to revolutionary new methods which make
somes is quite varied. In synchronized DNA rep- it possible to isolate mutants of specific genes of
lication, a specific genome segment is replicated interest. Although the details of gene technology
in the vast majority of the chromosomes at a are discussed in Section 3.6, some of the ap-
specific time. If mutation is induced by use of proaches to use for mutagenesis purposes are dis-
NTG pulses and the mutation frequency of cer- cussed here.
tain markers is subsequently plotted against time,
maxima are found at certain times during a rep- Deletions A circular DNA molecule that has
lication cycle (Figure 3.10). The time between only a single recognition site for a particular re-
start of replication and the appearance of such a striction endonuclease is linearized when it is
 3.2 MUTATION / 21

treated with this restriction enzyme. The single- Transposon Tn5 contains the gene for resistance
strand regions at the site of cutting are then di- to an aminoglycoside antibiotic which can be ex-
gested with the specific nuclease S1, producing pressed in a wide variety of both procaryotes and
blunt-end fragments. When the linear molecule eucaryotes. Several transposons have been in-
is introduced into a cell, recircularization can oc- ‘tegrated into plasmids (for instance, Tn1 and
cur, although the shortened ends are generally Tn3), others in either plasmids or chromosomes
removed by a polynucleotide ligase (Figure 3.11), (for example, Tn5). Thus, transposons are avail-
thus leading to the formation of a small deletion. able for a wide variety of purposes in gene tech-
Larger deletions are obtained if the DNA con- nology.
tains two recognition sites for the restriction en- Transposon mutagenesis offers a wide variety
zyme. For example, after separation of the frag- of advantages. A mutant phenotype with a very
ments the larger fragment can be closed into a low reversion rate can be obtained. In addition,
ring and cloned. insertion mutations are relatively easy to isolate,
since the transposons contain antibiotic-resist-
Insertions By use of a linker-adaptor approach, ance markers. All one needs to do is plate on a
an adaptor DNA ora linker DNA can be inserted medium containing the antibiotic; only cells con-
at the recognition site where the restriction en- taining the transposon will be able to grow and
zyme has acted. An adaptor molecule is a chem- form colonies. The integration of a transposon
ically synthesized double-stranded DNA that can causes an interruption in transcription, so that
be used to connect together the ends of two DNA transposon mutagenesis exhibits a polar effect.
molecules. A linker molecule is similar but pos- Because of this, the site at which the transposon
sesses a recognition site for a restriction enzyme. has been integrated into the operon can be read-
Transposon mutagenesis is another method ily determined by assaying for enzyme activity
for inducing mutation via insertion. Transposons or measuring the accumulation of an interme-
are known in both procaryotes and eucaryotes diate product.
and can insert at arbitrary sites in the genome.
Point mutants at specific locations in the DNA
Mae Gace CmiCG Ge 3)
Bisulfite mutagenesis can be used to convert
8 = CGGCIGGANGCCG—5: cytosine residues in the single-stranded DNA
into uracil residues, since treatment of the DNA
ad) with sodium bisulfite causes deamination of cy-
Se GCC GC CH CGGEC—3) tosine. After synthesis of the complementary
3h EGG E GGAG CCG. strand, a transition from GC — AT results. The
production of single-strand regions can be
{ (2) brought about using certain restriction enzymes
Sa GiCEeG CEG 3: which, in the presence of ethidium bromide, split
3G Gie GCG. only a single strand of the DNA. The single-
stranded site can be extended by treatment of the
I @)
DNA with an exonuclease enzyme.
Si GeCGCGG C3: Nucleotide analogs can be incorporated in
3'—-CGGCGCCG~—S'
vitro into either RNA or DNA, using an enzyme
which will replicate the nucleic acid in a syn-
Figure 3.11 Directed mutagenesis by site-specific dele-
tion in the region of the recognition site for the restriction chronous fashion. Suitable nucleotide analogs in-
enzyme Bgll in the genome of virus SV40. 1. Cut with clude N‘-hydroxycytidine triphosphate (N*-hy-
enzyme Bgll. 2. Removal of the single-stranded region droxy-CTP) or N*-hydroxydeoxycytidine
through the action of the single-strand specific nuclease
S1. 3. Joining of the DNA ends by a ligase. triphosphate (N*-hydroxy-dCTP). An example of
22 / CHAPTER 3 / STRAIN DEVELOPMENT

the use of this procedure is shown in Figure 3.12. second strand is formed. The completed double-
N--hydroxycytosine is incorporated by means of stranded heteroduplex DNA, containing a single
an in vitro RNA synthesis using the RNA repli- incorrect nucleotide, is then incorporated into a
case from bacteriophage Q8. The incorporated cloning vector such as E. coli by transformation.
N*-hydroxycytosine pairs in the next round of After replication of the cloning vector, either a
synthesis with both G and A, leading to the for- mutant or wild type is obtained. If the result is
mation of GC — AT transitions. a mutant, the mutation is at the desired location
in the gene.
Oligonucleotide mutagenesis The availability of
synthetic oligonucleotides makes possible a very
Phenotypic expression of mutations
specific method for mutagenesis, since a nucleo-
tide in any desired position in the DNA sequence Many mutations which result in increased for-
can be substituted with any of the other three mation of metabolites are recessive. When a re-
nucleotides. First the source gene is cloned into cessive mutation takes place in a uninuclear, hap-
a single-stranded vector such as bacteriophage loid cell (e.g. bacteria and actinomycete spores,
M13. To the system is added a synthetic oligo- asexual conidia of fungi), a heteroduplex results
nucleotide of 15 to 100 bases which contains a from it (Figure 3.13a); the mutant phenotype can
sequence complementary to the region of inter- only be expressed after a further growth step.
est, but with one base that is mismatched. The This also applies to exponentially growing bac-
added oligonucleotide then serves as a primer for terial cells, which can contain 2-8 chromosomes
the action of DNA polymerase and a complete (Figure 3.13b); not until several steps in repro-

fmet lys
SS
fS REE fS

---GAAACUUUGGGUCAAUUUGAUC AUG GCA AAA UAAGAGA--- QB-(+)-Strand

(—)-Strand synthesis with OH-CTP

HO OH
ie
---ACUAG: UAC CGU UUU--- Substituted(—)-strand

(+)Strand-synthesis

fmet lys
(toeee

---UGAUC AUG GCA AAA --- Wild type

Mutants

---UGAUC AUA ACA AAA ---

Figure 3.12 Mutagenesis through the incorporation of hydroxy-CTP during the synchronized synthesis of RNA by the
RNA replicase of bacteriophage Qé. Based on Taniguchi and Weissman (1978)
 3.2 MUTATION / 23

a a
Heteroduplex

Mutants
“eso å Cod)
(OOO)
b~ @OOO
;

NER O000Q

Mitotic (FE)
recombination <=
C FDD
Diploid cells Meiosis or FE)
or dikaryon haploidization

e ee

d
Sensitive ce)
ribosomes a
istan
Resistant
ribosomes

cand
Figure 3.13 Phenotypic ex-
pression of mutants (Clarke,
1975). For explanation see text
Sensitive
cells with
receptors Se Resistant
cells

duction have taken place do pure mutant clones peating this segregation process. Another way for
appear. attaining segregation is the preparation of pro-
With the filamentous actinomycetes, special toplasts containing one or few nuclei.
procedures for mutant expression must be used. In diploid or heterokaroytic eucaryotes, re-
In the course of strain development, actinomy- cessive mutations are allowed to undergo phen-
cetes can lose their sporulation ability. To obtain otypic expression after meiosis, haploidization,
cells for plating, the heterokaryotic mycelium or mitotic recombination (Figure 3.13c).
which results from mutagenesis is grown and Delays in expression which are not directly
then fragmented by ultrasonic treatment or shak- the result of genetic effects are observed, such as
ing with glass beads. After filtration through pa- mutations which cause changed ribosomes (Fig-
per, cotton, or an 8 um membrane filter, myce- ure 3.13d) or mutations resulting in the loss of
lium fragments containing only one or a few surface receptors (as in the development of bac-
nuclear bodies are used for plating. Homokar- teriophage resistance, Figure 3.13e). In both
yotic material can be ultimately selected by re- cases, the wild type structures must be diluted
24 / CHAPTER 3 / STRAIN DEVELOPMENT

out during growth before the mutation is rec- of specific mutant types (such as auxotrophy)
ognizable phenotypically. might be overlooked.
In addition to these strain-specific factors, the
treatment conditions have a critical effect on
Optimizing mutagenesis
mutagenesis. Such factors as the pH, buffer com-
Although the molecular mode of action of some position, mutagen concentration, exposure time,
mutagens is quite well known, what can never temperature, and growth phase of the organism
be predicted is the effect of a mutagen on a spe- may greatly affect the efficiency of the process.
cific gene or the effect of a mutation on a complex By plotting dose-response curves (Figure 3.14),
process, such as the biosynthesis of a secondary all of these factors may be optimized. Action on
metabolite. The appearance of mutants, that is, DNA causes not only mutation, but also killing,
strains in which a mutation has phenotypically due either to irreparable damage to the DNA or
resulted in a change, depends on several factors. to formation of lethal mutations. Therefore mu-
¢ The appearance of mutations is dependent on tants are sought from among the few surviving
the base sequence of the gene to be mutated. members of the population which had been ex-
Mutations are not distributed evenly around posed to a strong mutagenic treatment (death
rate >99%), because there is thus a certain prob-
the genome; there are areas with high mu-
tation frequency, the so-called hot spots. Dif- ability that each of the surviving cells carries one
ferent mutagens cause hot spots at different or several mutations. A high death rate alone,
sites in the genome.
e The repair systems of the cell also play a
100
role. In strains with partially defective repair
mechanisms, organisms may be killed with-
out having induced mutations, so that specific
mutagens can be ineffective.
e A gene activity which has become lost
through mutation can be restored at least par-
tially through a second mutation, a suppres- 0.05M
sor mutation.
Suppressor mutations act in several different
ways. Suppressors can occur in the same gene ai
ro)
@
that already carries the primary mutation (intra- =
2
genic suppressors). The primary missense mu- =! 4
Or
tation is compensated through the exchange of S e

an amino acid or an additional deletion or in-

sertion which corrects a primary frameshift mu- me e

tation so that the reading frame remains intact.

] 0.2M 01M
Suppressor mutations which occur in another
gene (extragenic suppressors) compensate the
primary mutation particularly at the level of
translation, by the formation of mutant transfer OOl dies. : ee
5 10 15 20 25 30
RNAs or ribosomes. In strains with suppressor
Incubation time (min)
mutations, the function of the enzyme in ques-
tion is restored up to 10% of the wild type ac- Figure 3.14 Killing of Micromonospora inyoensis by ni-
tivity. This is enough activity so that the existence trous acid
 3.3 SELECTION OF MUTANTS / 25

however, is no guarantee of the occurrence of costly, easily detectable changes such as muta-
mutations in specific genes. These mutations can tions for resistance or reversion to auxotrophy
only be reliably determined by assessing quali- are frequently used to optimize conditions for
tatively or quantitatively changes in the product mutagenesis. However, the results of these latter
of this gene. In the case of an antibiotic, pro- experiments need not have any bearing on op-
duction should be considered as a criterion for timal conditions for increased formation of a de-
mutagen influence. To assess this, a random se- sired product.
lection of survivors in a population treated with a

mutagens is assayed for antibiotic formation in

3.3 SELECTION OF MUTANTS
laboratory fermentations. The antibiotic titers of
mutagen-treated isolates are plotted in a histo- Besides an optimal mutagenesis, the method of
gram and compared to a control group. Figure selection is crucial for the effective screening of
3.15 clearly shows the differences in the varia- mutants. Looking for a desired mutant is anal-
bility of the population according to mutagen ogous to the famous “search for a needle in a
concentration. Since studies of this type are quite haystack’’. There are basically two ways of

Nitrous acid
0.2M
pH 4.5
10 20 min

Frequency
E
2]
(9
PO
@

Nitrous acid
0.1M
pH 4.5
20 min

Control
pH 4.5

Figure 3.15 Histogram of sisomicin

production by Micromonospora in- T mat =TE
yoensis (control strain = 100%) in re-
lation to mutagen treatment with ni- 50 100 150
trous acid Relative Sisomycin formation (%)
26 / CHAPTER 3 / STRAIN DEVELOPMENT

screening. A random selection of survivors from quired for a mutation-selection cycle, the avail-
a mutagenized population can be examined for able test capacity of the screening program, and
antibiotic production or other properties in a fer- the accuracy of the screening test (e.g. antibiotic
mentation process that closely mimics the large- assay). As a rule, mutants with high yields are
scale process. This procedure is very costly, but much rarer than those with only slight improve-
is often the only way to find mutants with in- ments. Moreover, the variability of mutagen
creased productivity in industrial strains. Wher- treated populations is quite high even when mu-
ever possible, a screening method is used in tagenesis is performed under identical condi-
which selective conditions are chosen which pro- tions. Thus it is usually more economical to
mote the growth or early detection of the mu- screen a small number of survivors (about 20-
tants. 50) after many different mutagen treatments and
to continue mutating strains having small yield
Random screening increases as quickly as possible, than it is to test
a large number of isolations after a few mutagen
The high yields obtained with industrial micro- treatments and to hope for a one-step large yield
organisms have been possible largely through increase.
the process of empirical selection after mutagen- The number of strains which must be
esis. After mutagen treatment and expression of screened to obtain mutants with a yield increase
induced mutations, a random selection of sur- depends on the strain, the conditions of muta-
viving clones is inspected for ability to produce genesis, the biosynthesis pathway, and the reg-
the product of interest. This is done in model ulation of the product which is being optimized.
fermentations which are carefully adapted to the
Normally, several hundred to several thousand
medium and fermentation parameters of the
isolates per mutation cycle must be tested. In
large-scale procedure, in order to maximize the
practice, with nonautomatic methods the number
likelihood that the strains will be suitable for in-
of isolates that can be tested per unit time is usu-
dustrial production. The best strains from such a
ally limited to 1000-2000 per week. Thus the
mutation cycle are repeatedly mutated and se-
screening capacity determines the speed of the
lected. A gradual increase in the yield is attained
progress to be expected. In the first stage of mu-
by continuing with these steps. In this mutation
tant screening, only one fermentation sample per
and selection program, study does not center
only on the strain exhibiting the best yield. This isolation is usually assayed, provided that the test
is because multiple mutations usually occur due error is smaller than than the yield increase ex-
to the high mutagen doses and in the course of pected. The best isolates of the first series (usu-
strain development these unrecognized muta- ally 10-30%) are then tested in a second fer-
tions can cause certain strains to show no rise in mentation. Since the best strains of this second
productivity. Therefore, depending on the ca- screening are then used in a still further mutation
pacity of the screening program, the 5-10 best cycle, the yield increase must be statistically sig-
strains of a mutation-selection cycle should be nificant when compared to the parent strain. The
used as parent strains for future mutagenesis. number of replicates from the reference strain
These strains are normally treated with mutagens and the mutants should be chosen statistically.
different from those used in the initial isolation. An optimal increase in yield per test period can
Many factors determine how many isolates must then be calculated, if the number of isolates re-
be screened to obtain strains with increased pro- quired to attain a specific yield increase can be
ductivity. Factors which influence the size of the tested within the time period needed for muta-
screening program are: frequency of mutation, genesis, colony selection, and assay of the iso-
extent of yield increases, the amount of time re- lates.
 3.3 SELECTION OF MUTANTS / 27

Several industrial companies are developing an excess of metabolites, in some cases through
ways to automate mutant screening procedures changed regulatory mechanisms (elimination of
to increase the screening capacity. allosteric inhibition; constitutive product forma-
tion). Table 3.2 shows some antimetabolites fre-
Selective isolation of mutants quently used in screening programs.
Several examples of the many selective methods
Isolation of auxotrophs By using certain blocked
used in strain development are mentioned here.
mutants, desired products such as amino acids
Isolation of resistant mutants A high cell density and nucleotides may be formed via branching
of a mutagenized population can be plated on a biosynthetic pathways (see Chapters 9 and 10).
selective medium containing a concentration of Auxotrophic mutations in antibiotic-producing
a toxic substance that prevents the wild type from organisms frequently result in reduced product
growing. Only the resistant clones can develop. formation. Improved strains can be obtained in
In this way, mutants may be isolated which are some cases (e.g. in tetracycline) by isolation of
resistant to antibiotics or antimetabolites. The prototrophic revertants (suppressor mutants)
antibiotic resistance character can not only be from auxotrophs. In addition, auxotrophic mu-
used as a genetic marker, but mutants isolated tations can frequently be used as genetic markers.
may also have an increased cell permeability or The isolation of auxotrophs is done by plating
a protein synthesis with a higher turnover, mak- of the mutagenized population on a complete
ing them useful for industrial purposes. agar medium, on which the biochemically defi-
Antimetabolite resistance can be used to se- cient mutants can also grow. By means of Led-
lect mutants which exhibit defective regulation. erberg’s well-known replica plating technique,
Altered regulation may occur in such mutants. the clones are transferred to minimal medium
Antimetabolites, because of their structural sim- where the auxotrophic colonies cannot grow.
ilarity to metabolites, may cause feedback inhi- These mutants are picked up from the master
bition, but are unable to substitute for normal plates and their defect is characterized. Since in
metabolites. Antimetabolites cause death of nor- this method a large number of plates must be
mal cells, but analog-resistant mutants can form observed, various procedures have been devel-

Table 3.2 Frequently used antimetabolites

Natural metabolite Antimetabolite Natural metabolite Antimetabolite

Adenine Psicofuranine Leucine 5,5,5-Trifluoroleucine

2,6-Diaminopurine 4-Azaleucine

Guanine 8-Azaxanthine Methionine a-Methylmethionine

Norleucine, ethionine
Uracil 5-Fluorouracil
Phenylalanine p-Fluorophenylalanine
p-Aminobenzoic acid Sulfonamide Thienylalanine
Nicotinic acid 3-Acetylpyridine Proline 3,4-Dehydroproline
Pyridoxine Isoniazid Tryptophan 5-Methyltryptophan
6-Methyltryptophan
Thiamine Pyrithiamine
Tyrosine p-Fluorophenylalanine
Arginine Canavanine
Threonine 6-Hydroxynorleucine
Histidine 2-Thiazolalanine
1,2,4-Triazol-3-alanine Valine a-Aminobutyric acid
Isoleucine
28 / CHAPTER 3 / STRAIN DEVELOPMENT

oped to enrich for auxotrophic mutants by re- rectly in colonies growing on plates by spraying
moving or killing prototrophic organisms. In a with suitable reagents or by incorporating indi-
process known as filtration enrichment, after cator dyes into culture medium. Antibiotically ac-
mutagenesis the spores of filamentous organisms tive substances may be detected by measuring
(actinomycetes, fungi) are allowed to develop in the inhibition of sensitive assay organisms. By
a liquid minimal medium. The developing mi- using this method, the antibiotic content of a so-
crocolonies of prototrophs are then separated by lution can also be determined. A frequently used
filtration, leaving behind in the filtrate spores of variant of this method is the “agar plug method”,
auxotrophs which have been unable to grow. The in which agar cylinders with single colonies are
filtrate is then plated and the resulting colonies | transferred to test plates after incubation in a
are checked for auxotrophic characteristics. moist chamber (Figure 3.16). The diameter of the
Another procedure for selection of auxo- resulting inhibition zones serves as a measure of
trophs, which can be used also for unicellular the antibiotic production of each strain. How-
organisms, makes use of the fact that penicillin ever, one problem with this agar plug method is
kills growing cells but not nongrowing cells. In that frequently there is only a slight correlation
this penicillin-selection procedure, growing between antibiotic formation in plate culture and
cells are selectively killed by antibiotic treatment, the antibiotic production in submerged fermen-
thus enriching for auxotrophs which cannot grow
on minimal medium. Depending on their mode
of action several inhibitors other than penicillin Spore suspension of
kasugamycin-producing strain
can also be used in this procedure: dihydrostrep-
Mutation
tomycin for Pseudomonas aeruginosa, nalidixic
acid for Salmonella typhimurium, colistin for the Plating (30-100 colonies/plate)
penicillin-resistant Hydrogenomonas strain H16,
and nystatin for Hansenula polymorpha, Penicil-
aS
lium chrysogenum, Aspergillus nidulans, and Sac-
Incubation
charomyces cerevisiae. 29 °C, 48 hr
An enrichment procedure with sodium pen-
tachlorophenolate makes use of the greater tox-
Agar cylinder (6 mm diameter)
icity of this compound against germinating is transfered to asterile
spores than against vegetative cells. The method ie dish

has been successfully applied with Penicillium

chrysogenum, Streptomyces aureofaciens, S. oliva- Ganon)
ceus, and Bacillus subtilis.
By these methods, enrichments for auxo- Henne, Incubationina
trophs of 10- to 100-fold can be attained, thus faoooo9 WJ} moist chamber,
| 29 °C, 96-120 hr
increasing the probability of obtaining mutants.
However, it should be remembered that the types
Transfer of the
of mutants present in the original population cylindertoa
may be shifted; for instance, an increased pro- test plate

portion of proline auxotrophs has been found in

Culture from
E. coli after auxotroph enrichment. cylinders with
inhibition zones

Other procedures The presence or absence of Figure 3.16 Use of the “Agar plug” method in kasuga-
specific enzyme activities can be observed di- mycin strain development (Ichikawa et al., 1971)
 3.4 RECOMBINATION / 29

tation. Strains which produce at high yields their increased productivity, there is the de-
when grown on plates may produce at only low velopment of inapparent mutations which
yields or not at. all in liquid culture. Therefore prevent a further increase in the metabolite
the procedures mentioned are suitable for pro- production through pleiotropic influences.
cesses where simply a differentiation between With genetic crosses, these unfavorable mu-
productivity and nonproductivity is sufficient, tant alleles may be replaced with alleles of
such as for detecting the formation of constitutive one of the parents in the cross.
enzymes. If screening is initiated using high- + High-yielding strains can actually increase
yielding strains, further increases in yield often the cost of the fermentation because of
cannot be detected by this method. changed physiological properties (greater
foaming, changed requirements for culture
medium, etc.). By crossing back to wild-type
3.4 RECOMBINATION strains, high-yielding strains with improved
The genetic information from two genotypes can fermentation properties may be formed.
be brought together into a new genotype through Hence an effective strain development ap-
genetic recombination, which is thus another ef- proach should involve the use of sister-strain,
fective means of increasing the genetic variability divergent strain, and ancestral crosses at specific
of a population. As an example of how genetic intervals, besides use of careful mutagenesis to
variability can be increased, consider the follow- ensure the maintenance of genetic variability.
ing: In mutant screening, each high-yielding mu-
tant is derived ultimately from the wild strain
after a series of mutation and selection steps. A Sexual and parasexual cycles in fungi
cell line with 10 mutations, for example, contains The fungi have two distinct types of genetic re-
10 new genotypes. By crossing the last high- combination processes that can be used in a
yielding mutant with the wild type strain, 21° (or strain improvement program. These are known
1024) different genotypes can be elicited. This as the sexual and parasexual cycles. We consider
same reasoning also applies to the crossing of each of these processes in turn.
high yielding strains from two different lines.
The advantages of genetic recombination are: Sexual recombination Some fungi used indus-
. Different alleles of the parent strains with in- trially (e.g. strains of the genera Aspergillus, Clav-
creased metabolite production can be brought iceps, Emericellopsis, and Saccharomyces) have a
together in one strain, so that the cumulative complete sexual cycle. In these organisms, nu-
effect of these mutations can be greater than clear fusion (karyogamy) results after fusion of
the effect of the single mutation. However, hyphae has led to a mingling of nuclei in the
the original hope of attaining a significant heterokaryotic mycelium. After diploid forma-
yield increase by merely recombining two tion, recombination takes place during the sub-
high-yielding mutants has only been fulfilled sequent meiosis process. A new genotype results
in a few cases. In most cases, the productivity either from the combination of parent chromo-
of the recombinants usually is intermediate somes or through crossing over as a result of
between the values of the parent strains (see segment exchange of paired homologous chro-
Chapter 13, for examples). matids.
+ In the course of strain development, there is
frequently a decline in the increase in yield Parasexual recombination Some of the most eco-
after each stage of mutation. Besides mutants nomically useful fungi, such as Penicillium chry-
which are selectively enriched because of sogenum (producer of penicillin) and Cephalospo-
30 / CHAPTER 3 / STRAIN DEVELOPMENT

rium acremonium (producer of cephalosporin), do produce strains with increased penicillin titers.
not have a sexual cycle. Fortunately, the discov- The amount of penicillin formed by the diploid
ery of parasexual processes in imperfect fungi has strains and their segregants was, however, no
led to the development of suitable breeding tech- greater than that of the parent strains. Only when
niques. In parasexuality, the fusion of two hy- haploid recombinants were used could stable
phae of equal or different polarity results in a strains with improved antibiotic production be
mycelium with nuclei of both parent strains. This isolated.
heterokaryon is normally stable with the nuclei
mingling but not interacting. However, in rare
Recombination in bacteria
cases (10°7-10°* in P. chrysogenum), nuclear fu-
sion occurs and a diploid nucleus is formed. In’ The parasexual mechanisms established in vivo
such diploid nuclei, mitotic crossing over be- in bacteria include: transformation, transduc-
tween chromatids of homologous chromosomes tion and conjugation. In each case only a frag-
may occur, resulting in genetic recombination ment of the genome of the donor cell is trans-
(Figure 3.17). To obtain a recombinant, the for- ferred into a recipient cell which thus becomes
mation of haploid cells or spores must occur. a partial diploid (merozygote). After homologous
Spontaneous haploidization is relatively rare pairing, recombination occurs, but not every
(107), but can be induced with p-fluorophenyl- DNA transfer results automatically in recombi-
alanine. Haploidy occurs not through meiosis but nation. In transformation, short pieces of DNA
through random distribution of the chromo- are taken up by competent recipient cells. In gen-
somes to the progeny nuclei. eralized transduction, temperate phage parti-
Table 3.3 shows the industrial fungi for which cles which have lost a piece of their own genomes
genetic recombination has been established. Het- transfer a DNA fragment of the host bacteria at
erozygotic diploids from parent strains with dif- the optimal rate of 10° per phage and per char-
ferent cell lines have been used in attempts to acteristic. Phage P1 is an example of a phage

Heterokaryotic

Splitting of the
heterokaryon

Parasexual
cycle

Haploid
recombinants
©
©
Nuclear
fusion

Diploid Heterozygotic
segregants diploid
Mitotic Figure 3.17 Parasexual cycle of Pen-
crossing over icillium chrysogenum (Sermonti, 1959)
 3.4 RECOMBINATION / 31
Table 3.3 Antibiotic-producing fungi in which
recombination has been discovered
Escherichia coli and Pseudomonas are Gram-
negative bacteria of industrial interest in which
Species Antibiotic Recombination
conjugation is well developed and in which
type
transduction systems are present. Pseudomonads
Aspergillus nidu- Penicillin G sexual, para-
have sex plasmids similar to F, the factor of Esch-
lans sexual
Cephalosporium Cephalosporin C _ parasexual erichia coli. Transformation systems exist for Ba-
acremonium Penicillin N cillus strains (B. subtilis, B. licheniformis, B. pum-
Emericellopsis sal- Penicillin N sexual, para-
ilus); in addition, transduction systems have been
mosynnemata Cephalosporin C sexual
Emericellopsis terri- Penicillin N sexual established for B. subtilis with phages PBS1 and
cola var. glabra Cephalosporin C SP 10.
Penicillium chryso- Penicillin G, O, V parasexual
genum
Penicillium patu- Griseofulvin parasexual Recombination in actinomycetes
lum Patulin
(Hopwood and Merrick, 1977) Among industrial microorganisms, actinomy-
cetes (filamentous Gram-positive bacteria) are
economically significant as antibiotic producers.
which brings about generalized transduction in In streptomycetes, transduction has been sought
Escherichia coli. In specialized transduction, unsuccessfully in S. olivaceus and S. griseus and
only the loci which are adjacent to the attach- transformation has been sought unsuccessfully
ment site of the phage in the bacterial chromo- in S. aureofaciens and S. griseus. In the thermo-
some are transferred. The insertion of the pro- phile Thermoactinomyces vulgaris, however,
phage into the chromosome results in the further transformation has been definitely shown.
incorporation of the attached piece of DNA into Conjugation is the most common form of ge-
the genome of the host cell. Phage A is an ex- netic exchange in actinomycetes in vivo (Table
ample of a phage which brings about specialized 3.4). On the average, one-fifth of the genome is
transduction. Conjugation generally involves transferred to the recipient cell; the frequency of
the participation of plasmids. In this process, sin- recombination is between 10° and 10°. It has
been determined that fertility factors are in-
gle-stranded DNA is transferred from the donor
volved in conjugation in three streptomycetes (S.
cell to the recipient cell after the two cells have
reticuli, S. rimosus and S. coelicolor). In S. coeli-
come into contact. In the Escherichia coli system,
color A3(2), the best understood actinomycete,
F* cells are donor cells in which plasmid exists
two plasmids have been intensively studied:
in free form. After contact with F- cells which do
SCP1 with a molecular weight of approximately
not contain the fertility factor, a copy of the F 100 X 10°, which contains the genes for meth-
factor is transferred. Thus an F- population is ylenomycin synthesis; and SCP2 (molecular
almost completely transformed into an F* type. weight 18-20 X 10°), of which the mutant form
After the F factor is integrated into the chro- SCP2 causes greater recombination frequency.
mosome, the resulting Hfr strains show a signif- Recombination has not been of great impor-
icantly higher frequency of recombination (up to tance in industrial strain development of acti-
107 compared to 10° previously). In conjuga- nomycetes. This is because parent strains with
tion, a fragment of the F factor is first transferred, selective markers must be used to identify low
then the bacterial genome, and finally the re- frequency recombinants and the construction of
maining fragment of the F plasmid. Since the doubly marked parent strains (such as auxotro-
conjugation process is almost always interrupted phy and antibiotic resistance) is time-consuming.
before completion, the recipient cell usually does Singly marked strains can not be used in a genetic
not become an Hfr cell. recombination study because the spontaneous
32 / CHAPTER 3 / STRAIN DEVELOPMENT

Table 3.4 Recombination in actinomycetes.

Protoplast fusion
Streptomyces spp.
. coelicolor (Actinorhodin, Methylenomycin) Protoplasts are cells from which the cell wall has
. achromogenes var. rubradiris (Rubradirin)
. acrimycini
been removed by enzyme treatment. For bacte-
. aureofaciens (Chlortetracycline) ria, the enzyme lysozyme is used, whereas for
. bikiniensis (Zorbamycin, Zorbonomycin) fungi, chitinase or cellulase is used. The proto-
. erythreus (Erythromycin)
fradiae (Neomycin) plasts must be stabilized against lysis by suspen-
glaucescens sion in a medium containing an osmotic stabi-
. griseoflavus lizing agent, such as sucrose. Recombination by
. griseus (Streptomycin)
. olivaceus protoplast fusion or protoplast transformation
. rimosus (Oxytetracycline) is one of the most important developments in
. scabies applied genetics in recent years. Protoplast fusion
. venezuelae (Chloramphenicol)
ANnNNNNHDHHHHHHHN
Nocardia spp. is normally rare because of the strong negative
N. erythropolis charge of the protoplast surface, but in the pres-
N. mediterranei (Rifamycin) ence of polyethyleneglycol (PEG) the protoplasts
Micromonospora spp.
M. chalcea ageregate and fusion occurs accompanied by
M. echinospora DNA exchange. Besides the use of PEG to bring
M. purpurea (Gentamicin) about fusion, the method of electric-field-in-
Mycobacterium smegmatis
Thermoactinomyces vulgaris duced fusion of protoplasts has been developed.
This method results in a considerably higher fre-
The antibiotic produced is indicated within the
parenthesis. In Thermoactinomyces, transformation is quency of protoplast fusion. After fusion, the cell
responsible for the genetic exchange; in all other cases, wall is allowed to regenerate. In the regenerated
a “conjugation” mechanism has been recognized, but progeny there is a significant number of recom-
fertility factors have not usually been detected. (Elander
et al., 1977) binants.
Protoplast fusion can be used for the follow-
ing: ‘
reversion rate of auxotrophs is in the same range
as the frequency of recombination. Moreover, ¢ Intraspecific recombination of strains which
there is the danger that further mutations with lack sexual or parasexual systems or whose
a negative effect on antibiotic production may frequency of recombination is too low.
occur unnoticed, since mutations to auxotrophy ¢ Interspecific hybridization to obtain com-
commonly reduce antibiotic production drasti- pletely new organisms capable of synthesis
cally. In addition, better results occur if the part- of modified metabolites.
ners used in the cross come from completely dif- Engineered genes in plasmids or virus DNA
ferent origins. Industrial strain development, can also be used to transform protoplasts.
which commonly begins with one particular An interesting alternative to PEG fusion is
strain, cannot fulfill this requirement. Thus, re- electrofusion, a technique developed by Zim-
combination can only be used at a later point in merman. When cells are placed in an alternating
strain development when distinct cell lines exist. current electrical field, transient holes develop in
Under these conditions, recombination can result the plasma membrane, promoting the process of
in strains with higher yields; kasugamycin and membrane merging and cell fusion. With elec-
tetracycline yields have been improved this way. trofusion, two or more protoplasts can be caused
Since the development of genetic engineering to fuse under microscopic control, or several cells
methods for the actinomycetes, in vitro recom- can be fused into one giant cell. Further, proto--
bination has become of increasing importance plasts can be induced to fuse artificial phospho-
(see Sections 3.6 and 3.7). lipid vesicles called liposomes. The fusion rate is
 3.4 RECOMBINATION / 33

around 80-90%, considerable higher than the

60% rate obtained using PEG as a fusogen. In
addition to its use in the fusion of plant proto-
plasts, electrofusion can also be used with yeast
and fungal protoplasts. However, the fusion of
small bacterial protoplasts is more difficult to ac-
complish.

Intraspecific recombination Good genetic recom-

bination systems exist for many industrial mi- Genotype Genotype Numberof
frequency pairing partners
croorganisms: For various strains of Bacillus, Lac-
tobacillus, Streptococcus, Corynebacterium, or pac+
Brevibacterium, in fungi such as Aspergillus, Pen- patu,
Dit Gu
icillium, Mucor, Claviceps, and Cephalosporium +dacu}

strains, in yeasts such as Candida, Saccharomyces, pa+ +

p+-C +
Kluyveromyces, and in actinomycetes such as Osea ae (Ul -2
gets Pe 3.2 x 10 2
Streptomyces, Micromonospora, and Nocardia. A
eu Czas
number of advantages arise from the use of these er eel)
newer methods to achieve intraspecific recom- DES ces y
bination: +a++ ; 3.4 x10 3
++C+

e Protoplast fusion is applicable in bacteria in + + + uJ

which other recombination procedures have +++ + 5x VOR 4

been successful. It is also applicable in Bacil-

lus strains, which do not have a natural con-
jugation system. Gram-negative bacteria such Figure 3.18 Protoplast fusion in S. coelicolor with 4 mat-
ing partners (Hopwood and Wright, 1978)
as E. coli and Providencia alcalifaciens have p = proAl; a = argAl; c = cysD18; u = uraAl
also been used although the frequency of pro-
toplast regeneration is rather low.
+ The exchange of genetic material does not binants with increased cephalosporin formation
require the presence of fertility factors, and were isolated after protoplast fusion.
DNA can be recombined from up to four pa- Protoplast fusion has resulted in dramatic in-
rental genotypes (Figure 3.18). creases in frequency of recombination in some
e The entire genome can be transferred, rather streptomycetes, as shown in Table 3.5.
After ultraviolet radiation (approximately
than only fragments as in conjugation, trans-
99% inactivation), a further enrichment of re-
duction, and transformation.
combinants can be attained in S. coelicolor, be-
e The frequency of recombination is signifi-
cause only viable cells remain when lethal le-
cantly increased.
sions are eliminated through recombination.
An apparent increase in the formation of het- Thus the original frequency of recombination is
erokaryons as a result of protoplast fusion has increased from 10 to 107.
been found in some fungi. This is especially sig- At these high recombination rates, a popu-
nificant in Cephalosporium acremonium, in which lation can be assayed for recombinants after pro-
the hyphal cells are mainly uninucleate, which toplast fusion without the cumbersome use of
hinders heterokaryon formation during anasto- selective markers. Hence, because of the devel-
mosis of hyphae. In this fungus, several recom- opment of high-efficiency protoplast fusion tech-
34 / CHAPTER 3 / STRAIN DEVELOPMENT

Table 3.5 Frequency of recombination after buligera is also quite low, around 10. Crosses
conjugation and protoplast fusion in streptomycetes
between Saccharomyces cerevisiae X Lipomyces
Strain Mechanism of Recombination kononkoae showed several fusion products which
recombination frequency
exhibited metabolic properties of both parental
S. coelicolor A3(2) Conjugation KENO strains, as well as the formation of nonparental
(GERIESER2s Protoplast fusion 4.7 X 107?
PT SERISSCR2}) segregants after haploidization.
S. coelicolor A3(2) | Conjugation eae Oar
(SGHIESEP25 Protoplast fusion 6.2 X 107 Transformation and transfection of ‘protoplasts
KESCPIES CP 27
S. parvulus ATCC — Conjugation Crs Oe Recombinant DNA technology made possible for
12434 Protoplast fusion 0.6 X 107? the first time the development of methods for
S. lividans 66 Conjugation i Ox transformation of protoplasts with plasmid, chro-
Protoplast fusion 6.0 X 10°
S. griseus CUB 94 Conjugation SEX EOS mosomal, or viral DNA. In the basic method,
Protoplast fusion 10:10 which is simple and generally applicable, pro-
(Hopwood et al., 1977) toplasts are treated with DNA in the presence of
PEG and Ca?**. In this way, all of the numerous
techniques of genetic engineering become avail-
niques, not only mutation and selection but also able for use with microorganisms of industrial
recombination and selection may be considered interest (fungi, actinomycetes, other bacteria):
equally useful methods of strain development. amplification of genes, restoration of metabolism
in deficient mutants, in vitro mutagenesis with
Interspecific hybridization This approach allows protein engineering. Thus, even microorganisms
genetic information from different species to be for which conventional host-vector systems are
combined in vivo in order to create new or mod- not available can be handled. For example, ex-
ified products. The use of protoplast fusion for cellent transformation systems have been devel-
this objective is being examined by different anti- oped for microorganisms of interest to the dairy
biotic manufacturers. industry such as Streptococcus lactis and various
Among the fungi, interspecific crosses have lactobacilli, for Staphylococcus carnosus and Ba-
been attempted between Aspergillus nidulans X cillus subtilis, and vitamin B,,-producing Pro-
A. rugulosus; A. nidulans X A. fumigatus; Peni- pionibacterium freudenreichii. Transformation
cillium chrysogenum X P. cyaneofulvum; P. cy- systems have also been developed for B-lactam-
aneofuluum X P. citrinum; Saccharomyces cere- producing strains of Streptomyces clavuligerus
visiae X S. diastaticus; Kluyveromyces lactis X K. and S. wadayamensis, vancomycin-producing No-
fragilis. Correct heterokaryons and recombinants cardia orientalis, erythromycin-producing S. ery-
have only been obtained in the cross between P. threus, and gentamicin-producing Micromonos-
cyaneofuluum X P. citrinum, in which a fusion pora purpurea subspecies luridus. Using virus
frequency of less than 10 was obtained as com- vectors, transformation (transfection) systems
pared to an intraspecific frequency of 40-60%. have been described for Streptomyces, Thermo-
Interspecific crosses among streptomycetes have monospora, Mycobacterium smegmatis, and Brev-
given similar results. The low frequency of het- ibacterium lactofermentum.
erospecific fusion may because of genome in-
homologies which prevent recombination from
taking place or because of the presence of re- 3.5 REGULATION
striction/modification systems that lead to phys- Both catabolic and anabolic processes are regu-
iological incompatibility. lated and metabolism is generally so efficient that
The fusion frequency of intergeneric crosses excess products are not formed. Strains with less
between Candida tropicalis X Saccharomyces fi- efficient regulation can be selected in a screening
 3.5 REGULATION / 35
process. It is well established that strain devel- be an excess of all end products for inhibition
opment and the optimization of fermentation to occur (a phenomenon called multivalent
conditions lead to a relaxation of regulation in inhibition),
the producing strains. A broad understanding of + Each end product of a branched pathway acts
biosynthesis, the enzymes involved in these pro- as an inhibitor; cumulative inhibition is the
cesses, and their regulation is necessary for de- effect of all the inhibitors.
veloping a rational approach to the alteration of
the regulation of a fermentation process. Examples of various feedback-inhibition re-
action types are given in Chapters 9 and 10.
Microbial metabolism is controlled by the
regulation of both enzyme activity and enzyme
Energy charge Feedback inhibition also regu-
synthesis. We discuss both types of regulatory
phenomena here. lates catabolic pathways in which ATP is the
main products. The relative concentrations of
AMP, ADP and ATP in the cell are used in the
Regulation of enzyme activity following formula to calculate the energy charge
(EC):
Extensive research over the past several decades
has shown that the activity of enzymes can be (ATP) + 0.5 (ADP)
controlled by different mechanisms. We discuss (AMP) + (ADP) + (ATP)
here those mechanisms which are thought to be
of significance for industrial process develop- The values of the EC calculated in this way lie
ment. between 0 and 1.0.
If the EC is high, the activities of enzymes
Feedback inhibition In an unbranched biosyn- involved in ATP synthesis are inhibited (for ex-
thetic pathway, the end product inhibits the ac- ample, isocitrate dehydrogenase is inhibited
tivity of the first enzyme of the pathway, a pro- when the energy charge is = 0.8). Conversely,
cess called feedback inhibition. A conformation the activities of anabolic enzymes (e.g., aspar-
change and hence inactivation (allosteric effect) tokinases), which consume ATP, are stimulated
occurs when an effector (end product) is attached by a high energy charge. Thus, an alteration in
to a specific site of the enzyme (allosteric site) the rate of catabolism, since it affects ATP level
The end product thus inhibits the activity of the and hence energy charge, may cause an increase
enzyme noncompetitively. or decrease in the activity of a variety of en-
In a branched biosynthetic pathway, feed- zymes.
back inhibition of the first common enzyme by
means of one of the end products would cause Breakdown of enzymes Enzymes which are no
more than one end product to be affected. In longer needed in metabolism may be broken
branched biosynthetic pathways, different kinds down through the action of highly specific pro-
of feedback inhibition are found: teases. One of the best-known examples is the
enzyme tryptophan synthetase in Saccharomyces
e The end product inhibits the first enzyme in cerevisiae, which is broken down specifically
each case after the branch point. when the cells go into the stationary phase.
¢ The first step in the common synthesis path
is catalyzed by several isoenzymes, each of Modification of enzymes The activity of some en-
which can be regulated independently. zymes (such as glutamine synthetase in Esche-
¢ The first common enzyme in a branched bio- richia coli) is controlled by conformational
synthetic pathway is influenced by each end changes, such as phosphorylation or adenylyl-
product only slightly or not at all; there must ation. >
36 / CHAPTER 3 / STRAIN DEVELOPMENT

KE Hf SR

Regulation of enzyme synthesis i

Minimal
At least three mechanisms have been detected |] supplementation
which regulate synthesis of enzymes. Note that with E
although feedback inhibition affects enzyme
molecules that have already been made, the fol- A A
lowing mechanisms control the actual synthesis
neste st
of enzyme protein. ae
ee
B
SE
AX bi B a

Induction Some enzymes are formed irrespec- al Series Y

tive of the culture medium; such enzymes are
| Bede a | | cite
SRY ohSeer
called constitutive. Many catabolic enzymes are’ VÆ ae ! i x eae x /
SEES.
induced: they are not formed until the substrate \ : D Fee 35/ en D F |/
hep Xe Vrå AV py
to be metabolized is present in the medium. The \
SSE GÅ SE Gå

product of one enzyme can in turn induce the

synthesis of another enzyme (sequential induc-
tion).

Repression Anabolic enzymes are generally

present only when the end product is absent. The |
| B
excess end product suppresses enzyme synthesis,
:
|
acting as a co-repressor.
ie ~ Minimal
Attenuation This is a further mechanism for the | wie aX supplementation
control of gene expression which is involved in Le G H With
the biosynthesis of amino acids in bacteria. At- af Ne x
tenuation can be the only regulatory step as in
the case of histidine biosynthesis in Salmonella Figure 3.19 Overproduction of primary metabolites by
typhimurium, or it can work in addition to a re- auxotrophic mutants. ***: Reaction step which is blocked
pressor-operator mechanism as with tryptophan due to auxotrophic mutation; -—— Feedback regulation.
in Escherichia coli. According to the attenuator » Primary metabolite produced in excess (Demain, 1972)
model, the transcription rate of an operon is reg-
ulated by the secondary structure of the leader production of strains which excrete excess pri-
sequence of the newly transcribed mRNA. The mary metabolites (amino acids, vitamins, purine
structure of this leader sequence determines nucleotides). This has been accomplished pri-
whether the transcription of the operon is con- marily by eliminating feedback inhibition.
tinued by the RNA polymerase or a termination
occurs. If termination occurs, the mRNA tran- ¢ The elimination of end product inhibition or
scription ceases and the enzyme or enzymes repression is achieved by using auxotrophic
coded for by that mRNA are not made. In the mutants that can no longer produce the de-
tryptophan situation, repression has a large effect sired end product due to a block in one of
on enzyme synthesis whereas attenuation has a the steps in the pathway. By adding the re-
more subtle, although still important, effect. quired end product in low amounts, growth
occurs but feedback inhibition is avoided. Ex-
cretion of the desired intermediate product
Excess production of primary metabolites
thus occurs. Both branched and unbranched
The growing understanding of the biochemistry pathways can be manipulated in this way
and genetics of microorganisms has led to the (Figure 3.19).
 3.5 REGULATION / 37

| |
* A second method is the selection of mutants
that are resistant to antimetabolites (see Sec-
tion 3.3). In this case either the enzyme struc-
ture is changed so that the corresponding en- Shikimic Malonyl-
acid CoA
zyme lacks the allosteric control site, or
mutations in the operator or regulator gene
(Os-, R-mutants) result in constitutive en- Tryptophan Chloram- Fatty acids Nystatin
Phenylalanine phenicol Griseofulvin
zyme production and thus overproduction. Tyrosine Myxin Tetracycline
+ In mutants with a block in an allosterically p-Amino- Bacilysin ' Cycloheximide
benzoic acid
regulatable enzyme, suppressor mutations
can lead to restoration of enzyme activity;
however, these enzymes are not allosterically
controllable. Mevalonic a-Amino-
acid adipic acid

Regulation and overproduction of

secondary metabolites Sterols Gibberellins Lysine Penicillin
Steroid - Cephalosporins
The methods described above, which were used antibiotics
(Fusidic acid,
first for primary metabolites, can be successfully Helvolinic acid,
Cephalosporin P),
applied to secondary metabolites as well (Table Carotenoids
Terpenes
3.6). When a branched biosynthetic pathway si-
multaneously leads to primary and secondary
Figure 3.20 Branched biosynthetic pathways which si-
metabolites (Figure 3.20), an auxotrophic muta- multaneously lead to primary and secondary metabolites
tion in the biosynthesis of the primary metabolite (Demain, 1972)
can lead to an increased production of the sec-
ondary metabolite. Production of secondary me-
tabolites is controlled by 5 different classes of
genes: 1. Structural genes, which code for enzymes in-
volved in secondary metabolite biosynthesis.
2. Regulatory genes, which control secondary
Table 3.6 Overproduction of antibiotics by feedback-
resistant mutants
metabolite synthesis.
3. Resistance genes, which keep antibiotic-pro-
Microorganism Antibiotic Characteristic
ducing strains immune to their own products.
Penicillium chry- Penicillin Reduced sensitiv- 4. Permeability genes, which control the up-
sogenum ity to feedback in- take and excretion of substances.
hibition by valine
S. griseus Candicidin Resistance to 5. Regulatory genes, which control primary me-
tryptophan tabolism and thus indirectly affect the bio-
antimetabolites synthesis of secondary metabolites.
S. lipmanii Cephamycin Resistance to
leucine Many genes are involved in the synthesis of
antimetabolites
S. viridofaciens Chlortetra- Suppression of a secondary metabolites. It has been estimated that
. cycline met” mutation 300 genes are involved in chlortetracycline bio-
S. antibioticus Actinomycin Suppression of a synthesis and approximately 2000 genes are di-
ilv- mutation
S. lipmanii Cephamycin Suppression of a rectly or indirectly involved in neomycin bio-
cys’ mutation synthesis. In such a complex system, a rational
S. viridofaciens Chlortetra- Reversion of a approach to increased yield is possible only in
cycline nonproducer
rare cases because there is insufficient data on
38 / CHAPTER 3 / STRAIN DEVELOPMENT

biosynthesis and regulation of secondary metab- Endproduct regulation It is known that anti-
olite production, on the supply of intermediates biotics inhibit their own biosynthesis (e.g. pen-
of primary metabolism for secondary metabolite icillin, chloramphenicol, virginiamycin, risto-
synthesis, and on the energy links between cata- mycin, cycloheximide, puromycin, fungicidin,
bolic, anabolic and amphibolic pathways. candihexin, streptomycin). The mechanism of
Regulatory mechanisms that affect the prod- feedback regulation has only been explained in
ucts of secondary metabolism are outlined below. a few cases: chloramphenicol represses arylam-
ine synthetase, which is the first enzyme in the
Induction In batch fermentations with readily biosynthetic pathway which branches off from
metabolizable carbon and nitrogen sources, sec- aromatic biosynthesis to chloramphenicol. With
ondary metabolites are formed primarily after chloramphenicol and penicillin, it has been
growth has ceased. The logarithmic growth shown that the concentration of the end product
phase is called the trophophase, and the sub- which inhibits corresponds to the production
sequent phase, in which the secondary metab- level. Thus, if strains could be isolated which
olite may be produced, is called the idiophase. were less sensitive to endproduct inhibition by
Secondary metabolites are therefore also some- these antibiotics, they might produce higher
times referred to as idiolites. In all cases studied yields.
thus far, the synthesis of enzymes involved in
secondary metabolism is repressed during the
Catabolite regulation Catabolite regulation is a
trophophase (Table 3.7). The composition of the
general regulatory mechanism in which a key
culture medium can however also be so arranged
enzyme involved in a catabolic pathway is re-
so that a significant fraction of a slowly meta-
pressed, inhibited, or inactivated when a com-
bolizable substrate is used, the organism thus
monly used substrate is added. Substrates which
growing under suboptimal conditions, leading to
have been found to bring about catabolite repres-
a situation where growth and secondary metab-
sion include both carbon and nitrogen sources.
olite formation occur in parallel.
Carbon sources: Biosynthesis of different
There is only scanty information on the na-
ture of the induction of enzymes of secondary secondary metabolites (antibiotics, gibberellins,
metabolism: Methionine induces cephalosporin ergot alkaloids) is inhibited by rapidly ferment-
and fosfomycin synthesis, factor A induces strep- able carbon sources, particularly glucose (Table
tomycin production (see Section 13.4) and tryp- 3.8). Depending on the organism and metabolite,
tophan is a precursor and regulator of ergot al- the basic mechanism of this carbon catabolite
kaloid biosynthesis. (see Section 14.6). regulation is different. One is the well-known
carbon catabolite repression found in many
bacteria, yeasts and molds which involves a ca-
Table 3.7 Key enzymes of secondary metabolism
which are induced at the end of the trophophase tabolite activator protein (CAP) that must com-
bine at the promoter site before RNA polymerase
Enzyme Secondary
metabolite
can attach. The CAP will only bind if it is first
complexed with cyclic adenosine monophos-
Amidinotransferase Streptomycin
Acyltransferase Penicillin
phate, cyclic AMP. Readily utilizable carbon
Phenylacetate-activating enzyme sources such as glucose stimulate an enzyme
Oxidoreductase : which causes the breakdown of cyclic AMP, thus
Transmethylase ANSOS MR
Synthetase I and II rendering CAP inactive. Thus, glucose inhibits
Gramicidin S
Phenoxazinone synthetase Actinomycin the synthesis of the mRNA for any enzyme re-
Dimethylallyl tryptophan quiring CAP for its biosynthesis.
synthetase Ergot alkaloids
Chanoclavin-I-cyclase
If readily utilizable carbon sources such as
glucose elicit catabolite regulation, how is it pos-
 3.5 REGULATION / 39
Table 3.8 Catabolite regulation in antibiotic
biosynthesis secondary metabolites. In a number of systems
studied, the highest P; concentration which al-
Carbon Source lows unimpeded production of secondary me-
Antibiotic Inhibitory Not inhibitory tabolites is about 1 mM; complete inhibition of
Penicillin Glucose Lactose production occurs at about 10 mM P;. Phosphate
Glucose-feeding regulation has been observed in the production
Cephamycin Glycerol Asparagine
of alkaloids (Section 14.6), gibberellins and par-
Cephalosporin C Glucose Sucrose
Actinomycin Glucose Galactose ticularly in several antibiotics (Table 3.9). The
Streptomycin Glucose ~ Mannan and phosphate regulation mechanism is not yet fully
L-Rhamnose understood. P; controls the metabolic pathways
Siomycin Glucose Maltose
Indolmycin Glucose Fructose which precede the first stage of secondary me-
Bacitracin Glucose Citrate tabolite formation, but also affects the biosyn-
Chloram- Glucose Glycerol thesis of secondary metabolites themselves.
phenicol
Mitomycin Glucose Glucose-feeding Several varying phosphate effects have been
Neomycin Glucose Maltose described. In one type of mechanism, phosphate
Kanamycin Glucose Galactose stimulates primary metabolism. The consump-
Butirosin Glucose Glycerol
Puromycin Glucose Glycerol tion of P; in the culture medium seems to be one
Novobiocin Citrate Glucose of the main causes for the metabolic change from
Candicidin Glucose Glucose-feeding the trophophase to the idiophase. By adding P;
Candihexin Glucose Glucose feeding
at the end of the trophophase, the idiophase can
Listed are carbon sources which cause catabolite be delayed; after addition of P, in the idiophase,
regulation and carbon sources which do not inhibit
product formation.
growth resumes and antibiotic synthesis stops.
In a second mechanism, phosphate inhibits or
represses phosphatases, enzymes which are in-
sible to use these substrates in an industrial fer- volved in the biosynthesis of secondary metab-
mentation? In the manufacture of antibiotics, car- olites. This mechanism has been demonstrated
bon sources other than glucose may be used or for the biosynthesis of the aminoglycoside anti-
glucose can be fed at low rates, to minimize ca- biotics. A third mechanism leads to a shift in the
tabolite repression.
Nitrogen sources: In several antibiotic fer- Table 3.9 Some antibiotics whose formation is
mentations it has been observed that ammonia inhibited by organic phosphate
or other rapidly utilizable nitrogen sources act as Antibiotic Producer P, range (mM)
inhibitors: The fundamentals of this regulation permitting
have not yet been completely understood, al- antibiotic
formation
though glutamine synthetase and glutamic de-
hydrogenase are considered key enzymes. In en- Streptomycin Streptomyces 57415
griseus
teric bacteria it has been established that Kanamycin S. kanamyceticus Pep =) Saif
glutamine synthetase has a regulatory function Bacitracin Bacillus One
in the synthesis of additional enzymes which are licheniformis
Gramicidin S B. brevis 10) S60)
involved in nitrogen assimilation. Amphotericin BS. nodosus sy
Candicidin S. griseus 055350
Phosphate regulation In a culture medium in- Chlortetracycline 5S. aureofaciens AN are SÆLG)
Vancomycin S. orientalis ee
organic phosphate (P;) is required within a range Actinomycin S. antibioticus 14-17
of 0.3-300 mM for the growth of procaryotes and Tetracycline S. aureofaciens 0.14- 0.2
eucaryotes. However, a much lower phosphate Cycloheximide S. griseus OWS ss
concentration inhibits the production of many (from Martin, 1977)
40 / CHAPTER 3 / STRAIN DEVELOPMENT

carbohydrate metabolism from the pentose phos- The industrial production of P,-regulated sec-
phate cycle or hexose monophosphate pathway ondary metabolites is carried out, as far as pos-
to glycolysis when excess phosphate is present. sible, under conditions of P, limitation. The stan-
NADPH, thus becomes a limiting factor in the dard culture media used for production often
synthesis of antibiotics. contain relatively high proportions of P, (around
Finally, it has been shown that phosphate re- 2.5 mM) and at these levels, phosphate can de-
stricts the induction of secondary metabolite pro- crease yields. The isolation of phosphate-deregu-
duction. For instance, dimethyl allyltryptophan lated (PD) mutants (as has been done for can-
synthetase, the first specific enzyme of ergot al- dicidin producing strains) seems to be a
kaloid biosynthesis, is not produced in the pres- worthwhile possibility for strain development.
ence of high P; concentrations (see Section 14.6). . PD mutants, all of which are less sensitive to
Some data indicate that phosphate regulation phosphate regulation, frequently produce can-
occurs via a control system at the transcription dicidin up to 70% better than the wild type in a
level. A model of negative control shown in Fig- normal medium.
ure 3.21 has been proposed for the induction of
candicidin synthesis. The repressor concentra- Autoregulation In some actinomycetes it has
tion decreases at the end of the trophophase, so been possible to show that differentiation and
that the transcription of genes that code for anti- secondary metabolism are subjected to a type of
biotic synthesis is possible. It has not yet been “self-regulation” from low-molecular weight
determined whether phosphate itself functions substances. For instance, in Streptomyces griseus
as a corepressor or whether it regulates the con- and S. bikiniensis the formation of streptomycin,
tent of intracellular effectors (CAMP; ATP; phos- the development of streptomycin resistance, and
phorylated nucleotides such as ppGpp or spore-formation are all affected by factor A, a
pppGpp). In the case of candicidin synthesis, an substance produced by the streptomyces them-
effector may be ATP. The intracellular ATP con- selves (see Figure 13.24 for the structure). It has
tent decreases sharply before the production of been shown that the streptomycin resistance
the antibiotic, but increases rapidly after adding property is due to the increased transcription of
PRit the gene for the enzyme, streptomycin phospho-
transferase, induced by the factor A. The effect
on streptomycin formation is thought to be due
Can Can
AOA
ms CoR
DNA DNA to a shift in the metabolism of the carbohydrate
SB R source: although the activity of the enzyme glu-
pol
| mRNA R cose-6-phosphate dehydrogenase is high in fac-
tor A-deficient mutants, this enzyme cannot be
… MY CoR(P
,ATP) demonstrated in high-yielding strains. Addition
~ Growth
VA of factor A to mutants leads to a strong decrease
vA in enzyme activity. It is assumed that when the
VA
N Rg EA '
pentose phosphate cycle is blocked through the
.
7.
7 Candicidin absence of glucose-6-phosphate dehydrogenase,
glucose is channeled into pathways involved in
the formation of streptomycin units.
In a sense, factor A can be considered anal-
ogous to a hormone. Autoregulatory mecha-
Figure 3.21 Model for control of candicidin synthesis
nisms similar to that of factor A have been found
through P; or ATP (Martin et al., 1979). in other actinomycetes. For instance, a factor is
RNA,,, RNA polymerase; CoR corepressor; R repressor hypothesized in S. virginiae which stimulates the
 3.6 GENE TECHNOLOGY / 41
formation of the antibiotic virginiamycin. In ri- Plasmid Foreign DNA
famycin-producing Nocardia mediterranei butyryl
phosphoadenosine has been characterized as a
regulatory factor. Two y-lactones (L factors) have GAATTC GAATT
SSP FESD]
been shown to be autoregulatory agents in leu- CASE CTTAAG
kaemomycin-producing S. griseus. t
|ese

“sticky
3.6 GENE TECHNOLOGY
AATIC sg 7 ends”
By gene technology is meant those techniques CTTAA

that permit the manipulation of genes as bio-

chemical entities. Gene technology includes in
vitro recombination, gene cloning, gene manip-
ulation, and genetic engineering. These tech-
niques permit the introduction of specific DNA Association
sequences into procaryotic or eucaryotic orga- via
sticky ends
nisms and the replication of these sequences; that GAATTC
is, to clone them. To carry out these procedures, 9
CTTAAG
[I]
CTTAAG

the following steps are necessary:

|DNA Ligase
e The DNA sequence to be cloned must be
available. |Transformation
¢ The sequence must be incorporated into a
Chromosome Hybrid
vector. plasmid
e The vector with the DNA insert must be in-
Transformed
troduced by transformation into a host cell, cell
where the vector must replicate the insert in
a stable manner.
e The clone which contains the foreign DNA
must be selectable in some manner. Figure 3.22 Method for the production of recombinant
DNA

Methods of gene technology

structures 4—11 nucleotides in length. Either
The basic procedures for the first steps in gene blunt double-stranded ends or short, cohesive,
cloning are outlined in Figure 3.22. Details can single-stranded ”sticky ends” develop, depend-
be found in specialized books on gene cloning ing on the enzyme involved:
(see References). 1 I
Xma I CCCGGG Sma I CCCGGG
(Xanthomonas GGGCCC (Serratia GGGCCC
Isolation of DNA sequences for cloning malvacearum) t marcescens) tT

Genome fragments Restriction endonucleases More than 600 restriction endonucleases are
are used to cut DNA. These enzymes belong to known in bacteria.
specific restriction and modification systems and If the sequence of the DNA to be cloned is
are used by the cell to protect itself from foreign unknown, it is possible to use a so-called “‘shot-
DNA. Restriction enzymes split double-stranded gun” approach. With this procedure, a gene bank
DNA at specific sites, usually at palindrome is produced by using suitable restriction enzymes
42 / CHAPTER 3 / STRAIN DEVELOPMENT

to fragment the total genome of the organism DNA (a DNA copy of the mature mRNA) (see
into pieces of about 20 kilobases in length. Each below).
of these fragments is linked to a vector (generally
a phage or cosmid) and cloned into a suitable Synthetic DNA In order to produce a specific
host. With an appropriate screening method the DNA fragment containing the coding region of
cultures containing the clones are selected, a protein, the DNA sequence is deduced by “re-
among which the target gene should be present. verse translation” from the amino acid sequence
There will be a 99% probability, assuming 20 kb of this protein. Using an automated DNA syn-
fragments, that in E. coli (total genome length of thesis machine, it is possible to produce DNA
about 4000 kb) all genes will be cloned if 900 fragments of 20 to 100 bases, which can be con-
separate clones are selected (in yeast, which has. nected together to make longer sequences. Ex-
a total genome size of 13,500 kb, 3100 clones amples of the use of this technique are the ar-
would be required). tificial synthesis of the gene for somatostatin, a
It is preferable if the initial cloning is carried peptide hormone with 14 amino acid residues
out with enriched fragments. Enrichment can be and the synthesis of the A and B chains of insulin,
done either by use of sucrose-gradient centrifu- which were cloned and expressed in E. coli. Since
gation, agarose-gel electrophoresis, column chro- the DNA synthesis procedure is completely un-
matography, or by use of specific gene probes. der the control of the investigator, it is also pos-
Gene probes are short DNA or RNA fragments sible to produce sequences in which one or more
that have been ”tagged” in some way which are bases have been changed, making possible the
able to hybridize with the specific genome se- production of highly specific mutations.
quence. Gene probes can be tagged with %P or
355 labeled nucleoside triphosphates, permitting
Production of complementary DNA (cDNA) Spe-
the probes to be detected by autoradiography.
cific mRNA molecules, isolated from mRNA-rich
An alternative procedure is the use of biotin-la-
cells or tissues, are used as templates in vitro with
beled DNA. Because of the strong affinity of bio-
the enzyme reverse transcriptase, to produce com-
tin for avidin (a glycoprotein obtained from egg
plementary DNA. An example of the use of this
white) or streptavidin (a protein obtained from
method for cloning the insulin gene from rats in
cultures of Streptomyces avidini) the presence of
E. coli is shown in Figure 3.23.
the biotin-labeled DNA can be detected using
avidin or streptavidin which has been marked
with a fluorescence dye, an enzyme, or an an- Vectors As vectors for the transfer of DNA, a
tibody. variety of genetic elements are available. Most
To obtain expression of the cloned gene, it is commonly used are plasmids, temperate phages,
necessary to understand the genetic organization and cosmids.
of procaryotes and eucaryotes. In eucaryotes, the Plasmids are defined as circular, extrachro-
coding regions of genes (exons) are generally bro- mosomal, self-replicating DNA molecules. They
ken with noncoding regions (introns). From the have molecular weights between 1.5 X 10° and
primary transcript from the nuclear gene, the in- 200 X 10°. Large plasmids and small plasmids
trons must be removed (RNA processing) before have different properties. Large plasmids have
the mature translatable mRNA is present. The an average molecular weight of 65 X 10; there
RNA processing steps cannot be carried out by are usually 1—2 copies per bacterial chromosome
procaryotes so that a correct translation of cloned in the cell and they can be transferred during
genes of eucdryotes is not possible. Therefore, conjugation. Small plasmids (about 5 million mo-
when bacteria are used as cloning hosts it is pref- lecular weight), of which there are more than 10
erable to use synthetic DNA or complementary copies per bacterial chromosome, are not con-
 3.6 GENE TECHNOLOGY / 43

HindIl

(A)3' K
Cas Sas (T)5 pMB9
(35Md)

HindIll Endo-
(A)3' ©) nuclease
ts. CH A (T)5' alk. Phosphatase

5'0HAGCT
| ———A3'
5 —— (A)3' 3'A —---— TTCGAOHS'
3 SS {T)5'
T4 DNA Ligase
©) |, DCCAAGCTTGG 3’
3'GGT TCGAACC 5'
5 CCAAGCTTGG- ——__—(ACCA AGCTTGG 3’
3'GGT TC GAACC ———————_(T)GGT TCGAACC 5’

SpAGCiIGG——————
{Acc a3’
3ACC————.—{1)GGTTCGAp5"

T, DNA Ligase

AAGCTTGG-————_({A) CCAAGCTT
T TC GAAC C ———————_(T)
GGT T CGAA
cDNA

Plasmid- DNA

Figure 3.23 Cloning rat insulin genes (Ulrich et al., 1977). 1: mRNA is isolated from an insulin-producing tumor of
the rat and transcribed with reverse transcriptase into cDNA. Simultaneously, the enzyme reverse transcriptase produces
a hairpin structure (fold-back region) which serves as a primer for the completion of the double strand. 2: RNA is
removed from the mRNA-cDNA-hybrid molecule by treatment with alkali. 3: The single-stranded cDNA becomes double-
stranded through reverse transcriptase. 4: The fold-back region of the hairpin structure is digested by S1-nuclease. 5:
Through T4 ligase, chemically synthesized nucleotide sequences containing the recognition sequence for the restriction
endonuclease HindIII are attached at both ends of the cDNA. 6: Through the action of HindIII, sticky ends are created
which permit the attachment to a similarly-treated plasmid DNA
44 / CHAPTER 3 / STRAIN DEVELOPMENT

jugative. Plasmids code for numerous and varied Cosmids are plasmids which contain the cos
cell functions: region of phage A, which makes it possible for
the plasmid to be packaged within bacteriophage
+ Fertility: ability to transfer genetic material particles and hence transfered by infection to E.
through conjugation. coli. Cosmids are capable of incorporating ex-
e Antibiotic resistance: resistance to one or tremely long sequences of DNA, 32-47 kb, and
more antibiotics; R-plasmids are known in hence are especially useful for the production
more than 50 species of bacteria. gene banks.
e Resistance to heavy metals: Cd?*, Hg. The foreign DNA is either incorporated into
+ Resistance to ultraviolet radiation. a specific restriction site of the cosmid (insertion
¢ Production of bacteriocins, substances which vector) or replaces a fragment of equivalent
inhibit or kill cells of the same species. length of the vector (substitution vector).
¢ Production of antibiotics: a plasmid has been Depending on the purpose, the following
shown to code for the antibiotic methyleno- kinds of vectors can be constructed:
mycin. Cloning vectors are used primarily for the
¢ Utilization of unusual carbon sources: break- amplification of foreign DNA in the host cell.
down of camphor, octane, and octanol by With certain plasmids, the copy number per cell
Pseudomonas, for example (see Table 3.10). can be increased by growth in the presence of
¢ Formation of toxins and surface antigens, the antibiotic chloramphenicol, which inhibits
such as enterotoxin and hemolysin. the replication of the host genome without af-
e Tumor induction in plants: formation of fecting the replication of the plasmid. In this way,
crown gall tumors by the Ti plasmid of Agro- recombinants plasmids can often be produced to
bacterium. as many as 1000 copies per cell.
¢ Involvement in sporulation in streptomy- Sequence vectors are those which contain an
cetes. increased number of recognitions sites for re-
striction enzymes (so-called multi-purpose clon-
Plasmids are capable of adding foreign genes
ing sites). When the foreign DNA is integrated
to the genetic material of the cell. If there is no
into such a plasmid, DNA fragments of various
homologous area in the bacterial chromosome
sizes can be produced and sequenced.
for these foreign genes, they cannot be ex-
Expression vectors contain not only the for-
changed during crossing over, but may be main-
eign gene but also promoter, operator, and ter-
tained through plasmid replication. Some plas-
minator sequences so that the gene can be effi-
mids exhibit the phenomenon of incompatibility,
ciently transcribed into mRNA. In addition,
in which two related plasmids are unable to be
expression vectors contain the ribosome-binding
maintained together in the same cell.
site so that efficient translation can take place.
The expression of foreign DNA in the host
Table 3.10 Catabolic plasmids from Pseudomonas organism is largely dependent on the system
Plasmid Substrate broken down Transmissibility used. In Escherichia coli, procaryote DNA is tran-

NAH Naphthalene Conjugative ”

scribed and expressed into protein. Under fa-
SAL Salicylic acid Conjugative vorable conditions as much as 30% of the dry
CAM Camphor Conjugative weight of the cell can consist of the heterologous
OGE Octane, hexane, decane Nonconjugative
protein.
TOKE p- or m-xylene, toluene Conjugative
pJP1 2,4-dichlorophenoxyacetic Conjugative The expression of genes of lower eucaryotes,
acid such as Saccharomyces cerevisiae or Neurospora
pAC25 3-chlorobenzoate Conjugative
crassa, has been successfully achieved in Esche-
pAC27 3- and 4-chlorobenzoate Conjugative
richia coli, but in more complex eucaryotes, DNA
 3.6 GENE TECHNOLOGY / 45

transcription and translation have not occurred known for E. coli and Bacillus subtilis, and for E.
correctly. These difficulties may be overcome by coli and yeast.
incorporating eucaryote DNA into a procaryote
gene containing appropriate regulatory signals, Incorporation of foreign DNA _ This is done dif-
thus permitting expression of the eucaryote ferently with blunt end or cohesive end frag-
DNA. This was first accomplished with soma- ments. With blunt-end fragments, the 5’ ends are
tostatin, which was incorporated into the lactose digested by \-exonucleases and the 3’ ends are
operon located on plasmid pBR322 (Figure 3.24). extended through action of a terminal transferase
The product synthesized by the Escherichia coli enzyme with ATP or TTP. In this process, a ter-
cell (a fusion protein) was 8-galactosido-soma- minal poly-A or poly-T sequence results. One
tostatin. Active somatostatin was cleaved from strand with a poly-A sequence can combine with
the chimeric protein in vitro by treatment with another strand containing a poly-T sequence to
cyanogen bromide, which splits peptides at me- form a circle as a hybrid plasmid. When there
thionine residues. The yield of somatostatin ob- are sticky ends, DNA fragments of any origin
tained was one-tenth of the value calculated from combine as long as the same restriction endo-
the plasmid copy number but was still relatively nuclease has been used in the creation of both
favorable (10 mg somatostatin protein per 100 g fragments.
Escherichia coli wet weight) considering the sim-
plicity of the Escherichia coli fermentation. Insertion of foreign DNA into the host The effi-
In the same way, synthetic genes of the A cient incorporation of foreign DNA into the tar-
and B chain of human insulin were incorporated get host cell is a critical step in gene manipula-
into the #-galactosidase region of the pBR322 tion. It is essential that the host is able to take
plasmid and cloned separately in Escherichia colli. up the engineered DNA. Host organisms include
The transformed bacteria synthesized the A and not only microorganisms but also plant, animal,
B chains separately; by mixing both compounds, and human cells. In E. coli transformation of na-
an active insulin preparation was developed and ked plasmid or phage DNA can be brought about
has now been marketed by the Eli Lilly Com- in the presence of CaCl,, which renders the cell
pany. wall and cell membrane permeable to free DNA.
Another insulin production method involved With plasmid DNA, 107-108 transformants can
use of Escherichia coli x1776. Complementary be obtained per ug DNA. An alternative proce-
DNA copies of preproinsulin-mRNA of the rat dure is to use a transduction system consisting
were cloned into the penicillinase gene of plas- of phage or cosmid DNA packaged into empty
mid pBR322. A penicillinase-proinsulin complex, phage heads. For gram-positive bacteria, acti-
penicillinase(24-182)—(Gly),-proinsulin(4-86), nomycetes, yeasts, and filamentous fungi, effec-
was formed by the bacteria. The synthesis rate tive transformation of plasmid or phage DNA can
was about 100 molecules per cell. Since Esche- be accomplished using the PEG system in pro-
richia coli penicillinase is an extracellular protein, toplasts (see Section 3.4).
the complex was excreted from the cell and could
be transformed into active insulin by splitting the Analysis of recombinant clones There are several
penicillinase residue from the insulin C chain. possible procedures that can be used to screen
In order for a vector to replicate in the host transformed cells and determine that the cloning
cell, it must contain the appropriate origin-of- and expression processes have been effective:
replication site (ori). Vectors containing ori of two
separate host systems are able to replicate in + Identification of host cells containing the for-
both. Such vectors, called shuttle vectors, are eign-DNA-containing vector;
46 / CHAPTER 3 / STRAIN DEVELOPMENT

Escherichia coli Lac Operon DNA Genetic code

Chemical DNA synthesis

Somatostatin gene
[ATG]GCT GGT TGT AAGAACTIC TIT Io
G
A
A
G
cA
- CTAGIGAT AGT] TGT GCT CTA CTT T
pBR322 Plasmid DNA

in vivo

B-Gal
HN DV INID ID IDIOM
et-Al'a-Gly — Cys— Lys —Asn-Phe —Phe

| Trp
|

| Lys

HO—Cys—Ser—Thr—-Phe "IT

in vitro
Bromocyanogen splitting

B-Gal- Fragment a5 HN —Ala-Gly-

Cys — Lys~Asn-Phe Phe
|
S hk
|
S Lys
Figure 3.24 Expression of somato-
statin gene sequence in Escherichia HO — Cys— Ser—Thr— Phe—Thr
coli (Itakura et al., 1977). See text for
explanation Somatostatin

+ Detection of the foreign DNA in the host stance, antibiotic resistance), one of which con-
cells; i tains the recognition site for the restriction en-
. Detection of the foreign DNA indirectly by zyme used in the cloning process. If the foreign
assaying for the expression product (foreign DNA becomes integrated into this antibiotic re-
protein). sistance gene, the activity of that gene is lost (in-
sertional inactivation). Host cells that lack the
To select transformed cells, the marker in- vector are sensitive to both antibiotics, host cells
activation technique can be used. Vectors are containing a vector lacking the foreign DNA are
used containing two selectable markers (for in- resistant to both antibiotics, whereas vectors with
 3.6 GENE TECHNOLOGY / 47

inserted foreign DNA are sensitive to the one

antibiotic into whose resistance gene the foreign Cloning and expression systems for
DNA has been inserted. various microorganisms
To assay a complete gene bank for the pres- The selection of a suitable cloning system de-
ence of the gene, it is anticipated that around pends to a great extent on the protein to be syn-
1000 recombinant clones must be tested. Further thesized. Low-molecular weight proteins with
tests must then be used to determine that the few disulfide bridges and which do not require
appropriate clone has been obtained. To dem- glycosylation for activity can be cloned and ex-
onstrate the presence of cloned DNA in the cell, pressed in bacteria. Bacteria are unable, however,
two types of procedures are used, colony hy- to glycosylate proteins, whereas yeasts prefer-
bridization and Southern blotting. entially glycosylate with mannose rather than
Colony hybridization is done by using the glucose (the preferred sugar in human proteins).
replica-plating technique to transfer around 200 To produce complex proteins, cell cultures are
colonies from agar plates to a nitrocellulose filter. most suitable. In such cultures, not only is the
The colonies on the filter are then lysed and the protein properly glycosylated, but it is also se-
released DNA denatured with 0.5 N NaOH. Af- creted in its native configuration.
ter removing excess protein with the enzyme As shown in Table 3.11, when foreign pro-
proteinase K, the single-stranded DNA is fixed teins are expressed, much of the synthesized pro-
to the filter by heating to 80°C. After hybridi- tein may be retained in the cells rather than being
zation with a °**P-labeled probe (either DNA or excreted into the medium. It may therefore be
RNA), the filter is subjected to autoradiography. preferable to begin with a system in which total
The colonies corresponding to the active spots
activity is lower in order to save costs of isolation
and purification.
can then be isolated from the original plate.
After isolation of hybrid DNA molecules, the
Gram-negative bacteria Because of its well-
specific DNA sequence can be identified by
known biology and genetics, E. coli is the pre-
means of the Southern blotting technique. Af-
ferred organism for experiments in genetic en-
ter treatment of the DNA with a restriction en-
gineering. A large number of heterologous genes
zyme, the resulting DNA fragments are sepa-
have been cloned and expressed in this organism.
rated by gel electrophoresis. The DNA bands are
However, wild type plasmids such as ColE1,
then transferred to paper or nylon filter by blot- pSC101, and RSF2124 presented a number of
ting. The filter provides a stable carrier for the disadvantages, so that suitable vectors have been
fragments, and the fragment containing the tar- constructed in vitro. A widely used vector is
get sequence is identified using a gene probe. pBR322, a ColE1-like plasmid, which contains
(The corresponding technique for RNA is called
Northern blotting, that for proteins is called
Table 3.11 Final yield of heterologous proteins in
Western blotting.) relation to protein secretion in various cloning systems
A different kind of procedure for detecting
Cloning system Amount of het- Protein Final yield
the desired clones involves seeking clones in erologous pro- excre- (mg/l)
which the gene product (protein) has been ex- tein (mg/1) tion
pressed. Since the expression efficiency is often E. coli 5000 No 250
quite low, a very sensitive method for detecting B. subtilis 100 Yes 30
Saccharomyces 10 Yes 3
the gene product is necessary. One of the most cerevisiae 2000 No 50
widely used methods is immunological, in which Animal cell culture 100 Yes 30
an antibody (marked by radioactivity or enzyme) Final yield after isolation and purification. Data of Davies
is used as a probe. (1986).
48 / CHAPTER 3 / STRAIN DEVELOPMENT

the ampicillin-resistance gene (Ap®) of RSF2124, purposes could also be derived from temperate
the tetracycline-resistance gene (Tc®) of pSC101, phages p11, #105, and NPO2. For the transfor-
and the origin of replication of pMP1. In addi- mation of bacilli, it is necessary to begin with
tion, pBR322 contains recognitions sites for 20 protoplasts and to use single-stranded DNA. One
restriction enzymes. Other suitable vectors for E. problem with B. subtilis is that plasmids are often
coli include phage A and single-stranded DNA unstable, leading to rearrangments of the gen-
phages M13, f1 and fd, which are especially suit- ome or deletions.
able for DNA sequencing. Details of cloning sys- Other gram-positive microorganisms of in-
tems for E. coli are widely available in manuals dustrial interest for which cloning systems have
and reference books (see References). been developed include streptococci (for the
Large-scale production of foreign proteins in dairy industry), clostridia (for acetone, butanol,
E. coli presents some difficulties, especially be- isopropanol, butyric acid, and acetic acid fer-
cause foreign proteins are often subjected to ex- mentations), Corynebacterium and Brevibacter-
tensive proteolysis or are produced in the cell in ium, for amino acid and nucleotide production.
the form of highly insoluble protein bodies (in- A number of plasmid and phage vectors have
clusion bodies). Purification of these atypically been developed for the industrially important
folded and frequently denatured proteins leads group of streptomycetes. Among others, these
in many cases to only a very tiny amount of the are pIJ61, a derivative of plasmid SLP1.2 of Strep-
correct product. tomyces lividans, SCP2, a low-copy-number plas-
To clone in other gram-negative bacteria, for mid of S. coelicolor A3(2), bacteriophage ¢C31 of
instance Pseudomonas, derivatives have been de- S. coelicolor, and plasmid pIJ101, a conjugative
veloped of the wild type conjugative plasmids multicopy plasmid of S. violaceoruber. Detailed
IncP and IncW as well as the nonconjugative procedures for cloning in Streptomyces have been
plasmid IncQ. These plasmids possess an ex- developed (see References). With these methods,
tremely wide host range and can be used in al- recombinant DNA techniques can be used in the
most all species of gram-negative bacterila. study of biosynthesis and regulation of secon-
dary metabolites in streptomycetes. With such
Gram-positive bacteria Species of the genus Ba- basic information available, intraspecific or in-
cillus have been used for many years in industry terspecific cloning can be used for strain im-
for the production of peptide antibiotics and ex- provement. Some successes in this area have al-
oenzymes. Their use in large-scale cultivation is ready been obtained. The O-methyltransferase
therefore well understood. An additional advan- gene of S. coelicolor A3(2), involved in the bio-
tage of the bacilli for gene technology is that ex- synthesis of undecylprodigiosin, has been
cellent excretion of foreign proteins takes place. cloned. Studies on glucose repression of glycerol
However, the native plasmids and phages of Ba- catabolism in S. coelicolor have led to clarification
cillus subtilis are not suitable as cloning vectors. of the mechanism of glucose catabolite regula-
In vitro construction of suitable plasmids was tion.
based on the utilization of antibiotic resistance Genes controlling the production of three an-
plasmids of Staphylococcus aureus. An artificial tibiotically active substances of S. coelicolor have
plasmid pHV11, containing tetracycline-resist- been cloned and expressed: actinorhodin (7 chro-
ance and chloramphenicol-resistance genes mosomal genes), undecylprodigiosin (5-6 chro-
(Tc®Cm®) was developed for B. subtilis which was mosomal genes), and methylenomycin (5-6 plas-
analogous to pBR322 of E. coli. Addition of the mid-controlled genes). In addition, genes
origin of replication of E. coli led to the devel- involved in antibiotic synthesis and antibiotic re-
opment of a shuttle vector for E. coli and B. sub- sistance have been cloned for a variety of other
tilis. Vectors suitable for various experimental streptomycetes (see Section 3.7). Because of the
 3.6 GENE TECHNOLOGY / 49

extreme complexity in the biosynthetic pathways + YRp vectors (yeast replicating plasmids) are
of antibiotics, much further basic research will be yeast sequences which have a chromosomal
needed before these newer methods will lead origin and contain an origin of replication.
readily to strain improvement. After incorporation into bacterial plasmids,
Because streptomycetes readily excrete pro- they can be used as vectors. They exhibit a
teins, S. lividans has been used as a model system relatively high frequency of transformation
for the expression of bovine somatotropin, hu- (10°-104 transformants per ug DNA).
man serum albumin, and human interleukin-2, ¢ YCp vectors (yeast centromere plasmids) are
although the use of streptomycetes as expression YRp vectors into which the centromere of
systems for heterologous proteins is still in its chromosome III has been incorporated. These
infancy. plasmids behave as circular minichromo-
somes, are stably inherited, and can be used
Eucaryotes The yeast Saccharomyces cerevisiae as vectors.
has replaced E. coli in a number of projects for
the expression of eucaryotic proteins. Large-scale Filamentous fungi Although a variety of anti-
production of yeast is well-established. Genes for biotics and enzymes are produced commercially
foreign proteins have been expressed and the with filamentous fungi, cloning systems for these
proteins secreted into the medium. A further ad- organisms are not nearly so well developed as
vantage for the production of genes from mam- for yeast. Plasmids resembling the YIp type have
mals is that yeast is able to glycosylate foreign been constructed by combining genes from Neu-
proteins. A whole series of mammalian proteins rospora with the origin of replication of pBR322
have been successfully expressed and correct of E. coli. Hybrids between pBR322 and mito-
splicing of mRNA and correct glycosylation do chondrial DNA (mtDNA) of the ascomycete Po-
not always occur. dospora anserina replicate in both E. coli and Po-
A range of vectors have been developed for dospora. Plasmids from N. crassa and P. anserina
Saccharomyces cerevisiae, mostly hybrids be- can perhaps be produced by insertion of the or-
tween yeast DNA and E. coli plasmids which can igin of replication from mtDNA sequences.
serve as shuttle vectors. The principle vectors are Various commercially grown aspergilli, such
summarized here: as Aspergillus niger, A. awamori, and A. oryzae,
excrete large amounts of exoenzymes. However,
¢ YIp vectors (yeast integrating plasmids) con- the species A. nidulans is the best known genet-
tain the ColE1 origin of replication of E. coli ically and has served as a model for the devel-
and chromosomal sequences from yeast. opment of effective transformation and expres-
They replicate as plasmids in E. coli. Al- sion systems. Biologically active bovin chymosin,
though they do not replicate in yeast they are human interferon a2, and human tissue plasma
able to integrate into the yeast genome, al- activator (tPA) have been expressed and secreted
though the integrated sequences are rela- in A. nidulans. However, it is not certain whether
tively unstable. the correct glycosylation has been obtained.
¢ YEp vectors (yeast episomal plasmids) are The procedures that have been developed in
circular plasmids (2 um in length, about 6 kb) yeast and aspergilli have also been adapted to
that are present in yeast cells in around 50- the important 6-lactam-producing fungi Penicil-
100 copies. When connected with selectable lium chrysogenum and Cephalosporium acremon-
yeast sequences and corresponding se- ium. In C. acremonium a cosmid gene bank has
quences from E. coli plasmids, they can be been been constructed which has permitted the
used as vectors. Vectors based on the 2 um isolation of the leuB gene which codes for 6-is-
DNA are, however, relatively unstable. opropylmalate dehydrogenase. A vector has
50 / CHAPTER 3 / STRAIN DEVELOPMENT

been constructed, pIT221, which contains, international conference at Asilomar, California

among others sequences from pBR322, a mtDNA in 1975. They became the basis for the rules of
fragment of C. acremonium, and an ARS region the U.S. National Institute of Health (NIH). Since
(autonomously replicating sequence) which per- that time, however, the risks have been consid-
mits replication in S. cerevisiae. Using the gene ered so slight that the NIH guidelines have been
coding for hygromycin B phosphotransferase suspended. This has been because of:
(HPT) of E. coli, it has been possible to transform «+ The development of suitable safety measures.
protoplasts of C. acremonium and demonstrate
«+ The use of weakened Escherichia coli strains
the presence of the HPT coding sequence. The
(e.g. x1776) with a probability of survival of
principal goal of this work is to increase the yield
107 outside of the laboratory.
of the enzyme. + Experimental evidence that there is only very
slight danger of the transfer of cancer genes
Acrasiomycetes Inthe continuing search for suit-
to humans.
able expression systems for mammalian proteins,
research has turned to the organism Dictyoste- Certain experiments are still prohibited or re-
lium discoideum, a eucaryote which has a life cy- quire authorization. Each country has its own
cle in which a free-living amoeboid form alter- specific guidelines for recombinant DNA re-
nates with a fruiting body in which resting cells search, and the operative regulations can be ob-
are produced. A functioning expression system tained by applying to the proper authorities.
for this organism has been created; for a partic-
ular foreign protein, yields 300 times greater than
3.7 USE OF GENETIC METHODS
that of an optimized E. coli system have been
obtained. Since Dictyostelium grows in the veg-
Strain optimization
etative phase as an amoeba, diffusion is not a
limiting factor in nutrient uptake so that contin- The primary goal of an industrial strain devel-
uous culture can be used to produce the desired opment program is the increase in the yield of
protein in an unlimited fashion. However, the desired product. The genetic techniques for
whether correct protein processing and glyco- accomplishing this goal have been discussed in
sylation occur has not been determined, and Sections 3.2-3.6. If the desired product, for ex-
scale-up of this organism to a large-scale indus- ample, an enzyme, is controlled by a single gene
trial process has not yet been accomplished. or a small group of genes, then high-yielding
strains can be produced by:
Risks in genetic engineering + Isolation of mutants resistant to inhibitors of
protein synthesis, which often overproduced
The creation of new types of viruses and the pos-
proteins;
sible cloning of the DNA of tumor viruses has
+ Manipulation of regulatory signals to increase
been perceived to be potentially dangerous.
transcription or translation by cloning the
Since Escherichia coli, which exists in the human
gene on an expression vector or inserting the
intestinal tract, has been used almost exclusively
gene into a transposon which has a strong
for the cloning of recombined DNA, the possi-
promoter;
bility of human infection cannot be ruled out.
¢ Modification of the gene by use of site-di-
Thus in 1974 numerous renowned American sci-
rected mutagenesis.
entists called for a voluntary moratorium on cer-
tain cloning experiments until potential risks By increasing the gene dosage (gene ampli-
were resolved. The first guidelines for work in- fication), the yield may be increased, provided
volving recombined DNA were established at an the expression of the duplicated genes is not re-
 3.7 USE OF GENETIC METHODS / 51

stricted by regulation. Gene amplification can oc- characteristics of the process. For instance, if the
cur in the following ways: yeast Saccharomyces cerevisiae contains an a-am-
ylase from Bacillus subtilis, ethanol fermentation
+ Increasing the number of DNA replication
sites in growing bacterial cells causes ampli- can occur at 40°C and a stronger starch hydrol-
fication of the genes situated near the origin ysis can take place. After transfer of the killer
of replication. factor from a wild type to an industrial mutant
of S. cerevisiae, wine, beer, sake, and alcohol pro-
+ Diploidization of fungi increases gene dos-
age, although the strains are usually unstable. duction was protected from undesirable wild
yeast contamination.
+ Isolation of hyperinduced strains, which have
been cultivated under selective conditions
over a long period. These strains are ex- Qualitative change in the spectrum of
tremely unstable, however, and are usually antibiotics
not suitable for commercial processes.
Many producers of secondary metabolites also
. If the gene has been characterized, the great-
produce a range of chemically related byprod-
est success is likely by use of genetic engi-
ucts. Mutants which produce only the desired
neering methods, for example, cloning and
antibiotic or only a small percentage of other sub-
amplification of the gene by means of a mul-
stances can simplify the product recovery con-
ticopy plasmid or a phage vector. For in-
siderably and thus lower its cost. Table 3.12
stance, by use of a cosmid system the for-
shows examples which are industrially relevant.
mation of the enzyme penicillin acylase in E.
coli has been markedly increased when com-
pared to the wild type. A whole series of in- Production of modified secondary
dustrial enzymes have been optimized in this metabolites
way.
One way to produce therapeutically effective
Difficulties abound when the goal is the in- antibiotics is to use mutants which synthesize
crease in yield of a multi-gene product such as derivatives of known antibiotics. Such modified
a primary or secendary metabolite, although antibiotics may be synthesized either directly or
some successes have been achieved. Amino acid through transformation of added precursors.
production has been increased by cloning the With the use of the protoplast fusion or recom-
whole genome, first in E. coli, later in production binant DNA techniques, hybrid antibiotics can
strains such as Corynebacterium, Brevibacterium, be produced by bringing together in one strain
or Serratia (see Chapter 9). the biosynthetic genes from several organisms.
For secondary metabolites such as antibiotics,
cloning and amplification of the rate-limiting en- Direct biosynthesis of modified antibiotics Strains
zyme of the biosynthetic pathway can be done. with mutations in antibiotic synthesis produce
As a first step in this direction, the genes for a substances whose structure is similar to the anti-
number of antibiotics have been isolated, cloned, biotic of the parent strain (e.g. demethyl com-
and in a few cases expressed. These include ac- pounds from tetracycline, chlortetracycline, ri-
tinorhodin, methylenomycin, and undecylpro- famycin). One of the best examples is the
digiosin (Streptomyces coelicolor), cephalosporin production of adriamycin (14-hydroxydauno-
(Cephalosporium acremonium), erythromycin (S. mycin) by construction of a mutant from S. peu-
erythreus), oxytetracylcine (S. glaucescens) and ty- cetius var. caesius. This derivative of daunomycin
losin (S. fradiae). is more effective as an antitumor agent than dau-
In addition to increase in yield, genetic meth- nomycin itself and is the most commonly used
ods have been used to improve the fermentation antitumor antibiotic today. Intermediate prod-
52 / CHAPTER 3 / STRAIN DEVELOPMENT

Table 3.12 Changes in byproducts of antibiotic- analogs of this missing component are added,
producing organisms through strain development
modified antibiotics may be produced, provided
Fermentation By-products Production the analog penetrates the cell and is recognized
product eliminated by strain
strain
by the respective enzymes. Table 3.13 shows sev-
development eral examples. Attempts have been made to use
Polymyxin B Other poly- Bacillus poly- mutasynthesis to produce modified macrolide
myxins myxa and 6-lactam antibiotics, as well as novobiocin
Cephalosporin C | Cephalosporin Cephalosporium analogs. Although several new compounds have
N and P acremonium
n-Butanol Acetone (varia- | Clostridium sp. been found, commercial use has not yet been
tion in possible due to the poor rate of precursor incor-
amount pro- poration and the difficulty of obtaining proper
duced)
Griseofulvin Mycelianamide | Penicillium gri- mutants,
seofulvum
Actinomycin D Other actinomy- | S. antibioticus Cosynthesis In a mixed culture containing two
cins
Oleandomycin A Oleandomycin B S. antibioticus mutants, the first mutant may accumulate an in-
Tetracycline Chlortetra- S. aureofaciens termediate (C in the diagram below) which is
cycline transformed by the second mutant (blocked ear-
Neomycin B Neomycin C, S. fradiae
Fradicin lier in the synthetic pathway) into the end prod-
Streptomycin Mannosido- S. griseus uct.
streptomycin,
Cyclohexi- A —— B — C //-—D ——E
mide, Vitamin y
Bi A B /} C D — E
Cycloheximide Streptomycin S. griseus
Antimycin A, Other antimy- Streptomyces sp.
cins This method, which has been primarily used in
Tobramycin (Ne- Remaining fac- S. tenebrarius the study of biosynthetic pathways, has also been
bramycin factor tors of Nebra-
successful in the isolation of modified antibiotics.
6) mycin com-
Apramycin plex (1,1’, For example, cosynthesis between two mutants
(= Factor 7) 2,3,4,5,5', of the 6-demethyl tetracycline producer S. psam-
6 and 7) moticus resulted in a new end product of tetra-
Levorin Levoristatin (re- |Actinomyces le-
duced propor- voris No. 28 cycline biosynthesis having a different spectrum
tion) of activity.
Leucomycin A,,A; Leucomycin A,— | Streptoverticil-
A lium kitasa-
| | taensis Hybrid antibiotics Genes for biosynthetic steps
in different organisms can be combined in the
(Perlman, 1973; with additions)
same organism, thus leading to the production
of new metabolites. By protoplast fusion, a
ucts of biosynthesis, which in wild strains may blocked mutant of the streptomycin-producing S.
be produced at concentrations too low to detect, griseus was combined with the istamycin-pro-
may accumulate in blocked mutants, thus lead- ducing S. tenjimariensis, leading to the produc-
ing to the detection of new products. For in- tion of the hybrid antibiotic indolizomycin. In
stance, a new tylosin analog was found in this another example, the genes for actinorhodin bio-
way in a tylosin-producing culture of S. fradiae. synthesis from S. coelicolor were cloned into a
producing strain of the same antibiotic class and
Mutasynthesis In mutasynthesis (mutational various new compounds were detected. Also, the
biosynthesis), mutants are used which cannot gene for oleandomycin-producing S. antibioticus
synthesize a part of the antibiotic molecule. If was transferred to a blocked mutant of eryth-
 3.7 USE OF GENETIC METHODS / 53
Table 3.13 Some examples of mutasynthesis
Production strain (antibiotic Block in the biosynthesis Analog compound added Mutasynthesis product
produced) of
S. fradiae (Neomycin) 2-Deoxystreptamine Streptamine Hybrimycin A,, A,
2-Epistreptamine Hybrimycin B,, B,
Bacillus circulans (Butirosin) 2-Deoxystreptamine 2,5-Dideoxystreptamine 5-Deoxybutirosamine
Streptamine 2-Hydroxybutirosin
Neamine 6'-N-Methylneamine 6'-N-Methylbutirosin
A,B
Micromonospora inyoensis (Si- 2-Deoxystreptamine Streptamine Mutamicin 1
somicin) 2,5-Dideoxystreptamine Mutamicin 2
2-Epistreptamine Mutamicin 4
Micromonospora sagamiensis 2-Deoxystreptamine Streptamine 2-Hydroxysagamicin
(sagamicin)
S. griseus (Streptomycin) Streptidine 2-Deoxystreptidine Streptomutin A

romycin-producing S. erythreus. Although new development is the competitive situation, since

compounds can undoubtedly be produced in this many companies are often pursuing research on
way, it appears unlikely that these more or less the same compound.
random approaches will lead to the formation of In the following, a brief summary of the cur-
useful new compounds. rent developments in use of recombinant DNA
Another approach to the detection of new products is given.
substances is the use of DNA sequences as gene
probes for the detection of new antibiotics of the Gene technology in basic research Genetic en-
same chemical type. gineering has revolutionized research in basic bi-
ology. The clarification of control methods in
both procaryotes and eucaryotes, embryological
Recombinant DNA products and specific
development and differentiation, sequence de-
applications of gene technology
termination of proteins has all made enormous
Advances in the use of recombinant DNA tech- advances. The first completely sequenced eu-
nology to produce pharmacologically interesting caryotic gene was the @-globin gene of rabbit.
proteins, enzymes, or amino acids is proceeding This led immediately to the discovery that eu-
at a rapid pace. A whole series of recombinant caryotic genes were often split into coding re-
DNA products are on the market, and more can gions (exons) and noncoding intervening regions
be anticipated in the near future. Applications are (introns). Also discovered was the fact that the
in medical diagnostics, agriculture, and environ- genome of an organism is not static but that
mental protection as well as in basic research. genes exist whose role is in the regulation and
The production of heterologous proteins by re- expression of other genes (dynamic and hierar-
combinant DNA technology has become wide- chical aspects of genetics).
spread. However, the high development cost of As further examples from microbiology: the
these products is often not supported by a suf- identification and characterization of antibiotic
ficiently large market. For some products, only resistance genes, as well as regulatory and struc-
kilograms are required on a world-wide basis. In tural genes of biosynthetic pathways, the study
general, the cost for developing a recombinant of genetic instability and strain degeneration (pri-
product may be as much as 100-fold higher than marily in streptomycetes), and genome analysis
that for development of a conventional phar- (for example, the demonstration that the archae-
maceutical product. A further handicap to rapid bacteria constitute a distinct group of organisms).
54 / CHAPTER 3 / STRAIN DEVELOPMENT

In medicine, the methods of gene technology ¢ The development of vaccines, especially so-
have made important contributions in cancer re- called “subunit vaccines” that represent the
search, immunology, circulatory system, as well antibody-recognition sites of pathogens; for
as in the nerve and endocrine systems. instance, surface proteins of viruses or path-
ogenic bacteria. Hepatitis B vaccine is already
Protein engineering The use of X-ray crystallog- on the market in this area.
raphy has permitted the determination of the ¢ The development of gene probes for use in
three-dimensional structure of proteins. By the diagnosis for virus, bacteria, and fungal dis-
use of computer-aided protein design, molecular eases. Such probes are of special use for those
models can be readily produced and their struc- disease agents for which culture methods are
ture /function relationships determined. Changes not possible, such as many of the viruses.
in amino acid sequence lead to changes in protein ¢ Monoclonal antibodies are immunoglobu-
structure, not always in a predictable way. By lins that are very specific for their antigens.
use of modelling, the intelligent selection of de- They are produced by the hybridoma tech-
sirable mutations can be made and these muta- nique, in which an antibody-producing lym-
tions can then be created by recombinant DNA phocyte is fused with a tumor cell (myeloma)
technology. The technique offers the possibility to produce the so-called hybridoma. The
of modifying enzyme sensitivity to inhibitors, value of the hybridoma is that it can be cul-
turnover rate, substrate specificity, stability, etc. tured indefinitely, permiting unlimited pro-
As an example of the potentialities: changes in duction of the particular immunoglobulin.
various domains of the protein tPA (tissue plas- Monoclonal antibodies have already been
minogen activator) led to increase in the half-life perfected for analytical and diagnostic pur-
of the enzyme in animal studies. In another ex- poses as well as for use in protein purification
(for example, interferon). It can be anticipated
ample, changing a single amino acid (aspartic
acid to serine at position 99) in subtilisin (Bacillus
that monoclonal antibodies will also play a
role in cancer diagnosis, as well as in virus
amyloliquefaciens) led to an alteration in the pH
therapy.
optimum.

Agriculture Advances in the production of cul-

Human therapy By the use of recombinant DNA
tivated plants, such as the production of disease-
technology, substantial quantitities of proteins
resistant plants, can be expected from genetic en-
can now be produced that previously were only
gineering. Genetic manipulation of plants has
available in extremely small quantitities, if they
conventionally been a slow and difficult task, but
were available at all. Such proteins can be used
has been greatly facilitated by the new genetic
therapeutically in those patients deficient in these methods. It is possible to use plant cell culture
proteins (for example, insulin for diabetics, hu- procedures to select clones of plant cells that are
man growth hormone for dwarfs, factor VIII for
genetically changed and then, with proper treat-
hemophiliacs). Another use is in the increase in ments, induce these cell cultures to make whole
an immune response, as for instance interferon. plants which can be propagated vegetatively or
Over 100 proteins of human origin have been by seeds. One approach to the introduction of
cloned and expressed in experimental cell sys- foreign genes into plants is the use of the bac-
tems. Several such products are now being mar- terium Agrobacterium tumefaciens, which con-
keted commercially (Table 3.14) or are in clinical tains a large plasmid (the Ti plasmid) which can
trials (Table 3.15). become integrated into the plant genome. The Ti
Recombinant DNA technology opens up fur- plasmid can thus be used as a vehicle for the
ther possibilities through: introduction of foreign genes into plant cells. In
 3.7 USE OF GENETIC METHODS / 55

Table 3.14 Recombinant DNA products on the commercial market

Substance Trade name Company Application
Human insulin Humulin T Eli Lilly/Genentech Diabetes
Interferon Alpha 2 Berofor Boehringer Ingelheim Antiviral /Antitumor
Intron A Schering Plough/Biogen
Interferon Alpha (Namalva) Wellferon Wellcome Laboratories
Interferon Beta Frone Serono/Interpharm
Hepatitis B vaccine (HBsAG) Recombivax HB Merck/Chiron Vaccine
Human growth hormone Protropin Genentech Dwarfism
Tissue plasma activator (tPA) Actilyse Boehringer Ingelheim/ Thromobolytic activity
Genentech ER) (myocardial infarction)

Table 3.15 Recombinant DNA products in clinical trial Environmental protection Although still far in
Substance Application the future, there is the possibility of the use of
Antithrombin III Inhibition of blood engineered microorganisms for the elimination
coagulation of toxic wastes. Many compounds produced in-
Atrial Natriuretic Factor Diuretic dustrially are highly persistent in the environ-
Blood coagulation factors Hemophilia
VIII and IX ment and, because of their real or potential tox-
Epidermal growth factor Healing of wounds icity, should be eliminated. Genetic engineering
Erythropoietin (EPO) Anemia in dialysis can be used to develop new strains of microor-
patients
Human gonadotropin Sterility ganisms capable of breaking down these per-
(hCG, hMG) sistent chemicals, opening up the possibility that
Human serum albumin Blood substitute these new microorganisms could play a role in
Interferon Gamma Antitumor therapy/
Arthritis environmental protection. However, much more
Lymphokines (especially Stimulation of immune information on the microbial ecology of these
interleukin 2) defenses (bacteria/virus new microorganisms will be needed before they
infections; antitumor
therapy) can be effectively and safely released into the
Proteins as antigens Vaccine production environment.
(herpes, malaria,
influenza, AIDS)
Superoxide dismutase Heart attacks
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 Substrates for
industrial
fermentation

Media used in the cultivation of microorganisms + The composition of culture media must con-
must contain all elements in a form suitable for stantly be adapted to the fermentation pro-
the synthesis of cell substances and for the pro- cess. New batches of substrate have to be
duction of metabolic products. In laboratory re- carefully evaluated in trial fermentations be-
search with microorganisms, pure defined chem- fore they can be used in production.
icals may be used in the production of culture ¢ In addition to product yield, product recovery
media, but in industrial fermentations, complex, must be examined in trial fermentations.
almost undefinable substrates are frequently ¢ If catabolite repression or phosphate
used for economic reasons. Depending on the repression cannot be eliminated by optimi-
particular process, from 25 to 70% of the total zation of the nutrient medium or suitable fer-
cost of the fermentation may be due to the car- mentation management (e.g. feeding), dereg-
bohydrate source. In many cases, media ingre- ulated mutants must be used as production
dients are byproducts of other industries and are strains.
extremely varied in composition. For strain de-
Besides material cost and product yield, it
velopment and fermentation control, the conse-
must be considered whether materials used are
quences are as follows:
readily available in sufficient supply without
¢ An optimally balanced culture medium is high transportation costs, and whether impurities
mandatory for maximal production. A sup- will hinder product recovery or increase cost of
plement of critical elements must be used if product recovery.
necessary. Using statistical methods in com- We discuss below some of the frequently
puter-based programs, culture media may be used substrates in industrial fermentation. More
optimized. details can be found in references cited at the

59
60 / CHAPTER 4 / SUBSTRATES FOR INDUSTRIAL FERMENTATION

end of the chapter and in chapters on the indi- individual sugar factory. In addition to conven-
vidual fermentations. tional molasses, the residue from starch sac-
charification which accumulates after the crys-
talization of glucose is also widely used as a
4.1 SUBSTRATES USED AS CARBON fermentation substrate. For example, ”hydrol”
SOURCES molasses is a byproduct of glucose production
Carbohydrates are traditional energy sources in from corn.
the fermentation industry. For economic reasons, Malt extract, an aqueous extract of malted
pure glucose or sucrose can seldom be used as barley, is an excellent substrate for many fungi,
the sole carbon source, except in processes which yeasts, and actinomycetes. Dry malt extract con-
demand exact fermentation control. Molasses, a sists of about 90-92% carbohydrates, and is com-
byproduct of sugar production, is one of the posed of hexoses (glucose, fructose), disaccha-
cheapest sources of carbohydrate. Besides a large rides (maltose, sucrose), trisaccharides
amount of sugar, molasses contains nitrogenous (maltotriose), and dextrins, as shown in Table
substances, vitamins, and trace elements. How- 4.2. Nitrogenous substances present in malt ex-
ever, the composition of molasses varies de- tract include proteins, peptides, amino acids, pu-
pending on the raw material used for sugar pro- rines, pyrimidines, and vitamins. The amino acid
duction. Table 4.1 shows a comparison of the composition of different malt extracts varies ac-
analysis of sugar beet and sugar cane molasses. cording to the grain used, but proline always
Considerable variation in the quality of molasses makes up about 50% of the total amino acids
occurs, depending on the location, the climatic present. Culture media containing malt extract
conditions, and the production process of each must be carefully sterilized. When overheating
occurs, the Maillard reaction shown in Figure 4.1
results, due to the low pH value and the high
Table 4.1 Composition of sugar beet and sugar cane
molasses proportion of reducing sugar. In this conversion,
the amino groups of amines, amino acids (es-
Composition Sugar beet Sugar cane
molasses molasses pecially lysine), or proteins react with the car-
+
bonyl groups of reducing sugars, aldehydes, or
Dry matter % Us =89 77 -84
Sucrose 48.5 33.4 ketones, which results in the formation of brown
Raffinose 1.0 = condensation products. These reaction products
Invert sugar 1.0 21.2
are not suitable substrates for microorganisms.
Miscellaneous organic | 20.7 19.6
materials The Maillard reaction is one of the main causes
N 0:2) 42.8 0:4:=515 of damage to culture media during heat sterili-
P50; 0.02 - 0.07 ON65 =) 2:0 zation, resulting in considerably reduced yields.
CaO 0.15 = 0.7 01911
MgO 0.01 - 0.1 0.03- 0.1
K,O Dep EN lo) 2.6 = 5.0 Table 4.2 Typical composition of malt extract
SiO, Wail’ <= OS =
ALO, 0.005— 0.06 - Component % of
Fe,O, 0.001- 0.02 = Dry weight
Ash 4. - 8 7 -11
Maltose SAS
Thiamine yg/100¢g 130 830 Hexoses (glucose, fructose) 19.1
Riboflavin dry 41 250 Sucrose 1.8
Pyridoxine weight 540 650 Dextrin 15.0
Niacinamide 5100 2100 Other carbohydrates 3.8
Pantothenic acid 130 2140 Nitrogenous materials 4.6
Folic acid 21 3.8 Ash 1
Biotin 5.3 120 Water content 2.0
(Rhodes and Fletcher, 1966; Imrie, 1969) pH (10% solution) = 5.5
 4.1 SUBSTRATES USED AS CARBON SOURCES / 61

a fermentation, and the fermentative production of

BLE CEO SCENER sære butanol, acetone, and isopropanol is also being
HSCSOHSENRE Roe HSC=OH H=EC=OH considered. Work is in progress to develop one-
HO-C-H HO-C-H SL H0-¢-H O
step processes for direct conversion of cellulose
to ethanol, using fermentative organisms which
H=ICSOH > C-0OH H-C-OH
produce cellulases.
H-C-OH H-C-OH H-C
Whey, a byproduct of the dairy industry, is
CH, OH CH,OH CH, 0H produced annually on a world-wide basis to the
amount of 74 million tons (containing 1.2 million
D-Glucose Schiff base N-substituted tons of lactose and 0.2 million tons of milk pro-
glycosylamine tein). Only about 56% of this product is used for
human or animal feed. The lactose is used pri-
Figure 4.1 Maillard reaction
marily for the production of ethanol or single-
cell protein, but also in the production of xanthan
gum, vitamin B,,, 2,3-butandiol, lactic acid, and
Starch and dextrins can be directly metab-
gibberellic acid. Because of storage and trans-
olized as carbon sources by amylase-producing
portation costs, whey is often not economical as
organisms. In addition to glucose syrup, which
a substrate.
is frequently used as a fermentation substrate,
Animal fats such as lard and animal and plant
starch has become more important as a substrate
oils are readily utilized by some microorganisms,
for ethanol fermentation. The largest use of car-
but are generally added as supplemental sub-
bohydrate for ethanol fermentation has been the
strates rather than as the sole fermentable carbon
National Alcohol Program begun in 1975 by the
source. For instance, in certain antibiotic fermen-
Brazilian government. This Program, which is
tations, soy, palm, and olive oils are used.
based on starch from the cassava plant, produced
With respect to its carbon content, methanol
about 9 million metric tons of ethanol in 1984. is the cheapest fermentation substrate, but it can
Sulfite waste liquors, sugar-containing be metabolized by only a few bacteria and yeasts.
waste products of the paper industry, which have Methanol has commonly been used as a substrate
a dry weight of 9-13%, are primarily used in the for single cell protein production (see Chapter
cultivation of yeasts. Sulfite liquors from coni- 16). Research has been carried out on processes
ferous trees have a total sugar content of 2-3%, for the production of glutamic acid, serine, and
and 80% of the sugars are hexoses (glucose, man- vitamin B,, using methanol as the sole carbon
nose, galactose), the others being pentoses (xy- source or as a cosubstrate.
lose, arabinose). Sulfite liquors from deciduous Ethanol is available in ample supply from the
trees contain mainly pentose sugars. fermentation of either saccharified starch or cel-
Because of its wide availability and low cost, lulose, and can be metabolized by many micro-
cellulose is being extensively studied as a sub- organisms as the sole carbon source or as a co-
strate for conversion to sugar or alcohol. The substrate. Acetic acid, for instance, is presently
world’s annual cellulose production is estimated made by the oxidation of ethanol. At the present
at 101! metric tons; much of it exists as waste, in time, the cost of ethanol is too high to make it
such forms as straw, corn cobs, wood wastes, utilizable as a general industrial carbon source.
peat, bagasse, and waste paper. It is usually not Alkanes with a chain length of C,, to C,; are
possible to use cellulose directly as a carbon readily metabolized by many microorganisms.
source, so it must first be hydrolyzed chemically The use of alkanes as an alternative to carbo-
or enzymatically. The sugar syrup formed from hydrates depends on the price of petroleum (see
cellulose hydrolysis has been used for ethanol Section 16.2).
62 / CHAPTER 4 / SUBSTRATES FOR INDUSTRIAL FERMENTATION

largely converted to lactic acid (9-20%) by lactic

4.2 SUBSTRATES USED AS NITROGEN
acid bacteria.
SOURCES Yeast extracts are excellent substrates for
Many large-scale processes utilize ammonium many microorganisms. They are produced from
salts, urea, or gaseous ammonia as nitrogen baker’s yeast through autolysis at 50-55°C or
source. A nitrogen source which is efficiently me- through plasmolysis in the presence of high con-
tabolized is corn steep liquor, which is formed centrations of NaCl. Yeast extract contains amino
during starch production from corn. The concen- acids and peptides, water-soluble vitamins, and
trated extract (about 4% nitrogen) contains nu- carbohydrates. The glycogen and trehalose of
merous amino acids, such as alanine, arginine, yeast cells are hydrolyzed to glucose during yeast
glutamic acid, isoleucine, threonine, valine, | extract production—Table 4.3 shows some typical
phenylalanine, methionine, and cystine. The analyses. The composition of yeast extract varies,
sugar present in corn steep liquor becomes partly because the substrates used for yeast cul-
tivation affect the quality of the yeast extract.
Peptones (protein hydrolysates) can be uti-
Table 4.3 Composition of yeast extract lized by many microorganisms but they are rel-
Yeast extract produced atively expensive for industrial application.
by means of
Autolysis Plasmolysis Table 4.4 Compositions of some typical peptones of
with NaCl different origins
Composition (%) N-Z- HY-Case? Soy
Dry matter 70 80 ine? i
Total nitrogen 8.8 7.4 nine wheat OS xSeige
Protein (N X 6.25) 55 46 Starting material Casein Casein Soy meal
NaCl 1 18 Hydrolytic method Enzymatic Acid Enzymatic
Amino acids (% of total) Composition (%)
Alanine 3.4 23 Water content 4.8 SLD 4.0
Aminobutyric acid 0.1 0.1 Ash 5.8 39.7 14.0
Arginine 2.1 Lal Total nitrogen 12.8 8.3 92
Asparagine 3.8 3.1 Amino nitrogen 6.7 6.4 1.8
Cystine 0.3 0.2 NaCl 3.0 38.0 529)
Glutamic acid Tied: 5.1 Carbohydrate = - 29.0
Glycine 1.6 1.6 ; 2
Histidine
Isoleucine
0.9
20
0.8
16
FregeAlanine
"Amino acids MS 68)16.8 14.1 Si)
reine 29 23 Arginine 30.2 5.0 1.1
Lysine 32 29 Aspartic acid 8.7 33.2 2.4
Methionine 0.5 0.5 Cystine 2 0.7 QL
Outihine 03 0.9 Glutamic acid 38.6 79.6 5.6
Phenylalanine 1.6 1.6 Glycine 4.4 9.3 2.3
Proline 1.6 1.5 Histidine 13:33 4.6 del
Garine 1.9 15 Isoleucine 29:9 10.7 2.8
Threonine 19 1.4 Leucine ZAC BOY 9.4
Tyrosine 0.8 0.5 Lysine 61.1 12.8 7.9
Valine One 1.9 Methionine Deg 112 BB:
Phenylalanine 33.0 97 87
Vitamin content (ppm) Proline 7.4 34.5 0.8
Thiamine 20-30 10=15 Serine 28.7 19.8 4.8
Riboflavin 50-70 50-70 Threonine ZS 13.1 19
Pyridoxine 25—35 20-30 Tryptophan 8.6 0.05 1.6
Niacinamide 600 100 Tyrosine 14.0 4.9 0.7
Pantothenic acid 200 350 Valine 36.4 16.7 3.8
(Data from Ohly Inc., Hamburg) (Data from Sheffield Chem. Co.)
 REFERENCES / 63
Sources of peptones include meat, casein, gelatin, Dale, B.E. and J.C. Linden. 1984. Fermentation substrates
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Imrie, F.K.E. 1969. Fermentation media. Sugar and mo-
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Holz und Stroh, Natiirlich Ole und Fette, Alkohole fir
Bridson, E.Y. and A. Brecker. 1970. Design and formula- Kraftstoffe. (Recyclable raw materials. Wood and
tion of microbial culture media. Methods in Microbiol., straw, natural oils and fats, alcohol as energy source.)
vol. 3A, pp. 229-295. Verlag J. Kordt, Bochum.
 Methods of
fermentation

In fermentation processes, engineering is

5.1 INTRODUCTION
only an aid in the development and regulation
The goal of biotechnology (bioengineering) is to of biological processes and the microorganism is
obtain useful metabolic products from biological the center of attention. All methods, such as ge-
material. Biotechnology encompasses two dis- netic manipulation, regulation of metabolism by
tinct phases: fermentation and product recovery. optimizing culture media, and provision of ad-
Fermentation procedures must be developed for equate oxygen supply under sterile conditions,
the cultivation of microorganisms under optimal are only ways of directing the process toward the
conditions and for the production of desired me- desired product. The more precisely metabolic
tabolites or enzymes by the microorganisms. processes during growth and product formation
Product recovery or downstream processing in- are understood, the greater is the possibility of
volves the extraction and purification of biolog- operating fermentation processes rationally
ical products. Biochemical process recovery dif- rather than empirically. However, a fully auto-
fers from chemical recovery primarily in that matic, computer-run fermentation process to ob-
biological materials are frequently much more la- tain a metabolite such as an antibiotic has not
bile. Although many of the techniques used in yet been realized at the industrial scale.
biological or biochemical product recovery over-
lap with those used for strictly chemical pro-
5.2 GROWTH KINETICS OF
cesses (for instance, separation, distillation, heat-
MICROORGANISMS
ing, cooling, and drying), there is increasing use
of methods specifically designed for biological A clear understanding of microbial growth ki-
products, such as chromatography and electro- netics is necessary if a large-scale process is to
phoresis. be properly managed. Growth kinetics are

64
 5.2 GROWTH KINETICS OF MICROORGANISMS / 65

treated differently for conventional batch pro- nisms adapt to their new environment. Because
cesses than for continuous processes. of this transfer to a new medium, several param-
eters will probably be altered for the inoculum
cells: change in pH value, increase in supply of
Batch fermentation
nutrients, decrease of growth inhibitors. New
A batch fermentation can be considered to be a transport systems for nutrients must be induced
“closed system.” At the time T = 0 the sterilized within the cells. Essential cofactors may diffuse
nutrient solution in the fermenter is inoculated out of a cell and enzymes of primary metabolism
with microorganisms and incubation is allowed must be adjusted to the new conditions.
to proceed under optimal physiological condi- Moreover, the physiological condition of the
tions. In the course of the entire fermentation, inoculum is crucial to the length of the lag phase.
nothing is added except oxygen (in the form of If a culture for use as inoculum is still in the log
air), an antifoam agent, and acid or base to con- phase (see below), a lag may not occur and
trol the pH. The composition of the culture me- growth may begin immediately. However, in-
dium, the biomass concentration, and the me- oculum taken from a culture in which growth
tabolite concentration generally change has stopped due to substrate limitation needs
constantly as a result of the metabolism of the more time to adapt to the new nutrient solution.
cells. After the inoculation of a sterile nutrient The concentration of the inoculum also has an
solution with microorganisms and cultivation influence on the lag phase.
under physiological conditions, four typical The proper cultivation and transfer of inoc-
phases of growth are observed: lag phase, log ulum are essential for the later production of both
phase, stationary phase and death phase (Figure primary and secondary metabolites. Although
SL): the regulation of product formation is not yet
fully understood, other preculture media and cul-
Lag phase When cells are transferred from one ture conditions often have to be designed for op-
medium to another, there is initially no increase timal yields. However, the kinetics of product
in the number of cells, although cell weight may formation are not necessarily correlated with the
change. During the lag phase, the microorga- length of the lag phase.

Log phase By the end of the lag phase, the cells

have adapted to the new conditions of growth.
Growth of the cell mass can now be described
quantitatively as a doubling of cell number per
unit time (yeasts and bacteria) or a doubling of
biomass per unit time (filamentous organisms
such as streptomycetes and fungi). By plotting
oO
oO
Qa
0
oO
the number of cells or biomass against time on
iS oO
s
>
3
n
oO
a semilogarithmic graph, a straight line results,
=
ie
DD
fs
(72)
iS
==
c
c
GE
hence the term “log phase”.
BD = a. z = Although the cells alter the medium through
cpg,
a4 Ar
g
oa
so
(79)
bedt
a uptake of substrates and excretion of metabolic
a products, the growth rate remains constant dur-
+s ————— ing the log phase. Growth rate is independent of
Time substrate concentration as long as excess sub-
strate is present. The rate of increase in biomass
Figure 5.1 Growth curve of a bacterial culture is correlated with the specific growth rate 4 and
66 / CHAPTER 5 / METHODS OF FERMENTATION

the biomass concentration X (g/l), whereas the mass which is present by the end of the log
rate of increase in cell number is correlated with phase, the substrate is quickly exhausted so that
u and cell density N (1/1) the period of time during which the substrate
concentration is near that of the K, is very short
dX
a
— —
u-X
.
[1]
1 and the stationary phase is approached abruptly.
Maximum specific growth rates are of con-
or siderable industrial importance. The 4, is de-
pendent on the organism and on the conditions
dN
RES
Ti uN 2
[2] of fermentation. The values for several fungi are
listed in Table 5.1. The specific growth rates vary
The specific growth rate, 4, is generally found to between 0.090-0.61 h7.
be a function of three parameters: the concen- Since an organism needs extra energy to split
tration of limiting substrate, S, the maximum long-chain substrates, the specific growth rate for
growth rate u,,, and a substrate-specific constant simple substrates is greater than for long-chain
Ks. molecules. One example in Table 5.2 shows the

M = um HULDA
K+ SE, 3
[3] Table 5.1 Maximal specific growth rates (u,,) of some
fungi on glucose
Organism FE (HE) Doubling time
This equation is generally called the Monod (h)
equation since the relationship expressed was
Aspergillus niger 30 0.20 3.46
first described by Jacques Monod. The constant Aspergillus nidulans 20 0.090 TM
K, is the substrate concentration at which half 25 0.148 4.68
the maximum specific growth rate is obtained (u 30 0.215 3.23
37 0.360 1.96
= 0.5 u,,). K, is equivalent to the Michaelis con- Penicillium
stant in enzyme kinetics. If several limiting sub- chrysogenum 25 0.123 5.65
strates are present, eq. (3) can be expanded. Mucor hiemalis 25 0.17 4.1
Fusarium
avanaceum 25 0.18 3.8
s k, S, k, 8, kj Sj 1 Fusarium
biepane(Stet eet ee sie at graminearum 30 0.28 2.48
Verticillium
.
J=1
Kj
agaricinum 25 0.24 Zo
Geotrichum
If there is an excess of all substrates, then uw = candidum 25 0.41 1%
Geotrichum
Hw and the culture is in the log phase at its max- candidum 30 0.61 1,1
imal growth rate. If one substrate has been ex- Neurospora sitophila 30 0.40 1.73
hausted, and another substrate is still present, (Anderson et al., 1975)
there may be a second log phase during the me-
tabolism of this second substrate with a second
Table 5.2 Effect of glucose chain length on the
specific growth rate pu. maximal specific growth rate (,,) in Fusarium
The value for K, is generally very low; in graminearum at 30°C
Escherichia coli, K, values of 1.0 mg/l have been Substrate Number of Mm Doubling time
measured for glucose and 1.1 mg/l for trypto- glucose units (h)
phan. Such low values also account for the fact Glucose 1 0.28 2.48
that substrate levels equivalent to the K, value Maltose 2 0.22 345
are not obtained in normal batch fermentations Maltotriose 3 0.18 3:83
during the log phase. Because of the large bio- (Anderson et al., 1975)
'
 5.2 GROWTH KINETICS OF MICROORGANISMS / 67
growth of Fusarium graminearum with glucose, at the end of the log phase or before the death
maltose (2 glucose units) and maltotriose (3 glu- phase begins.
cose units).
The Monod equation for the correlation of
specific growth rate and substrate concentration Fed-batch fermentation processes
does not hold true when the intracellular sub-
strate concentration is reduced during fast In the conventional batch process just described,
growth, even though adequate substrate is still all of the substrate is added at the beginning of
available in the medium. Additional mathemat- the fermentation. An enhancement of the closed
ical models have been developed for handling batch process is the fed-batch fermentation,
such situations, in which the concentrations used
which is used in the production of substances
are not those of the medium but of the intra- such as penicillin. In the fed-batch process, sub-
strate is added in increments as the fermentation
cellular milieu.
progresses. The formation of many secondary
When complex nutrient solutions are used,
metabolites is subject to catabolite repression by
two log phases frequently occur separated by a
high concentrations of glucose, other carbohy-
second lag phase. This process is called diauxy
drates, or nitrogen compounds (see Section 3.5).
and arises because one of the substrates (usually
For this reason, in the fed-batch method the crit-
glucose) is catabolized preferentially (see the dis-
ical elements of the nutrient solution are added
cussion of catabolite regulation in Section 3.5).
in small concentrations at the beginning of the
The presence of this one substrate represses the
fermentation and these substrates continue to be
break down of the other substrates. The catabolic
added in small doses during the production
enzymes for the other substrates are induced
phase.
(during the second lag phase) only after the first
Since it is usually not possible to measure the
substrate has been completely metabolized.
substrate concentration directly and continu-
ously during the fermentation, indirect param-
Stationary phase As soon as the substrate is me-
eters which are correlated with the metabolism
tabolized or toxic substances have been formed,
of the critical substrate have to be measured in
growth slows down or is completely stopped.
order to control the feeding process. For instance,
The biomass increases only gradually or remains
in the production of organic acids, the pH value
constant during this stationary phase, although
may be used to determine the rate of glucose
the composition of the cells may change. Due to
feeding. In fermentations with critical osmotic
lysis, new substrates (carbohydrates, proteins)
values, feeding can be regulated by monitoring
are released, which then may serve as energy
the pO,-value or the CO,-content in the exhaust
sources for the slow growth of the survivors. The
air.
various metabolites formed in the stationary
phase are often of great biotechnological interest.
Continuous fermentation
Death phase In this phase the energy reserves
of the cells are exhausted. A straight line may be In continuous fermentation, an open system is
obtained when a semilogarithmic plot is made of set up. Sterile nutrient solution is added to the
survivors vs. time, indicating that the cells are bioreactor continuously and an equivalent
dying at an exponential rate. amount of converted nutrient solution with mi-
The length of time between the stationary croorganisms is simultaneously taken out of the
phase and the death phase is dependent on the system. Among the diverse kinds of continuous
organism and the process used. In commercial fermentation, two basic types can be distin-
processes, the fermentation is usually interrupted guished:
68 / CHAPTER 5 / METHODS OF FERMENTATION

D=u [5]
Product Product Product
dX Growth rate [6]
w= 1/X- “dt Cell concentration in bioreactor

The rate of flow D is defined as the volumetric

flow rate (in and out) through the fermenter vol-
A C ume.
By using the Monod equation describing de-
pendence of the specific growth rate on the lim-
iting substrate concentration (equation 3) and the
yield constant (equation 7), the cell density X
(equation 8) and substrate concentration S (equa-
Nutrient Nutrient Nutrient
solution solution solution tion 9) in the bioreactor can be determined.

Figure 5.2 Continuous fermentation in the chemostat x _ Biomass (g)

(A), turbidostat (B), and plug-flow reactor (C) S Substrate consumption (g) id

X
ak
= Yg
D-Ks
(s0- ruger 5) [8]
1. Homogenously mixed bioreactor. This is run
as either a chemostat or a turbidostat. In the D-K
chemostat in the steady state, cell growth is
controlled by adjusting the concentration of
one substrate (Figure 5.2 A). Any required The substrate concentration in the incoming
substrate (carbohydrate, nitrogen compound, medium is designated as S,. Equations 8 and 9
salts, O,) can be used as a limiting factor. In show the dependence on flow rate. At a lower
the turbidostat, cell growth is kept constant flow rate, the substrate is almost completely used
by using turbidity to monitor the biomass con- up by the cells (S — 0). The cell concentration
centration and the rate of feed of nutrient so- is then X = S,/Y. If D increases, X decreases
lution is appropriately adjusted (Figure 5.2 B). slowly at first in a linear fashion and then at D
2. Plug Flow Reactor. In this type of continuous = u,, it drops sharply to 0. S increases slowly at
fermentation, the culture solution flows
first as D increases and approaches S, at D = um
When X is zero in equation 8, the wash-out point
through a tubular reactor without back mix-
D,, has been reached.
ing. The composition of the nutrient solution,
the number of cells, mass transfer (O,-supply) Hm * So
and productivity vary at different locations Prt Ke#5, ss
within the system (Figure 5.2 C). At the en-
So
trance to the reactor, cells must continuously KS urne Gk une [11]
be added along with nutrient solution (usually
as a back flow in a bypass from the fermenter Above D,, a steady state is not possible. If the
outlet or from a second continuous fermen- flow rate is only slightly lower than D,, the sys-
tation). tem is very sensitive to external influences. Ex-
treme changes in biomass may result from small
In a continuous process under steady state deviations in feeding or cell removal. Figure 5.3
conditions, cell loss as a result of outflow must shows the interdependence of rate of flow, sub-
be balanced by growth of the organism. strate concentration, cell concentration and rate
 5.2 GROWTH KINETICS OF MICROORGANISMS / 69

chrysogenum the production of penicillin ceases

below a minimal growth rate, and conidiospores
are formed.
In continuous fermentation at a constant flow
rate, steady state conditions do exist and the sub-
strate content, the biochemical reactions within
the cells, and the rate of product formation do
not change. However, there are differences in
X(g/l) (g/l)
DX metabolism at a constant flow rate depending on
the substrate limitation. Table 5.3 shows the in-
fluence of substrate limitation on the amino acid
7 — pool and glutamic acid dehydrogenase in Sac-
Z ees ee = — —
charomyces cerevisiae.
025 0.50 075 100 Furthermore the rate of product formation
Dilution rate (1/h) is dependent on the flow rate. This can be applied
not only to individual enzymes, but to the ex-
Figure 5.3 Effect of flow rate on substrate concencentra- cretion of primary and secondary metabolites as
tion (S), cell concentration (X), Doubling time t, and cell
formation rate (D-X). well (Figure 5.4). When the idiophase (product
formation phase) is separate from the tropho-
phase (growth phase), product formation cannot
of cell formation (u,, = 1h", K, = 0.2 g/l, Y = be carried out continuously in an optimally
0.5). mixed bioreactor. Fermentation must then be
The maximum specific growth rate is chosen done using a series of fermenters as a cascade or
in industrial processes so as to obtain the largest in a tower reactor using single reaction chambers
yield in the smallest fermenter volume and the separated by sieve plates.
shortest time. Continuous fermentation processes have
The Monod model is not usually applicable been developed for the production of single-cell
to continuous fermentation at very low and very protein, antibiotics, organic solvents, starter cul-
high dilution rates. At mean dilution rates the tures and for cellulose decomposition (in labo-
molar relationship between biomass produced ratory studies). Pilot plants or production plants
and CO, given off is constant. At low dilution have been installed for continuous production of
rates, the proportion of CO, becomes higher. single-cell protein based on n-alkanes, C,-com-
This is because more of the energy source is used pounds and starches (see Chapter 16).
for maintenance of cell structure, for osmotic reg-
ulation, or for motility. On the other hand, at
Table 5.3 Effect of growth conditions on the amino
high flow rates, a part of the added carbon is acid metabolism of Saccharomyces cerevisiae
incompletely oxidized (to products such as py- Dilution rate = 0.1 hot, 30°C
ruvate, acetate or tricarboxylic acids) and pro- Limiting Amino acids (mM) Glutamic acid
ductivity is thus reduced. With nitrogen as the substrate dehydrogenase
limiting substrate, reserve materials such as poly- Total Glu- (mMol/min/mg
tamic acid protein)
saccharides are formed at low flow rates, which
results in an increase in cell size and hence in Ammonium 144 35.6 1730
ion
biomass. Glutamic acid 180 49.2 510
The production of metabolites is also not Glucose 381 135.4 620
well described by the Monod model at very low Phosphate 386 121.4 465
or high flow rates. For example, with Penicillium (Brown and Stanley, 1972)
70 / CHAPTER 5 / METHODS OF FERMENTATION

Continuous cultures also find considerable

use in research on ecological processes. Growth
at low-substrate levels in a continuous culture is
analogous to growth at low-substrate levels in
nature, so that the continuous cultures provide
excellent models for analysis of ecological pro-
cesses.

weight
Dry
% Classification of fermentation processes
We have previously subdivided fermentation
processes into three types: batch, fed-batch, and
continuous. Another means of classification has
been developed in which fermentations are clas-
0.2 0.4 0.6 0.8 sified according to the dependence of product
Dilution rate (1/h) formation on energy metabolism.

Figure 5.4 Effect of flow rate on the production of poly- TypeI Ina type I process, the product is derived
hydroxybutyric acid (PHB e e) and glycogen
(X — X) with limited carbon, and PHB with excess ace-
directly from the primary metabolism used for
tate (A — 0). (Wilkinson and Munro, 1967) energy production. The product can also be bio-
mass itself. Growth, carbohydrate catabolism,
and product formation run almost in parallel
Industrial processes using continuous fer-
(Figure 5.5 A) and trophophase and idiophase
mentation include waste-water treatment (see
are not separated from each other. The reaction
Chapter 17) as well as the production of beer,
can be expressed as follows:
glucose isomerase, and ethanol.
Although many fermentations for metabolite
Substrate A —> Product
production work well as continuous processes at or Substrate A +B —C — Product
a laboratory scale, only a few processes have
proved useful for practical application for several Processes for the production of single-cell pro-
reasons: tein, ethanol, and gluconic acid all belong to this
e Many laboratory methods operate continu- category. Continuous fermentations in reactors
ously for only 20 to 200 hours; for industrial without a cascade system are classified here as
use the system must be stable for at least 500 well.
to 1000 hours.
e Maintaining sterile conditions on an indus- Type II Here the product is also derived from
trial scale over a long period of time is dif- the substrate used for primary energy metabo-
ficult. lism, but production takes place in a secondary
e The composition of substrates must be con- pathway which is separate from primary metab-
stant in order to obtain maximal production. olism.
The composition of industrial nutrient solu-
tions varies (e.g. corn steep liquor, peptone, Substrate A >B >C —D —Primary metabolism

starch).
E >F >
Product
e When high-yielding strains are used, reverse
mutants arise, which can overgrow the pro- In batch fermentations of this type there are two
duction strains in continuous culture. maxima. At first, good growth occurs accom-
 5.2 GROWTH KINETICS OF MICROORGANISMS / 71

TypeIl Type Ill

a Cc Time (hr)

Figure 5.5 Fermentation categories defined by Gaden (1959). Specific growth rate ( ), specific carbohydrate con-
sumption (- — >=), specific product formation rate (-""")

panied by high substrate consumption and little relation of mycelium production and changes in
or no product formation. Then growth slows mycelium structure with antibiotic production is
down and product formation begins, accom- complex. The stages in oxytetracycline formation
panied by a high substrate consumption rate with the mycelium-forming organism Strepto-
(Figure 5.5 B). In type II processes, the tropho- myces rimosus are shown in Table 5.4.
phase and idiophase are separated in time. Citric
acid, itaconic acid, and some amino acids are pro-
duced by this type of process. Table 5.4 Stages in the oxytetracycline fermentation
with Streptomyces rimosus
Type II In the type III processes, primary me- Stage Fermentation process
tabolism and product formation occur at com-
1 Lag phase Duration 90 minutes for low inocu-
pletely separate times. The product is not derived lum. No metabolism evident.
from catabolism, but from amphibolic pathways. 2 Growth of Duration 10-25 h, depending on in-
In this type of fermentation, primary metabolism primary oculum. Hyphae dense. Respira-
mycelium tion, nucleic acid synthesis and
functions first, accompanied by substrate con- some other enzymatic conversions
sumption and growth. Afterwards, the product are high. Pyruvate content reaches
is formed by the reactions of intermediary me- the maximum. No antibiotic pro-
duction.
tabolism (Figure 5.5 C). Many antibiotics and vi- 3 Fragmentation Duration 10 h, no growth during this
tamins are produced by this type of fermentation. of primary time. Respiration and nucleic acid
This classification cannot be applied to all fer- mycelium synthesis decline. Pyruvate content
falls to low value.
mentation processes. Depending on metabolite 4 Growth of Duration 25 h, mycelium volume is 2
production, composition of culture medium, and secondary to 4 times that of Stage 2, hyphae
regulation in the strain used, there may be in- mycelium thin, good antibiotic production.
Nucleic acid production resumes,
termediate forms. For example, the production of but respiration is low. Carbohy-
lactic acid falls between types I and II, and ami- drate and ammonium content fall
to zero. Pyruvate content can begin
noglycoside antibiotic production falls between
to increase again.
types II and III. 5 Stationary Growth ceases, metabolism is low,
Production involving mycelium-producing phase antibiotic production continues but
at a low rate.
microorganisms can not be classified among
these simple fermentation types, since the cor- (Bailey and Ollis, 1977)
72 / CHAPTER 5 / METHODS OF FERMENTATION

The fermentation conditions directly affect

Productivity and specific rate of productivity. Figure 5.7 shows the productivity
production of a single-cell protein process in relation to the
The productivity of a fermentation is defined as substrate (hydrocarbon) content. There is a linear
increase in productivity as the substrate content
Er Product concentration /1 rises until the concentration is reached at which
Productivity (P) = 7 3
Fermentation time the petroleum hydrocarbon becomes the contin-
= Units/hr'l uous phase (oil and water are not miscible).
Above this concentration, productivity then de-
In evaluating the cost-effectiveness of a process, creases.
several factors must be considered: the produc- In continuous fermentations, productivity
tion time of the fermentation, the time required can be expressed as:
to clean and set up the fermenter, the sterilization
time, and the duration of the lag phase. In Figure P=D-X(g/&-h) [12]
5.6, the slope of the tangent of the product for-
mation curve is a measurement of maximal pro- When the relationship of equation 8 is used, the
following equation results:

ES MEET É…,
ductivity and the slope of the straight line to the
end of the product formation curve indicates total
D-K
productivity.
Whether the fermentation process is termi-
nated at the time of maximal productivity or later In continuous fermentations, maximal productiv-
depends on the operating costs, which include ity is equal to total productivity since set-up time
energy, overhead, labor, and the capacity of the and lag phase can be disregarded. For instance,
system. By using a graph such as that of Figure if the initial set-up time in a continuous fermen-
5.6, an estimate can be made of how to optimize tation requires 24 hours, and the continuous fer-
the process. In short-term fermentations (8—70 h) mentation itself is run for 24 hours with a rate
the set-up time is significant, whereas in long- of flow of 0.1 h7, 24 hours with 0.15 h” and at
term fermentations (> 3 days) the set-up time is least 72 hours with 0.2 h>, the total productivity
less crucial. for product X is calculated as shown in Table 5.5.

Maximal productivity Å
Total
productivity

Product formation
Productivity

Termination

Å Å Å Time (h )
må

Clean-up Filling Å Production phase

Figure 5.6 Total productivity and
Lag phase maximal productivity
 5.2 GROWTH KINETICS OF MICROORGANISMS / 73

Table 5.5 Total productivity of a continuous

fermentation
Time period Flow rate Total productivity
h X(g/1)"D(h™)
WwW
0-24 Batch =
24-48 0.1 X:0.05
48-72 0.15 X:0.08
nO 2272 0.2
300 0.2 X-0.171
1000 0.2 X:0.191
5000 0.2 i X-0.198
(g/I/h)
Productivity
—

converted or to energy released or energy con-

sumed. However, yield coefficients are not con-
20 40 60 80 100 stants, since they are dependent on biological pa-
rameters (X, u) and chemical parameters (pO,,
Gas oil concentration (%)
C/N ratio and P content of the medium).
Figure 5.7 Productivity in relation to gas oil concentra- Yield coefficients have been classified in three
tion in a single-cell-protein process groups. The first group of coefficients (Ys, Yo,,
Yxa) gives information about the technical pro-
cess and thus about cost-effectiveness. The sec-
The specific rate of production q, is derived ond group (Yc, Yp, Yn, Yave-) describes catabolism,
from equation 1 in the following way: amphibolism, and anabolism. The third group
Cpe includes Yp, a coefficient which describes the
Gr =%°X [14] energy relations of the cell. Some authors use
other yield coefficients, which must be desig-
The value P, is the concentration of product and nated properly, as shown:
the specific rate of production q, is equivalent to
_ Mol CO, formed
the specific growth rate u in equation 1. In type Yo? ~ Mol substrate consumed (17)
II or III processes, however, there is no direct
correlation between u and q,.

Heat production
Yield coefficients
In order to obtain optimal yields, fermentations
Monod originally defined the yield coefficient, Y,
must be carried out at constant temperature. We
as the ratio of cells produced to substrate con- now discuss the parameters affecting the heat
sumed: balance of a fermentation process.
dX dS
The rate of heat production due to stirring
dt dt [15] and due to the metabolic activity of the micro-
organisms must be balanced by the heat loss re-
or sulting from evaporation and radiation plus heat
_ Biomass (8) 16] removal by the cooling system (the jacket of the
Substrate consumption (g)
fermenter or cooling coils).
The heat production during stirring can be
Today general yield coefficients are used to char- calculated from the energy consumption of the
acterize fermentation processes, i.e. the relation- motor minus the losses through the seals, bear-
ship of cells produced to individual substrates ings, and drive shaft.
74 / CHAPTER 5 / METHODS OF FERMENTATION
Table 5.6 Yield coefficients for bacteria with different
Heat production of the microorganism can be
substrates
determined using the yield coefficient Y,..i:
Substrate Yica(g cells/kcal)

Ys Malate 0.30
real” foe Vue. [18] Acetate 0.21
Glucose 0.42
Methanol 0.12
The yield coefficient Y,.., is given in grams of Ethanol 0.18
cells/kcal energy released; Y, indicates the ratio Isopropanol 0.074
n-Paraffin 0.16
of grams of cells/gram of substrate utilized; the Methane 0.061
heats of combustion of the substrate H, and of
the biomass H, are measured in kcal/g. Al:
though there are tables giving the heats of com- The rate of microbial heat production per unit
bustion of different substrates, the heat of com- time, which along with the heat produced by
bustion of the cells (H.) must be determined stirring must be removed from the bioreactor, is
experimentally or calculated by using various as- calculated from the rate of cell production (equa-
sumptions. tion 1), the reactor volume and the heat coeffi-
One possibility for calculating H, makes use cient:
of the electron theory of valence. In the transfer
dX
of an electron equivalent, 26.05 kcal are released. Growth
rowth rat
rate —=
aH -X [1]
Since four valence electrons are shared by oxy-
gen, 104.20 kcal/M O, are produced. One cal- Heat production rate Qw=V-4X [22]
Ykcal
culation using Pseudomonas fluorescens assumed
that when the biomass is burned, CO,, N, and
H,O are produced.
5.3 FERMENTER SYSTEMS
Ca.41 Ho,3 No.s6 O1.19 + 5-64 O, > 4.41 CO, + 0.43 N,
Pseudomonas + 3.65 H,O [19] In a bioreactor, production of metabolites must
be accomplished with maximum emphasis on re-
The heat of combustion is calculated as: liability for the process and a minimum of capital
investment and operating cost. Reliability is more
: 5.64 - 104.2 difficult to achieve in a microbiological than in a
Hc = 741-1247. -1.0+0.86-14+1.19 -16 chemical process so that bioreactors are more ex-
= 6.44 kcal/g [20] pensive to design and construct than chemical
reaction vessels.
The value obtained must be corrected by about Microbiology must be the focus of all consid-
10% due to the ash content of the cells. erations concerning construction of a fermenter
system. It must be decided in the planning stages
Hc = 6.44 - 0.90 = 5.8 kcal/g [21] whether the fermenter is to be used for a special
The heat production coefficients for some bac- process with one organism or for a variety of
teria on different substrates are listed in Table processes with different microorganisms. A num-
5.6. For the more highly oxidized substrates (e.g. ber of different bioreactors for aerobic fermen-
glucose and acetate), less heat is produced per tation are diagrammed in Figure 5.8. They may
biomass than with the more reduced substrates be classified in four groups with respect to gas
(methane and methanol). In general, Y,… values
distribution.
increase linearly in relation to the specific growth 1. Gas distribution by stirring. The stirred ves-
rate u of the cultures. sel (Figure 5.8, part 1) is the best understood
 5.3 FERMENTER SYSTEMS / 75

Gas distribution by stirring

Turbine stirring installation Stirred vessel with draft tube Stirrer with automatic
suction tube

Gas distribution by pumps

Fritted disk with recycling Forced water jet Water jet aerator

LY

Are

VU &
5 6

Gas distribution by overpressure of gas

J Sieve plate
Fritted disk system Airlift system Pressure cycle reactor Giant tube reactor cascade system

8 9 10

Continous gas phase

Trickling film reactor Surface film reactor Blade wheel reactor

Figure 5.8 Basic construction types for bioreactors

76 / CHAPTER 5 / METHODS OF FERMENTATION

and most widely used bioreactor and is quite

flexible. Loop reactors with draft tubes (2) are
also suitable for mass production. A system
in which air is sucked through the tube by the
low pressure on the lee side of the stirrer
blades is used in vinegar production (3). The
various surface aerators used in waste-water
treatment also belong to this group.
2. Gas distribution through pumps. In a mod-
ified airlift system (4), the pump primarily
transports liquid and the air is distributed.
more or less independently through the spar-
ger. In the more efficient systems (5, 6), there
is a direct mixing of air and fluid by means of
a water jet pump.
3. Gas distribution by means of pressurized
air. In most of these systems, no moving parts
are present in the sterile area. The “pressure
cycle fermenter” (9) was the first model of this
kind used in biotechnology for single-cell pro-
tein production on an industrial scale.
4. Continuous gas phase. In these processes, air
circulates over a film of microorganisms. In
the trickle-film reactor (12), the organism Figure 5.9 Laboratory fermenter
grows on a solid, inert carrier material. In the
surface reactor (13), it floats on or in the nu-
trient solution or grows in a semi-solid or solid struction ‘is of stainless steel. Figure 5.10 illus-
culture medium. In the blade-wheel reactor trates the design of a large scale fermenter.
(14), it grows on movable blades or drums and Up to the 3000 | scale, fermenters can be stan-
is dipped alternately in nutrient solution and dardized. However, the geometry of production
in gas phase as the wheel turns. Reactors used fermenters changes as the volume and the pro-
in waste-water treatment, for the leaching of cess requirements change. The height and width
ores, and for vinegar or citric acid production, relationship may vary from 2:1 to 6:1 and the
as well as the culture flasks on laboratory stirrer may be top or bottom driven.
shaking machines, are included in this group.
Baffles Baffles bring about the transfer of tur-
bulence to the fermenter wall. Four baffles are
Stirred bioreactors
commonly installed, with a width 1:10 or 1:12
For industrial use, especially in the pharmaceu- of the reactor diameter. If heat dissipation is a
tical industry, the most versatile bioreactor is the problem, as it often is in large production fer-
simple stirred, aerated’ fermenter. However, no menters (> 100 må), up to 12 baffles can be used
single system which adequately meets the needs as heat exchangers.
of all biological systems can be constructed. Fer-
menters for laboratory experiments requiring Foam separators Foaming is frequently a prob-
volumes of up to 20 I are made of glass or stain- lem in large-scale aerated systems. Antifoam
less steel (Figure 5.9); for larger volumes con- chemical agents cannot always be used for the
 5.3 FERMENTER SYSTEMS / 77

Steam mmm — PH controller

Acid/base reservoir

Exhaust

Impeller

Cooling
water
out

Cooling
jacket

Cooling
water in

Sparger
(air bubbles)

—<—— Sterile air

Figure 5.10 An industrial fermenter,

illustrating the construction and fa-
cilities for aeration and process con-
trol. = Harvest

reduction of foam, since they may have inhibi- Types of stirrers Only specific types of stirrers
tory effects on the fermentation. There are sev- developed for chemical technology can actually
eral methods by which the foam in the sterile be used for microbiological processes. Figure 5.12
area can be destroyed by mechanical methods. shows the most important ones. The disc stirrer
The simplest devices have rakes mounted on the is the most common type; 4-8 radial blades pro-
stirrer. In the Frings system for vinegar produc- ject out beyond the edge of a disc of appropriate
tion and the Fundafom system (Figure 5.11), both diameter. In the turbine-type stirrer, blades are
of which are currently used industrially, foam is curved. Compared with the disc stirrer, the tur-
destroyed by centrifugal force. The nutrient so- bine type requires 50% less air for the same yield
lution held in the foam flows back into the bio- and energy consumption. Two other types con-
reactor and the air released from the foam leaves sist of stirring arms with blades attached at an
the sterile system. angle. Compared with both of the above-men-
78 / CHAPTER 5 / METHODS OF FERMENTATION

tioned stirrers, the MIG and INTERMIG stirrers

(Figure 5.12) require 25% and 40% less energy
respectively for equivalent yields.

Maintenance of sterility There should be a min-

imum number of openings in the fermenter to
favor maintenance of sterility. Small openings
must be made leakproof with O-rings, larger
openings with flat gaskets. Wherever a movable
shaft penetrates the fermenter wall, special prob-
lems of sterility maintenance arise. Double me-
chanical seals on the agitator shaft are currently
used and present noticeable advantages in com-
parison to the more conventional stuffing-box
seal. If possible, the joints of all parts connected
within the sterile area as well as all of the pipes
both inside and outside the fermenter should be
welded. There should not be any direct connec-
tion between the nonsterile and sterile area; that
Figure 5.11 Mechanical foam separator (Fundafom, is, sampling devices and injection ports must be
Chemap). a) Foam entrance, b) Gas exit, c) Lubrication, covered with steam-sterilizable closures. Sterile
d) Double seal, e) Packing, f) Drive, g) Intermediate flange,
h) Rotating plate pipes must be slanted to collect the condensate
and to drain it.
Although the fermenter vessel is the main
focus of interest in the overall fermenter system,
in an actual installation a large number of ad-
ditional devices must be provided in order to ob-
tain a functioning fermenter system. Figure 5.13
shows the layout for the installation of a fer-
menter system.

Reactors for immobilized enzymes or cells

An important type of large-scale biological re-
actions involves the attachment of the biocata-
Disc stirrer Turbine stirrer lyst, enzyme or cell, to an inert support through
which the substrate is passed. The product is then
present in the effluent and can be directly puri-
fied. Bioreactors for use with immobilized en-
zymes or cells must be so constructed that that
es JL. 22 — rate of movement between substrate and the bio-
catalyst is not rate-limiting. Internal mass trans-
fer can only be influenced by the method by
MIG Stirrer INTERMIG Stirrer which the catalyst is immobilized. Figure 5.14
summarizes the various types of bioreactors for
Figure 5.12 Impeller systems for fermenters immobilized systems. Two basic types are used:
 5.3 FERMENTER SYSTEMS / 79

Ss Exhaust air Nutrient reservoir

SW SW SW

x
VY VY VV VY V
AA 4 & LS

><] — 0%
bp. % 2

S RS

; (LS epee (8ae 8

\/ W LV, \/ V
f\ (4 A £\ £\

@ @
LA
CV

Cc Cc
p|
Filter ep. XY X XY a

‘\
@ @ 6
Incoming air

><

VW
O .
A Cooling
og circuit
C)

><] S

byd bod
dd BEG Harvest
LS ON

\/

ee
S W C Weight
load cell

Figure 5.13 Installation of a fermenter. S, steam; C, condensate; W, water; A, air. The steam lines permit in-place
sterilization of valves, pipes, and seals. The input air is sterilized by both incineration and filtration
80 / CHAPTER 5 / METHODS OF FERMENTATION

Reactors for immobilized

enzymes or cells

Reactors for
particle-bound Membrane/enzyme
enzymes reactors

Stirred reactors
Tube reactors Reactors with Reactors with
encapsulated membrane-bound
Bed reactors enzymes enzymes
Fixed-bed reactors
Stirred reactors Reactors with adsorbed,
Fluidized-bed reactors encapsulated, or
Ultrafiltration chemically bound
Cyclone reactors membranes enzymes
Dialysis membranes

Figure 5.14 Types of systems for immobilized enzymes/cells

those in which the enzyme is incorporated into

a membrane and those in which the enzyme is
bound to an inert particle.

Particle-bound systems For systems that used

particle-bound enzymes, stirred reactors or those
mixed by pumps are used (see Figure 5.8, parts
1,2, and 4) if high aeration or careful pH control
is necessary, or if the fluid has a high viscosity.
A disadvantage of the use of a stirred reactor is
that the shearing force of the stirrer or pump is
high, thus raising the possibility that the catalyst
may be damaged.
Another approach is the use of a system in Figure 5.15 Bed reactions for immobilized enzyme re-
which the catalyst is immobilized in a bed, such actions: a) Fixed bed; b) Fluidized bed; c) Cyclone
as a fixed-bed, fluidized bed, or cyclonic reactor
(Figure 5.15). The fixed-bed reactor adapts itself
well to large-scale operation; because of the reacted substrate; regulation of temperature and
dense packing of the immobilized material a high PH is difficult. In the fluidized-bed reactor the
rate of substrate conversion per unit time is-ob- height of the catalyst must be somewhat larger
tained. Since the flow is from the bottom to the than that of the substrate solution. Because of
top, there are gradients of concentrations of sub- the plug-flow nature of fluid movement, the cat-
strate and product. Disadvantages of the up-flow alyst is kept in suspension. Recycling of the fluid
fixed-bed reactor are: gas formation may reduce is not possible, the flow-rate cannot be varied,
the contact surface between substrate and en- and scale-up is quite difficult.
zyme; channels in the fixed bed may develop, Cyclone reactors can be well mixed, either by
leading to the appearance in the product of un- installation of a stirrer or by use of a gas under
 5.4 STIRRING AND MIXING / 81

pressure. Because of the broad distribution of res- Modern ultrafiltration membranes made of
idence times of the substrate, complete conver- polyamides (nylon) or polysulfones offer excel-
sion of substrate to product is not possible. lent molecular-weight separation in the range of
500-300,000 Daltons). They are also quite stable
Membrane/enzyme reactors In membrane/en- to temperature, pressure, and chemical attack
zyme reactors the soluble enzyme and substrate and are also resistant to microbial degradation.
In order to reduce the tendency for clogging,
are introduced on one side of an ultrafilter mem-
asymmetrical membranes can be used with
brane. By means of a pump, the product is forced
smaller pores on the high-pressure side. Such
through the membrane under 0.5 to 5 atmos-
membranes are also readily cleanable by back-
pheres pressure; the membrane holds back the
washing. For industrial-scale operation, the sep-
enzyme (Figure 5.16). Enzyme/membrane re-
aration is not carried out in the bioreactor itself
actors have several advantages:
but in a bypass in which the membrane is in-
¢ The expense of immobilization is eliminated stalled. Instead of ultrafiltration, it is also possible
and the loss of enzyme is reduced. to separate product from enzyme and substrate
+ By periodic addition of enzyme to make up by dialysis.
that lost by denaturation, the rate of reaction Bioreactors have also been developed in
can be maintained at a constant rate. which the enzyme is adsorbed onto or incorpo-
+ Multi-enzyme systems can be used. rated within the membrane, either through phys-
e By recycling of the reaction mixture internal ical or chemical means. In such systems the sub-
and external mass transfer can be optimized. strate is converted to product as it passes through
+ Substrate and enzyme can be easily replaced. the membrane. For instance, a cross-linked sul-
+ Scale-up presents no particular problems. fonic acid cellulose has been developed which
can be fabricated into paper sheets and wrapped
The main disadvantages of enzyme/mem- into cartridges. In a radial reactor of this type,
brane systems are the shearing stress from the the enzyme mutase was used to quantitatively
fluid flow, which tends to denature the enzyme, convert a solution of sucrose to isomaltulose. By
and the relatively large surface area of membrane passage through the cartridge the substrate was
required. completely converted to product.

5.4 STIRRING AND MIXING

A microbial fermentation can be viewed as a
three-phase system, involving liquid-solid, gas-
solid, and gas-liquid reactions.

Substrate —= 1. The liquid phase contains dissolved salts,

substrates, and metabolites. A second liquid
Enzyme phase may occur in some cases if there is an
pea ae —- ——— —}Membrane
insoluble substrate, e.g. alkane fermentations.
2. The solid phase consists of the individual
cells, pellets, insoluble substrates, or precipi-
tating metabolic products.
Product
3. The gaseous phase provides a reservoir for
Figure 5.16 Use of ultrafilter membrane to separate en-
oxygen supply, for CO, removal, or for the
zyme and substrate from product adjustment of pH with gaseous ammonia.
82 / CHAPTER 5 / METHODS OF FERMENTATION

The transfer of energy, substrate, and metab- The Reynold's number describes the flow only
olite within the bioreactor must be brought about at the periphery of the stirrer. To distribute the
by a suitable mixing device. The efficiency of the turbulence homogeneously within the entire re-
transport of any one substrate may be crucial to actor, an impeller of appropriate shape and di-
the efficiency of the whole fermentation. ameter must be used. The flow rate is crucial to
For the three phases, the stirring of a bio- the distribution of turbulence. However, as tur-
reactor brings about the following: bulence may damage filamentous organisms,
there are frequently limitations as to how fast a
¢ Dispersion of air in the nutrient solution.
system can be stirred. The mixing time, då, is the
¢ Homogenization to equalize the temperature
time needed for homogenization to the required
and the concentration of nutrients through-
degree of homogeneity in the entire reactor.
out the fermenter.
e Suspension of microorganisms and solid nu-
trients. Power number
e Dispersion of immiscible liquids. The power number (N, = Newton's number =
Ne) has been defined as a dimensionless param-
Reynold’s number eter relating to the energy required by stirred re-
actors. It is calculated as follows:
Stirring ensures the transport of nutrients within
Imposed force
the culture liquid. In turbulent flow, two mole- P ower nu mber = Np = Iner
cules of liquid move in relation to each other. tial force

The relative velocity between the nutrient solu- Po

tion and the individual cell should be about 0.5 TAROT ROSE [24]
m/sec. Turbulent flow can be characterized by
P, = Stirring power (kW)
the dimensionless Reynold’s number, well dis- N = Stirring speed (sec”')
cussed in texts on fluid mechanics. The Reynold’s D; = Stirrer diameter (cm)
number is calculated as follows p = Density of the medium (g/cm?)

D?-N-p The power number has been correlated with the

Reynold’s number R, = Np, = ————— [23] Reynold’s number for several types of stirrers.
Figure 5.17 shows this relationship with six types
Stirrer diameter (cm)
Stirrer speed (sec!)
of stirrer blades.
Density (g/cm?) In the laminar flow range of mixing speed
Dynamic
ououu
38220 viscosity (g/cm - sec) (Nae < 10) the Reynold’s number is correlated
with the power number as follows:
The following Reynold's numbers have been cal-
culated for the penicillin fermentation with Pen- Np = K, - (Np,)™ [25]
icillium chrysogenum. Note how much lower the
K, = Constant dependent upon the container
values are than for pure water:
geometery and the shape of the stirrer, but not
dependent upon the reactor size
m
Small Production
fermenter fermenter
Water With laminar flow, the power required for stirring
(n = 107? g/cm - sec Re 4.0 - 10° 6.9 - 10° is not dependent on the density, but is correlated
= 1 centi Poise)
with fermentation parameters as follows:
Culture medium
(n= 5 g/cm - sec Re 8.0 - 10? 1.4 - 107
= 500 centi Poise)
Po ~N’-D?-n [26]
 5.4 STIRRING AND MIXING / 83

500

4 5 6
Fo oOco
3.D;5
eN
sf a NY,
(NS
w/D=1/5 w/D=1/5 |w/D=1/8 w/D=1/8 |w/D=1/8| w/D= 1/8
w = blade width
D = stirrer diameter

uw

number
Power

2
1 10 10 10 10 10
D2: N-0
Reynold’snumber N,.. Re = Ul

Figure 5.17 Correlation between power number and Reynold’s number in Newtonian solutions for various impellers
(Bates et al., 1963)

In the turbulent flow range of mixing speed (N,. N Fr Praia

g [28]
> 104), the power number is independent of the
Reynold’s number and is constant g = Acceleration due to gravity

Np = K,=Constant m=0 Power number, Reynold’s number and Froude's

number are correlated with each other
Under these conditions the power number is in-
dependent of the viscosity Np = K, - (Npe) Ng)” [29]

Py =K,-N?-Dé-p [27] No vortex is formed in bioreactors with baffles,

so that Froude’s number is not relevant in these
In the transient range of mixing speed (N,. = cases.
10-10), there is no simple correlation between
the power number and the Reynold’s number.
Effect of viscosity
Disc impellers have a higher power number than
slanted blade or propeller stirrers. Nutrient solutions can be subdivided into two
In bioreactors where a vortex is formed, an- groups according to the way they behave when
other dimensionless number, Froude’s number, stirred: viscous solutions with Newtonian and
is useful. It is described as follows non-Newtonian properties; and viscoelastic so-
84 / CHAPTER 5 / METHODS OF FERMENTATION

lutions, in which normal liquid-state properties

are not observed in stirred vessels.
There are only a few examples which fall into
the second group (polysaccharides and certain
antibiotic fermentations). During agitation of
such solutions, the liquid does not flow tangen-
tially, but rises up along the impeller, a behavior
called the Weissenberg effect. With such solu-
tions, scale-up calculations are difficult to make.
Most fermentation solutions fall into the first
category. Uninoculated solutions and bacterial
cultures often behave as simple Newtonian liq-
uids. In such liquids, the dynamic viscosity (n) of
a nutrient solution is a constant at a constant
temperature, and it is dependent on the ratio of
the shearing stress (T) to the rate of shear (vy).

Newton’s law of friction

20 40 60 80
T=n-¥ [30] Fermentation time (hr)

Figure 5.18 Progress of kinematic viscosity in Micro-

Newtonian fluid monospora fermentations (1cSt = 10°* m?/sec, D = 10s)
T
ihe FRE Constant [31]
various types of non-Newtonian solutions are
T = Shear stress (kp/m?) shown in Figure 5.19, which illustrates the de-
y = Shear rate (sec) pendence of shearing stress on rate of shear.
For solutions which exhibit pseudoplastic
With many mycelial organisms, changes occur behavior, the apparent viscosity decreases as the
during the fermentation not only in the amount rate of shear increases. With dilatant nutrient
of mycelium, but in the characteristics of the nu- solutions, the apparent viscosity increases as the
trient solution. Substrates are taken up during rate of shear increases. Bingham-plastic behav-
metabolism and the proportion of undissolved ior is exhibited by nutrient solutions which will
substrates is reduced. At the same time, metab- not flow unless a stress T, is imposed. The law
olites are excreted, thus affecting the viscosity of is stated as follows
the solution.
Figure 5.18 presents data on a fermentation T-T (y
=n = Constant
system with non-Newtonian properties. In this
figure, the dynamic viscosity of a Micromonospora
culture is plotted against time (v = dynamic vis- Table 5.7 lists the type of viscosity of some mi-
cosity/density; 1 Stokes = 1 St = 10-*m?/sec). crobial mycelia.
In this case, the viscosity increases by a factor of It is more difficult to determine the amount
100 during mycelium formation. of energy required for non-Newtonian solutions
In non-Newtonian nutrient solutions, the dy- than for Newtonian solutions. The rate of shear
namic viscosity is dependent not only on the changes with changes in the impeller speed, tem-
temperature, but also on the rate of shear. The perature, and time. The curves in Figure 5.19 are
 5.4 STIRRING AND MIXING / 85

7 Shearing stress ment decreases. The correlation between power

requirements of aerated and unaerated fermen-
ters is given below:
Bingham plastic
23) . T3\ 0.45
rok (i mn |
Q .
[32]
Pg = Power requirement of the aerated bioreactor
Dilatant NEMIADL (horsepower) '
K = Constant (function of fermenter geometry)
P, = Power requirement of the unaerated fermenter
(horsepower)
Pseudoplastic N = Stirring rate (rpm)
Dj = Stirrer diameter (cm)
Q = Aeration rate (vvm)

This calculation describes both Newtonian and

Rate of shear non-Newtonian fermentation systems. This
equation has been shown to hold for a series of
Figure 5.19 Correlation between shear rate and shear
stress in nutrient solutions with Newtonian and non-New-
tonian properties

50
Table 5.7 Viscosity of mycelium-producing
microorganisms
Microorganism Use Viscosity
[=]
Penicillium Penicillin Pseudoplastic
chrysogenum
Coniothyrium Steroid Bingham
hellbori hydroxylation
Streptomyces noursei Nystatin Newton
Aspergillus niger Bingham
Streptomyces niveus Novobiocin Bingham så
Så i

Streptomyces griseus Streptomycin Bingham

Streptomyces sp. Newton and
pseudo-
plastic 2%,
Endomyces sp. Glucoamylase Pseudoplastic
Oo ee
|el

not linear and the Bingham-plastic line does not

cross zero. Mycelial suspensions which are taken
from a bioreactor to determine viscosity may (aerated);
requirement
Energy
horsepower
change their behavior within minutes without itv T T T T T T T pr
constant gaseous conditions, stirring, and tem- BEDE EO 00 ae? to ae
perature. (P2ND2/089 x 10% [(HP2)(RPM)(CM)?/
(L/MIN )°°5]x10°°
Power requirement in aerated bioreactors Figure 5.20 Michel and Miller correlation of energy re-
quired in geometrically similar aerated and unaerated bio-
During aeration, as the effective density of the reactors (Taguchi et al., 1968). e 30 1, O 100 1, 4 5000 1,
gas-liquid mixture is reduced, the power require- and 0 50,000 | fermenters
86 / CHAPTER 5 / METHODS OF FERMENTATION

fermenters from 20 1 to 30,000 I for the gluco- Table 5.8 Energy demand in pilot plant and
amylase fermentation by Endomyces sp. (Figure production fermenters
5.20). Fermenter size Power
(m?) kWh/m?
Table 5.8 shows data for actual power re-
quirements for some industrial fermenters in 0.1 11-13
1 5 4— 7
both pilot-plant and production-sized systems.
15 -120 1- 3
Small fermenters are usually constructed so as to
be oversized in power, in order to accommodate (Einsele, 1978)

future needs. The power requirements of large

fermenters are similar to those in the chemical
industry. ] tion increases in the gas phase, the O, proportion
of the nutrient solution increases. Consequently,
5.5 GAS EXCHANGE AND MASS the highest O, partial pressures are attained dur-
TRANSFER ing aeration with pure oxygen. Compared to the
value in air (9 mg O,/1), 43 mg O,/1 dissolves in
One of the most critical factors in the operation water when pure oxygen is considered. Henry’s
of a large-scale fermenter is the provision of ad- constants for various gases are summarized in
equate gas exchange. Oxygen is the most im- chemical handbooks for standard conditions.
portant gaseous substrate for microbial metabo- As temperature rises, the O, solubility de-
lism, and carbon dioxide is the most important creases
gaseous metabolic product. When oxygen is re-
quired as a microbial substrate, it is frequently a 468
*=
limiting factor in fermentation. Because of its low . 316.430 [34]
solubility, only 0.3 mM O,, equivalent to 9 ppm,
T = Temperature (°C)
dissolves in one liter of water at 20°C in an air/
water mixture. Due to the influence of the culture
ingredients, the maximal oxygen content is ac- Oxygen transfer
tually lower than it would be in pure water. The
solubility of gases follows Henry’s Law in the For oxygen to be transfered from a gas bubble to
gas pressure range over which fermenters are op- an individual cell, several independent partial re-
erated. sistances must be overcome (Figure 5.21): 1) re-
sistance within the gas film to the phase bound-
ary; 2) penetration of the phase boundary
Henry’s law between gas bubble and liquid; 3) transfer from
Henry’s law describes the solubility of O, in nu- the phase boundary to the liquid; 4) movement
trient solution in relation to the O, partial pres- within the nutrient solution; 5) transfer to the
sure in the gas phase. surface of the cell.
For fermentations carried out with single-

h
C+ = cS [33] celled organisms such as bacteria or yeasts, the
resistance in the phase boundary between gas
bubble and liquid is the most important factor
In this equation, C* is the oxygen-saturation con- controlling the rate of transfer.
centration of the nutrient solution, p, is the par- Microbial cells near gas bubbles may absorb
tial pressure of the gas in the gas phase and H oxygen directly through the phase boundary and
is Henry’s constant, which is specific for the gas the rate of gas transfer to such cells is increased.
and the liquid phase. As the oxygen concentra- In cell agglomerates or pellets, the O, transfer
 5.5 GAS EXCHANGE AND MASS TRANSFER / 87

Gas-fluid phase boundary Fluid-cell phase boundary

2 | 4

Figure 5.21 Resistances for Cz

oxygen transfer from the air | Gas
Gas bubble Fluid film Fluid Fluid film! Cell
bubble to the microbial cell film

within the agglomerate can become the limiting Table 5.9 Oxygen transfer rates (OTR) in bioreactors
factor. Reactor —_Impeller Method of OTR
volume assay mM O,/I-h
Mass transfer of oxygen into the liquid can m3?

be characterized as follows:
0.1 Turbine Sulfite 100-223
0.8 Turbine Sulfite 94
12 Turbine Sulfite 64
Na =ky -a-:(C* — Cy) = OTR [35] 5.0 Turbine Sulfite 45=—72
47.7 Turbine Sulfite 42
NA = Volume-dependent mass transfer (mM O,/ Q-h) 34.2 Waldhof Yeast 16—22
kj = Transfer coefficient at the phase boundary 58.5 Vogelbusch Yeast 26- 43
a = Specific exchange surface
ky -a = Volumetric oxygen transfer coefficient (h7’)
(Hatch, 1975)
ce = Saturation value of the dissolved gas in the phase
boundary
CL Concentration of the dissolved gas (mM/2) The volumetric oxygen transfer coefficient is
OTR = Oxygen transfer rate (mM O,/8 - h) dependent on the following fermentation con-
ditions:

Table 5.9 summarizes several oxygen transfer kya =k - (Py/V)°* (Vs) (INDSE [36]
rates for three impeller systems. k = Constant
The volumetric oxygen transfer coefficient V = Bioreactor volume
Vs = Gas exit speed (cm/min)
has been thoroughly examined as a critical pa-
rameter for bioreactor function. The coefficient is
dependent on the diameter, capacity, power, aer- The k, value is frequently substituted for the k,a
ation system, and aeration rate of the bioreactor value in multi-level systems, as described in the
and on the density, viscosity, and composition following relationship:
of the nutrient solution, the structure of the kg = (2.0 + 2.8 Nj) . (P,/V)°°8 4 Veo? - N27. 107 [37]
microorganism, the antifoam agent used, and the
temperature. N; = Stirrer number
88 / CHAPTER 5 / METHODS OF FERMENTATION

The value of the combined parameters k,a can value decreases gradually as the pellet increases
be directly calculated, but k, and a are difficult in size, there is a much steeper decline with loose
to calculate individually. The interfacial area a is forms (Figure 5.22),
usually unknown since it depends on bubble The gas bubbles are replenished in locations
size. Table 5.10 shows some values for the in- of the bioreactor where there is negative pres-
terfacial area a. sure, such as behind the agitator blades. As the
Surface-active substances such as antifoam aeration rate increases, various conditions can be
agents reduce the value of k,a. In pure water, the characterized. At low aeration rates, large gas
bubble surface is constantly renewed through vi- bubbles form behind individual turbine blades
bration and oscillation. As soon as surface-active and smaller bubbles are spun off centrifugally
substances are added, the renewal of the bubble into the nutrient solution. As the aeration rate is
surface by bubble movement ceases. Microor- increased, gas bubbles collect behind all turbine
ganisms themselves have an effect on the oxygen blades and continue to accumulate. The energy
transfer by acting as a barrier, thus inhibiting the input is one-third less than that used in unaer-
O, transfer. With filamentous organisms, there ated systems. In this intermediate stirring range,
are variations depending on whether the myce- gas dispersion is the best. At very high aeration
lium is in loose form or in pellets. While the k,a rates, many large gas bubbles adhere to each
other and the impeller is flooded with gas, re-
sulting in sharply lowered gas dispersion.
Table 5.10 Values of the specific exchange coefficient a
in some bioreactors
Oxygen as a substrate
Bioreactor Aeration Aeration Specific Ex-
type rate energy change As previously mentioned, growth in a microbial
vvm demand co-
kW/m? efficient
culture with limited substrate is calculated as fol-
a (m”) lows:
Stirred vessel 1 120
1 300
A
1 400
50 4
2 600
Bubble Fritted disk 0.63 0.6 650
column
Frings 152 1000 40 KR Cnet
[
\ Af Fre
bioreactor
SEERE SE STER
Stirred vessel 4 1000 N ne . Far ER;
30 ie & 30 ae
10 1100
Shas SiS <a e
Tubular loop Ejector 0.9 1300
reactor 0.9 1300 nS Be SS > = =o — VS
1.8 1700 200 NR BRASS
R ~.
1.8 1800
= ee går rr
7.2 2000 r=,

Bubble Fritted disk 0.56 0.9 2000 ig re

column aq
=)

Stirred vessel 10 2200 3

Frings 0.48 Doli PRN; T i i T | ee

bioreactor 2 4 6 8
Tubular loop Ejector WP 2500
Cell dry weight (g/l)
reactor
Bubble Ejector 0.42 15 6000
column Figure 5.22 Relation of the k,a value to the mycelium
Bubble Ejector 1.4 22 8000 concentration (Miura, 1976). Impeller rotations per min
column are given as follows: O — O 480, e — e390, and 4 — a
290; —— mycelium in pellet form, — — — mycelium with-
(Schtgerl and Lucke, 1977) out pellet formation
 5.5 GAS EXCHANGE AND MASS TRANSFER / 89

S tion rate increases and the O, content in the broth

Beas [3]
decreases until it becomes limiting. Thereafter,
with unicellular bacteria the O, absorption rate
With oxygen as substrate, S = C,, which is the is constant until another substrate becomes lim-
current oxygen concentration. When the specific iting. In mycelial (streptomycete and fungal) fer-
growth rate in the steady state is correlated with mentations, however, the O, absorption rate de-
the specific oxygen absorption rate, the equation creases when O, becomes limiting, due to the
can be stated increase in mycelium volume and the related vis-
cosity increase.

Co," Om" KO+E a

Ko = Michaelis-Menten constant for O, Critical oxygen concentration
Specific O, uptake rate/cell dry weight
(mM O,/g - h) Critical oxygen concentration is the term used
Qm = Maximum specific uptake rate to indicate the value of the specific oxygen ab-
sorption rate which permits respiration without
The specific maximal O, uptake is designated as hindrance. Table 5.12 shows several examples of
the specific O, requirement; some examples are critical oxygen concentrations. They are 5-25%
compiled in Table 5.11. of the oxygen saturation value in cultures.
When the Q,, value is correlated with bio- At oxygen absorption rates which are lower
mass/liter, one obtains the gas absorption rate than the critical concentrations, respiration rate
or oxygen uptake rate which must be achieved is correlated with the O, concentration in the so-
during a fermentation: lution. Above this value, no dependence between
respiration rate and dissolved oxygen has been
X - Qm = kya : (CX — Cy) (mM O,/2
- h) [39] observed. In Newtonian fluids, such as those oc-
curring in yeast and bacterial fermentations, the
X = Cell concentration (dry weight g/1) critical oxygen concentration is constant and is
not affected by fermentation conditions. In non-
The gas absorption rate is not constant dur- Newtonian solutions, such as those occurring
ing the fermentation, since if a substrate other with filamentous organisms (e.g., during novo-
than O, becomes limiting, such as at the end of biocin production with Streptomyces niveus), the
the log phase, the value can be reduced. critical oxygen concentration has been shown to
During the fermentation of single-celled and be dependent on fermentation conditions. Table
mycelium-producing organisms, there is a char- 5.13 shows an experimental result in which the
acteristic difference in the oxygen absorption rate critical oxygen concentration in a strain varied
(Figure 5.23). During log growth, the O, absorp- (within the error of measurement) between 0 and
55% of the saturation value.
Table 5.11 Specific O, requirements of some
microorganisms
Organism Q,.(mM O,/g cells-h) Determining the oxygen uptake rate
Aspergillus niger 3.0 There are four acceptable methods of determin-
Streptomyces griseus 3.0 ing the O, uptake rate: the dynamic method, the
_ Penicillium chrysogenum 39
Klebsiella aerogenes 4.0 sulfite method, the direct measurement of the
Saccharomyces cerevisiae 8.0 volumetric O, transfer rate, and calculation from
Escherichia colt 10.8 measurements of the growth of the microorga-
(Brown, 1970) nisms.
90 / CHAPTER 5 / METHODS OF FERMENTATION

une 0, limited

—å
O
N
je)
Ze)
S
re Figure 5.23 O, uptake rate and con-
Uptake
0, of centration of dissolved oxygen under
ran) O, limitation. (a) Bacterial culture, (b)
Fungal fermentation; O, absorp-
Time Time tion rate, — — — C,, dissolved O, con-
b centration

Table 5.12 Critical oxygen concentrations of some

organisms dc
FH = kia: (C* — C1)m — Q, "X [40]
Organism Cy, (mg/l)
Escherichia coli 0.26 or
Penicillium chrysogenum 0.40
Saccharomyces cerevisiae 0.60
Pseudomonas ovalis 1.10 a 1 dCi, ) a
Torulopsis utilis 2.00
ea (art* 20,*X)m+C [41]
(Brown, 1970)
m = average value over the bioreactor

Table 5.13 Dependence of the critical oxygen When the decline in O, concentration in the
concentration of Streptomyces niveus on fermentation broth is plotted against time after interruption of
parameters
aeration, a straight line results with a slope of
Fermenter Impeller D; Impeller Critical O, —Qo,"X. When the change in the O, concentra-
Volume (cm) speed Concentration
(1) (rpm) (% saturation) tion is plotted against time according to equation
1 (Figure 5.24), the slope of the straight line
20 7.0 400 0
20 12.2 275 0 equals the reciprocal of the volumetric transfer
20 21.0 180 50 coefficient k,a.
250 15.8 175 5 The advantage of this procedure is that only
250 15.8 220 18
250 23.7 175 22. one parameter must be calculated. The disad-
15,000 76.8 164 55 vantages are that the results are inaccurate due
15,000 90.5 124 55 to the long response time of the oxygen elec-
15,000 118 82 55
trodes and due to oxygen transfer from the head
(Wang and Fewkes, 1977) space of the fermenter, thus requiring a gas ex-
change with nitrogen. In some cases the value of
C, is so low that an exact measurement in the
Dynamic method In batch fermentations, the ac- fermenter is not possible. Also there may be a
tual dissolved O, concentration C, can be deter- production deficit whenever the slightest inter-
mined by measuring the speed of the C, decrease ruption of aeration occurs (as is found in acetic
after interrupting the aeration, thus determining acid production).
the volumetric oxygen transfer coefficient. The
following expresses the change of the current O, Sulfite method The sulfite method is frequently
content: used to obtain a quantitative measure of the O,
 5.5 GAS EXCHANGE AND MASS TRANSFER / 91

sulfite value (oxygen transfer rate, OTR) is a char-

acteristic of the bioreactor and gives some infor-
mation about the aeration device but gives no
indication of the current oxygen energy supply
in a fermentation broth. In addition, the cost of
analysis materials needed for use of this method
in a large bioreactor is considerable.
dissolved
0,
Direct measurement of the volumetric O, transfer
rate A direct measurement of oxygen transfer
is the most exact method, but it is time-consum-
ing and requires precise analytical devices. In this
method, the O, content in incoming and out-
dC
eeOPS Units going air is measured to determine the O, transfer
dt
rate. The exact oxygen absorption can be deter-
Figure 5.24 Determination of mass transfer coefficient mined by multiplying the aeration rate by the
using the dynamic method difference in O, content (in and out) and taking
the absolute temperature and pressure into con-
sideration. The oxygen content in the air is meas-
transfer in bioreactors. In the presence of 10-°M
ured specifically and very accurately by its par-
Cu?" or Co?" ions, sodium sulfite is oxidized with
amagnetic properties or with a mass
dissolved oxygen according to the formula
spectrometer, so that no other gases interfere.
Cu?2* or Co?*

Na,SO, + =O,2 2 Na, SO, [42] Q (Yin BE Yout) a Na [46]

Q = Aeration rate
Since this reaction takes place in a very short Yin = Oxygen content of the input
Y out = Oxygen content of the exit
time, if there is excess sodium sulfite, the O, con-
centration in the solution equals zero and the O,
transfer from the gas phase into the solution is Determining the O, transfer rate via measurement
the limiting factor. Nonoxidized sulfite is deter- of microbial growth The amount of oxygen re-
mined by adding a sample to an excess iodine quired can be calculated from thermodynamic
solution and by titrating the residual iodine with considerations, if it is assumed that a single car-
sodium thiosulfate. bon substrate is oxidized to carbon dioxide, water
and ammonia by an organism. Under aerobic
conditions the oxygen requirement C is calcu-
Na,SO, + I, +H,O — 2 Nal + H,SO, [43]
lated per gram of dry biomass produced:
I, +2 Na,S,0, — 2 Nal + Na,S,0, [44]
1 mM Na,S,0, = 0.25 mM O, [45]
C=—-B [47]
If two samples are taken at intervals, the O, ab- C = Oxygen demand/dry weight biomass
sorption per unit time can be calculated from the (mM O,/g cell dry weight)
rate of sulfite decrease. A = Oxygen required for oxidation of 1 g of substrate
to CO,, H,O, NH,
The sulfite procedure has a disadvantage: The B = Oxygen required for oxidation of 1 g biomass
process is performed as a chemical reaction in an to CO,, H,O, NH,
aqueous solution without further additives. The Yg = Cell yield/substrate (g/g)
92 / CHAPTER 5 / METHODS OF FERMENTATION

Table 5.14 Cell yields obtained with various substrates

Substrate Y, Bacteria Y, Yeast

Acetate 0.36 0.37

Glycerol 0.51
Glucose 0.51 0.50 mM O,
k = Correction factor 31.3
Methanol 0.40 gO,
Ethanol 0.68
Alkanes 1.03 1.00
Methane 0.62
For the cell yield per substrate, Yo = g cells/
g oxygen, the following equation is used:
The bacterial data are from Abbott and Clamen (1973)
and the yeast data from Bronn (1966).
I (2C#H2 0,0, CaN eee
Yo 1° Yo-M> 1600 600 933 200 ae
C,H, O = Number of carbon, hydrogen, and oxygen atoms
in substrate
The value of A can be calculated from the the- C’,H',O',N’' = Percent of carbon, hydrogen, oxygen, and
oretical oxidation of the substrate. For B the bio- nitrogen in biomass
mass must be analyzed (in yeast B = 934 ml O,/ M = Substrate molecular weight

g dry weight).
Some examples of cell yields (Y,) in yeast and Effects of carbon dioxide on the fermentation As
bacteria are given for several substrates in Table mentioned earlier, carbon dioxide is the most im-
5.14. portant gaseous metabolite produced in fermen-
Experimental values for the amount of O, tation. Although much study has been devoted
needed are listed in Table 5.15. Since these are to the effects of oxygen, too little attention has
the total amounts needed, they do not provide been given to the correlation between carbon
any information about when during a fermen- dioxide and microbial metabolism. The negative
tation the O, is needed. In order to determine influence of CO, on penicillin formation has been
the amount of O, required in a culture per hour, studied; erythromycin and rifamycin B formation
the specific growth rate u (h!), the biomass X (g/ rates are also reduced by excess CQ,,.
1), and the Y, value as grams of cells per gram Figure 5.25 and Table 5.16 show the effect of
of oxygen (g/g) must be known. CO, on production of the aminoglycoside anti-
biotic sisomicin. When 1% CO, is added to the
incoming air in a 300 | fermenter, substrate is
metabolized more gradually, mycelium is formed
Table 5.15 Oxygen requirements of some slowly, and the sisomicin yields are 33% less
microorganisms than in the control.
O, required
(ml O,/
Microorganism Substrate g dry weight) 5.6 SCALE UP
Aerobacter aerogenes Glucose-NH, 515
Escherichia coli Glucose-NH, 400- Significance of scale up
Pen. chrysogenum Glucose-NH, 410
Sacch. cerevisiae Molasses 500 The conversion of a laboratory procedure to an
Sacch. cerevisiae Molasses 420-590 industrial process is termed scale up. It is well
Sacch. cerevisiae Molasses 430-550
Rhodotorula glutinis |Glucose-peptone 510
established in the field of industrial microbiology
Candida utilis Glucose-peptone 550 that a process which works well at the laboratory
Candida utilis Glucose-urea 460 scale may work poorly or not at all when first
Candida utilis Glycerol-peptone 480
attempted at large scale. It is generally not pos-
(Bronn, 1966) sible to take fermentation conditions that have
 5 GE SCALE JUR 7 93

oO =)

Mm

Mycelium
(%)
volume

(%)
disappearance
Carbohydrate

Fermentation time (hr)

Figure 5.25 Sisomicin formation during aeration with a 1% CO, air mixture. ——— CO,/air mixture, — control,
e antibiotic, 4 carbohydrate, X mycelium wet volume

Table 5.16 Influence of CO, on yield of the antibiotic e Construction of a completely new fermen-
sisomicin
tation plant, a rare occurrence.
CO, content of Relative
incoming air (%) sisomicin yield While laboratory microbiologists are primar-
ily interested in yield based on weight of bio-
0 100
1 66 mass, units per ml broth, or maximal yield per
2 15 unit time, success in scale up is evaluated on the
Be 0 basis of maximal yield for minimal operating cost
4 0
and time.
Comparing fermenters with similar geome-
tries, Table 5.17 shows that at different fermenter
worked in the laboratory and blindly apply them sizes not all parameters can be kept constant. If
to industrial-scale equipment. All of the skills of the impeller increases in diameter by a factor of
the biotechnologist must be brought into play in five, the fermenter volume increases by a factor
order to develop a successful large-scale process. of 125, from 80 liters to 10,000 liters. If one of
Scale up is necessary in the following circum- several criteria is kept constant in scale up, e.g.
stances: energy consumed/volume or Reynold’s number,
e A new process is implemented in the plant the other parameters are quite different from the
+ Mutants with 10-20% greater yield are to be values obtained with the small fermenter.
introduced into large-scale production as Although many parameters have been tested
soon as possible for use as scale up criteria, there is no general
94 / CHAPTER 5 / METHODS OF FERMENTATION

Table 5.17 Interdependence of scale-up parameters

Scale up criterion Designation Small fermenter 80 1 Production fermenter 10,000 I

Energy input Ie 125 3125 25 0.2

Energy input/volume BSN 1.0 25 0.2 0.0016
Impeller rotation number N 0.34 1.0 0.2 0.04
Impeller diameter D; 5.0 5.0 5.0 5.0
Pump rate of impeller E 42.5 125 25 5.0
Pump rate of impeller/volume FAV: PRP
PPE
ey
SVS
GIK 0.34 1.0 0.2 0.04
Maximum impeller speed
(max. shearing rate) N/D; 187, 5.0 1.0 0.2
Reynolds number ND?p/n 8.5 25.0 5.0 1.0
joo
(Oldshue, 1966)

formula because of the variation in fermentation in many production fermenters. Mixing time is
processes. The most important methods are: not suitable as a scale-up parameter because it
e Constant power consumption per unit of
increases as reactor size increases. It can be seen
broth in Table 5.18 that mixing time is approximately
e Constant volumetric oxygen transfer rate related to the cube root of the fermenter volume.
In reality, scale up is not usually done with
Some metabolites (for example, phenylala- geometrically similar fermenters in laboratory,
nine, valine, leucine, and capreomycin) are pilot plant and production scale. There is usually
formed best at oxygen concentrations below the no means nor any necessity of scaling up while
critical level. Figure 5.26 shows the use of the keeping one parameter constant.
critical O, concentration to scale up the formation
of nikkomycin, an acracide, from shake flasks to
40 m?° fermenters. Scale up with constant power
An additional criterion may be the impeller consumption per volume
tip velocity, which is in the range of 5-7 m/sec
In this simple method, one of the factors in the
determination of energy input in aerated bio-
reactors is the previously mentioned correlation
(see Section 5.4) between energy consumption
(P,) and fermenter parameters:

Py = K(P? 4 N Di Qs

Table 5.18 Mixing time in relation to fermenter

volume
Nikkomycin
(Units/I)
Fermenter Impeller Mixing
volume speed time
] rpm sec
3 750 3
120 180 9 2,000 3
Time (hr) 100 230 6.6
300 350 5
Figure 5.26 Scale up of nikkomycin production using the 1,000 200 25
critical O, concentration as the scale-up criterion. (C crit 3,000 180 20
24,000 30 66
1.5 mg O,/1). (Crueger et al., 1982).
 5.6n SCALE UP*/ 95

Studies have been done on the power input in a’ fite values, as described earlier. But this calcu-
penicillin fermenter with an energy input of 1.5— lation does not give any information on oxygen
3.0 horsepower/m? and in a streptomycin fer- transfer in highly viscous non-Newtonian fer-
menter with 2 horsepower/m’. mentation solutions (such as found in many anti-
In Figure 5.27, novobiocin titers are plotted biotic fermentations) or under conditions of high-
against energy input for three impeller types. The speed stirring.
curves are parallel and maximal yields are ob- Figure 5.28 shows the production of baker’s
tained above 1.5 horsepower/m‘’. Below 1 horse- yeast in vessels ranging in volume from a small
power/m? considerably lower yields are ob- shake flask up to 114 m’, plotted as the sulfite
served. This graph also shows that at equivalent value. In this study, neither the fermenter type
power inputs, antibiotic biosynthesis is depen- nor the volume were crucial in determining yeast
dent on the diameter of the impeller. growth. Above 125 mM O,/I-h, optimal yields
In another example, the flavomycin process were attained. Scale up of penicillin and strep-
was transferred from a 4 m° to a 40 m° fermenter tomycin processes has been successful in 15 |
by keeping constant the P/V relationship. through 100 I and 3 m? up to 63 m? fermenters.
In a comparative study of many production Instead of the k,a value, a modified k, value
plants, no constant P/V ratio was found. Rather, could be used as the constant parameter.
the following relationship was observed: Figures 5.29 shows scale-up tests by Jarai
(1972) on the value of k,a for the secondary me-
P,/V ~(vy°?” [50] tabolites (nystatin and fumagillin). For both these
antibiotics and several industrial enzymes, there
was a good agreement between k,a values and
Scale up with constant oxygen transfer the yields in 6 1 and 3000 | fermenters.
rate
Scale up is most commonly performed on the
basis of k,a data, which are calculated from sul- Å
120 ]

8 8
_~ 1004
ie) e 2 & eo
800 = . re) 8—e ra ee x

5 80 4 Sat ee ee
=E ef a eee eee = x
(AN oOo
aS (—
== a SX 60 4 :
600 a A
= ue) 0

2
= Å > 40 |
oO
re)
400 | 5
3 Stirrer 20 4

ZA Oy Dy/De = 3:42
sai T T T Sa sae
200 e 2.54 Standard
50 100 150 200 250
a 2.02
Sulfite value (mMO,/I-h)

l T T 7 >i T Teed Figure 5.28 Results of the scale up of baker's yeast pro-
as 1.0 15 20 25 30 duction (Strohm et al., 1959). Reactor volume: X 190 ml
Energy requirement (PS/m?) shaker ;0 19 1, 600 rpm, impeller flooded; e 19 1, 800 rpm;
Em 265 1, 550 rpm; O 265 I, unstirred, fritted disk with small
Effect of impeller geometry and energy input holes; 4 265 1, unstirred, fritted disk with large holes;
Figure 5.27
a 114 m3, unstirred
on novobiocin biosynthesis (Maxon, 1959)
96 / CHAPTER 5 / METHODS OF FERMENTATION

LE The ratio of N,/N is the inactivation factor, the

100 4 > °
pee, z- eH-0-~
a—0 ratio of N/N, is the survival factor and the In of
ic
©
x
Le
e
N./N=V is the design criterion, a parameter
E Wie ea which encompasses the contamination level of
eal MAIC
XR 7 A the medium to be sterilized, N,, and the desired
= PS
sterility level, N.
@
>
ys The ideal curve expressing the exponential
507
decline in survivors with time is plotted in Figure
T T T= T ee
5.30. This line has a negative slope and in Figure
10 20 30 40
k_ aValue(min') 5.31 part II corresponds to the death curve for
vegetative cells of Escherichia coli at 54—60°C.
Figure 5.29 Nystatin and fumagillin titers in relation to This type of killing kinetics is referred to as a
K,a values during scale-up (Jarai, 1972). -O-—®—O— logarithmic death curve. In actuality, this curve
nystatin, —A—a—A— fumagillin; O, 4 6 1, @, 4 3000 | is not always linear, as shown for the death curve
of Bacillus stearothermophilus spores (Figure 5.31,
part I). Mathematical models for such nonloga-
5.7 STERILIZATION OF GASES AND rithmic destruction curves have been developed.
NUTRIENT SOLUTIONS In the above equations, k is a constant which
In virtually all fermentation processes, it is man- expresses the specific death rate. It increases
datory for a cost-effective operation to have con- sharply with temperature and can be experimen-
tamination-free seed cultures at all stages, from tally determined for an organism using equation
the preliminary culture to the production fer- 53. According to the Arrhenius equation the fol-
menter. A bioreactor can be sterilized either by lowing connection between temperature and k
destroying the organisms with some lethal agent value exists:
such as heat, radiation, or a chemical, or by re- dink E
moving the viable organisms by a physical pro- REE ia,
cedure such as filtration.
The process of destroying a population gen-
erally follows first-order kinetics. Using the ini-
tial number of cells N,/ml, the number of de-
stroyed organisms N’ at time t (min), and the
NS
surviving cells N, the death rate can be calculated OE:e
as follows

dN
FH
oOo,
— a7 =k(No-N’) =KN [51]
fo.)
(k is the specific death constant /min) oO.
Survivors
N/N
When integrated between N, at time t = 0
and N at time t = t, the following equation is
obtained

N
kt = In—— or [52] 50 100
Sterilization time (min)
In ——=— kt [53]
Figure 5.30 Theoretical death curve of a bacterial culture
 5.7 STERILIZATION OF GASES AND NUTRIENT SOLUTIONS / 97

destruction rate is thus not a straight line, but a

curve, as shown in Figure 5.32 for a culture con-
sisting of two strains.
During fermentation the following points
must be observed to ensure sterility:
¢ Sterility of the culture media
+ Sterility of incoming and outgoing air
+ Appropriate construction of the bioreactor for
ZC ASS MOSE sterilization and for prevention of contami-
nation during fermentation.
0 5 10 15 20 25
Sterilization time (min)
Sterilization of culture media
I B. stearothermophilus
Nutrient media as initially prepared contain a
variety of different vegetative cells and spores,
derived from the constituents of the culture me-
dium, the water, and the vessel. These must be
eliminated by a suitable means before inocula-
tion. A number of means are available for ster-
ilization, but in practice for large-scale installa-
tions, heat is the main mechanism used.

Heat sterilization This is the most useful method

for the sterilization of nutrient media. A number
of factors influence the success of heat sterili-
0 2 4 6 8 10 zation: the number and type of microorganisms
Sterilization time (min)

II E. coli

Figure 5.31 Nonlogarithmic and logarithmic death rates

R is the gas constant (J/Mol°C), T the absolute

temperature (°K) and E a specific constant for the
population (activation energy, J/Mol).
If the experimentally determined In k value Survivors
from this equation is plotted against the recip-
rocal temperature value, a straight line should be
obtained from which the k value can be calcu-
lated for a desired temperature.
| a K T T T

10 20 30 40 50
The Arrhenius relationship has been ob-
served only for pure cultures. The populations Sterilization time (min)
which normally exist in unsterile solutions are
Figure 5.32 Death curve of a mixed culture (C). The
generally nonhomogeneous mixed cultures con- straight lines (A) and (B) indicate the death rates of both
taining organisms of varying heat resistance. The pure cultures
98 / CHAPTER 5 / METHODS OF FERMENTATION

present, the composition of the culture medium, that inhibition of the fermentation organism
the pH value, and the size of the suspended par- could occur from the residual chemical.
ticles. Vegetative cells are rapidly eliminated at
relatively low temperatures, as shown in Figure Mechanical removal of organisms Alternatives
5.31, but for destruction of spores, temperatures such as centrifugation, adsorption to ion exchan-
of 121°C are needed. gers, adsorption to activated carbon, or filtration
Spores of Bacillus stearothermophilus are the are possible. Filtration is the only method in prac-
most heat resistant. Therefore they are used as tical use. Filter sterilization is often used for all
assay organisms for testing the various proce- components of nutrient solutions which are heat
dures used to sterilize equipment. Table 5.19 pro- sensitive and would thus be denatured through
vides data on the relationship between temper- the steam sterilization process normally used in
ature, k value, and design criterion for this industrial fermentation. Vitamins, antibiotics or
organism. A list of sterilization times and tem- blood components are examples of heat-labile
peratures for various organisms is given in Table compounds which must be sterilized by filtra-
5.20. tion. Such materials must be completely dis-
solved before filtration, otherwise they would be
filtered out of the mixture with the microorga-
Radiation (UV, X Rays, or y rays) Although oc-
nisms. Deep filters (plate filters) are sometimes
casionally used in the food industry, these agents
used to filter complex nutrient solutions. Two
are not used in industrial fermentation.
disadvantages of filtration are: 1) certain com-
ponents of the nutrient solution may be adsorbed
Chemical methods Although a number of chem- on the filter material, and 2) high pressures must
ical disinfectants are known, they cannot be used be used (up to 5 bar), which are undesirable in
to sterilize nutrient media because there is a risk industrial practice.
One approach which is cost-effective is the
Table 5.19 Relationship of temperature, k-value and filtration of just the water which is to be used in
the design criterion in Bacillus stearothermophilus the preparation of the culture medium. For in-
Te k(min“) Vv stance, in steroid bioconversion processes, a con-
centrated nutrient solution is sterilized by heat
100 0.019 v
115 0.666 3.154 in the fermenter and is then diluted to the normal
118 1.307 6.341 concentration with water which has been filter-
121 2.538 12.549 sterilized.
130 17.524 90.591
140 135.9
150 956.1
Batch sterilization

Most nutrient media are presently sterilized in

Table 5.20 Sterilization time and sterilization batch volumes in the bioreactor at 121°C. Ap-
temperature of various groups of organisms proximate sterilization times can be calculated
Cells Sterilization Sterilization from the nature of the medium and the size of
time min temperature °C the fermenter. Not only the nutrient media, but
Vegetative cells 5-10 60 also the fittings, valves and electrodes of the fer-
Fungus spores /yeast 15 80 menter itself must be sterilized. Therefore, actual
spores
Streptomyces spores 5-10 60-80 sterilization times are significantly longer than
Bacterial spores, general 5 121 calculated ones and must be empirically deter-
Spores of Bacillus 15 121 mined for the specific nutrient solutions in the
stearothermophilus
fermenter. One method of sterilization is to inject
'
 5.7 STERILIZATION OF GASES AND NUTRIENT SOLUTIONS / 99

steam into the fermenter mantle or interior coils. for heating must subsequently be removed in or-
(indirect sterilization). Another method is to in- der to cool the fermenter and if the hot water
ject steam into the nutrient solution itself (direct obtained during the cooling cannot be put to
procedure), in which case pure steam (free of some use, batch sterilization becomes very costly.
chemical additives) is a prerequisite. Many in- Another disadvantage of heat sterilization
dustrial steam supplies contain potentially toxic (and from the standpoint of microbiology the
chemicals derived from anti-corrosive additives most significant shortcoming) can be seen in Fig-
used in the steam-manufacturing process. With ure 5.33. The heating, sterilization and cooling
direct steam injection, condensate accumulates phases not only kill microorganisms but also se-
within the fermenter and the volume of liquid verely alter nutrient solutions. Discoloration and
thus increases during the sterilization process. changes in the pH value result from carameli-
zation and Maillard reactions (see Chapter 4).
The drawbacks of the heat-sterilization pro-
Vitamins are destroyed and the quality of the
cess are shown for a typical sterilization of a 3000
culture medium deteriorates. The extent to which
1 batch fermenter in Figure 5.33. It takes 2-3
the subsequent fermentation is affected depends
hours to reach the sterilization temperature of
on the organism and the process.
121°C, depending on the steam conduction and
fermenter size. Once the proper temperature has
Continuous sterilization
been reached, another 20-60 minutes are re-
quired for the actual killing process, followed by The two main disadvantages of batch steriliza-
cooling for about one hour. The energy required tion just mentioned, culture medium damage and

X——
= — — =X——-—---—- X—=---- KER
— i 0 X~ A

5 X
2= 08 +66 6 x
es A

E 6.2 z
<o 0.6 fa
i$?)
St °

E PÅ så)
9 oO
oO
‘O wh og
E 04 - at o=
® Be ge
3 Mite =
c bose ®
KS, of fet

(= Maximum change in extinction ©

x for continuous sterilization
Ww
—_—
ef
—
T Toa Naas "JER rr oe la

40 80 120 160 200

Time (min)

Figure 5.33 Temperature profile and culture medium change in a batch sterilization for a 3000 1. fermenter. O— O
°C, @ — @ extinction (1:5 diluted, 436 nm), X —— X pH value
100 / CHAPTER 5 / METHODS OF FERMENTATION

high energy consumption, can be largely avoided of this method is that with some nutrient solu-
by use of a continuous sterilization procedure. tions, insoluble salts (e.g., calcium phosphate or
Although continuous sterilization is the logical calcium oxalate) are formed and crusts appear in
preliminary step for continuous fermentations in the first heat exchanger, due to the extreme tem-
industrial scale, it is also of value in batch fer- perature differences between the sterilized nu-
mentations, making greater yields possible for trient solution and the cold incoming solution.
the time and space allotted. The reason for this The heat transfer coefficient is calculated as fol-
is because of the exponential relationship be- lows:
tween death rate and temperature, making the
time required for the complete elimination of life NO
shorter if higher temperatures are used. While ESA
batch sterilization is carried out in 30—60 minutes
at 121°C, continuous sterilization is normally ac- K = Heat transfer coefficient
Q = Heat consumed
complished in 30-120 seconds at 140°C. A = Transfer surface
The heating of culture media for continuous ATm = Average temperature gradient
sterilization can be done either by injection of
steam or by means of heat exchangers. Sterili-
zation with steam injection is done by injecting If precipitation occurs, the heat transfer coeffi-
steam into the nutrient solution. The temperature cient decreases and the system must then be
is raised quickly to 140°C and is maintained for stopped, treated with cleaning agents (acid or
30-120 seconds. Due to the formation of con- base), and resterilized. By sterilizing the critical
densate, the nutrient solution becomes diluted; components of the nutrient solution separately,
to correct this, the hot solution is pumped the value of k can be kept constant and the useful
through an expansion valve into a vaporizer and period can be extended for weeks.
the condensate is removed via vacuum pumps Starch-containing solutions which become
so that the sterilized nutrient solution has the viscous when heated are difficult to use in con-
same concentration after the cooling process as tinuous sterilization processes. Before the actual
before. The disadvantage of this process is the sterilization, a liquefaction and partial hydrolysis
sensitivity it exhibits to changes in the viscosity through acids or amylases must be carried out.
of the medium and to pressure variations. Moreover, if there are suspended particles in the
In the continuous process using heat exchan- nutrient solutions, the short sterilization times in
gers (Figure 5.34), the nutrient solution in the first the continuous process may not be sufficient for
heat exchanger is preheated to 90-120°C within the heat to permeate them thoroughly. The heat-
20-30 seconds by the exiting previously steri- ing time for 1 mm particles is 1 second; for 1 cm
lized nutrient solution. Then in the second heat particles it is 100 seconds. Therefore the particle
exchanger, it is heated indirectly with steam to size should be restricted to 1-2 mm in continuous
140°C. This temperature is maintained for 30- sterilization processes.
120 seconds in a holding pipe before it is placed
in the first exchanger for preliminary cooling and
Sterilization of fermentation air
then in a third exchanger for cooling to the tem-
perature of the fermenter. The cooling phase is Most industrial fermentations are operated under
only 20-30 seconds. Figure 5.35 shows the tem- vigorous aeration and the air supplied to the fer-
perature profile of the nutrient solution during menter must be sterilized. The number of par-
sterilization. ticles and microorganisms in air varies greatly
In the process using heat exchangers, 90% of depending on location of the plant, air move-
the energy input is recovered. The disadvantage ment, and previous treatment of the air. On the
‘
 5.7 STERILIZATION OF GASES AND NUTRIENT SOLUTIONS / 101

Cooler Figure 5.34 Diagram of continuous

sterilization via spiral heat exchan-
Cooling gers
Cleaning cycle Sterile medium
to fermenter

Recovery of
residual heat

Heater

Medium
reservoir
150

average, outdoor air has 10—100,000 particles per

m’ and 5-2,000 microorganisms/m:. Of these, 100 Temperature
C)
(°
50% are fungus spores and 40% are Gram-neg-
ative bacteria.
Fermenters generally work with aeration
rates of 0.5-1.0 vvm (air volume/liquid vol-
ume:minute. A fermenter having a working vol- 50

ume of 50 m? with an aeration rate of 1 vvm

needs 3000 m: sterile air per hour. The critical
importance of air sterilization in industrial mi-
crobiology can be seen from these values. Heat
maintenance
unit Recovery
of
heat
residual
The methods available for sterilizing gases
include filtration, gas injection (ozone), gas
Stage
scrubbing, radiation (UV), and heat. Of these,
only filtration and heat are practical at an in- Figure 5.35 Temperature profile of nutrient solution dur-
dustrial scale. For many years, air was sterilized ing a continuous sterilization process
102 / CHAPTER 5 / METHODS OF FERMENTATION

filtration involves inertial effects, blocking ef-

Electrostatic é
charge — 122 fects, diffusion, gravity separation, and electro-
static attraction. The last two mechanisms have
Inertial effect a minimal effect on the removal of particles. The
disadvantages of glass wool filters are shrinkage
and solidification during steam sterilization.
Glass fiber filter cartridges, which do not have
Direct these shortcomings, have replaced glass wool fil-
interception Diffusion ters.
Gravity New cartridge filter systems using pleated
membranes are now widely available. The ad-
vantage of these filters is that they are substan-
Figure 5.36 Mechanism by which particles are removed
by a depth filter tially smaller than glass wool or activated carbon
towers. Operating the systems has become much
simpler; because of the removable cartridge con-
struction, replacing used filter elements is easy.
by passing it over electrically heated elements, Constructed of cellulose ester, polysulfone, or
but due to the high cost of electricity, this process nylon, these membrane filters have the same
has been replaced by filtration. structure as depth filters, but because they have
In industrial systems, the air is sterile fil- a membranous structure they have an absolute
tered. In older systems pure depth filters such filter effect. Figure 5.37 shows an example of the
as glass wool filters were installed, in which par- manner by which a filter cartridge is constructed.
ticles would be trapped by a combination of Figure 5.38 shows a fermenter installation
physical effects. As shown in Figure 5.36, particle with incoming and outgoing air filters. The dis-

Figure 5.37 Structure of a filter cartridge

 5.8 FERMENTATION PROCESSES / 103

RS

| al
|
E© || EoO
= S lp 2 @)
Se ey, | At
_ IN CX
= | LA KS
pS lice
=
oO
D> ><}
BS
RR
xe
’
= = (od a

| 2 Air heater i DK
= £| = X A Exhaust
3© 8}° Cle Å airir filter
fi
ep) O ><} | D (2
Y Back flow
ge ue SE sa peor
Incoming x
air filter X
@

Fermenter

Figure 5.38 Installation of an air filter system (incoming and outcoming) in a fermenter

advantage of most systems installed today is that

there is not yet any absolute filter for bacteri-
ophages in industrial use. Bacteriophages can
cause total failure in a system: for example, in
Stage Course of the fermentation
the production of glutamic acid with Corynebac-
terium glutamicum or in the production of peni- I Inoculum preservation
cillin acylase with Escherichia coli. II Inoculum build-up a. 1-2 Shake flask cul-
tures

5.8 FERMENTATION PROCESSES b. Spore formation of

solid medium
An overall scheme of a fermentation process for
Ill | Prefermenter culture 1-3 Preculture fermen-
the production of primary or secondary metab- tations
olites is given in Figure 5.39. The various stages
Production fermenter a. Batch fermentation
of this scheme are discussed below.
b. Continuous fermenta-
tion
Stage I: Inoculum preservation
The preservation of production strains over a Figure 5.39 General course of a fermentation in the pro-
long period is a basic requirement for a practical duction of primary and secondary metabolites
104 / CHAPTER 5 / METHODS OF FERMENTATION

fermentation. The mere survival of strains is not cultivation in special media. The addition of pro-
the main objective. Microorganisms can easily be tective agents (such as skim milk or sucrose) re-
kept viable through periodic transfer, but it is duces the lethality during the lyophilization pro-
their capability for product formation which must cess. Special equipment can be purchased which
be preserved. High-yielding strains have often reduces the difficulties involved in freezing,
become damaged in primary metabolism during drying, and aseptically sealing the ampules. Ly-
the strain selection process, and such strains fre- ophilization is the method of choice in large cul-
quently degenerate during successive transfers, ture collections because the cultures can be sat-
probably as a result of spontaneous mutation. isfactorily maintained for an essentially
The objective of preservation is thus to main- unlimited amount of time.
tain strains as long as possible without cell di-
vision. ‘’Master strains’ should not be cultivated Stage II: Growth of the Inoculum
more than once in two years and activity levels
must be checked with each usage. Depending on The preserved culture is initially revived by
the strain, selection procedures must be under- growth in shaken liquid culture or on solid me-
taken periodically. ‘Working strains’ are derived dium (if spore formation is needed). The condi-
from ‘master strains’. Working strains should be tions used in the initial culture (medium, tem-
inspected for sterility and capability of product perature of incubation, etc.) will depend upon the
formation, and then stored until used. specific process. Standard growth times can be
The optimal method of preservation must be expected as follows:
worked out for each process, i.e., each strain. The
following three techniques are most commonly
Lyophilized cultures 4-10 days
used: Frozen cultures
Bacteria 4—48 hours
Storage at low temperatures (2-6°C) This method Actinomycetes 1— 5 days
is the easiest, but also the least secure. Micro- Fungi 1— 7 days
organisms are kept as stab cultures on agar or in Refrigerated cultures
liquid culture in the refrigerator. There is a rel- Bacteria 4-24 hours
atively high risk of contamination and reverse Actinomycetes 1- 3 days
mutation through frequent transfer (normally Fungi 1- 5 days
every 8-16 weeks, at least once annually).
In order to obtain sufficient inoculum for small
Frozen storage The most common method is fermenters, a second series of shake cultures is
freezing at —18°C or —80°C in freezers or at usually made in more flasks. In some fermen-
—196°C under liquid nitrogen. Freezing down tations, the large-scale inoculum must consist of
to —196°C must be done gradually (1°C/min), spores. To obtain a spore crop, the preserved cul-
although rapid freezing can be used if protective ture is cultivated on a solid substrate in 2-10 liter
substances are added to prevent the formation of glass vessels under conditions of constant tem-
crystals. Frozen cultures may be kept for several perature and sterile aeration for 8-24 days. The
years. The proportion of survivors is critical be- substrate for the production of large amounts of
cause up to 95% of the microorganisms are gen- spores is generally a granular material such as
erally killed during freezing and subsequent bran, peat, rice, or barley. In order to ensure con-
thawing. tinued aeration, the substrate must be shaken
daily, which makes maintenance of aseptic con-
Lyophilization The best method of strain pres- ditions difficult. Further, many of the spores pro-
ervation is freeze-drying (lyophilization) after duced may be incapable of germinating.
 5.8 FERMENTATION PROCESSES / 105

For inoculation, the total culture (spores plus Table 5.21 Fermenter sizes for various processes
culture medium) is suspended with the aid of a Size of Product
surface-active agent (e.g., Tween 80) and trans- fermenter m3
ferred into the fermenter. 1- 20 Diagnostic enzymes, substances for
The proper cultivation of inoculum is vital for molecular biology, recombinant or-
ganisms
optimal titers in the later production-scale pro- 40- 80 Some enzymes, antibiotics
cess. For optimal yields, not only the number of 100-150 Penicillin, aminoglycoside anti-
cells and spores have an influence but also the biotics, proteases, amylases, steroid
transformations, amino acids
nutrient medium used for the inoculum, the tem- —450 Amino acids (glutamic acid), single-
perature of growth, and the inoculum age. In- cell protein
duction or repression phenomena in the culture
used for inoculum may affect the rate of pro-
duction. how the nutrient medium is prepared. Several
parameters which must be optimized are:
Stage III: Fermenter preculture ¢ Composition of ingredients, quality, carbon /
nitrogen relationship, impurities, variability
Fermenter precultures must be made in order to
from batch to batch.
have enough inoculum for a large fermenter. If
. Order of solution or suspension of ingredi-
a production fermenter is started with too little
ents, pH value before and after sterilization,
inoculum, growth is delayed and the product for-
effect of sterilization on the entire nutrient
mation rate can be unsatisfactory (see Section
solution or on individual components.
5.2). The optimal inoculum concentration for the
e Changes in the sterilized nutrient solution be-
production fermenter determines the number of
fore inoculation due to increase in tempera-
stages of fermenter preculture that are needed.
ture and aeration.
In general the following inoculum concentrations
are required: The most important parameters during the
fermentation are:
Bacteria 0.1- 3.0%
Actinomycetes 5-10 7% Temperature Fermentations are run either in the
Fungi 5 -10 % mesophile range (temperature optimum 20-
"Spore suspension 1-5-10°/] 45°C) or thermophile (>45°C) range. The ap-
culture solution propriate temperature must be chosen to achieve
maximum growth on the one hand and optimal
At times, the production culture medium is used product formation on the other hand. In some
for the last stage of inoculum build-up in order fermentations, higher temperatures are used to
to induce product formation. obtain increased growth of the culture and then
the temperature is decreased at the onset of the
idiophase. As an example of how important tem-
Stage IV: Production fermentation
perature control is, an increase of 1°C above the
Depending on the fermentation, reactors of var- optimum produces a 20% lower yield in the pen-
ious sizes are used and no general scheme for icillin acylase fermentation!
the inoculation of a production fermenter can be
given. Table 5.21 gives the sizes of various pro- Aeration The aeration rate is 0.25—1.0 vvm (air
duction fermenters in actual use. volume/liquid volume-minute). The aeration
Nutrient media for production must be op- rate must be adjusted to the amount of O, re-
timized not only in the ingredients used but in quired.
106 / CHAPTER 5 / METHODS OF FERMENTATION
Table 5.22 Parameters that can be measured in
Pressure In order to minimize the risk of con-
fermentation processes
tamination, an overpressure of 0.2-0.5 bar is
Physical Chemical Biological
used. The hydrostatic pressure also has to be
parameters parameters parameters
taken into account in large fermenters, since this
Temperature pH Biologically active
influences the O, and CO, solubility in the nu-
product
trient solution. Pressure Dissolved O, Enzyme activity
Power O, and CO, in DNA and RNA
consumption waste gas content
Stirring Depending on construction, the follow- Viscosity Redox potential _NADH, and ATP
ing stirring rates are used with disc impellers. content
Flow rates (air Substrate Protein content
Fermenter size (m°) Impeller speed (rpm) and liquid) concentration
Turbidity Product
0.02 250-450 concentration
0.2 250-350 Weight of Ionic strength
18-20 120—180 fermenter
40 —150 120—150
450 60—120

The installation of a continuous drive system is ing these gases are well-developed and function
desirable in industrial fermenters in order to be with few interruptions. But the mass spectrom-
able to precisely adjust the stirring rate to the eter is more versatile, since it can also measure
process. N,, NH,, methanol, and ethanol simultaneously
as well as give qualitative and quantitative in-
formation on exchange of O, and CO,. By the
5.9 INSTRUMENTATION
use of gas-permeable membranes, it is also pos-
To carry out measurements during fermentation sible to measure dissolved gases in nutrient me-
for data analysis and control of the process, spe- dia. Devices have been developed which analyze
cial sensors, which differ somewhat from those up to eight gases in the fermentation simulta-
in the chemical industry, have been developed neously.
for bioreactors: 1. All sensors located in the sterile Equipment for making accurate pH measure-
area must be sterilizable. 2. Some sensors must ments is readily available. Combination elec-
be specifically adapted to biochemical needs. The trodes (glass electrode, reference electrode, and
physical and chemical parameters listed in Table temperature compensator in a single unit) are
5.22 can either be measured directly at many available which are able to withstand steriliza-
pilot plant or production fermenters or can be tion temperatures, pressures, and mechanical
measured off-line in the laboratory. stresses. Response time and sensitivity of these
The biological parameters listed must all be electrodes is satisfactory for the usual fermen-
measured outside of the fermenter, with the ex- tation requirements. However, although elec-
ception of the NADH, measurement, which can trodes are available for measuring many other
be done on-line using a fluorescent method. inorganic ions, they are not as sensitive as those
There are interesting developments in the field for the hydrogen ion (see the paper by Fiechter
of enzymatic electrodes, so-called biosensors et al., 1987).
(see Section 11.11). Such sensors, however, can- CO, electrodes and oxygen electrodes have
not be sterilized. been used in commercial operations with varying
A standard procedure is to determine O, and success. The electrodes are of the amperometric
CO, in the ingoing and outgoing air separately type (galvanic or polarographic).
through the paramagnetic property of O, and the The current and not the voltage is altered by
infrared absorption of CO,. Sensors for measur- the oxygen concentration. In the polarographic
 5.10 USE OF COMPUTERS / 107

electrode, oxygen is reduced at the cathode and temperature, pressure, viscosity, fermenter
silver is oxidized at the anode: weight, power uptake, aeration rate, and O, and
CO, content in the gas stream. Other data can
Cathode (Pt) 0,+2H,0+4e—>40H be obtained from laboratory measurements and
Anode (Ag) 4 Ag+4 CI >4AsCl+4e
fed into the computer off-line, e.g. biomass con-
centration, nutrient content, metabolite forma-
Overall reaction 4 Ag+ O, +2H,O+4 Cl —4 AgCl+4OH™ tion. This information can be entered as raw data
and can be converted by the computer to stan-
dard units; for example, to adjust volumes for a
The current produced in this reaction is propor-
standard temperature, temperature-correction
tional to the oxygen partial pressure.
data can be used to calculate the true aeration
Commercial oxygen electrodes suitable for
rate for a production system.
fermentation processes are widely available.
An alarm system can be hooked up to the
However, the durability of these electrodes var-
data-acquisition system to inform the attendant
ies. Failure rates after sterilization lie above 50%,
when deviations from standard values occur.
depending on the nutrient medium.
Data about the course of fermentation can be
stored, retrieved, and printed out and product
5.10 USE OF COMPUTERS calculations can be documented.

Computers can serve a variety of functions in

Data analysis The data entered or measured is
fermentation process control and analysis:
used in calculations such as CO, formation rate,
1. Optimization via computer. Computers are O, uptake rate, respiratory quotient, specific sub-
used in scale up to store and evaluate fer- strate uptake rate, yield coefficient, heat balance,
mentation parameters and to measure the ef- productivity, volume-specific energy uptake, and
fects of individual parameters on the meta- Reynold’s number. When biomass is not contin-
bolic behavior of cultures.
2. Control via computer. Computers can con- A
trol fermentation processes. On-line fermen- 8 4
tation control is widely used in the production
scale in many companies. ee .
6 4 e
Computer applications in biotechnology are
not yet as widespread as in the chemical industry Le)
a= YoXo = 2g x
for several reasons: sensors suitable for use in Ø 2 g 0,

sterile systems are not yet reliable enough to take a 01290

=
fo}
Miva
x gXh
;
advantage of computer capacity; biosynthesis
jaa)
and regulation of metabolite formation are not pan
yet fully understood; fermentation cost reduction
by using computers is difficult to calculate.
Thus, in biotechnology, computers are used
T = TE GE
primarily for data acquisition, data analysis, and 8 16 24 a2 40

development of fermentation models. Fermentation time (hr)

Figure 5.40 Comparison of experimentally determined

Data acquisition Data can be acquired directly Thermoactinomyces sp. concentrations (®) with growth
at the fermenter with on-line sensors. The in- curve generated from model calculations (Zabrieskie,
formation acquired can be data such as pH, pO,, 1976)
108 / CHAPTER 5 / METHODS OF FERMENTATION

200 h

220h
230h

240 h

250 h

Production
Figure 5.41 Isoproduction and iso-
time curves for the erythromycin pro-
duction process in relation to tem-
perature and pH. The percentages are
6.4 6.6 6.8 7.0 WA based on the lowest yields obtained
pH (Cherny and Durand, 1979)

uously measured, the biomass concentration can faster and more cost-effectively. The use of
be calculated through the O, uptake rate. It is models can also aid the development of better
assumed that the yield constant and the propor- control strategies for fermentations. A large num-
tion of O, needed for maintenance metabolism ber of models exists for batch and continuous
are known. The calculations must be adjusted if, fermentations. However, each model is only ap-
for example, secondary metabolites are formed plicable to a specific process and cannot be used
or the yield constant changes during the fer- for other processes.
mentation. Figure 5.40 shows the agreement be-
tween the measured biomass of Thermoactino-
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M is the amount for maintenance metabolism. Abbott, B.J. and A. Clamen. 1973. The relationship of sub-
As a further example of data analysis, the strate, growth rate, and maintenance coefficient to sin-
gle-cell protein production. Biotechnol. Bioeng. 15:
optimization of erythromycin biosynthesis is il- 117127:
lustrated in Figure 5.41. After fermentation at dif- Anderson, C., J. Longton, C. Maddix, G.W. Scammell and
ferent pH and temperature levels, ”isoproduc- G.L. Solomons. 1975. The growth of microfungi on
carbohydrates, pp. 314-329. In: Tannenbaum, S.R. and
tion” and ”isotime curves” are computed. The D.I.C. Wang (eds.), Single cell protein, vol. II. MIT
yields are given as percentage of the minor pro- Press, Cambridge, MA.
duction compared with the maximum erythro- Bailey, J.E. and D.F. Ollis. 1977. Biochemical engineering
fundamentals. McGraw-Hill Book Comp., New York.
mycin titer. Optimal productivity (production/ Bartholomew, W.H. 1960. Scale-up of submerged fer-
fermentation time) for a given set of operating mentation. Adv. Appl. Microbiol. 2:289-300.
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amination of some geometric parameters of impeller
power. I.A.E.C. Process design and development.
Development of fermentation models By using 2:310-314.
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Brauer, H. (editor). 1985. Biotechnology, Volume 2. Fun-
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»
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Bronn, W.K. . 1966. Sauerstoffbedarf aerober Mikroorgan-" Gaden, E.L. 1959. Fermentation process kinetics. J.
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gen requirement of aerobic microorganisms and meas- Gekas, V.C. 1986. Artificial membranes as carriers for the
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Brown, D.E. 1970. Aeration in the submerged culture of technologische Prozesse in Ruhrfermentern (Basic con-
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Ribbons (eds.), Methods in microbiology, vol. 2. Ac- Crueger, W., K. Esser, P. Prave, M. Schlingmann, R.
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Charles, M. 1978. Technical aspects of the rheological nologie 1986/87. Hanser Publishers, Munich.
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Cherny, A. and A. Durand. 1979. Optimization of eryth- Terui, G. (ed.), Fermentation technology today. Ya-
romycin biosynthesis by controlling pH and temper- mada-Kami, Osaka.
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Biotechnol. Bioeng. Symp. 9:303-320. in bioprocesses. pp. 5-56 In: Moo-Young, M. (editor).
Cooper, C.M., G.A. Fernstrom, and S.A. Miller. 1944. Per- Comprehensive Biotechnology, Volume 2. Pergamon
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Chem. 36:504-509. Kim, J.H.,J.M. Lebeault, and M. Reuss. 1983. Comparative
Crueger, W. 1973. Beliiftungsfilter in der Fermentation, studies on rheological properties of mycelial broth in
Fragen zur Bakteriophagen-Dichtigkeit (Air filters in filamentous and pelleted forms. Appl. Microbiol. Bio-
fermentation; resistance to passage of bacteriophage technol. 18:11-16.
particles), pp. 181-186. In: Dellweg, H. (ed.), 3rd Laine, B.M. and J. du Chaffaut. 1975. Gas-oil as a substrate
Symp. Technische Mikrobiologie. Verlag Versuchs- for single-cell protein production, pp. 424-437. In:
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Crueger, W., H.-J. Henzler, and M. Schedel. 1982. Scale protein, vol. II. MIT Press, Cambridge, MA.
up of nikkomycin. 13th International Congress of Mi- Lengyel, Z.L. and L. Nyiri. 1966. Studies on automatically
crobiology, Boston. aerated biosynthetic processes II. Occurrence and elim-
European Federation of Biochemistry. 1983. European ination of CO, during penicillin biosynthesis. Biotech-
Federation of Biochemistry Working Party of Immo- nol. Bioeng. 14:337-352.
bilized Biocatalysts. ‘Guidelines for the characteriza- Lin, S.H. 1979. Residence time distribution of flow in a
tion of immobilized biocatalysts.” Enzyme Microb. continuous sterilisation process. Proc. Biochem. July:
Technol. 5:304-307. 23-27
Egerer, P., W. Crueger, and G. Schmidt-Kastner. 1985. Luong, J.H.T. and B. Volesky. 1983. Heat evolution during
Continuous technique for the enzymatic production of the microbial process—estimation, measurement, and
isomaltulose. German Patent 3528752. applications. Advances in Biochem. Engineering /Bio-
Einsele, A. 1978. Scaling-up of bioreactors. Proc. Biochem. technology 28:1—40.
July: 1-14. Malek, I., J. Ricica, and J. Votruba. 1984. Continuous cul-
Fiechter, A., M. Meiners, and D.A. Sukatsch. 1987. Biol- tivation of microorganisms—ecological significance of
ogische Regulation und Prozessfiihrung (Biological physiological state studies. In: Dean, A.C.R., D.C. Ell-
regulation and process control). pp. 189-228 In: Prave, wood, and C.G.T. Evans (editors). Continuous culture
P., U. Faust, W. Sittig, and D.A. Sukatsch (editors). 8: Biotechnology, Medicine, and Environment. Ellis
Handbuch der Biotechnologie. Oldenbourg Publishers, Horwood Publishers, Chichester.
Munich. Mateles, R.J. 1971. Calculation of the oxygen required for
Fox, R.I. 1984. Computers and microprocessors in indus- cell production. Biotechnol. Bioeng. 13:581-582.
trial fermentation. pp. 125-174 In: Wiseman, A. (edi- Maxon, W.D. 1959. Aeration-agitation studies on the no-
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Fukuda, H., Y. Sumino, and T. Kanzaki. 1968. Scale-up Michel, B.J. and S.A. Miller. 1962. Power requirements of
of fermentors. I. Modified equations for volumetric ox- gas-liquid agitated systems. A.I.Ch.E. (Amer. Inst.
ygen transfer coefficient. J. Ferment. Technol. 46:829- Chem. Eng.) Journal. 8:262-266.
837. Miura, Y. 1976. Transfer of oxygen and scale-up in sub-
Gaden, E.L. 1955. Fermentation kinetics and productivity. merged aerobic fermentation. Adv. Biochem. Eng. 4:3-
Chem. Ind. 154-159. 40.
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Monod, J. 1942. Recherches sur la croissance des cultures Tsao, G.T. 1979. Elementary principles of microbial re-
bacteriennes. Herrman and Cie, Paris. action engineering, pp. 223-241. In: Peppler, H.J. and
Moo-Young, M. and H.W. Blanch. 1981. Design of bio- D. Perlman (eds.), Microbial technology, vol. II. Aca-
chemical reactors. Mass transfer criteria for simple and demic Press, New York.
complex systems. Adv. Biochem. Engineering 19: 1— Tsao, G.T., A. Mukerjee, and Y.Y. Lee. 1972. Gas-liquid-
69. cell oxygen transfer in fermentation, pp. 65-71. In:
Oldshue, J.Y. 1966. Fermentation mixing scale-up tech- Terui, G. (ed.), Fermentation technology today. Ya-
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Perkowski, C.A. 1983. Fermentation process air filtration Wallhauser, K.H. (editor). 1984. Praxis der Sterilisation,
via cartridge filters. Biotechnol. Bioeng. 25:1215-1221. Desinfektion—Konservierung, Keimidentifizierung—
Prokop, A. and A.E. Humphrey. 1970. Kinetics of disin- Betriebshygiene (Practical aspects of sterilization, dis-
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using immobilized whole cells. Enzyme Microb. Tech- and geometry parameters on the behavior of non-
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Strohm, J., R.F. Dale, and HJ. Peppler. 1959. Polaro- of Penicillium chrysogenum pellet suspensions. Appl.
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Taguchi, H., T. Imanaka, S. Teramoto, M. Takatsu, and in microbial processes. Advances in Biochem. Eng./
M. Sato. 1968. Endomyces sp. glucoamylase. Scale-up Biotechnology 30:147-184.
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Tempest, D.W. and O.M. Neijessel. 1984. The status of nent balancing and culture fluorescence. In: Armiger,
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486. nology. Academic Press, New York.
 Product
recovery

specific purification processes had to be perfected

6.1 INTRODUCTION
for biological materials. The bioreactor is no
One of the most critical aspects of an industrial longer considered in an isolated manner from the
fermentation process is the recovery and purifi- apparatus used in purification. Today, the bio-
cation of the product. The selection of the ap- reactor and the purification techniques are con-
propriate purification steps depends on the na- sidered as an integrated system, all components
ture of the end product, its concentration, the side of which must be optimized together.
products present, the stability of the biological Further, the work of the biochemical engineer
material, and the necessary degree of purifica- has been considerably expanded by the devel-
tion. opment of processes for expressing foreign genes
When discussing product recovery, one must of eucaryotes in procaryotes. As discussed in
distinguish between the concepts of purification Chapter 3, eucaryotic gene products are often
and concentration. A recovery step changes formed in procaryotes in the form of highly in-
either the purity or the concentration of a me- soluble inclusion bodies, frequently in inactive
tabolite. In the ideal recovery process, both of granules held together by disulfide bridges. Such
these parameters are optimized. In the early days inclusion bodies must be released and purified
of industrial microbiology, the techniques used and eventually converted into active proteins.
in product recovery (for instance, extraction, dis-
tillation, dialysis, crystallization, precipitation,
6.2 UNIT OPERATIONS IN PRODUCT
drying) were taken over more or less directly
RECOVERY
from chemical engineering without more than an
approximate attempt to adapt them to biological The microorganism itself can be the desired end
materials. Gradually it came to be realized that product, as, for example, the ICI Pruteen process

111
112 / CHAPTER 6 / PRODUCT RECOVERY

for the production of biomass as single-cell pro-

Flocculation and flotation
tein (SCP). In this case, if the cells are heated
they aggregate into large clumps which can be Single cells in the size range of 1 to 10 um settle
readily separated from the fermentation broth by only very slowly and are difficult to bring down
sedimentation. However, in most large-scale pro- even with the centrifuge. In some cases (for ex-
cesses, the desired product is a metabolite, which ample, SCP production and sewage treatment),
is present either intracellularly or extracellularly. flocculation can be used to produce large aggre-
Examples of intracellular metabolitles include gates which will settle more readily. In most
nucleic acids, vitamins, enzymes, and certain cases, a flocculating agent is added, such as an
antibiotics, such as sisomicin and griseofulvin. inorganic salt, an organic polyelectrolyte, or a
Examples of extracellular metabolites include mineral hydrocolloid. Depending on the agent
amino acids, citric acid, alcohol, some enzymes used, the flocculation process can be either re-
(for example, amylases and proteases) and most versible or irreversible. The flocculation process
antibiotics (for example, penicillin and strepto- is also influenced by the nature of the cells and
mycin). In a few cases, metabolites are found the ionic constituents and their concentrations.
both in the cells and the culture filtrate (for ex- The reverse of flocculation is flotation, which
ample, flavomycin, vitamin B,,). is most readily accomplished by introducing gas
The first step in product recovery is therefore into the liquid. The cells become adsorbed to the
the separation of cell biomass and insoluble nu- gas bubbles and rise to the foam layer at the top
trient ingredients from the supernatant. For this of the vessel, where they can be collected and
purpose, several methods are available, includ- removed from the bioreactor.
ing flocculation, flotation, filtration, or centrifu-
gation. If an intracellular metabolite is to be iso- Filter systems
lated, it must be liberated from the cells.
Once the metabolite has been separated from Most commonly, the first step in process recov-
the cells, the selection of further purification
ery, separation of the biomass and the culture
steps will depend upon the desired product. Fig- filtrate, is carried out by some sort of filtration
process. Two major types of filters are so-called
ure 6.1 summarizes various purification pro-
cesses, arranged according to the separation prin- depth filters and absolute filters. A depth filter
is constructed of a filamentous matrix, such as
ciple for particles of various sizes. The last stages
glass wool, filter paper, or asbestos, the filtration
in process recovery involve precipitation, crys-
process occurring because the particles to be re-
tallization, and/or drying.
moved are trapped within the matrix. The par-
Fermentation products are present in culture
ticles being filtered are often smaller than the
solutions primarily at quite low concentrations
spaces in the filter, but are removed anyway as
(Table 6.1). In order to keep costs down, it is
they pass through the contorted interstices of the
desirable in the very first stages of purification
filter. Absolute filters, on the other hand, are
to reduce the volume to the smallest possible.
membranes, in which the pore sizes are smaller
than the particles being filtered. The particles are
Table 6.1 Concentrations of some metabolites at the therefore removed on the surfaces of the filters.
end of fermentation
Filamentous fungi are most commonly filtered
Metabolite Yield (g/1) through depth filters such as cloth, sometimes in
Vitamin B,, 0.06 the presence of a filter aid. Bacterial cultures, on
Riboflavin 0.1 - 7 the other hand, must be filtered with absolute
Antibiotics 0.2 — 60
Amino acids 2 —100
filters. In either case, the efficiency of filtration
is influenced by numerous factors, such as the
 6.2 UNIT OPERATIONS IN PRODUCT RECOVERY / 113

Separation princip le
err racens A}
embrane filter Depth filter
ae ees
Ultrafiltration
FEER cee
Gel chromatography

Dialysis
ERE ERE
Electrodialysis
he, kat

Vapor pressure Distillation, Freeze drying

re ed
Solubility Solvent extraction

==
Ultracentrifugation
Flota tion

Centrifugation
Weight Re 4
Counter fluid flow
ele 4
Sedimentation
re
serende 5
=f} -3 =2 -1 0 1 2 3
Separation range 10 10 10 10 10 10 10 10 pm

Figure 6.1 Concentration processes using different chemical and physical properties of particles and molecules

size of the organism, its morphology, the pH, maintain the efficiency of filtration, an automatic
viscosity, presence of slimes, temperature, and knife is used to continuously scrape the biomass
presence of other organisms as possible contam- plus a thin layer of the filter aid from the filter.
inants. As the cell material piles up on the filter, By arranging the drum in segments, the filter aid
a filter cake is formed which reduces the filtration can be washed as the drum turns. Typical filtra-
rate. tion efficiencies for arrangements of this type are
. In the classical purification process used in shown in Table 6.2. Vacuum drum filters are es-
the antibiotic industry, biomass is separated from pecially good for suspensions which contain a
the culture filtrate on a vacuum rotating drum high concentration of solids (20-60% mycelial
filter. A cloth or rope is wound around the out- volume).
side of the apparatus and a vacuum is pulled Another type of filter, better suited for so-
from the inside of the drum. To increase the ef- lutions in which the solid concentration is lower,
ficiency of the filtration process, a filter aid such is the filter press, in which a stack of flat porous
as diatomaceous earth is generally used. To plates are used as supports for a filter, either cloth
114 / CHAPTER 6 / PRODUCT RECOVERY

Table 6.2 Typical filtration rates for antibiotic Depending on the sizes of particles being fil-
fermentations
tered, three major types of filtration process are
Antibiotic Organism Filtration rate recognized: reverse osmosis, for particles of
1/h:ft?
0.0001-0.001 um; ultrafiltration, for particles of
Penicillin G P. chrysogenum 130-170 0.001-0.1 um; and microfiltration, for particles of
Kanamycin S. kanamyceticus 8
Lincomycin S. lincolnensis 35 0.1-10 um. The capabilities of the ultrafiltration
Neomycin S. fradiae 12 and reverse osmosis processes are generally
(Belter, 1985) given in terms of the nominal molecular weights
of substances separated (molecular-weight cut-
off). Reverse osmosis generally involves sub-
or membrane. The volume of the chamber within stances of molecular weight less than 1000, ul-
the plates determines the biomass capacity of this
trafiltration molecular weights greater than 1000.
The microfiltration process concerns mainly
type of filter.
the separation of cells or cell fractions. Mem-
Although mycelium (from either fungi or ac-
branes made of cellulose esters, polyvinylfluor-
tinomycetes) can be removed with a drum filter,
ides, polycarbonates, polysulfones, and cellulose
single-celled bacteria are generally separated
are used. Membranes are arranged either in cas-
from the medium by use of membrane filters.
ettes, as spiral-wound modules, as bundles of
Two types of membrane filtration processes,
tubes of 1-2 cm diameter, or as capillary bundles
static and cross-flow, are compared in Figure 6.2.
(Figure 6.3). Numerous parameters must be con-
Because the filtration process is strictly depen- sidered when selecting a filtration system.
dent on the size of the pores and the size of the Among these are the pore size and particle se-
particles, clogging of filters is a problem. The lectivity, the rate at which fluid can be passed
cross-flow method was developed in order to re- through when viscous solutions are being fil-
duce the tendency for clogging. In this method, tered, the ease with which the filter can be
the solution is pumped in a crosswise fashion cleaned, and the number of times the filter can
across the membrane. The filtrate passes through be reused. Other factors to consider are the cost
the membrane and the biomass is washed off the of the filter system, the membrane surface area
filter and carried out with the retentate. With the presented, the dead volume within the filter
cross-flow method, an increase in filtration rate (which determines how much of the product will
of 100-fold can be obtained in comparison to be lost), and the sterilizability of the system. An-
static filtration. other factor that often reduces the efficiency of

Culture solution

o °
—f O °
° °
Culturem—— o Ona o >, ™ Concentrate
—f 0 ° o © °°
Membrane
— Filtrate

5 Filtrate

Figure 6.2 Comparison of static and

Static Filtration Cross-flow filtration cross-flow filtration
 6.2 UNIT OPERATIONS IN PRODUCT RECOVERY VÆRE Ka)

Retentate input Permeate

Retentate exit
Filtrate

Permeate
Retentate input
Retentate exit

Figure 6.3 Comparison of a casette system and a cross-flow cartridge module

a filter is the presence of antifoam agents, which cess itself. They can function in providing sterile
are often used in large-scale fermentations. air, and they can be used in other ways in the
The following data are typical for an indus- fermenter (Figure 6.4).
trial process for the concentration of Escherichia
coli from a culture broth: fermenter volume, 6
Centrifugation
må; working volume, 4 m3; filtration rate, 25—80
1/h-m2; process time, 12 h; concentration factor, The sedimentation rate of a particle in a gravi-
1:10. Table 6.3 presents data for filtration pro- tational field can be represented by Stoke’s law:
cesses with several organisms with various types
of filters. eres d-(5,—8)
ENS aU ae
18n “
The following are typical costs that must be
incurred for filtration in a large-scale industrial where v = the sedimentation rate (m/s), d =
process: Production of 1 m? retentate: mem- the diameter of the particle (m), 6, = the density
branes ($420), capital investment ($1600), labor of the particle (kg/m), 6, = the density of the
($70), energy ($230). Production of 1 m: filtrate: liquid (kg/m), r = the radius of the centrifuge
membranes ($14), capital investment ($60), labor head (m), w = the angular velocity (rad/s), and
($3), energy ($8). n = the dynamic viscosity (Pa’s).
In addition to their use in filtration, mem- Centrifugation is used not only for separating
branes also play a role in the fermentation pro- solid particles from the liquid phase (fluid/par-
 116 / CHAPTER 6 / PRODUCT RECOVERY

Table 6.3 Concentration of microorganisms by use of ticle separation) but also for fluid/fluid and
cross-flow filtration
fluid/fluid/particle separation. Fluid/particle
Organism Type of Concentra- Average separation is of most significance, although
membrane tion flow
% cell wet rate fluid/fluid separation is used in penicillin pro-
volume 1/h-m2 duction (for the separation of the antibiotic-ex-
Bacillus cer- Capillary tubes 10 — 30 124 tracting solvent from the aqueous phase by
eus polypropylene means of a two-stage continuous countercurrent
0.3 um extractor; see Section 13.2).
B. cereus Capillary tubes 0.8 — 15 47
polysulfone Two distinct types of centrifuges are used,
10° dalton filter- and sieve centrifuges, and baffle centri-
Brevibacter- Casette Sh) oY Sy fuges. In the filter- or sieve centrifuge the sep-
ium sp. polyacryl
0.2 wm
aration occurs as the particles are forced against
Escherichia Capillary tubes 4.0 — 40 15 a filter material. In the baffle centrifuge the sep-
coli polysulfone aration occurs because of density difference be-
10° dalton
E. coli Casette 4.2 — 48 16 tweeen the particles and the liquid. The product
PVDF can be removed either continuously or batch-
0.45 wm wise. A wide variety of centrifuges are marketed
Candida boi- Capillary tubes 10 — 40 56
dinii polypropylene for large-scale centrifugation processes, and the
0.45 um capabilities of the major types are given in Table
Klebsiella Capillary tubes 2 — 58 73 6.4. In the case of yeast, machines capable of
pneumoniae polycarbonate
2 X 10 dalton handling volumes as large as 300 m°/h have
Lactobacillus Cassette 1.5 — 28 28 been developed.
casei PVDF Figure 6.5 shows cross-sectional diagrams of
0.45 wm
two major types of baffle centrifuges, a plate sep-
Kroner et al. (1984) arator and a decanter. The major considerations

/ Sterile nutrient solution

Sampling for analysis

Substrate
Removal of inhibitors
return feed

Biomass
return feed
Enzyme return feed

To concentration stage
Dialysis filtration

To concentration stage

Figure 6.4 Various locations where

Removal of membrane systems can be used in a
residual cells bioreactor unit
 6.2 UNIT OPERATIONS IN PRODUCT RECOVERY / 117

in the selection of a centrifuge for biotechnol- For use in the pharmaceutical industry, sep-
ogical processes depend on the task at hand. For arators are available that are capable of with-
fluid/particle separation, factors to consider are: standing temperatures of 121°C and hence can
1) the needed purity of the fluid phase; 2) the be completely sterilized. An example of such a
needed recovery of the fluid phase; 3) the needed machine is shown in Figure 6.6.
recovery of the particle phase; 4) the needed per-
missible moisture content of the particles; 5) the Disintegration of microorganisms
specific density of the particles. For fluid/fluid
separation, the major factors to consider are: 1) In some cases, the recovery of product requires
the needed purity of the lighter or heavier liquid; that the microorganisms be fragmented, either by
and 2) the needed recovery of the lighter or heav- chemical, physical, or biological means. A sum-
ier phase. mary of methods for disintegrating microorga-
nisms is given in Figure 6.7. The selection of a
method depends principally on the nature of the
Table 6.4 Comparison of a separator, a decanter, and
a tube centrifuge cells. Although the cell membrane offers no spe-
cial resistance to breakage, cell walls vary widely
Parameter Separator Decanter Tube
centrifuge in how readily they are broken. Gram-positive
bacteria and yeasts are much more difficult to
Solids, % 1-30 5-80 1-5
Maximal 5000-15,000 1500-4500 13,000- break than gram-negative bacteria or filamentous
centrifugal force 17,000 fungi. Even within a given organism, cells vary
(8) significantly in sensitivity to breakage depending
Dewatering Average Average Good
capacity
upon their physiological state. It is also impor-
Removal of small Good Moderate Very good tant, when selecting a disintegration method,
particles that the target biomolecule not be destroyed. For
Cleanability Good Good Very good
instance, acid can be used to break many orga-

d i n a a s e s ; es 135 FR rs

|=
‘i :

Figure 6.5 Construction details for a decanter (left) and a separator. 1 = inflowing stream; 2 = removal of particles;
screw; 6 = fluid level; 7 =
3 = zone of low water content; 4 = particle sedimentation and drum cover; 5 = decanter
clear liquid effluent; 8 = drive shaft
118 / CHAPTER 6 / PRODUCT RECOVERY

through the action of glass beads. The cells and

the glass beads are mixed together and subjected
to high-speed mixing in a reaction vessel. Break-
age occurs when a cell is forced against the wall
of the vessel by a glass bead. The efficiency of
the process depends on the following param-
eters: 1) the cell concentration (optimal at about
40% cell wet volume); 2) rate of flow through
the mill; 3) orientation of the apparatus (hori-
zontal or vertical); 4) the cell/glass bead ratio; 5)
the size of the glass beads (generally 0.5-0.8
mm). The procedure must, of course, be optim-
ized for the particular process. One can expect a
maximal breakage rate of about 80% of the cells.
Another approach is the use of homogeniz-
ers. In such devices, the biological material is
placed under a high hydrostatic pressure (around
500 atmospheres) and the pressure suddenly re-
leased by allowing the liquid to exit through a
valve. A cross section of the valve arrangement
is shown in Figure 6.8. The operating conditions
must be carefully optimized for each situation.
Depending on the organism and the desired me-
Figure 6.6 Sterilizable separator. This is type BTPX 20s tabolite, from 10-60% of the cells can be broken.
manufactured by Alfa Laval. It contains 80 plates, an in- In order to achieve 90% breakage, two or three
terior volume of 3.1 liter, a maximal centrifugal force of passes of the material through the homogenizer
12,800 X gravity, a maximal rotation of 9,650 revolutions
per minute, a throughput for biotechnological processes must be carried out. As seen in Figure 6.9, the
of around 400-500 liters/hour. efficiency of the process depends on the applied
pressure. In general, the best pressure to use is
between 450 and 600 atmospheres.
nisms but cannot be used if the product is acid
labile.
Chromatography
For the extraction of yeast cells, autolysis is
often used. Endogenous autolytic enzymes of the Some of the most expensive stages in product
yeast cells themselves bring about the destruc- recovery involve the use of chromatographic
tion of the cell wall. Addition of a sodium chlo- methods. Such methods are especially value for
ride, ethyl acetate, or chloroform, and incubation the purification of biologically sensitive materials
for 24 h at 45°C increases the rate of the autolytic and find widespread use in the preparation of
process. Another technique is plasmolysis, fol- pharmaceuticals, diagnostic reagents, and re-
lowing the addition of a high concentration of search materials. In many cases, several separate
NaCl. Ultrasonic disintegration is widely used types of chromatographic methods must be used,
in the laboratory, but because of the high cost it one following the other (Figure 6.10). Separation
is not suitable for large-scale industrial processes. generally occurs in a column: a stationary phase
Industrially the most widely used methods of (generally a resin) is used to adsorb the product
cell disintegration involve mechanical action. which is then eluted with a mobile (liquid) phase.
Ball mills bring about the breakage of cells The elution liquid can be at a single density or
 6.2 UNIT OPERATIONS IN PRODUCT RECOVERY / 119

Microbial cell breakage

Physical methods Chemical methods Biological methods

-

Liquid medium | Solid medium ] Lyophilization Detergents Cell-wall digesting

ST enzymes

Ultrasound Presses Osmotic pres- Extraction

Gaulin/Manton sure change with
French press acetone/toluene’

Freeze/Thaw

Mills Agents which

Dyno mill inhibit cell wall
Colloid mill synthesis

[Treatment with acid]

Ball mill Presses

X Press
Hughes press

Figure 6.7 Methods of opening cells to isolate microbial metabolites

a gradually changing density (density gradient) are various chromatographic methods that are
can be used. By use of a suitable detector at the widely used.
exit of the column, the eluted fractions which
contain the desired product can be determined. Gel filtration (molecular sieve chromatography)
Suitable detectors use ultraviolet or visible light Molecules of different sizes can be separated by
absorption or conductivity. Summarized below passage through gels with different pore sizes.
Large molecules do not penetrate the gel and
pass directly through the column, exiting with
Broken cells the elution front. Small molecules penetrate
more or less deeply into the gel and their mobility
is hence retarded from the solvent front. Stan-
dard curves can be developed which relate mo-
lecular weight to the elution position. At the in-
Input of cell dustrial scale, gel filtration is used mainly to
concentrate
remove salts and to separate low-molecular-
weight impurities. At a smaller scale, gel filtration
is used to fractionate and purify protein mole-
cules (for instance, insulin or interferon). The
most widely used gels for gel filtration are the
Biogel types P and A from BioRad and the Seph-
Figure 6.8 Outlet valve for a homogenzier adex series S, G, and LH from Pharmacia.
120 / CHAPTER 6 / PRODUCT RECOVERY

Ion exchange chromatography Macromolecules

with ionic groups can be separated by ion ex-
00
==
change chromatogra phy. The macromolec ule ad-
2 560 bar sorbs to the carrier and is eluted by a solution of
—A—S—_ 470 bar
the ionic
80 De es defined ionic strength. Depending on
We nature of the molecule and the ionic strength of
—*— 300 bar the eluting solution, the separation can be quite
60 ete specific. Ion exchange chromatography finds

i=
wide use for purification of antibiotics from fer-
om 200 bar
—
mentation broths. It also is widely used in large-
40 scale purification of proteins. The most widely
used ion materials for cation exchange are Dowex

"a Er
e— 100 bar
HCR and OCR, Amberlite IR and IRC, and Le-
20
Se gl watit S. Anion exchangers used are Dowex SAR
and MSA, Amberlite IRA, and Lewatit M.
cells)
protein/g
(mg
release
Protein
Affinity chromatography Affinity chromatogra-
13203 SEERE phy is an absolutely specific method for purifi-
Number of treatments cation of biological materials. The desired ma-
terial binds specifically and reversibly to a ligand
Figure 6.9 Protein release from yeast cells with a pres- which has been fixed to an inert carrier. For ex-
sure homogenizer (Whitworth, 1974) ample, nucleotide adenine dinucleotide (NAD)
can be purified by allowing it to bind to a carrier
containing a dehydrogenase enzyme. The anti-
Adsorption chromatography In adsorption chro- biotic bacitracin can be used as a ligand to isolate
matography, separation involves hydrophilic or Asp-, Ser-, Cys- and metalloproteases from a
hydrophobic interactions (van der Waals forces) crude mixture.
between the carrier and the biological material.
Elution and fractionation are accomplished by Isoelectric focussing By means of isoelectric fo-
means of solutions of higher or lower polarity or cussing, proteins can be purified by use of pH
ionic strength. Adsorption materials include sil- gradients in association with ion exchange gels.
icates, alumina, activated carbon, cross-linked A linear pH gradient is constructed and the pro-
dextrans, and hydroxylapatite. teins are separated according to their isoelectric

Culture solution Lewatite SC 104

Lewatite MP 62

Lewatite S 100 Lewatite MP 62 Lewatite TSW 40

Cationic deionization pH value > 3.0 Column chromatography

Lewatite M 600 Lewatite S100 and M600 sele filtration Acarbose

pH 5.0 Deionization, pH 6.0 Drying (52% yield)

Figure 6.10 Flow diagram for the purification of a particular product, the invertase inhibitor Acarbose® (From Rauen-
busch and Schmidt, 1978).
 6.2 UNIT OPERATIONS IN PRODUCT RECOVERY / 121

points. The separation of proteins by this method

is very good, but only small volumes can be han- Drying
dled. i For biological materials, it is essential to dry the
final product in such a way that its activity is not
lost. Drying essentially involves transfer of heat
Extraction to the wet product, and removal of the moisture
Many products can be purified by use of solvent in a stream of gas. Heat transfer can be either by
extraction. A two-phase system is set up, using direct contact, by convection, or by radiation.
a solvent which is immiscible with the aqueous Numerous types of apparatus are commercially
fermentation broth. After the desired product is available for carrying out the drying process.
concentrated in the solvent phase, it can be fur- They include convection dryers (such as pulver-
ther purified. Ideally, the product will transfer izing, rotating, and band dryers) and contact
quantitatively to the solvent phase. Solvent ex- dryers (such as thin layer and chamber dryers).
traction has been widely used in the antibiotic In some industries, the final product is a liv-
industry, for antibiotics such as penicillin, using ing microbial culture (for instance, starter cul-
organic solvents such as amyl or butyl] acetate. tures in the dairy industry). To prepare dry cul-
For the purification of enzymes in the active tures, the freeze-dry technique must be used,
state, organic solvents cannot be used, but two- since in this way the living organism is never
phase aqueous systems can be used, prepared in subjected to heating. Freeze drying is also used
a salt solution with hydrophilic polymers. In both in the preparation of some pharmaceutical prod-
phases, water is the main component, but the ucts (for instance, penicillin is freeze dried di-
two phases are not miscible. Cells remain in one rectly in ampules).
of the phases and the enzyme is transferred to
the other phase without any loss of activity. Hu- 6.3 YIELD
man fibroblast interferon can be concentrated 70-
Recovery losses depend on the sensivitity of the
fold with 100% yield using this method, at the
substance to the process and on the number of
same time that a four-fold reduction in volume
purification stages. Table 6.5 shows typical yield
is achieved.
losses. Further details will be given in the dis-
An interesting variant of this procedure is ex-
cussions of particular processes later in this book.
traction with supercritical solutions. This ap-
The fraction of the total cost attributed to pu-
proach is used in the extraction of caffeine from
rification is on the average around 20%, but in
coffee beans and hops for brewing, but can also
extreme cases with certain kinds of intracellular
be used for the extraction of pigments and flavor
metabolites can be as high as 90%.
ingredients from biological materials.
In Table ‘6.6, the data for glutamic acid pu-
rification demonstrate the extent to which recov-
ery methods must be combined and optimized.
Crystallization and precipitation
Once the metabolite has been extracted, it can Table 6.5 Recovery losses in typical fermentation
be further concentrated and purified by crystal- processes
lization, either by evaporation or by transfer to Fermentation product Recovery loss
low temperature. Low-temperature crystalliza-
tion is a very gentle way of purification. In the Single cell protein 5
case of precipitation, a chemical agent is some- Antibiotics 20-50
times added to promote the concentration reac- Extracellular enzymes 10
Intracellular enzymes 90
tion.
122 / CHAPTER 6 / PRODUCT RECOVERY

Table 6.6 Glutamic acid purification process

Process Direct crystalization Yield | Anion exchange process Yield | Cation exchange process were
% % 0
Stages of | Culture solution 100 Culture solution 100 Culture solution 100
process | Filtration 90 | Filtration 90 | Filtration 90
pH 7.5-8.0 pH 7.5-8.0 pH 7.5-8.0
Filtrate evaporation 98 Adsorbtion to anion 95 Adsorbtion to cation 98
exchanger exchanger
pH adjustment Elution with acid 98 Elution with base 95
Crystalline acid 73 Crystalline acid 90
Crystalline Na salt 88 Crystalline Na salt 93 Crystalline Na salt 93
Chemicals | 3 Equivalents of acid 3 Equivalents of acid 3 Equivalents of acid
used 1 Equivalent of NaOH 3 Equivalents of NaOH 2 Equivalents of NaOH
Total 56.6 70.0 78
yield
(Wolf, 1974)

The final yield is only one factor, since the cost Kula, M.R. 1985. Recovery operations, pp. 725-760. In:
Rehm, H.J., and G. Reed (eds.). Biotechnology. Vol. 2.
of chemicals, equipment, and wages must also
VCH Verlag, Weinheim.
be considered in the overall calculation. Kula, M.R., K.H. Kroner, and H. Hustedt. 1982. Purifica-
tion of enzymes by liquid-liquid extraction. Adv.
Biochem. Eng. Biotechnol. 24:73-118.
REFERENCES Kula, M.R., K. Schtigerl, and Ch. Wandrey (eds.). 1986.
Technische Membranen in der Biotechnologie. (Mem-
Alt, C. 1972. Filtration, pp. 154-198. In: Ullmanns En-
cyklopadie der technischen Chemie, Vol. 2. Verlag
branes for large-scale biotechnological processes.) GBF
Chemie, Weinheim. Mongraphien 8. VCH Verlag, Weinheim.
Atkinson, B., and I.S. Daoud. 1976. Microbial flocs and Mellor, J.D. 1978. Fundamentals of Freeze Drying. Aca-
flocculation in fermentation process engineering. Adv. demic Press, New York.
Biochem. Eng. Biotechnol. 4:41-124. Moo-Young, M. (ed.) 1985. Comprehensive Biotechnol-
Belter, P.A. 1985. Filtration of fermentation broths, pp. ogy. Vol. 2. The Principles of Biotechnology: Engi-
347-350. In Moo-Young, M. (ed.). Comprehensive Bio- neering Considerations. Pergamon Press, Oxford.
technology. Vol. 2. Pergamon Press, Oxford. Mullin, J.W. 1972. Crystallisation. 2nd Ed. Butterworth,
Cheryan, M. 1986. Ultrafiltration Handbook. Technomic London.
Publ., Lancaster, PA. Rauenbusch, E., and D. Schmidt. 1978. Verfahren zur Is-
Edebo, L. 1983. Disintegration of cells by extrusion under olierung von 0-(4,6-Dideoxy-4[[1S-(1,4,6/5)-4,5,6-
pressure, pp. 93-114. In: Lafferty, R.M. (ed.). Enzyme trihydroxy-3-hydroxymethyl-2-cyclohexen-1-yl]
Technology. Springer Verlag, Berlin. amino]-a-D-glucopyranosyl)-(1-4)-0-a-glucopyrano-
Egerer, P. 1986. Chromatographische Methoden in der syl-(1—4)-D-glucopyranose aus Kulturbriihen. (Tech-
Aufarbeitung von Naturstoffen (Chromatographic nique for the isolation of 0-(4,6-Dideoxy-4[[1S-(1,4,6/
methods for the purification of natural products). pp.
5)-4,5,6-trihydroxy-3-hydroxymethyl-2-cyclohexen-1-
125-187. In: Crueger, W., K. Esser, P. Prave, M.
yl] amino]-a-D-glucopyranosyl)-(1-4)-0-a-glucopyra-
Schlingmann, R. Thauer, and F. Wagner (eds.). Jahr-
buch Biotechnologie 1986/87. Hanser Verlag, Munich. nosyl-(1—4)-D-glucopyranose from culture broths.)
Fritz, J.S., D.T. Gjerde, and C. Pohlandt. 1982. Ion Chro-
DE-OS 2719912.
matography. Hiithig Verlag, Heidelberg. Scouten, W.H. (ed.). 1981. Affinity chromatography. Bio-
Hustedt, H., K.H. Kroner, N. Papamichael, and U. Menge. selective adsorption on inert matrices. J. Wiley and
1987. Verteilung zwischen wassrigen Phasen unter Sons, New York.
Mikrogravitat. (Use of microgravity in the partitioning Trawanski, H. 1972. Zentrifugen und Hydrozyklone (Cen-
between aqueous phases.) BioEngineering, 1/1987:12- trifuges and hydrocyclones), pp. 204-224. In: Ullmans
29! Encyklopadie der technischen Chemie, vol. 2. Verlag
Kroner, K.H., H. Schiitte, H. Hustedt, and M.R. Kula. 1984. Chemie, Weinheim.
Cross-flow filtration in the down stream processing of Verrall, M.S. (ed.) 1985. Discovery and Isolation of Mi-
enzymes. Process Biocem. April 67-74. crobial Products. Ellis Horwood Ltd., Chichester.
 REFERENCES / 123

Vogelpohl, A. and E.U. Schliinder. 1972. Trocknung fester ” Whitworth, D.A. 1974. Assessment of an industrial ho-
Stoffe (Drying of solids), pp. 698-721. In: Ullmans En- mogenizer for protein and enzyme solubilization from
cyklopadie der technischen Chemie, vol. 2. Verlag spent brewery yeast. Compt. Rend. Trav. Lab. Carls-
Chemie, Weinheim. berg. 40: 19-32.
Weiss, J. 1985. Handbuch der Ionenchromatographie. Wolf, F.J. 1974. Outline for fermentation technology. MIT,
VCH Verlag, Weinheim. Cambridge, MA.
 Organic
feedstocks
produced by
fermentation

Use of mixed cultures in order to catabolize

7.1 GENERAL
different substrates and to convert them into
The chemical industry makes use of a variety of the desired metabolite.
simple organic compounds as feedstocks, that is, Use of thermophilic strains to save costs for
raw materials, for synthetic processes. Due to the cooling, to bring about higher conversion
low cost of petrochemical products in the post- rates, and to reduce contamination risks.
World War II era, little attention was given to Due to the high energy demand for aeration,
studies involving the microbial production of or- anaerobic processes are preferable.
ganic feedstocks from plant substances. Since the Amenable to a continuous process.
mid-1970's, due to political and economic fac- Low recovery and concentration costs.
tors, petroleum and natural gas have become
scarce and more attention has turned to microbial 7.2 ETHANOL
production of feedstocks. Of the 180 10° tons
of plant material annually produced on earth, For thousands of years, ethanol has been pro-
only 1—2% is utilized for animal and human nu- duced for human consumption, and for at least
trition and 1% for energy and fiber production. a thousand years it has been possible to make
The requirements for a cost-effective fermen- concentrated alcoholic drinks by means of dis-
tation of organic feedstocks from plant carbo- tillation. Ethanol for use as a chemical feedstock
hydrates are as follows: was produced by fermentation in the early days
of industrial microbiology; however, for many
+ Low transportation costs of raw materials. years it has been obtained by chemical means
¢ Low costs to convert polymers (wood, cel- instead, primarily through the catalytic hydration
lulose, hemicellulose, starch) to usable mono- of ethylene. In recent years, attention has turned
and disaccharides. again to the production of ethanol for chemical

124
 7.2 ETHANOL / 125

and fuel purposes by fermentation. For example, " most widely used organism is Zymomonas mob-
in three major countries the following amounts ilis. Saccharomyces cerevisiae is the most com-
of industrial ethanol were produced by fermen- monly used yeast but Kluyveromyces fragilis has
tation in 1986: United States, 2.5 X 10? liters; also been employed.
Federal Republic of Germany, 1 X 108 liters; Bra- Under aerobic conditions and in the presence
zil, 1.1 X 10 liters. In countries with large ag- of high glucose concentrations, Saccharomyces
ricultural areas, such as Brazil, South Africa, and cerevisiae grows well, but produces no alcohol.
the United States, intensive studies are being Under anaerobic conditions, however, growth
conducted on the production of ethanol from car- slows and pyruvate from the glycolytic pathway
bohydrates such as sucrose and starch. The ob- is split with pyruvate decarboxylase into acetal-
jective of such research is to use ethanol for au- dehyde and CO, (Figure 7.1). Ethanol is then
tomobile fuel (generally mixed with gasoline). In produced from the acetaldehyde by reduction
some countries, fermentation ethanol is also used with alcohol dehydrogenase.
to produce ethylene and other petrochemicals. Batch systems for ethanol production are
The efficiency of energy conversion by started aerobically to obtain maximum biomass,
ethanol fermentation varies considerably de- since if anaerobic conditions begin too early, the
pending on the starting material. For instance, population density is not high enough to obtain
under optimal conditions the efficiency of energy a good conversion rate. Forced aeration may even
yield (ratio of energy demand to energy pro- be necessary for a short time in order to avoid
duced) is as follows: sugar beet, 86%; potatoes, yield losses. In continuous processes, optimal
59%; corn, 25%; cassava, 50%; sugar cane, 66%. yeast growth and ethanol production are carried
Ethanol is more expensive than petroleum when out under sugar limitation (< 1 g/l) and in a
used as a motor fuel but some countries, for po- microaerobic environment (0.2-5 mg O,/g dry
litical or internal economic reasons, subsidize substance-h). Theoretically, from one gram of
ethanol production. glucose, 0.511 grams of ethanol can be obtained.
In Brazil, as early as 1982, about 30% of pe- When pure substrates are fermented, the yield is
troleum imports were replaced by ethanol pro- 95% and reduces to 91% when industrial-grade
duction from sugar cane (5.2 X 10° m’), involv- starting materials are used. One hundred grams
ing 60-80 separate fermentation plants. In 1986 of pure glucose will yield 48.4 grams of ethanol,
in the United States, 65 separate fermentation 46.6 grams of CO,, 3.3 grams of glycerol, and
plants were in operation, with a total capacity of
2.6 X 10% m? per year, using corn starch as the
starting material. The capacity of these plants Glucose
varied from 20,000 to 570,000 m3. However, the |Glycolysis
total capacity was only about 25% of that which
Pyruvate
had been planned and some of the plants which
had opened were unsuccessful and closed. The Pyruvate decarboxylase
Mg2*, Thiamine pyrophosphate
main problems were lack of technical and mi-
crobiological expertise and the high price of corn
starch (which constituted 60-70% of the total Acetaldehyde + CO,
production cost). Alcohol dehydrogenase
NADH,
‘Biosynthesis of ethanol
Ethanol
Both yeasts and bacteria have been used for the
production of ethanol. Among the bacteria, the Figure 7.1 Biosynthesis of ethanol
126 / CHAPTER 7 / ORGANIC FEEDSTOCKS PRODUCED BY FERMENTATION

1.2 grams of biomass (yeast cells). If corn starch rameters for Zymomonas and Saccharomyces
is used, 100 kg starch (corresponding to 180 kg shows the following: ethanol formation rate (g/
of corn) ferments to 51.5 grams of ethanol. gh) 2.9 times higher, growth rate (u) 2.4 times
Ethanol is inhibitory at high concentrations higher, and glucose uptake rate 2.6 times higher.
and the alcohol tolerance of the yeast is critical In order to increase even more the produc-
for high yields. The ethanol tolerance of different tivity, both the bacteria and yeast processes are
strains varies considerably. As the concentration being carried out in large scale by continous fer-
of ethanol increases, the growth rate is first re- mentation. The optimal amount of cell recycling
duced, whereas at higher concentrations the bio- is under study. Maximal productivity with glu-
synthesis of ethanol itself is inhibited. However, cose as carbon source that has been reached is
yeast is more sensitive to endogenously pro- about 82 g/I-h for yeast and 120 g/1-h for Zym-
duced ethanol than to that added from external omonas. A further possibility is the use of im-
sources to the fermentation system. Growth gen- mobilized cells. In a direct comparison of yeast
erally ceases at 5% ethanol (volume ethanol in using molasses as substrate, the following results
volume water = vv) and the production rate is have been obtained: batch process, 2.0 g/l-h
reduced to zero at 6-10% (vv). Using molasses ethanol; continuous process, 3.35 g/I*h; immo-
(12% sucrose, weight per volume water = wv), bilized cells under continuous conditions, 28.6
sucrose is normally converted to 6% (vv) ethanol g/l-h. The immobilized cell process has already
within 36 hours. been placed in large-scale production in Japan.
With pure sugar solutions, the ethanol con-
centration can reach as high as 10%, although
Ethanol production process
ethanol-tolerant mutants have been isolated that
can produce 12-13% ethanol and are currently Ethanol is produced in three steps, each of which
being scaled up to full production. The bacterium must be optimized: 1) Preparation of the nutrient
Zymomonas mobilis has in recent years come un- solution; 2) fermentation; 3) distillation of
der increasing study because it has a number of ethanol. Figure 7.2 shows the stages in the pro-
potential advantages: duction of ethanol from corn meal.
e Osmotic tolerance to higher sugar concentra-
Preparation of the nutrient solution Three types
tions (up to 400 g/l).
of substrates are used in the ethanol fermenta-
Relatively higher ethanol tolerance (up to 130
tion: 1) starch-containing roots, tubers, or grains;
g/l). 2) molasses or juice from sugar cane or sugar
e Higher specific growth rate than yeast
beet; 3) wood or waste products from processed
(growth rate, u of 0.27 compared to 0.13 for
wood. The production of alcohol from milk whey
yeast; laboratory culture studies).
has been tested but is not yet in use commer-
e Anaerobic carbohydrate metabolism is car-
cially.
ried out through the Entner-Doudoroff path-
The most important root starch is derived
way, where only one mole of ATP is pro-
from the tropical plant Manihot esculenta, from
duced per mole of glucose used, thus
which cassava, manioc or tapioca flour is ob-
reducing the amount of glucose that is con-
tained. In Brazil, 28 10° tons of this plant are
verted to biomass rather than ethanol.
harvested annually.
The pH optimum of the bacterial ethanol fer- Since Saccharomyces cerevisiae has no amy-
mentation is considerably broader (pH 5-7) and lases, the starch must be hydrolysed. The roots
the temperature optimum is higher (30°C). Even (containing 20-35% starch and 1-2% protein),
at 37°C the yield of ethanol from glucose is 97% are first ground, squeezed, and dried. The starch
of theoretical. A comparison of the kinetic pa- is liquefied by boiling under pressure, and then
 7.2 ETHANOL / 127

»
Corn meal/water Molasses, a byproduct of sugar crystalliza-
oOa 15-30% solids, —— a-Amylase
pH 6.0-6.5 tion, is commonly used today as an ethanol
| -
U 7-7;
H,0
om
source, but may become less important in the
Heat (60-80°C) " future since it has better uses as animal feed or
Steam —= in other fermentations.
Cooler (heat recovery) —— When molasses is made from sugar beets, the
juice is extracted from the beet chips with hot
water. With sugar cane, the cane juice is released
Liquefaction (60-80°C)
by means of presses. The residue from the press-
ing of sugar from sugar cane stems is called ba-
:
|
|

gasse. Eighty percent of the bagasse formed can

core be burned as an energy source in the distillation

process and 20% more can be fermented after
chemical hydrolysis. In the Federal Republic of

ii
Separation =
estat} 2 Germany a hybrid plant which is a cross between
sugar beet and turnip is being developed as a
|
Distillation source of carbohydrate for ethanol production.
Wood has not yet been used in the commer-
Ethanol (94.5% wiv) cial production of ethanol. Because of the large
amount of waste wood available, the direct fer-
Figure 7.2 Steps in ethanol production from a substrate mentation of wood which has been hydrolyzed
containing starch with cellulases would be of considerable impor-
tance.
Sulfite waste liquor is another potential
hydrolysed enzymatically. By adding cellulose- source of sugar. This is formed during production
splitting enzymes of different microbial origin, of paper from conifers and contains fermentable
the proportion of reducing sugars can be in- hexoses. Sulfite waste liquor from deciduous
creased in the cassava paste. trees cannot be used for commercial processes
The starch-containing grain used in Asia is because of the high fraction of the sugar which
is in the form of pentoses rather than hexoses.
rice, in the United States mainly corn but also
millet in some areas, in Europe potatoes in ad-
Fermentation Continuous fermentations are
dition to other grains. Grain can be used whole
only slowly being brought into large-scale op-
or, in the case of corn, coarsely ground; the ker- eration. In the United States, among 10 large-
nels are soaked at 40-50°C for several hours, scale plants using yeast, four are operating con-
then steeped and liquefied. tinuously, one of which also employs cell recy-
Continuous liquefaction and saccharification cling. A few continuous plants have also been
processes using first steam injection (3 min at started in Brazil. A Danish system has been de-
150°C) and then vacuum cooling are used in scribed in detail: The nutrient solution was mo-
modern plants. Some a-amylase is added before lasses with diammonium phosphate additive.
heating in order to decrease the viscosity which The pH value was adjusted to 5 with H,SO, and
develops after the steeping process. For glucose pasteurization was then carried out. The fermen-
production, glucoamylase and a-amylases are tation temperature was 35°C and the yeast pro-
added after the heating and cooling process. Ap- duction was 10 g/l. After the fermentation, the
proximately 1 1 commercial a-amylase and 3.5 | cells were separated by centrifugation and chan-
glucoamylase are added for 1 ton of starch. neled back into the first fermenter. After 10.5
128 / CHAPTER 7 / ORGANIC FEEDSTOCKS PRODUCED BY FERMENTATION

hours, the yield in the first fermenter was 6.1%

Waste gas
(vv) alcohol with 1% residual sugar, whereas in
the second fermenter the yield was 8.4% (vv)
with 0.1% residual sugar. Mixing
In the Federal Republic of Germany, ethanol zone
production with Zymomonas mobilis is being car- Waste
liquid
ried out in two parallel bioreactors, each of which
has a volume of 70 m’. The fermentation operates
continuously but without cell recycling, using as
a starting material a starch fraction which was a
byproduct of enzymatic glucose production. The
ethanol concentration ranged from 7-8%. The
process could be operated for up to three weeks Substrate
before a lactobacillus contaminant became estab- addition

lished and the process had to be restarted from Pump and

scratch. OD homogenizer

Batch fermentation is more commonly em-

Yeast paste
ployed for ethanol production. Production is car-
ried out in batch processes (fermenter volume of
Figure 7.3 ALCO-FLOC system using air suction and cell
600 m?) with either starch hydrolysate (up to flocculation (Meyrath, 1979)
20% dry weight) or molasses, using a 3% inoc-
ulum (cell density 3X10°/ml). Within 12 hours,
Energy balance of the ethanol fermentation Since
Saccharomyces produces 10% (vv) ethanol with
ethanol is produced primarily as a fuel and en-
10-20 g cell dry weight/l when the process is
ergy source, the energy balance of the whole pro-
carried out at 35-38°C, pH 4.0—4.5; the maximal
cess determines its economic viability. A sum-
productivity was 1.9 g/l-h. The short fermen-
tation time was accomplished by use of cell re- mary of the energy requirements of the different
cycling; 80% of the cells were removed in a sep- stages in the process for ethanol production from
arator and brought back again into the fermenter. various substrates is given in Table 7.2.
When high quality molasses is used, the maximal It can be seen that the product-recovery stage,
yield is 95% of the theoretical. Table 7.1 shows the distillation of ethanol, is the single most en-
a comparison of the operating parameters of ergy-demanding step of the whole process. Be-
three different types of fermentation systems, cause of this, improvements in the distillation
batch, continuous, and continuous with cell re- process will have more impact on the success of
cycling. the process than will improvements in the fer-
Before distillation, the cell mass is separated mentation itself. If the energy yield of the ethanol
by centrifugation or sedimentation. Figure 7.3 produced is related to the total energy inputs
shows the layout of a modern system. from the various stages of the process, it is found

Table 7.1 Comparison of several kinds of fermentation systems for ethanol production
Fermentation process Sugar Cell Ethanol Specific Volume-specific
concentration concentration concentration productivity productivity
% 8/1 8/1 g/g cells-hr g/\-h
Batch 16.7 21:3 85.6 0.42 11:8
Continuous 15.6 1957 Dil 0.72 14.1
Continuous with 16.7 100 85.6 0.42 42.5
cell recycling
(Maiorella et al., 1984)
 7.3 ACETONE/BUTANOL FERMENTATION / 129
Table 7.2 Energy (MJ/1 of pure ethanol) required to
World War I, the product of interest was acetone
produce absolute alcohol
used for the production of the explosive trinitro-
Process Stage Substrate
toluene (TNT), but after the war butanol became
Beets Cane | Starchy more important, used primarily for the produc-
raw tion of nitrocellulose lacquers. After World War
materials
II, petroleum-based processes replaced biological
Digestion/
fermentation processes, and the majority of
hydrolysis,
batch 4. —5 - 7 -8 plants with fermenters less than 200 m in vol-
continuous - - 2 ume were closed. However, some plants of this
Cane mill - — 1.5 - size are still functioning today in countries where
Extraction 0.8 - 1 - 3 =
Fermentation, economic, political, geographical, or climatic fac-
batch 0.06 0.06 tors are favorable, such as in Taiwan and South
continuous 0.1 0.1 Africa.
Distillation,
single-stage 10 -13 =13 10° 13 Butyric acid, butanol, acetone, and isopro-
optimized SEE = 7 ery panol are obtained through clostridial fermen-
Process, tation of starch, molasses, sucrose, wood hy-
conventional 16 19 drolysates, and pentoses. The relative
optimized 7 8 proportions of each of these products in the fer-
fe eee
(Misselhorn, 1979) mentation depends on the bacterial strain used
and on the fermentation conditions. Three fer-
mentation types can be categorized, according to
that there is actually either an approximate bal- their fermentation products:
ance or a net energy loss. This emphasizes the
importance in optimizing all steps in the process + Acetone-butanol fermentation with Clostri-
to the utmost. Because the profit margin is so low, dium acetobutylicum
such optimization is economically much more ¢ Butanol-isopropanol fermentation with Clos-
important for the production of an energy source tridium butylicum
than it is for the production of a fine chemical + Butyric acid-acetic acid fermentation with
or antibiotic. Ethanol with a purity of 92.4% is Clostridium butyricum.
used as a solvent in the cosmetic, pharmaceutical,
and chemical industry and at 99.2% purity as a Biosynthesis
motor fuel. In order to produce the virtually
water-free product, better distillation methods Figure 7.4 shows the biosynthesis of the main
would be desirable. fermentation products: acetone, butanol, butyric
acid, isopropanol, and acetic acid. The butanol
referred to here is entirely n-butanol. Hydrogen
7.3 ACETONE/BUTANOL
and carbon dioxide are also produced. In addi-
FERMENTATION
tion, ethanol can be produced through the re-
Pasteur first observed the production of butanol duction of acetaldehyde. The organism forms a
by bacteria in the 19th century. Before World War net yield of 2 ATP in the steps from glucose to
I, microbial processes were developed for the pyruvate and this is the total energy released
purpose of obtaining butadiene for synthetic when no acetic acid is produced.
rubber, at which time Chaim Weizmann per- Only the acetone-butanol fermentation is of
formed fundamental research on the fermenta- current economic interest. Butanol at a concen-
tion of Clostridium acetobutylicum for the pro- tration of less than 0.5% has no influence on the
duction of acetone, butanol, and ethanol. During cells, whereas at higher concentrations it causes
130 / CHAPTER 7 / ORGANIC FEEDSTOCKS PRODUCED BY FERMENTATION

4 Glucose 6 CH3COSCOA Fatty acid degradation

NADH,

8 CH,COCOOH
/ VA0
= 3) COM CHz-C -SCoA
8 Ha |
3 CH,COCH,COSC
0 Arr H3C=C>OH
Acetoacetyl-CoA CH, COOH
8CO,|a"
a
/
7 2H B-Hydroxy- -methyl-
/ glutaryl-CoA
8 CH3COSCoA
| Acetyl-CoA
|
| CH,CHOHCH,COSCoA
R B-Hydroxy-butyry!-CoA
\
2 CoA
H20 CH 3 COCH COOH

2157
Acetoacetic acid

ek
2 CH,CO00-®) CH3CH=CHCOSCOA
Acetyl-P Crotonyl-CoA CH 3CHOH CH,COOH
ADP B-Hydroxy-butyric acid CH3COCH3
72 Acetone
ATP.
2H
ey
2 CH,COOH CH3CH,CH,COSCoA CH3CHOHCH3
Acetic acid Butyryl-CoA CH,COOH Isopropanol

NADH
NAD
HSCoA
CH3COSCOA +
CH3 CH2CH>CHO 3 CH3CH2CH » COOH
Butyraldehyde Butyric acid

NADH5
NAD

CH3CH2CH2CH 2 OH Figure 7.4 Pathway of biosynthesis

of butanol, butyric acid, acetic acid,
Butanol
acetone, and isopropanol

damage to the phospholipids of the cell mem-

Production process
brane; at concentrations above 1.3% butanol.pro-
duction ceases. In addition, depending on the One of the few remaining systems for acetone-
strain and the fermentation conditions, autoly- butanol production still in use is in South Africa.
sins may be produced that cause the cells to lyse. Stock cultures of Clostridium acetobutylicum are
Laboratory studies are underway to develop stored as spores in sand for up to 30 years. Both
a continuous fermentation process for butanol, the production of the spores and the preparation
using either cell suspensions or immobilized of the inoculum are critical. Although production
cells. fermenters must be inoculated only in the low
 7.4 GLYCEROL/ 131

inoculum ratio of 1:3000, build-up of the inoc- - Table 7.3 Yields from a 90m? fermenter
ulum over several stages has the following ad- Product % of converted sugar Amount
vantages: greater resistance to contamination, produced
shorter fermentation times, greater yields, greater Butanol 1053 kg
fermentable sugar concentrations. Acetone | 30 (ratio 6:3:1) 526 kg
Ethanol 175 kg
After sterilization, the fermenters (12 X90 m3
CO; 50 2900 kg
fermenters) are gassed with CO,, and before and H, 2 117 kg
after inoculation the fermenter contents are (Spivey, 1978)
stirred with CO,. The base for the fermentation
is molasses plus corn steep liquor with a begin-
ning pH of 5.8-6.0 at 34°C. Contamination due to bacteriophages and
The 36-hour fermentation has three phases lactobacilli is a common problem and absolute
(Figure 7.5): sterility is therefore a necessity. Table 7.3 shows
typical yields of a 90 m® fermenter containing
1. During the first 18 hours, the pH value de- 5.85 tons of fermentable sugar.
creases to 5.2 due to the formation of acetic CO, produced during the fermentation is re-
acid and butyric acid. covered and converted to liquid CO, or dry ice.
2. During the next 18 hours, the pH value in- Acetone, butanol, and ethanol are recovered
creases through the metabolism of these acids through continuous distillation and fractionation.
to acetone and butanol. When excess NADH, The residue left after the distillation may be dried
is present, the cells take up butyric acid and and utilized as animal feed.
reduce it to butanol.
3. In the next stage, growth and solvent pro-
duction stop. The pH value (5.8) remains con- 7.4 GLYCEROL
stant. The products can now be recovered. Glycerol has wide uses in commerce and is also
a starting material for explosives manufacture.
During both world wars, glycerol was produced
by microbial processes, but today these processes
are no longer of any commercial value. However,
the glycerol fermentation is of theoretical interest
in demonstrating how modification of fermen-
tation conditions can lead to modification of
product type.
Glycerol is formed by yeast along with
ethanol during the alcoholic fermentation (Figure
7.6). Normally, the amount of glycerol formed is
tiny, but by modifying the fermentation balance
the amount of glycerol produced can be greatly
increased. This is done in the following way: An
units
Relative intermediate in the ethanol fermentation is acet-
aldehyde, and if sodium bisulfite is added, an
9 18 27 36 acetaldehyde-sulfite complex is formed. Nor-
Fermentation time (hr) mally, the NADH, formed during the first part
of glycolysis is reoxidized by acetaldehyde to
Figure 7.5 The kinetics of the acetone-butanol fermen- form ethanol, but if the acetaldehyde is removed
tation (Spivey, 1978), solvent," titratable acids, — — —
gas production, — - — pH value in this sulfite complex, the NADH, is available
132 / CHAPTER 7 / ORGANIC FEEDSTOCKS PRODUCED BY FERMENTATION

Glucose concentration of its environment. The higher the

external salt concentration, the higher is the in-
y
ee Fructose -1,6-P) tracellular glycerol concentration. However, if
Ye CH20H the salt concentration is suddenly reduced, the
| —— esis
H-C=08 ore excess glycerol is excreted by the alga.
|
H;-C-0-© Neen as ee In the glycerol production process, the alga
f
O" 0-®
- NADH,
| is first cultivated photosynthetically in a medium
E
|
H2-C—OH containing a high salt concentration. After
A=C—OR
|
fe el
growth is completed, the cells are concentrated
H>-C—O-
2 f © “s-ONa Hz-C—O-
2 ©
and transferred to a low-salt medium. During a
nto
Cc” NaHS0;
o| ÆRE
H=C—OH
short incubation period in the low-salt medium,
| H-C-OH | the glycerol is excreted in a fairly concentrated
CH; | H-C—OH
CH3 |
H,-C—OH
form and purification can be carried out.
H,C—OH
| Glycerol
CH,
REFERENCES
Figure 7.6 Glycerol biosynthesis Bringer, S. and H. Sahm. 1985. Jetzt industriereif: Ethanol-
Produktion durch Bakterien. (Now ready for large-
scale application: ethanol production by bacteria.)
for the reduction of dihydroxyacetone phosphate BioEngineering 1/85:30-36.
(also formed during glycolysis) via the enzyme Drawert, F., W. Klisch, and G. Sommer. 1987. Garungs-
verfahren-Ethanol Wein, Bier. (Ethanol fermentation
glyceraldehyde phosphate dehydrogenase to processes: wine, beer.), pp. 321-362. In: Prave et al.
glycerol phosphate (which is dephosphorylated Handbuch der Biotechnologie. Oldenbourg Verlag,
to glycerol). Through the dismutation of two ace- Munchen.
Frick, C. and K. Schugerl. 1986. Continuous acetone-bu-
taldehyde molecules into acetic acid and ethanol,
tanol production with free and immobilized Clostri-
some glycerol enrichment is also attained by add- dium acetobutylicum. Appl. Microbiol. Biotechnol.
ing alkali (e.g. Na,HPO,). 25:186—193.
Jones, D.T. and D.R. Woods. 1986. Acetone-butanol fer-
The balance of the glycerol process is as fol- mentation revisited. Microbiol. Rev. 50:484—524.
lows: Klyosov, A.A. 1986. Review. Enzymatic conversion of cel-
lulosic materials to sugar and alcohol. The technology
and its implications. Appl. Biochem. Biotechnol. 12:249
ff
Kosaric, N., A. Wieczorek, G.P. Cosentino, R.J. Magee, and
J.E. Prenosil. 1983. Ethanol fermentation, pp. 257-385.
Since additional byproducts are formed, yields In: Rehm, H.J. and G. Reed (eds.). Biotechnology 3.
never exceed 20-30% of the sugar used, in fer- Verlag Chemie, Weinhem.
menters up to 1000 m°, during a fermentation Linden, J.C., A.R. Moreira, and T.G. Lenz. 1985. Acetone
and butanol, pp. 915-931. In: Moo-Young, M. (ed.).
running for 2-3 days. Osmotolerant yeasts are Comprehensive Biotechnology 3. Pergamon Press, Ox-
best for glycerol production and the strains Sac- ford.
charomyces rouxii, Torulopsis magnoliae and Pi- Maiorella, B.L. 1985. Ethanol, pp. 861-914. In: Moo-
Young, M. (ed.). Comprehensive Biotechnology 3. Per-
chia farinosa are commonly used. gamon Press, Oxford.
An interesting process for glycerol production Maiorella, B.L., H.W. Blanch, and C.R. Wilke. 1984. Bio-
has been developed in recent years in Israel, us- technology Report. Economic evaluation of alternative
ethanol fermentation processes. Biotech. Bioeng.
ing the halophilic alga Dunaliella salina. This 26:1003-1025.
alga, which is commonly present in hypersaline Meyrath, J. 1979. Das ALCO-FLOC-System,
eine kontin-
lakes and salty lagoons, synthesizes glycerol as uierliche und zuverlassige Methode zur Athanol-pro-
duktion mit extrem hoher Produktivitat (The ALCO-
an intracellular solute which balances the os- FLOC system, a continuous and reliable method for
motic pressure brought about by the high salt the manufacture of ethanol at extremely high produc-
 REFERENCES / 133

tivity), pp. 107-116. In: Dellweg, H. (ed.), 4th Symp. Schlote, D. and G. Gottschalk. 1986. Effect of cell cycle
Technische Mikrobiologie. Verlag Versuchs- und Lehr- on continuous butanol-acetone fermentation with
anstalt ftir Spiritusfabrikation, Berlin. Clostridium acetobutylicum under phosphate limitation.
Misselhorn, K. 1979. Athanolherstellung unter energie- Appl. Microbiol. Biotechnol. 24: 1-5.
wirtschaftlichen Aspekt (Ethanol production under en- Spencer, J.F.T. and D.M. Spencer. 1978. Production of
ergy-efficient conditions). Stand der Technik, pp. 47- polyhydroxy alcohols by osmotolerant yeasts, pp. 393-
55. In: Dellweg, H. (ed.), 4th Symp. Technische Mi- 425. In: Rose, A.H. (ed.), Economic microbiology, vol.
crobiologie. Verlag Versuchs- und Lehranstalt ftir Spir- 1, Primary products of metabolism. Academic Press,
itusfabrikation, Berlin. London.
Murtagh, J.E. 1986. Fuel ethanol production—the U.S. ex- Spivey, M.J. 1978. The acetone/butanol/ethanol fermen-
perience. Proc. biochem. April, 61-65. tation. Proc, Biochem. 13: 2—-4,25.
Rogers, P.L., K. J. Lee, M.L. Skotnicki, and D.E. Tribe. Stewart, G.G. and I. Russell. 1985. Modern brewing tech-
1982. Ethanol production by Zymomonas mobilis. Adv. nology, pp. 335-381. In: Moo-Young, M. (ed.), Com-
Biochem. Eng. 23:37-84. prehensive Biotechnology 3. Pergamon Press, Oxford.
 Organic acids

which are not used commercially, either due to

8.1 INTRODUCTION
lack of sufficient need for the acid or for economic
Organic acids find wide use both as additives in reasons. Table 8.1 lists several acids that have
the food industry and also as chemical feed- been produced microbially or for which pro-
stocks. Fermentation processes play a major role cesses have been extensively developed. In this
in the production of most organic acids. All acids chapter, we will discuss the most important pro-
of the tricarboxylic acid cycle can be produced cesses of commercial interest.
microbiologically in high yields. Some acids de-
rived indirectly from the Krebs cycle, such as ita-
8.2 CITRIC ACID
conic acid, can likewise be produced. Other or-
ganic acids are derived directly from glucose (for Citric acid has been known as a natural plant
example, gluconic acid), or they are formed as substance since the end of the nineteenth cen-
end products from pyruvate or ethanol (for ex- tury, and since 1893 scientists have known that
ample, lactic acid and acetic acid). it is produced by filamentous fungi. In 1923, the
Except for the production of citric acid which first practical fermentations for the production of
is manufactured entirely by fermentation, there this organic acid were started, using microorga-
is frequently great competition between micro- nisms growing in surface culture. Fermentation
biological and chemical processes for production using deep-vat fermenters began in the 1930’s.
of the various organic acids. For some acids (lactic Although in South America and Mexico there are
acid, acetic acid) microbiological and chemical some small factories where citric acid is isolated
methods for preparation are simultaneously in from unripe citrus fruits (lemons contain 7-9%
use. For others (a-ketoglutaric acid, malic acid), citric acid), today over 99% of the world’s output
fermentation processes have been developed of citric acid is produced microbially.

134
 8.2 CITRIC ACID / 135
Table 8.1 Practical microbiological processes for organic acid production
Organic acid Organism Commer- Substrate Process Yield Use of References
cially % product
produced
Fumaric acid Rhizopus Yes Glucose 3 days, 33°C 65 Resin Buchta, 1983b
spy
Candida n-Alkane 7 days, 30°C 84

Propionic Propioni- Yes Lactose 8-12 days 60 Perfume Buchta, 1983b

acid bacterium Glucose se Fungicide
Starch å
Malic acid Leuconostoc No Fumaric 24h 99 Food Buchta, 1983b
brevis, acid
Candida n-Alkane 72
a-Keto- Candida, No n-Alkane 40h 67 Rose, 1978
glutaric acid Aerobacter Glutamic 46
acid
5-Keto- Acetobacter Yes Glucose 5-6 days 85 L-Tartaric | Buchta, 1983b
gluconic acid suboxydans acid
2-Keto- Serratia Yes Glucose 16h 95- Isoascorbic Buchta, 1983b
gluconic acid marcescens 100 acid

The following amounts of citric acid have industry, pure metals are produced as metal cit-
been produced microbially: rates,
Citric acid is being used more and more in
1929 5,000 tons the detergent industry because it can replace
1953 50,000 tons polyphosphates. The higher cost of citric acid as
1976 200,000 tons compared to polyphosphates formerly restricted
1979 220,000 tons its use; however, detergents containing poly-
1982 350,000 tons phosphates have been prohibited in some loca-
tions and the polyphosphates have been com-
The fermentation processes are carried out either pletely replaced with citric acid.
in surface culture or in fermenters up to 220 m°
in volume. Citric acid is marketed as citric acid-
1-hydrate or as anhydrous citric acid. Most citric Strains for citric acid production
acid (60% of the total) is used in the food and Many strains excrete traces of citric acid as a me-
beverage industry. The flavor of fruit juices, fruit tabolite of primary metabolism. Examples are As-
juice extracts, candy, ice cream, and marmalade pergillus niger, A. wentii, A. clavatus, Penicillium
is enhanced or preserved by the addition of citric luteum, P. citrinum, Mucor piriformis, Paecilo-
acid. myces divaricatum, Citromyces pfefferianus, Can-
The pharmaceutical industry (10% of total dida guilliermondii, Saccharomycopsis lipolytica,
usage) uses iron citrate as a source of iron and Trichoderma viride, Arthrobacter paraffineus, and
citric acid as a preservative for stored blood, tab- Corynebacterium sp. However, only mutants of
lets, ointments, and in cosmetic preparations. In Aspergillus niger are used for commercial pro-
the chemical industry (25% of total usage), citric duction. Compared to Penicillium strains, the as-
acid is used as an antifoam agent, as a softener, pergilli produce more citric acid per unit time.
and for the treatment of textiles. In the metal Moreover, the production of undesirable side
136 / CHAPTER 8 / ORGANIC ACIDS

products, such as oxalic acid, isocitric acid, and PEP

gluconic acid, can be more efficiently suppressed
in these mutants.

Pyruvate JEthano!
Biosynthesis
mn
Citric acid (2-hydroxypropane-1,2,3,-tricarbox-
ylic acid) is a primary metabolic product and is HRK
formed in the tricarboxylic acid cycle. Glucose is saae TT

STEEL DU-CoA
the main carbon source used for citric acid pro-
duction. In many organisms, 80% of the glucose
used for citric acid biosynthesis is broken down sie
by reactions of the Embden-Meyerhof-Parnas
ny ase
: Citrate
(EMP) pathway and 20% by reactions of the pen-
tose phosphate cycle. During the growth phase,
the relationship between these two pathways is Fumarate
LN
eee
Glyoxylate 5 Isocitrate
2:1. In citric-acid producers, the enzymes of the
Succinate
EMP-pathway are present throughout the fer- ai
NN EL ae
mentation process and the activity of the path-
way is regulated in a positive manner with
phosphofructokinase and in a negative manner 1. PEP Carboxylase 4. Pyruvate carboxylase
by pyruvate kinase. When pyruvate is decarbox- 2,PEP Carboxykinase 5 Malate enzyme
ylated with the formation of acetyl-CoA, the ace-
3PEPCarboxytrans- 6 Glyoxylate cycle
tate residue is channeled into the tricarboxylic phosphorylase
acid cycle. During the idiophase, all enzymes of
the Krebs cycle are expressed except a-ketoglu- Figure 8.1 Anaplerotic reactions which connect with the
tarate dehydrogenase. The citrate synthase activ- tricarboxylic acid cycle
ity (condensing enzyme) is increased by a factor
of 10 during the production of citric acid, while
the activities of the enzymes which catabolize first anaplerotic enzyme present in Aspergillus is
citric acid, such as aconitase and isocitrate de- a pyruvate carboxylase (reaction 4, Figure 8.1),
hydrogenase, are sharply reduced as compared which converts pyruvate and CO, into oxalace-
to their activity in the trophophase. One of the tate, inorganic phosphate, and ADP (while con-
three isocitrate dehydrogenase isozymes, a mito- suming ATP). The reaction is dependent on Mg?"
chondrial enzyme that is specific for NADP, is and K* ions; acetyl-CoA is not needed for the
inhibited by glycerol that accumulates during the reaction, in contrast to its requirement in the met-
spore germination process. In addition, citric acid abolic reactions of other microorganisms. Pyru-
production is inhibited by high intracellular con- vate carboxylase is the key enzyme for citric acid
centrations of ammonium ion. production.
Citrate synthase cannot be solely responsible The second anaplerotic sequence involves a
for maintaining the activity of the tricarboxylic phosphoenol pyruvate carboxykinase (reaction
acid cycle, since the cycle would cease if citric 2, Figure 8.1), which converts PEP and CO, into
acid were removed. Distinct sequences which re- oxalacetate and ATP in the presence of ADP. The
plenish the tricarboxylic acid cycle intermediates system requires Mg?" or Mn?* and K* or NH,7.
(anaplerotic sequences) must thus exist in the If acetate or higher aliphatic compounds such
production phase, as outlined in Figure 8.1. The as n-alkanes (C,-C,,) are used as carbon source,
 8.2 CITRIC ACID / 137
a third anaplerotic sequence is present in Asper- . sates, glucose syrup from saccharified starch, su-
gillus niger. In the absence of glucose, the gly- crose of different levels of purity, sugar cane
oxylate cycle (reaction 6, Figure 8.1) is operable: syrup with two-thirds of the sucrose converted
isocitrate lyase is induced and malate synthase into invert sugar, sugar cane molasses, sugar beet
is present. If glucose is added, the glyoxylate cy- molasses.
cle is then repressed, although isocitrate lyase is
If starch is used, amylases formed by the pro-
still partially active. ducing fungus or added to the fermentation broth
hydrolyze the starch to sugars. If hydrolysates or
Yields syrup are used, a preliminary treatment with
either precipitants or cation exchangers must be
In the trophophase, part of the added glucose is
carried out to remove cations. For elimination of
used for the production of mycelium and is con-
metals, molasses is generally treated before ster-
verted through respiration into CO,. In the idi-
ilization with calcium hexacyanoferrate or is pur-
ophase, the rest of the glucose is converted into
ified by cation exchangers. Although the effect
the organic acids and during this phase there is
of the hexacyanoferrate is primarily to bring
a minimal loss through respiration. Table 8.2
about metal precipitation, there are also studies
gives data on citric acid yields in various pro-
which show a direct influence on the fermenta-
cesses. The theoretical yield is 123 g citric acid-
tion, since an excess of hexacyanoferrate over
1-hydrate or 112 g anhydrous citric acid per 100
that needed to complex metals increases the citric
g sucrose. However, such yields are not obtained
in practice, due to the loss during the tropho- acid yield.
phase. Production strains are optimized based on the
carbon sources used. A strain which produces
well with one carbohydrate source generally can-
Nutrient media not be used with another starting material with-
The media used for citric acid production have out a substantial yield reduction.
been highly perfected over the many years that There are no analytical methods that permit
the commercial process has been underway. an evaluation of a molasses source for its use in
citric acid production; each batch of molasses
Carbohydrate source A 15-25% sugar solution must be given a preliminary fermentation test.
is converted during fermentation. A variety of Firms which use molasses as starting material
starting materials can be used as carbohydrate generally optimize molasses treatment and the
sources: Starch from potatoes, starch hydroly- make-up of the culture media in model fermen-

Table 8.2 Citric acid yields in different fermentation processes

Microorganism Raw material Process Actual yield Theoretical % of Theoretical
(Sucrose, %) 2 ; maximum yield maximum yield
kg % kg % kg%/kg Carbon
Raw material Carbon Sode
source
A. niger Beet molasses (50%) Surface 42 84 61.4 68.4
A. niger Beet molasses (50%) Submerged 41 82 61.4 66.8
A. niger Beet molasses (54%) Submerged 46 85 66.3 69.4
Yeast n-Alkane Submerged 165 82 247.0 66.8
Yeast Methanol Submerged 40 45 109.4 36.6
Yeast Ethanol Submerged 60 48.4 145.8 41.1
(Siebert and Schulz, 1979)
138 / CHAPTER 8 / ORGANIC ACIDS

tations of up to 30 m%, before the molasses is ing the trophophase, the pH is generally started
used in production. at 5. In the first 48 hours during the trophophase,
the pH falls to below 3.0 as a result of the me-
tabolism of ammonium ions. For better control
Trace elements Extensive research on the role
of yield in submerged cultures, the trophophase
of trace elements in the citric acid fermentation
and idiophase can be separated by reducing the
was begun in the 1940’s and has continued as
pH to under 2 once growth has ceased.
analytical methods have improved. Copper,
Another benefit of the low pH is the de-
manganese, magnesium, iron, zinc, and molyb-
creased risk of contamination, since at these low
denum are necessary in the ppm range for op-
pH values only Penicillium and yeast cause con-
timal growth. However, if optimal concentrations
tamination. Especially in surface fermentations,
are exceeded, there may be a toxic effect.
the frequency of contamination is a significant
The role of iron is especially interesting.
economic factor.
While optimal growth requires higher iron con-
centrations (among other things as a cofactor for
aconitase), only 0.05-0.5 ppm is needed for max- Production processes
imal production of citric acid. Optimal iron con-
Citric acid is produced by both surface and sub-
centration is dependent on the starting material
merged processes. Surface processes can be fur-
used. For instance, with pure sucrose, 2.0 ppm
ther subdivided according to the state of the cul-
of iron is optimal for growth, whereas when raw
ture medium used: solid or liquid. Two types of
materials such as invert sugar or starch hydrol-
submerged processes are used, stirred fermenters
ysates are used, growth without acid production
or airlift fermenters (Figure 8.2). The surface pro-
is obtained by adding 0.2 ppm iron, due to the
cess employing solid culture medium is in use
fact that the iron content of the raw material so-
solely in small plants and a maximum of 500 tons
lutions is so high. The sensitivity of strains to
per year are produced, but the other three pro-
heavy metals such as zinc, iron, manganese, and
cesses are widely used. Several factors affect the
copper decreases with a decrease in the temper-
choice of production type: availability of invest-
ature. In addition, copper reverses the inhibitory
ment capital, energy availability, cost of labor
effect of iron (Table 8.3).
and training, and availability of techniques for
Besides the composition of the nutrient so-
measurement and regulation.
lution (sugar content, metal-salt concentration,
and phosphate and nitrogen contents) the pH of
Production of inoculum The material used as in-
the medium influences the yield. During the idi-
oculum for citric acid production is a spore sus-
ophase, the pH must be below 3 in order to sup-
pension. Spores are produced in glass bottles on
press oxalic and gluconic acid formation, but dur-
solid substrates at 25°C with incubation times of
10-14 days. Besides the total numbers, the via-
Table 8.3 Reversal by copper of the inhibition of citric bility of the spore crops is critical. When a sub-
acid formation by 10 ppm Fe" merged fermenter is to be inoculated, the spores
Copper (ppm) Citric acid are induced to germinate in a preliminary fer-
% Yield ~
mentation. A nutrient solution containing 15%
0 = sugar (from molasses) is used in this seed fer-
5.0 77.8
10.0 77.4
menter, and to induce the formation of mycelium
20.0 75.0 in the form of pellets, cyanide ions are added. If
30.0 75.6 too little cyanide is added, growth proceeds well
50.0 74.0
but the citric acid yields in the later production-
(Noyes, 1969) scale fermenter are low. The spores germinate at
 8.2 CITRIC ACID / 139

Industrial processes

Submerged

Solid Liquid Stirred Airlift

Figure 8.2 Various processes media media reactor reactor
for citric acid manufacture

32°C and form pellets 0.2-0.5 mm in diameter Table 8.4 shows the composition of a typical
within 24 hours. During this period, the pH falls nutrient solution; in surface processes, the su-
to 4.3. These pellets are then used as the inoc- crose is supplied as beet molasses instead of
ulum for production fermenters. Conversion rate sugar cane molasses. In modern systems, contin-
and efficiency in the production fermenter are uous sterilization of the medium is used.
extremely dependent on the manner in which the Figure 8.3 shows the layout of a typical fer-
spores and pellets are produced. mentation system. The sterilized nutrient solu-
tion automatically flows over a distribution sys-
Surface processes tem onto the trays. Inoculation is done in the
incubation chamber at 30-40°C by blowing dry
Surface processes employing solid substrates spores (2-5 X10’ spores/m?) or by spraying
may use either wheat bran or pulp from sweet spore suspensions.
potato starch production as a culture medium. In The temperature is kept constant at 30°C dur-
this process, the A. niger strains are not as sen- ing the fermentation by means of an air current.
sitive to trace elements as in the other processes. Ventilation is also important for gas exchange
The pH of the bran is reduced to 4-5 before ster- because the rate of citric acid production falls if
ilization; after sterilization the material is inoc- CO, in the atmosphere increases to > 10%.
ulated with spores, spread on trays in layers 3— Within 24 hours after inoculation, the germinat-
5 cm thick and incubated at 28°C. Growth can ing spores form a thin cover of mycelium on the
be accelerated by adding a-amylases, although
the fungus can hydrolyze starch with its own
amylase. The solid surface process takes 90 Table 8.4 Composition of nutrient solution for a
hours, at the end of which the entire solution is surface culture
extracted with hot water to isolate the citric acid. Substrate g/1 Substrate
Surface processes using liquid nutrient solu- Sucrose 160-200,
tions are the oldest production methods and ac- ca. 320-400 g molasses
NH,NO, 1.6-3.2,
count for 20% of the world’s supply of citric acid. Not needed with
These processes are still in use because of the molasses
low investment (for the bioreactor), the low en- CaH,PO, 0.3-1.0
MgSO,°7 H,O 0.2-0.5,
ergy cost for the cooling system, and the simple Not needed with
technology. The labor costs, however, are sub- molasses
stantially greater than for the deep vat processes ZnSO, 0.01-0.10
Calcium hexacyanoferrate 0.4-2.0
because of the manpower needed to clean the
pipes, trays, and walls of the system. (Schultz and Rauch, 1975)
140 / CHAPTER 8 / ORGANIC ACIDS

E
+ 2
Water Salts
CAR
fin te 8
Trace elements << iS Steam Water
4)

ty

Myce-

e
Sterili-
lial
wash
zation >

ES Filtrate an
Fermentation
chamber

@) @)

Sulfuric
lon
Oxalic BL, Citric acid Charcoal
exchange
acid acid
removal precip- Figure 8.3 Block diagram of
itation Evaporation a citric acid process using the
Crystallization surface method

surface of the nutrient solution. As a result of the presses are also used to obtain more citric acid
uptake of ammonium ions, the pH in the culture from the cells.
liquids falls to 1.5-2.0. Figure 8.4 shows the
course of the fermentation during the first 60 Submerged processes
hours. After 30 hours, the idiophase begins. If
too much iron is present, oxalic acid is produced Eighty percent of the world’s supply of citric acid
and a yellowish pigment is formed which later is produced by submerged processes. The sub-
hinders the recovery process. The fully devel- merged process, although taking a longer time
oped mycelium floats as a thick convoluted white (8 days), has several advantages: lower invest-
layer on the nutrient solution. Through evapo- ment for construction by a factor of 2.5, 25%
ration, the temperature can be maintained con- lower total investment, and lower labor costs.
stant, but the culture then loses 30-40% of its The disadvantages are the greater energy costs
original volume. The fermentation is stopped af- and the more sophisticated control technology
ter 8-14 days. which requires more highly trained personnel.
Yields from the surface process using liquid Three factors are especially important for pro-
nutrient solution amount to 1.2 to 1.5 kg citric duction in submerged processes: a) quality of the
acid monohydrate/m? of fermentation surface material used to construct the fermenter, b) my-
per hour. The heat production is 12,500 kJ/kg of celium structure, c) oxygen supply.
citric acid produced. Fermenters for citric acid production must be
During recovery, the mycelium and nutrient either protected from acids or constructed of
solution are removed from the chambers. Due to stainless steel. At pH values between 1-2 the
its volume, the mycelium must be carefully heavy metals leached from normal steel fermen-
washed in sections. In some cases, mechanical ter walls can inhibit the formation of citric acid.
 8.2 CITRIC ACID / 141

Although Aspergillus niger requires relatively

400 fo
little oxygen, it is sensitive to oxygen deficiency.
Throughout the entire fermentation period, there
must be a minimum oxygen concentration of 20-
25% of the saturation value. Short interruptions
in the oxygen supply cause production to cease
irreversibly. The aeration rates in deep vat fer-
menters should be 0.2-1 volume/volume/min-
ute (vvm) during the acid production phase. Due
to the low viscosity, stirring is not necessary.
i
Thus, although some plants use stirred fermen-
ters, air-lift bioreactors can also be used.
Most fermenters for citric acid production are
(g/l)
Mycelium
constructed in the range of 120-220 m3. Sub-
merged systems are somewhat less sensitive than
b -— surface systems to variability in the medium
(mM/l)
concentration
Metabolite composition and in molasses composition (which
is notoriously variable), so that they can be op-
erated much more reproducibly than surface sys-
tems.

Sl Foaming is a problem in submerged fermen-

tations. A foam chamber 1/3 the size of the fer-
0 10 20 30 40 50 60
Fermentation time (hr) menter volume is needed in both air-lift and
stirred bioreactors. Antifoam agents, such as lard
Figure 8.4 Kinetics of the citric acid fermentation (Siebert
oils, can be added at frequent intervals, and me-
and Schulz, 1979)
1, Sucrose; 2, Glucose; 3, Fructose; 4, Mycelium; 5, Citric chanical anti-foam devices can also be used.
acid; 6, Gluconic acid Continuous fermentation for citric acid pro-
duction has not yet been accomplished. The com-
plexity of pellet formation and the control re-
With small fermenters (up to 1000 1), even stain-
less steel chambers must have plastic liners be- quirements of the idiophase could only be
cause of the large surface/volume ratio. In large satisfied by a multistage system with fermenters
fermenters, the lining is not necessary if stainless connected in a series; for economic reasons, how-
steel is used. ever, such a system would not be sensible, even
The structure of the mycelium that forms in if the microbiological and sterility problems
the submerged culture during the trophophase is could be solved.
vital to a successful production process. If the
mycelium is loose and filamentous, with limited
Citric acid production with alkane-utilizing micro-
branches and no chlamydospores, little citric acid organisms Several processes for the production
is produced in the idiophase. Mycelium for op- of citric acid from C,-C,, hydrocarbons have
timal production rates consists of very small solid been described, using both yeast and bacteria.
pellets. The ratio of iron to copper in the medium The best producer is Candida lipolytica, which
determines the mycelium structure. Since Asper- can be employed in batch, semicontinuous, or
gillus is genetically unstable, only a minimum continuous fermentations. Compared to carbo-
number of preliminary culture steps should be hydrate fermentations, the specific yields are
performed. In some cases, production fermenters measured as higher with hydrocarbons (145% g
are inoculated directly with spores. citric acid/g paraffin), but there are difficulties
142 / CHAPTER 8 / ORGANIC ACIDS

with the low solubility of the alkanes and the

increased amount of isocitric acid produced,
dt e—*
which must later be separated from the citric acid
(Table 8.5). Studies by Ermakova et al. (1986)
and Finogenova et al. (1986) have focussed on
a yo
|
the regulation of anaplerotic pathways and the
proportion of citric acid versus isocitric acid in
Candida lipolytica when different substrates are 80 -
used. In some cases, the proportion of isocitric
acid can be up to 50% of the citric acid content.
Figure 8.5 shows citric acid production from hy-
drocarbons by both batch and semicontinuous (g/l)
acid
Citric
fermentations.

Product Recovery
If oxalic acid should be formed as a side product
due to suboptimal fermentation control, it is pre-
cipitated as calcium oxalate at a low pH, leaving
behind the citric acid in solution as the mono- T Fi BER
calcium citrate. Rotating filters or centrifuges are
80 120 160
then used to separate the mycelium and the pre-
cipitated calcium oxalate from the liquid. At pH Fermentation time (hr)
7.2 + 0.2 and 70-90°C, citric acid is precipitated,
which can in turn be separated by means of ro- Figure 8.5 Citric acid production with Candida lipolytica
ATCC 8661 in batch (0 —— O) and semicontinuous
tating filters, and dried. For some uses the citric (® — 6€) fermentation (Gledhill et al., 1973)
acid is further purified by adding sulfuric acid to
dissolve the citric acid, with a calcium sulfate
precipitate forming. The subsequent recovery foods must of course be purer than that needed
steps include treatment with activated carbon, for industrial purposes.
treatment with cation and anion exchangers, and
crystallization as citric acid or citric acid mono-
8.3 GLUCONIC ACID,
hydrate. Above 40°C citric acid crystallizes as the
GLUCONOLACTONE, AND
anhydrous acid, and below 36.5°C as the mono-
GLUCOSE OXIDASE
hydrate.
The required purity of the product depends Gluconic acid is used in the manufacture of
on its intended use. Citric acid for addition to metal, leather, and food; sodium gluconate is
used as a sequestering agent in many detergents;
Table 8.5 Citric acid and isocitric acid production in 6-gluconolactone functions as a baking powder
relation to substrate additive; and calcium gluconate is used in med-
Substrate Citric acid Isocitric acid Isocitric acid icine. The production of gluconic acid from glu-
g/l g/l % cose by means of glucose oxidase is a simple re-
Glycerol 1255 11.4 8.3 action which can be carried out by many
Glucose 89.4 7.0 TED microorganisms (Figure 8.6). The gluconolactone
n-Paraffin 92.0 49.0 34.8 formed in the first step hydrolyzes either spon-
(Treton et al., 1978) taneously or enzymatically to gluconic acid. Dur-
 8.4 ACETIC ACID / 143

Table 8.6 Relationship between gluconic acid

FN OOH production and pressure
CH20H H- -OH ! H-C-OH
O. OH HO-C-H H20 HO-C-H Pressure (bar) Yield by weight
HO OH H-C-OH Ve H-C-OH
42.5
OH FAD FADH2 H-C H-C-OH 80.4
H203 0» CH20H CH20H 82.4
81.3
WOR
Ook 86.1
D-Glucose 6 -D-Gluconolactone Gluconic acid
(May et al., 1934)
Figure 8.6 Production of gluconic acid from glucose

Production
ing the transfer of hydrogen from FADH, to ox- The fermentation, carried out at pH 4.5-6.5, re-
ygen, hydrogen peroxide (H,O,) is produced and quires a growth medium in which both phos-
is immediately split into water by the enzyme phorus and nitrogen are limiting. If calcium glu-
catalase. Because the activity of the enzyme leads conate is to be produced, no more than 13-15%
to the formation of hydrogen peroxide, which glucose can be added as substrate because of the
has antimicrobial activity, glucose oxidase has low solubility of calcium gluconate (4 g/l at
also been identified as an antibiotic in fermen- 30°C); higher substrate concentrations would al-
tation broths and has thus been known under low higher calcium gluconate levels, which
the names notatin and penicillin B. would spontaneously crystallize, making purifi-
Gluconic acid has had a long history in in- cation difficult. In the production of sodium glu-
dustrial microbiology. In 1911 Alsberg described conate (solubility 396 g/l), a glucose concentra-
the production of gluconic acid with Pseudo- tion as high as 28-30% can be used.
monas. The first process with a fungus, a surface The fermentation runs 20 hours at 28-30°C
process using Penicillium luteum-purpurogenum, with a high aeration rate (1-1.5 vvm). By raising
was begun in 1928. In this process the yields the pressure in the system, the oxygen solubility
and therefore the gluconic acid yields can be in-
amounted to 80-87% of theoretical.
creased (Table 8.6). Commercial yields today are
Today only submerged processes are used,
90-95%.
with either the fungus Aspergillus niger or the
bacterium Acetobacter suboxydans. In these pro-
cesses, gluconic acid, sodium and calcium glu- 8.4 ACETIC ACID
conate, and glucose oxidase are manufactured.
The production of acetic acid from alcoholic liq-
Other organisms which have been optimized to
uids has been known as long as the production
produce gluconic acid but which have not been of wine (about 10,000 years). The Romans and
used commercially include the fungi Penicillium, Greeks, who used diluted vinegar as a refreshing
Scopulariopsis, Gonatobotrys, Endomycopsis, and drink, produced vinegar by leaving wine open to
Pullularia and the bacteria Vibrio and Pseudo- air. Vinegar was produced only for local con-
monas. sumption until the Middle Ages. The first in-
A process for immobilizing cells or glucose dustrially manufactured vinegars were produced
oxidase has been described which leads to yields in flat open vats. These were slow processes, in
of 93% when pure oxygen is used. There is con- which a film of bacteria floated on the surface of
siderable competition between the microbiolog- the wine. In the nineteenth century, surface fer-
ical process and chemical methods, which also mentations were developed into more rapid pro-
produce high yields. cesses. One of these, the trickling generator pro-
144 / CHAPTER 8 / ORGANIC ACIDS

cess is still used today. Beginning in 1949 specific alcohol dehydrogenase. Then there is a
submerged processes were developed. Both hydration to acetaldehyde hydrate and a second
types of process, the trickling generator and the oxidation with acetaldehyde dehydrogenase to
submerged fermenter, are used worldwide, the acetic acid (Figure 8.7). During the oxidation, 1
older methods still being used because of the bet- mole of acetic acid is produced from 1 mole of
ter flavor of the product. The submerged process ethanol. From 1 liter of 12% (v/v) alcohol, 1 liter
has been extensively improved by the H. Frings of 12.4% (w/v) acetic acid is produced.
Company in Germany. As an example of the For optimal production, sufficient oxygen is
scale of the process, in 1980 production of vi- required, which is reduced by means of the res-
negar with 10% acetic acid amounted to 356 X piratory chain. Six ATP are produced per mole
10° liters in the European Economic Community, of acetic acid produced. If sufficient oxygen is not
469 X 10° liters in the United States and 1600 available, at high acetic acid and ethanol con-
X 10° liters worldwide (excluding China and the centrations the cells die. At a concentration of
Soviet Union). 5% acetic acid plus ethanol, 34% of the bacteria
Although acetic acid is produced by many die after a 2-minute interruption of aeration,
fermentative bacteria, only members of a special whereas at a total concentration of 12% acetic
group, the acetic acid bacteria, are used in com- acid plus ethanol, the same killing occurs after
mercial production. The acetic acid bacteria can only 10-20 seconds.
be divided into two genera, Gluconobacter and Both acetic acid and ethanol must be present
Acetobacter, the first group oxidizing ethanol for optimal growth of Acetobacter. Ethanol sup-
solely to acetic acid and the second group (the ply is critical and with less than 0.2% (v/v) in
overoxidizers) able to oxidize ethanol first to solution the death rate increases. However, the
acetic acid and then further to CO, and H,O. maximal ethanol content should not exceed 5%
Members of the genus Acetobacter are Gram-neg- in conventional processes. Today high-yielding
ative and acid tolerant. Strains used commer- strains produce 13-14% acetic acid.
cially belong to the species Acetobacter aceti, A.
pasteurianus or A. peroxidans. Members of the ge-
Production of vinegar
nus Gluconobacter are not overoxidizers. The spe-
cies Gluconobacter oxydans (formerly Acetomonas Starting materials with low alcohol content, such
oxydans) and several subspecies of this species as wine, whey, malt, or cider, do not require any
are used commercially. Mixed cultures appear additional components to constitute a complete
during production, even when the inoculum is nutrient solution. However, if potato or grain
assumed to be pure, particularly in surface pro- spirits or technical alcohol is used, nutrients must
cesses. Production strains seem to lose their high- be added in many cases to obtain optimal growth
yielding character if they are transported to anew and acetic acid production (Table 8.7). In sub-
location on agar. To avoid this problem, pro- merged fermentation, concentrations of nutrients
duction strains are shipped to new vinegar plants must be five times higher due to the increased
in transportable small fermenters. biomass which is taken out of the reactor with
the vinegar. The inoculum can be either pure
Biosynthesis cultures or vinegar from previous batches.

Acetic acid production is an incomplete oxida- Surface processes Despite the success of the sub-
tion rather than a true fermentation, because the merged process, the trickling generator is still
reducing power which is produced is transferred widely used in vinegar production (Figure 8.8),
to oxygen. The first oxidation step from ethanol especially for making household vinegar. The
leads to acetaldehyde with a NAD- or NADP- wooden bioreactor has a total volume of up to
 8.4 ACETIC ACID / 145

2x3 x H20

USR sr=)
alte EEN

NAD(P) NAD(P)H, NADP NADPH,

H,0 :
CH,CH,OH CH,CHO Seer CH, CH(OATS CH,COOH

Ethanol Acetaldehyde Acetaldehyde Acetic acid

hydrate

Figure 8.7 Oxidation of ethanol to acetic acid

60 m? and is filled with beechwood shavings. The such low-yielding processes, aeration was not
starting material is sprayed over the surface and critical, but in present-day high-yielding pro-
trickles through the shavings containing bacteria cesses, which produce 13% acetic acid in quan-
into a basin in the bottom, where the partially tities of up to 50 må, aeration must be highly
converted solution is cooled and pumped back regulated. The fermenters (Figure 8.9) resemble
up to the top. other bioreactors (see Chapter 5). The tanks are
Of the alcohol added, 88-90% is converted constructed of stainless steel and are stirred from
to acetic acid in the trickling generator process. the bottom. The aeration apparatus consists of a
The rest of the alcohol is used for primary me- suction rotor with the incoming air coming down
tabolism or it escapes with the waste air. The
temperature in the upper part of the system is
about 29°C and is about 35°C in the lower part.
The time needed to produce 12% acetic acid in
this process is about three days.

Submerged processes Fruit wines and special

aan
mashes with low total concentrations of alcohol
were first used in submerged processes. With
a

Table 8.7 Additives for acetic acid fermentation

Gram/m nutrient solution

ee
Surface Submerged
process process
Sugar solution from grain 200 1000
_ hydrolysate
Ammonium phosphate 96 480
Magnesium sulfate 24 120
Calcium citrate 24 120
Calcium pantothenate 0.24 12
Figure 8.8 Diagram of a trickling generator for vinegar
146 / CHAPTER 8 / ORGANIC ACIDS
Mechanical
foam separator
FUNDAFOM?
Electro motor
Nozzle for
effluent gas

Socket for
inoculation Separator cone nest.

Air-suction pipe Substrate feed-line

Reactor vessel

Heat exchanger plates

Central suction tube

EFFIGAS® turbine
Bearing

Harvest-valve

Electro motor

Figure 8.9 Submerged fermenter for acetic acid production (Chemap)

through a pipe from the top of the fermenter. during the process and when the concentration
Heat exchangers control the temperature and sinks to 0.05-0.3% (about 35 hours), 50-60% of
mechanical foam eliminators must usually be in- the solution is removed and replaced with a new
stalled. Household vinegar (13% acetic acid) is mash containing 0-2 g acetic acid/100 ml and
produced in a semicontinuous, fully automatic 10-15% ethanol. Fully continuous processes
process, under continuous stirring and aeration, with yields of 98% at 40°C have also been de-
beginning with a starting material that contains scribed.
7-10 g acetic acid/100 ml and 5% ethanol. The With the submerged process, the production
ethanol concentration is measured continuously rate per m? is 10 times higher than with the sur-
 8.5 LACTIC ACID / 147

face fermentation and about 5% higher than with — an NAD-dependent lactate dehydrogenase to py-
the trickling generator process. Other advantages ruvate, which is reduced stereospecifically to
are: lower capital investment per production L(+) or (D—) lactic acid (Figure 8.10).
amount, only 20% of the plant area required for Theoretically, two moles of lactate are pro-
the installation, capability of conversion to other duced from 1 mole of glucose. Over 90% of this
mashes in a short time, and low personnel cost theoretical yield is actually attained in practice.
due to fully automatic control. The organisms used are Lactobacillus del-
brueckii and L. leichmannii when glucose is used
Recovery The acetic acid obtained in the sub-
as substrate, Lactobacillus bulgaricus with whey,
merged process is turbid due to the presence of
and Lactobacillus pentosus with sulfite waste li-
bacteria and the product must be clarified by fil-
quor. These organisms are facultative rather than
tration. Plate filters and filter aids are generally
obligate anaerobes, and therefore the bioreactors
used. Once the filtrate is obtained, K,(Fe(CN),) is
need not be run with complete oxygen exclusion.
used to decolorize the final product when desir-
In addition to 12-13% glucose and
able.
(NH,),HPO, (0.25%), the fermentation medium
must contain B vitamins. Production is carried
8.5 LACTIC ACID out in 25-120 m3 fermenters at 45-50°C with an
The first microbial production of an organic acid excess of CaCO, added to maintain the pH be-
was the production of lactic acid, carried out in tween 5.5 and 6.5. The fermentation time is
1880. Today chemical methods for the produc- about 72 hours. Since free lactic acid is toxic to
tion of lactic acid are very competitive at the same the organism, procedures for removing the prod-
cost as biological processes. Two kinds of lactic uct continuously have been studied. In one ap-
acid bacteria are recognized, heterofermentative proach, electrodialysis has been used. In another
and homofermentative. The heterofermentative procedure, continuous culture was done in a
organisms produce a great number of byproducts membrane reactor, leading to the production of
and are not suitable for commercial purposes. 80 g/I-h.
With homofermentative organisms, as little sub- Only L+ lactic acid is produced commer-
strate as possible is converted into cell material cially. When the fermentation has completed, the
plus byproducts and as much as possible is me- broth is heated to dissolve the calcium lactate.
tabolized into lactic acid. The biosynthesis of lac- The heated broth is filtered and the calcium pre-
tic acid from glucose proceeds via glyceralde- cipitated by addition of sulfuric acid. After con-
hyde-3-P, 1,3-di-P-glycerate and pyruvate. The centration of the lactic acid, it is further purified.
reducing power produced during the oxidation Total microbial production per year amounts to
of glyceraldehyde phosphate is transferred with about 20,000 metric tons.

Glucose
i
I
U Lactate
Glyceraldehyde-3-P
P NAD
Glyceraldehyde Lactate
phosphate dehydrogenase
dehydrogenase NADH,
Pyruvate
1,3-di-P-Glycerate
Figure 8.10 Lactic acid pro-
duction from glucose ewer
148 / CHAPTER 8 / ORGANIC ACIDS

CH,OH O itaconic acid acrylonitrile copolymer is more

Die teER
OH Aspergillus HO MN readily dyed than some other polymers.
Oryzae
Itaconic acid is produced by way of the tri-
HO O CH20H carboxylic cycle from cis-aconitic acid via decar-
OH boxylation. The enzyme cis-aconitic acid decar-
boxylase has been purified and characterized
6 -D-Glucono- Kojic acid (Figure 8.12). Another biosynthetic pathway
pyranose from pyruvate through citramalic acid, citraconic
acid, and itatartaric acid also results in itaconic
Figure 8.11 Biosynthesis of kojic acid acid.
The undesirable by-products succinic acid
and itatartaric acid are produced from itaconic
8.6 KOJIC ACID acid (Figure 8.13). Calcium inhibits the enzyme
itaconic acid oxidase and hence calcium additions
Kojic acid (5-hydroxy-2-hydroxymethyl-4-py- increase the yield of the desired product. By the
rone) has found minimal commercial use as a
feedstock in the plastics industry. For a long time,
it has been produced by the direct fermentation HO-CH-COOH
of glucose. Yields of 70-90% can be obtained
oe oe D-lsocitric acid
from glucose with Aspergillus flavus and A. oryzae
CHz COOH
(Figure 8.11). This acid can also be produced
from pyruvate, glycerol, acetate, or ethanol. The
|. Aconitic acid hydrolase
production using glucose is very similar to that HO

of the citric acid and itaconic acid processes. By

joes
changing the fermentation parameters, such as
C—COOR cis-Aconitic acid
stirring or aeration rate, some strains can be con-
CHz COOH
verted from citric acid or itaconic acid production
to kojic acid production.
Il. Aconitic acid decarboxylase

CH;COOH
8.7 ITACONIC ACID
‘imap Itaconic acid
In 1931, itaconic acid was first shown to be a CH
metabolic product of Aspergillus itaconicus. In the
same decade, it was discovered that some strains Figure 8.12 Biosynthesis of itaconic acid out of the tri-
carboxylic acid cycle
of A. terreus also excrete itaconic acid. Mutants
of both strains are used today for commercial
production.
At present there are only a few commercial CH, Itaconic acid
: CH,OH
systems which produce itaconic acid: 2 in West- Il oxidase |
age OST a OIA 100)
=
ern Europe, one each in America, USSR and Ja-
CHzCOOH CH>COOH
pan. The main use of itaconic acid is in the plastic
industry. Itaconic acid forms copolymers with its
Itaconic acid Itatartaric acid
esters and other monomers; these are used in the
paper industry for wall paper and other paper Figure 8.13 Destruction of itaconic acid by conversion to
products and in the production of adhesives. An itatartaric acid
 REFERENCES / 149

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A. terreus is used only in batch submerged Ju, N. and S.S. Wang. 1986. Continuous production of
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porous disk bioreactor. Appl. Microbiol. Biotechnol.
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of citric acid accumulation in Aspergillus niger. Enzyme
Microb. Technol. 8: 258-259.
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to produce citric and isocitric acid. II. Synthesis of citric In: Rose, A.H. (ed.). Primary products of metabolism.
and isocitric acid by C. lipolytica mutants and pecul- Academic Press, New York.
iarities of their enzyme systems. Appl. Microbiol. Bio- Schulz, G. and H. Rauch. 1975. Citronensaure (Citric acid),
technol. 23: 378-383. pp. 626-636. In: Ullmanns Encyklopadie der techn-
Fukui, S. and A. Tanaka. 1980. Production of useful com- ischchen Chemie., Vol. 9.
pounds from alkane media. Adv. Biochem. Eng. 17: 1- Siebert, D. and H. Hustede. 1982. Citronensaure-Fermen-
30: tation. Biotechnologische Probleme und Moglichkeiten
Gledhill, W.E., I.D. Hill. and P.H. Hodson. 1973. Citrate der Rechnersteuerung. (Biotechnological problems and
production from hydrocarbons by use of a nonsterile, possibilities for computer control of the citric acid fer-
semicontinuous cell recycle system. Biotechnol. mentation.) Chem. Ing. Technik 54: 659-669.
Bioeng. 15: 963-972. Siebert, D. and G. Schulz. 1979. Citric acid production by
Greenshields, R.V. and E.L. Smith. 1974. The tubular re- fermentation. Intern. Microbiol. Food Ind. Congr.,
actor in fermentation. Proc. Biochem. 9: 11-17, 28. Paris.
Hardy, G.P.M.A., M.J. Teixeira de Mattos, O.M. Neijssel, Sodeck, G., J. Modl, J. Kominek, and W. Salzbrunn. 1981.
. and D.W. Tempest. 1987. Effect of the growth condi- Production of citric acid according to the submerged
tions on gluconate and 2-ketogluconate production fermentation process. Proc. Biochem. 16: 9-11.
from glucose by three pseudomonads in continuous Treton, B., M.-T. Le Dall, and H. Heslot. 1978. Excretion
culture. Poster 9th ISCC “Continuous culture in bio- of citric and isocitric acids by the yeast Saccharomy-
technology and environment conservation.” 19-24. copsis lipolytica. Europ. J. Appl. Microbiol. Biotechnol.
July, Hradec Kralove, CSSR. 6: 67-77.
 Amino acids

The most well-known manufacturer of L-lysine

9.1 INTRODUCTION
is Eurolysine Co. (France) and of L-aspartic acid
The taste-enhancing properties of glutamic acid the two French firms Orsan S. A. and Rhåne-
were discovered in 1908 in Japan, and commer- Poulenc S. A.
cial production of sodium glutamate from acid In 1985 the world production of biochemi-
hydrolysates of wheat and soy protein began cally produced amino acids was 460,000 tons, of
soon after. In 1957, L-glutamic acid was discov- which 370,000 tons per year were sodium glu-
ered as a product in the spent medium of Co- tamate and 70,000 were L-lysine. It is anticipated
rynebacterium glutamicum, and this organism that increased demand will result in yearly in-
subsequently became the major source of sodium creases of around 10%.
glutamate. This development was an enormous Further increases in the production of amino
boost to the fermentation industry in Japan, and acids are expected because of the following de-
fermentative processes for the production of velopments:
amino acids as well as nucleotides (see Chapter
10) have been almost exclusively Japanese de- + The isolation of improved production cul-
velopments. i tures through the addition of recombinant
Thirteen of the 17 major manufacturers are DNA and protoplast fusion techniques to the
situated in Southeast Asia (Japan, Korea, Tai- currently used mutation methods to isolate
wan), the foremost Japanese firms being Ajino- derepressed strains.
moto Co., Kyowa Hakko Kogyo Co. and Tanabe Combining chemical synthesis with microbial
Seiyaku Co. The most important western man- and enzymatic processes.
ufacturers of sodium glutamate are Stauffer Adaptation of existing processes to the use of
Chemical Co. (USA) and Orsan S. A. (France). less expensive starting materials.

150
 9.3 METHODS OF PRODUCTION / 151

¢ Optimization of scale-up conditions. proteins with those essential amino acids which
occur at suboptimal levels. Aspartame® (L-a-as-
9.2 COMMERCIAL USES OF AMINO partyl-L-phenylalanine methyl ester) is a major
ACIDS product in the soft-drink industry, serving as an
artificial sweetener in so-called ”diet” soda. The
Amino acids have extensive industrial applica- sweetness level of aspartame in aqueous solution
tions. About 66% of the amino acids produced is 200-times higher than a 3-4% sucrose solu-
are used in the food industry, 31% as feed ad- tion. As the starting material for the industrial
ditives, in 4% in medicine and cosmetics and as production of aspartame, L-phenylalanine and L-
starting materials in the chemical industry. In the aspartic acid are used, both of which are products
food industry amino acids are used alone or in of industrial microbiology. The significance of as-
combination to enhance flavors. The flavor-en- partame can be seen from the fact that during
hancing effect of sodium glutamate has been the period from 1981 to 1985 L-phenylalanine
mentioned; sodium aspartate and D,L-alanine production rose from 50 to over 3000 metric tons,
are added to fruit juices to round off the taste, and similar rates of increase occurred for aspartic
and glycine is added to foods containing swee- acid. By means of genetic engineering it has been
teners. L-Cysteine improves the quality of bread possible to obtain an organism capable of pro-
during the baking process and acts as an antiox- ducing an alternating copolymer of phenylala-
idant in fruit juices. L-Tryptophan combined nine and aspartic acid, which can be converted
with L-histidine also acts as an antioxidant and into asp-phe dipeptide by enzymatic splitting.
is used to keep powdered milk from getting ran- The latter is esterified to produce aspartame.
cid. Aspartame® (L-aspartyl-L-phenylalanine Many amino acids are used in medicine, par-
methyl ester), which is made from L-phenylal- ticularly as ingredients of infusion solutions in
anine and L-aspartic acid, is used as a low-calorie post-operative treatment.
sweetener in soft drinks. In the chemical industry, amino acids are
Plant proteins are often deficient in essential used as starting materials for the manufacture of
amino acids such as L-lysine, L-methionine, L- polymers, such as polyalanine fibers and lysine
threonine, or L-tryptophan. The lysine contents isocyanate resins. Poly-y-methylglutamate is
of some foods and feeds are given in Table 9.1. used as a surface layer in the manufacture of
Lysine is added to bread in Japan, and in some synthetic leather, and the N-acyl derivatives of
countries soy products are enhanced by the ad- some amino acids are used in the manufacture
dition of methionine. L-Lysine and DL-methio- of cosmetics and as surface-active substances.
nine are used to upgrade animal feeds, and the Urocanic acid, used as a suntanning agent, is pro-
addition to foods of L-threonine and L-trypto- duced by the biotransformation of histidine. An-
phan is being developed. In view of the scarcity other amino acid, glycine, is used as a starting
of nutritious food in the Third World, there will material for the production of the herbicide gly-
be an ever-increasing need to supplement plant phosate, and threonine serves a similar purpose
for azthreonam (see Section 13.2).
Table 9.1 L-lysine content of several foods and feeds
Food/feed Lysine Food/feed Lysine 9.3 METHODS OF PRODUCTION
content content
(%) (%) Amino acids that are currently in commerical
Corn 0.21 Soy meal 2.90
production are summarized in Table 9.2. Fer-
Oats 0.50 Dry yeast 3.40 mentation processes have been developed for all
Barley 0.40 Skim milk powder 2.50 amino acids except glycine, L-cysteine and L-cys-
Wheat 0.60 Meat-meal 2.60
tine, but not all are in commercial use. In the
152 / CHAPTER 9 / AMINO ACIDS

Table 9.2 World production of amino acids; production processes and applications
Amino acid Annual production Production methods Application
(metric tons)

L-Alanine 130 il, Bre Flavor enhancer

D,L-Alanine 700 2 Flavor enhancer

(beverage production)
L-Arginine 1000 1, 3a Infusions
Therapy (liver diseases)
Cosmetics
L-Aspartic acid 4000 il, Se Therapy
Flavor enhancer
Aspartame® production
L-Asparagine 50 1,2 Therapy
L-Cysteine 700 1 Bread production
Antioxidant
Therapy (bronchitis)
L-Glutamic acid 370,000 3a Flavor enhancer
L-Glutamine 500 3a Therapy (ulcer)
Glycine 5000-6000 2 Component of sweeteners
L-Histidine 200 3a,1 Therapy (ulcer)
L-Isoleucine 150 3a Infusions
L-Leucine 150 1,3a Infusions
L-Lysine 70,000 3a, 3c É Feed additive
Infusion solution
DL-Methionine 70,000 2 Feed additive
L-Methionine 150 3c Therapy
L-Ornithine 50 3a, 3c Liver therapy
L-Phenylalanine 3000 3a, 3c Infusions
Therapy
Aspartame® production
L-Proline 100 3a Infusions
L-Serine 50 3a, 3b Cosmetics
L-Threonine 160 3a Feed additive
L-Tryptophan 200 3a, 3c Infusions
L-Tyrosine 100 i 136 Infusions
Precursor for L-DOPA
synthesis
L-Valine 150 3a, 3c Infusions
Production methods: 1. Extraction of protein hydrolysates; 2. Chemical synthesis; 3a. Direct fermentation;
3b. Microbial transformation of precursors; 3c. Use of enzymes or immobilized cells
(Soda et al., 1983; Kinoshita, 1987)
 9.3 METHODS OF PRODUCTION / 153

commercial manufacture of amino acids there are DL-R-CHCOOH

three rival processes: ee aes H,0
| NHCOR’
1. The extraction of amino acids from protein |
hydrolysates. This method is used to obtain |
L-cysteine, L-cystine, L-leucine, L-asparagine Aminoacylase
and L-tyrosine. (Aspergillus
2. Chemical synthesis. The production of gly- KØAGEN SL oryzae)
cine, D,L-alanine, D,L-methionine and D,L-
tryptophan always involves chemosynthesis. |
|
Chemical synthesis is cheaper than microbial
production, but the chemical product is the
optically inactive mixture of the D- and L-
| L-R- CHCOOH
R'COOH
isomers. NH,
3. Microbiological production, discussed be- |
low. | +
There are three approaches to microbiological |
production. The first, outlined in Table 9.3, is by SAR BR SEC OD I
direct fermentation of amino acids using dif- NHCOR'
ferent carbon sources, such as glucose, fructose,
molasses, starch hydrolysate, n-alkanes, ethanol, Figure 9.1 Use of an enzyme for the separation of a ra-
glycerol, acetate, propionate, etc. Fermentations cemic mixture in the manufacture of L-amino acids (R=
side chain of an amino acid; R'= e.g. -CH,)
using methanol as a starting material have been
studied extensively because of the low cost of
this substrate, but have not yet been used com-
L-valine (monthly production of 5—20 metric
mercially due to the low yields.
tons).
The second approach is by converting in-
e L-a-amino acids can be produced by reduc-
expensive intermediate products via biosyn-
tive amination of a-ketoacids using amino
thesis. For example glycine, which is inexpen-
acid dehydrogenases. The most commonly
sive, can be converted to L-serine (see Table 9.4).
used dehydrogenase is an enzyme from Ba-
The third approach is by the use of enzymes
cillus strains such as B. megaterium, which re-
or immobilized cells, sometimes in continuous
quires NADH as coenzyme. L-alanine, L-leu-
processes involving enzyme-membrane reactors
cine, and L-phenylalanine are produced in
(see Section 5.3). The most common reactions of
this way either with immobilized cells or in
this type are:
enzyme-membrane reactors in combination
. Use of a stereospecific amino acylase from with a NADH regeneration system.
Aspergillus oryzae to selectively split a chem- + D,L-5-monosubstituted hydantoins, which
ically synthesized a D,L-amino acid that had are easily produced chemically, can be con-
been chemically acetylated. In this way, the verted by means of stereospecific enzymatic
L form of the amino acid is selectively lib- cleavage to optically active D- or L-amino
erated (see Figure 9.1). A German company, acids. The hydantoin cleavage by either D-
Degussa AG, has been using an enzyme- or L-hydantoinase leads to the production of
membrane reaction technique of this type for D- or L-N-carbamoyl-amino acids. The car-
many years to produce L-alanine, L-methi- bamoyl residue is chemically or enzymati-
onine, L-phenylalanine, L-tryptophan, and cally removed with the corresponding car-
154 / CHAPTER 9 / AMINO ACIDS

Table 9.3 Production of amino acids by fermentation

Amino acid Strain used Genetic Yield Carbon source
characteristics (8/1)
D,L-Alanine Microbacterium ArgHx' 60 Glucose
ammoniaphilum
L-Alanine Pseudomonas No. 483 Wild type 17.5 Glucose
L-Arginine Serratia marcescens Transduction 50 Glucose
AT 428 (aru argR2 Canavanine’
argA2)
L-Aspartic acid Escherichia coli K1-1023* 56 Fumaric acid
L-Glutamic acid Corynebacterium Wild type 100 Glucose
glutamicum
Brevibacterium 98 Acetate
flavum
Arthrobacter 82 n-Alkanes
paraffineus
L-Glutamine Corynebacterium Wild type of 58 Glucose, with
glutamicum glutamic acid high biotin and
producer NH,Cl content
L-Histidine Serratia marcescens rDNA 40 Sucrose
L 120 (pSH 368)
L-Isoleucine B. flavum Eth: 30 Acetate
L-Leucine Brevibacterium Ile- Met” TA: 28 Glucose
lactofermentum
L-Lysine B. lactofermentum AEC'Ala- 70 Glucose
AJ 11204 CCL'ML'FPs
B. flavum Hom':*y Thre- 75 Acetate
L-Methionine C. glutamicum Thr- Eth MetHx: 2 Glucose
KY 9276
L-Ornithine C. glutamicum Arg” 26 Glucose
L-Phenylalanine B. lactofermentum SMT'PFP'Dec« 25 Glucose
Tyr'Met-
L-Proline C. acetoacidophilum Uncharacterized mutant 108 Glucose +
Glutamic acid
L-Serine B. lactofermentum SG 4.5 Glucose
L-Threonine E. coli rDNA 55 Sucrose
VL 344 (pYN7)
L-Tryptophan E.coli JP 4114 rDNA 2335 Glucose
L-Tyrosine C. glutamicum Phe- PEP: PAP: PAT: 18 Glucose
Pr-20 TyrHx«
L-Valine Brevibacterium TA: 31 Glucose
lactofermentum
No. 487
“Technique used for the production of L-aspartic acid between 1960 and 1973. Resistanc
e: AEC S-(6-Aminoethyl)-L-
cysteine; ArgHx Arginine hydroxamate; CCL, a-Chlorcaprolactam; Dec, Decoyinine; Eth Ethionine; FP,
B-Fluoropyruvate; MetHx Methionine hydroxamate; ML , y-Methyl-L-lysine; 5 MT 5-Methyltryptophan; PAP p-
Amino- phenylalanine; PAT p-Aminotyrosine; PFP p-Fluorophenylalanine;
SG Sulfaguanidine; TA 2-Thiazolalanine;
TyrHx Tyrosine hydroxamate. Auxotrophs: Ala, alanine; arg, arginine; hom, homoserine; ile, isoleucine; met,
methionine; phe, phenylalanine; thr, threonine; tyr, tyrosine. aru: block in arginine degradation; argR: biosynthetic
enzyme is derepressed; argA: N-acetylglutamate synthesis is insensitive to feedback inhibition
by arginine.
 9.3 METHODS OF PRODUCTION / 155

Table 9.4 Enzymatic production of amino acids

Amino acid Enzyme Enzyme- Reaction Yield Efficiency
producing (8/1) (%)
microorganism

L-Alanine Aspartate Pseudomonas L-Aspartate—L-Alanine + CO, 600 100

decarboxylase (RC) dacunhae
L-Aspartic acid Aspartase (IC) Escherichia coli Fumarate +NH,*— 560 99
L-Aspartic acid
L-Cysteine Cysteine desulfhydrase Bacillus 6-Chloro-L-alanine + Na,S— ‘ 70 80—85
(RC) sphaericus L-Cysteine + NaCl + NaOH
L-DOPA Tyrosinase (RC) Erwinia herbicola Pyrocatechol + Pyruvate +NH,*— 59 95
(L-3,4-Di- L-DOPA + H,O
hydroxyphenyl-
alanine)
L-Leucine L-Leucine Bacillus a-Ketoisocapronic acid+ NH,*+ 42 max.
dehydrogenase (EMR) sphaericus NADH-L-Leucine +H,O0+ NAD* 21015997
L-Lysine D-Amino- Achromobacter DL-Aminocaprolactam + H,O— 100 100
caprolactam racemase, obae, L-Lysine
L-Amino- Cryptococcus
caprolactam hydrolase laurentii
(RC)
L-Phenyl- L-Phenylalanine Brevibacterium Phenylpyruvate + NH,* 37
alanine dehydrogenase (EMR) sp. —L-Phenylalanine+H,O glade

Acetamidocinnamate Bacillus Acetamido cinnamic acid— 8 94

amidohydrolase (RC) sphaericus L-Phenylalanine
Phenylalanine Rhodotorula trans-Cinnamic acid+ NH,” 18 70
ammonia lyase (RC) glutinis —L-Phenylalanine
Aspartate phenyl- E. coli L-Aspartate + Phenylpyruvate 30 98
alanine transaminase —L-Phenylalanine + Oxalacetate
(EMR)
D-Hydroxyisocaproate Lactobacillus D,L-Phenyllactate
dehydrogenase casei —Phenylpyruvate
L-Hydroxyisocaproate Lactobacillus
dehydrogenase confusus
L-Phenylalanine Rhodococcus Phenylpyruvate + NH," 28
dehydrogenase (EMR) sp. —L-Phenylalanine + H,O nt fo br

L-Serine Serine hydroxymethyl Klebsiella Glycine+ HCHO 450 88

transferase (CCE) aerogenes —L-Serine

L-Tryptophan Serine hydroxymethyl Klebsiella Glycine + HCHO 200 95 based

transferase aerogenes —L-Serine on indole

Tryptophanase (EMR) E. coli L-Serine+ Indole

—L-Tryptophan + H,O
L-Tyrosine Serine hydroxymethyl Klebsiella Glycine + HCHO
transferase aerogenes —L-Serine
B-Tyrosinase (RC) Erwinia L-Serine + Phenol 26 61 based
herbicola —L-Tyrosine+ H,O on glycine

Aida et al. (1986); Groeger and Sahm, 1987; Schmidt et al., (1987)
cell extract.
EMR, enzyme /membrane reactor; IC, immobilized cells; RC, resting cells; CCE, crude
156 / CHAPTER 9 / AMINO ACIDS

bamolyase. L-hydantoinases have been acids, since cell metabolism regulates production.
found in only a few bacteria, for example Fla- In strain development, several methods of elim-
vobacterium ammoniagenes, whereas D-hy- inating the regulation control are available.
dantoinases are found in numerous bacteria,
e The use of auxotrophic mutants which can
actinomycetes, yeasts, and some strains of As-
no longer form the regulatory effector or co-
pergillus. In addition to the use of this ap-
repressor (usually the end product), and ex-
proach for the production of L amino acids,
crete the intermediate of the biosynthetic
it can also be used for the manufacture of D-
pathway which is just in front of the block.
N-carbamoyl amino acids and D amino acids,
+ The use of regulatory mutants. For instance,
which find a role as side-chain precursors for
if an excess of the end product of an un-
the production of semi-synthetic penicillins
branched biosynthetic pathway is to be pro-
and cephalosporins.
duced, mutants with a feedback-insensitive
In some manufacturing processes, several en- key enzyme are selected either from anti-
zymatic reactions are coupled. For example, L- metabolite-resistant strains or from a popu-
alanine is produced by sequential conversion of lation of revertants from auxotrophs.
fumarate to L-aspartic acid with immobilized e Use of genetic recombination to combine
cells of E. coli and Pseudomonas dacunhae. This auxotrophy or regulatory mutants from dif-
process, which has been in commercial operation ferent strains into a single hybrid strain. In
by Tanabe Seiyaku in Japan since 1982, involves Serratia marcescens transduction with phage
the connection of two reactors in series. PS20 has been used for creating suitable hy-
Enzymes or immobilized cells are used in the brids. Protoplast fusion has been used in Co-
production of L-alanine, L-aspartic acid, L-di- rynebacterium glutamicum, Brevibacterium lac-
hydroxy phenylalanine (DOPA), L-lysine, L-me- tofermentum, and B. flavum.
thionine, L-phenylalanine, L-tryptophan, L-ty- e Use of recombinant DNA technology to am-
rosine, and L-valine. The most important plify the gene dosage of the rate-limiting en-
approaches used are summarized in Table 9.4. zyme. This procedure leads to yield increases
The current interest in the production of L-phen- if the amount of enzyme synthesis rises in
ylalanine as a precursor for aspartame produc- parallel with the gene copy number. This ap-
tion has led to the development of numerous proach has been used with E. coli, albeit with-
competing biotransformation reactions, which out significant advantages. In this case, the
have not as yet replaced the direct fermentation cloning vector pBR322 was used to increase
process. the gene dosage of genes for the production
of the following amino acids: L-glutamic acid,
L-histidine, L-lysine, L-phenylalanine, L-pro-
9.4 STRAINS FOR AMINO ACID
line, L-threonine, and L-valine. The yields
PRODUCTION
were in most cases significantly lower than
Although microorganisms which excrete glu- the yields of commercial production strains
tamic acid are easily obtained from nature, it has of coryneform bacteria. However, cloning
been more difficult to obtain natural isolates systems developed for these commercial
which excrete other amino acids for large-scale strains have permitted more success for sev-
production. Extensive screening has led to the eral Japanese companies. Kyowa Hakko has
isolation of only a few strains which excrete D,L- used recombinant DNA technology to create
alanine and L-valine. Figure 9.2 shows the over- hybrid strains of Corynebacterium glutamicum
all biosynthetic pathways for the other amino for the production of L-cysteine, L-histidine,
acids. Natural isolates rarely excrete these amino L-isoleucine, L-phenylalanine, L-serine, and
 9.5 PROCESS CONTROL / 157

Glucose (C,)

Histidine (C.N,) <— Pentose(C, )

Glycine(C,N)
Tetrose(C, )

Triose (C, ) ——_————» Serine(C,N)

Phenylalanine (C,N)
'
Tyrosine (C,N) ae RK mete Cysteine (C,NS)
c
Tryptophan (C..N.)
1—2
i

Alanine (C,N) «—_—_——— Pyruvat

(C,) e—» (C,) -»Valine(C.N)

Lysine (C,N,) <+——— DAPIC,N,)

Acetyl (C,) Leucine(C,N)

Methionine (CNS)
!
Aspartate (C,N) =—Oxalacetate(C,)
Citrate (C,)
Threonine (C,N)

Isoleucine (C,N) Proline (C.N)

a-Ketoglutarate(C, )———»Glutamate(C.N)
'
Arginine (C,N_)

Figure 9.2 Biosynthesis of amino acids using glucose as the carbon source

L-threonine; Ajinomoto has modified Brevi- netic characteristics, and the yields obtained.
bacterium lactofermentum for the production Processes using various carbon sources have
of L-histidine, L-phenylalanine, L-proline, L- been developed for different production strains.
threonine, and L-tyrosine; Tanabe has altered In this list, only the processes with the highest
Serratia marcescens for the production of L- published yields have been given and the actual
" histidine, L-proline, and L-threonine. industrial yields are assumed to be significantly
higher.
Mutants producing excess amounts of amino
acids have also been isolated by combining sev-
9.5 PROCESS CONTROL
eral of the above methods.
Table 9.3 lists the strains available for the Amino acid production runs for 2—4 days in batch
microbial production of amino acids, their ge- processes in vessels containing up to 450 mi.
158 / CHAPTER 9 / AMINO ACIDS

Methods using continuous processes have been tured economically for use as feed additives, the
developed, but have not yet been implemented demand for L-threonine and L-tryptophan can-
in commercial plants. not yet be filled due to the low yields of existing
Infections due to bacteriophages may cause processes.
considerable losses in production. Phage-resis- In order of their importance, the processes for
tant mutants and chemical agents which inhibit the production of L-glutamic acid, L-lysine and
phage reproduction may be used to avoid phage L-tryptophan are discussed below. Further de-
infection. Intensive research on the maintenance tails for these and other amino acids are available
of pure cultures (sterilization of equipment, me- in the references at the end of this chapter.
dia, and air) is underway; the sterilization of nu-
trient solutions usually takes place continuously.
Because of the high rate of sugar breakdown
9.8 L-GLUTAMIC ACID
and the high respiratory activity during the fer-
mentation, a high oxygen requirement is exhib-
Production strains
ited. The optimal aeration rates for individual
processes depend on the strain, the substrate, L-glutamic acid is manufactured predominantly
and the biosynthetic pathway. Excess heat must by microbial means, although it is also manu-
be simultaneously dissipated, since the processes factured chemically. Japanese researchers began
are carried out at temperatures between 28- developing a direct fermentation process because
38°C. The pH is kept constant between 6.8-8.0, the D,L-glutamic acid which is formed by chem-
depending on the process; gaseous NH, is fre- ical synthesis is the racemic mixture. In screening
quently used for pH control and is simultane- about 2,000 microorganisms on different media,
ously metabolized as a nitrogen source. During
L-glutamic acid production was found to occur
the fermentation of L-glutamic acid, such critical
in a wide variety of bacteria, streptomycetes,
parameters as aeration rate, temperature, pH,
yeasts, and fungi. The isolation of Corynebacte-
and antifoam dosage are all automatically reg-
rium glutamicum (synonym: Micrococcus glutam-
ulated and in a few cases are under computer
control. Biosensors have been developed which icus) was accomplished in 1957. It was imme-
permit continuous measurement of the amino diately used industrially by Kyowa Hakko
acid concentration in the fermenter. For instance, because of its high excretion of glutamic acid.
a glutamic acid sensor has been prepared using Other industrially important strains with L-glu-
the glumatine synthase enzyme obtained from a tamic acid excretion of at least 30 g/1 belong to
thermophilic organism, Bacillus stearothermophi- the genera Corynebacterium, Brevibacterium, Mi-
lus. crobacterium, or Arthrobacter. Morphologically
and physiologically, these glutamic acid-produc-
9.6 PRODUCT RECOVERY ing strains resemble C. glutamicum: They are usu-
ally Gram-positive, nonsporulating, nonmotile
Centrifugation is used at the end of the fermen- bacteria. Moreover, all glutamic acid producers
tation process to separate the cell material. The
require biotin, lack or show little activity of a-
amino acids are obtained after acidification
ketoglutarate dehydrogenase, and show in-
through precipitation at the isoelectric point, ion
creased activity of glutamate dehydrogenase. In
exchange chromatography, electrodialysis, or ex-
addition, some Brevibacterium and Corynebacte-
traction with organic solvents.
rium mutants have lower isocitrate lyase activity.
Further strain development has led to the iso-
9.7 PRODUCTION OF INDIVIDUAL
lation of mutants which overproduce glutamic
AMINO ACIDS
acid even in the presence of high concentrations
The demand for amino acids is increasing. While of biotin. For instance, a lysozyme-sensitive mu-
L-lysine and D,L-methionine can be manufac- tant of C. glutamicum is able to convert 40% of
 9.8 L-GLUTAMIC ACID / 159

the added carbon source to L-glutamic acid even " During glutamic acid formation in the presence
in the presence of 100 ug/1l biotin. of 4CO,, the a-carboxyl group of glutamate is
Successful hybrids have also been con- labeled radioactively. Oxalacetate carboxylase
structed by cloning Brevibacterium or Corynebac- and the NADP-dependent malate enzyme are in-
terium DNA into Brevibacterium or Corynebacte- volved with the CO, fixation process. The malate
rium recipients. enzyme catalyzes the carboxylation of pyruvate
to malate (Figure 9.3). These anaplerotic se-
Biosynthesis of glutamic acid - quences complete the TCA cycle with C, dicar-
boxylic acids. Malate is then trahsformed via ox-
The glucose carbon source is broken down into alacetate into citrate and isocitrate, which either
C, and C, fragments by glutamic-acid-producing serve as preliminary stages to the glutamic acid
microorganisms through the Embden-Meyerhof- formation or are channeled into the glyoxylate
Parnas (EMP) pathway and the pentose-phos- cycle. Particularly with acetate as a carbon
phate-cycle, and the fragments are channeled source, the energy gain and the formation of in-
into the tricarboxylic acid (TCA) cycle. The EMP termediates are carried out chiefly via the gly-
pathway is more common under conditions of oxylate cycle for C. glutamicum (Figure 9.4). Thus
glutamic acid production. The key precursor of there is competition between a) the isocitrate
glutamic acid is a-ketoglutarate, which is formed lyase reaction, which forms succinate and gly-
in the TCA cycle via citrate and isocitrate and oxylate (necessary for optimal growth) and b) the
then converted into L-glutamic acid through re- isocitrate dehydrogenase reaction which leads to
ductive amination with free NH,” ions (Figure the key precursor a-ketoglutarate. The stoichi-
9.3). This last step is catalyzed by the NADP- ometry for glutamic acid formation from glucose
dependent glutamate dehydrogenase. The or acetate as a carbon source would be as follows:
NADPH, required at this stage of the reaction is
furnished through the preceding oxidative de- C,H,,0, + NH, + 1.5 0,-> C,H,O,N+ CO, + 3 H,0,
carboxylation of isocitrate to a-ketoglutarate by
the enzyme isocitrate dehydrogenase. The 3 C,H,O, + NH, + 1.50,—> C,H,O,N+CO,+3H,0
NADPH, is then regenerated by the reductive
One mole of glutamic acid is produced from 1
amination of a-ketoglutarate:
mole of glucose or from 3 moles of acetate. Ex-
periments with resting cells have shown that the
Isocitrate NADP L-Glutamate
actual conversion rate is between 50-70 mole%.
Some part of the yield reduction is due to the
reversibility of the malate enzyme reaction and
the decarboxylation of oxalacetate to CO).
CO, + a@ -Keto- a -Ketoglutarate
glutarate NADPH, + NHj
Effect of permeability on glutamic acid
The strain used commercially for glutamic acid production
production has a block in a-ketoglutarate de-
Production and excretion of excess glutamic acid
hydrogenase. In the absence of NH," ions, a-ke-
is dependent upon cell permeability. Increased
toglutarate accumulates because of the interrup-
permeability in glutamic-acid-producing bacteria
tion of the TCA cycle. Thus efficient anaplerotic
can be attained in several different ways:
sequences are necessary to provide TCA cycle
intermediates which are required for other cell e Through biotin deficiency
reactions. Studies on the pathway of biosynthesis ¢ Through oleic acid deficiency in oleic acid
have been carried out using labeled compounds. auxotrophs
160 / CHAPTER 9 / AMINO ACIDS

Glucose

Glucose-6-P

Shale Ge:

pa
ae 3-P <— — —-—--—— Pentose-5-P
|

måki ak ni Acetyl-CoA

ae

Citrate
Acetyl -CoA
a

\ Isocitrate
Glyoxylate — NADP
Fumarate oy ee

uccinate
= a-Ketoglutarate
NADPH, +NH,
© NADP

Glutamine ae Glutamate
Acetyl-CoA
ADP» P.
CoA

N-Acetylglutamate
Figure 9.3 Biosynthesis of L-glutamic acid using glucose as the carbon source. Glyoxylic acid cycle, thin lines; pentose
phosphate cycle, broken lines. 1, Malic enzyme; 2, Oxalacetate carboxylase; 3, Isocitrate dehydrogenase; 4, Isocitrate
lyase; 5, Glutamate acid dehydrogenase; 6, Glutamine synthetase
(Modified according to Kinoshita and Nakayama, 1978)
 9.8 L-GLUTAMIC ACID / 161

Acetate dry weight is accumulated intracellularly, so that

biosynthesis is stopped because of feedback in-
hibition. On the other hand, growth in a biotin-
Acetyl-P
deficient medium causes membrane damage
through reduction of phospholipid synthesis,
Acetyl-CoA leading to a changed ratio of saturated to unsat-
urated fatty acids. Under these conditions, intra-
cellular glutamic acid can be excreted. It has been
postulated that a biotin-containing acetyl-CoA-
carboxylase, which catalyzes the first stage of
fatty acid synthesis from acetyl-CoA to malonyl-
Oxalacetate Citrate CoA, is involved in fatty acid biosynthesis. Sat-
urated fatty acids (C,,-C,,) repress this acetyl-
Acetyl-CoA CoA-carboxylase. Hence it is understandable
that oleic acid auxotrophs or strains to which sat-
cis- Aconitate urated fatty acids have been added synthesize
Malate |
cell membranes with lower phospholipid con-
tent; such strains also excrete L-glutamic acid in
K Glyoxylate the presence of biotin.
Isocitrate The addition of penicillin in the logarithmic
Fumarate growth phase promotes significant excretion of
L-glutamic acid, even in the presence of biotin.
Succinate Penicillin is added to fermentations in media
containing large amounts of biotin 8-12 hours
after inoculation of the fermenter. A penicillin
« -Ketoglutarate

|
concentration is selected (between 5-300 units/
ml) so that the bacterial growth rate is reduced
Glutamate to a level corresponding to the rate in low-biotin
media.
Figure 9.4 Biosynthesis of L-glutamic acid using acetate The use of penicillin or saturated fatty acids
as the carbon source makes possible the commercial use of inexpen-
sive culture medium components, such as sugar
¢ Through the addition of saturated fatty acids cane or sugar beet molasses, which otherwise
(C16-C;8) or fatty acid derivatives cannot be utilized due to their high biotin con-
+ Through the addition of penicillin tent. The discovery of the role of cell permeability
¢ Through glycerol deficiency in glycerol aux- in the production of glutamic acid has thus made
otrophs possible some rational approaches to the indus-
trial production of this important amino acid.
Ali glutamic-acid-producing strains have a
growth requirement for biotin, an essential coen-
Conditions of manufacture
zyme in fatty acid synthesis. In the presence of
biotin concentrations >5 ug/l, increased oleic Under optimal culture conditions, glutamic-acid-
acid synthesis results in a high phospholipid con- producing bacteria convert about 50-60% of the
tent of the cell membrane. Cells with high phos- added carbon source to L-glutamic acid. If less
pholipid content are incapable of excreting glu- favorable fermentation conditions are used so
tamic acid; up to 25-35 ug L-glutamic acid/mg that low glutamic acid production is obtained, an
162 / CHAPTER 9 / AMINO ACIDS
increase of cell mass and an excretion of lactate, fermentation when these inexpensive carbon
succinate, a-ketoglutarate, glutamine and N-ace- sources are used. In Europe, only beet molasses
tylglutamine have been observed. Factors which is considered an inexpensive carbon source (pro-
affect glutamic acid fermentation are described portion of manufacturing cost: 26%) but in other
below. areas of the world cane molasses is chiefly used.
In Japan, where acetate is inexpensive and read-
Carbon sources A wide variety of carbohydrates ily available in large quantities, extensive studies
can be used as carbon sources in the fermentation have been carried out to use this carbon source
process. Among the monosaccharides, glucose but for industrial production, cane sugar molas-
and sucrose are frequently used, and fructose, ses or starch hydrolysate are still the main carbon
maltose, ribose, and xylose find some role. Of sources used.
the unrefined carbohydrate sources, sugar cane Processes using methanol, ethanol, acetal-
and sugar beet molasses are most important, but dehyde, or n-alkanes have also been developed,
starch hydrolysates are also frequently em- but the cost-effectiveness of these processes de-
ployed. Since molasses has a high biotin content pends largely on the price of petroleum. Figures
(0.4-1.2 mg/kg for cane molasses; 0.02-0.08 9.5 and 9.6 show the progress of typical fermen-
mg/kg for beet molasses), penicillin or fatty acid tations with glucose and acetate as carbon
derivatives (e.g., Tween 60) must be added to the sources.

9g Urea additions
ag
Qa

fhe
sore
‘N04
ec
5
= cS)
©) (0)
Pe 2
52 13)
=
® a)
=
eS
TU
=
2_
© 9
O 2
Do
SJ £ro)
®
x
1 8

fe) 3
Si oO
= ©
v 2
5 E
o a
=0L3
O oO Figure 9.5 Production of L-glu-
72 96 tamic acid with Corynebacterium
glutamicum No. 541 using glucose
Fermentation time (hr) as the carbon source
 9.8 L-GLUTAMIC ACID / 163

Nitrogen sources In addition to ammonium salts, - growth factor; for media based on an n-alkane,
ammonia (gaseous or in aqueous solution) can supplementation with thiamine may be neces-
be used as a nitrogen source. In the industrial sary.
manufacture of glutamic acid, ammonia feeding
permits pH control and obviates the problem of O, Supply Optimal glutamic acid yields are ob-
ammonia toxicity. Most glutamic acid-producing tained at a K, value of 3.5X10-° mole:O,/
bacteria possess urease activity, so that urea is atm-min-ml. The oxygen concentration should
also frequently used as a nitrogen source, In the be neither too low nor too high. Under oxygen
acidic pH range with excess ammonia, glutamine deficiency, excretion of lactate and succinate oc-
is produced instead of glutamic acid. curs, whereas excess oxygen in the presence of
an ammonium ion deficiency causes growth in-
Growth factors The optimal biotin concentration hibition and production of a-ketoglutarate; in
is dependent on the carbon source used. In media both cases glutamic acid yields are low.
with 10% glucose, it is 5ug/l, in media with
lower glucose concentrations it is considerably
Production processes
lower, and for acetate it is between 0.2-1.0 ug/
1. Some strains require L-cysteine as an additional A typical fermentation from glucose with Brev-

Acetic acid addition

{oo 1 | Papa USE

raønh
9 iI opatn abs/l aus ME tel
E ja
I \ iS)

if d eS o
8 | IO 13
°
| ibeanon &
£ |=]
oa Se

=20 {One| conse

3 g |3 3
3S e
O
6/9 SGSERED
°
STER
i
S +30 +06 3
iD
€ \ | iS)

= |
oe
G! | |
| 20 4, Obes
dl
a
ag
<
5 10 02 +100

Figure 9.6 Production of L-glu-

0 70 40 60 a a
tamic acid with Brevibacterium fla-
vum No. 2247 using acetate as the
carbon source (Tsunoda et al.,
1961) ° Fermentation time (hr)
164 / CHAPTER 9 / AMINO ACIDS

ibacterium divaricatum (NRRL B-231) runs as fol- acid yields with different carbon sources are
lows (Miescher, 1975): listed in Table 9.5.
. Seed culture: glucose 40 g; K,HPO, 1.0 g;
MgS0,:7 H,O 0.5 g; yeast extract 1.0 g; urea
8 g; tap water 1 1; 16 h incubation at 35°C.
9.9 L-LYSINE
¢ Main culture: glucose 121 g; ammonium ace-
tate 5 g; molasses from starch saccharification Lysine is an amino acid essential for animal and
6 g; KH,PO, 1.2 g; K,SO, 1.2 g; MgSO, (an- human nutrition. It occurs in plant proteins only
hydrous) ser hesO7- 7 ho OUG6eppm, in low concentrations; addition of lysine can
MnSO,°H,O 6 ppm; antifoam agent Hodag therefore increase the quality of plant foods. The
K-67 0.1 ml; tap water 1 1. Inoculum volume: market for lysine is increasing. Lysine is pro-
6%. duced today only by microbial processes and a
variety of approaches for its production have
At the beginning of the fermentation, 0.65
been developed.
ml/1 of oleic acid is added. The pH is set at 8.5
with ammonia and is automatically maintained
at 7.8 during the course of the fermentation. After
Lysine production via diaminopimelic acid
beginning growth of the culture (about 14 hours),
the temperature is increased from 32-33°C to Lysine-histidine double auxotrophic mutants of
38°C. After metabolism of the glucose down to Escherichia coli (ATCC 13002) produce diami-
a level of 0.5-2%, glucose feeding is done until nopimelic acid (DAP) on a molasses medium
the fermentation is completed; 160 g/l are fed with a yield of 19-24 g/l. The entire fermenta-
on the average. Aeration is controlled so that the tion solution, including the cell material, is sub-
CO, content in the exhaust gas does not exceed sequently incubated with Aerobacter aerogenes
4.5 vol%. The glutamic acid content is analyzed (ATCC 12409) at 35°C. After 20 hours, the DAP
hourly. As a rule, the fermentation is stopped has been quantitatively decarboxylated to L-ly-
after 30-35 hours with a glutamic acid yield of sine. One complication with this procedure is
about 100 g/l. If molasses from starch sacchar- that the DAP formed during the fermentation is
ification is substituted for glucose, the glutamic a mixture of meso- and LL-forms; since only
acid yield is 94 g/1 after 36 hours. The glutamic meso-DAP can be decarboxylated into L-lysine,

Table 9.5 Processes for glutamic acid production with different carbon sources
Carbon source Organism Yield (g/l)
Sugar beet molasses C. glutamicum >100
Glucose + Ammonium acetate Brevibacterium divaricatum 100
Acetate B. flavum 98
Ethanol Brevibacterium sp. 136 59
n-Alkanes Arthrobacter paraffineus : 62
Corynebacterium hydrocarboclastus 84
C. alkanolyticum (Glycerol-) 72
Benzoic acid Brevibacterium sp. 80
Methanol Methylomonas methylovora M12-4 i
 9.9 L-LYSINE / 165

the LL-DAP must be transformed into the meso portant lysine-excreting strains are listed later in
form by racemization before the decarboxylation this chapter (Table 9.7). The development of
step. high-yielding strains by mutation to auxotrophy
and to antimetabolite resistance has been carried
Conversion of DL-a-amino caprolactam out with B. lactofermentum.
Protoplast fusion between high-yielding
The enzymatic transformation of DL-amino cap- strains and wild strains of B. lactofermentum, Co-
rolactam into L-lysine takes place according to rynebacterium, and Brevibacterium mutants has
the following scheme: led to strains with improved growth properties
or higher efficiencies.
Racemization with
Achromobacter obae Cloning studies with E. coli, using plasmid
D-a-Amino- SEE
oe
L-a-Amino- pBR322 as a vector, have shown that only in
caprolactam caprolactam
(D-Aminocaprolactam
transformed strains that contain the dapA gene
racemase) does a significant increase in lysine production
Hydrolysis with occur (6.5 g/l). The enzyme coded by dapA, di-
Cryptococcus
laurentii hydrodipicolinate synthase (DDPS) is therefore
(L-Amino- indicated as a rate-limiting step for lysine bio-
caprolactam synthesis. Studies have also been carried out on
hydrolase)
the transformation of C. glutamicum with plasmid
L-Lysine pAC2 as vector for the DDPS-encoding gene.
Yeasts, such as Candida periculosa, Saccharo-
A 10% DL-amino caprolactam solution (pH 8.0) myces cerevisiae or Saccharomycopsis lipolytica
is added to 0.1% (w/v) acetone-dried cells of have been studied extensively for lysine produc-
Cryptococcus laurentii and of Achromobacter obae. tion. Lysine accumulates intracellularly in these
A conversion efficiency of 99.8% is obtained at organisms at concentrations up to 20% of the dry
40°C after 24 hours. The enzymatic process has weight. However, these yeasts cannot be used
been commercialized in Japan by the Toray Com- successfully in industrial processes because ly-
pany, which has a capacity of over 4000 tons per sine is not excreted into the medium.
year. In the Toray process, the enzyme D-a-ami-
nocaprolactam racemase from Achromobacter
obae has been expressed in a lysine auxotrophic Biosynthesis and regulation Lysine is synthe-
mutant of E. coli. sized in microorganisms either via the diami-
nopimelic acid pathway or the aminoadipic acid
pathway. However, in any single organism, only
Direct fermentation one of the two alternatives is used: Bacteria, ac-
Direct fermentation processes are now used tinomycetes, cyanobacteria (blue-green algae),
world-wide for the production of L-lysine. some phycomycetes, and protozoa use the DAP
pathway. Some phycomycetes, all ascomycetes,
Production strains Efficient L-lysine producers all basidiomycetes, and eucaryotic algae use the
are found among glutamic-acid-producing mu- aminoadipic acid pathway.
tants of Corynebacterium and Brevibacterium Biosynthesis via the DAP pathway in bacteria
which are homoserine auxotrophs or among me- is shown in Figure 9.7. Although two organisms
thionine-threonine double auxotrophs. High-ly- may use the same pathway, the manner in which
sine-producing strains are also found among or- this pathway is regulated may differ, as shown
ganisms resistant to the lysine antimetabolite S- by the comparison of Escherichia coli and the ly-
(8-aminoethyl)-L-cysteine (AEC). The most im- sine-producer Corynebacterium glutamicum in
166 / CHAPTER 9 / AMINO ACIDS
Methionine
CODE: ATP A OOF NADPH» B Ge

CHNH, coun og NH,—* Threonine -Isoleucine

CH CH, Cie
| 2 ADP | NADP Fi | Cc Pyruvate

COOH COO-P CHO 2 H40

Aspartate B-Aspartyl-P Aspartyl- p -
semialdehyde

SSS
ZA
COOH HOOC> =N=s COOH
I
CO 2,3-Dihydrodi -
| picolinate

(CH),
CHCOOH D RE G2
ie SuccinylCoA NADP
|
CO
| NZ
CH, a-Keto- CH, .coa HOOE COOH
| glutarate |
2 oh A'- Piperidine-2,6-
| COOH dicarboxylate
COOH
N-Succinyl - N-Succinyl- & -
LL- a,€-di- keto-L- & -
aminopimelate aminopimelate
COOH
|
Succinate CHNH,
|
COOH GODE
|
HC-NH, 4 NHS CH
| |
(CH>)3 ne (CH)3
| |
HC-NH, HC-NH, CH,
| | |
COOH COOH NH,
LL- a,€£ - Diamino- meso- « € -Di- Lysine
pimelate aminopimelate

Figure 9.7 Biosynthesis of L-lysine via the DAP pathway. A, Aspartokinase; B, Aspartic semialdehyde dehydrogenase;
C, Dihydrodipicolinate reductase; D, Dihydrodipicolinate synthase; E, Succinylketoaminopimelate synthase; F, Succi-
nyldiaminopimelate aminotransferase; G, Succinyldiaminopimelate desuccinylase; H, Diaminopimelate epimerase; I,
meso-Diaminopimelate decarboxylase
 9:9 LYSINE: 7167

® Lvgaiyi ae@
Asp pr Ase? === ASA Hp Hom — > Hom-P-—©Thr 2
| lle
|

| ee
|| hay
DAP !

:| rage Lysii “-—Met

Corynebacterium glutamicum

if feces a 4
ok See rons OSocean
ae cara inst eS Tel RS. TieFH
Sc eyeees a La era = aes

Asp; Asp-P—> ASA FRE Hom + =Hom-P-—Thr =lle

fia Be i |
Figure 9.8 Regulation of L-lysine hea | as ; io tbe geeta
and L-threonine biosynthesis in Esch- bet É .
erichia coli and C. glutamicum. 1, As- FN | |
partokinase; 2, Homoserine dehydro- Ret | | DAP |
genase. Dashed line, feedback | | | { |
inhibition; dashed and dotted line, | witht = ere, L._Met
repression: (From: Kinoshitavand Na= 9) 04 | = «= a: ona S saad
kayama, 1978) keyi GS eg ne ee TE a Escherichia coli

Figure 9.8. In Escherichia coli three distinct reg- In contrast to Escherichia coli, the regulatory
ulatory processes are involved: mechanism for lysine-producing strains, such as
Corynebacterium glutamicum or Brevibacterium
* Two isoenzymes of homoserine dehydrogen- flavum, is much simpler (Figure 9.8). There is
ase exist which are repressed by L-methio- only one aspartokinase and one homoserine de-
nine or L-threonine. hydrogenase. Aspartokinase is regulated via
¢ There are three isoenzymes of aspartokinase, multivalent feedback inhibition from L-threonine
one showing repression by L-methionine, the and L-lysine, as the experimental data in Table
second showing multivalent repression by L- 9.6 show.
threonine and L-isoleucine in addition to In both organisms, feedback inhibition due to
feedback inhibition by L-threonine, and the L-threonine and repression due to L-methionine
third showing feedback inhibition and regulate the homoserine dehydrogenase. Biosyn-
repression by L-lysine. thesis from aspartate semialdehyde to L-lysine
e Dihydrodipicolinate synthase, the first spe- has already been accomplished in wild strains
cific enzyme of lysine biosynthesis, shows without regulatory control. Thus good lysine-
feedback inhibition due to L-lysine. producers are classified among three mutant
types:
For all three of these enzymatic reactions,
regulatory mechanisms must be eliminated to ob- + The flux of aspartate semialdehyde to thre-
tain the overproduction of L-lysine which is nec- onine is reduced in homoserine auxotrophs
essary for its commercial preparation. which have a block in the homoserine de-
168 / CHAPTER 9 / AMINO ACIDS

Table 9.6 Effect of various amino acids on

Relative activity
aspartokinase activity
Added amino acid Relative B. flavum
(10 umol/ml each, aspartokinase
L-form) activity (%)
C. glutamicum Se Ql see
yee
-e—o—_ i
paces
aE ae
(Homoserine”) 100 : SE
Control 100 100 80 SSG ‘0
Threonine
Lysine
123
94
99
80
6 Nr. 2247 SS
A EOS FA3+I5
\,Be
Homoserine 94 102
Methionine 109 102
Threonine + Lysine 52 44 20 KR
Homoserine + Lysine 95 74 0 \\— Ui LS Be ee
Threonine + Methionine 129 114 0.2 05 2 5 10°) -20 50

(Nakayama, 1972) L-Lysine + L-Threonine (mM)

Figure 9.9 Effect of lysine and threonine on aspartoki-

hydrogenase. Because of the low threonine nase activity. No. 2247: wild type Brevibacterium flavum.
FA 1-30 and FA 3-115: AEC mutants (From Hirose et al.,
-content in the cell, the multivalent feedback
1978)
inhibition of aspartokinase is prevented de-
spite increased lysine formation.
e The same effect is obtained with a mutant in urease activity. Growth factors L-homoserine or
which homoserine dehydrogenase is super- L-threonine and L-methionine must be added,
sensitive to feedback inhibition through thre- but in suboptimal concentration to avoid unde-
onine. sirable regulatory effects. Soy protein hydroly-
e In comparison to the wild type, the aspar- sates or other inexpensive protein sources are fre-
tokinase of AEC-resistant mutants is insen- quently used. The biotin content in the medium
sitive to multivalent feedback inhibition, as must be over 30 ug/1 for optimal lysine produc-
Figure 9.9 shows. Excess aspartate semialde- tion. The biotin content in sugar cane molasses
hyde flows into lysine biosynthesis, because is usually high enough, but if sugar beet molasses
the conversion to L-threonine is prevented or starch hydrolysates are used, biotin must be
through feedback inhibition. In this simply added. Table 9.7 shows lysine production from
regulated system, strains with increased ly- different carbon sources.
sine formation can be developed in just a few A typical fermentation is shown in Figure
mutation steps. 9.10. The lysine yield is 44 g/l and the conver-
sion rate in relation to the sugar used approaches
Lysine excretion is accomplished through ac-
a maximum of 30-40%.
tive transport. In some organisms, a loss of lysine
A typical fermentation based on sugar cane
through decarboxylation to cadaverine occurs,
molasses with C. glutamicum (Strain No. 901;
but this destruction does not occur in Coryne-
(Hom) takes place as follows:
bacterium or Brevibacterium strains.
° First seed culture: glucose 20 g; peptone 10
Conditions for commercial production Sugar cane 8; meat extract 5 g; NaCl 2.5 g; tap water 1
molasses is primarily used as a carbon source in liter.
industrial production; acetate, ethanol or alkanes e Second seed culture: sugar cane molasses 50
can also be used. Gaseous ammonia or ammo- 8; (NH,),SO, 20 g; corn steep liquor 50 g;
nium salts are used as nitrogen sources; urea is CaCO, 10 g; tap water 1 1.
also used if the producing microorganism has + Main culture: Sugar cane molasses 200 g
 9.10 L-TRYPTOPHAN / 169

Table 9.7 Lysine production on media with different carbon sources

Carbon source Organism Genetic Yield
characteristics (g/l)
Glucose C. glutamicum Hom Leu 34
Glucose C. glutamicum Hom” Leu” AEC" 39
Glucose B. lactofermentumAJ11204 AEC'Ala-CCL'ML'FPs 70
Acetate B. flavum Hom!eky Thr- 75
Ethanol B. lactofermentum AEC: 66
n-Alkanes (C,,-C,;) Nocardia alkanoglutinosa AEC: 29

(ACE: Resistance to S-(8-aminoethyl)-L-cysteine; CCL, a-chlorcaprolactam; ML, y-methyl-L-lysine; FPs, 6-

fluoropyruvate sensitive. Auxotrophs: Ala, alanine; Hom, Homoserine; Leu, Leucine; Thr, Threonine)

50

cooOo

ne
{eo

2 Growth
o—o

£7De 40 ,
nm)
562
(0.D. pe. ge
a
fo}

= ae
60 +12 atte

Won
af
ay
(g/l)
acid
Acetic
s—s
30 i 40-08

— —~p

—
8 Oo

20 =

|
oO
ee
a
(g/l)
L-Lysine
e—e
ee
ee
bd

SITE
ee
.

T
————

12 24 36 48
Fermentation time (hr)

Figure 9.11 Production of L-lysine with Brevibacterium

o—ae flavum using acetate as the carbon source (From Tanaka
et al., 1971)
(g/l)
weight
dry
Cell
(%)
Glucose
Oe 0 20) 7.30) =40" 250°.
-60 70
duction medium is as follows: acetic acid 7 g;
Fermentation time (hr)
KH,PO, 0.4 g; MgSO,°7 H,O 0.4 g; FeSO,"7 H,O
Figure 9.10 Production of L-lysine with Corynebacterium 0.01 g; soy protein hydrolysate 35 g; glucose 30
glutamicum No. 901 using glucose as the carbon source g; biotin 50 wg; thiamine HCI 40 ug; tap water 1
(From Nakayama, 1972) 1. The pH is kept at 7.4 through acetic acid feed-
ing (60% acetic acid and ammonium acetate in

(based on sugar content); soy protein hydrol- molar ratio of 100:25; 3% glucose). After 48
ysate 18 g; tap water 11. The pH is kept neu- hours at 33°C the lysine content is 75 g/l; a con-
version rate of 29% is obtained based on acetic
tral with aqueous ammonia. Duration of fer-
acid and glucose used.
mentation, 60 hours. Impeller speed 150 rpm;
aeration 0.6 vvm; temperature 28°C.
9.10 L-TRYPTOPHAN
Figure 9.11 shows a fermentation by Brevi-
bacterium flavum (Hom'*, Thr’) with acetate as Tryptophan has traditionally been produced
the carbon source. The composition of the pro- through chemical synthesis or through fermen-
170 / CHAPTER 9 / AMINO ACIDS

tative or enzymatic conversion of chemically syn- g

&

thesized intermediates. The procedures have in- 218

Å Sts
volved primarily the use of immobilized cells or I: sie]
215]
immobilized enzymes. To optimize the process, $ : 55
recombinant DNA technology has been used. ke
ce
o
oO

The disadvantage of the enzymatic process is the

oOo Cc

2 ie 50
high cost of the starting materials, indole, serine, = =—

© 5
pyruvate, or anthranilic acid. Because of this, ex- = 60 + 45
tensive work has been carried out to develop a < meg e

direct fermentation of L-tryptophan. Because of / “poe

the complex regulatory mechanism of trypto- el Ea ° 40

phan biosynthesis, these studies have not led to
= oOo wa
titers sufficiently high so that L-tryptophan could
be used in the food industry, although direct fer-
wo
mentation can be used to produce this amino acid
for medical uses.
nNw

Table 9.8 Production of tryptophan by transformation

of precursors
ead
ee
Organism Precursor Amount Yield
used (g/l)
S648 S60 723 58494-96
(2/1)
Fermentation time (hr)
Hansenula Anthranilic 4.2 SA
anomala acid
Bacillus subtilis Anthranilic 5.0 bys)
Figure 9.12 Production of L-tryptophan from anthranilic
(Anthranilate-) acid acid by Hansenula anomala. Cultured on the following me-
Candida utilis Anthranilic 4.2 6.4 dium (%): glucose 5; NH,NO, 0.3; KH,PO, 0.05;
acid
Na,HPO,°12 H,O 0.2; NaH,PO,°2 H,O 0.1; MgSO,°7 H,O
Claviceps purpurea Indole 13 165 0.05; NaCl 0.01; FeSO,°7 H,O 0.001; yeast extract 0.1;
Escherichia coli Indole + 3.0 anthranilic acid 0.2; CaCO, 2.0 (From Terui, 1972)
DL-Serine 6.0 SER
Bacillus subtilis Anthranilic 1575 15.6
(SFT‘AG’) acid (feeding)
(SFT, 5-fluorotryptophane; AG, 8-azaguanine)
From Kinoshita and Nakayama (1978), with additions.

Table 9.9 Enzymatic processes for the production of L-tryptophan

Enzyme Organism Reaction

D-Tryptophan hydantoin racemase Flavobacterium DL-Tryptophan hydantoin + H,O—

L-Tryptophan hydantoin hydrolase aminogenes L-Tryptophan + CO, + NH,
N-Carbamoyl-L-tryptophan
hydrolase
Tryptophanase Proteus rettgeri Indole + Pyruvate + NH,—L-Tryptophan + H,O
Achromobacter Indole + L-Serine—L-Tryptophan + H,O
liquidum
E. coli
Tryptophan synthetase E. coli Indole + L-Serine—L-Tryptophan + H,O
 9.10 L-TRYPTOPHAN / 171

Fermentative conversion of intermediates Table 9.10 Mutants for the production of tryptophan
by direct fermentation with glucose
stages
Organism Genetic Yield
The most important fermentation processes are characteristics (g/l)
listed in Table 9.8. Figure 9.12 shows a typical Bacillus subtilis SFT", Arg” or Leu: 6
fermentation sequence with Hansenula anomala.
Enterobacter SMT'THA'Str'Tyr 10
Starch hydrolysate or molasses is used as a car- cloacaeTA 599
bon source and the feeding of sugar/NH,NO,- Corynebacterium Phe Tyr" SMT" 12
solution and anthranilate begins after the culture glutamicum TrpHx: 6FT: 4MT: PEP"
is grown. Px-115-97 PAP"
TyrHx' PheHx"
Brevibacterium SFT'AZ'SG'PFP'Tyr- 19
Enzymatic process for tryptophan flavumS-225

Table 9.9 lists enzymatic processes for L-tryp- Resistance: 5FT 5-fluorotryptophan; PFP p-fluoro-
phenylalanine; 5MT 5-methyltryptophan; TrpHx
tophan production. The methods include either Tryptophan hydroxamate; 6FT 6-fluorotryptophan; 4MT
the stereoselective hydrolysis of hydantoin com- 4-methyltryptophan; PAP p-aminophenylalanine; TyrHx
pounds derived by chemical synthesis or the bio- Tyrosine hydroxamate; PheHx Phenylalanine
hydroxamate; AZ azide; SG sulfaguanidine; THA
conversion of intermediates. thienylalanine; Str streptomycin.
In the hydantoinase technique, an enzyme Auxotrophy: Arg arginine; Leu leucine; Phe
phenylalanine; tyr tyrosine.

Erythrose- Phospho- COOH COOH COOH =

4-P enolpyruvate NH, N

— — — oe — --—— |

HO “OH “O-c CH,

| OH OH Coo Anthranilate Tryptophan HNH,

Shikimate Chorismate |
COOH
| COOH COOH

coe |COOH bo
ee co
NH
CHN H

oe
ee HO_ HOOC___CH, vie
CH,
| ——_— —_—_> —__
aes i x
= OH Phenyl- Phenyl-
BR OH OH pyruvate alanine
H-C-OH 5-Dehydro- Prephenate ola COOH
CH.OG quinic acid co CHNH,

|
3-Deoxy-D-arabino- CH, H,
heptulonic acid-7-P

p-Hydroxy- Tyrosine
phenyl-pyruvate
OH OH

Figure 9.13 Pathways for the biosynthesis of tryptophan, phenylalanine and tyrosine
172 / CHAPTER 9 / AMINO ACIDS

from Flavobacterium aminogenes which hydro- formed, the yield can be even higher (83.3 g/l),
lyzes D,L-tryptophan hydantoin is used. This resulting in a molar conversion efficiency of 96%.
strain has a reduced tryptophan-degrading abil- In a related process, an enzyme reactor contain-
ity and the enzyme is induced by the addition ing tryptophanase from E. coli has been used to
of D,L-tryptophan hydantoin to the medium. A convert indole and L-serine, resulting in a tryp-
100% conversion of the hydantoin to L-trypto- tophan concentration of 200 g/1 and an efficiency
phan is obtained (starting material, 5% D,L-tryp- of 95%.
tophan hydantoin; 40°C; 100 hours), the tryp- Another process involves the use of the en-
tophan formed being removed from the reaction zyme tryptophan synthetase which transforms
mixture by complex formation with inosine. indole and L-serine to L-tryptophan. In a mixed
The most widely used commercial process is culture of E. coli and Pseudomonas putida, using
the tryptophanase technique, which involves D,L-serine as the starting material, the racemi-
the conversion of indole using Proteus rettgeri. zation of the D-form of tryptophan leads to yields
The reaction mixture contains, per liter culture of 23 g/l (81% efficiency).
solution, 60 g indole (dissolved in 100 ml meth-
anol), 80 g sodium pyruvate, 80 g ammonium
Direct fermentation processes
acetate, 0.01 g pyridoxal phosphate, and 1 g so-
dium sulfate. After incubation at 34°C for 48 The possibilities for a direct synthesis of L-tryp-
hours, 75 g of L-tryptophan is produced. By pre- tophan starting with inexpensive carbon sources
cipitation of the tryptophan with inosine as it is are outlined in Table 9.10.

Partialy<_ L-Tyrosine
Fle
FI 4
p-Hydroxyphenyl-
\ pyruvate
PEP ad @s
+ —— DAHP -----= Chorismate ——_» Prephenate
EPS | NO
Ng Act
\ Phenylpyruvate

L-Tryptophan
oS Phenvlala
nine

Fl

Figure 9.14 Regulation of aromatic amino acid biosynthesis in Corynebacterium glutamicum. The main sites of regulation
are numbered, and the lines and symbols indicate the type of regulation. 1, DAHP synthetase; 2, Chorismate mutase;
3, Anthranilate synthase; 4, Prephenate dehydrogenase; 5, Prephenate dehydratase.
FI, Feedback inhibition; Rp, Repression; Act, Activation.
EP, Erythrose-4-P; PEP, Phosphoenolpyruvate; DAHP, 3-Deoxy-D-arabinoheptulonic acid-7-P.
(From Nakayama et al., 1976)
 REFERENCES / 173

The tryptophan biosynthetic pathway illus- " REFERENCES

trated in Figure 9.13 has been found in many
Aida, K., I. Chibata, K. Nakayama, K. Takinami, and H.
microorganisms, but there are considerable dif-
Yamada (eds.). 1986. Progress in industrial microbi-
ferences in the regulation of the individual or- ology. Vol. 24. Biotechnology of amino acid produc-
ganisms. In Corynebacterium glutamicum, for in- tion. Kodansha Ltd., Tokyo.
stance, tryptophan biosynthesis is regulated by Groeger, U., and H. Sahm.1987. Microbial production of
L-leucine from a-ketoisocaproate by Corynebacterium
the activity of DAHP synthetase and anthranilate glutamicum. Appl. Microbio. Biotechnol. 25: 352-356.
synthase (Figure 9.14). DAHP synthetase, which Hamilton, B.K., H.Y. Hsiao, W.E. Swann, D.M. Anderson,
catalyzes the condensation of erythrose-4-phos- and J.J. Delente. 1985. Manufacture of L-amino acids
with bioreactors. Trends Biotechnol. 3: 64-68.
phate and phosphoenol pyruvate, shows con-
Hirose, Y., K. Sano, and H. Shibai. 1978. Amino acids.
certed feedback inhibition by phenylalanine and Ann. Rep. Ferm. Proc. 2: 155-189.
tyrosine; tryptophan increases the inhibition so Hirose, Y., H. Enei and H. Shibai. 1985. L-Glutamic acid
that the activity of DAHP synthetase is inhibited fermentation. pp. 593-600. In: Moo-Young, M. (ed.).
Comprehensive biotechnology. Pergamon Press, Ox-
up to 90% in the presence of all three amino ford.
acids. Anthranilate synthase, the first enzyme of Kamiryo, T., S. Parthasarathy, and S: Numa. 1976. Evi-
tryptophan biosynthesis after the branch point, dence that acyl coenzyme A synthetase activity is re-
quired for repression of yeast acetyl coenzyme A car-
shows feedback inhibition and repression due to boxylase by exogenous fatty acids. Proc. Natl. Acad.
L-tryptophan. The DAHP synthetase of C. glu- Sci. USA 73: 386-390.
tamicum Px-115-97 is completely sensitive to reg- Kinoshita, S. 1987. 30 years of amino acid fermentations.
Vortrag. 4th European Congress on biotechnology,
ulation by phenylalanine and tyrosine. The max- Amsterdam.
imum yield is determined not only by the effect Kinoshita, S. and K. Nakayama. 1978. Amino acids. pp.
of regulatory mechanisms but also by the specific 209-261. In: Rose, A.H. (ed.). Economic microbiology,
Vol. 2. Academic Press, London.
activities of the individual enzymes of the bio-
Martin, J.F., R. Santamaria, H. Sandoval, G. del Real, L.M.
synthetic chain. More yield increases are to be Mateos, J.A. Gil, and A. Aguilas. 1987. Cloning sys-
expected from the application of recombinant tems in amino acid-producing Corynebacteria. Bio/
DNA technology to the development of dere- Technol. 5: 137-146.
Nakanishi, T., T. Azuma, T. Hirao, K. Hattori, and M.
pressed strains of tryptophan producers, follow- Sakurai. 1986. Process for producing L-lysine by fer-
ing the methods that have already been perfected mentation. U.S. patent 4,623,623.
with E. coli. The genes for the biosynthetic path- Nakayama, K. 1972. Lysine and diaminopimelic acid. pp.
369-397. In: Yamada, K., S. Kinoshita, T. Tsunoda and
way from anthranilic acid to tryptophan are pres-
K. Aida (eds.). The microbial production of amino
ent in a single cluster. A tryptophanase-negative acids. Kodansha, Tokyo.
strain with a deletion of the trp operon has been Nakayama, K. 1985. Lysine, pp. 607-620. Tryptophan,
transformed with a plasmid containing the trp pp.621-631. In: Moo-Young, M. (ed.). Comprehensive
biotechnology, Vol. 3. Pergamon Press, Oxford.
operon in which the gene for anthranilate syn- Schmidt, E., D. Vasic-Racki, and C. Wandrey. 1987. En-
thase was resistant to feedback inhibition by zymatic production of L-phenylalanine from the ra-
tryptophan. The E. coli strain Tna (pSC101 trp cemic mixture of D, L-Phenyllactate. Appl. Microbiol.
Biotechnol. 26: 42-48.
115-14), whose plasmid contains five copies per Soda, K., H. Tanaka, and N. Esaki. 1983. Amino acids,
genome, forms 4-5 g/l tryptophan when using pp. 479-530. In: Rehm, H.J. and G. Reed (eds.). Bio-
glucose as carbon source. By the addition of a technology, Vol. 3. VCH Publishers, Deerfield Beach,
FL
multicopy plasmid with the trp operon, it has Syldatk, C, D. Cotoras, A. Måller, and F. Wagner. 1986.
also been possible to obtain yields of 23.5 g/l Microbial, enantioselective hydrolysis of D, L-l-5-mon-
with E. coli strain JP 4114. osubstituted hydantoins for the production of D- and
L-aminoacids. BTF-Biotech-Forum 3: 9-18.
Tanaka, K., T. Suzuki, and S. Okumura. 1971. Production
of sugars and amino acids from hydrocarbons and pe-
trochemicals by microorganisms, pp. 165-170. 8th
World Petroleum Congress, Proceedings No. 5. Ap-
plied Science Publ., London.
174 / CHAPTER 9 / AMINO ACIDS

Teh, J., D. Leigh, G. Allen, H. Burrill, P. Cowan, and H. Tsuchida, T., K. Miwa, S. Nakamori, and H. Momose.
Camakaris. 1985. Direct production of tryptophan by 1984. Method for producing I-glutamic acid by fer-
Escherichia coli from simple sugars. Biotech 85 Asia 3: mentation. U.S. Patent 4,427,773.
399-402. Tsunoda, T., I. Shiio, and K. Mitsugi. 1961. Bacterial for-
Terui, G. 1972. Tryptophan, pp. 515-531. In Yamada, K., mation of L-glutamic acid from acetic acid in the grow-
S. Kinoshita, T. Tsunoda, and K. Aida (eds.), The mi- ing culture medium. (1) Cultural conditions. J. Gen.
crobial production of amino acids. Kodansha, Tokyo. Appl. Microbiol. 7: 18-29.
 10
Nucleosides,
nucleotides,
and related
compounds

The process of producing 5'-IMP and 5'-

10.1 INTRODUCTION
GMP through enzymatic hydrolysis of yeast
Microbially produced nucleosides and nucleo- RNA was developed in Japan in 1959 and was
tides have found wide use as flavor-enhancers commercialized in 1961. The development of di-
for food. The use of synthetic flavor-enhancers rect fermentation processes for nucleotide pro-
developed first in the Orient and has spread duction was accomplished at about the same
throughout the world. Since the beginning of the time, also in Japan. The discovery of synergism
seventeenth century, the Japanese have used dif- between the flavor-enhancing properties of so-
ferent substances (fungi, dried fish, seaweed, etc.) dium glutamate and the sodium salts of 5’-IMP
to enhance the flavor of foods. L-Glutamic acid and 5'-GMP was another research advance.
(see Chapter 9) and the histidine salt of inosinic IMP-Na,, and GMP:Na,, as well as mixtures of
acid were identified in 1908 and 1913 as flavor- sodium and calcium salts of both nucleotides, are
enhancing components of these substances. Fur- approved food additives and are frequently used
ther studies showed that a number of other sub- in combination with sodium glutamate in pre-
stances, all purine ribonucleoside-5'-monophos- pared food products. To enhance the flavor of
phates, also improve flavor. In descending order soups and sauces, for example, as little as 0.005-
of effectiveness, these are: guanylic acid (5'- 0.01% 5’-ribonucleotide additives is sufficient;
GMP), inosinic acid (5'-IMP) and xanthylic acid moreover, addition of these additives can help
(5'-XMP). Substances which have no effect are eliminate undesirable properties, such as the me-
5'-AMP, and the 2’ and 3’ isomers, as well as the tallic taste of cans. The amount of seasoning mix-
5'-deoxyribonucleotides, nucleosides and pyrim- ture consumed per meal is 0.1—-0.2 g; the mixture
idine nucleotides. contains 8-12% 5'-IMP and 1.5-2.0% 5’-GMP

175
176 / CHAPTER 10 / NUCLEOSIDES, NUCLEOTIDES, AND RELATED COMPOUNDS

added in combination with sodium glutamate

10.2 STRUCTURE AND BIOSYNTHESIS
(based on the Na glutamate content). Both hy-
drolytic RNA-splitting and direct fermentation The structures of the economically relevant com-
processes are about equally used in Japanese pounds are shown in Figure 10.1.
commercial production, for an annual production As seen in Figure 10.2, the biosynthesis of
of 3000 tons. purine-5’-ribonucleotides proceeds from 5’-
In addition to their use in the food industry, phosphoribosylpyrophosphate (PRPP), which is
nucleotides, nucleosides and related compounds formed from ribose-5-phosphate and ATP. The
are being studied for therapeutic purposes. The first complete purine derivative, inosinic acid (5'-
antibiotic and cytostatic effects of purine analogs IMP), is produced after several reaction steps and
(8-azaguanine, 6-mercaptopurine) are being 5'-IMP is then the precursor of 5'-AMP, 5’-XMP
tested, particularly for cancer chemotherapy. 9- and 5'-GMP. AMP biosynthesis involves the ac-
B-D-arabinofuranosyladenine is effective against tion of adenyl succinate lyase (step M, Figure
herpes simplex and herpes zoster in humans. 10.2), a bifunctional enzyme which also catalyzes
The substances which are microbially pro- reaction H to AICARP.
duced in Japan and their applications are given In GMP biosynthesis, IMP is oxidized (re-
in Table 10.1. The following Japanese companies action N) to XMP and aminated (reaction O) to
are producers of 5'-ribonucleotides and nucleo- GMP.
sides: Ajinomoto Co., Asahi Chemical Ind.,
Kyowa Hakko Kogyo Co., Takeda Chemical Ind.
Ltd. and Yamasa Shoyu Co. Ltd. R;
io N
Table 10.1 Nucleotides, nucleosides, and related
compounds manufactured in Japan
5 ys sd
Substance Amount Application
manufactured
(tons/year)
5'-IMP 2000 Flavor enhancer
5'-GMP 1000 Flavor enhancer
Inosine 25 Heart ailments
ATP 6 Muscular dystrophy
CDP-Choline 2 Stroke
FAD Small amounts Liver or kidney diseases
NAD Small amounts Liver or kidney diseases
Adenine il Leukopenia
Adenosine 5 Coronary deficiencies,
angina pectoris,
arteriosclerosis, high
blood pressure
5'-AMP ? Circulation problems,
rheumatism
CAMP ? Diabetes, asthma, cancer
therapy, biochemical
reagent
Orotic acid 20 Liver diseases
6-Azauridine 2 Cancer chemotherapy
Ara A (9-B- 2 Antiviral
D-arabino-
furanosyladenine)
(Yamada, 1977, with additions) Figure 10.1 Structure of the purine nucleotides
 10.2 STRUCTURE AND BIOSYNTHESIS / 177

&ocHz 0 ® Jee

ee | ey
Gin Glu OH OH ings Olea

PRPP PRA aS 5')

NNO
Me thy nyl- ©
Fe eH
“ADP ATP H
4 na) a Gln+ ATP
CHO eh
saat N i
ag ~CHO
ADP+Glu oF i

Rib-S® ue 5) Rib-5-®)
AIRP FGAM FGAR
©
. i COOH
HO |\ AspealP CH, 9 Fumarate Q
HN N aa ae ‘ eed H2N N
| ADP COOH | » © | S
Ri b-5'E) H2N ) HON Å
Rib-5-) Rib-5-P)
CAIRP SAICARP AICARP
N'° Formyt-
per
folat THFS

ig HN

N ~ LS
HoN

HCL
i
N

N
|
© a bes å Rib-5-P)
ea sp
OQ nao FAICARP
NAD
GTP
OH

SAMP<en
nee
H
Crs N
|
XMP
raerie/ ees
Rib54P)
©
SØ GMP

Figure 10.2 Biosynthesis of AMP and GMP.

PRPP = 5-Phospho-a-D-ribosylpyrophosphate; SAICARP = 1-(5'-Phosphoribosyl)-4-(N-Succinocar-

PRA = 5-Phospho-6-D-ribosylamine; boxamide)-5-aminoimidazole;
GAR = 5’ Phosphoribosylglycinamide; AICARP = 5-Amino-1-(5'-phosphoribosyl)-imidazole-
FGAR = 5'-Phosphoribosyl N'-Formylglycinamide; 4-carboxamide;
FGAM = 5’-Phosphoribosyl-N’ formylglycinami- FAICARP = 5-Formamido-1-(5’-phosphoribosyl)-im-
dine; idazole-4 carboxamide;
AIRP = 1-(5'-Phosphoribosyl)-5 aminoimidazole; SAMP = Adenylsuccinate
CAIRP = 1-(5'-Phosphoribosyl)-5 aminoimidazole-
4-carboxylate;
178 / CHAPTER 10 / NUCLEOSIDES, NUCLEOTIDES, AND RELATED COMPOUNDS

If an excess of AMP or GMP is formed, GMP 5-Phosphoribosyl-

can be transformed through GMP reductase into pyrophosphate (PRPP)
IMP, and AMP through AMP deaminase into
IMP, or through several steps into AICARP. oe
Phosphoribosylamine

10.3 REGULATION
The overproduction of purine nucleotides in wild |
type bacteria is prevented by feedback regula- SAICARP
tion. For commercial production, the regulatory
mechanisms must be partially eliminated. One ©
approach is to isolate auxotrophic mutants and
add the required end product in growth-limiting AICARP
concentration, so that feedback inhibition and
repression are prevented. Another alternative is
to isolate mutants with resistance to purine an-
alogs. IMP.
The regulation of GMP and AMP synthesis
in Bacillus subtilis (Figure 10.3) has several pe- a @
culiarities. IMP, the precursor of AMP and GMP,
is transformed mainly into GMP. IMP dehydro- XMP Succino- AMP
genase, the first enzyme of this biosynthetic
pathway, has a 10- to 30-fold greater specific ac-
tivity than adenyl succinate synthetase. Feedback
© AS
inhibition and repression of IMP dehydrogenase GMP | AMP
is caused by GMP and XMP; the last enzyme of
the chain, GMP synthetase, is only slightly af- Figure 10.3 Regulation of purine nucleotide biosynthesis
fected by GMP. Under these conditions the pro- in Bacillus subtilis. 1, PRPP amidotransferase; 2, IMP de-
hydrogenase; 3, Adenylsuccinate synthetase; 4, GMP syn-
duction of AMP is increased. AMP synthesis is thetase; 5, Adenylsuccinate lyase (From Shiio, 1979)
chiefly regulated by feedback inhibition of the
first common enzyme, PRPP amidotransferase.
There is asymmetric control by AMP, but the a. hydrolysis of yeast RNA by microbial en-
regulatory effect of GMP is considerably lower: zymes
50% of the inhibition of PRPP amidotransferase b. breakdown of cellular RNA by endogenous
is accomplished by 0.2 mM AMP or 2 mM GMP. cell enzymes, leading to excretion of 5'-mon-
Both enzymes of AMP biosynthesis, adenyl suc- onucleotides
cinate synthetase and adenyl succinate lyase, are c. chemical hydrolysis of yeast RNA into nu-
regulated by AMP. cleosides with subsequent chemical phospho-
rylation.
2. Direct fermentation using mutants with a
10.4 PRODUCTION block in nucleotide biosynthesis, in which

Different methods are used to produce nucleo- endproduct regulation is eliminated (as dis-
cussed above).
tides:
a. manufacture of nucleosides by fermentation
1. Enzymatic or chemical breakdown of nu- and their chemical phosphorylation
cleic acids through b. direct manufacture of 5'-nucleotides
 10.4 PRODUCTION / 179

through fermentation g'mole:O,/atm:min'ml), using either sulfite

c. microbial conversion of bases of nucleosides waste liquor or molasses. In Japan, a large pro-
to 5'-nucleotides. duction plant may produce 10,000-20,000 tons
of yeast cells per year.
Methods 1a, 1c, 2a and 2b are used for in-
dustrial production of 5'-IMP and 5'-GMP. The cell mass produced in this process is 35
g/l, with an RNA content of 10% (sulfite waste
liquor) or 15% (molasses). The cell material is
Production of 5’-IMP and 5'-GMP by separated and dried by means,of yeast separa-
enzymatic hydrolysis of RNA tors, and the RNA is then extracted with hot al-
kaline saline (5-20% NaCl, 8 h, 100°C). After
The breakdown of RNA was the first method
separating the cell residue, the RNA is precipi-
used to produce 5’-nucleotides commercially. As
tated by adding HCI or ethanol and is dried. The
late as 1975, 50% of the 5’-nucleotides in Japan
crude preparation, which has an RNA content of
were still obtained using this method. The multi-
70-90% with a molecular weight distribution of
stage process consists of the following steps: 1)
10,000-150,000, can be used directly for enzy-
growth of yeast cells containing a high RNA con-
matic hydrolysis.
tent; 2) extraction of RNA from cell material; 3)
Another process for the production of nu-
production of enzymes used in RNA hydrolysis;
cleotides makes use of acidophilic, methanol-uti-
4) enzymatic hydrolysis of RNA; and 5) isolation
lizing bacteria such as Methylobacter acidophilus.
and purification of 5'-IMP and 5'-GMP.
Enzymatic hydrolysis As Figure 10.4 shows, sev-
Obtaining RNA The nucleic acid content of dif- eral distinct enzymatic reactions can be used for
ferent microorganisms varies considerably: the hydrolysis of RNA. For optimal production
of 5’-IMP and 5'-GMP, organisms must be used
Microorganisms DNA content (%) RNA content (%)
which have higher activities for the enzymes
Bacteria 0.37-4.5 5 -25 which catalyze steps a—d (Figure 10.4) than for
Yeast 0.03-0.52 2.5-15
steps e-g, in order to avoid the production of
Fungi 0.15-3.3 0.7-28
undesirable substances. Two organisms, Penicil-
lium citrinum and Streptomyces aureus, have been
The RNA of the cell consists of 5% messenger found to contain enzymes which meet these re-
RNA, 10-15% transfer RNA and 75-80% ribo- quirements, and enzymatic RNA hydrolysis on
somal RNA. Because of their relatively low DNA an industrial scale is carried out exclusively with
content, yeasts are the best sources of RNA. these two organisms.
Moreover, there are well-developed fermenta- One process makes use of hydrolysis with
tion and recovery techniques for yeast. The yeast nuclease P, from Penicillium citrinum. A pig-
Candida utilis is most commonly used, followed ment-free mutant from P. citrinum is cultivated
by Saccharomyces cerevisiae. The RNA content of on wheat bran for 5 days at 30°C. The aqueous
yeast cells is very dependent on culture condi- extract contains the thermostabile nuclease P,,
tions. A high proportion of RNA is found in the which quantitatively splits the 3’-5'-phospho-
early logarithmic phase, particularly in media diester linkage of polynucleotides and the 3'-
with a low carbon/nitrogen ratio. The RNA con- phosphomonoester linkage of mono- and oli-
tent of cells growing on molasses or glucose can gonucleotides. The undesired phosphomonoes-
be further increased by adding zinc ions (0.25 terase activity, which produces 3’-nucleotides,
ppm) and phosphate (0.15%). The yeast fermen- can be almost completely inactivated by heat
tation is generally done in continuous culture in treatment. To eliminate effects of the residual
an airlift fermenter (aeration rate, K,>20X10~° phosphatase activity (optimal temperature
180 / CHAPTER 10 / NUCLEOSIDES, NUCLEOTIDES, AND RELATED COMPOUNDS

Oligonucleotide
(5'-terminal Phosphate) Nucleoside
Deaminase
d
Endo- Exo-
nuclease nuclease Phosphatase
a b e
5'- AMP
Nucleotide
Exonuclease D N-ribosidase Base

BRA
ee
5'-UMP a
ae
Ribose-5-P
Polynucleotide 5'-CMP
ee SEA Nucleotide
Nucleoside pyrophosphorylase

diphosphatase g

Nucleoside-5- Base+ PRPP

diphosphate

Figure 10.4 Enzymes which are involved in the production of GMP and IMP via RNA hydrolysis (see text for expla-
nation)

45°C), hydrolysis of a solution of 2% RNA is zyme activities of different mutants compared

performed in 4 hours at pH 5.0 at the optimal with the wild type. Mutant A-5 is the best mutant
temperature of nuclease P, (65°C). In this reac- because of its clearly reduced phosphatase activ-
tion, 5'-GMP, 5'‘-AMP, 5’-CMP, and 5'-UMP are ity. The production of the enzymes takes place
produced. The 5’-AMP is deaminated by adeny]l during a 30-hour incubation in a submerged cul-
deaminase from Aspergillus oryzae to 5’-IMP. ture (Ky=2.72X10° g-Mol-O,/atm:min:ml) at
A process with immobilized nuclease P, has 28°C on a medium containing starch hydroly-
also been described. sate, soy meal, and corn steep liquor. Typical
Separation of the 5’-nucleotides formed progress of a 6 m? fermentation of Streptomyces
through this enzymatic hydrolysis is performed aureus is given in Figure 10.5. Because of the high
by means of anion exchange chromatography. 5'-nucleotidase activity of the S. aureus enzymes,
Another process involves hydrolysis with nu- RNA hydrolysis is carried out at a different tem-
cleases from Streptomyces aureus. S. aureus forms perature and pH. A 0.5-1.0% RNA solution is
a mixture of endo- and exonucleases; any 5’- hydrolyzed for 10 hours at 42-65°C in a pH
AMP is thus deaminated by AMP deaminase to range between 7.0-8.0.
5'-IMP. By isolating appropriate mutants, re- The purification of 5'-GMP and 5’-IMP is
searchers have attempted to reduce the amounts done by means of a preliminary adsorption with
of a 5’-nucleotidase and an unspecific alkaline activated carbon, then ion exchange chromatog-
phosphatase which occur in the enzyme mixture raphy, followed by a fractional precipitation with
produced by S. aureus. Table 10.2 shows the en- methanol.
 10.4 PRODUCTION / 181

Table 10.2 Enzyme production by Streptomyces aureus mutants relative to the wild type
Strain Endonuclease Exonuclease AMP deaminase 5'-Nucleotidase Alkaline
phosphatase
Wild type 100 100 100 100 100
K-1 310 180 110 160 20
A-5 400 210 150 180 10
S-8 100 80 100 60 10

produced via an intermediate 2',3’-cyclic phos-

A
4
<Q phodiester. However, nucleosides can be pro-
a
8 4
duced quantitatively from RNA by heating to
130°C for 3-4 hours in calcium hydroxide. The
|400) 4
oOo
chemical phosphorylation of these nucleosides to
= NMN o
E ees Ta 5'-IMP and 5'-GMP then can be done, after the
= E deamination of adenosine to inosine.
2 oD
2 =a.

Polls:
S 300+
H
e +30 Production of 5’-IMP by direct
2
pad
fermentation
(=
=
5'-IMP can be produced fermentatively by var-
ious methods:
¢ Through microbial production of inosine and
subsequent chemical phosphorylation to 5'-
IMP
+ Through direct fermentation to 5'-IMP
¢ Through fermentative production of adeno-
sine or 5'-AMP and subsequent chemical or
enzymatic conversion into 5’-IMP
¢ Through microbial conversion of chemically
synthesized hypoxanthine into 5’-IMP.

(units/ml)
Phosphatase
5-Nucleotidase
alk.
deaminase
5-AMP
x—
Exonuclease
—1,
s—a
e—e,
For economic reasons only, the first two pro-
cesses are used commercially.

Fermentation time (hr)

Fermentative production of inosine The cell
membrane is not permeable to nucleotides but is
Figure 10.5 Fermentation process for nuclease produc- permeable to the dephosphorylated derivatives,
tion with Streptomyces aureus the nucleosides. When a cell synthesizes 5'-IMP,
it cannot be excreted through the cytoplasmic
membrane; however, it can be dephosphorylated
Production of 5'-IMP and 5'-GMP through by the cell and then can be excreted, so that it
chemical hydrolysis of RNA accumulates in the medium as inosine. Because
In the chemical hydrolysis of RNA under alkaline of the regulatory effect of AMP and ADP on
conditions, 5’-nucleotides are not produced di- PRPP amidotransferase in Bacillus subtilis, the
rectly, but a mixture of 2'- and 3’-nucleotides is adenine nucleotide content of the cells must be
182 / CHAPTER 10 / NUCLEOSIDES, NUCLEOTIDES, AND RELATED COMPOUNDS

kept at a low level to produce IMP. This is ac- optimal pH range is 6.0-6.2, the optimal tem-
complished through a block in the AMP biosyn- perature 30-34°C. Maximal inosine accumula-
thesis (absence of SAMP-synthetase). Reduced tion is obtained with an oxygen transfer coeffi-
nucleosidase activity, absence of IMP-dehydro- cient of K,=5.8-7.0X10-* g:Mol:O,/
genase and/or an increase in 5'-nucleotidase ac- atm-min-ml. At the same time, the CO, content
tivity also favor the accumulation of inosine. In- of the medium must be low. The progress of an
osine-producing microorganisms were first inosine fermentation with Brevibacterium am-
found among adenine auxotrophs of different moniagenes is illustrated in Figure 10.6.
strains of the genera Bacillus, Brevibacterium, Co- The final stage is the phosphorylation of in-
rynebacterium, Streptomyces, and Saccharomyces. osine. At pH 11, inosine is precipitated from a
Corynebacterium petrophilum (Ade-) accumulates culture filtrate and crystallized. The chemical
1.6 g/linosine on a medium with n-alkanes (C,,— phosphorylation of inosine is carried out using
C,,). Various strains of Bacillus subtilis, Brevibac- trialkyl phosphate with PCl,. The amount of un-
terium ammoniagenes, and Microbacterium have desired 2'(3')5'-phosphodiester can be reduced to
been developed for commercial production, as < 10%; under these conditions the 5'-monoester
shown in Table 10.3. content is 90%.
In a typical fermentation process with Bacil-
Direct fermentation of 5'-IMP Mutants for the
lus, starch hydrolysate is most commonly used
direct fermentation of 5'-IMP should have the
as the carbon source, but glucose can also be
following properties:
used. Dry yeast or crude RNA serves as the
source of adenine. Ammonia is added both to
regulate the pH and as a nitrogen source. The E
2 S
S E,s
E
[o.)
518
2 D
Table 10.3 Mutants used for the inosine fermentation E© g | ow3
~
Mutants Genetic Inosine ic oT $j
@ JAN) fer0]
of =8| 9°
characteristics production = o ®
(6) a
(8/1) w oO 60 + 12
Bacillus subtilis
strain ao

No. 2 Ade 0.21

B-4 Ade His 4.46
C-30 Ade His Tyr 10.5
RDA-16 Ade” Trp- Red” Dea” 18.0
8AG:
Brevibacterium
ammoniagenes
KY 13714 Ade 6MG' Gua- 13.6
KY 13761 Ade" 6MG' 6MTP: Gua- 30.0
Microbacterium
sp.
No. 250 Bio- 6MP: 8AG* MSO: 35.0
6TG:

Auxotrophy: Ade Adenine; Bio Biotin; Gua Guanine;

His Histidine; Tyr Tyrosine; Trp Tryptophan; Red~ GMP
reductase is absent; Dea" AMP deaminase is absent.
Resistance: 8AG 8-Azaguanine; 6MG 0 10 20 30 40 50
6-Mercaptoguanine; 6MTP 6-Methylthiopurine; MSO Fermentation time (hr)
Methionine sulfoxide; 6TG 6-Thioguanine; 6MP
6-Mercaptopurine Figure 10.6 Inosine fermentation with Brevibacterium
(Furuya, in Ogata et al., 1976) ammoniagenes KY 13761 (From Kotani et al., 1978)
 10.4 PRODUCTION / 183

e Absence of SAMP synthetase to eliminate .

Å Å
AMP regulation at the level of PRPP ami-
dotransferase, E
ae 3
oD

° Low activity of the enzymes which break E 4 ER

down 5’-IMP, SØ SENERE SENG mn NM E
= 2
ae \ [52E
e Increased permeability of the cytoplasmic aoe
iS Å
membrane to bring about 5'-IMP excretion. \ 2
=o ex
| Beene tee Se aes
Mutants with reduced nucleotidase were first ==4[-—-—9== = EGE I

isolated among inosine-producing B. subtilis Ie aT T

5
His

10 2030
T T T

50
i

100
BSN

200 500 1000

strains. Mutants able to grow on adenine but not
Mn**addition ( jig/l)
on AMP showed a lower incidence of nucleotide
breakdown. A further inhibition of nucleotidase Figure 10.7 Effect of Mn?” on IMP and hypoxanthine
activity was obtained in mutant A-1-25, which production. Mutant KY 13102 (Mn?* sensitive): e———e
is listed in Table 10.4 along with the other 5’- IMP; O-~—-—O Hypoxanthine. Mutant KY 13105 (Mn?*
resistant): e —— e IMP; O — O Hypoxanthine
IMP producers. Industrial production of 5'-IMP
is carried out with Brevibacterium ammoniagenes.
The isolation of permeability mutants whose fies ‘ Å
IMP-accumulation is not affected by Mn?* was . = 497 5
an important advance. In the mutants KY 7208 | E = i
a + = ee ee =>
and KY 13102, which are sensitive to Mn?*, the =Ens rel 7 EE
optimal Mn?" content of the medium is 0.01-0.02 ao 3 al E
mg/l; at higher levels, IMP production decreases. E 20 +10 & Wea 6+ 100 =
Figure 10.7 illustrates the relationship of the IMP = € . 2
rae) rs. eyes
accumulation of both mutants to the Mn” con-
centration.
Bt Wiens søbetE
= 4. e sv P r @
In the fermentation by strain KY 13102 (Fig-
oO | a
Oo $ x
T 3 Poe
ure 10.8), the production of hypoxanthine con-
tinues during the first 2-3 days. Then it decreases iO abs = ZA - 50

ve f
i [
Table 10.4
Mutants
Microorganisms producing 5'-IMP
Genetic 5’-IMP 690 ;rå
il LEG SS x
|
i
characteristics Yield 354 ele a OLS >
(2/1) Ze ote eal f

Oe T T T T T T T i Fay 0

B. subtilis Ade Nuc 0.6 0 2 4 6 8

A-1-25 Fermentation time (days)
Corynebacterium Ade” 6MP" 2.0
glutamicum
Brevibacterium
Figure 10.8 5'-IMP fermentation with B. ammoniagenes
ammoniagenes KY 13102 (From Furuya et al., 1968)
KY 7208 Ade 5.0
KY 13102 Ade 12.8
KY 13105 Ade Mn?+ 19 as IMP accumulation begins. With Mn?" as a lim-
insensitive iting factor, abnormal cells with reduced perme-
KY 13369 Ade Mn?* 20-27
insensitive
ability are formed. They excrete hypoxanthine,
Gua™ which is extracellularly phosphorylated to 5’-
Ade” Adenine-requiring; Nuc” Nucleotidase-negative;
IMP, as shown in Figure 10.9. Moreover, 5’-IMP
6MP: 6-Mercaptopurine-resistant. is synthesized de novo in the subsequent fer-
184 / CHAPTER 10 / NUCLEOSIDES, NUCLEOTIDES, AND RELATED COMPOUNDS

PRPP Nucleotide
pyrophosphorylase
PP

Purine base 5'-Nucleotide

ADP
Nucleoside Nucleoside
phosphorylase kinase

ATP
Nucleoside
phosphotrans-
ferase

Nucleoside
P-NPP Figure 10.9 Extracellular conversion
—U of purine bases to purine nucleotides

mentation phase and is excreted directly into the

Fermentative production of 5'-GMP
culture medium.
For fermentations with B. ammoniagenes, it is The excretion of sizable amounts of 5’-GMP in
important that the phosphate and MgSO, con- wild type strains is rare, due to regulatory effects
centrations be kept at 2 and 1%, respectively. The primarily at the levels of PRPP amidotransferase,
typical composition of the culture medium for IMP dehydrogenase, and GMP synthetase.
Mn?'-insensitive B. ammoniagenes is as follows Moreover, 5'-GMP can be broken down by 5’-
(measured in g/l): KH,PO, 10.0; K,HPO, 10.0; nucleotidases or nucleosidases or can be con-
MgS0,7 H,O 10.0; CaCl:2:H,O 0.1; FeSO,°7 verted to IMP by GMP reductase. Various means
H,O 0.01; urea 6.0; cysteine 0.02; thiamine 0.05;
of production have therefore been developed.
nicotinic acid 0.05; Ca pantothenate 0.01; biotin + Fermentation from AICAR (5-amino-4-im-
3X10; MnCl,°4 H,O 0.001; adenine 0.04; glu- idazole carboxamide riboside, Figure 10.2),
cose 100.0; pH 8.3. which is chemically converted to 5'-GMP.
The Ajinomoto Company has developed a e Microbial production of guanosine with sub-
process for IMP production that uses a nitroso- sequent chemical phosphorylation to 5'-
guanidine-induced mutant of Brevibacterium am- GMP.
moniagenes. Inosine (50-2000 mg/1) or hypoxan- e Fermentative production of xanthine or 5’-
thine (25-1000 mg/l) is added during the XMP and enzymatic conversion into 5'-GMP.
fermentation. This process greatly increases the e Direct fermentation of 5’-GMP.
amount of 5'-IMP production from sugars such Only the first two processes are used for in-
as glucose, fructose, sucrose, or starch hydroly- dustrial production of 5'-GMP. In mutants used
sate. in production, intracellular accumulation is as
 10.4 PRODUCTION / 185

GMP, but the nucleoside guanosine is actually - production. Sporulation can be suppressed by in-
excreted. hibitors such as butyric acid, or by reducing the
O, supply. If good aeration is provided during
Production from AICAR AICAR is commercially the period about 8-12 hours after the start of the
produced as the starting material for production fermentation, sporulation occurs and AICAR
of 5'-GMP and as an intermediate for the en- production is reduced. However, if O, supply is
zymatic production of 5-amino-4-imidazole car- suboptimal, especially during this period, spor-
boxamide (AICA), which is used as the starting ulation is suppressed and AICAR production is
material for the chemical synthesis of purine de- increased. ‘
rivatives. The final step is the chemical conversion of
Escherichia coli, B. subtilis, B. megaterium, and AICAR to 5'-GMP. The AICARP formed is ex-
Brevibacterium flavum have been examined as AI- creted into the fermentation medium as the de-
CAR producers. Purine auxotrophs exhibit es- phosphorylated compound, AICAR. The isola-
pecially significant AICAR production, and the tion of AICAR can be accomplished with a 90%
purine auxotroph B. megaterium No. 366 (ATCC yield through several purification stages. For the
15117) and its mutants have been used in in- transformation into 5'-GMP, AICAR is first con-
dustrial production. Under optimal fermentation verted to guanosine in several chemical steps and
conditions, this strain produces 16 g/l AICAR then phosphorylated.
from 80 g/l glucose. The genetic properties that
make this strain suitable for AICAR accumula- Production of guanosine by direct fermentation
tion are: 1. The strain is a purine auxotroph and Another cost-effective process for the production
has a block in the AICARP formyltransferase re- of 5'-GMP is the production of guanosine by fer-
action, which causes AICARP to be converted mentation, followed by chemical phosphoryla-
into FAICARP (see Figure 10.2). 2. The strain has tion. Guanosine-excreting strains should have
no enzyme activities causing the hydrolysis of the following properties: 1. SAMP synthetase-
AICA-riboside. 3. The enzymes which catalyze negative, 2. GMP reductase-negative, 3. reduced
AICAR biosynthesis, especially PRPP amido- nucleosidase activity, and 4. enzymes of GMP
transferase, are insensitive to regulation by in- biosynthesis unregulated, particularly PRPP ami-
tracellular purine nucleotides. dotransferase, IMP dehydrogenase, and GMP
Starch hydrolysate is the carbon source used synthetase.
with mutant B. megaterium No. 366. Fifty percent Microorganisms which possess the ability to
of the total AICAR synthesis occurs after the me- excrete guanosine include Bacillus subtilis, B.
tabolism of glucose is complete. As an interme- pumilus, B. licheniformis, Corynebacterium petro-
diate step in glucose breakdown, gluconic acid philum, C. guanofaciens, and Streptomyces griseus.
accumulates and is then subsequently metabo- Mutants of B. subtilis, also an inosine producer,
lized during AICAR synthesis. For maximal AI- are chiefly used for commercial production (Ta-
CAR production, the medium must contain a pu- ble 10.5).
rine source, such as dry yeast or yeast RNA, in IMP dehydrogenase is regulated by 5’-GMP.
suboptimal concentration. Soy protein hydroly- B. subtilis AJ 1993 is a mutant which is resistant
sate and NH,CI are used as nitrogen sources and to the purine analog 8-azaguanine. It shows IMP
mineral salts (MgSO,°7 H,O, FeSO,°7 H,O, dehydrogenase activity three times greater than
MnSO,°'H,0O) are also used. Gaseous ammonia is the initial strain, as well as reduced GMP reduc-
used both as a nitrogen source and pH regulator. tase activity. Mutation to resistance to methio-
The AICAR yield during the fermentation is nine sulfoxide (a glutamine analog) in mutant
directly affected by the sporulation process; spor- MG-1 causes another increase in the IMP de-
ulating cultures have a strongly reduced AICAR hydrogenase activity. In mutants which are re-
186 / CHAPTER 10 / NUCLEOSIDES, NUCLEOTIDES, AND RELATED COMPOUNDS

Table 10.5 Guanosine-excreting B. subtilis mutants

Mutants Genetic Guanosine AQ
characteristics production Pe ieee Bas 6 pH
(2/1
uo
B. subtilis AJ 1993 Ade” Red” 8AG' 4.3 15 VÆRE
No. 30-12 Ade Trp Red-8AX: 5
MG-1 Ade-His'Red- MSO' 16
PsirDec
10 -a
For the mutant designations, see Table 10.3; Resistance:
0.6
8AX 8-azaxanthine; Psi Psicofuranine; Dec Decoyinine RES avy
(Psicofuranine and Decoyinine are inhibitors of GMP
synthetase)

sistant to nucleoside antibiotics such as psico- (g/l)

sugar
Residual
Adenine,
Adenosine,
furanine and decoyinine (which inhibit
0 40 80 120
production of GMP), the GMP synthetase activ-
ity is clearly increased, while regulation by GMP Fermentation time (hr)
is completely eliminated. The high guanosine ex-
Figure 10.10 Adenosine fermentation with Bacillus sp.
cretion of mutant MG-1 is thus due to several strain P 53-18. e —-e Adenosine; O —— O Adenine;
factors: adenine auxotrophy, the absence of GMP = — E Residual sugar; 4 — 4 Growth (Absorbance at 655
reductase, the elimination of the feedback reg- nm); crosses, pH (From Haneda et al., 1971)
ulation by GMP, and the increased activity of
IMP dehydrogenase and GMP synthetase. Another organism, a xanthine auxotroph of Brev-
The production medium for B. subtilis AJ ibacterlum ammoniagenes, carries out a direct syn-
1993 has the following composition (g/l): glu- thesis of 5'-AMP to a level of 2.1 g/l.
cose 70; adenine 0.3; soy protein hydrolysate Adenosine triphosphate, ATP, can be ob-
0.48; NH,Cl 15; KH,PO, 1; MgSO,°7 H,O 0.4; tained through phosphorylation of adenine,
FeSO,°7 H,O 0.01; MnSO,°H,O 0.01; CaCO, 25. adenosine, and ATP by various microorganisms
The fermentation temperature is 30°C. Under in the presence of glucose or methanol plus phos-
these conditions, 4.3 g/l guanosine and 3.1 g/l phate. Protoplasts of the yeast Candida boidinti
inosine are produced. produce ATP at a yield of 100 g/l (77.4% effi-
ciency) from a mixture of adenine and methanol.
Production of adenosine and adenine Cyclic adenosine-3',5'-monophosphate,
nucleotides cAMP, can be produced either chemically or mi-
crobially. cAMP production has been shown in
The production of adenosine and adenine nu- a variety of bacteria, including E. coli, Coryne-
cleotides has gained significance due to the use bacterium murisepticum, Microbacterium sp., and
of these substances as starting materials for the Brevibacterium liquefaciens. A mutant of Micro-
synthesis of medically important adenine deriv- bacterium sp. resistant to 8-azaguanine, azaser-
atives. ine, methionine sulfoxide, and 6-mercaptoguan-
For adenosine production, the Bacillus subtilis ine has been shown to synthesize cAMP de novo,
mutant P 53-18 (His Thr Xan 8AX’) was isolated. producing as much as 16.6 g/l in a medium con-
This mutant has a block in adenosine deaminase, taining glucose and inorganic salts. If 3 g/l of
GMP reductase, and IMP dehydrogenase. An adenine or hypoxanthine is added, the produc-
adenosine accumulation of 16 g/1 was obtained tion is increased to 20.5 or 21.3 g/l respectively.
under optimal fermentation conditions. Progress Studies on cloning the gene for adenyl cy-
of a typical fermentation is given in Figure 10.10. clase in E. coli and Salmonella typhimurium have
 REFERENCES / 187

been undertaken. Although the activity of the. resentative examples are discussed in Section
enzyme in S. typhimurium increased by a factor 19,7;
of 20, there was only a 60% increase in cAMP
formation.
REFERENCES
Fermentative production of other
Dale,B.E. and D.A. White. 1979. Degradation of ribonu-
substances related to nucleotides cleic acid by immobilized ribonuclease. Biotechnol.
Bioeng. 21:1639-1648.
Other substances related to nucleotides are pro- Demain, A.L. 1978. Production of nucleotides by micro-
duced by fermentation in Japan for medical pur- organisms, pp. 187-208. In: Rose, A.H. (ed.), Primary
poses, although the market for such products is products of metabolism, vol. 2. Academic Press, Lon-
don.
limited (see also Table 10.1). Production strains
Enei, H., H. Shibai, and Y. Hirose. 1985. 5’'Guanosine non-
and yields of these processes are listed in Table ophosphate, pp. 653-658. In: Moo-Young, M. (ed.).
10.6. Comprehensive Biotechnology, Vol. 3. Pergamon
An enzyme/membrane reactor has been de- Press, Oxford.
Furuya, A., S. Abe, and S. Kinoshita. 1968. Production of
veloped for the production of coenzymes nucleic acid-related substances by fermentative pro-
NADP/NADPH, substances that find consid- cesses. XIX. Accumulation of 5’-inosinic acid by a mu-
erable value in analytical biochemistry. tant of Brevibacterium ammoniagenes. Appl. Microbiol.
16:981-987.
A group of pharmacologically interesting Haneda, K., A. Hirano, R. Kodaira, and S. Ohuchi. 1971.
compounds are the purine arabinosides. Sub- Accumulation of nucleic acid-related substances by mi-
stances which possess antibiotic, antiviral, or an- croorganisms. II. Production of adenosine by mutants
derived from Bacillus sp. Agr. Biol. Chem. 35:1906-
titumor activity include guanosine derivatives
1912:
and adenosine analogs such as arabinosyladen- Hirose, Y., H. Enei, and H. Shibai. 1979. Nucleosides and
ine. A major site of action of these compounds nucleotides. Ann. Rep. Ferm. Proc. 3:253-274.
is the enzyme S-adenosylhomocysteine hydro- Kotani, Y., K. Yamaguchi, F. Kato, and A. Furuya. 1978.
Inosine accumulation by mutants of Brevibacterium am-
lase, a key enzyme in biochemical systems for moniagenes, strain improvement and culture condi-
biological transmethylation. S-adenosylme- tions. Agr. Biol. Chem. 42:399-405.
thione and S-adenosylhomocysteine, interme- Kuninaka, A. 1986. Nucleic acids, nucleotides, and related
compounds, pp. 71-114. In: Rehm, H.J. and G. Reed
diates in the transmethylation pathway, have (eds.). Biotechnology, Vol. 4. VCH-Verlagsgesellschaft,
been studied clinically as sedatives and for the Weinheim.
treatment of mental illness. Both microbiological Nakao, Y. 1979. Microbial production of nucleosides and
nucleotides, pp. 311-354. In: Peppler, H.J. and D. Perl-
and enzymatic processes for the production of man (eds.). Microbial technology. Vol. I. Academic
these substances are under study. Press, New York.
Over 100 antibiotics have been described Ogata, K., S. Kinoshita, T. Tsunoda and A. Aida (eds.).
which are derived from nucleosides. Several rep- 1976. Microbial production of nucleic acid related sub-
stances. Kodansha Ltd., Tokyo.
Perlman, D. 1977. Fermentation industries . . . quo vadis?
Table 10.6 Production by fermentation of substances Chem. Techol. 7:434-443.
related to nucleic acids Schitz, H.J., M.R. Kula, and C. Wandrey. 1986. Ein en-
zymatischer Weg zur kontinuierlichen Produktion von
Product Organism Yield phosphorylierten Nicotin-Adenin-Dinukleotiden. (An
(g/l) enzymatic process for the continuous production of
phosphorylated nicotine adenine dinucleotides.) BTF-
FAD Sarcina lutea 1 Biotech Forum 3:98-102.
NAD Brevibacterium ammoniagenes 19 Shibai, H., H. Enei, and Y. Hirose. 1978. Purine nucleo-
Coenzyme A Brevibacterium ammoniagenes 2 sides fermentations. Proc. Biochem. Nov. 1978, pp. 6-
IFO 12071
8452:
Orotic acid Arthrobacter paraffineus 20
Shiio, I. 1979. Microbial production of nucleotides.
CDP-choline Saccharomyces carlsbergensis 17
Congrés international microbiologie et industrie ali-
(Ogata et al., 1976; Hirose et al., 1979) mentaire. Paris, 10, Oct.
 UNDS
188 / CHAPTER 10 / NUCLEOSIDES, NUCLEOTIDES, AND RELATED COMPO

Shimizu, S. and H. Yamada. 1984. Microbial and enzy- Yamada, K. 1977. Bioengineering report. Recent advances
matic processes for the production of pharmacologi- in industrial fermentation in Japan. Biotechnol. Bioeng.
cally important nucleosides. Trends in Biotechnol. 19: 1563-1621.
2:137-141.
 Enzymes

such detergents are much less widely used in the

11.1 INTRODUCTION
United States.
The first enzyme produced industrially was the The development of amylases and amylo-
fungal amylase takadiastase, employed as a glucosidases for the production of glucose from
pharmaceutical agent (for digestive disorders) in starch has led to a new industrial application of
the United States as early as 1894. Otto Roehm's enzymes. Also, the use of glucose isomerase for
patented ”laundry process for any and all cloth- the production of fructose has become wide-
ing via tryptic enzyme additives” was announced spread since 1967.
in 1915. By 1969, 80% of all laundry detergents Microbial rennin is next in order of signifi-
contained enzymes, chiefly proteases. Along cant enzymes. It has been used instead of calf's
with these, additional enzymes such as lipases, rennin in cheese production since 1965.
amylases, pectinases, and oxidoreductases were Gross world sales for enzymes in 1987 was
used experimentally in the detergent industry. $450 million. The following enzymes are cur-
Due to the occurrence of allergies among pro- rently produced commercially:
duction workers and consumers, the use of pro- e Enzymes used in industry, such as amylases,
teases in detergents was drastically reduced in proteases, catalases, isomerases, and penicil-
1971 and the world sales fell from $150 million lin acylases;
to one-third this amount. Only when special pro- Enzymes used for analytical purposes, such
cessing techniques, such as microencapsulation, as glucose oxidase, galactose oxidase, alcohol
were developed could dustless protease prepa- dehydrogenase, hexokinase, muramidase,
rations be produced that were risk-free to work- and cholesterol oxidase;
ers and consumers. Today 95% of laundry de- Enzymes used in medicine, such as aspara-
tergents in Europe contain proteases, although ginase, proteases, lipases, and streptokinase.

189
190 / CHAPTER 11 / ENZYMES

These three areas of application require vary- Table 11.2 Production of industrial enzymes
ing levels of quality and quantity (Table 11.1). Enzyme Enzyme Sales, percent
On a tonnage basis, the most important are the preparation of total
tons/year
industrial enzymes (about 1200 tons of pure pro-
tein/year) (Table 11.2). The United States and Bacillus proteases 6200 45
Glucoamylases 4500 13
Western Europe produce about 40-45% of the Bacillus amylases 4200 5
world market, and nine firms account for 90% Glucose isomerases 1900 6
of the total market. Industrial enzymes are pro- Rennin (microbial) 1500 10
Amylases (fungal) 800 4
duced in 120 m? fermenters, while enzymes for Pectinases 100 3
analytical or medical purposes can frequently be Proteases (fungal) 100 2
produced in pilot-plant-sized fermenters. Others = 12
The low concentration of enzymes which are (Aunstrup et al., 1979)
normally produced by wild strains is a consid-
erable hindrance for enzyme production. Al-
though in 1985 about 2500 enzymes were
known, only 250 were marketable, mostly in
amounts around 10 g, some even in mg amounts.
Figure 11.1 shows the overall picture of com-
mercial enzyme production.
The prospects for enzyme application have
30%
improved due to developments in the following
areas:
e Microbial genetics. High yields can be ob-
20%
tained by genetic manipulation. For instance,
the yeast Hansenula polymorpha has been ge-
netically modified so that 35% of its total pro-
tein consists of the enzyme alcohol oxidase.
10%
e Optimization of fermentation conditions via
induction of enzyme production, use of low
industry
Detergent
cost nutrients, optimal utilization of compo-
nents in nutrient solution, and introduction Distilling
andindustry
Fruit
industry baking,etc.
juiceBrewing,
of fed-batch fermentations.
. Release of enzymes from cells by means of Figure 11.1 Percentage of industrial enzymes used in
new cell-breaking methods. various markets

Table 11.1 Differences in enzyme qualities

Industrial enzyme Analytical enzyme Clinical enzyme
Scale of application Tons Milligrams—grams Milligrams—grams
Degree of purification Crude Pure crystalline Pure crystalline
Secondary activities Present Usually none. If yes, then Only isoenzymes
defined
Origin Microbial, usually Microbial, animal, plant, Microbial, animal, plant,
extracellular usually intracellular usually intracellular
Multiple applications Possible in part Possible in part Not yet possible
Production costs Low Middle-high High
 11.2 AMYLASES / 191
+ Modern purification processes such as coun- ”
all |
tercurrent distribution, ion-exchange chro-
matography, molecular-sieve chromatogra-
phy, affinity chromatography, and | fe Labor, energy, overhead
precipitation.
+ Development of processes for the immobili- HM Manufacturing cost
Zation of enzymes and for their recycling. The
|Z Raw materials
amount of enzyme needed per amount of
substrate converted is thus considerably de- Relative
(%)
cost :

creased. The proportion of enzyme cost in

some processes becomes only a few percent | Batch, conventional
(Figure 11.2). Il Immobilized cells,
continuous
* Continuous enzyme production in special re-
actors. The investment cost for a new system
Figure 11.2 Reduction in the cost of L-aspartic acid pro-
can be minimized in a continuous operation, duction through use of cell immobilization techniques
since smaller-sized equipment is used. (From Chibata and Tosa, 1977)

In this chapter, we will discuss those reac-

tions which involve more or less purified en-
zymes (either intracellular or extracellular). Ad- sweetener sucrose. The most important enzymes
ditional enzymatic processes involving living or in the starch-saccharification process are a-amy-
dead cells will be discussed in Chapter 15 (Trans- lases, B-amylases, glucoamylases, glucose iso-
formation). merases, pullulanases, and isoamylases. Figure
11.3 shows the manner of action of these en-
zymes on starch.

11.2 AMYLASES
a-Amylases
Starch, a glucose polymer, is one of the most
widely available plant polysaccharides. Amy- a-Amylases (1,4-a-glucan-glucanohydrolases)
lases are enzymes which hydrolyze starch. One are extracellular enzymes which hydrolyze a-
of the main uses of amylases is in the production 1,4-glycosidic bonds. These enzymes are en-
of sweeteners for the food industry. The hy- doenzymes, splitting the substrate in the interior
drolysis of starch with amylase results first in the of the molecule. Their action is not inhibited by
production of short-chain polymers called dex- a-1,6-glycosidic bonds although such bonds are
trins, then the disaccharide maltose, and finally not split.
glucose. Maltose syrup (>80% maltose), which a-Amylases are formed by many bacteria and
is produced primarily in Japan, is of low viscosity, fungi. They are classified according to their
is weakly hygroscopic, not crystallizable, only starch-liquefying and/or saccharogenic effect,
slightly sweet, but has good heat stability and pH optimum, temperature range, and stability.
does not undergo browning reactions. Glucose is Saccharogenic amylases produce free sugars,
not nearly as sweet as its isomer fructose, so the whereas starch-liquefying amylases break down
next step is the conversion of glucose to fructose the starch polymer but do not produce free sug-
using the enzyme glucose isomerase (discussed ars. Many organisms produce several a-amy-
in Section 11.3). Commercial sweeteners based lases.
on fructose have some economic and manufac- Bacteria which produce a-amylases are: Ba-
turing advantages over the more widely used cillus subtilis, B. cereus, B. amyloliquefaciens, B.
192 / CHAPTER 11 / ENZYMES
Table 11.3 Molecular weight of some a-amylases from
different microorganisms
Organism Molecular weight
10°
Aspergillus oryzae 51— 52
A. niger 58- 61
Bacillus acidocaldarius 68
B. amyloliquefaciens 49
B. subtilis 24-100
Thermomonospora curvata 62

(Fogarty, 1983)

tophan in the enzyme protein and most require

calcium as a stabilizer.
Reducing end The most important a-amylases are produced
by Bacillus amyloliquefaciens, Bacillus lichenifor-
mis, and Aspergillus oryzae. Bacillus amylases are
used much more extensively than those of As-
—<=— o-Amylases
pergillus. The most important areas of application
=—0 Glucoamylases for these two enzymes are shown in Table 11.4.
Pullulanases ,
lsoamylases Production of bacterial a-amylases Bacterial am-
—— + £-Amylases ylase production involves the function of the nor-
mal cell machinery for protein synthesis. This is
Figure 11.3 Mechanism of starch hydrolysis by amylases shown by experiments involving the addition of
antibiotics. If the specific antibiotics are used to
inhibit protein synthesis in B. subtilis during the
coagulans, B. polymyxa, B. stearothermophilus, B. production of a-amylases, both growth and pro-
caldolyticus, B. acidocaldarius, B. subtilis var. amy- duction of amylase cease (Figure 11.4). Com-
losaccharaticus, B. licheniformis, Lactobacillus, Mi- pared to the mRNA of intracellular enzymes (sta-
crococcus, Pseudomonas, Arthrobacter, Escherichia, bility about 2 minutes for Escherichia coli), the
Proteus, Thermomonospora, and Serratia. mRNA for the production of extracellular hydro-
Three very similar strains producing sac- lases has an extremely long lifetime. When ac-
charogenic a-amylases are B. subtilis Marburg, B. tinomycin D is added to the amylase-producing
subtilis var. amylosaccharaticus, and B. natto. The culture to inhibit RNA synthesis, both RNA syn-
strain B. amyloliquefaciens differs from these in thesis and growth cease after 30 minutes but the
that it produces a liquefying a-amylase. The sub- production of amylase continues (Figure 11.5).
strate concentration determines the extent to For industrial production, a-amylases are
which the enzymes act in a liquefying or sac- produced either in batch or in fed-batch fermen-
charogenic manner. tation. The enzyme formation rate is very low
Some a-amylase-producing fungi are from during exponential growth in many production
the genera Aspergillus, Penicillium, Cephalospo- strains, but just before the growth rate decreases
rium, Mucor, Candida, Neurospora, and Rhizopus. and spore formation begins, amylase production
The molecular weights of various a-amylases increases (Figure 11.6).
do not differ considerably (Table 11.3). They all The production of a-amylases is regulated by
contain a large proportion of tyrosine and tryp- several genes, which have been only partially
 11.2 AMYLASES / 193
Table 11.4 Important applications of a-amylases
Industry Source Application
Bacillus Aspergillus
Starch industry oT Liquefaction of starch for production of glucose, fructose, maltose
Milling g ul Modification of a-amylase-deficient flour
Alcohol a he Liquefaction of starch before the addition of malt for
saccharification
Baked goods + Increase in the proportion of fermentable carbohydrates
Brewing a Barley preparation, liquefaction of additives
+
Improved fermentability of grains, modification of beer
characteristics
Paper an Liquefaction of starch without sugar production for sizing of paper
Textiles ste Continuous desizing at high temperatures
Feed industry + Improvement of utilization of enzymatically treated barley in
poultry and calf raising
Sugar Hg Improvement of filterability of cane sugar juice via breakdown of
starch in juice
Laundry and + Increase in cleansing power for laundry soiled with starch, additive
detergent in dishwasher detergents

NS
e
A En
RR
e

Extinction

f=)foo) oN oOo Å Oe

100 is DE
oOo (ej
oOo o

co(2) e e

SD=}
Oc ne

(units/ml)
Amylase
20m o=—— 0 ° e ° ° O96
Figure 11.4 Effect of chloramphen-
icol and puromycin on a-amylase
production in B. subtilis. O — O anti- FF T mg T T sare T FE, T FE SEER

biotic added (10 ng/ml chloram- 60 120 min 60 120

phenicol, 100 ug/ml puromycin);
e — e control. (From Terui, 1973) Chloramphenicol Puromycin
194 / CHAPTER 11 / ENZYMES

5 S ' =JE
800 |3 2 70
as x (=
SE e wi 5
60, 2 E a9 + A AE BES
2
© ©
ars vA &
E
>

4004 085 J 2 BORES

I ss
O

Figure 11.5 Effect of actinomycin D

on growth, RNA synthesis, and a-
30 60 30 60 amylase production. O —— O actino-
mycin D (0.6 ug/ml); e — e control.
RNA synthesis Growth Amylase formation (From Terui, 1973)

characterized. Whereas single-step mutations in- tutively and excreted into the medium where its
crease yields by a factor of 2—7, mutants have action on starch leads to the production of low-
been selected after 5 steps which produce yields molecular-weight inducers. Catabolite repres-
250 times greater than the wild strain. sion is significant in the production of most ex-
Since starch is a macromolecule, it cannot be tracellular enzymes. Glucose promotes the best
taken up by the cells and hence cannot act as an growth compared to other substrates, but is the
inducer of a-amylase synthesis. It is assumed that least favorable in terms of amylase production.
a small amount of enzyme is produced consti- In continuous fermentation with carbohydrate as
a limiting factor, growth is inversely proportional
to the a-amylase production. When nitrogen is
used as a limiting factor with excess glucose, only
E
=) traces of amylase are produced.
020 . 40 & axe aa
é eA aes It seems unlikely that amylase is excreted by
SS pure diffusion, since the molecule is quite large
NE
= a

Ho 20. pels pel

and is hydrophilic. There is good evidence that
£ BAR SEA
2 2 FE
o— e——4— 0
the biosynthesis as well as the steps leading to
6 44000 as excretion of extracellular proteins such as amy-
we a
NØ
—F lases and proteases takes place at the outer mem-
as
2 120002
MØRE. Sedawe
a
brane of the cell. As it is formed, the protein
contains an additional peptide, the signal peptide,
E = . EG
x 2 a cA x which participates in the transfer of the nascent
protein from the ribosome through the mem-
7 ee

om eee
eo? nere
r = 3 T —— T T 5 _ brane. Only after the protein has passed through
the membrane does it fold into its tertiary con-
Fermentation time (hr) figuration and the signal peptide is removed.
A medium for the production of a-amylases
Figure 11.6 Amylase production in Bacillus amylosolvens.
in a 100 m° fermenter with B. subtilis consists of:
u specific growth rate, & specific enzyme production rate,
X cell concentration, E enzyme concentration (From 5% starch, 0.56% NH,NO,, 0.28% sodium cit-
Terui, 1973) rate, 0.13%. KH,PO,, 0105% MgSO,°7 H,0,
 11.2. AMYLASES /.195
0.01% CaCL-2 H,O, 0.5% peptone, 0.2% yeast . for stabilization and activation of bacterial B-am-
extract; pH 6.8. ylase.
The effect of temperature on enzyme pro- In the future, it is likely that B-amylases will
duction and growth rate is given in Figure 11.7. be used for the production of maltose syrup.
At 45°C, the maximal specific enzyme formation
rate is reached with the test strain after 18 hours;
Glucoamylases
however, the maximal amount of enzyme pro-
duced (up to 3000 units/ml) is obtained at con- Glucoamylases (a-1,4-glucan-glucohydrolases)
siderably lower temperatures (27 to 30°C). Ther- act on starch by splitting glucose units from the
mophilic strains are used in newer processes. nonreducing end. Maltose is broken down only
Thermomonospora, isolated from compost, has a slowly, while 1,6-bonds in the branched poly-
temperature optimum for growth and amylase saccharides are hardly attacked. Thus glucose,
production at 53°C. Figure 11.8 shows the nar- maltose and limit dextrins are the end products
row pH optimum for amylase production by this of glucoamylase action.
strain. Microorganisms used to produce glucoam-
ylases are Aspergillus niger, A. oryzae, A. awamori,
a-Amylase production from fungi The production Rhizopus niveus, R. delemar, R. formosaensis, and
of fungal amylases is constitutive, but as with R. javanicus. Production strains in western Eu-
other enzymes, it is repressed by regulators. For rope and in the United States are mutants of A.
amylase production using Aspergillus oryzae, the niger and R. niveus. Today, industrial fermenta-
following nutrient solution can be used: 8% tion of glucoamylase is carried out almost exclu-
starch, 1.2% NaNO,, 0.1% K,HPO,, 0.1% sively in submerged fermenters up to 150 m? in
MgsO,, 0.05% KCl, 0.003% FeSO,, 0.08% volume. Due to the increasing demand for gluco-
Mg(NO;),, 0.05% Mg(H,PO,),, 2.0% malt extract. amylase in fructose-syrup production, smaller
The medium composition must be optimized, be- systems which involve growth on the surface of
cause as seen in Table 11.5, a marked shift in liquid or solids are no longer economical. How-
enzymatic activities is observed when different ever, in genetic studies, strains are frequently iso-
carbohydrates are used. lated which produce higher yields in surface cul-
The optimal temperature lies in a narrow ture than in submerged culture (Table 11.6).
range between 28-30°C, and the duration of the Experiments with batch-fed fermentations have
fermentation process is 3-4 days. been successfully carried out over periods as long
as 320 hours.
Frequently, several glucoamylase isoenzymes
6B-Amylases
are produced by one strain; in addition, the en-
B-Amylases (a-1,4-glucan-maltohydrolases) are zymes may be modified during the fermentation
usually of plant origin, but some microbial pro- process. A. awamori var. kawachi produces three
ducers are also known: Bacillus polymyxa, B. cer- glucoamylases, only one of which hydrolyzes
eus, B. megaterium, Streptomyces sp., Pseudomonas crude corn starch. Due to the action of proteases
sp., and Rhizopus japanicus. Although yields in and glycosidases, which are also present under
wild strains are usually low, mutants have been various experimental conditions, one of the other
discovered which produce 200 times more en- two enzymes may be formed instead of the
zyme than the wild type. glucoamylase which hydrolyzes crude corn
Bacterial G-amylases have greater heat re- starch. These other enzymes can only act on corn
sistance (>70°C) than plant 6-amylases and the starch which has been swollen by heat or chem-
pH optimum is also higher (about pH 7.0). In ical treatment. Therefore the changes in gluco-
contrast to a-amylase, calcium is not necessary amylase activity must be carefully examined
196 / CHAPTER 11 / ENZYMES

NISE SAG 100 Sne

é °
al 80 -L 2000 ark ER
fo)
E fe)
60 = / |
ee = O
> 5 O
(units/ml/hr)

pare
06 £0 7 000 iO

03 20 2 3) 3

oa Na
re 20 Lohr A 3099 20 40 hr
s O
Få 30786 o
/ is 2h °
OR wae

12 ve
ts
Ovens 80 | 2000
f
SE
gg

Oowo a (op)eS

Ps units/ml/hr)
( units/ml FR
ers

06 "740 421000 sy
‘Bit

20 40 hr 20 40 hr

Figure 11.7 Relationship of growth and a-amylase production to temperature. See Figure 11.6 for symbols. (Modified
from Terui, 1973)
 11.2 AMYLASES / 197

Table 11.6 Glucoamylase production in some mutants

ipodoO
of Rhizopus formosaensis
SS
Strain Submerged Surface Surface
culture culture culture
units/100 ml (liquid) (solid)
units/100 ml units/g

A 1.0 R 13-5 298 2745 858

R 13-59 491 3067 1322
E ° R 13-591 1595 3788 2160
=@ Ng R 13-59136 4432 7425 5120
3o taE
n ox
= = (Lin, 1972) nhs
5E AN vey RS e a
3
< \ = 5
‘
a.
oO
~
oO
= Either starch or dextrins induce enzyme pro-
10 SÅ 2 >
== a duction; nutrient solutions for enzyme produc-
tion are generally based on starch. Glucose, glu-
tamic acid, and lactose cause catabolite
repression. Fermentation time varies from 3-5
days, depending on the organism, and the fer-
mentation temperature can range from 28-35°C.
In addition to glucoamylase, a-amylase may also
be produced in the same fermentation.
The quality of the spores used as inoculum
has an effect on the enzyme yield in gluco-
amylase production with A. niger. When spores
> i are produced on a rich agar medium, such as one
T
8 pH containing a supplement of malt extract, the
glucoamylase formation rate of later cultures de-
Figure 11.8 Effect of pH on a-amylase production, ex- creases with each transfer, although the spore
tracellular protein and growth. O — O a-amylase; e
protein; 4 —— 4 cell dry weight (From Glymph and Stutz- formation rate does not diminish. After 6 trans-
enberger, 1977) fers only 50% of the original enzyme activity is
produced. However, if the inoculum spores are
produced on a minimal medium (Czapek Dox),
Table 11.5 Effect of carbon source on amylase and
protease production in Trichoderma viride the enzyme formation rate remains constant, but
the spore formation rate decreases.
Carbon source Amylase Protease
units/ml units/ml
Corn starch 235 351
1,6-Glycoside-splitting enzymes
Maltose 179 175
Glucose 52. 243
Enzymes which can split the side chains of amy-
Sucrose 17 350
Lactose 3 175 lopectin via hydrolysis of a-1,6-bonds are im-
Control 0 324 portant in commercial processes. These enzymes
(Upton and Fogarty, 1977) can be subdivided into two groups: a) those
which directly affect amylopectin, such as pul-
lulanases and isoamylases, and b) those which
when high-yielding strains are isolated and have an indirect effect after enzymatic modifi-
when fermentation conditions are being optim- cation of the substrate. Pullulanases and isoa-
ized. mylases hydrolyze amylopectin. Moreover, pul-
198 / CHAPTER 11 / ENZYMES
lulan, the a-glucan from the fungus Pullularia process, the viscosity of a 20% solution increases
pullulans, is split by means of pullulanases. This significantly to 3000 centipoise. Acid treatment
latter reaction cannot be carried out with isoa- at 120-140°C (pH 1.8-2.0) or treatment with a-
mylases. Table 11.7 shows the effect of pullu- amylases cause starch liquefaction in the next
lanase on starch hydrolysis by 6-amylase. step; an alternative method is a combined acid/
Late in the log phase, Aerobacter aerogenes enzyme liquefaction. The disadvantages of acid
ATCC 15050 excretes pullulanase into the me- treatments are generally low glucose yields,
dium. With inducible strains, either maltose or higher purification costs, risk of deterioration,
pullulan can be used as a carbohydrate source and corrosion of the system used.
for enzyme induction, but constitutive mutants Enzymatic liquefaction has therefore gener-
are now generally used instead. Both batch fer- ally displaced acid treatment. Liquefaction is car-
mentations and continuous processes are in use. ried out at 110°C with amylases from B. subtilis
Several other pullulanase producers besides which are added to the solution before conver-
Aerobacter aerogenes have been described: Bacil- sion to paste. When a-amylases from A. niger or
lus polymyxa, Pseudomonas saccharophila, Strep- A. oryzae are used, the process is carried out at
tococcus sp., and Streptomyces sp. Among the iso- 55°C and the hydrolysis continues to the point
amylase-producers, Pseudomonas amyloderamosa of a maltose /maltotriose mixture without the ad-
is the most important strain. In addition, there dition of other enzymes.
are other strains: Agrobacterium, Erwinia, Staph- Starch solution which has been liquefied with
ylococcus, Serratia, Nocardia, Bacillus, Pediococcus, a-amylases can be saccharified with glucoamy-
Lactobacillus, and Leuconostoc. lases at a pH of 4.0-5.0 and a temperature of 55—
60°C without an intermediate purification stage.
Pullulanases or isoamylases may also be added
Starch hydrolysis
at this stage to bring about splitting of bonds
In the food industry, starch hydrolysates are used other than the 1-4 linkages. The specificity of
as additives in the manufacture of candies, baked enzymes used and the conditions of conversion
goods, canned goods, and frozen foods. Syrups determine the composition of the end products.
used can contain maltose or glucose (after sac- Figure 11.9 shows a diagram of the glucose
charification), or fructose (after isomerization, syrup production process.
Section 11.3). Starch hydrolysates affect the taste,
sweetness, and texture of the food products.
To produce a hydrolysate, starch must first 11.3 GLUCOSE ISOMERASES
be converted to paste at 65-90°C. During this
Glucose isomerase (D-glucose ketoisomerase)
causes the isomerization of glucose to fructose.
Table 11.7 Conversion of 10% potato starch (pre- The reaction is reversible and a mixture of glu-
treated with a-amylase) via pullulanase and
B-amylase cose and fructose is produced, the ratio of the
components depending on the enzyme used and
Type of process Maltose Glucose Malto- Dextrin
% % triose % the reaction conditions, such as temperature. The
%
enzyme has become of commercial value because
25 units 6-Amylase/ 65.5 1 Ske) 29.9 the price of sugar has increased compared to that
g starch; 16 hr 45°C of starch. In the Western world, the current su-
10 units Pullulanase + 90.5 0.4 12 7.9 crose consumption rate is 50 kg per person per
25 units $-amylase/ year. In the United States in 1985, 4X10 tons
g starch; 16 hr 45°C
of isosyrup (glucose/fructose mixture) were pro-
(Hayashibara, 1970) duced. As an example of European usage, the
 11.3 GLUCOSE ISOMERASES / 199

Corn starch (40% w/v) Sucrose Starch

Deionized water (Sugar beets, Sugar cane) (Wheat, Corn, Potatoes)
Ca? (20 ppm), &-Amylase (0.15%)
!
Hydrolysis
5 min, 105° C (Enzymatic, Acid)

60 min, 95°C '

Invertase
|
Glucose

pH 4.5, 60° C, Filtration

Glucose isomerase
! A
Dilution (10-20% dry weight)
Glucose/Fructose Glucose/Fructose
Glucoamylase (0.15 units/g starch) ~50:50, Invert sugar ~/ 60:40, Isosyrup

48 h, 60° C
Figure 11.10 Use of biochemical methods for the pro-
duction of high-fructose syrup
Glucose (98% yield)

Figure 11.9 Production of glucose solution from starch

(Novo Industries, 1977) D-Glucose D-Man nose

nt H alae)
VÆ \ \ 74
size of the German fructose production is esti-
mated at 10,000-20,000 tons per year.
H-C-OH
oleae
COOH HOF CH
il
Starch can be converted to glucose by either
SEE es For SENE ES As
acid or enzyme hydrolysis (see Section 11.2). The
advantage of starch hydrolysis over direct sugar H-C-OH H-C-OH H -C-OH

production is that the initial materials used, such H-C-OH H -C-OH H -C-OH
as wheat, corn, cassava, and to some extent po- CH,OH CH,OH CH,OH
tatoes, are nonperishable, but sugar beets (the
only direct source of sugar in temperate climates)
are only available about 100 days per year. ke emek CH2OH
Glucose has 70-75% the sweetening strength C=O C-OH C0
of beet sugar (sucrose) but 6-D-fructopyranose HO =C-H HO 0 =H -¢-0H
(fructose), the sweetest monosaccharide, has
HÆCOH HÆCOH HEÆC-OH
twice the sweetening strength of sucrose. Thus,
processes for the manufacture of fructose are of H-C-OH H-C-0H H-C-OH

considerable value. Fructose can be produced CHOH CH.OH CH,OH

biochemically from sucrose or starch by way of
D-Fructose D-Psicose
glucose production, as shown in Figure 11.10. It
can also be produced chemically from glucose at Figure 11.11 Chemical isomerization of glucose to fruc-
high temperatures under alkaline conditions, but tose
byproducts are formed such as psicose which
cannot be metabolized in the body (Figure
11.11). Thus, chemical production of fructose is glucose isomerase used must fulfill the following
not acceptable to the food industry. criteria: low pH optimum to avoid side reactions,
The glucose isomerases in use today are ac- high specific activity, high temperature optimum.
tually D-xylose-isomerases which have addi- A large group of bacteria and some yeasts are
tional activity with D-glucose and D-ribose. The known which carry out the direct isomerization
200 / CHAPTER 11 / ENZYMES

(Table 11.8). There are other bacteria (Escherichia mentation. In batch process, B. coagulans does not
intermedia, E. freudii, and Aerobacter aerogenes) form the enzyme during the log phase. As soon
which have a somewhat different glucose isom- as the glucose content in the nutrient solution
erase activity. The enzyme produced by these approaches zero, growth ceases. In a typical
bacteria is actually a glucose phosphate isom- diauxy, additional carbon sources present in the
erase. These organisms are not usable industri- medium are then metabolized, and enzyme pro-
ally, however, since their enzymes require ar- duction begins. Maximal enzyme activity is ob-
senic for maximal activity, and arsenic cannot be tained after 24 hours’ incubation.
used in food production. Cobalt and magnesium are prerequisites for
The most important glucose isomerase pro- maximal enzyme production in wild strains, but
ducers are Bacillus coagulans (Sweetzyme®, mutants have been isolated which produce op-
Novo Industri), Streptomyces rubiginosus (Opti- timal enzyme titers in the absence of cobalt.
sweet®, Miles-Kali), Actinoplanes missouriensis Either yeast extract or corn steep liquor can be
(Ketozyme®, Universal Oil Products; Maxa- used as the nitrogen source. As in the case of
zyme®, Gist Brocades), Flavobacterium arbores- many other fermentations, the choice of a nitro-
cens (Taka-Sweet®, Miles Laboratories). gen source is vital for the yield and the concen-
tration must be optimized for each individual
Glucose isomerase from Bacillus process.
The difficulties that arise when attempting to
Novo Industries has developed glucose isomer- optimize the nitrogen source for glucose isom-
ase from B. coagulans for commercial use. This erase production are illustrated in Table 11.9 by
immobilized enzyme process is used today in results for Actinoplanes missouriensis. As seen,
many plants in the United States, Europe, Japan there is wide variation in yield using various
and Korea. Since this glucose isomerase is pri- complex nitrogenous substrates. Because it is so
marily a xylose isomerase, xylose must be added difficult to standardize the nitrogen source, the
for induction of the enzyme with all wild strains. fermentation yield may vary considerably from
Xylose is quite expensive, however, and may be batch to batch.
replaced by xylan or wheat bran, which also con-
tain xylose. Constitutive mutants have also been
isolated. Table 11.9 Effect of organic nitrogen sources on
glucose isomerase production in Actinoplanes
Catabolite repression regulates the produc- missouriensis
tion of glucose isomerase. Glucose acts as a re-
Nitrogen source Manufacturer Activity
pressor in both batch process and continuous fer-
GI/ml %

Table 11.8 Glucose isomerase producers Corn steep liquor Anheuser-Busch 17.8 100
O. M. peptone Amber Lab. 13:45 575
Bacillus coagulans Casein hydrolysate Amber Lab. IO. 73
Streptomyces phaeochromogenes (Amber EHC)
Streptomyces rubiginosus Bacto-soytone Difco 11.8 166
Streptomyces olivochromogenes Yeast extract (BYF-100) | Amber Lab. 1055858
Arthrobacter sp. Distiller's solubles National 7.9 44
Actinoplanes missouriensis Distillers
Microbispora rosea Yeast extract (BYF-300) | Amber Lab. 9.833
Micromonospora coerula Bacto-peptone Difco Lab. 52 skt 29
Microellobospora flavea Yeast extract (BYF 50 X) Amber Lab. 4.3 24
Nocardia asteroides Malt extract Difco Lab. 2.5 14
Nocardia dassonvillei Atlantic Menhaden Haynie Products 1.1 6
Brevibacterium imperiale peptone (fish)
Flavobacterium arborescens
(Activity with corn steep liquor = 100%)
 11.3 GLUCOSE ISOMERASES / 201

In a continuous fermentation process, the. a

growth-limiting substrate must be glucose. For 0.10 4
; Aa
optimal enzyme yields, oxygen limitation is
necessary since microaerophillic conditions
also SØ NY
in-
Fd
4a
e a

side the cells stabilize the system. 'e

bg —-0 —0.
Ny
AM
(=) oOa i

Glucose isomerase from Streptomyces

Glucose isomerases from Streptomyces strains are (mg/ml
Fructose
min)
002
LØ
de =>
a
- RR
a

produced only in batch processes today. There

are mutants which require neither xylose nor co- 30 40 ED GO" 70 © ad's gC
z se I ee
balt. Production is begun with a spore inoculum 6 7 8 9 iO 12
pH
of the working strain, which is cultivated for 24—
48 hours at 30°C in shaken culture. The culture Figure 11.13 Effect of temperature and pH on glucose
medium used contains soy meal, yeast extract, isomerase activity. The enzyme was derived from Strep-
glucose, starch, phosphate, and deionized water. tomyces albus. e——e Na,HPO,-KH,PO, buffer; 0 — oO
Na,HPO,-NaOH buffer; 0 O NaOH-glycine buffer;
A number of such shake flasks are used to in- 4—— 4 Na,CO,-NaHCO, buffer; X —— X temperature
oculate (5% inoculum) the production fermenter experiment with phosphate buffer 0.05 M (From Takasaki
(which is 80-150 m3 in volume). Streptomyces oli- et al., 1969)
vaceus NRRL 3588, produces glucose isomerase
at pH 8.5 and between 60-70°C. A typical fer-
mentation with Streptomyces albus is shown in Immobilization of glucose isomerase
Figure 11.12.
The commercial process for production of fruc-
Figure 11.13 shows the pH and temperature
tose from glucose became feasible only when
optima of the glucose isomerase produced in this
procedures for immobilization of the enzyme
process.
were developed (see Section 11.11), since this
permitted the same batch of enzyme to be used
Å many times. Since glucose isomerase is formed
20, 4 5 ss intracellularly in most strains, many commercial
mane Lg
2% 7 ° processes are carried out with immobilized cells
£ 16 4 o> e ee = 0-0 Pea or by addition of partly broken cells.
‘©
=
a + 6 The Novo process uses glutaraldehyde to
g 12 4 o cross-link cell suspensions of B. coagulans. The
SK € cross-linked material is processed into 0.3-1.0
Ba 8 4 an. ek ; 2
S
mm diameter pellets and used either in a batch
8 1 x 6 [ 2
reaction or continuously with several bioreactors
EY
(za;
Nees
|2
ANS
connected in series. Figure 11.14 shows a system
4 oOo
which produces 100 tons of fructose per day with
T T T År
2 parallel rows, each with three 2.2 m? columns.
15 20 25 30 35
Fermentation time (hr)
The starting material is glucose syrup with a
dry weight of 40-45% containing 93-96% glu-
Figure 11.12 Industrial production of glucose isomerase cose. The solution must first be purified by fil-
with Streptomyces albus. 22 m? fermenter, temperature tration, carbon treatment, and ion exchange to
30?C, impeller speed 150 rpm, aeration rate 0.33 vvm.
e— e pH; X — X cell growth; O — O glucose isom- prevent loss of enzyme activity during isomeri-
erase (From Takasaki et al., 1969) zation. Magnesium is added as an activator and
202 / CHAPTER 11 / ENZYMES

Charcoal
treatment
From the
hydrolysis
process

Concentration

Alkali

Figure 11.14 Flow chart of a process for fructose syrup production (100 tons/day)

the reaction is run at pH 8.5 and a temperature same conversion temperature, a considerably
of 60-65°C. The time required for isomerization better product is obtained in a continuous system
is 0.8-4 hours. in 20% of the reaction time and with only 4%
When compared to the batch process, at the of the reactor volume (Table 11.10).
In the recovery process, the converted solu-
tion is first acidified and then passed over acti-
Table 11.10 Comparison of batch and continuous vated carbon and ion exchange resins to remove
methods for production of fructose salts and colored materials. Fructose syrup is then
Parameters Batch process Continuous concentrated to about a 70% solution (dry weight
conversion basis), stored, and used in this form in the food
Bioreactor volume 750 m3 30 m3 industry. Since di- and trisaccharides are not con-
Enzyme consumption 17-20 tons 10 tons verted by glucose isomerase, a typical product
MgsO,°7 H,O- 4.3 tons 2.2 tons has the following composition:
consumption
CoSO,°7 H,O0- 2.2 tons 0
consumption Fructose 42 % Dry weight
Color formation 0.05-0.10 0-0.02 Glucose 53 % Dry weight
OD eon
Psicose formation 0.1% 0.1% Oligosaccharide 5 % Dry weight
Purification Carbon treatment, Carbon Psicose 0.1 % Dry weight
cation and anion treatment Ash 0.05-0.1% Dry weight
exchangers
Dye value 0.003
(Zittan et al., 1975) pH 4-5
 11.5 “PROTEASES / 203

11.4 L-ASPARAGINASES the pharmaceutical industry, the leather indus-

try, the manufacture of protein hydrolysates, the
L-Asparaginases (L-asparagine amidohydro- food industry, the film industry, and waste pro-
lases) are used as antitumor agents in the treat- cessing.
ment of some leukemias and lymphomas. The Proteases are produced commercially both
principle behind the use of this enzyme as an from bacteria and fungi. An important distin-
antitumor agent is as follows: The metabolism of guishing feature is the pH optimum of the pro-
these tumor cells requires L-asparagine, and tease. The proteases on the market include al-
since asparagine is split into aspaftic acid and kaline, neutral, and acid protéases.
ammonium ions by asparaginase, these malig-
nant cells can be destroyed.
The enzyme is produced by several bacteria, Akaline proteases
but for commercial production (80 m? fermenters) Many bacteria and fungi excrete alkaline pro-
only mutants of Escherichia coli ATCC 9637, Ser- teases. The most important producers are Ba-
ratia marcescens ATCC 60, and Erwinia carotovora
cillus strains such as B. licheniformis, B. amyloli-
are used. An appropriate medium is 3% corn
quefaciens, B. firmus, B. megaterium, and B.
steep liquor, 0.6% sodium acetate, and 0.2% am-
pumilis; Streptomyces strains such as S. fradiae, S.
monium sulfate at pH 7.0, used in reactors up to
griseus, and S. rectus; and the fungi Aspergillus
80 m’ in volume. Oxygen supply is critical during
niger, A. sojae, A. oryzae, and A. flavus. Enzymes
the 16- to 40-hour fermentation. Table 11.11
used in detergents are mainly proteases from Ba-
shows that the prerequisite for a high enzyme
cillus strains (Bacillopeptidases). The best known
titer is a low pQ,.
proteases are Subtilisin Carlsberg from B. lich-
At the end of the fermentation process, the
eniformis, and Subtilisin BPN and Subtilisin
cells are broken open and the enzyme is crys-
Novo from B. amyloliquefaciens. These enzymes
tallized.
contain serine at the active site of the molecule
and are not inhibited by EDTA (ethylene diamine
tetraacetic acid), but are inhibited by DFP (diiso-
11.5 PROTEASES
propyl fluorophosphate).
After amylases, the second most important in- The proteases of this type have many features
dustrial enzymes currently are the proteases. of value for use as detergent enzymes:
About 500 tons of these enzymes (based on the
pure protein) are produced per year. Proteases + stability at high temperature
are used primarily in the detergent industry and + stability in the alkaline range (pH 9-11)
in the dairy industry (rennin, see Section 11.6). ¢ stability in association with chelating agents
Other areas in which proteases are used include and perborates.

Table 11.11 L-asparaginase formation with Erwinia aroideae at 28°C in a small fermenter
Aeration rate Baffles Impeller speed Cell dry weight L-Asparaginase
vvm rpm g/10 1 Units/g
0.5 = 300 14 960
1.0 + 300 16 580
LS ar 300 18 340
2.0 al 300 18 230
(Peterson and Ciegler, 1969)
204 / CHAPTER 11 / ENZYMES
However, their stability in the presence of Fermentation process Sterility is mandatory for
surface-active agents is low, thus limiting their protease production, as with other enzyme fer-
shelf life. mentations. Cultures are stored in the lyophi-
lized state or under liquid nitrogen. Initial growth
Screening Because the enzymes must be stable is carried out in shaken flasks and small fermen-
under alkaline conditions, screening for better ters at 30-37°C. For production, 40-100 m° fer-
producers is done using strongly basic media. menters are used. Production of extracellular pro-
Single colonies have been tested on protein-agar teases is chiefly regulated by the medium
plates at pH 10.0. Figure 11.15 shows the results composition.
of screening with a large number of strains. As The fed-batch process is generally used in or-
seen, strains of B. licheniformis and B. subtilis der to keep down the concentration of ammo-
showed optimal growth in the pH range of 6-7, nium ions and amino acids, since these nitroge-
but some new strains had maximal growth rates nous materials repress protease production.
at pH 8-9 and grew somewhat at pH 11. Although continuous processes have been de-
Since the enzyme yields of wild-type strains scribed, they are not used commercially.
are insufficient for industrial utilization, exten- High oxygen partial pressure is generally nec-
sive genetic studies have been carried out to in- essary for optimal protease titers. Aeration rates
crease the yield. The genes for the formation of are 1 vvm and the time span of the fermentation
several proteases have been cloned. Protein en- is 48-72 hours, depending on the organism.
gineering has been used to develop modified Ba- Proteases must be converted into particulate
cillus subtilopeptidases with altered amino acid form before they are added to detergents, since
sequences and corresponding changes in enzy- if dry enzyme powder is inhaled by production
matic properties such as substrate specificity, pH workers or users, allergic reactions may result.
optimum, and stability to bleaching agents. Enzyme concentrates are marketed in a microen-
capsulated form. To make a suitable encapsu-
lated product, a wet paste of enzyme is melted
Strain
Es] at 50-70°C with a hydrophobic substance such
as polyethylene glycol and then converted into
cheniformis
wn =btilis tiny particles. These solidified spherical particles
are not hazardous when added directly to the
detergent. A further development is the immo-
bilization of enzymes in fibrous polymers and in
granules.

fF
NP
Wn
pn
—
Neutral proteases
WwW

TU
a=<UUJU>P
PPU
TUN
DDOWDDDDDDCWDWO
IFN
HS
—WHDMNn
@~aOWN
Neutral proteases are excreted by both bacteria
and fungi. Producing organisms include: Bacillus

joule subtilis, B. cereus, B. megaterium, Pseudomonas

aeruginosa, Streptomyces griseus, Aspergillus ory-

| 1
zae, A. sojae, and Pericularia oryzae.
Neutral proteases are relatively unstable and
pH 5 6 7 Cc wo 10
calcium, sodium, and chloride must be added for
maximal stability. The pH range of activity is
Figure 11.15 Screening for alkaline proteases. Within the
indicated pH range, at least 90% of the maximal growth fairly narrow and these enzymes are not very
rate is attained. (From Aunstrup et al., 1972) stable to increased temperatures (Figure 11.16).
 11.6 RENNIN / 205

100 possibility for curd production, salting out, is

rarely used. A third possible method is heat pre-
cipitation but this method is used for only a few
processes. A fourth method, the addition of the
8
3 milk-coagulating enzyme rennin, has been
known for a long time and is used in most cases
= i=) today (alone or in combination with pH precip-
Activity
(%)
itation).
nNoO In modern processes, starter cultures are
added which convert lactose to lactic acid and
a lower the pH. The most commonly used strains
10 20 30 40 50 60
Test period (min) include Streptococcus lactis, S. cremoris, S. ther-
mophilus, Lactobacillus helveticus,and L. bulgari-
Figure 11.16 Temperature stability of a neutral protease cus. These organisms are cultured industrially
(Neutrase*, Novo, 1978) and the culture selected depends upon the de-
sired curd structure and flavor, a minimal gas
The neutral proteases are also quickly inactivated production during fermentation, a low sensitivity
by alkaline proteases. Because of these limita- to bacteriophages, and the extent of acid pro-
tions, they have restricted industrial application, duction.
but do find some uses in the leather industry and Rennet has been used for generations as a
in the food industry for the manufacture of crack- milk-coagulating enzyme. It is an extract from
ers, bread, and rolls. the fourth stomach of 3- to 4-week-old calves
which have been raised on milk. The purified
enzyme is called rennin, chymase, or chymosin.
Acid proteases The main binding site of rennin on x-casein is
the phenylalanine residue at location 105 and the
In this category are rennin-like proteases from
methionine at location 106. The kinetics of milk
fungi which are chiefly used in cheese production
coagulation with microbial rennins has been ex-
(see Section 11.6). In addition, other acid pro-
tensively studied.
teases which are similar to mammalian pepsin
Due to the increasing production of cheese
are also on the market. These enzymes have a
(in 1974, six million tons were manufactured
pH optimum of 2-4. Acid proteases are used in
worldwide) and a decline in the number of
medicine, in the digestion of soy proteiln for soy
slaughtered calves, intensive research has been
sauce production, and to break down wheat glu-
underway since 1960 to develop rennin products
ten in the baking industry.
of. microbial origin.
Enzyme systems must meet the following re-
11.6 RENNIN quirements:

In earlier centuries, curds for cheese production ¢ Good coagulation of casein without hydrol-
were produced by the action on milk casein of ysis
microorganisms which were naturally present in * Good odor and structure of the cheese
milk. These organisms produced organic acids, ¢ No unpleasant odor of its own
primarily lactic acid, which lowered the pH to ¢ Nontoxic
the isoelectric point of casein, leading to a natural e Low proteolysis in order to prevent the de-
precipitation of casein in the form of curds which velopment of bitterness in the ripening pro-
could easily be removed from the whey. Another cess
206 / CHAPTER 11 / ENZYMES
¢ Low lipase activity, to avoid the development pH 6.8. The fermentation takes 48 hours at 28°C.
of rancidity in the cheese. At the end of the fermentation, after removal of
the mycelium, the extracellular enzyme is con-
A variety of fungi and bacteria have been iso- centrated and precipitated in an evaporation pro-
lated as producers and have been examined for cess,
the above characteristics. Several Mucor miehei strains produce usable
The following are the most important genera rennin. The enzyme is stable around pH 4.5 and
of bacteria: Alcaligenes, Bacillus, Corynebacterium, the molecular weight is 38,000-41,800. A typical
Lactobacillus, Pseudomonas, Serratia, Streptococ- medium for production is 4% potato starch, 3%
cus, and Streptomyces. The Bacillus strains (B. sub- soy meal, 10% ground barley, 0.5% CaCO. The
tilis, B. polymyxa, and B. mesentericus) have been fermentation takes 5-6 days at 40°C.
marketed but have not proved commercially suc- After the curd has formed, most of the en-
cessful. zyme remains in the whey and is therefore lost.
There are several genera of fungi which pro- Because of this, studies have been carried out to
duce milk-coagulating enzymes: Aspergillus, immobilize the enzyme, but so far these studies
Candida, Coriolus, Endothia, Enthomophthora, Ir- have been unsuccessful. Microbial rennins are
pex, Mucor, Penicillium, Rhizopus, Sclerotium, and actually too temperature stable, remaining active
Torulopsis. in the curd after precipitation and subsequently
Only three strains of fungi are used world- causing harmful proteolysis. Research is thus
wide in production. They are subdivided into 2 being done to clone the gene for calf rennin into
groups: microorganisms. Complementary DNA (cDNA)
Type I Mucor pusillus var. Solid surface from calf prorennin has been successfully ex-
Lindt culture pressed in Escherichia coli, making possible the
Type II Endothia parasitica Submerged culture first commercial production by a microorganism
Mucor miehei Submerged culture of the calf rennin enzyme.

It has not yet been possible to cultivate Mucor

11.7 PECTINASES
pusillus successfully by the submerged culture
process; in a stirred bioreactor, only about half Pectinase preparations contain at least six en-
as much rennin activity is formed as in the sur- zymes, splitting pectins at different sites of the
face process. Moreover, there are substantial side molecule. The basic structure of pectin is a-1,4-
products in submerged fermentation, such as linked galacturonic acid, with up to 95% of its
proteases and lipases, which prohibit the direct carboxyl groups esterified with methanol. Pec-
use of enzyme preparations without extensive tinases are classified according to their point of
purification. The enzyme, which is produced at attack on the pectin molecule (Figure 11.17). The
30°C in 70 hours, is sold commercially as a liquid methyl ester is split by pectinesterase and the
preparation. The rennin produced by Endothia glycosidic bonds of the pectin chain are split by
parasitica was the first rennin marketed com- hydrolysis with endo-polygalacturonases or exo-
mercially, placed on the market in 1967. The en- polygalacturonases. Another method of splitting
zyme is an acid protease, stable at pH 4.0-5.5, is transelimination by means of bacterial endo-
with a molecular weight of 34,000-37,500. pectate lyases, or fungal exo- and endo-pectin
Within the designated pH range at 50°C, only lyase (Figure 11.18).
30% of the activity is lost within 30 minutes. A A number of commercial firms produce fun-
nutrient solution for enzyme production contains gal pectinases using Aspergillus niger or A. wentii
3% soy meal, 1% glucose, 1% skim milk, 0.3% and one fermentation plant uses Rhizopus. Both
NaNO,, 0.05% K,HPO,, 0.025% MgSO,°7 H,O; surface and submerged processes are used. Pec-
 TES LIPASES) /2207

ee atone Pectinases are used primarily to clarify fruit

BEDE juices and grape must, for the maceration of veg-

COOCH 3 COOCH3 COOCH3

etables and fruits, and for the extraction of olive

oil. Pectin-like substances are present at concen-
LER age ee lyase
trations ranging from 0.2-1% in grapes to 30%
Pectin-
esterase _ in sugar beet pulp. By treatment with pectinase
(1-2 hours at 54°C or 6-8 hours at 18°C) the
COOH COOH COOCH3 COOH COOH COOH
fo) 0. 0 0. 0. 0. R yield of fruit juice during pressing is considerably
OK on OK on OK on OK OH OK on OK ou oO increased. ’
OH OH OH OH OH OH
Polygalacturonase
Pectate lyase
11.8 LIPASES
Figure 11.17 Pectin hydrolysis with various individual
pectinases Lipases (glycerol ester hydrolases) split fats (glyc-
erol esters) into di- or monoglycerides and fatty
acids (Figure 11.19). They are usually extracel-
COOH aca lular enzymes. Fungi producing lipases include
0 j .
“KOH OK 0H is Aspergillus, Mucor, Rhizopus, Penicillium, Geotri-
OH chum, and the yeasts Torulopsis and Candida. Bac-
linens teria producing lipases include Pseudomonas,
Achromobacter, and Staphylococcus. Of these or-
COOH COOH COOH COOH ganisms, only Aspergillus, Mucor, Rhizopus, and
0 0 i ) Va ee Candida are used commercially.
“KOH OK OH ore KOH + KOH O°
OHH In most cases, enzyme production must be
OH OH OH OH
induced by adding oils and fats. However, there
Pectate lyase
are some cases in which fats have no effect on

Eo — Re De
lipase production, and with Penicillium roque-
COOCH, COOCH, COOCH,
forti, they actually repress enzyme production.
Glycerol, a product of lipase action, represses li-
pase formation. This is also true for glucose, so
Pectin lyase that lipase production in Geotrichum candidum is
first induced at the end of the log phase (around
Figure 11.18 Splitting of glycosidic bonds in pectin
through hydrolysis with polygalacturonase and through
15 hours). Lipases are generally bound to the
transelimination with pectate lyase and pectin lyase cells and hence inhibit an overproduction, but
addition of a cation such as magnesium ion lib-
erates the lipase and leads to a higher enzyme
tinases have been discovered in other fungi and titer in the production process. Isoenzymes with
also in bacteria, protozoa, insects and higher
plants.
The fermentation with Aspergillus niger runs Q
AYVWAAVNAVC-O=CH2 HOS
for 60-80 hours in fed-batch cultures at pH 3-4 | Lipase )
PNYNNAYNINACE OE CH Sg AVIVAOCH
and 37°C, using 2% sucrose and 2% pectin. The
purification of the enzyme is simple: the biomass NVMAAAVC=0-CH7 + HO- CH,
is removed by filtration or centrifugation, stabi- 2 NVAVWA5-0H
O
lizing agents are added, the enzyme is precipi-
tated with organic solvents, and the crude protein Figure 11.19 Splitting of fats into monoglycerides and
dried. fatty acids with lipases
208 / CHAPTER 11 / ENZYMES
Table 11.12 Temperature and pH optima of several trations by Penicillium chrysogenum when the or-
microbial lipases ganism is grown in a medium without addition
Organism Optimal Optimal of phenylacetic acid as a precursor. This process
pH temperature for production of the basic ring system cannot
Penicillium chrysogenum 6.2—6.8 37 currently be commercially utilized due to its high
Pseudomonas fragt 1205722 32 cost. 6-APA can also be produced by the split-
Rhizopus delemar 5.6 30
Aspergillus niger 5.6 35 ting-off of the acyl side chain of microbially pro-
Penicillium roqueforti 8.0 37 duced penicillin G with the enzyme penicillin
Staphylococcus aureus 8.5 45 acylase (also called penicillin amidase or ben-
Geotrichum candidum 8.2 37
Achromobacter lipolyticum 7.0 37 zylpenicillin amidohydrolase). It is essential that
with the enzyme used the 6-lactam ring not open
(Shahani, 1975)
(Figure 11.20). Penicillin acylases thus have com-
mercial use in the production of semisynthetic
varying pH optima, temperature optima, or sub- penicillins.
strate specificities are frequently produced.
Temperature and pH optima for lipases are
given in Table 11.12. Classification of penicillin acylases
The commercial use of lipases has been lim- Penicillin acylases are produced by yeasts, fungi,
ited. They are primarily marketed for therapeutic and bacteria. They can be subdivided into 2
purposes as digestive enzymes to supplement types. Type I acylases, also called the fungal type,
pancreatic lipases. The enzymes also find some split phenoxymethy! penicillin (penicillin V) but
use in the dairy industry. Since free fatty acids attack benzyl penicillin (penicillin G) much less
determine the odor and taste of cheese, and the efficiently. Known producers of these acylases
cheese ripening process is affected by lipases, mi- are Penicillium sp., P. chrysogenum, Aspergillus
crobial affects during the aging process can be ochraceus, Trichophyton mentagrophytes, Epider-
due to lipase action. For instance, in the produc- mophyton floccosum, Cephalosporium sp., and Fu-
tion of roquefort cheese (blue cheese) spores of sarium semitectum. Aside from fungi, the bacte-
Penicillium roqueforti are added and serve as a rium Streptomyces lavendulae also produces this
sort of lipase preparation. In the soap industry,
the lipase from Candida cylindraceae is used to
hydrolyze oils. A potentially important future
use of lipases is for the synthesis of esters from CS -crgco-ne S. CH,
acids and alcohols in nonaqueous media. An-
CH
other potential use is improvement of fat quality salar
by the exchange of one fatty acid by another. O COOH
| Penicillin acylase
11.9 PENICILLIN ACYLASES
As we will describe in Chapter 13, many peni-
H2N Sker
cillin antibiotics used in medicine are produced (|\-cxgcoot + | i Sox,
4
semisynthetically by chemical modification of Oo COOH
the basic penicillin ring structure. The starting
material for chemical modification is the non-
Phenylacetic acid 6-APA
acylated thiazolidine-6-lactam ring system of
penicillins, 6-aminopenicillanic acid (6-APA, Figure 11.20 Splitting of penicillin G into 6-aminopen-
Figure 11.20). 6-APA is excreted in low concen- icillanic acid and phenylacetic acid
 11.9 PENICILLIN ACYLASES / 209

type of acylase. In addition to penicillin V, pen- - The cells are concentrated 20-fold on a sep-
icillin K (heptyl penicillin), dihydro penicillin F arator and the enzyme is then released by use of
(pentyl penicillin), and a number of synthetic a pressure disintegrator (500 kg/cm? pressure).
penicillins are split. These acylases, which are The crude enzyme preparation so obtained is
usually extracellular, have a pH optimum of 10 subsequently purified by conventional biochem-
and a temperature optimum of 50°C. ical procedures.
Type II acylases, also designated as the bac- The enzyme yield in commercial production
terial type, are produced by Aerobacter, Alcali- has been substantially improved by optimizing
genes, Bordetella, Cellulomonas, Corynebacterium, the culture medium and by use of classical ge-
Erwinia, Escherichia, Flavobacterium, Micrococcus, netic techniques. Recombinant DNA techniques
Nocardia, Proteus, Pseudomonas, Salmonella, Sar- promise to increase the yields even more. For
cina, and Xanthomonas. The temperature opti- instance, by cloning the penicillin acylase gene
mum is 40°C and the pH optimum is pH 8, which on a multicopy plasmid, strains have been ob-
is lower than that of the Type I acylases. Phenyl- tained having a 28-fold increase in enzyme for-
acetic acid acts as a competitive inhibitor and 6- mation rate in noninduced fermentation and a
APA as a noncompetitive inhibitor. further 6-fold increase in induced fermentation
The extracellular and intracellular acylases of (Table 11.13).
various microorganisms differ in substrate spec- There are 3 methods used commercially for
ificities. The substrate specificity is determined producing 6-APA from penicillin G. A strictly
by the nature of the acyl moiety and not by the chemical method involves converting penicillin
6-APA. G in a series of steps to N-iminomethoxypeni-
cillin ester, which is hydrolyzed with aqueous
ammonia to 6-APA. Two microbial methods are
Penicillin acylase from Escherichia coli used for the enzymatic conversion, one using a
bacterial slurry and the other using carrier-bound
Penicillin acylases from Escherichia coli are used penicillin acylase.
almost exclusively for 6-APA production. The
production strains are mutants of Escherichia coli Enzymatic splitting with whole bacteria The orig-
ATCC 11105 and Escherichia coli ATCC 9637. inal process uses cell-bound penicillin acylase.
Intracellular acylase production is induced in Escherichia coli slurry is added as a crude enzyme
wild-type strains by addition of phenylacetic solution directly to a penicillin G solution in a
acid. Glucose represses enzyme production and batch process which has been run at pH 8.0 and
must therefore be present in the culture medium 37°C. At the end of the reaction, the cells are
only in low concentrations. The production rate removed by filtration and discarded. The filtrate
is also strongly affected by the O, partial pres- is adjusted to pH 2.0 and the 6-APA separated
sure. Although E. coli can grow under anaerobic
conditions, enzyme yields are low; however, en- Table 11.13 Penicillin G-acylase formation in hybrid
zyme production is also suppressed by high aer- strains
ation rates. Escherichia coli Specific activity
A suitable nutrient medium consists of 2%
= Not induced Induced
corn steep water at pH 7.0. The inoculum consists
ATCC 11105 0.02 0.12
of 0.25% of an 18-hour preculture. To induce
5 K pHM6 0.56 0.70
enzyme formation, after 8 hours of fermentation 5 K pHM7 — 0.44
at 24°C, 0.1% of a sterile ammonium phenyl- 5 K pHM8 — 0.36
5 K pHM11 — 0.28
acetate solution is added at hourly intervals for
the next 13 hours. (Mayer et al., 1979)
210 / CHAPTER 11 / ENZYMES
from phenylacetic acid and nonconverted peni- ing an enzyme from Pseudomonas sp. (BN-188),
cillin by extraction with methylisobutyl ketone. which is closely related to Pseudomonas putida.
6-APA is then precipitated at its isoelectric point
(pH 4.3) and the crystals are washed and dried.
11.10 LACTASES
Carrier-bound penicillin acylase Several Lactase, also called 6-galactosidase, splits lactose
hundred tons of 6-APA are produced each year into glucose and galactose. The enzyme is intra-
using this widely applied process. Its advantages cellular in bacteria and yeast, but it is excreted
are: conservation of the penicillin acylase by re- by many fungi. The genetics of some bacterial 6-
cycling, greater purity of the 6-APA, and fewer galactosidase systems have been intensively
losses due to side reactions. There are processes studied, but the enzymes produced commercially
for the immobilization of whole cells, in which are obtained from fungi or yeasts and are less
acrylamide monomers are polymerized with well known. Lactases of commercial interest in-
cells. However, methods in which the purified clude those from the fungi Aspergillus oryzae and
penicillin acylase is immobilized are more widely A. niger, and those from the yeasts Kluyveromyces
used. lactis, K. fragilis, and Torula cremoris and Bacillus
In one method, penicillin acylase from Esch- sp. (in immobilized form). The enzymes differ
erichia coli is rendered insoluble by formation of mainly in the following:
a covalent bond with a polymer. Penicillin
(100,000 units/ml) can be quantitatively split at Fungi Yeasts
38°C and pH 7.8 in 6 hours when substrate and pH optimum 2.5-4.5 6.0-—7.0
enzyme are used in a proportion of 5X 10¢ units Temperature optimum 55°C S50
of substrate (1670 International Units = 1 mg
penicillin G) per unit of penicillin acylase. One The yeasts are cultured in a submerged process,
unit of enzyme is defined as the activity which but the aspergilli must be cultivated on the sur-
hydrolyzes 1 wMole penicillin G in one minute face of wheat bran at 30°C. Lactose is generally
to 6-APA and phenylacetic acid at 37°C. With added to induce enzyme formation.
this method, 6-APA can be isolated with a re- Lactases are used as digestive enzymes in
covery of 87% of the theoretical yield and at 97% cases of lactose-intolerance (usually due to lac-
purity. tase deficiency) and as a feed additive to increase
the nutritive value of some animal feeds. In the
dairy industry, lactases are used to break down
Cephalosporin acylases lactose in milk. For example, in Europe lactose-
Cephalosporins are antibiotics with a 6-lactam low milk is produced by pasteurization and then
ring similar to but different from that found in treating with yeast lactase (4 hours at 35°C,
the penicillins. Semisynthetic cephalosporins are which results in 70-80% hydrolysis of the lac-
also produced in a manner analogous to the pen- tose). The resulting product is then sterilized by
icillins. In the past, the side chain L-a-aminoad- the ultra-high temperature process and mar-
ipic acid was chemically split to produce the ring keted. Lactases are also used commercially to
structure needed for the synthetic process, since process whey.
microbiological methods resulted only in low
yields. Today, however, there is a process which
11.11 STABILIZATION OF ENZYMES
allows the microbiological splitting of cephalo-
AND CELLS
sporin C into the 7-acyl side chain and 7-ami-
nocephalosporanic acid (7-ACA). A solution of Purified enzyme preparations generally cannot
35% cephalosporin C can be split in 7 hours us- be stored for long periods without losing their
 11.11 STABILIZATION OF ENZYMES AND CELLS / 211

effectiveness. Native enzymes are subject to in- ° 4. Incorporation within semipermeable mem-
activation by chemical, physical, and biological branes. Encapsulated enzymes are separated
factors, and the inactivation can occur either in from the surrounding substrate and product
storage or during use. There is thus a need to by a semipermeable membrane. Cells with
stabilize enzymes because of the high cost of en- enzymatic activity can also be encapsulated.
zyme production. Ideally, enzymes should be The activity of the enzyme is not affected as
stable for months with as much activity as pos- a result of the encapsulation process and the
sible under varied conditions. Moreover, it preparation can be used repeatedly or contin-
should be possible to stabilize enzymes in com- uously. i
mercial processes in such a way that they can be
used over and over again and so that conversion
of substrate to product can be carried out con- Stabilization of soluble enzymes
tinuously.
In some cases, the enzyme cannot be immobi-
If a cofactor such as NAD or an energy-rich lized but must be used in soluble form. Examples
substrate such as ATP is needed in the process,
include the enzymes used in liquid detergents,
it is essential that regeneration be possible. Un-
as diagnostic reagents, and as food additives. In
der such conditions, it is more cost-effective to
such cases, some procedure for stabilization of
use immobilized cells rather than immobilized
the enzyme is necessary in order to prolong the
enzymes. The technique for immobilizing cells is
shelf life of soluble enzymes. Following are some
actually very analogous to that for immobilizing
methods of stabilization:
enzymes.
There are four basic methods of achieving
enzyme stability: Substrate stabilization The active site of an en-
zyme is responsible for its specific activity and
1. Stabilization of soluble enzymes. Stabili- this site can be stabilized by adding the substrate.
zation of soluble enzymes is accomplished by For instance, a-amylase is stabilized by adding
additives or by chemical modification. This starch and glucose isomerase is stabilized against
improves the stability of enzymes against heat damage by addition of glucose. Figure 11.21
physical and chemical agents without de-
creasing their solubility. Such stabilized en-
zymes can be successfully stored but since
they are still soluble they cannot be recycled
after use.
80
2. Stabilization by cross-linkage (immobili-
zation) of enzyme molecules or cells. Cross-
linked enzyme molecules are linked to each 60
other in such a way that their activity is not
affected. They are no longer soluble and can 40
be repeatedly or continuously used.
3. Bonding to carriers. In these procedures, the 20
“enzyme molecules are not bound to each 1
Residual
(after
hr)
activity
other, but to a carrier. Immobilization is also
= wa sa SST et
possible within the original microbial cell. The
4 6 8 10 12
fixation is done in such a way that it does not Starch (%)
affect the enzyme activity, and the prepara-
tion can be used repeatedly or continuously Figure 11.21 Stabilization of a-amylase by its substrate
as a carrier-bound enzyme. starch (Novo, 1970)
212 / CHAPTER 11 / ENZYMES
shows the stabilization of a bacterial amylase at polyglycyl enzymes. Another method is acyla-
pH 7.0 and a temperature of 80°C using starch. tion with acetyl, formyl, propionyl, or succinyl
On the other hand, there are enzymes such groups. Asparaginase in blood can be consider-
as pyruvate dehydrogenase whose activity is de- ably protected by acylation. In other enzymes,
creased when substrate is added. such as a-amylases, the temperature sensitivity
and sensitivity to proteases can be reduced.
Solvent stabilization Enzymes can also be sta- By means of bifunctional or multifunctional
bilized by adding solvents; many of these can additives, enzymes can also be polymerized in
cause denaturation at high concentrations but af- such a way that they remain soluble.
ford considerable stabilization at low concentra-
tions. Figure 11.22 shows several examples of the
Stabilization by means of immobilization
stabilizing effect of solvents on benzylalcohol de-
hydrogenase. The bonding of an enzyme to another enzyme
or to a carrier must take place without changing
Stabilization by means of salts Cations such as the three-dimensional structure at the active site
Ca, Cu, Fe, Mn, Mo, and Zn have an effect on of the molecule. Neither the substrate specificity
the stability and activity of metalloenzymes. For nor the specificity of the reaction must be lost as
example, calcium helps to stabilize the tertiary a result of the immobilization process.
structure of a-amylases of Bacillus caldolyticus Functional groups on the enzyme molecule
and proteases. that are suitable for use in the immobilization
process are free a-, B-, or y-carboxyl groups, a-
Stabilization by means of polymer additives Nat- or B-amino groups, and phenyl, hydroxyl,
ural or synthetic polymers such as gelatin, al- sulfhydryl, or imidazole groups of the appropri-
bumin, fatty alcohol ethylene oxide adducts, or ate amino acids. The groups used must not be
polyethylene glycols can increase the heat sta- critical for the activity of the enzyme.
bility of enzymes. The three major approaches to enzyme im-
mobilization are illustrated in Figure 11.23 and
Stabilization by chemical means In addition to are summarized below.
enzyme-bonding to soluble carriers, stabilization
can be accomplished by chemical modification Cross-linked enzymes Several examples of this
without a loss of solubility. One method is the method for immobilizing enzymes can be given.
formation of enzymes with polyamino side Glutaraldehyde is commonly used as a polymer-
chains, such as the production of polytyrosy] or izing agent, and Figure 11.24 shows how this

100

>80
=
© 60
©
ZA Figure 11.22 Stability of benzyl al-
3 cohol dehydrogenase in organic sol-
2 a vents. X —— X without solvent;
A— A 2% acetone; O——O 5% ace-
tone; O—O 10% acetone; 4a——a
— 2% ethanol; m B 5% ethanol;
10 15 20 25 e— e 10% ethanol (From Katagiri et
Test period (hr) al., 1967)
 11.11 STABILIZATION OF ENZYMES AND CELLS / 213

Carrier-bound Cross-linked reacts with enzymes by peptide bond formation

enzyme enzyme (Figure 11.25).

Carrier-bound enzymes The bonding of an en-

zyme to a carrier through adsorption, ionic bond-
ing, or covalent bonding is the oldest method of
enzyme immobilization, and it is the most fre-
quently used commercial method. The structure
of the carrier, the type of functional bonding
sites, the ratio of hydrophilic to hydrophobic
groups, the particle size, and the surface /volume
ratio must be suitable for the individual enzyme
and for its eventual commercial use.
Bonding by means of adsorption causes the
least damage or conformation alteration of the
protein. Fixation is easily accomplished and the
method is applicable to a wide variety of en-
zymes. In addition, the carrier can be regenerated
Enzyme inclusion Microcapsule in most cases. However, the bonding strength is
low, and this is a disadvantage of adsorption pro-
Figure 11.23 Procedures for the immobilization of en- cedures; any temperature changes in the bio-
zymes (From Chibata, 1978) reactor cause elution of enzyme from the carrier
and hence activity loss. Both organic and inor-
ganic carriers can be used for the adsorption pro-
=N -Enzyme-N=CH-(CH,),-CH cess and several types are listed in Table 11.14.
| ll Ion exchangers can be used as enzyme car-

i
CH
1
Enzyme
riers, the enzyme being held to the carrier by
ionic bonds. This method overlaps in part with
the adsorption method. Ionic bonding is an easily
|
(CH,); N accomplished method with low activity loss, al-

-CO-NH- Enzyaie -NH-CO- NH{CH,)g NH- CO

NH he
= N—Enzyme—N= CO —NH- Enzyme
NH NH
Figure 11.24 Cross linkage of enzymes with glutaral-
| |
dehyde
(CH
NE

compound reacts with amino groups of the en- se

zymes (for example, amylases and alcohol de- NH
hydrogenase) in the formation of Schiff bases. |
—NH-Enzyme-NH—
Immobilization of cells is also frequently accom-
plished with glutaraldehyde. Another cross-link- Figure 11.25 Cross linkage of enzymes with hexame-
ing agent is hexamethylene diisocyanate, which thylene diisocyanate
214 / CHAPTER 11 / ENZYMES

Table 11.14 Adsorption carriers for immobilization of CH, OH NH,-NH,

enzymes Cellulose-O-CH; COOH ToS oa OS EOS CE OLN Cn Es

Type of Carrier Enzyme

NaNo,
carrier used ARIEL TL AR gO SS FE Eo lulose-O-CH;CO-N,

Inorganic Aluminium oxide Glucoamylase

Enzyme
Bentonite Invertase Cellulose-O-CH,-CO-NH- Enzyme
Glass Lipase
Ca phosphate gel Aspartase
Figure 11.26 Immobilization of enzymes with carboxy-
Organic Activated carbon Glucose oxidase methylcellulose
a-Amylase
6-Amylase
Glucoamylase
CO-NH, MUG)
Invertase |
Starch a-Amylase Ber Ca cre -CH ea =
Tannin aminohexyl Glucose
cellulose isomerase
Aminoacylase
:
CO CO-NH
ah
CO

ve AM
CH, Enzyme Ch,
|
NH NH NH,
|
though changes in pH or ionic strength can result CO NZeO CO CO
in enzyme loss from the carriers. A variety of | | |
- CH>CH eng -CH; CH-CH;CH-
CH; CH -
commercially available ion exchangers can be
used as carriers. CO-NH -
Immobilization by covalent bonding has
Figure 11.27 Enzyme incorporation into a polyacrylam-
the advantage of strong bonding forces. In this ide gel
procedure, the side chain of one or more amino
acids of the protein is modified so that it forms
a covalent link with the carrier. The preparation sponding azide derivative is produced which
of a covalently bonded enzyme is expensive and reacts with the amino groups of the enzyme.
difficult to carry out because amino acids of the
active center may also become involved in the
Encapsulation of enzymes
reaction. Covalent bonding is the most widely
used commercial process. The kinds of covalent When enzymes are physically enclosed in gels,
bonds used include peptide bonds and diazo microcapsules, or fibrous polymers, there must
bonds, as well as alkylation and isourea bond be pores which are so small that the enzyme
formation with BrCN-activated carbohydrates. molecules cannot be washed out, yet which are
For enzyme bonding to occur, the functional still large enough to permit the unimpeded dif-
groups of the carrier must be activated. For the fusion of low-molecular-weight substrates and
production of peptide bonds with primary amino products across the barrier. Unfortunately, there
groups of the enzyme protein, the carboxyl may be considerable yield loss in the production
groups of the carriers can be activated through of an encapsulated enzyme, but the finished
the production of azide or anhydride derivatives. products are very stable.
An example of covalent linking is that shown
with carboxymethyl] cellulose (CM cellulose) in Gel enclosure Cross-linked, water-insoluble
Figure 11.26. Carboxymethyl cellulose is first polymers such as polyacrylamide, polyvinyl al-
converted to the methylester and subsequently cohol or starch can be used to surround the en-
treated with hydrazine. The resulting hydrazide zyme. The method most frequently used involves
then reacts with sodium nitrite and the corre- incorporation into a polyacrylamide gel (Figure
 11.11 STABILIZATION OF ENZYMES AND CELLS / 215

11.27). Acrylamide and the enzyme are polymer- - of the use of immobilized cells is in the trickling
ized within minutes and at room temperature generator used for vinegar production.
into a gel by means of a cross-linking compound A number of processes have also been intro-
(e.g. NN’-methylene bisacrylamide), a starter duced in more recent times. Among these are the
(sodium persulfate), and an accelerator (6-di- use of immobilized cells containing glucose
methylamino propionitrile). The gel can then be isomerase for the production of high-fructose
cut up and used in a batch or column process. syrup (see Section 11.3). The half-life of the im-
mobilized isomerase in large-scale operation
Microcapsules The advantage of immobilization ranges from 70-120 days. World-wide produc-
of enzymes using microcapsule preparations is tion with this method amounts to 7.5 X10 tons
that free enzyme or free cells can be surrounded per year, of which about half is in the United
by a semipermeable polymer membrane. Pro- States. Glucose amylase for the production of
duction takes place by means of a polymerization glucose from starch is now frequently used in an
reaction at the surface of aqueous enzyme drop- immobilized state. The enzyme $-galactosidase
lets which are suspended in a non-water-soluble from yeast is now being immobilized in some
organic phase. Microcapsulated enzymes have countries (for instance, Italy) for the splitting of
not yet found commercial application, but they lactose to glucose plus galactose. Some studies
appear promising for future clinical and analyt- are under way to use this process to produce a
ical uses. sweet syrup from whey. A large-scale process for
the immobilization of invertase has been de-
veloped for the splitting of sucrose to glucose
Immobilization in fibrous polymers This method
plus fructose. The enzyme a-galactosidase splits
of immobilization is similar to the method using
the trisaccharide raffinose to galactose plus su-
microcapsules. An emulsion of aqueous enzyme
crose. Raffinose is present in sugar beet juice at
solution and organic water-immiscible solvent is
a concentration of around 0.1% and reduces the
extruded into a precipitation bath in which the
yield of sucrose during sugar beet production be-
fibrous polymer forms and encloses the enzyme.
cause of its effect on the crystallization process.
Cellulose triacetate and other cellulose deriva-
A large-scale process is under study to immo-
tives serve as suitable polymers.
bilize the a-galactosidase from the fungus Absidia
sp. for use in the sugar beet industry. A number
Industrial application of immobilized of processes for amino acid production have
enzymes and cells been developed using immobilized enzymes or
cells (see Section 9.3).
The first large-scale applications of immobilized In addition to the food industry, immobilized
cells were optimized empirically and have been systems have also been developed for fine-chem-
in use for many years. The best example is the ical production. The process using penicillin ac-
use of the trickling filter for sewage treatment (see ylase has already been discussed in Section 11.9.
Chapter 17). A modern application of this pro- In another example, malic acid is being produced
cedure is the immobilization of cultures of an- from fumaric acid using the fumarase of Brevi-
aerobic bacteria on sintered glass particles, which bacterium ammoniagenes. In this process, an en-
makes it possible to increase the rate of anaerobic zyme/membrane reactor has been developed
sewage treatment. For instance, in the effluent which carries out the following reaction:
from a cellulose processing plant, 84% of the
HOOC—CH=CH—COOH + H,0 =>
chemical oxygen demand (COD) was removed
HOOG—CHOH—CH,GOOH
in a 12 hour holding time with a loading of 88
kg COD/m?/day. Another well-known example In this process, a 1 m? column reactor has been
216 / CHAPTER 11 / ENZYMES

Product

I
Electrode
Thermistor

Immobilized
enzyme
Thermistor
Micro- Immobilized
calorimeter enzyme

Figure 11.28 Biochemical assays us-

ing immobilized enzymes or cells. A.
Enzyme/thermistor. B. Enzyme elec-
A Substrate trode

Table 11.15 Examples of biochemical assays carried used, with a flow rate of 0.3 h” at pH 7.5 and
out by means of the enzyme/thermistor method
37°C. The half-life of the enzyme in this process
Substrate Enzyme Concentration was about 160 days.
(mMol/1)
In Japan, the large-scale production of
Ascorbic acid Ascorbic acid oxidase 0.05-0.6 ethanol has been carried out using growing, im-
ATP Hexokinase 1-8
Cholesterol Cholesterol oxidase 0.03-0.15 mobilized cells of the yeast Saccharomyces. The
Cephalosporin Cephalosporinase 0.005—10 cells are immobilized by photochemical reaction
Ethanol Alcohol oxidase 0.01-1 with a cross-linked gel. The bioreactor, which
Galactose Galactose oxidase 0.01-1
Glucose Hexokinase 0.5-25 produces more than 1.5 mol/l-h ethanol, has
Lactose Lactase/Glucose 0.05-10 been operated successfully over long periods of
oxidase /Catalase time. A process for the production of glycerol
has been developed using immobilized Saccha-
romyces cerevisiae, but is still at the laboratory
stage.
Special bioreactors for use in immobilized
processes were discussed in Section 5.3.

Table 11.16 Electrode sensors employing immobilized

enzymes or cells Uses of immobilized cells and enzymes
for analytical biochemistry
Substrate Strain/enzyme Measurement
principle An immobilized system can be used in the de-
Sucrose Invertase/Mutarotase O, uptake velopment of a precise and sensitive biochemical
Glucose oxidase
assay. The basic principle is that the enzyme acts
Monoamine Monoamine oxidase O, uptake
Cholesterol Cholesterol oxidase O, uptake on the substrate and a decrease in substrate con-
Acetic acid Trichosporon brassica O, uptake centration, increase in product concentration, or
Ethanol Trichosporon brassica O, uptake
Nystatin Yeast O, uptake
change in cofactor concentration can be followed,
(as a result using one of many assay methods. The reaction
of cell death) can be followed spectrophotometrically, polari-
Cephalosporin _—_Citrobacter pH
with cephalosporinase measurement
meterically, photometrically, or by mass spec-
troscopy. Additionally, as shown in Figure 11.28
 REFERENCES / 217

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cephalosporins, pp. 110-131. In: Wiseman, A. (ed.), III, Pergamon Press, Oxford.
Topics in enzyme and fermentation technology, vol. 1 Wingard, L.B., W. Katchalski-Katzir, and L. Goldstein
Ellis Horwood, Chichester. (eds.). Applied Biochemical Bioengineering. Volume 1,
Novo Industries. 1977. Production of high-purity glucose Immobilized enzyme principles (1976); volume 2, En-
syrups. U.S. Patent 4,017,363. zyme technology (1976); volume 3, Analytical appli-
Novo 1978. Novo Enzyme Information JB163cGB. cations of immobilized enzymes and cells (1981); vol-
Oestergaard, J. and S.L. Knudsen. 1976. Use of Sweetzyme ume 4,-Immobilized cells (1983). Academic Press, New
in industrial continuous isomerization. Various process York.
alternatives and corresponding product types. Die Woodward, J. (ed.). 1985. Immobilized cells and enzymes,
Starke 28: 350-356. a practical approach. IRL Press, Oxford.
Peterson, R.E. and A. Ciegler. 1969. L-Asparaginase pro- Zittan, L., P.B. Poulsen, and S.H. Hemmingsen. 1975.
duction by Erwinia aroideae. Appl. Microbiol. 18: 64- Zweetzyme-a new immobilized glucose isomerase.
67. Die Starke 27:236-241.
Shahani, K.M. 1975. Lipases and esterases, pp. 181-217.
In: Reed, G. (ed.), Enzymes in food processing. Aca-
demic Press, New York.
 Vitamins

12.1 INTRODUCTION possible, is not economic under large-scale con-

ditions.
Microorganisms can be used for the commercial
production of certain vitamins, such as thiamine,
12.2 VITAMIN B,,
riboflavin, folic acid, pantothenic acid, pyridoxal,
vitamin B,,, and biotin. Also, microorganisms
Occurrence and economic significance
(including algae) synthesize 6-carotene, which is
provitamin A. Ergosterol (provitamin D,), pro- In 1926, G. R. Minot and W. B. Murphy deter-
duced with a yeast fermentation, can occur in mined that liver extracts cure human pernicious
certain Saccharomyces strains at concentrations as anemia, a discovery for which they were
high as 0.1-10% of the cell weight. In addition awarded the Nobel Prize in medicine in 1934.
to direct fermentation, certain vitamins can also Independently, E. L. Ricke and L. Smith isolated
be produced by combined chemical/microbio- crystalline vitamin B,, from liver extracts in 1948.
logical means, the microorganism carrying out Vitamin B,, (cyanocobalamin) is a vitamin that is
specific reaction steps (so-called biotransforma- synthesized in nature exclusively by microor-
tion, see Chapter 15). Biotransformation plays an ganisms; because it is required by animals it is
important role in the production of ascorbic acid present in every animal tissue in very low con-
and tocopherol. In Japan, a wide variety of vi- centrations (e.g. 1 ppm in the liver). Although
tamins are produced by microbial fermentation. the substance isolated from tissues is cyanoco-
However, on a worldwide basis only the pro- balamin, in the cell only the coenzyme form (ad-
duction of vitamin B,,, riboflavin, and ascorbic enosyl- or methylcobalamin) is present, cyano-
acid have any major economic significance. The cobalamin not appearing until the product
microbial production of 6-carotene, although recovery stage. The vitamin B,, needs of animals

219
220 / CHAPTER 12 / VITAMINS

are covered by food intake or by absorption of Figure 12.1) is linked to the heterocyclic base 5,6-
vitamin B,, produced by intestinal microorga- dimethylbenzimidazole, which in turn occupies
nisms. However, humans obtain vitamin B,, only another coordination site of the cobalt next to
from food, since the B,, synthesized by micro- CN in the final cyanocobalamin product. Vitamin
organisms in the large intestinal tract cannot be B,, analogs having other heterocyclic bases (pu-
assimilated. rines or substituted benzimidazoles) are either
The concentrations of vitamin B,, which are spontaneously produced by microorganisms or
present in animal tissues are too low for use in are produced after the addition of these sub-
commercial production. Activated sludge from stances to the culture medium. However, they
sewage treatment contains 4-10 mg B,,/kg, but are less biologically effective in vertebrates than
isolation from this source is expensive due to the the corresponding 5,6-dimethylbenzimidazole
problem of separating the various B,, analogs. compounds. It is noteworthy that vitamin B,, de-
Chemical synthesis is also impractical, since it rivatives with a purine base are ineffective in hu-
requires 70 reaction steps. Vitamin B,, was first
mans.
obtained commercially as a byproduct of strep-
tomycete fermentations for the production of the
antibiotics streptomycin, chloramphenicol, or
neomycin, with a yield of about 1 mg/l. As the
demand for vitamin B,, increased, fermentation
processes were developed with higher-yielding
strains. Commercial production is currently car-
ried out entirely by fermentation. The most im-
portant manufacturers of vitamin B,, are: Farm-
italia S.p.A. (Italy); Glaxo Lab., Ltd. (England); chil) BS!
vw

Merck & Co., Inc. (United States); Rhé6ne Poulenc RE

S.A. (France); Roussel UCLAF (France); G. Rich- BE

a a
fo} o
ter Pharmaceutical Co. and Chinoin (both Hun- SKO

gary). The current annual world production of

vitamin B,, is estimated at about 12,000 kg. Ap-
proximately 3500 kg cyanocobalamin, 2000 kg
SØ itera
hydroxocobalamin, 1000kg coenzyme B,, and a tee | 5,6 -Dimethylbenz-
small amount of methylcobalamin is supplied to || HO-CH O H | imidazol

the pharmaceutical industry; the remainder goes

to the animal feed industry. For swine and poul- R Designation

try feeds, 10-15 mg vitamin B,, is added per ton -CN Vitamin By
of feed, since animal protein can be replaced with (Cyanocobalamin)
z OH Vitamin By,
less-expensive vegetable protein if the vegetable (Hydroxocobalamin )
protein is fortified with vitamin B,,. =CH3 Methylcobalamin
OH OH

Structure Coenzyme By
(5'-Deoxyadenosyl -
Cobalamine, Cobamamide)
Corrin, the base structure of vitamin B,,, consists
of a tetrapyrrole ring which differs from the por-
phyrin ring system in that the methene bridge
between rings A and D is missing. The C-1 of
ribose from cobamide (see structural formula, Figure 12.1 Structure of cobalamins
 12.2 VITAMIN B,, / 221

Biosynthesis (3.3 mg/1); Micromonospora sp. (11.5 mg/1); Kleb-

siella pneumoniae (0.2 mg/1). Higher yields have
The biosynthesis of vitamin B,,, which runs par- been obtained from Propionibacterium freuden-
allel with the biosynthesis of porphyrins and reichii (19 mg/l), Propionibacterium shermanii
chlorophyll up to the formation of uroporphyr- (30-40 mg/1), and (in a process using sugar cane
inogen III, is diagramed in Figure 12.2. The con- molasses) Pseudomonas denitrificans (60 mg/l).
version of uroporphyrinogen III into the corrin The use of modern genetic engineering tech-
ring system is not yet fully understood. niques is being applied to vitamin production
also. A hybrid strain called Rhodopseudomonas
protamicus, made by the protoplast fusion tech-
Vitamin B,, production based on media
nique between Protaminobacter ruber and Rho-
containing carbohydrates
dopseudomonas spheroides, produces 135 mg/l vi-
Most of the B,, fermentation processes use glu- tamin B,, using glucose as carbon source without
cose as a carbon source. Several producing the addition of 5,6-dimethylbenzimidazole.
strains are known, listed here with their yield of
B,,: Bacillus megaterium (0.45 mg/l); Butyribac- Processes with Propionibacteria Propionibacter-
terium rettgeri (5 mg/l); Streptomyces olivaceus ium freudenreichii ATCC 6207 and P. shermanii
ATCC 13673, as well as other variants and mu-
Glycine
tants, are used in a two-stage process with added
cobalt (10-100 mg/l). In a preliminary anaerobic
Succinyl- 5 - Amino- Porpho-
CoA levulinic acid bilinogen phase (2-4 days), 5’-deoxyadenosylcobinamide
is mainly produced; in a second, aerobic phase
(3-4 days) the biosynthesis of 5,6-dimethylbenz-
Uroporphyrinogen III
imidazole takes place, so that 5’-deoxyadeno-
| sylcobalamin (coenzyme B,,) can be produced.
Only traces of other cobamides are synthesized
CH3 Groups i
Co?" '
in this process. As an alternative to the two-stage
Coprogen Ill Cobyrinic acid batch process in a fermenter, both stages can also
1-Amino-2 - be operated continuously in two tanks operated
propanol
NH3 in cascade fashion.
Protoporphyrin IX During the recovery process, the cobalamins,
5'- Deoxy- which are almost completely bound to the cell,
adenosin
are brought into solution by heat treatment (10-
5'- Deoxyadenosyl -
cobinamide
30 min at 80-120°C, pH 6.5-8.5). They are then
——_—_—_—————_+)
converted chemically into the more stable cyano-
cobalamin. The raw product (80% purity) is used
S'-Deoxyadenosyl -
Hemoglobin
Cytochrome
Chlorophyll
cobinamide guanosine- as a feed additive. Additional purification stages
Catalase diphosphate
yield a medically usable preparation (95-98%
purity). The total yield is 75% of the theoretical
ar a-Ribazol- 5-P—»

I
5,6-Dimethyl - 5'- Deoxyadenosyl- value.
benzimidazol cobalamin phosphate
4

Processes with Pseudomonas Pseudomonas deni-

(|

Riboflavin 5'-Deoxyadenosyl- trificans has been found to be the most produc-

cobalamin
(Vitamin Bx) tive species among the different vitamin B,,-pro-
ducing pseudomonads. In this one-stage process,
Figure 12.2 Biosynthesis of cobalamins vitamin B,, is produced during the entire fer-
222 / CHAPTER 12 / VITAMINS

mentation. Cobalt and 5,6-dimethylbenzimida- Vitamin B,, production with other carbon
zole must be added as supplements. It has also sources
been found that additions of the compound be-
taine result in increased yield; sugar beet molas- In the last 10 years, studies have been underway
ses is used as a low-cost betaine source. Although with a variety of strains to determine if various
the mode of action is not known, betaine is as- alcohol and hydrocarbon substrates could be
sumed to cause an activation of biosynthesis or used for vitamin B,, production (Table 12.1). Hy-
drocarbon and higher-alcohol fermentations re-
an increase in membrane permeability. The pro-
sulted in low yields, but those using methanol
cess shown in Figure 12.3 is one of the most cost-
seem to have considerable promise.
effective processes. After 12 years of strain de-
velopment, the yields from this process have
been increased from 0.6 mg/l to 60 mg/l. 12.3 RIBOFLAVIN

Occurrence and economic significance

Pseudomonas Riboflavin (also called lactoflavin or vitamin B,)
Inoculum storage: lyophilized in
denitrificans
MB 2436
dry milk was first isolated from whey by Kuhn, Gyorgy
and Wagner-Jauregg in 1933; the structure was
confirmed by synthesis by Kuhn and Karrer in
Inoculum Agar slant with Medium A. In-
cultivation cubation: 96 h, 28°C 1935. Riboflavin is present in milk as free ribo-
flavin, but is present in other foods (liver, heart,
kidney, or eggs) as part of flavoproteins which
11 Erlenmeyer flask with 150 ml
Preculture Medium B. Incubation: 72 h, contain the prosthetic groups FMN (flavin mon-
28°C, shaker. onucleotide) or FAD (flavin adenine dinucleo-
tide).
5 1 Fermenter with 3.3 1 Medium A riboflavin deficiency in rats causes stunted
C, sterilization 75 min, 120°C. growth, dermatitis, and eye damage. Ariboflav-
Production
Inoculum: 150 ml preculture. In- inosis is a disease in humans caused by riboflavin
culture
cubation: 90 h, 29°C; stirring 420
rpm; aeration 1 vvm deficiency. Appearing as a type of dermatitis, this
disease can be counteracted by administering ri-
boflavin at a daily dosage of 1 mg. Riboflavin is
Medium A (g/l): Sugar beet molasses 60; Yeast extract
1; N-Z-Amine 1; (NH,),HPO, 2; MgSO, available in various preparations for human and
- 7H,O 1; MnSOQ, - H,O 0.2; ZnSO, - veterinary application. In the United States ri-
7 H,O 0.02; Na,MoO, - 2 H,O 0.005; boflavin, as well as thiamine and nicotinic acid,
Agar 25; Tap water; pH 7.4
is frequently added to flour for use in the pro-
Medium B (g/l): Medium A, without agar duction of vitamin-enriched bread.
Riboflavin is produced industrially by several
Medium C (g/l): Sugar beet molasses 100; Yeast extract
2; (NH,),HPO, 5; MgSO, - 7 H,O 3; processes:
MnSQ, - H,0 0.2; Co(NO,), - 6 H,O
0.188; 5,6-Dimethylbenzimidazol 0.025; + Chemical synthesis, primarily for pharma-
ZnSQ, - 7 H,0 0.02; Na,MoQ, - 2 H,O ceutical use (20% of world-wide production).
0.005; Tap water; pH 7.4
+ Biotransformation of glucose to D-ribose by
Figure 12.3 Laboratory scale production process for vi-
mutants of Bacillus pumilus and subsequent
tamin B,, using Pseudomonas denitrificans. (Merck and Co., chemical conversion of ribose to riboflavin
Iney 971) (about 50% of world-wide production).
 12.3 RIBOFLAVIN / 223
Table 12.1 Vitamin B,, formation with alcohols and carbohydrates as carbon sources
Microorganism Vitamin B,,
Carbon source yield (mg/l)
Bacteria from activated sludge Methanol 0.3-1.2% (w/v) 35
Methanobacterium soehngenii Methanol 0.8% (w/v) 8.0
Methanobacillus omelianski Methanol 0.8% (w/v) 8.8
Protaminobacter ruber Methanol 1.2% (w/v) 25
Methanosarcina barkeri Methanol,
fed-batch culture 0.8% (w/v) 42
Arthrobacter sp. Isopropanol, fed-batch culture ‘ 131
Arthrobacter hyalinus Isopropanol 1% (v/v) 0.56
Nocardia MT 2003 1,2-Propanediol 5% (w/v) 0.78
Klebsiella sp. 101 Ethanol 2% (v/v) 0.02
1,2-Propanediol 2% (v/v) 0.06
Methanol 2% (v/v) 0.16
Methanol,
fed-batch culture 0.5% (v/v) 3
Pseudomonas sp. ATCC 14718 Ethanol 1% (v/v) 0.05
1,2-Propanediol 2% (v/v) 0.12
Methanol 2% (v/v) 0.16
Methanol,
fed-batch culture 0.7-0.8% (v/v) 3:2
Strain XF Methanol 2% (v/v) 0.23
Pseudomonas ovalis n-Decane 1% (v/v) 0.08
Pseudomonas aureofaciens n-Dodecane 1% (v/v) 0.04
Nocardia gardneri n-Hexadecane 2% (w/v) 4.5
Mixed culture of
Corynebacterium sp. and
Rhodopseudomonas spheroides n-Alkanes 10% (w/v) 23
(Florent and Ninet, 1979; Kamikubo et al., 1978)

«+ Direct fermentation (about 30% of world- Table 12.2 Microorganisms making riboflavin and the
effect of iron on biosynthesis
wide production).
Microorganism Riboflavin Optimal iron
formation concentration
Total production of riboflavin on a world-
(mg/l) (mg/I)
w
wide basis is around 2000 tons per year.
Clostridium acetobutylicum 97 1=3
Mycobacterium smegmatis 58 No effect
Mycocandida riboflavina 200 No effect
Direct fermentation Riboflavin is synthesized by Candida flareri 567 0.04—0.06
many microorganisms, including bacteria, yeasts Eremothecium ashbyii 2480 No effect
Ashbya gossypii 6420 No effect
and fungi. High-yielding organisms which could
be used in production are listed in Table 12.2. (Perlman, 1979)

However, only the two ascomycetes listed are

important in commercial production. Originally
Eremothecium ashbyti was used (yield 2 g/l), but Structure
after 1946, a process with genetically stable Ash- Riboflavin is an alloxazine derivative which con-
bya gossypii was introduced. The riboflavin titer sists of a pteridine ring condensed to a benzene
of this process is 10-15 g/l. Despite this yield, ring. The side chain consists of a C;-polyhydroxy
there is stiff competition between this microbi- group, a derivative of ribitol. The structure of
ological process and the two other processes. riboflavin (6,7-dimethyl-9-(D-1'-ribityl)-isoallox-
224 / CHAPTER 12 / VITAMINS

O azine) is given in Figure 12.4. The isoalloxazine

I ring acts as a reversible redox system, as de-
HC \— 4 SNH scribed in textbooks of biochemistry and micro-
i biology.
H,C N~ SN So
"CH, Biosynthesis
H safes OH The biosynthetic pathway shown in Figure 12.5
ly is postulated on the basis of experiments done
HE CEO primarily with yeast and Ashbya gossypii. The ef-
I, j fect of iron on riboflavin synthesis is not yet
H- — OH understood. The overproduction of riboflavin by
HC OH Eremothecium ashbyii and A. gossypii is not af-
fected by iron, but riboflavin production in clos-
Figure 12.4 Structure of riboflavin tridia and yeasts is inhibited even by very low
concentrations of iron. In clostridia, 1 ppm iron
causes a 75% inhibition of synthesis. It is as-
sumed that an iron-flavoprotein acts as a re-
pressor of riboflavin synthesis. The overpro-

NADPH
0 HCO3 0 NAD fe) oO

cry <= WT ae ee
HN NH 2 HN NH 2 HN NH 2
HN Pp HN” N NH H20 NH, 0 N Ni 0 N NH>
Ribose-PPP Ribose-P Ribityl-P
GTP PRP ADRAP-P Diaminouracil

2P

NH
ry ADRAP
: OPP N~N SN-Ribi
N ibit 2

HCHO(?) 0
x
OP NON CH 0% “N7 "NZ >CH we NY
| : ewe | FT\CH-CH.OH
RAS
Ribit Ribit Rint
Riboflavin DMRL MERL

Figure 12.5 Biosynthetic pathway for riboflavin (from Brown and Williamson, 1982). Abbreviations: GTP, Guanosine
triphosphate; PRP, 2,5-Diamino-6-keto-4-(5'-phosphoribosylamino)-pyrimidine; ADRAP, 5-Amino-2,5-dioxy-4-(5'-
phosphoribitylamino)-pyrimidine; MERL, 6-Methly-7-(1',2'-dihydroxyethyl)-8-ribityllumazine; DMRL, 6, 7-Dimethyl-8-
ribityllumazine; Ribit, Ribitol.
 12.3 RIBOFLAVIN / 225
duction by E. ashbyii and A. gossypii could be' as is the use of a small inoculum (0.75-2%) of a
explained by the presence of constitutive ribo- 24- to 48-hour-old actively growing culture. The
flavin-synthesizing enzymes. fermentation takes 7 days with an aeration rate
of 0.3 vvm at 28°C. For foam control, silicone
Production process antifoam is applied at first, and soy bean oil,
which is also metabolized, is added later.
The fermentative production is currently carried Riboflavin is present both in solution and
out with Ashbya gossypii NRRL Y-1056. The high bound to the mycelium in the fermentation
yields of more than 10-15 g/l were attained by broth. The bound vitamin is released from the
strain development as well as by optimization of cells by heat treatment (1 hour, 120°C) and the
the nutrient solution, the cultivation of inoculum, mycelium is separated and discarded. The ribo-
and the fermentation conditions. Originally the flavin is then further purified.
fermentation used a medium with glucose and Production of riboflavin with an aliphatic hy-
corn steep liquor; sucrose and maltose were other drocarbon (C,.—C,,) as carbon source has been
suitable carbon sources. When lipids were also reported using Pichia guilliermondii, but yield in-
used as energy sources, yields markedly in- formation was not given. Using Pichia miso, 51
creased. Riboflavin production on what is now mg/l riboflavin was obtained on a medium with
the basic medium (corn steep liquor 2.25%, com- n-hexadecane, corn steep liquor, and urea. Stud-
mercial peptone 3.5%, soy bean oil 4.5%) has ies have been carried out on the use of a meth-
been further stimulated by the addition of dif- anol-utilizing organism, for example, Hansenula
ferent peptones, glycine, distiller’s solubles, or polymorpha, for the formation of riboflavin in
yeast extract. By simultaneous feeding of glucose batch or continuous culture.
and inositol the rate of formation of riboflavin Crystalline riboflavin preparations of high
can be further increased. A careful sterilization purity have been produced using Saccharomyces
of the culture medium is critical for high yields, fermentation with acetate as sole carbon source.

Table 12.3 Production processes for several carotenoids

Carotenoid Organism Composition of medium Length of Yield
fermentation (mg/1)
(days)
6-Carotene Mixed culture of Blakeslea see Figure 12.9 8 3000
trispora NRRL 2456(+) and
NRRL 2457(—)
Lycopene Streptomyces chrestomyceticus Starch, soy meal, (NH,),SO, 6 500
subsp. rubescens
Mixed culture of Blaskeslea Cotton seed meal, corn meal, soy 2) 400
trispora NRRL 2895(+-) and bean oil; addition of triethylamine
NRRL 2896(—) after 48 hours (suppresses
carotenoid cyclization)
Zeaxanthin Flavobacterium sp. Glucose, corn steep liquor, 335
palmitate ester, methionine,
pyridoxine, Fe? salts

Mixture of Mycobacterium phlei Sugar beet molasses, urea; vi 300

various exposure to light required
Cee iad S. chrestomyceticus Glucose, yeast extract 5 500

Yeast Sugar, ethanol or alkanes eB

(Ninet and Renaut, 1979)

226 / CHAPTER 12 / VITAMINS

12.4 B-CAROTENE trinaxanthine, 3-5 tons per year) or in the salmon

industry (astaxanthine). Chemical synthesis is
Occurrence and economic significance currently the only commercially feasible means
of obtaining carotenoids; xanthophylls may also
Carotenoids are found in many animal and plant
be obtained by extraction of plant material. Ca-
tissues, but originate exclusively from plants or
rotenoids are produced by many microorganisms
microbes. 6-Carotene (provitamin A) is con-
(including algae), but fermentative production is
verted into vitamin A in the intestinal mucous
not economical in the present market, since the
membrane and is stored in the liver as the palmi-
yields of currently available processes (Table
tate ester. In human beings, a deficiency of vi-
12.3) do not compete with cost-effective chemical
tamin A (daily requirement 1.5-2 mg) causes
synthetic processes. However, depending on the
night blindness and changes in the skin and mu-
market it appears that a competitive process
cous membranes; it is particularly necessary for
might be successful, using the halophilic alga
the normal biosynthesis of mucopolysaccharides.
Dunaliella salina (see Borowitzka et al., 1986).
The demand for 8-carotene is about 100 tons
per year, primarily for use as a food coloring
agent. The demand for provitamin A is very low. Acetyl-CoA
Other carotenoids, such as lycopene or xantho- 2x
Acetoacetyl-CoA
phylls, which do not have a provitamin A activ-
ity, are used as food coloring agents (e.g. mar- Acetyl-CoA
garine, cheese, egg products), for improving color 3-Hydroxy-3-methyl-
in meat or egg yolk in the poultry industry (ci- glutaric acid
|

Mevalonic acid-5-PP

QTY YY YY WY NY YH NYS
Isopentenyl-5-PP => _34,8 -Dimethylallyl-PP

a-Carotene
Geranyl-PP (Cyo)

STS OST SY SO NY YH NY SS
Farnesyl-PP

p- Carotene Geranylgeranyl-PP (C20)

sn
Phytoene(C,, )

Phytofluene
$- Carotene

& -Carotene

Neurosporene

Lycopene
Lycopene B-Zeacarotene a-Zeacarotene

5-Carotene 5-Carotene

p-Carotene a-Carotene
HO Zeaxanthin
Zeaxanthin Lutein
Figure 12.6 Structures of several carotenoids that can be
produced by fermentation Figure 12.7 Carotenoid biosynthesis
 12.4 B-CAROTENE / 227

HC ER COOK I ols Ch of both sexual forms, (+) and (—) strains, are
mixed, a significant increase in carotene produc-
structures of some carotenoids produced by mi-
crobial fermentation. Only compounds with the

isse B-ionone structure (the ring structure found at

each end of the 6-carotene molecule) are effective
O as provitamin A. Thus, 2 molecules of vitamin A
can be formed from §-carotene; only one mole-
Figure 12.8 Structure of trisporic acid C
cule of vitamin A can be formed from a- and y-
carotene since each of these molecules has one
B-ionone ring.
Structure
Biosynthesis
Carotenoids are highly unsaturated isoprene de-
rivatives. Naturally occurring carotenoids are tet- Biosynthesis of 6-carotene has been studied
raterpenoids consisting of 8 isoprene residues. mainly in plants and fungi and takes place as
Over 400 naturally occurring carotenoid com- shown in Figure 12.7. 6-Carotene and related ca-
pounds are known today. Figure 12.6 shows the rotenoids are produced primarily by fungi and
impact on process development. When cultures algae, xanthophylls by bacteria and algae.

B. trispora Inoculum storage: spores in sterile soil

B. trispora
NRRL 2456 (+)
NRRL 2457 (—)

Culture on Culture on Incubation: 168 h, 27°C

agar slant agar slant

2 1 Erlenmeyer flask with 400 ml Medium A. Incu-

Preculture Preculture
bation: 48 h, 26°C, shaker.

1701 Fermenter with 120 1 Medium A. Inoculum: 400

ml of each preculture. Incubation: 40 h, 26°C; stirring
170 rpm; aeration 1.1 vvm

800 I Fermenter with 320 1 Medium B. Sterilization

Production 55 min, 122°C. Inoculum: 32 | of mixed preculture.
culture Incubation: 185 h, 26°C; stirring 210 rpm; aeration
1.3 vvm

Medium A (g/l): Corn steep liquor 70; Corn starch 50; KH,PO, 0.5; MnSO, - H,O 0.1; Thiamin HCI 0.01; Tap water

Medium B (g/l): Distiller’s soluble 70; Corn starch 60; Soy bean meal 30; Cottonseed oil 30; Antioxidant 0.35; MnSO,
- H,O 0.2; Thiamin HCI 0.5; Isoniazid 0.6; Kerosene 20 ml; Tap water; pH 6.3. Isoniazid and kerosene
are sterilized separately. After 48 h 1 g/l 8-ionone and 5 ml/l kerosene are added; glucose feeding
(total addition 42 g/l) until the end of the fermentation.

Figure 12.9 Production process for 6-carotene using Blakeslea trispora (From Ninet and Renaut, 1979)
228 / CHAPTER 12 / VITAMINS
Production processes for $-carotene REFERENCES

The highest yields have been obtained with the Beytia, E.D. and J.W. Porter. 1976. Biochemistry of poly-
Blakeslea trispora process. The observation that isoprenoid biosynthesis. Ann. Rev. Biochem. 45: 113-
142.
carotene production occurs during the process of Borowitzka, L.J., T.P. Moulton, and M.A. Borowitzka.
zygospore formation in this organism has had an 1986. Salinity and the commercial production of beta-
impact on process development. When cultures carotene from Dunaliella salina. Nova Hedwigia
83:224—229.
of both sexual forms, (+) and (—) strains, are Brooke, A.G., L. Dijkhulzen, and W. Harder. 1986. Reg-
mixed, a significant increase in carotene produc- ulation of flavin biosynthesis in the methylotrophic
tion in the (—) strain is achieved. Production is yeast Hansenula polymorpha. Arch. Microbiol. 145:62—
70.
induced by trisporic acids (Figure 12.8), a mixture Brown, G.M. and J.M. Williamson. 1982. Biosynthesis of
of closely related substances. Trisporic acids are riboflavin, folic acid, thiamine, and pantothenic acid.
not precursors of $-carotene but rather they act Adv. Enzymol. 53:346-353.
Ciegler, A. 1965. Microbial carotenogenesis. Adv. Appl.
as (+)-gamones (sexual hormones). They are de- Microbiol. 7: 1-34.
rived biosynthetically from 6-carotene, as shown Florent, J. 1986. Vitamins, pp. 115-158. In: Rehm, HJ.
and G. Reed (eds.). Biotechnology, Vol. 4, VCH Pub-
by labeling experiments. The production of 6-
lishers, Deerfield Beach, FL.
carotene (Figure 12.9) is carried out in a sub- Florent, J. and L. Ninet. 1979. Vitamin B,,, pp. 497-519.
merged fermentation using a mixed culture of In: Peppler, H.J. and D. Perlman (eds.), Microbial tech-
nology, vol. I, 2nd edition. Academic Press, New York.
(+) and (—) strains. The proportions of the two
Kamikubo, T., M. Hayashi, N. Nishio, and S. Nagai. 1978.
strains need not be equal and since it is the (—) Utilization of non-sugar sources for vitamin B,, pro-
strain which produces the 6-carotene, this strain duction. Appl. Environ. Microbiol. 35: 971-973.
Karlson, P. 1980. Kurzes Lehrbuch der Biochemie ftir Med-
can be present in great excess. Another activator iziner und Naturwissenschaftler (Short textbook of bio-
of 6-carotene synthesis is isoniazid, particularly chemistry for medicine and biology), 11th edition.
in combination with B-ionone. Alone, G-ionone Georg Thieme Verlag, Stuttgart.
Malzahn, R.C., R.F. Phillips, and A.M. Hanson. 1959. U.S.
is toxic to the production organism, but in the Patent 2,876,169.
presence of plant oils it promotes carotene pro- Mazumder, T.K., N. Nishio, M. Hayashi, and S. Nagai.
duction. The B-ionones themselves are not in- 1986. Production of corrinoids including vitamin B,,
by Methanosarcina barkeri growing on methanol. Bio-
corporated into carotene but affect the synthesis technol. Letters 8:843-848.
of various of the enzymes involved in the bio- Merck and Co., Inc. 1971. French Patent 2,038,828.
synthetic pathway. They can be replaced by a Ninet, L. and J. Renaut. 1979. Carotenoids, pp. 529-544.
In: Peppler, H.J. and D. Perlman (eds.), Microbial tech-
number of other substances, such as terpenes or nology, vol. I, 2nd edition. Academic Press, New York.
cyclohexane and cyclohexanone and their tri- Perlman, D. 1977. Fermentation industries, quo vadis?
methyl derivatives. The addition of purified ker- Chem. Technol. 7: 434-443.
osene to the medium doubles the yield by in- Perlman, D. 1978. Vitamins, pp. 303-326. In: Rose, A.H.
(ed.), Economic microbiology, vol. 2, Primary products
creasing the solubility of the hydrophobic of metabolism. Academic Press, London.
substrate. Perlman, D. 1979. Microbial process for riboflavin pro-
Because of the low stability of 6-carotene duction, pp. 521-527. In: Peppler, H.J. and D. Perlman
(eds.), Microbial technology, vol. I, 2nd edition. Aca-
within the cells, the addition of an antioxidant is demic Press, New York.
necessary for the fermentation process. Renz, P. 1984. Untersuchungen zur Biosynthese von Vi-
The carotenoid-rich mycelium can be used tamin B,, (Studies on the synthesis of vitamin B,,). GIT
Fachz. Lab. 28:884-892.
directly as a feed additive. To obtain pure (-car- Speedie, J.D. and G.W. Hull. 1960. U.S. Patent 2,951,017.
otene, the mycelium is removed, dehydrated (for Upjohn Co. 1965. Netherlands Patent 64/11 184.
example, with methanol), extracted with meth- Yamada, K. 1977. Bioengineering report. Recent advances
ylene chloride (75-92% yield), and the crude in industrial fermentation in Japan. Biotechnol. Bioeng.
19: 1563-1621.
product is further purified.
 Antibiotics

8000. In addition, around 3000 antibiotically ac-

13.1 INTRODUCTION
tive substances have been detected in lichens,
Antibiotics are products of secondary metabolism algae, higher animals, and plants. Each year,
which inhibit growth processes of other orga- about 300 new antibiotically active materials are
nisms even when used at low concentrations. detected, of which 30-35% are secondary com-
Growth inhibition of one microorganism by an- ponents from fermentations with known anti-
other in mixed culture has been known for a long biotics.
time. The most famous example is the growth Of the large number of known antibiotics of
inhibition which was observed by Alexander microbial origin, only 123 are currently produced
Fleming in 1929, when staphlococcal growth on by fermentation. In addition, more than 50 anti-
a petri plate was inhibited by a contaminating biotics are produced as semisynthetic com-
Penicillium notatum culture. The Penicillium con- pounds, and three antibiotics, chloramphenicol,
taminant produced the antibiotic penicillin. Dur- phosphonomycin, and pyrrolnitrin, are produced
ing World War II, the demand for chemothera- completely synthetically.
peutic agents to treat wound infections led to the The significance of antibiotic production for
development of a production process for peni- the producing strain is unclear. Antibiotic pro-
cillin and the beginning of the era of antibiotic duction could be of ecological significance for the
research. This continues to be the most important life of the organism in nature, but solid research
area of industrial microbiology today. Intensive to support this hypothesis is very limited. As sec-
screening programs in all industrial countries ondary metabolites, antibiotics could serve reg-
continue to increase the number of described ulatory roles during differentiation, perhaps act-
antibiotics: 513 antibiotics were known in 1961, ing as temporary inhibitory agents. To date, most
4076 in 1972, 7650 in 1985, and currently around new antibiotics have been detected strictly by

ARD
230 / CHAPTER 13 / ANTIBIOTICS

empirical screening methods, with little attention classification. Table 13.2 shows a simplified clas-
to their possible roles for the producing strains. sification according to chemical structure. In this
chapter, primarily the economically important
compounds are covered.
The microbial groups producing
antibiotics
Table 13.2 Classification of antibiotics according to
Antibiotics are produced by bacteria, actinomy- their chemical structure. An example of each is given in
cetes and fungi; their distribution within the tax- parentheses.
onomic groups is shown in Table 13.1. 1. Carbohydrate-containing antibiotics
In the fungi, only the antibiotics produced by Pure sugars (Nojirimycin)
the Aspergillaceae and Moniliales are of practical Aminoglycosides (Streptomycin)
Orthosomycins (Everninomicin)
importance. The compounds isolated from bas-
N-Glycosides (Streptothricin)
idiomycetes have not had any practical use. Only C-Glycosides (Vancomycin)
10 of the known fungal antibiotics are produced Glycolipids (Moenomycin)
commercially and only the penicillins, cephalo- 2. Macrocyclic lactones
sporin C, griseofulvin, and fusidic acid are clin- Macrolide antibiotics (Erythromycin)
ically important. In the bacteria, there are many Polyene antibiotics (Candicidin)
taxonomic groups which produce antibiotics. The Ansamycins (Rifamycin)
Macrotetrolides (Tetranactin)
greatest variety in structure and number of anti-
3. Quinones and related antibiotics
biotics is found in the actinomycetes, especially
in the genus Streptomyces. Another important Tetracyclines (Tetracycline)
Anthracyclines (Adriamycin)
group of substances are the peptide antibiotics, Naphthoquinones (Actinorhodin)
produced by bacteria of the genus Bacillus. A Benzoquinones (Mitomycin)
number of new compounds have also recently 4. Amino acid and peptide antibiotics
been isolated in other taxonomic groups (see Amino acid derivatives (Cycloserine)
Chapter 2). 6-Lactam antibiotics (Penicillin)
The significance of antibiotic production for Peptide antibiotics (Bacitracin)
Chromopeptides (Actinomycins)
producer strains is still unclear. The status of the Depsipeptides (Valinomycin)
controversy can be found in the references of Chelate-forming (Bleomycins)
Zahner and Barabas cited in the bibliography. peptides
5. Heterocyclic antibiotics containing nitrogen
Nucleoside antibiotics (Polyoxins)
Classification of antibiotics
6. Heterocyclic antibiotics containing oxygen
Antibiotics can be classified according to their Polyether antibiotics (Monensin)
antimicrobial spectrum, mechanism of action,
7. Alicyclic derivatives
producer strain, manner of biosynthesis, or
Cycloalkane derivatives (Cycloheximide)
chemical structure. There is some overlap in each Steroid antibiotics (Fusidic acid)
8. Aromatic antibiotics
Table 13.1 Numbers of antibiotics peed by major
Benzene derivatives (Chloramphenicol)
groups of microorganisms
Condensed aromatic (Griseofulvin)
Taxonomic group Number of antibiotics antibiotics
Aromatic ether (Novobiocin)
Bacteria, other 950
than actinomycetes 9. Aliphatic antibiotics
Actinomycetes 4600 Compounds containing (Fosfomycins)
Fungi 1600 phosphorous
Berdy (1985) (Berdy, 1985)
 13.1 INTRODUCTION / 231

Applications of antibiotics Antibiotics as food preservatives Government

regulations in each country directly control the
Chemotherapeutic antibiotics can be either use of antibiotics as food preservatives. The fol-
broad-spectrum antibiotics, active against many lowing compounds are available: pimaricin, a
organisms, or narrow-spectrum antibiotics, ac- fungicide applied to food surfaces; tylosin (ef-
tive against only a restricted range of organisms. fective against Bacillus spores) and nisin (effec-
Most antibiotics are manufactured as antimicro- tive against clostridia), both used in the canning
bial agents for chemotherapy, but some have industry; chlortetracycline, used to maintain
other applications, as outlined bélow. freshness in fish, meat and poultry by incorpo-
ration into ice (5 ppm) or by addition to an im-
Antitumor antibiotics Such antibiotics are clin- mersion bath (10 ppm). Of these, pimaricin and
ically used as cytostatic agents (Table 13.3). Al- nisin are currently used commercially.
though generally toxic, with careful control of the
dose certain of these antibiotics are effective in
the treatment of certain kinds of tumors. Antibiotics used as animal growth promoters and in
veterinary medicine Animal feed is more effi-
Antibiotics for plant pathology Antibiotics may ciently processed in the animal’s digestive system
be more useful than synthetic chemicals in the if an antibiotic additive is used in subtherapeutic
control of plant diseases for the following rea- concentrations (1-10 mg/kg feed). Weight gain
sons: they may be applied selectively in low con- may also be accelerated. The cause of this growth
centrations, they are only slightly toxic to warm- promotion may be traced to changes in the mi-
blooded animals and beneficial insects, and they croflora of the gastrointestinal tract. This is fur-
are normally easily broken down by soil micro- ther confirmed by the fact that the growth of
organisms. Initially, medically useful antibiotics animals raised in germ-free conditions is not
were also used on plants (especially streptomycin stimulated by antibiotics. Originally, large quan-
to combat plant disease caused by Pseudomonas tities of therapeutically useful antibiotics (such as
sp. and Xanthomonas oryzae). Now antibiotics penicillins, tetracyclines, erythromycins, bacitra-
have been developed which are used exclusively cin, or streptomycin) were added to feed. Because
for plant application (Table 13.4). such extensive use of common antibiotics en-
couraged the risk of rapid development of anti-
biotic resistance, government regulations have
Table 13.3 The most important antitumor antibiotics
been established to eliminate the parallel use of
Antibiotic Category Organism antibiotics in human medicine and in animal
producing
feed. Some new-generation antibiotics which are
Aclacinomycin Anthracycline S. galilaeus used solely for nutritional purposes are listed in
Actinomycin C,, | Chromopeptide — S. antibioticus
CAD: Table 13.5.
Adriamycin Anthracycline S. peucetius In veterinary medicine, the use of antibiotics
Daunomycin Anthracycline S. peucetius is similarly regulated. Antibiotics which are used
Chromomycin A, C-Glycoside S. griseus
(oligosaccharide solely for veterinary work include hygromycin B,
with aromatic thiostrepton, tylosin, and the coccidiostatic
; chromophore) agents monensin, lasalocide, and salinomycin.
Mithramycin C-glycoside S. plicatus,
(oligosaccharide $. argillaceus,
with aromatic S. atroolivaceus
chromophore)
Antibiotics as tools in biochemistry and molecular
Mitomycin C Benzoquinone S. caespitosus biology The use of antibiotics as selective in-
Bleomycin A,, B, |Glycopeptide S. verticillus hibitors has made a vital contribution to the un-
Neocarzinostatin Peptide S. carzinostaticus
derstanding of certain cell functions, such as
232 / CHAPTER 13 / ANTIBIOTICS

Table 13.4 Antibiotics of use in plant pathology

Antibiotic, chemical type in () Organism producing Uses

Blasticidin S (Nucleoside) S. griseochromogenes Rice fungicide against Piricularia oryzae (rice burn);
relatively toxic

Mildiomycin (Nucleoside) Streptoverticillium Fungicide for mildew

rimofaciens
Polyoxin (Nucleoside) S. cacaoi var. asoensis Multi-purpose fungicide
Prumycin (Nucleoside) S. kagawaensis Fungicide against Botrytis and Sclerotinia species
Cycloheximide (Amino acid) S. griseus Leaf fungicide, highly toxic to warm-blooded animals;
aids harvesting
Kasugamycin (Aminoglycoside) S. kasugaensis Rice fungicide against Piricularia oryzae
Validamycin (Aminoglycoside) S. hygroscopicus var. Fungicide against Rhizoctonia solani (leaf dropping and
limoneus stalk diseases); vegetable crops
Tetranactin (Macrotetrolide) S. flaveolus Insecticide (mites)

Table 13.5 Antibiotics used in animal feed

Economic significance of antibiotics
Antibiotics Classification Organism producing
Enduracidin Peptide S. fungicidicus World-wide antibiotic production is over 100,000
antibiotic tons per year and estimated gross sales for 1980
Mikamycin Peptide S. mitakaensis were $4.2 billion. The annual gross sales in the
antibiotic
Siomycin Peptide S. sioyaensis United States alone is $1 billion, with cephalo-
antibiotic sporin in the leading position, followed by am-
Thiopeptin Peptide S. tateyamensis picillin and the tetracyclines. Feed-additive anti-
antibiotic
Thiostrepton Peptide S. azureus biotics are believed to have a world market of
antibiotic $100 million annually.
Virginiamycin Peptide S. virginiae
antibiotic
Macarbomycin Phospho- S. phaeochromogenes Objectives of antibiotic research
glycolipid
Moenomycin Phospho- S. bambergiensis Before 1960, about 5% of the newly isolated anti-
glycolipid
Quebemycin Phospho- S. viridans
biotics were therapeutically useful. In the follow-
glycolipid ing years new antibiotics were discovered at an
Tylosin Macrolide S. fradiae approximately constant rate, but the percentage
Mocimycin Heterocyclic S. ramocissimus
compound
of the new antibiotics which actually came on
the market decreased from 2.6% in 1961-1965
to 1% in 1966-1971. This is primarily because
of severe cost increases in development and clin-
DNA replication, transcription, translation, and ical testing, so that manufacturers produce only
cell wall synthesis. The uses of antibiotics in those compounds which clearly show promising
these areas is discussed in textbooks of biochem- therapeutic progress. On the average, it takes
istry and microbiology. In addition, some anti- around 8-10 years to develop a new antibiotic,
biotics (for example, gentamicin) have been at an average cost of $10,000,000 to $20,000,000.
added to animal cell cultures to control contam- Considering the very large number of known
ination. compounds, it may seem questionable whether
 13.2 B-LACTAM ANTIBIOTICS / 233

the search for new antibiotics should continue... chemical or genetic means (mutasynthesis, pro-
The reasons for continued research are: toplast fusion, recombinant DNA technology—
+ In many cases the properties of natural anti- see Chapter 3). However, antibiotics with en-
biotics are not optimal for therapeutic appli- tirely new basic structures can be expected only
cation. The following improvements are from screening, especially by the use of new test
needed: greater activity with unchanged or procedures and by research on new groups of
diminished toxicity, decreased side effects, microorganisms (see Chapter 2).
broader antimicrobial range, greater selectiv-
ity against certain pathogens, improved phar- 13.2 8-LACTAM ANTIBIOTICS
macological properties.
As shown in Figure 13.1, the B-lactam antibiotics
+ Suitable antibiotics are not available in many
fields of human medicine or in nonmedical can be divided into five distinct classes. The pen-
areas, as shown in Table 13.6. Numerous
icillins and the cephalosporins belong to the most
tests have been made of new and semisyn- effective of all therapeutic agents for the control
thetic substances, but no significant break-
of infectious diseases. In addition to the devel-
opment of numerous semisynthetic 6-lactams
throughs have been made.
based on the known §-lactam rings, antibiotics
. Since the beginning of chemotherapy, the
with completely new 6-lactam ring systems have
number of resistant strains has increased.
been isolated in the past few years using new
Multiple- and cross-resistance can occur, i.e.,
specific and sensitive screening methods.
if resistance develops to one antibiotic it may
simultaneously develop to others having the
same mode of operation or uptake mecha- Penicillins
nism. Careless use of antibiotics has been re-
Penicillin was described by Fleming in 1929. A
sponsible for much increase in resistance, but
research group at Oxford under Florey and Chain
even with careful use in chemotherapy, re-
isolated it from surface cultures of Penicillium no-
sistance still develops, albeit at a slower rate.
tatum in 1940 and the first clinical application of
Currently, the only alternative for overcom-
penicillin was in 1941. Penicillins are produced
ing the resistance problem is the discovery of
by many fungi, particularly Penicillium and As-
new antibiotics.
pergillus species. Natural penicillins are effective
Improved antibiotics can be obtained by against numerous gram-positive bacteria. They
modifying known compounds using either are labile in acid and may be inactivated by split-

Table 13.6 Possible applications for antibiotics

Application Many products Some products Products needed

Medicine Gram-positive bacteria Gram-negative bacteria, Systemic mycoses

including multiple Viruses
resistance Protozoa
Dermatophytes Parasites
Tumors

Nonmedical areas - Plant pathology Plant pathology

(phytopathogenic fungi) Phytopathogenic bacteria
Animal nutrition and viruses
Insects and mites
Nematodes
Food preservatives

(Zahner, 1978)
234 / CHAPTER 13 / ANTIBIOTICS

Basic structure Antibiotic Most important producing strains

Penam Penicillins Penicillium chrysogenum
Se EN seecHs Aspergillus nidulans
| CH3 Cephalosporium acremonium
07 COOH Streptomyces clavuligerus

Ceph-3-em R* Cephalosporins Cephalosporium acremonium

aon S 7-Methoxycephalosporins Nocardia lactamdurans
aalicg Sl Streptomyces clavuligerus
of “\ SCHR x

COOH

cae
Clavam QX R Clavulanic acid S. clavuligerus

4
Carbapenem R i Thienamycins S. cattleya
| R Olivanic acids S. olivaceus
o2—N Coon ithei
Epitheinamycinsi S. flavog
flavogriseus

Monolactam Nocardins Nocardia uniformis

subsp. tsuyamenensis
0
ae -OH
os i COOH
Monobactams Gluconobacter sp.
ge Chromobacter violaceum
? R Å g É
grobacterium radiobacter
Botan Pseudmonas acidophila
0 NL 592 H Pseudomonas mesoacidophila
Flexibacter sp.
Acetobacter sp.

Figure 13.1 The basic structures of the naturally occurring $-lactam antibiotics

ting the 6-lactam ring with penicillin-8-lactam- mation, and PBP4 and 5 are carboxypeptidases.
ases (see Figure 15.11). Because of its low tox- A further point of attack of the B-lactam anti-
icity, large doses of penicillin can be used; only biotics seems to involve phospholipid synthesis.
a small percentage of patients develops allergies
(0.5-2%). Chemical structure The basic structure of the
B-Lactam antibiotics are specific inhibitors of penicillins is 6-aminopenicillanic acid (6-APA),
bacterial cell wall (peptidoglycan) synthesis. which consists of a thiazolidine ring with a con-
They combine specifically with the so-called densed $-lactam ring. The 6-APA carries a var-
penicillin-binding protein (PBP) of the bacte- iable acyl moiety in position 6, as illustrated in
rial cell and, by inhibiting the enzyme activity of Figure 13.2. If the penicillin fermentation is car-
this protein, bring about cell death. In E. coli it ried out without addition of side-chain precur-
has been shown that PBPla and 1b are trans- sors, the natural penicillins are produced. From
peptidases involved in the cross-linking of the this mixture, only benzylpenicillin is therapeu-
peptidoglycan, PBP3 plays a role in septum for- tically useful; the other compounds must be re-
 13.2 6-LACTAM ANTIBIOTICS / 235
|
|

G
1H
R—C+Nas 1 CH
SER. BIOSYNTHETIC PENICILLINS

OF H” ‘H mk Benzylpenicillin (Pen. G) CH; CO—

acid labile, $- lactamase sensitive, «
low activity against Gram-negative Phenyl acetic acid
og
10 N4 H anne
COO? (nd e K®) bacteria

-Lactam Thiazolidine
Phenoxymethylpenicillin
(Penicillin V)
¢ \-o-cHrco-
ring ring acid stable, other properties like
Phenoxy acetic acid
penicillin G
Acyl residue | 6-Aminopenicillanic acid
;
Allylmercaptomethylpenicillin H,C=CH-CH,-S- CH,- CO-
(Penicillin 0) Allyl mercapto acetic acid
reduced allergenic properties

Designation N-Acyl residue R SEMI-SYNTHETIC PENICILLINS

NATURAL PENICILLINS
Propicillin
acid stable,
Benzylpenicillin
I Vor cor B-\actamase sensitive
(Penicillin G)

2-Pentenylpenicillin CH; CH,- CH=CH-CH,-CO— Methicillin

(Penicillin F) acid stable,
B-\actamase resistant
n-Amylpenicillin CH3(CH,),
—CO —
( Penicillin-Dihydro F)
n-Heptylpenicillin CH,(CH),— CO—
(Penicillin K ) Oxacillin
acid stable,
p-Hydroxybenzylpenicillin
(Penicillin X)
wot \-cupco- B-lactamase resistant

Ampicillin
Penicillin N (Synnematin B) ®00C~ CH - (CH3),~CO — broadened spectrum of activity (espe- CH-CO—
(D-4-Amino-4-carboxy- NH, D cially against Gram-negative bacteria), | DI-)
n-butylpenicillin } e acid stable, NH,
B-lactamase sensitive

Isopenicillin N ®00C- CH - (CH,);- CO—

(L-4-Amino-4-carboxy- '
NH L Carbenicillin SCO
broadened spectrum of activity (espe- ¢\- ra CO
n-butylpenicillin) e 3 cially against Pseudomonas aeruginosa), cooe Na®
acid stable but ineffective orally,
Methylpenicillin CH,-CO— B-\lactamase sensitive

Figure 13.2 Structure of the known natural penicillins, the most important biosynthetic penicillins, and several semi-
synthetic penicillins

moved at the product recovery stage. The fer- their improved characteristics (acid stability, re-
mentation can be better controlled by adding a sistance to plasmid or chromosomally coded $-
side-chain precursor, so that only one desired lactamases, expanded antimicrobial effective-
penicillin is produced. Over 100 biosynthetic ness), semisynthetic penicillins have come to be
penicillins have been produced in this way. In extensively used in therapy. Because of their
commercial processes, however, only penicillin broadened action spectrum, these semisynthetic
G, penicillin V, and very limited amounts of pen- penicillins are sometimes compared with the so-
icillin O have been produced. In order to produce called third generation cephalosporins.
semisynthetic penicillins, penicillin G (some- About 38% of the penicillins produced com-
times penicillin V is used instead) is chemically mercially are used in human medicine (see be-
or enzymatically split to form 6-APA, which is low), 12% in veterinary medicine, and 43% are
chemically recycled to make yet another peni- used as starting materials for the production of
cillin derivative. (See Section 11.9 for a discus- semisynthetic penicillins. In the early 1980’s,
sion of the enzyme penicillin acylase). Due to world-wide production of penicillins amounted
236 / CHAPTER 13 / ANTIBIOTICS

to 12,000 tons, an exceedingly large amount con- product of biosynthesis, is excreted in the ab-
sidering how highly active these agents are. sence of a side chain precursor.
Several regulatory mechanisms are known in
penicillin biosynthesis. The amino acid lysine is
Biosynthesis and regulation The 6-lactam-thia-
synthesized from a pathway that involves L-a-
zolidine ring of penicillin is constructed from L-
aminoadipic acid, so that penicillin and lysine
cysteine and L-valine. Biosynthesis occurs in a
share a common branched biosynthetic pathway.
nonribosomal process by means of a dipeptide
Lysine inhibits penicillin synthesis because it is
composed of L-a-aminoadipic acid (L-a-AAA)
a feedback inhibitor of homocitrate synthase, an
and L-cysteine or a breakdown product of cys-
enzyme involved in L-a-AAA synthesis. If L-a-
tathionine. Subsequently, L-valine is connected
AAA is deficient, penicillin cannot be synthe-
via an epimerization reaction, resulting in the for-
sized. However, feedback regulation by lysine
mation of the tripeptide 6-(L-a-aminoadipyl)-
does not seem to be a rate-limiting step in pen-
cysteinyl-D-valine. The first product of the cy-
icillin biosynthesis.
clization of the tripeptide which can be isolated
Penicillin biosynthesis is affected by phos-
is isopenicillin N, but the biochemical reactions
phate concentration and also shows a distinct ca-
leading to this intermediate are not understood.
tabolite repression by glucose, in addition to a
Benzylpenicillin is produced in the exchange of
regulation by concentration of ammonium ion,
L-a-AAA with activated phenylacetic acid (Fig-
the latter by an unexplained mechanism. Because
ure 13.3). 6-APA, which is not an intermediary
of the glucose repression, penicillin fermenta-
tions were originally done only with the slowly
metabolizable sugar lactose.
HOOC-CH-CH;- CHy-CHs COOH
NH2
Strain development The penicillin production of
© DM L-a-Aminoadipic acid (a-AAA)
Fleming’s isolate was about 2 International
SE ahr L-Cysteine

COOH units/ml; today’s processes yield a penicillin titer

of about 85,000 units/ml. This is an increase
CH a -AAA-Cys
ee L-Valine
from 0.0012 g/l to about 50 g/l and well illus-
HN-CH © trates the value and power of a strain-selection
|
COOH program.
SH) GH; The initial culture improvement came in 1943
a-AAA-NH=CH—CH, CS 4-(L-
a-Amino-
CH; adipyl) cysteinyI- with the isolation of Penicillium chrysogenum
CO—NH—2'CH D-valine
COOH (LLD-Tripeptide) strain NRRL 1951. This organism was better
| Cyclization
suited for submerged production than the orig-
| in 2 steps inal P. notatum strain. Through further mutagen-
esis, strain Wis Q 176 was isolated, the original
a FAAA-NH= CH
| Isopenicillin N
strain in the famous Wisconsin culture line. Fig-
0
Ss N
COOH
ure 13.4 shows the genealogy of these strains
CHsCO-CoA
over a time period of about 10 years. Wis Q 176
Penicillin-
transacetylase was adopted by most penicillin manufacturers
a -AAA + CoA SH and was used as the original strain for the various
s commercial strain improvement programs, about
CY cH7co-NH=cH
fe)
eel COOH
Benzyl -
penicillin which little has been published. Newer data have
been released only by Panlabs Inc., a corporation
Figure 13.3 Biosynthesis of penicillin in Penicillium chry- which since 1973 has specialized in commercial
sogenum strain development (Table 13.7).
 13.2 B-LACTAM ANTIBIOTICS / 237

NRRL 1951 [120]

ls
NRRL 1951 B 25 [250]
|x
X- 1612 [500]
| Uv-I
WIS.Q176 [900]
| UV-I
BL3-D10°

47-636 47-650 47-638 1980] 47-762 47-91}

|s |s |s |s
47-1327 47-1380 47-1564 [1357] 47-1040

UV-I
S

48-749 48-701 11365] 48-786

N S UV-II
48-1655 48-1372 [1343]
|S |UV-I1
49-133 [2230] 49-482 49-901
N |S | UV-II
49-2695 49-2429
|s |UV-II
49-2166 49-2105 50-529 50-724
|N [2266] |s | UV-I
50-25 50- 1247 1506] 50-1583
IN |s |uv-1
50-935 51-616 51-825

N S S 52-85
51-70 |UV-1
51- 20 [2521] 51-1113 53-533 52-817
|uv-I
S S 53-174
52-318 |uv-I
| 53-844 [1846]
S
52-1087
S 51-20A 51-20B 51-20F
|s Is |s
51-20A, 51-20B3 F3 121401
IS
F,-64
53-399 53-414 [2493]
[2658] [2580]

Figure 13.4 Genealogy of the Wisconsin strain of Penicillium chrysogenum. S, stages of selection; X, X-ray treatment;
UV I, ultraviolet radiation at 275 nm; UV II, ultraviolet radiation at 253 nm; N, treatment with nitrogen mustard. Square
brackets show yields in International units/ml. a = pigment-free mutant (From Backus and Stauffer, 1955)
238 / CHAPTER 13 / ANTIBIOTICS

Table 13.7 Increase of penicillin formation in such as more stable sporulation and improved
Penicillium chrysogenum.
growth for inoculum production.
Strain Penicillin G Penicillin yield in Productivity Some of the genes involved in penicillin syn-
potassium glucose equivalents P (b)
(mg/ml) (g Pen.G-K/ thesis in P. chrysogenum have been identified by
g gluc. equiv.) (a) analysis of mutants blocked in penicillin synthe-
P=32 9.0 0.05 0.72 sis (npe). Twelve npe mutants have been mapped
P-,7 16.1 0.09 1.8 into 5 loci (npe V, W, X, Y, and Z) by means of
P-11 21.6 0.12 2:3 complementation experiments with heterozy-
P=13 27.0 0.09 2.6
P=15 29.4 0.12 Bez gous diploids. Cosynthesis studies showed a de-
fect in the LLD-tripeptide synthesis for npe X, Y,
(a): (kg fat/oils X 2.5) = kg glucose equivalents.
Carbon sources in medium: sugar and plant oils and Z, a block between the tripeptide and iso-
(b): kg product/1000 I total fermenter capacity X day penicillin N in npe W, and a block between is-
Results of strain development and culture medium openicillin N and penicillin G in npe V. The func-
optimization at Panlabs Inc. (Swartz, 1979) tion of npe Y was elegantly demonstrated as
follows: Protoplasts of the various mutants were
fused with liposomes into which the tripeptide
Yield increases have been the main objective had been preloaded, and penicillin synthesis was
of strain development, but other factors which obtained.
have an effect on fermentation and efficiency of The use of recombinant DNA technology to
product recovery have also been optimized. Ma- increase the formation of rate-limiting enzymes
jor advances in processing have resulted through via gene amplification or improved transcription
empirical screening of mutants. Until the mid- has not yet been possible in P. chrysogenum, due
1960’s, the most frequently used mutagens were to the absence of precise biosynthetic data and
X-rays, methylbis-(6-chloroethyl)amine (nitro- the absence of good host-vector systems. How-
gen mustard), and short-wave ultraviolet radia- ever, a gene bank for P. chrysogenum has been
tion. More recently, nitrosoguanidine, alkylating constructed, and a transformation system has
agents, and nitrite have been used as mutagens. been developed.
In the early 1970's, strain improvement by
using mere mutation had reached its limit. The Production methods Penicillin G and V are pro-
discovery of a parasexual cycle in P. chrysogenum duced using submerged processes in 40,000-
provided a means of utilizing genetic recombi- 200,000 liter fermenters. Due to difficulties with
nation for strain development. Heterozygous the O, supply, larger tanks cannot be employed.
diploids were described which had penicillin ti- Penicillin fermentation is an aerobic process with
ters above those of the haploid parent strains. a volumetric oxygen absorption rate of 0.4-0.8
Several such diploids have found use as pro- mM/1-min. The required aeration rate is between
duction strains. However, the highest percentage 0.5-1.0 vvm depending on the strain, on the bio-
of strains with increased penicillin production reactor, and on the impeller system; various tur-
arose from haploid segregants of crosses between bine impellers are used for mixing (120-150
mutants of different strain lines. rpm). Some manufacturers use Waldhof fermen-
The protoplast fusion technique has opened ters or air-lift fermenters, but this is only possible
up a new approach to the development of high- in mutants which generate low viscosity. De-
yielding strains. Yield increases of 8% have been pending on the production strain used, the op-
obtained over those obtained by mutation and timal temperature range is between 25-27°C.
selection. In addition, some of the resulting A typical flow chart for penicillin production
strains have had better growth characteristics, is shown in Figure 13.5.
 13.2 B-LACTAM ANTIBIOTICS / 239

Feeding of substrates
(carbon source, nitrogen source,
phenylacetic acid)

Filtrate

Cooling

tt ill
tank

Mycelium
Lyophilized Inoculum Prefermenter Production
culture cultivation fermenter
(Spores)

Figure 13.5 Flow chart of the penicillin fermentation (From Swartz, 1979)

The inoculum is started using lyophilized

spores. Because of the great variability of high-
yielding strains, careful strain maintenance is
necessary. Spore concentration (optimal 5 X 10°/
ml) and pellet formation are crucial for the sub-
sequent yield. If an optimal penicillin formation Glucose f, 4. 45 g/l/h —ap Steps 154
feeding
rate is to be achieved, pellets must grow not as Nitrogen
‘eadieg k-18mg/l/h >
compact balls, but in a loose form.
After several stages of growth the production Lactose =Penicillin
culture is ready. In the typical penicillin fermen-
tation (Figure 13.6), there is a growth phase of
about 40 hours duration, with a doubling time
of 6 hours, during which time the greatest part
of the cell mass is formed. The oxygen supply (g/l
10)
x Oo
(ep)
oO
(=) all
I

in the growing culture is critical, since the in- =

=~3)
creasing viscosity hinders oxygen transfer. This
“~Biomass
can be resolved by engineering changes in the Penicillin
WwoO
fermenter or by the use of mutants which de- =
velop reduced viscosity. After the growth phase NOoO
Je

the culture proceeds to the actual penicillin pro- Biomass

Carbohydrate,
(g/l),
Ammonia,
duction phase. In cases of high penicillin pro- 10 74 Ammonia

duction, growth is sharply reduced (u = 0.01 REED SEES

Q T ST + Aj T 7
h”). By feeding with various culture medium
O 20) 40m. 60) SO) 100k ZO 40
components, the production phase can be ex-
Fermentation time (hr)
tended to 120-180 hours.
The medium of a typical fed-batch culture Figure 13.6 Penicillin fermentation with Penicillium
may vary depending on the strain and usually chrysogenum (From Swartz, 1979)
240 / CHAPTER 13 / ANTIBIOTICS

consists of: corn steep liquor (4—5%, dry weight),

Cephalosporins
which in present processes may be replaced by
other nitrogen sources (e.g., a commercial nitro- Cephalosporins are 6-lactam antibiotics contain-
gen source called Pharmamedia); an additional ing a dihydrothiazine ring with D-a-aminoadipic
nitrogen source, such as soy meal, yeast extract, acid as acyl moiety (Figure 13.7). Cephalosporin
or whey; a carbon source (such as lactose); and C was discovered in culture filtrates of Cepha-
various buffers. The pH is kept constant at 6.5. losporium acremonium in 1953. The strain isolated
Phenylacetic acid or phenoxyacetic acid is fed by Brotzu in 1945 was later classified as Acre-
continuously as a precursor (0.5-0.8% of the to- monium chrysogenum and produces several anti-
tal). Processes with glucose or molasses feeding biotics: cephalosporin C, penicillin N (a 6-APA
are also successful: feeding rates of 1.0-2.5 derivative with D-a-AAA as side chain), and the
kgem”h” with a glucose concentration of 500 steroid cephalosporins P,-P;. The cephalosporin
kg:m”. About 65% of the metabolized carbon antibiotics are also produced by other fungi, such
source is used for maintenance energy, 25% for
as Emericellopsis and Paecilomyces. In 1971, in a
growth, and only 10% for penicillin production.
screening program designed to discover B-lactam
When it is considered that 50% of the production
antibiotics, the first cephamycins (7-methoxy-
cost derives from the medium ingredients, it is
cephalosporins) were found, which are produced
obvious that control of carbon metabolism offers
an important point of attack for further optimi-
zation of the production process. Yield increases
sin
of 25% have been reported by adding glucose Ri ay SS
and acetic acid. Critical parameters in these fed-
ats
batch systems are the rate of sugar utilization and OF, taeCH,— R>
COOH
the oxygen supply.
Production with continuous penicillin fer- B-Lactam Dihydro-
ring thiazine ring
mentation has been attempted, but has been dif-
ficult due to the instability of the production Designation

strains. A “batch fill and draw” system has been 7-Aminocephalo-

suggested as an alternative. In this procedure, sporanic acid (7-ACA)

20-40% of the fermentation contents is drawn

off and replaced with fresh nutrient solution; this Cephalosporin C =0=C0-CH;

process may be repeated up to 10 times without Deacetyl-3'- carbamoy|-

cephalosporin C
yield reductions. 7-Meth oxy- - 0-CO-Ch, - OCH;
cephalosporin C
Extensive research is being carried out to pro-
Cephamycin A -OCO-C=CH + Vo - OCH3
duce penicillin with immobilized cells. In one SOH

laboratory-scale study, the advantage of this ap- Cephamycin B -OCH,

proach over the use of a fed-batch system was
demonstrated, but this technique has not as yet Cephamycin C

been introduced commercially. SEMI-SYNTHETIC CEPHALOSPORINS

Penicillin is excreted into the medium and Cephalotin E JcHco| -0-co-cH,

HN
less than 1% remains mycelium-bound. After
separation of the mycelium, product recovery is Cephalexin

accomplished by means of a two-stage contin-

uous countercurrent extraction of the fermenter
broth with amyl or butyl acetate at 0-3°C and Figure 13.7 Structure of cephalosporin C, cephamycins,
pH 2.5-3.0. The yield is around 90%. and several semi-synthetic cephalosporins
 13.2 B-LACTAM ANTIBIOTICS / 241
from various Streptomyces species, such as S. lip- L-a-AAA+ L-Cys + L-Val
manii, S. clavuligerus, or Nocardia lactamdurans.
1
Cephalosporins are valued not only because
of their low toxicity but also because they are LLD-Tripeptide
broad-spectrum antibiotics, comparable in action
to ampicillin. With about 29% of the antibiotic
market, the cephalosporins are the single most Isopenicillin N
(L-a- AAA-APA)
important group of antibiotics. Therapeutically
|Racemase
only semisynthetic derivatives of cephalosporin
and cephamycin are used. For oral use, the first Penicillin N
generation cephalosporins have been modified (D-a-AAA-APA)

so that they have improved stability against 6- 1 Ring formation

| stimulated by
lactamases. These second generation antibiotics Fe?" ascorbate, ATP
(D)
were then further modified so that they were i H

more active against gram-negative bacteria. Fur- N

ther modifications have led to the commercial O G SE
COOH
development of third generation antibiotics with
Deacetoxycephalosporin C
excellent 6-lactamase stability and a broadened
(0)2 Dioxygenase, stimulated by
action spectrum. Fe£", ascorbate, a -Ketoglutarate
Thirteen therapeutically important semisyn- (D) H S

thetic cephalosporins are commercially pro-

N
duced. These have been synthesized by chemical og SF SCH20H
splitting to form 7-aminocephalosporanic acid COOH
Deacetylcephalosporin C
(7-ACA) with subsequent chemical acylation, as
well as by modifications on the C-3 site. Ceph- CH,C— CoA
alosporins are as economically significant as pen- 0
(D) H
icillins. "i esald

Biosynthesis and regulation As in the case of 67 TNH 0CO'CH,

benzylpenicillin, the first stages of cephalosporin COOH
Cephalosporin C
biosynthesis (Figure 13.8) proceed from 6-(a-ami-
noadipyl)-L-cysteinyl-D-valine to isopenicillin Figure 13.8 Biosynthesis of cephalosporin C by Cepha-
N. In the next stage, penicillin N is produced by losporium acremonium
transformation of the L-a-AAA side chain into
the D-form, via the action of a very labile race-
mase. After ring expansion to deacetoxycephal- methoxycephalosporin or cephamycin C. In con-
osporin C by the so-called ”expandase” reaction, trast to penicillin, the D-a-AAA moiety cannot
hydroxylation via a dioxygenase to deacetylce- be changed with precursor feeding.
phalosporin C occurs. The acetylation of ceph- Cephalosporin production is affected by
alosporin C by an acetyl-CoA dependent trans- phosphate regulation as well as by nitrogen and
ferase is the end point of the biosynthetic carbohydrate catabolite regulation. Rapidly me-
pathway in fungi. In streptomycetes, further tabolizable carbon sources, such as glucose, mal-
transformations occur: cephalosporin C or the tose, or glycerol, reduce the formation of ceph-
carbamoyl derivative of deacetylcephalosporin C alosporin. The repression of the “Expandase”’
is converted in a two-step reaction with molec- seems to be the most significant effect. The ad-
ular oxygen and S-adenosylmethionine to 7- dition of lysine in low concentrations promotes
242 / CHAPTER 13 / ANTIBIOTICS
cephalosporin production (Figure 13.9), whereas fur analogs, exhibit an increased cephalosporin
the inhibition caused by higher amounts of lysine production or utilize inorganic sulfate as well as
can be overcome by addition of DL-a-AAA. Me- methionine. Parasexual recombination with my-
thionine stimulates cephalosporin synthesis in C. celium of C. acremonium is difficult because the
acremonium, but not in the streptomycetes. compartments of the hyphae are almost always
In the biosynthesis of cephamycins by strep- mononuclear. By using protoplasts of different
tomycetes, it should be noted that a-AAA is not strains in fusion experiments, haploid recombi-
a precursor of L-lysine, because streptomycetes nants have been isolated which showed a 40%
increase in cephalosporin C production, as well
synthesize lysine via the diaminopimelic acid
pathway. L-lysine is converted by an L-lysine a- as increased growth and improved sporulation.
ketoglutarate-6-aminotransferase into a-AAA Protoplast fusion techniques have been com-
bined with parasexual reproduction to develop
via L-1-piperidine-6-carboxylate as intermediate.
an overall map of the biosynthetic genes for
cephalosporin C. The gene for isopenicillin N
Strain development Strain development has synthetase from C. acremonium has been cloned
been conducted with the original Brotzu strain, in E. coli and an effective transformation system
C. acremonium CMI 49 137, using mutation, se- for C. acremonium has been developed, making
lection, and parasexual techniques. Mutants have it likely that the key steps in cephalosporin pro-
also been isolated with defective sulfur metab- duction will become subject to analysis and mod-
olism. Some of these regulatory mutants, which ification.
were isolated because they were resistant to sul- The cephalosporin C titer of contemporary
high-yielding strains, in which the proportion of
penicillin N is reduced, is about 20 g/l.

100
Production method The fermentation of cepha-
losporin is similar to that of penicillin. Complex
Lysine media with corn steep liquor, meat meal, sucrose,
concentration
glucose and ammonium acetate are used. Fer-
=O Img/ml
E
mentations are carried out at as fed-batch pro-
oO cesses with semicontinuous addition of nutrients
= . at pH 6.0-7.0 and at temperatures between 24-
o 60 ee 28°C. In the main growth phase, a high aeration
c WA rate is necessary, but during the production
6
(os phase (48-160 h), O, consumption decreases
n
LEO - sharply.
Le}
We The chemical synthesis of cephalosporin by
o ring-expansion of penicillin has been developed
a ih ih pgs a Control in recent years. This process has become espe-
Sam gym cially attractive because of the low price of the
” dlr
ae a ae,x—x —x 10 mg/m
/ml penicillin starting material. An example of how
this approach has been commercialized is the use
Al T T airs SS of pencillin V to produce oraspor, an orally active
4 6 8 10
cephalosporin.
Fermentation time (days)
New £-lactam ring systems
Figure 13.9 Effect of lysine concentration on cephalo-
sporin C formation in Cephalosporium acremonium (From New £-lactam ring systems have been discovered
Mehta et al., 1979) since the 1970’s by the introduction of screening
 13.3 AMINO ACID AND PEPTIDE ANTIBIOTICS / 243

methods such as using as test organisms $-lactam . a wide range of 6-lactamases in concentrations
hypersensitive strains or using enzymatic tests to under 0.1 ug/ml. The inhibition is irreversible;
detect 8-lactam inhibitors. The basic structures of in combination with 6-lactamase-sensitive pen-
these new @-lactams are shown in Figure 13.1, icillins and cephalosporins, this compound
and some specific examples are given in Figure causes a distinct increase in activity of these anti-
13.10. Because they may be active as either biotics, even against bacteria which are normally
broad-spectrum antibiotics or 8-lactamase inhib- resistant to 6-lactam antibiotics. Clavulanic acid
itors, these new compounds, especially their has been marketed in combination with amox-
semisynthetic derivatives, can be expected to find ycillin. i
considerable use in chemotherapy. Thienamycins and olivanic acids have a $-

Clavulanic acid, a 6-lactam-oxazolidine ring lactam-pyrroline ring system in common. Thien-

amycin, the first in a series of carbapenems with
system, is produced along with cephalosporins
very similar structures, was isolated in 1976 from
by Streptomyces clavuligerus. Clavulanic acid is
Streptomyces cattleya. Thienamycin acts as a $-
only slightly effective antibacterially but inhibits
lactamase inhibitor and as a broad-spectrum anti-
biotic against Gram-positive and Gram-negative
bacteria, including Pseudomonas aeruginosa, but
NH> NOHO H it is extremely unstable.
eal |
CH-(CH;0 tilt lal The semisynthetic N-formimidoylthienamy-
COOH N cin, resistant to dehydropeptidases, has good sta-
07
bility. It is the 6-lactam with the broadest spec-
trum and is undergoing clinical trials.
Nocardicin A The olivanic acids and the closely related
epithienamycins show a lower activity against
Pseudomonas aeruginosa and Staphylococcus au-
i H H 3 CH20H reus.
i : OH Nocardicins are monocyclic 6-lactams. From
a mixture of seven components, nocardicin A has
Cian ae been isolated as the most active compound. It is
COOH
weakly active against gram-negative bacteria, but
Clavulanic acid research is being carried out to improve its ac-
tivity by formation of chemical derivatives.
Monobactams, which are also monocylcic 6-
OH
s\ HoH lactams, have been isolated from bacterial cul-
fs = SS-NNH, tures. However, they are only weakly antibac-
ys N terial. Compounds with a 3a-methoxy group are
y COOH resistant to G-lactamases, whereas the rest of the
Thienamycin
monobactams show varying degrees of sensitiv-
ity to B-lactamases. Azthreonam, a semisynthetic
monobactam made from threonine, has low tox-
NH> i CH3 4 OCH3 icity and excellent activity against gram-negative
HOOC —C-(C
. (CH, 5SCENEN
; saaies see
pathogens; it is in clinical trials.
H mi ee) } NS
(9) SO 13.3 AMINO ACID AND PEPTIDE
ANTIBIOTICS
Sulfazecin
This group of antibiotics includes amino acid de-
Figure 13.10 Newer $-lactam antibiotics rivatives such as cycloserine and azaserine, most
244 / CHAPTER 13 / ANTIBIOTICS

of the 8-lactam antibiotics, which have already alanine racemase, the enzyme responsible for
been covered in Section 13.2, chromopeptide production of the D-alanine moiety of the pep-
antibiotics, depsipeptide antibiotics, and linear or tidoglycan. Cycloserine is very effective against
cyclic peptide antibiotics. Over 400 peptide anti- mycobacteria, particularly M. tuberculosis, and is
biotics are currently known. used in the treatment of tuberculosis in combi-
nation with isonicotinic acid hydrazide (INH)
and rifampicin or streptomycin.
D-cycloserine
Cycloserine, a D-4-amino-3-isoxazolidone (Fig-
Chromopeptide antibiotics
ure 13.11 A), may be synthetically produced or
may be isolated from cultures of Streptomyces or- This group includes the actinomycins (Figure
chidaceus, S. lavendulae, S. garyphalus, or S. ro- 13.11 B), which are produced as a mixture of very
seochromogenes. D-Cycloserine, a D-alanine an- similar substances by different streptomycetes,
alog, inhibits cell wall synthesis by inhibiting especially Streptomyces antibioticus and S. chry-
somallus. The following actinomycins have been
described: A, B, C;, Cx, i-C,, C/D, Fi, Zp-Z,.. All
L- MeVal Bee
actinomycins which have thus far been isolated
Sar Sar |
| have the same phenoxazone-chromophore (ac-
O | - O
GOERE Migs Pas tinocin), which is linked to two pentapeptide lac-
| | Xe Ve tones. These two lactones have varied amino acid
OG Cw |
NE =O L-Thr -Thr sequences, and by adding different amino acids
|
CO CO to the fermentation broth, 30 distinct biosyn-
A. Cycloserine Nw NH, thetic actinomycins have been produced.
The production of actinomycin from 2 mol-
O =O ecules of 3-hydroxy-4-methylanthranilic acid
Gils CH,
(MeVal = N-Methylvaline, Sar =Sarcosine)
pentapeptide lactone is catalyzed by phenoxa-
zine synthase (Figure 13.12). This enzyme is pro-
Actinomycin X4 X>
duced only after the transition into the idiophase
ActinomycinC, D-Val D-Val and shows pronounced catabolite regulation by
ActinomycinC, D-Val D-a- |leu
Actinomycini-Cz D-a-lleu D-Val glucose.
ActinomycinC3 D-a-lleu D-a-lleu
The phenoxazone rings of actinomycins in-
B. Actinomycins tercalate at a GC pair in 5'-T-G-C-A-3’ palin-
dromes of DNA, thus blocking DNA-dependent
RNA-polymerases. Actinomycins are very toxic,
FLE
3 Ci 3 ees)
ay (Cal 3
causing liver and kidney damage, but are ben-
MEE eficial in tumor treatment. The most important
BsHEHREST
0 Che
glOHHcoils© Sclap
CH
| actinomycin used in cancer chemotherapy is dac-
EX tinomycin (actinomycin C,).
pKowelels?
D-Valine L-Lactate L-Valine D-Hydroxy- 3
isovalerate Depsipeptide antibiotics
In depsipeptides, the subunits (amino acids, hy-
droxy acids) are usually linked with alternating
C. Valinomycin
ester and acid amide bonds. Valinomycin (Fig-
Figure 13.11 Structure of D-cycloserine (A), several ac- ure 13.11 C), a cyclododecadepsipeptide pro-
tinomycins (B) and valinomycin (C) duced by Streptomyces fulvissimus, has no eco-
‘
 13.3 AMINO ACID AND PEPTIDE ANTIBIOTICS / 245

CHy- CH-COO” CO-CH;CH-COO® CO-R

|
NH, — =
N
se
NH, ° NH,
OH
L-Tryptophan Kynurenine
Hydroxykynurenine

Fr Methionine

COR COR Phenoxazine - y

NS NH, Rn CO-R CO-R
synthase

(oe) SQ SE HC NH; H3C NH> i

CH, CH, OH OH
3-Hydroxy-4- 3-Hydroxy-4-
Actinocin R=OH methylanthranilic methylkynurenine
Actinomycins R= Penta- acid (R = OH)
peptidolactone

Figure 13.12 Actinomycin biosynthesis in Streptomyces antibioticus

nomic significance but finds wide use in contain other components in addition to the
biochemical reasearch as an agent for uncoupling amino acids (Figure 13.15). The peptide anti-
oxidative phosphorylation. It is also useful as a biotics produced by bacilli are formed at the on-
potassium carrier because of its selective binding set of sporulation and seem to play a regulatory
of K* ions. role in the sporulation process. Table 13.8 lists
Antibiotics of the virginiamycin family also the peptide antibiotics which are produced com-
have a depsipeptide structure. They consist of a mercially. Bialophos, a tripeptide formed by S.
mixture of structurally different macrocyclic lac- hygroscopicus from two L-alanine and the L-glu-
tone rings (type A and B). Figure 13.13 shows
the structure of virginiamycin S (type B).
Antibiotics of the virginiamycin group are iS
OH

chiefly effective against Gram-positive bacteria ae

and are widely used as growth promoters for NH Rs
poultry, swine, and calves. Seventeen com- H.C=— CH=
|
Ca
|
CO— N= CH —CO- N gs
pounds are commercially produced; the most im-
| CO
portant are the mikamycins and virginiamycins, O
|
|
N-R,
which are produced by Streptomyces mitakaensis I
” CH-NH (29 CO re
or S. virginiae.
CH

Linear and cyclic peptide antibiotics

R3
The majority of the antibiotics formed by species
of the genus Bacillus are peptides; other peptide R, RBR 22
antibiotics are produced by streptomycetes. The Virginiamycin S, CoH; CH; Pkeleplpecole
aci
molecular weights of peptide antibiotics are be- Virginiamycin S, CH, CH; “Kelcoigscaiiols
tween 270 (bacilysin) and 4500 (licheniformin). aci
Virginiamycin S, CH, H A Uyeroxy bipecotn
Some peptide antibiotics are linear (Figure Virginiamycin S3 C3H. CH3 eget
cele
ees
ls
aci
3-Hydroxy-4-keto-
13.14), but most are cyclic compounds. They may pipecolinic acid

consist of either amino acids alone (often with

D-amino acids and rare amino acids) or they may Figure 13.13 Structures of the virginiamycin antibiotics
246 / CHAPTER 13 / ANTIBIOTICS

HCO-X-Gly-L-Ala-D-Leu-L-Ala-D-Val— L-Val — D-Val —L-Trp — B, C, D,E, F, F,, F,, F, and G. Bacitracin A is the
tele 3 4 5 6 7 8 9 main component of the mixture (about 70%) and
has the greatest biological activity. It is a dodec-
—D-Leu —Y — D-Leu —L-Trp — D-Leu — L-Trp -NHCH,CH,OH
10 11 12 13 14 15 apeptide consisting of 8 L- and 4 D-amino acids
Gramicidin and contains both a cyclic hexapeptide and a thi-
azoline ring structure (see Figure 13.15).
x Y
|
Valine-Gramicidin A L-Val L-Trp
Mechanism of action Bacitracin inhibits the for-
Isoleucine-Gramicidin A L-lleu L-Trp mation of the bacterial cell wall at the level of
Valine-Gramicidin B L-Val L-Phe
Isoleucine-Gramicidin B L-lleu L-Phe
peptidoglycan biosynthesis. It prevents the de-
Valine-Gramicidin C L-Val L-Tyr phosphorylation of C,;-isoprenylpyrophosphate
Isoleucine-Gramicidin C L-Ileu L-Tyr
to C,,-isoprenylphosphate.

B-Tyr— B-Ser — DAPA — DAHAA— Gly — Spermidine

Biosynthesis and Regulation Peptide formation
can occur by either ribosomal or nonribosomal
EdeinA

Figure 13.14 Linear peptide antibiotics. Above, grami- D-Phe —*L-Pro —L-Val > L-Orn —*LLeu
cidins. Below, edein A (edein B: guanylspermidine).
DAPS, Diaminopropionic acid; DAHAS, Diaminohydrox- L-Leu «— L-Orn <— L-Val=— L-Pro =— D-Phe
yazelain acid

Gramicidin S
tamic acid analog phosphinothricin, is being
field-tested as an herbicide.

BE
S—

Therapeutic application of peptide anti- Sale

biotics is limited, due to their toxicity. Gramicidin CH, NH, N= — CO ken

S, linear gramicidins, tyrocidin, and bacitracin \ His -D-AspNH 2

ei eer D-Glu
are used topically (for wounds, burns, skin trans- D-Phe LAsp
plants, etc.). Polymyxins are used for infections L-lleu Ltys =—Llleu
TS G
caused by Gram-negative pathogens (including D-Orn
Pseudomonas), and viomycin and capreomycin
Bacitracin A
are used in tuberculosis therapy.
NH2
DAB — ®) —+t-Leu
Bacitracin ®—-LDAB+LThr —L-DAB see
NH,
Bacitracin is the most economically important of NH, NH, |
L-Thre—L-DAB-L-DAB-—NH,
the peptide antibiotics. In addition to its use as
®Q® ®
a topical antibiotic, it is also used as a growth
promoter in animal feeds. As a feed supplement, Polymyxin B, MOS D-Phe
it can be used as the zinc or manganese salt, as Polymyxin B, 10S D-Phe
Polymyxin €,(ColistinA) MOS DtLeu
the lignin-bacitracin complex, or as bacitracin
Poly myxin E,(Colistin B) 10S D-Leu
methylene disalicylate. Total production of baci-
tracin world-wide was estimated at 500 tons in Polymyxins
1976.
Figure 13.15 Cyclic peptide antibiotics: gramicidin S,
bacitracin A, and the polymyxins.
Bacitracin structure Various strains of B. lich-
DAB, Diaminobutyric acid; MOS, 6-Methyloctanoic acid;
eniformis produce a mixture of bacitracin A, A’, IOS, Isooctanoic acid
 13.3 AMINO ACID AND PEPTIDE ANTIBIOTICS / 247
Table 13.8 Commercially produced peptide antibiotics _
thiol groups in thioester linkages. The amino acid
Antibiotic Organism producing Activity sequence of the subsequent peptide is deter-
Application: Human Therapy mined by the arrangement of the thiol groups on
Amphomycin Streptomyces canus Gt the enzyme. The racemization to the D-enan-
Bacitracin Bacillus licheniformis GG)
Capreomycin S. capreolus My
tiomers probably takes place at this level. The
Gramicidin A B. brevis G transfer of the growing peptide chain (growth
Gramicidin S (J) _ B. brevis G from the N-terminal end toward the C-terminal
Iturin A B. subtilis- G*G AF
end) to the next amino acid and to the next en-
ituriensiens ,
Polymyxin B B. polymyxa G zyme sub-unit is catalyzed by 4'-phosphopan-
Tyrothricin B. brevis G* tetheine, a cofactor of each sub-unit. The cycli-
(bacitracin-tyro-
zation of the peptide chain takes place in the
cidin complex)
Tyrocidin B. brevis GE multi-enzyme complex, but the formation of the
Viomycin S. floridae My thiazoline ring is not yet fully understood.
Application: Feed additive
Bacitracin B. licheniformis GG)
Enduracidin A S. fungicidicus G*My Production methods Bacitracin was initially ob-
Parvulin S. parvulus G tained from surface cultures; today its production
Siomycin S. sioyaensis G occurs in aerobic submerged culture, like that of
Thiopeptin S. tateyamensis Gt
Thiostrepton S. azureus eu all other antibiotic processes. The production
process for bacitracin is shown in Figure 13.17.
Application: Food preservative
Nisin Streptococcus cremoris G The yields of this process were increased from
about 13 mg/l to about 9 g/l through strain de-
G*: Gram-positive; G-: Gram-negative bacteria; AF:
antifungal; My: mycobacteria. velopment and medium optimization. A contin-
(Perlman, 1979; Kleinkauf and von Dåhren, 1985) uous process has also been developed for baci-
tracin production using immobilized cells.

mechanisms. The biosynthesis of high molecu-

lar-weight antibiotics such as nisin and neocar- Chelate-forming peptide antibiotics
zinostatin involves ribosomes, mRNA, and
tRNA. However, the biosynthesis of most pep- Siderochromes and bleomycins are peptide anti-
tide antibiotics does not involve this protein-syn- biotics which form chelates with metals. Sider-
thesizing machinery, but takes place instead on ochromes are iron-containing or iron-binding
a multi-enzyme complex. This nonribosomal natural substances containing hydroxamic acid
peptide synthesis process, which has also been groups, and bring about the formation of ferri-
called the ‘‘thiotemplate’’ mechanism, has been hydroxamate complexes (Figure 13.18). Albo-
described for gramicidin S, tyrocidin, and the lin- mycins and ferrimycins have been isolated as an-
ear gramicidins, as well as for bacitracin. tibiotically active siderochromes (sideromycins),
Bacitracin synthetase consists of subunits A, but there is no known industrial application for
B and C, with molecular weights of 200,000, these substances. Sideramines are siderochromes
210,000 and 380,000, respectively. A simplified which act as growth stimulants, and ferrioxamine
version of the process of bacitracin synthesis is B, a sideramine from Streptomyces pilosus ETH
given in Figure 13.16. The first step, the activa- 21748, is commercially produced. It is used in
tion of amino acids in the presence of ATP and human therapy as the desferri compound for the
Mg?* to aminoacyl adenylates, takes place at a treatment of diseases involving iron storage and
specific activation site for each amino acid on the also in agriculture in such applications as the
enzyme complex. In a transfer reaction, the ac- elimination of soil iron deficiencies in peach or-
tivated amino acids are then bound to specific chards.
248 / CHAPTER 13 / ANTIBIOTICS

(c) (fdd)
Figure 13.16 Model of nonribosomal peptide synthesis according to the ”thiotemplate” model for the formation of
bacitracin through the action of bacitracin synthetase in B. licheniformis (From Zimmer et al., 1979)
 13.4 CARBOHYDRATE ANTIBIOTICS / 249

Inoculum Spore suspension in sterile soil’

preservation or agar slant

4 X 11 Erlenmeyer flask with

Shake culture 200 ml tryptone or peptone
culture medium
OC-(CH,)=¢=0 Fe ONE-(CH),-NH
18-24 hr årg
af37C
(Hs CH
/

Prefermenter
(800 1) Same as for shake culture
NH
6 hr at 37°C with /
intensive CO
aeration
Pr [Sos Hige2°s"\ |
Prefermenter Soy meal 5%; Sucrose 1.2%;
(3,000 1) (NH,),SO, 0.2%; CaCO, 0.2%
Growth to
log phase
OH A. Ferrimycin A
VE
Production
fermenter
Soy meal 5%; Sucrose 2.4%;
(NH,).SO, 0.2%; CaCO, 0.2%
(90,000 1)
30-hr;372C yd &
Purification a. For pharmaceutical uses: OCICH)= c a reek NH
Extraction with n-Butanol, Q,
extraction of the organic
phase with buffer, /
concentration of the (CH) CH,
aqueous phase, ion- NH>
exchange chromatography
b. For use in animal feeds:
Spray drying of the whole B. Ferrioxamine B
fermentation broth
Figure 13.18 Structure of siderochromes
Figure 13.17 Flow scheme for the production of bacitra-
cin by B. licheniformis (From Perlman, 1979)

Nojirimycin (Figure 13.19 A), shows anti-

Bleomycins are isolated as Cu(II) complexes bacterial activity against Sarcina lutea and Xan-
from the culture broth of Streptomyces verticillus. thomonas oryzae, and is of interest because of its
The glycopeptide exhibits some antitumor action high inhibitory activity against a- and 6-gluco-
and is marketed as a mixture of bleomycin A, sidases and amylases.
and B,. Vancomycin, a broad-spectrum antibiotic,
consists of the amino sugar vancosamine, two 6-
13.4 CARBOHYDRATE ANTIBIOTICS hydroxychlortyrosine moieties, three phenylgly-
Carbohydrate antibiotics include the therapeu- cine derivatives, N-methyl leucine, and aspartic
tically important aminoglycosides, the orthoso- acid. It is effective against Gram-positive orga-
mycins, and various sugar derivatives. nisms, particularly staphylococci. However, it
has adverse side effects in humans and thus is
Glycosides and sugar derivatives used only to treat infections caused by pathogens
Several commercially important antibiotics from which are resistant to other antibiotics or with
this heterogenous group are listed in Table 13.9. patients who are hypersensitive to penicillin. No
250 / CHAPTER 13 / ANTIBIOTICS

Table 13.9 Carbohydrate antibiotics. Sugars, glycosides development of resistance to vancomycin has yet
and other commercially important sugar derivatives
been observed in therapy.
Group Antibiotic Organism Lincomycin (Figure 13.19 B) is used in the
producing
treatment of staphylococcus, streptococcus, and
5-Amino sugar Nojirimycin Streptomyces pneumococcus infections; only minor side effects
roseochromo-
genes are observed, but during therapy, resistance is
S. lavendulae acquired rapidly. Clindamycin (7-chlor-7-deoxy-
S. nojiriensis lincomycin), a semisynthetic derivative with im-
Glycopeptide Vancomycin S. orientalis proved pharmacological properties, is principally
Sugar amide Lincomycin S. lincolnensis marketed today. In protein synthesis, the bond-
var. lincolnensis. ing of aminoacyl-tRNA to the A-binding site on
Phospho- Moenomycin S. bambergiensis the ribosome is inhibited by lincomycin, proba-
glycolipid A,C,D,E,F,G,H S. ederensis bly through interaction with the ribosomal pro-
(= Bambermycin) S. ghanaensis tein L6 of the 50S subunit.
S. geysiriensis
Moenomycin is a phosphoglycolipid which
shows activity against Gram-positive bacteria as
well as Gram-negative bacteria containing a re-
sistance plasmid. Moenomycin A and the related
A antibiotics macarbomycin and quebemycin are
CH,OH 4H marketed for use as nutritional feed supplements.
: N Phosphoglycolipids inhibit bacterial cell wall
HO
HO R2 synthesis by affecting the transglycosylation re-
OH action in peptidoglycan biosynthesis.
R
Nojirimycin
Orthosomycins
(5-Amino-5-deoxy-
D-glucopyranose ) The orthosomycins are a class of antibiotics
R, = OH; R,= H (60°%s) newly discovered in the late 1970’s. They are
Ry = H; R,=OH (40%)
characterized by an oligosaccharide structure and
two noncarbohydrate ester groups (Figure 13.20).
The following antibiotics have been described:
B
the avilamycins (produced by Streptomyces
CH;
viridochromogenes), the curamycins (S. curacoi),
CH,CH,CH, the everninomicins (Micromonospora carbona-
ceae), and flambamycin (S. hygroscopicus). The
CH3 avilamycins are being tested for use as animal
feed supplements.
H O=C—NH-FH
OH
0 Aminoglycoside antibiotics
HO Aminoglycosides are oligosaccharide antibiotics
OH and consist of an aminocyclohexanol moiety (e.g.
SCH,
deoxystreptamine, streptidine), which is glyco-
Lincomycin sidically linked to other amino sugars. Over 100
aminoglycosides are known, of which those pro-
Figure 13.19 Structure of nojirimycin and lincomycin duced industrially as well as some newer com-
 13.4 CARBOHYDRATE ANTIBIOTICS / 251

Me OMe

Me Me Me Me CH20Me 0
MeO 0 0 (9) MeO 0 0 5
E o MeO OMe O
2 =; aS o—_—_

| 0 0
Me OH Me OH n
(ej
|
co
MeO Me

Cl OF

OH

Figure 13.20 Everninomicin B

pounds are listed in Table 13.10; Figure 13.21 a result of a screening program, the new group
shows the most important structures. of fortimicins was discovered. This is a group of
Aminoglycosides are primarily used against pseudodisaccharides with a broad spectrum of
Gram-negative bacteria in a wide range of ap- activity, particularly against aminoglycoside-re-
plications. Although the semisynthetic cephalo- sistant bacteria. The kanamycin derivatives ami-
sporins are also now widely used for Gram-neg- kacin and dibekacin (1-N-L(—)-y-amino-a-hy-
ative infections, in severe infections the droxybutyrylkanamycin A and 3',4'-
aminoglycosides are often the antibiotics of dideoxykanamycin B), and netilmicin (1-N-
choice. Although streptomycin and dihydro- ethylsisomicin) are semisynthetic compounds
streptomycin are broad-spectrum antibiotics, with partial resistance to inactivating enzymes.
they are primarily used to treat tuberculosis. These compounds have already been introduced
They are considered reserve antibiotics, mainly for clinical use. The compound 5-epi-sisomicin,
due to the fact that resistance can develop rapidly isolated by mutasynthesis, has been tested clin-
and in a single step by a chromosomal mutation. ically because of its action against sisomicin-re-
In 1976 the world market for aminoglyco- sistant organisms. However, for cost reasons it is
sides (not including the Soviet Union and China) being produced chemically rather than by fer-
was estimated at $500 million, with a yearly in- mentation.
crease of 10-15%. Besides the antibiotics noted above which are
All of the aminoglycoside antibiotics cause useful for human therapy, kasugamycin and val-
kidney damage and deafness (nephro- and oto- idamycin are used as important antibiotics in rice
toxicity) as side effects. In addition to resistance cultivation.
development by chromosomal mutation, plas-
mid-determined resistance due to the formation Mode of action The mode of action of amino-
of inactivating enzymes is known (see Section glycosides is on protein synthesis in sensitive
15.4). Compounds with reduced toxicity and/or bacteria. Streptomycin, the aminoglycoside
increased effectiveness against resistant orga- which has been studied the most, binds to pro-
nisms are being sought via screening, mutasyn- tein S12 of the 30S ribosome and causes mis-
thesis, and formation of chemical derivatives. As reading of the code and hence inhibition of pro-
252 / CHAPTER 13 / ANTIBIOTICS

Table 13.10 Classification of the most important aminoglycoside antibiotics according to the aminocyclohexanol
moiety. Production strain, spectrum of activity, and application are given for commercially produced antibiotics
Group Antibiotic Organism producing Spectrum of Application
activity"

I Streptamine derivatives
Streptidine Streptomycin Streptomyces griseus G*G My Human therapy
Dihydrostreptomycin — S. humidus® G*G-My Human therapy
Actinamine (N,N- Spectinomycin® S. spectabilis G* Human therapy
dimethylstreptamine) S. flavopersicus (Penicillin-resistant
gonococci)
Bluensomycin (S. bluensis)
II 2-Deoxystreptamine (DOS) derivatives
A. 4,5-Disubstituted DOS
Neomycin group Neomycins B, C S. fradiae GG Human therapy
(Pseudotetrasaccharides) Paromomycins I, II S. rimosus forma GG Human therapy
paromomycinus Protozoa
Lividomycins A, B S. lividus GG Human therapy
Ribostamycin group Ribostamycin S. ribosidificus GG Human therapy
(Pseudotrisaccharides) Butirosins (Bacillus circulans)

B. 4,6-Disubstituted DOS
Kanamycin group Kanamycins A, B, C S. kanamyceticus G*G My Human therapy
Tobramycin S. tenebrarius GG Human therapy
(= Nebramycin-
factor 6)
Seldomycins 1, 2,3,5 (S. hofunensis)
Gentamicin group Gentamicins C,, C,,, C, Micromonospora purpurea GG Human therapy
M. echinospora
Sagamicin= (M. sagamiensis, etc.) GG Human therapy
Micronomicin
Sisomicin M. inyoensis GG Human therapy
Verdamicin (M. grisea)
C. Monosubstituted Hygromycin B S. hygroscopicus GG F Feed additive
DOS worms
Destomycins A, B,C (S. rimofaciens) G*G My Feed additive
Apramycin worms
(=Nebramycin- (S. tenebrarius)
factor 5)

III Fortamine Fortimicin M. olivoasterospora GG Human therapy

derivatives
Sannamycins A, B (S. sannanensis sp. nov.)
Sporaricins A, B (Saccharopolyspora hirsuta
subsp. kobensis)
Istamycins A, B (S. tenjimariensis nov. sp.)
IV Other Validamycins S. hygroscopicus GGF Plant pathology
aminocyclohexanol var. limonensis
derivatives
Kasugamycin S. kasugaensis G*GF Plant pathology
2 Gt: antibiotically effective against gram-positive organisms, G~: against gram-negative organisms; My: against
mycobacteria; F: antifungal antibiotics.
> Dihydrostreptomycin is almost exclusively produced via chemical reduction of streptomycin.
© Spectinomycin has unusual status: it contains one aminocyclitol moiety but no amino sugar.
(Berdy, 1985)
 13.4 CARBOHYDRATE ANTIBIOTICS / 253

NH
I biosynthesis. Over 30 separate enzymatic steps
HN=C NH2
0. NH CeNH are known. One of the principal components of
CH 3 OH OH NH
H
streptomycin, streptidine, is synthesized from
me OH - VED
HON 5 0 ay glucose-6-phosphate via myo-inositol. A number
HN OH 0 Streptidin
Lae OH OH HO/CHOH
K RON of enzymatic steps, involving oxidation, amina-
| tion, phosphorylation, carbamidinylation, and
COOH HO
Kasugamycin Streptomycin dephosphorylation, lead to the formation of
streptidine-6-P. The second dephosphorylation
CH,NH, NH, step takes place during the final biosynthetic step
CH, OH (0) NH» to streptomycin synthesis. Although the pathway
HO
OH
HO 0
DOS for streptose biosynthesis is known (see Figure
HO
13.22), that for N-methyl-L-glucosamine is still
NH unclear. The combination of the three subunits
OH

OH
involves two steps. Only in the last step is the
phosphate moiety removed, leading to the for-
HocH, | — CH,OH mation of the biologically active streptomycin
from the biologically inactive streptomycin-
OH on 3 phosphate.
OH HO DOS
In the DOS-aminoglycosides (see Table
ValidamycinA HOH,C 13.10), the biosynthesis of 2-deoxystreptamine
occurs from glucose (Figure 13.23).

Neomycin B Regulation of biosynthesis Only few data are

available on the induction and regulation of ami-
Figure 13.21 Aminoglycoside antibiotics (structure of noglycoside biosynthesis. Streptomycin-produc-
validamycin according to Suami et al., 1980). For the struc- ing strains of S. griseus and S. bikiniensis show
ture of neomycin/ribostamycin see Figure 15.9; genta-
micin complex see Figure 15.10 regulation via the so-called factor A, 2 (S)-iso-
capryloyl-3-(S)-hydroxymethyl-y-butyrolactone,
shown in Figure 13.24. This compound controls
tein synthesis. S12 is a component of the A site streptomycin biosynthesis, streptomycin resist-
at which the amino-acyl-tRNA and fMet-tRNA ance, and sporulation. It is active at the extremely
bind. 2-Desocystreptamine-containing anti- low concentration of 10°°M. Factor A~ mutants
biotics inhibit the translocation process of the that are unable to produce the antibiotic can be
peptidyl-tRNA. In addition, aminoglycoside restored to streptomycin production by addition
antibiotics cause damage to the bacterial cell of factor A.
membrane, thus bringing about loss from the cell In addition, streptomycin production is influ-
of low-molecular weight constituents. enced by nitrogen and carbohydrate catabolite
regulation, as well as phosphate regulation.
Biosynthesis of aminoglycosides As shown by la-
beling experiments, glucose is the precursor of Strain development Strain-development pro-
all the subunits of those aminoglycoside anti- grams have been conducted to increase yields for
biotics which have been examined. Although a all industrially produced aminoglycoside anti-
number of enzymatic reactions have been un- biotics, since yields of wild strains range only
covered, the exact biosynthesis of any aminogly- from 10-200 ug/ml, which is not sufficient for a
coside antibiotic is not yet fully understood. Fig- cost-effective process. The following yields have
ure 13.22 shows the pathway of streptomycin been published: kanamycin 2000 ug/ml; tobra-
254 / CHAPTER 13 / ANTIBIOTICS

D-Glucose

D-Glucose-6-P

myo-Inositol-1-P D-Glucose-1-P D-Glucosamine-6-P

op |
myo-Inositol

of
2-Keto-myo-inositol 4-Keto-6-deoxy- Methylation
D-glucose-1-dTDP
A (Gin) { (Methionine)
scyllo-Inosamine

Pt
scyllo-Inosamine-4-P 4-Keto-L-Rhamnose-1-dTDP
CA (Arg) | q----------------------
N-Amidino-scylio-inosamine-4-P UDP-N-Methyl-
D-glucosamine-6-P
DP
N-Amidino-scyllo-inosamine
Oo
N-Amidino-3-keto-scyllo-inosamine
A (Ala) I
N-Amidino-streptamine
Pp
N-Amidino-streptamine-6-P
CA (Arg) {
Streptidine-6-P L-Dihydro-streptose-1-dTDP

ern
deen
essere
renee
rrr
nr
en
nn

4-(O-Dihydrostreptosy})- NDP-N-Methyl-
streptidine-6-P L-glucosamine

Dihydrostreptomycin-6-P

[Srepienyen
]—22— DP
Oo
Streptomycin-6-P

Figure 13.22 Streptomycin biosynthesis (based on Florent, 1985). O, oxidation; A, amination; P, phosphorylation; CA,
carbamidinylation; DP, dephosphorylation; Ala, alanine; Arg, arginine; Glu, glutamine.

mycin 1500 ug/ml; neomycin 2000 ug/ml; gen- opment; there have also been some cases in
tamicin 1400 ug/ml. Streptomycin yields have which recombination (conjugation and proto-
been increased from 100-200 ug/ml to about 15 plast fusion) has been employed. The strain de-
mg/ml by strain-improvement techniques. The velopment of kasugamycin is a typical example.
current yields of production processes are prob- Using the “agar disk” method (see Section 3.3),
ably far more than the published values in many in one year 650,000 isolates were screened after
cases, mutagenesis, resulting in a yield increase from
Mutation and selection are the principal 200 ug/ml to about 7000 ug/ml. Conjugation of
methods which have been used in strain devel- auxotrophs produced hybrid strains, whose off-
‘
 13.4 CARBOHYDRATE ANTIBIOTICS / 255

CH,OH 0) NH> tants not subject to catabolite regulation has

(eg) nx OH OH
made it possible to use glucose. In several pro-
cesses, continuous feeding of glucose has been
HO OH ~ HO HO
OH OH OH successfully used. Soy meal is an ideal nitrogen
D-Glucose A B
source for aminoglycoside production because of
its slow catabolism. NaCl (1-3 g/l) must be
added to the streptomycin fermentation. The
H
2-Deoxy- NH2-
production of gentamicin, sisomicin, and forti-
streptamine Kon = Kou >=0 micin A is significantly accelerated by addition
HO HO of Co%, which is active in the methylation steps
during biosynthesis. Addition of Zn?* increases
the yield of nebramycin and addition of Mg?*
Figure 13.23 Biosynthesis of 2-deoxystreptamine in Ba-
cillus circulans S-11. A, 2-Deoxy-scyllo-inosose; B, 2-
stimulates kanamycin and neomycin production.
Deoxy-scyllo-inosamine (From Fujiwara et al., 1980) Aminoglycoside processes are typical sec-
ondary metabolite fermentations with tropho-
and idiophases, as shown in Figure 13.26 for the
CH, butirosin fermentation.
OH

CH; 15
ore’
Figure 13.24 Structure of Factor A from S. griseus
) ONS

spring in one case produced up to 26% more 10

kasugamycin (9300 ug/l) than the initial proto-
troph strain (Figure 13.25). Cell fusion and re-
combinant DNA methods have been used in sev-
eral cases to produce higher-yielding strains.
Protoplast fusion has been used to increase yield
of sisomicin producers and amplification of a re-
sistance gene, 6’-N-acetyltransferase, has in-
creased production of kanamycin and neomycin.
DEG
of
(number
strains
Frequency
Production methods Aminoglycoside antibiotics
are produced in fermenters with volumes up to
150,000 1. Optimal oxygen supply is required
o095
0
(aeration rate 0.5-1.0 vvm) and temperatures are
held between 28-30°C with the pH in the neutral 2 4 6 8 10 12
range. The length of fermentation is between 4- Kasugamycin( ug/ml x 1073)
7 days, depending on the strain.
Figure 13.25 Effect of genetic recombination on kasu-
Glucose in combination with starch or dextrin gamycin formation. O, prototrophic original strain (ka-
serves as the carbon source. Originally, the anti- sugamycin formation, 7300 ug/ml; P,, auxotrophic (ilv)
biotic butirosin could be produced only with mating partner (4400 ug/ml); P,, auxotrophic (ser) mating
partner (2900 ug/ml); H,, prototrophic hybrid strain (ilu
glycerol as carbon source, due to catabolite X ser) (5100 ug/ml); VS176, selection from H, (9300 pug/
repression by glucose, but the isolation of mu- ml) (From Ichikawa et al., 1971)
256 / CHAPTER 13 / ANTIBIOTICS
bered lactone rings with 1-3 sugars glycosidically
HE linked with the aglycone (lactone ring) and with
2
c each other. The sugars are amino sugars and/or
sl|Siler3 eiroan ei:
Besa
6-deoxyhexoses.The structures of erythromycin
and oleandomycin are examples of 14-membered
| i BN ele
6007 S |» macrolides (Figure 13.27); the 16-membered
5504 a macrolides leucomycin and tylosin are illustrated
0-4 AR in Figure 13.28. Macrolides are produced by 1-
20 | 450-_ ris
3% of all actinomycete isolates; the compounds
listed in Table 13.11 are commercially available.
30 4 4007 4.0 +16
Macrolides are very effective against Gram-
ae 3504 AN g i Fi positive bacteria, particularly staphylococci, and
50 7 300 , 2 30 12 L 80 mycoplasmas and were frequently used against
604 280 eee L10 275 penicillin-resistant organisms, but because of the
a 200 20 +08 ie development of the newer 6-lactam antibiotics
807 150 | Los +65 (see Section 13.2), the macrolides are no longer
90+ 100 + 1.0 Low
of as great medical significance. The aglycone
and sugar moieties are both necessary for anti-
an 504 ix HR 02
it 0
2Sameer
40 80
Sesss
120 160 Desosamine

Fermentation time (hr) HG CH,

ven
Figure 13.26 Butirosin formation with Bacillus circulans. Frags S
Medium: glycerol 4%; soy meal 1%; Wilson’s peptone TR:
tuo, htØg
1.75%; NH,Cl 0.4%; CaCO, 0.5% (Howells et al., 1972)

Most of the aminoglycoside antibiotics are ex- oO

oO=F
(2)
Geer oO

creted and are present in the culture supernatant,

from which they are removed by adsorption to
Ry: CH3 =Cladinose Erythronolide
ion-exchange columns. Cell-bound aminogly- Ro: H = Mycarose
cosides, such as gentamicin, sisomicin, and for-
timicin, must first be released by acidification to Erythromycin A | OH CH 3 (Main components)
pH 2-2.5 with H,SO,. BH CH
CuNsOh AH
By) tal H
13.55 MACROCYCLIC LACTONE Ba. NiCH. ye
ANTIBIOTICS
HO a ;
0 <
The macrolide group of antibiotics consists of OSCE Øg ;
compounds with large lactone rings. In addition
to those antibiotics conventionally called mac- OSECH3 we
O
rolides, this group includes polyenes, macro- (9) oe FAR

tetrolides, and a heterogeneous group of atypical OCH3

macrolides. Oleandrose Oleandolide

Oleandomycin
Macrolide antibiotics
Figure 13.27 Structure of erythromycin and oleando-
Macrolides are hydrophobic, usually basic com- mycin. Straight arrow, acetate residue; bent arrow, pro-
pounds. They consist of 12-, 14-, 16-, or 17-mem- pionate residue
 13.5 MACROCYCLIC LACTONE ANTIBIOTICS / 257

BRS ders
CH3 CHO 40 N se
HO 0 0” “CH3
O° CH3 Mycarose
VAH.CO Mycaminose
OR,

LT CH3 \5
O
Leucomycin ‘

Leucomycin A, COCH2CHICH 312

A3 Uosamycin] COCH COCH.CHICH 3),
A, COCH3 }COCH2CH2CHz
AS
As coc
OCH
Ee COCK.
cH,
As COCH3 COCH3

CH3 CH2CHO LL dd
Mycarose
O= Mycami
ycaminose \ (7x A
ig NEDE
OH erb sen
”; NR

OCH, A Ne
Figure 13.28 Structure of leucomy- HO OCH3 AE) NY 3 “Oo rene
cin and tylosin. Straight arrow, ace-
tate residue; bent arrow, propionate
residue; upper right bent arrow, bu-
RecemOrnal ?
Mycinose
tyrate residue; dotted region, position
of C-3, 4 unknown Tylosin Leuconolide

Table 13.11 Commercially produced macrolide antibiotics

Macrolide antibiotic Organism producing Activity Application
Carbomycin A Streptomyces halstedii G Was marketed for several
years but discontinued due to
ineffectiveness
Erythromycin S. erythreus Se Human therapy, feed
additive
Josamycin (= Leucomycin A;) S. narbonensis var. josamyceticus Gt Human therapy
Leucomycins A,-Å, (= Kitasamycins) Streptoverticillium kitasatoensis Gt Human therapy
Maridomycin S. hygroscopicus G*
Midekamycin S. mycarofaciens G*
Oleandomycin S. antibioticus Gt Human therapy
S. olivochromogenes
_ Spiramycins I, II, Ill S. ambofaciens Ce Human therapy
Tylosin S. fradiae GG Feed additive,
S. hygroscopicus Myco- veterinary medicine
plasmas
258 / CHAPTER 13 / ANTIBIOTICS

bacterial activity. Therapy with macrolides re- Erythromycin biosynthesis is regulated at

sults in a rapid buildup of resistance and there various levels. For instance, erythronolide B in-
is also cross-resistance between the individual hibits its own production, the synthesis of 6-
antibiotics. Because of the low toxicity, efforts are deoxyerythronolide B, and presumably also the
being made to isolate new compounds of this activity of the lactone synthase. End-product in-
class, to chemically modify known macrolides, hibition of the transmethylase by erythromycin
and to modify known macrolides through bio- A has been observed. This enzyme catalyzes the
transformation. methylation of mycarose to cladinose as a last
All macrolide antibiotics are inhibitors of pro- step of the biosynthetic pathway. In high-yield-
tein synthesis, binding to the 50 S subunit of ing strains used for industrial production, this
bacterial ribosomes. Erythromycin is known to regulation is eliminated.
inhibit the elongation-factor G-dependent re- When propanol (0.2-0.5%) is added to the
lease of deacylated tRNA from the P site of the fermentation broth of S. erythreus, it induces the
ribosome. synthesis of acetyl-CoA-carboxylase and causes
a 100% increase in erythromycin production.

Biosynthesis and regulation The aglycone moiety Production method Macrolide antibiotics are
of macrolides is built either of propionate sub- produced in aerobic submerged fermentations.
units or of a combination of acetate and pro- Yields in large-scale industrial processes are
pionate subunits, sometimes with the participa- around 20 g/l. A typical medium for S. erythreus
tion of butyrate or related compounds. One of contains glucose 50 g, soy meal 30 g, (NH,),SO,
the first intensively studied macrolides was 3 g, NaCl 5 g, CaCO, 6 g, tap water 1 1, pH 7.0.
erythromycin; the biosynthesis of this antibiotic Complex culture medium ingredients may also
was studied by labeling experiments and from be used, such as starch, corn steep liquor, yeast
the behavior of mutants of Streptomyces erythreus extract, and oils (e.g., soy bean oil). Fermentation
blocked in antibiotic biosynthesis. Analogous to temperatures for most macrolides are 25-28°C
fatty acid synthesis, erythronolide production and for erythromycin 33°C. The length of the
proceeds from propionyl-CoA as a starting ma- fermentation is 3-7 days.
terial, onto which 6 molecules of 2-methylma- The macrolide tylosin can be produced in
lonyl-CoA condense. This polyketide synthesis either batch or continuous fermentation. The
takes place on a multi-enzyme complex called specific production rate is increased to 1.1 mg/
lactone synthase, which is very similar to the fatty g biomass-h if glycerol is used instead of glucose
acid synthetase of S. erythreus. After cyclization and if the feeding rate of sodium glutamate is
to the aglycone, the first free compound, 6-deox- optimized at a low specific growth rate of 0.03/
yerythronolide B, can be isolated. The remaining h (27% of the u,,,,)-
glycosylation steps are diagrammed in Figure
13:29:
A number of observations have been made Polyene macrolide antibiotics
about the regulation of macrolide biosynthesis. Polyenes consist of 26- to 38-membered lactone
Biosynthesis of the macrolide tylosin is stimu- rings with 3-7 conjugated double bonds. De-
lated by long-chain fatty acids; glucose inhibits pending on the length of the chromophore, poly-
the metabolism of fatty acids and tylosin pro- enes are classified as trienes, tetraenes, pen-
duction, probably by inducing an acetyl-CoA de- taenes, hexaenes, or heptaenes. Many polyenes
ficiency. Phosphate regulation is also observed also contain a glycosidically-bound amino sugar
with many macrolide antibiotics, as well as ni- (mycosamine or perosamine). Some heptaenes
trogen (NH,") catabolite regulation. carry p-aminoacetophenone or N-methyl-p-ami-
 13.5 MACROCYCLIC LACTONE ANTIBIOTICS / 259

OR TS ATP-C03H20 ADP +R

pee lbh CoA 2-Methylmalonyl-CoA

Hz ne CH3 CH,
CHSCH-re-tic- a aie ¢—CH-C~CH-C —S-=R
ioe Polyketide
6 6 0 0 GO
ANE SEE y 5) te ae ai (enzyme-bound)

CH;
Os dTDP-Glucose 2
H3C CH3
HO OH 6-Deoxy -
erythronolide B
HC CHz (R=H)
OH CH3
O
CRY sears 0
9 O=( OH
(I O - dTDP
Erythronolide B OH
dTDP-L-Mycarose
(R=OH) ne. © dTDP-4-Keto-6-
deoxy -D-glucose

3-a-L-Mycarosyl-
erythronolide B
rat dTDOP-D-Desosamine

CH,

noe Cc CH sell Eryth i D

rythromycin
(Ry =Rz =H)

Methylation
ee
SAM Hydroxylation

SAM
Erythromycin B Erythromycin C ——————_+F rythromycin A
Trans-
(R4=H, Ro =CH3 ) (Ry = OH, Ro=H) methylase (Ry = OH ,R2=CH3)

Figure 13.29 Biosynthesis of erythromycin in S. erythreus. SAM: S-adenosyl-L-methionine

noacetophenone as an aromatic moiety on the membrane sterols of sensitive eucaryotes, caus-

carbon atom which is involved in lactone for- ing a change in membrane permeability. Some
mation. The structures of two polyenes are given polyenes are relatively toxic (nephro- or hepa-
in Figure 13.30. totoxic) but they are widely used commercially
More than 90 polyenes have been character- for topical application because no better antifun-
ized, primarily from streptomycetes. These anti- gal antibiotics are available.
biotics are active against fungi and yeast but they Polyenes are not suitable for plant-disease
are ineffective against bacteria. Polyenes affect control because they are sensitive to light and
260 / CHAPTER 13 / ANTIBIOTICS

OH OH OH OH OH OH erization. The chromophore consists solely of

acetate units. The amino sugar is derived from
H OH ORSA glucose, with thymidine diphosphate-4-keto-6-
HOt N\ANAW4AnRI47 deoxy-D-glucose as an intermediate. Polyene
OH
biosynthesis shows phosphate regulation, catab-
olite repression, and feedback inhibition.
Filipin
Production methods Glucose is usually the best
carbon source for polyene production. Most fer-
mentations using glucose are continuous, be-
cause catabolite repression occurs at the required
glucose concentration (for candicidin 7-9.6%).
The candicidin production phase can be ex-
tended to 300 h by glucose feeding. The addition
Amphotericin B
of acetate, propionate, malonate, or n-propanol
Figure 13.30 The polyene antibiotics filipin (pentaene) stimulates polyene production, and soy meal or
and amphotericin B (nonaromatic heptaene) soy peptone can be used as nitrogen sources.
For maximal polyene production, careful con-
trol of aeration is essential. During the growth
heat. Table 13.12 lists the commercially pro- phase, the required oxygen transfer rate is around
duced polyenes. 0.8 mmole O,/I-min, but during the production
phase, O, concentration should not exceed 40%
Biosynthesis and regulation Like macrolides, po- of saturation. Figure 13.31 shows the relation of
lyene antibiotics are produced from C, and C, candicidin production to various fermentation
units via the polyketide biosynthetic pathway. parameters.
Among the aromatic polyenes, p-aminobenzoyl- Since polyenes are not water-soluble, they
CoA, the precursor of p-aminoacetophenone and accumulate as crystals or microparticles at the
N-methyl-p-aminoacetophenone, seems to func- surface of the cell or in the medium, from which
tion as the starting point for polyketide polym- they can be extracted with appropriate solvents.

Table 13.12 Commercially produced polyene macrolide antibiotics

Antibiotic Organism producing Polyene type Application
Amphotericin B Streptomyces nodosus Heptaene, M F, Histoplasmosis
Candicidin B S. griseus Heptaene, M, AAP F, Prostate hypertrophy
Candidin S. viridoflavus Heptaene, M
Filipin S. filipinensis Methylpentaene
Fungimycin (=Perimycin) S. coelicolor var. aminophilus Heptaene, P, MAAP
Hamycin S. primprina Heptaene, M, AAP
Levorins S. levoris Heptaene, M, AAP , Prostate hypertrophy
Mycoheptin Streptoverticillium Heptaene, M loo
Fryd
rd
Fry
rr]
mycoheptinicum, S. netropsis
Nystatin A,, A,, A, S. noursei Tetraene, M F, Candida albicans; slightly
toxic
Pimaricin S. natalensis Tetraene, M F, Food preservative
Trichomycin A, B S. hachijoensis Heptaene, M, AAP F, Trichomonas vaginalis
M=Mycosamine; AAP=p-Aminoacetophenone; P=Perosamine; MAAP=N-Methyl-p-aminoacetophenone;
F=Antifungal activity.
 13.5 MACROCYCLIC LACTONE ANTIBIOTICS / 261

HC R, i

O O O
z | 0o= Ry
x8 = 2 CH pa Q
ve I Rs ‘ =O
EE i O O O
= E64 2
~~ 8 I
SE go | O R; CH
&
2/3 |e
a
ås
+45 30:
c
5
— a

ES
= 5 ig
gs Nonactin S.werraensis R,=R,=R3=R,=CH3
(5; < zoa
Bel ler a Monactin S.sp.ETH 23112 R, =R,=R3=CH3 R, =CoHs
= Spe
3Si 2x Dinactin S.sp.ETH 23112 R,=R2=CH3 R3=R,=C2Hs
n oOo
= Trinactin S.sp.ETH 23112 R,=CH, R2=R3=R,
SHS
es]oO

I Tetranactin S. flaveolus Ry =R2=R3=R, =CoHs

2 E
i=, 60
Oo
1200
Ey
| =| Figure 13.32 Macrotetrolides
= za
E i Page)
E o Iso
a
24 _ a OCH3
=2 |&
= ~E3} 40 HO. OCH3 Ry H
= | £
8 8 - Os
£204 § 3
c
Hf. <9
pate, = £120
oe ae 2 HE oA
& (2;
Hoe
30 60 90 120

Fermentation time (hr)

Figure 13.31 Relationship of candicidin formation to dif-

ferent fermentation parameters

Avermectin R, R, R,

Macrotetrolides Ana SGH: CH,

Ay CH; CH,
Macrotetrolides are cyclic polylactones which Ay, OH GER CH,
A» OH CH CHE
function as ionophores. These sugar-free com- BR GE H
pounds are synthesized from four tetrhydrofur- Bip CH H
anyl hydroxy acids (Figure 13.32). Tetranactin, Bx OH SGH: H
By OH CH, H
produced by $. flaveolus, is used as an insecticide.
In those avermectins without an R, group, a double
bond is present.
Atypical macrolides
Figure 13.33 Structures of the avermectins
This is a heterogeneous group, classified together
because all structures contain the lactone ring.
Examples include avermectin, milbemycin, ven- veterinary medicine because of its high activity
turicidin, bafilomycin, and irumamycin. Aver- against nematodes and arthropods. Irumamycin
mectin, whose structure is shown in Figure 13.33, is undergoing field trials in agriculture as a fun-
is produced by S. avermitilis. It has found use in gicide.
262 / CHAPTER 13 / ANTIBIOTICS

ethylbarbiturate. Rifamycin B is completely in-

Ansamycins
active but during its breakdown or via chemical
Ansamycins are macrolactam antibiotics with an modification, the biologically active rifamycins
aromatic chromophore to which an aliphatic O, S, and SV are formed. Rifamycin S and SV
polyketide derivative is bound at both ends, via are biosynthetic precursors to rifamycin B. Mu-
amide, ether, and/or carbon-carbon bonds, the tants have been isolated which excrete rifamycin
so-called ”ansa chain” (Figure 13.34). Ansamy- SV due to a block in the subsequent steps. The
cins are distinguished by their chromophore. ansa chain of rifamycin is formed from two ma-
Those compounds with a benzene ring, such as lonyl-CoA and eight methylmalonyl-CoA units
geldanamycin or ansamitocin, act mainly on eu- and methionine. The polyketide condensation
caryotes, whereas those with a naphthalene process proceeds from an aromatic C, unit. The
structure are antibacterially effective. In this lat- biosynthesis of the naphthohydroquinone
ter class are the rifamycins, halomycins, and chromophore branches off from the aromatic bio-
streptovaricins. synthetic pathway between 3-deoxy-D-arabino-
Rifamycins are important in chemotherapy. heptulose-7-P and shikimate.
They are very effective against Gram-positive Rifampicin, a semisynthetic compound de-
bacteria and mycobacteria, but less effective rived from rifamycin SV, is widely used in the
against Gram-negative bacteria; they show no treatment of tuberculosis. It is a specific inhibitor
cross resistance with other antibiotics. They are of bacterial DNA-dependent RNA polymerase. It
produced by Nocardia mediterranei. Approxi- binds with the 6-subunit of the polymerase, and
mately 20 different rifamycins have been isolated RNA polymerases of higher organisms are not
from culture solutions of Nocardia mutants, and affected.
genetic studies have shown that the genes for
the last steps in the biosynthetic pathway lie in
a single cluster. The wild strain produces rifa- 13.6 TETRACYCLINES AND
mycin B as a major component and smaller ANTHRACYCLINES
amounts of the so-called rifamycin complex (A,
C, D, E). The production of this complex can be Tetracyclines
largely suppressed by the addition of sodium di-
The tetracyclines are important antibiotics with
widespread medical use. Chlortetracycline, the
first antibiotic of this group, was isolated from
OH cultures of Streptomyces aureofaciens in 1945. Tet-
rs NH racycline itself was first prepared by dehalogen-
ation of chlortetracycline but was subsequently
found to be produced by cultures of S. viridifa-
OH ciens as well. Presently about 20 streptomycetes
Rifamycin SV are known which produce a mixture of different
FinelI tetracyclines. Several strains are listed in Table
: NH 13513.
The basic structure of the tetracyclines con-
eh sists of a naphthacene ring system. Figure 13.35
O shows the structures of several therapeutically
Rifamycin B RifamycinS important compounds. There are also semisyn-
thetic derivatives on the market as well as the
Figure 13.34 The rifamycins B, S and SV fermentatively produced tetracyclines.
 13.6 TETRACYCLINES AND ANTHRACYCLINES / 263
Table 13.13 Streptomycetes producing tetracycline
mydias. There is a marked cross-resistance be-
Species Products? tween different tetracyclines. After cephalospo-
S. alboflavus CTC, TC, OTC, actinomycin rin and penicillin, the tetracylcines are the most
S. antibioticus OTTE widely used antibiotics. Chlortetracycline and
S. aureofaciens CTESTE
S. aureus TC oxytetracycline are commonly used in human
S. californicus TC, actinomycin and veterinary medicine. In many countries they
S. cellulosae OTC, actinomycin are also widely used as nutritional supplements
S. feofaciens ne
S. flaveolus OTC, TC, actinomycin
in poultry and swine production and in some
S. flavus CTC, TC, OTC, actinomycin countries they are also used to preserve fish, meat
S. lusitanus ETS TE and poultry.
S. parvus OTC, TC, actinomycin
S. platensis OTC
Tetracyclines act as inhibitors of protein
S. rimosus OTC, TC, rimocidin synthesis. Their site of action is the 30S ribo-
S. sayamaensis NE CTE some, where binding of aminoacyl-tRNA to the
S. vendargensis OTC, vengacide
S. viridifaciens CTC Me
ribosomal A-site is inhibited.
»
CTC 7-chlortetracycline; TC tetracycline; OTC oxy-
tetracycline Biosynthesis and regulation Chlortetracycline
(Perlman, 1979) and oxytetracycline are the tetracyclines most
frequently produced by the streptomycetes, tet-
racycline itself usually being only a minor com-
HC js
ponent. However, S. aureofaciens mutants with a
R, R3 RoR, H ie
block in the chlorination reaction excrete tetra-
cycline as the major product. A simplified version
HOH li ie of chlortetracycline biosynthesis is illustrated in
Figure 13.36. The biosynthesis can be subdivided
Tetracyclines R, R, R3. R, | Production strain
=
into three sections:
Tetracycline H OH CH; H S. aureofaciens (in chloride-
free medium) or through e Glucose is converted into acetyl-CoA.
chemical modification of
chlortetracycline + Malonyl-CoA is produced in the transfor-
7-Chlortetracycline
(Aureomycin)
H OH CH, Cl | S. aureofaciens mation of acetyl-CoA by means of acetyl-
5-Oxytetracycline OH OH CH; H S. rimosus CoA-carboxylase. After transamination to
(Terramycin)
malonamoyl-CoA, which is bound to an en-
6-Demethyl-7-
chlortetracycline
H OH H Cl S. aureofaciens
(+ inhibitor) zyme complex (“anthracene synthase’’), this
(Declomycin)
6-Deoxy-5-hydroxy- OH FH CH, H | Semisynthetic compound condenses with 8 molecules of
tetracycline
(Doxycylcine) malonyl-CoA. The polyketide presumably
7-Dimethylamino-6- H H H N(CH ,),Semisynthetic
demethyltetracycline formed from this condensation has not been
(Minocyclin) \
6-Deoxy-6-demethy!-6- OH =CH, H Semisynthetic isolated. An alternative pathway to malony]l-
demethyl-6-methylene
5-hydroxytetracycline
(Methacycline)
CoA is via the production of oxalacetate cat-
alyzed by phosphoenol pyruvate carboxyl-
ase, oxalacetate being converted to malony]l-
Figure 13.35 Structure of clinically important tetracy-
clines CoA by oxidative decarboxylation. Cycliza-
tion into the tricyclic intermediate also takes
place on the enzyme anthracene synthase.
Tetracyclines are broad-spectrum antibiotics, e The step-by-step transformation of the as-:
which are effective against Gram-positive and sumed tricyclic intermediate, which has not
Gram-negative bacteria, as well as rickettsias, yet been isolated, into the variety of known
mycoplasmas, leptospiras, spirochetes, and chla- tetracyclines.
264 / CHAPTER 13 / ANTIBIOTICS

232 COS-E
Pyruvate —m Acetyl-CoA= Malonyl-CoA ean eS
COOH
Neale Glutamate

NAD H-SCoA 2-Keto-

CO glutarate OSTE CO>,H50

Phosphoencl- “=Ora H.C

vate acetate
fae ‘co NH,

ofcCoA

He ef COS-E COS-E
CO-CHy ee CONH, CONH,
3 H20

ElFeder:
~. + 000.
see
CH

Ho (Q)OH OH OH

IW)
NAD CH3

fee:

mer Om EFCOCK se © OH On

Cl Hy N(CH AR NoPE & OH N(CH ab

os ‘GeCONH, OL .SECONH,

Figure 13.36 Biosynthesis of chlortetracycline by S. aureofaciens. a, 6-methylpretetramide; b, 4-Hydroxy-6-methylpre-

tetramide; c, 6-Deoxytetramide green; d, 4-Oxyanhydrochlortetracycline; e, Anhydrochlortetracycline; f, Dehydrochlor-
tetracycline; g, Chlortetracycline; I, Acetyl-CoA carboxylase; II, Phosphoenolpyruvate carboxylase (From Vanek et al.,
1971; HoStalek et al., 1979)
 13.6 TETRACYCLINES AND ANTHRACYCLINES / 265
The scheme of chlortetracycline biosynthesis, Considering also the dearth of knowledge of bio-
still mainly hypothetical, has been deduced from synthesis and regulation in this pathway, it is
studies of mutants blocked in tetracycline bio- understandable that a rational approach to strain
synthesis and from cosynthesis studies with S. improvement has not been possible. So far, all
aureofaciens. There are 72 intermediate products; developments of higher-yielding strains have
27 substances have been characterized but 45 come from empirical mutagenesis studies.
have not yet been isolated and the chlorination Ultraviolet radiation, alone or in combination
reaction itself is not yet understood. with other mutagens, has brought about the
There is a clear correlation between tetracy- greatest number of higher-yielding strains using
cline production and carbohydrate catabolism. S. aureofaciens and S. rimosus. Table 13.14 shows
High-yielding strains have a lower glycolysis rate the effect of different mutagens on the variability
than strains with low tetracycline production. of chlortetracycline production, and Figure 13.37
The addition of 0.5-3 ug/ml benzylthiocyanate, shows the increase in chlortetracyline production
an inhibitor of sugar uptake, causes a 50% in- with each step in the course of strain develop-
crease in chlortetracycline production; simulta- ment. Strains with increased tetracycline pro-
neously, the activity of the pentose-phosphate duction have been isolated by selecting rever-
cycle increases. In the case of tetracycline bio- tants of mutants blocked in antibiotic synthesis
synthesis, phosphate regulation is well under- and from prototrophic revertants of auxotrophs.
stood. The addition of P; to the producing culture They may also be isolated from mutants showing
decreases the rate of tetracyline formation by in- increased resistance to their own product. The
hibition of ATC oxygenase. The rate of tetra- highest published yield for tetracycline is 25 g/
cycline synthesis is directly proportional to the liter.
activity of this enzyme, which is only detectable Hybridization has not resulted in strains with
in the culture after phosphate has been com- increased product formation, but in combination
pletely depleted. with mutation it has caused an increase in strain
variability. Protoplast fusion has been used to
Strain development Studies on the genetics of S. increase yields.
aureofaciens have shown that over 300 genes are A genetic map has been compiled for the wild
involved in the biosynthesis of chlortetracycline. type S. rimosus (ATCC 10970), as illustrated in

Table 13.14 Spontaneous and induced variability of chlortetracycline production in S. aureofaciens

Mutagen? Distribution of productivity Frequency of strains with improved
(% of control)? productivity (%)*
Total range Most frequent class
— 40-110 80-100 5:3
Ultraviolet radiation 0-130 90-110 17.4
X-rays 0-110 70- 90 7.1
y-Rays 0-110 0- 10 1.8
Nitrosoguanidine 0-120 40- 80 9.4
N-Mustard 0-110 80-100 5.8
2 Research was conducted with conidia at death rates >99%. Ultraviolet 50 sec; X-rays and y-rays 100 kR;
N-methyl-N’-nitro-N-nitrosoguanidine 1 mg/ml, 60 min, 0.02 M phosphate buffer, pH 6.7; N-mustard 0.01 M,
30 min, 0.15 M phosphate buffer, pH 8.0.
> Productivity of initial strain=100%.
© Percent of the tested isolates.
(HoStalek et al., 1974)
266 / CHAPTER 13 / ANTIBIOTICS

77 (600) Production methods The production of tetracy-

Ie " clines is carried out in stirred fermenters with
volumes up to 150,000 I. Figure 13.39 illustrates
536 (1000) 546 (1000 )
a typical process for chlortetracycline produc-
lear
tion. If glucose is used, continuous feeding is nec-
122 (1200) essary; Starch can be used as an additional carbon
| E—UV source. Because of phosphate repression, tetra-
cycline fermentations are run with phosphate
Cee (1400) Binion es
xX X— UV
limitation.
For maximal yield, an optimal oxygen supply
205 (1700) 134 (1700) is important, particularly in the first hours (6-12
pre h) of the fermentation. Aeration rates of 1 vvm
are customary; yield increases result when oxy-
16 (2200)
gen-enriched air is used.
bak
There are several routes to tetracycline:
542 (2260)
E ¢ By achemical process using chlortetracycline.
¢ By fermentation in chloride-free culture me-
542-2(2460)
dium, which can be made by pretreatment
X—E—UV
with ion exchangers.
e By adding chlorination inhibitors to the me-
2185 (3000) 2201(3500)

Figure 13.37 Genealogy of the chlortetracycline pro-

ducer S. aureofaciens . UV, ultraviolet radiation; PR, pho-
toreactivation; E, ethyleneimine treatment; X, X-rays. In
(), Chlortetracycline titers in ug/ml (From HoStalek et al.,
1974)

Figure 13.38. This map has some similarities to

that of S. coelicolor, the streptomycete best
known genetically. The genes for the first steps
of oxytetracycline biosynthesis are located be-
tween ribB and cysD in a narrow area of the S.
rimosus chromosome. The genes for the steps
from anhydrotetracycline to oxytetracycline are
located in a second cluster between proA and
adeA. Location of the genes in clusters facilitates
genetic engineering research, and recombinant
DNA methodology has been developed for .ox-
ytetracycline-producing strains of S. rimosus. Al-
though plasmids of S. rimosus have been detected
which function as fertility factors and which code
for self-resistance, the role of extrachromosomal Figure 13.38 Outer circle, genetic map of S. rimosus R 7
(ATCC 10970). Inner circle, genetic map of S. coelicolor.
DNA in the regulation of tetracycline biosyn- The correct orders for the sites outside the dotted lines
thesis is not yet clear. have not yet been determined (From Alacevit, 1976)
 13.7 NUCLEOSIDE ANTIBIOTICS / 267

Inoculum O OH O
Il ll
preservation (spores
on agar slant or in CHR
soil)
i Se
Agar plates
H3;CO O OH HO
2% Meat extract; 0.05%
Asparagine; 1% Glucose;
Spores as 0.5% K,HPO,; 1.3% Agar
inoculum

Shake flask 2% Corn steep liquor (50%

solids); 3% Sucrose; 0.5%
CaCO,

Same as for shake culture

5% Daunomycin R=H
Inoculum
19-24 hr Adriamycin R= OH
pH 5.2-6.2
Figure 13.40 Anthracyclines. Structure of adriamycin
1% Sucrose; 1% Corn steep and daunomycin
liquor; 0.2% (NH,),HPO,;
0.2% KH,PO,; 0.1% CaCO,;
0.025% MgSO, - 7 H20;
0.005% ZnSO, - 7 HO; intercalate into DNA and inhibit DNA-depen-
0.00033% CuSO, - 5 H,O; dent DNA polymerase in DNA replication.
0.00033% MnCl, - 4 H,O
pH 5.8-6.0 Daunomycin (daunorubicin) and adriamy-
cin (doxorubicin), produced by Streptomyces peu-
Purification from cetius, are used clinically in cancer treatment. The
the clear broth after
removal of the
structure of both compounds is diagramed in Fig-
mycelium ure 13.40. Daunomycin acts against certain forms
of leukemia; adriamycin, which has low toxicity,
is effective in treating various tumors. However,
Figure 13.39 Production chart for chlortetracycline with
S. aureofaciens (Perlman, 1979) these compounds have significant side effects
and research is under way, using mutasynthesis
and protoplast fusion as well as by the formation
dium, such as mercaptobenzothiazole, 2- of chemical derivatives, to produce compounds
thiouracil, or thiourea. Bromide inhibits chlo- with decreased problems.
rination in some cultures, but causes forma-
tion of 7-bromtetracycline in other strains. 13.7 NUCLEOSIDE ANTIBIOTICS
e With mutants which are blocked in the chlo-
About 200 antibiotics with nucleoside-like struc-
rination reaction.
tures are known. Because of the diversity of
structures, marked differences in biological ac-
tivity are seen. Aminoacyl nucleosides used as
Anthracyclines fungicide antibiotics in plant protection include
Anthracyclines are glycosides which consist of blasticidin S from Streptomyces griseochromog-
7,8,9,10-tetrahydro-5,12-naphthacenequinone as enes, mildiomycin, from Streptoverticillium ri-
an aglycone combined with 1-3 sugar residues. mofaciens, and the polyoxins from S. cacaoi var.
Anthracyclines are very toxic compounds. They asoensis. Polyoxins (Figure 13.41 A) inhibit the
268 / CHAPTER 13 / ANTIBIOTICS

HC, CH
0 N Chloramphenicol

Been
on SQ a.
Chloramphenicol (Figure 13.42) is produced by
Streptomyces venezuelae, S. phaeochromogenes var.
R,OC
chloromyceticus, S. omiyaensis, and other strep-
O=CHNCH HOCH 6
H2N=CH
tomycetes. Chloramphenicol is a broad-spectrum
HC-Rz antibiotic which acts on Gram-positive and
HO-cH
|
OH OH Bott Gram-negative bacteria, actinomycetes, rickett-
CH OCNH
ae :
re
Hed-chy—{)-0-cH
sias, and chlamydias. It causes significant side
| effects, particularly bone-marrow damage, but
RY R2 NH2
risk of toxicity can be reduced if therapy is con-
PolyoxinA |CH,0H CH N- OH
ducted carefully. Because of its potential toxicity,
CHs COOH
Polyoxin B |CH2OH HO OH chloramphenicol has not been as widely used as
Polyoxin E |COOH HO H would be expected from its antimicrobial spec-
A. Polyoxins B. Puromycin
trum and it is presently considered a reserve anti-
biotic. Nevertheless, chloramphenicol is indis-
Figure 13.41 Nucleoside antibiotics pensible in the treatment of persistent Salmonella
infections.
Chloramphenicol binds specifically to the
incorporation of N-acetyl-D-glucosamine into 50S subunit of 70S ribosomes and blocks the
the chitin moiety of fungal cell walls by com- peptidyl transferase reaction, causing a prema-
petitive inhibition of chitin synthase. ture chain break. In molecular biology, chlor-
The dapiramines as well as nikkomycin, amphenicol is used as a research tool to inhibit
which also act as chitinase inhibitors, are the translation process in procaryotes without af-
undergoing field tests as possible herbicides. Sev- fecting the synthesis of nucleic acids.
eral compounds with cytostatic or antiviral ac- The pathway for chloramphenicol biosyn-
tivity are under development; for example, ne- thesis is via the aromatic pathway from chorismic
planosin A from cultures of Ampullariella acid. However, chloramphenicol has been pro-
regularis. duced by chemical synthesis since about 1950
Puromycin (Figure 13.39 B) from S. alboniger and even improved fermentation processes have
has found wide use as a tool in studying ribo- not succeeded in replacing this chemical process.
somal function in protein biosynthesis but has
no clinical significance. Puromycin resembles the
aminoacyl end of transfer RNA. In both procar- Dichloracetylamino moiety
yotes and eucaryotes it binds to the A-site of the
ribosome and is transferred to the growing poly- |
li
peptide chain, thus causing a break in the po- | NH—C —CHCl,
lymerization process.

ON CHE gh SCR OM
|
13.8 AROMATIC ANTIBIOTICS OH
This is a heterogenous group of antibiotics with p-Nitrophenyl- Propandiol moiety
aromatic rings in the molecule. The biosynthesis moiety

of individual compounds differs considerably.

Three antibiotics of this group are produced com-
mercially. Figure 13.42 Chloramphenicol
 13.8 AROMATIC ANTIBIOTICS / 269

Griseofulvin source. The addition of potassium chloride (0.1-

0.3%) stimulates production. The fermentation
Griseofulvin, a benzofuran derivative (Figure take place at 25°C for 7-9 days within an optimal
13.43), is produced by Penicillium griseofulvum, pH range between 6.8-7.2. High aeration (1 vvm
P. patulum, and other species of Penicillium. Gris- O,) is required when media with high carbon or
eofulvin is a fungistatic antibiotic but acts only nitrogen concentrations are used. The entire cul-
on fungi with chitinous cell walls. In humans, ture must be processed for recovery, since gri-
griseofulvin is administered orally for the treat- seofulvin is only partially mycelium-bound.
ment of fungal skin infections (dermatomycoses). Uj

Although the antibiotic has been used in plant

pathology in the treatment of leaf diseases due Novobiocin
to Botrytis, Alternaria solani, and species of mil- Novobiocin is a coumarin glycoside (Figure
dew-causing fungi, it is currently not in wide use 13.44), which can be isolated from culture fil-
here because of the high cost (due to the high trates of Streptomyces spheroides, S. niveus, S. gris-
production costs). Griseofulvin affects the mor- eus, or S. griseoflavus. Novobiocin is effective
phogenesis of many fungi and in concentrations against Gram-positive bacteria, particularly
of 10-1000 ppm causes a characteristic ”curling” staphylococci, against Gram-negative meningo-
effect of the hyphae. The exact mechanism of cocci and gonococci, and also against Haemo-
action has not yet been determined, but mitosis philus and some Proteus strains. Novobiocin
and chitin biosynthesis are presumed to be af- shows no cross resistance with other antibiotics,
fected. but organisms do develop rapid resistance, a
The biosynthesis of griseofulvin occurs on a marked disadvantage for its medical use. No-
multi-enzyme complex. The antibiotic is pro- vobicin acts as a specific inhibitor of DNA gyrase
duced from acetate, 6 molecules of malonate, and during DNA replication.
2 methyl groups derived from methionine. An The biosynthesis of the coumarin moiety is
intermediate stage in the biosynthetic process is via aromatic metabolism. L-Tyrosine is incor-
a 14-member polyketide, from which the anti- porated into ring A and B; ring C is derived from
biotic is produced via cyclization, chlorination, glucose. The fermentation is carried out in a me-
and methylation reactions. dium with glucose or sucrose as a carbon source.
The chemical synthesis of griseofulvin has Organic acids, such fumaric acid, can be added,
been achieved, but production by fermentation and peptone and meat extract are favorable ni-
with P. patulum is more cost-effective. Griseo- trogen sources. p-Aminosalicylic acid can be
fulvin production is carried out as an aerobic sub- used as a precursor and addition of tyrosine and
merged process in a glucose-rich medium. In phenylalanine stimulate the formation of the
some processes, glucose feeding is used. Starch, antibiotic. Fermentations take place over about
lactose, or sucrose can also be used as carbon 90 h at 25-28°C and at a slightly alkaline pH
sources and usually NaNO, is used as a nitrogen range, with aeration rates of up to 1.5 vvm.

H5CO 0 OCH, OH
CH, [| NH— 0¢
HCO re TC bes
ING ae SÆR 0 00 OH ~CH,
QO OH CH;
OC-NH,

Figure 13.43 Griseofulvin Figure 13.44 Novobiocin

270 / CHAPTER 13 / ANTIBIOTICS

13.9 OTHER COMMERCIALLY

PRODUCED ANTIBIOTICS
Some antibiotics are discussed here which are
used in therapy or have other uses. Their struc-
HO CH20H
tural affiliation with certain classes of antibiotics
was given in Table 13.2.

Fusidic acid

Among the steroid antibiotics, only fusidic acid

(Figure 13.45) is therapeutically important. This
compound, isolated from culture filtrates of Fu- O Citrulline
sidium coccineum, inhibits the ribosomal trans- N
location process during protein synthesis in both
procaryotes and eucaryotes. Fusidic acid finds
special use for the treatment of staphylococcal IQ Methionine
Pyruvate
infections, especially wound infections and those |

of the skin.

Polyether antibiotics
Mitomycin A
Due to the numerous tetrahydropyran and tetra- Mitomycin B
furan moieties in these antibiotics, they are called Mitomycin C
polyethers. Over 150 different compounds are
known. Their biosynthesis is derived from a pol-
yketide derivative which is produced on a multi- Figure 13.47 Structure of mitomycins A-C and biochem-
ical precursors of mitomycin C
enzyme complex. Monensin (Figure 13.46),
commercially the most important compound of
this group, consists of 5 acetate, 4 propionate and Antibiotics in this group which are commer-
3 butyrate units. Because of their ability to trans- cially prepared include monensin, produced by
port mono- or divalent cations through biological Streptomyces cinnamonensis, lasalocide, pro-
or artificial membranes, these antibiotics are clas- duced by S. lasaliensis, and salinomycin, pro-
sified with the ionophores. duced by S. albus. Salinomycin yields of 60 g/1
are attained on media containing a fatty acid.
Polyethers are active against coccidia (protozoa),
as well as Gram-positive bacteria, mycobacteria,
and fungi. They are added to poultry feed (about
100 ppm) to prevent coccidioses. Monensin,
which was first introduced in 1971, has about
80% of the market; current annual gross world
sales are estimated at $100 million.

Mitomycins
The mitomycins, produced by Streptomyces caes-
Figure 13.45 Fusidic acid pitosus, are benzoquinones; Figure 13.47 gives
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Swartz, R.W. 1979. The use of economic analysis of pen- Zimmer, T.L., Ø. Froyshov, and S.G. Laland. 1979. Pep-
icillin G manufacturing costs in establishing priorities tide antibiotics, pp. 123-150. In: Rose, A.H. (ed.), Eco-
for fermentation process improvement. Ann. Rep. nomic microbiology, vol. 3. Secondary products of me-
Ferm. Proc. 3: 75-110. tabolism. Academic Press, London.
 Ergot
alkaloids

tant alkaloid producers are listed in Table 14.1.

14.1 OCCURRENCE AND
Ergot alkaloids have also been found in higher
SIGNIFICANCE
plants, but they seem to be limited to several
Alkaloids, some of which have therapeutic ap- species of the plant family Convolvulaceae.
plications, are generally derived from plants, but Some pharmacological properties of ergot
the ergot alkaloids are produced by fungi. Ergot alkaloids include the stimulation of the sympa-
alkaloids were first obtained from the sclerotium thetic nervous system, an effect on the smooth
of the parasitic ascomycete Claviceps purpurea, muscles (uterus, artery walls), and antagonism to
which develops on rye and other grasses. The adrenalin and serotonin. Chronic ergot poisoning
term ergot is used to refer both to the fungus (ergotism), which used to occur frequently due
developing in the rye plant and to the alkaloids to consumption of bread made from grain con-
which are produced by the fungus. Two related taining ergot alkaloid, results in cramps, vomit-
groups of ergot alkaloids are known, those based ing, and gangrene, and can be fatal in severe
on lysergic acid and those based on clavin (see cases, In the Middle Ages, ergotism was common
later). Presently there are over 40 known ergot in northern Europe, where it was called St. An-
alkaloids produced by various Claviceps strains. thony’s Fire, but it has been eliminated by mod-
Although tests have been conducted on over ern methods of grain cleaning.
1000 different species of various filamentous Some lysergic acid alkaloids are used thera-
fungi, lysergic acid alkaloids have been found peutically in obstetrics, including ergotamine, er-
only in species of the genus Claviceps. However, gobasine, and the semisynthetic methylergoba-
clavine alkaloids were found in species of the sine, all of which act to stimulate labor and cause
genera Aspergillus, Penicillium, and Rhizobium, as the uterus to contract. They are also employed
well as in Claviceps. Several of the most impor- during the period after birth to contract the post-

274
 14.3 STRUCTURE / 275
Table 14.1 Several producers of ergot alkaloids and the alkaloids produced by them
Organism Plant host Alkaloid content (%) Alkaloids produced
(main components)
Claviceps purpurea Rye, wheat, barley and others 0:1 -0:5 Ergotoxine group:
Ergocornine
Ergocryptine
Ergocristine
Ergotamine group:
Ergotamine
Ergosine
C. fusiformis Pennisetum typhoides 0.3 Ergometrine
Agroclavine
Elymoclavine
Chanoclavine
C. gigantea Zea mais 0.03 Festuclavine
Pyroclavine
Dihydroelymoclavine
Chanoclavine

C. paspali Paspalum spp. 0.003 D-Lysergic acid

a-Hydroxyethyl-
lysergamide
(Mantle, 1975)

partum uterus. Hydrogenation of the A®!°-double tion of conidia stops, the filamentous mycelium
bond in the D-ring reduces the contracting action is replaced with a plectenchymous, nonsporu-
on the smooth muscles; dihydroergotamine is lating tissue, and the sclerotium appears. It is
thus used to treat migraine headaches. Also, a during this latter developmental phase that al-
mixture of dihydro compounds from ergocristine, kaloids are produced, usually as part of a mixture
ergocryptine, and ergocornine is used for treat- of different substances. The sclerotia survive un-
ment of disturbances of the peripheral and cen- til the host plant blooms again; hyphae grow out
tral circulation system. Finally, a few naturally from the sclerotia and long-stalked stromata
occurring alkaloids, particularly the semisyn- grow on the ends of the hyphae. The stromata
thetic compound lysergic acid diethylamide contain perithecia with asci; the products of this
(LSD), are very effective hallucinogens. sexual stage are 8 needle-shaped ascospores per
ascus. The ascospores germinate on the plant and
14.2 DEVELOPMENTAL CYCLE OF the cycle begins again, as illustrated in Figure
CLAVICEPS 14.1.

The Claviceps fungus lives as a parasite on

14.3 STRUCTURE
grasses. During flowering of the host plant, the
infection cycle begins. Hyphae from germinating The active ingredients of ergot were character-
ascospores or conidia grow into the plant ovary ized in 1935 as derivatives of lysergic acid and
and within a week the ovary tissue is penetrated the structural formulas were confirmed through
by a filamentous mycelium (sphacelia), which synthesis in 1954. Ergot alkaloids are classified
produces mononuclear conidia for asexual repro- among the indole alkaloids and are derived
duction. No alkaloids are produced during this from the tetracyclic ergoline ring system (Figure
stage. About two weeks after infection, produc- 14.2).
276 / CHAPTER 14 / ERGOT ALKALOIDS

Two main classes of ergot alkaloids have

been recognized. The clavine alkaloids or clav-
ines have ergoline as a basic structure, but con-
tain no peptide components. Some substances of
this group are intermediates in lysergic acid bio-
synthesis (see Figure 14.5). Various Claviceps
species produce clavines, particularly in culture
away from the plant, but traces of different clav-
ines mixed with lysergic acid alkaloids have also
been found in parasitic growth. Despite their
pharmacological effect, clavines have not been
used therapeutically.
The other class of alkaloids are the lysergic
acid alkaloids or ”Ergo” alkaloids. These are the
classical alkaloids which are isolated from scler-
otia. In these compounds, D-lysergic acid (or its
stereoisomer D-isolysergic acid) is linked with a
tricyclic peptide or an amino alcohol by an amide
bond. The best known member of the latter
group is ergometrin, which has 2-aminopro-
panol-(1) as a side chain. The structure of this
Figure 14.1 Development cycle of Claviceps purpurea. A, compound, as well as the structures of several
ascospore (1100 um); B, infection of grass flower; C,
other of the simpler lysergic acid alkaloids, are
growth of filamentous mycelium in the ovary; D, asexual
spores excreted with honey dew cause additional infec- shown in Figure 14.3.
tions but no alkaloid formation; E, filamentous mycelium Two groups of peptide alkaloids have been
is replaced by plectenchymatic tissue; alkaloid formation
is initiated; F, the sclerotium ripens within 2 months; G,
isolated, the classical ergopeptins and the more
sexual stage with perithecium formation (From Mantle, recently discovered ergopeptams. The ergopep-
1975) tins consist of a lysergic acid molecule connected
by a peptide bond to a tricyclic peptide which
consists of an a-hydroxy-y-amino acid derived
from L-valine or L-alanine, with another amino
acid and L-proline (Table 14.2; Figure 14.4). The
8 first ergopeptams were only isolated in 1982.
9 They are ergocristine, a-ergocryptine, and er-
10 D NH
gocornine. In these compounds, the tripeptide
moiety, an acyclic lactam, is composed of two
amino acids (which can be identical to the amino
acids of the ergopeptins) and D-proline.

HN B | Lysergic acid and its derivatives epimerize

easily at C-8, leading to the formation of the
pharmacologically ineffective D-isolysergic acid
Ergoline compounds. They are distinguished by the end-
ing ”-inine”, so that ergocristinine is the C-8 epi-
Figure 14.2 Structure of the ergoline ring system mer of ergocristine.
 14.4 BIOSYNTHESIS / 277

R Le, R 2

CH

H R;

Name R Name R, Rø R,

Ergotamine H H Chita
Ergometrine nite
(Ergobasine) | Ergosine H H CH,CH(CH3)-
CH.OH
Ergocristine CHER ECH cH,—€ )
a -Hydroxyethyl- CH.
| a -Ergocryptine | CH, |CH, |CH,CHICH,),
lysergamide [SN CH=-0H
B -Ergocryptine | CH, |CH, |CH(CH,)CH,CH,
Lysergic acid = OH
Ergocornine CH, |.CH;”| CHICHE),

om)
8,9 å 5
A -Lysergic acid | —OH
Ergostine H Crs

Figure 14.3 Structure of several simply constructed ly-

sergic acid alkaloids (x = double bond in the A®? position)
Figure 14.4 Naturally occurring ergot alkaloids of the
peptide type (ergopeptins)

Table 14.2 Amino acid sequence of the tripeptide

moiety in ergotoxine and ergotamine alkaloids 14.4 BIOSYNTHESIS
(ergopeptins)
Alkaloid synthesis occurs upon the endoplasmic
Alkaloid Amino acid sequence of the tripeptide reticulum in Claviceps when it is grown in sub-
Ergotoxine- merged culture. About 95% of the clavines and
alkaloids: the simple lysergic acid derivatives are excreted
Ergocristine —L-Valine—L-Phenylalanine—L-Proline
Ergocornine —L-Valine—L-Valine—L-Proline into the medium; the peptide alkaloids remain
a-Ergo- —L-Valine—L-Leucine—L-Proline mostly mycelium-bound.
cryptine
The formation of the ergoline ring proceeds
B-Ergo- —L-Valine—L-Isoleucine—L-Proline
cryptine from mevalonic acid and tryptophan, as outlined
in Figure 14.5. In a series of reactions analogous
Ergotamine-
alkaloids: to those involved in isoprenoid synthesis, mev-
Ergotamine —L-Alanine—L-Phenylalanine—L-Proline alonic acid is transformed via mevalonic acid py-
Ergosine —L-Alanine—L-Leucine—L-Proline
rophosphate and isopentenyl pyrophosphate
278 / CHAPTER 14 / ERGOT ALKALOIDS

COOH CH, CH. NH,

| CH-COOH
HO CH, HG C=CH, C=CH;
= \ 4 |
@ HC år :
C
PAN mp Ge EN xe i
CH, CH, H CH,0®® CH,0 GG a \
CH,OH
I II III IV
CH, 7
GREN GEL
N-CH, NH,
—=_
COOH

HN |
V
Ergotoxine CH;
chy Færre page CO—NH-CH XI]

ct
CO-NH— CH CO-NH-CH
fell
CH; CH-COOH ba COOH ee CH,OH
NH,
IX X XI

Figure 14.5 Biosynthesis of the ergoline ring system. I, Mevalonate; II, Isopentenylpyrophosphate; III, Dimethylallyl-
pyrophosphate; IV, Tela ee V, 4-Dimethylallyltryptophan; VI, Chanoclavin-I; VII, Agroclavine; VIII, Elymoclavine;
IX, A8®-Lysergic acid;X, Lysergic acid; XI, Lysergylalanine; XII, a-Hydroxyethyllysergamide; XIII, Ergometrine

into dimethylallyl pyrophosphate. The first re- lysergic acid, which in contrast to lysergic acid
action specifically for alkaloid synthesis is the occurs freely in relatively high concentrations in
linkage of dimethylallyl pyrophosphate with L- the medium.
tryptophan (in position C-4), tryptophan being Biosynthesis of tripeptide moieties of the pep-
converted into 4-dimethylallyl tryptophan. In the tide alkaloids is not fully understood. Attempts
next intermediate step, chanoclavin-I, a clavine to incorporate ‘C-labeled precursors showed
with an open D-ring, is produced. The methyl that lysergic acid and various amino acids are
group on the nitrogen in position 6 is derived incorporated as single compounds. From results
from methionine. The ring closes after oxidation of experiments with inhibitors, ergopeptin for-
to chanoclavin-I-aldehyde and the tetracyclic mation is postulated to take place on a multi-
compound agroclavine is formed. Hydroxylation enzyme complex, analogous to the biosynthesis
of the methyl group of agroclavine to form ely- of peptide antibiotics (see Section 13.3). The pro-
moclavine is carried out by a peroxidase or ox- posed biosynthetic pathway, which begins with
ygenase in the presence of cytochrome P-450. the linkage of L-proline to the enzyme, is illus-
The further transformation from elymoclavine trated in Figure 14.6. It has been possible to ob-
into derivatives of lysergic acid occurs via A®?- tain incorporation of the appropriate amino acids
 14.5 PRODUCTION OF ERGOT ALKALOIDS / 279

L-Proline L-Valine

E—SH re AN
E-S= € == Hø
@

Re; t==0
Ze
HO CH-CH;
CH;
L-Valine

ATP

D-Lysergic acid

ps)

agfi 0
Wea UR NO en
=0 HN em DE ie O
BER WwW
ZS H aa CH, 5 “CH—CH;
H ‘CH-
| CH, CH, H = = Øg CH;
CH, CH,

E-SH :
Ergocornine
H.C. ‘A2CH. HC ‘oh
CH,
Oy sl) J O ake J
SNe C N Lys—N---C~ ~SC N
oo ca eg ee Ho pees)
Figure 14.6 Hypothetical scheme fay 50 Saar eine NowC=O
for the formation of the peptide side ne S \CH-CH H% ~~“CH—CH
chain of ergopeptines on a multi-en- | 3 :
zyme complex (From Floss, 1976) CH, CH;

into the respective ergopeptin in a cell-free sys- plants, by microbial fermentation, either in sur-
tem in ergosine or ergotamine-producing strains face culture or with immobilized cells. Culture
of Claviceps purpurea, and experiments are un- on the host plant or in submerged culture are the
derway to characterize the ‘‘cyclol-synthetase processes most used commercially.
complex” which is involved. It is likely that er-
gopeptams are formed by irreversible epimeri- Chemical synthesis
zation as intermediate products before ring clo-
Total chemical synthesis of ergot alkaloids is pos-
sure.
sible, but currently this is not cost-effective. Ly-
sergic acid which is produced fermentatively can
14.5 PRODUCTION OF ERGOT be chemically transformed into the desired al-
ALKALOIDS kaloids. Ergoline derivatives used medically are
produced commercially using this method, with
Annual world production of peptide alkaloids
the exception of peptide alkaloids.
was 4000 kg in 1976, and lysergic acid produc-
tion was 12,000 kg.
Obtaining alkaloids from sclerotia
There are a number of methods of obtaining
ergot alkaloids: by chemical synthesis, by cul- In 1976, more than 95% of the peptide alkaloids
ture of Claviceps strains on the respective host were prepared by extraction from sclerotia,
280 / CHAPTER 14 / ERGOT ALKALOIDS

chiefly in Switzerland and eastern European Homogenized

countries. In this process, rye flowers are me- mycelium of a 5- to
chanically inoculated with conidia. The fungus 10-day agar slant
culture
grows in the ovary tissue and forms conidia
which then serve as the inoculum for an exten-
sive plant infection. About 200—500 kg of scler- Growth in shake Conidium suspension
flask
otia can be harvested from 1 hectare (2.47 acres)
of rye. Disadvantages of this process are that the
harvest is only possible once a year and the ex- Preculture Medium (g/1): Mannitol 40;
Succinate 10; KH,PO, 1.0;
tent of infection is extremely dependent on the MgSO, + H,O 0.8; Ca(NOs),
weather. Since 1978, this production method has - 4H,O 0.3; NaNO, 0.1;
been replaced in Switzerland with the fermen- FeSO, - 7 H,O 0.05; MnSO,
- H,O 0.01; ZnSO, - 7 H,O
tation method. 0.005; CuSO, - 5 H,O 0.005.
pH adjustment with NH,OH
to 5.2
Production by fermentation
Three species of Claviceps are currently used in Production Medium (g/l): Mannitol 50;
the production of alkaloids by fermentation: culture Succinate 30; KH,PO, 1.0;
MgSO, - 7 H,0 0.8;
Ca(NO;), - 4 H,O 0.03;
Claviceps paspali a-Hydroxyethyl lysergamide,
NaNO, 0.1; FeSO, - 7 H,O
A82-Lysergic acid, ergometrine
0.05; MnSO, - H,O 0.01;
C. fusiformis Clavines (chiefly agroclavine)
ZnSQ, - 7 H,O 0.005;
C. purpurea Ergotamine, ergosine, ergocor-
nine, ergocristine, ergocryptines
CuSO, - 5 H,O 0.005.
pH adjustment with NH,OH
to 5.1-5.4
Research with C. paspali at the Italian pharma- Temperature: 24-25°C
ceutical company Farmitalia led to the initial Fermentation time: 8-12 days
breakthrough in fermentative production of ergot
Figure 14.7 Flow scheme for the production of a-hy-
alkaloids. Whereas the initial isolate grown in droxyethyl lysergamide with C. paspali (From Kelleher,
submerged culture produced about 20 mg alka- 1970)
loid/1, strain development and culture medium
optimization resulted in a commercial process
which had alkaloid titers of 5 g/l, with a-hy- Processes for the production of therapeuti-
droxyethyl lysergic amide as the main product. cally important peptide alkaloids have also been
The stages of the fermentation process are developed. Table 14.3 lists commercially pro-
outlined in Figure 14.7. After hydrolytic splitting, duced compounds.
D-lysergic acid can be used in the production of Alkaloid composition is affected by the strain
semisynthetic alkaloids. Chemical conversion to used and the culture medium, as shown in Table
the peptide alkaloids has also been accom- 14.4. In C. purpurea the relative proportions of
plished. ergocornine and ergocryptine can be altered from
A second simplified process for the produc- 2:1 to the more desirable 1:1 by addition of L-
tion of lysergic acid has been developed by the valine to the fermentation medium. Good alka-
Swiss company, Sandoz. An isolate of C. paspali loid formation seems to be linked to the ability
produces high yields of A*?-lysergic acid which to metabolize high sucrose and citrate concen-
can be rearranged by isomerization into D-ly- trations, but at the same time, the medium must
sergic acid (with the double bond in the A®?° po- not contain any phosphate. Synthesis proceeds
sition). Production of the alkaloid agroclavine is in parallel with lipid and sterol biosynthesis. The
possible with C. fusiformis, which in semicontin- progress of a typical fermentation is illustrated
uous culture can achieve yields of 6g/. in Figure 14.8 for C. purpurea.
 14.6 REGULATION OF ALKALOID PRODUCTION IN CULTURES / 281
Table 14.3 Production of ergot alkaloids by
fermentation tions. The advantage of this process is that a
higher proportion of the more desirable ergot-
Alkaloid Manufacturers
2 3
amine and ergotoxine alkaloids are formed.
Ergocornine
Ergocristine Semicontinuous transformation with
Ergocryptine ++4]- +++)" immobilized mycelium
Ergotamine
Ergometrine +
+++ A process has been developed for semicontin-
Lysergic acid ++++
+
A®°-Lysergic acid $ ‘ aw uous alkaloid production using immobilized my-
celium of Claviceps purpurea and C. fusiformis. In
Manufacturers: 1. Biochemie GmbH, Kundl (Austria); 2.
Farmitalia S.p.A. (Italy); 3. Gedeon Richter (Hungary); C. purpurea CBS 164.59, which forms ergometrin
4. Sandoz-Wander AG (Switzerland). and a mixture of chanoclavin, agroclavin and ely-
(Rehacek, 1980)
moclavin, the best results have been obtained by
immobilization in 4% calcium alginate gel. At
concentrations of alginate up to 8%, the overall
Scale-up has been a problem, since the pro-
duction strains are sensitive to the shearing ac- yield of alkaloids was increased by 35%, but be-
tion of the impeller but at the same time exhibit cause of the reduced O, diffusion into the algin-
a high oxygen requirement. Maintenance of ste- ate beads, the main product was agroclavin. To
rility is also difficult for long fermentation pe- prevent contamination problems during the long
riods. Very slight alterations in the composition incubation period with the immobilized my-
of the medium or the addition of antifoam agents celium (200-400 days), an antibiotic such as
usually cause lower yields. In addition, the de- chloramphenicol was added.
generation of high-yielding strains frequently oc-
curs, so that a careful strain maintenance pro-
gram is necessary. 14.6 REGULATION OF ALKALOID
PRODUCTION IN CULTURES
To produce alkaloids, the medium must always
Surface culture
contain an organic acid of the TCA cycle or a
A process has been described for the large-scale related compound as well as a carbohydrate.
production of ergot alkaloids using surface cul- Mannitol and succinate serve for the production
tures of C. purpurea grown under sterile condi- of lysergic acid, and sucrose and citrate for the

Table 14.4 Ergot alkaloid production by various strains of C. purpurea

Characteristics 275 Fl FI 32/17 FI 43/14 FI S 40
Alkaloid produced Ergotamine Ergocryptine Ergocornine Ergocristine
Ergotamine Ergosine
Alkaloid yield (mg/1) 1150 1800 1000-1100 1000-1100
Culture medium 1253 18252 75252 Bok
Sucrose consumed (g/1) 160 78 145 100
Citric acid consumed (g/1) 14 15 6
Lipid production (g/1) 14 24 47 18.5
Sterol production (mg/1) 316 444 393 510
Fermentation time (days) 14 16 12-14 8-10
2 Medium T 25 (g/l): Sucrose 300; citric acid 15; KH,PO, 0.5; MgSO,°7 H,O 0.5; KCl 0.12; FeSO,°7 H,O 0.007; ;
ZnSO,°7 H,O 0.006; yeast extract 0.1. Tap water; pH adjusted to 5.2 with NH,OH. Sterilization at 120°C, 20 min.
> Medium TS (g/l): Sucrose 100; asparagine 10; KH,PO, 0.5; MgSO,°7 H,O 0.3; FeSO,°7 H,O 0.007; ZnSO,°7 H,0
0.006; yeast extract 0.1. Distilled H,O; pH adjusted to 5.2 with NaOH. Sterilization at 110°C, 20 min.
(Amici et al., 1969)
282 / CHAPTER 14 / ERGOT ALKALOIDS

£
SS
= a
Dirs= SES: fo)
Once =
~~! ‘= sø

SES EG
ep =e v
= tm
aa} c E oe
o rote te =
440 ire ‘i en D £
i)
KS, E ha onal) e O
og | ts
oO)
CS ae S
E
400 x e x FA Oo ra mn?

| <x == Ps = Oo 2
wn ! & za ew © es,
g
360 4 1800 42 ! La OO xe) a o I

eam ee ese
1

:
g tem = oO)
=
foul
320 1600 / ae ieee rh.
60 67 an S
/1 @ 240 - 15 +30 + 60
280 4 1400 ee ;
al baited ETS Te DOO) F 125 825 3
240 4 1200 50 a ] aes Mates /
nope
=i 1000 4 40 i

160 4 800
30 3 |

120 4 600
(mg/ml)
weight
ey
20
80 400

40 200

OSSE: AAG AAEG 1081214 6

Fermenation time (days)

Figure 14.8 Fermentation kinetics of the ergocryptine/ergotamine producer C. purpurea FI 32/17 in medium T 25
(composition in Table 14.4)

production of ergotamine or ergocristine. Both tabolism appear, such as 4-dimethylallyl tryp-

carbon sources are metabolized simultaneously tophan synthase, which is induced by trypto-
without any evidence of a diauxy effect, although phan or trytophan analogs (such as 5-
glucose itself represses alkaloid production. methyltryptophan). The phosphate inhibition
Ergot alkaloid production exhibits a typical mentioned above can be counteracted by the ad-
phosphate regulation, as shown in Figure 14.9. dition of tryptophan. During the transition from
In the trophophase (3-4 days), after free phos- the trophophase to the idiophase, another key
phate has been used up growth ceases and the enzyme of secondary metabolism, chanoclavin-
culture enters the idiophase. Tryptophan, which I-cyclase, appears.
induces alkaloid synthesis and simultaneously Measurements of the enzyme activities of in-
serves as a precursor, accumulates. At the same termediary metabolism have indicated the pre-
time, the inducible enzymes of secondary me- dominant role of the TCA cycle at the beginning
 14.7 STRAIN DEVELOPMENT / 283

phase showed there was an increase in aspara-

; : ginase activity at the same time that an accu-
mulation of ammonium ions occurred. The ini-
tiation of alkaloid formation is coupled with a
significant decrease in the intracellular ammo-
nium concentration brought about by ammo-
(Ce nium assimilation via glutamine synthetase.
Since glutamine is the amino group donor, this
lead to an increase in the intracellular concen-
10 e STER
tration of tryptophan, an ergolin precursor.
production
Alkaloid
weight)
dry
(mg/g e fæ DYSTER
4 Se
FER
ORPO gg gg
0 Zon rh BYSREE NOs) BAA SAGT 16 200-5 -22 14.7 STRAIN DEVELOPMENT
Fermentation time (days)
In nature the production of ergot alkaloids is
Figure 14.9 Clavine formation with Claviceps SD 58 after linked exclusively to the sexual phase of the de-
a single addition of phosphate (concentration in the me- velopmental cycle, which does not occur in vitro.
dium: 1.1 g/l KH,PO,) at different times: e — e control; In submerged culture, the alkaloid-producing
O — 0 addition after 3 days; a — a addition after 5 days;
4 — 4 addition after 7 days; & — m addition after nine mycelium has morphological similarities with the
days; and 0 — 0 addition after 11 days (From Robbers et hyphae in sclerotia. These thick-walled, isodi-
al., 1978) ametric cells have a high fatty acid content. The
composition of the culture medium affects the
of the submerged fermentation process. In con- differentiation into sclerotic cell types. Increasing
trast, the glyoxylate cycle prevails during the al- the sucrose concentration from 10 to 30% or add-
kaloid production phase. Acetyl-CoA can be pro- ing citrate, (NH,),SO,, or high Ca?* concentra-
duced via an anaplerotic sequence (glycerate tions promotes the formation of the sclerotial cell
pathway) of the dicarboxylic acid cycle. The ac- types. Most alkaloid-producing strains are hom-
tivity of phosphofructokinase, a key enzyme of ocaryotic. In several strains that produce pri-
glycolysis, is reduced. The changeover from the marily peptide alkaloids, heterocaryosis leads to
TCA to the glyoxylate cycle is stimulated by the an increase in product formation. Plasmids have
addition of citrate, succinate, or malate. More- been demonstrated so far only in wild type
over, excess citrate causes inhibition of citrate strains. These plasmids are linear genetic ele-
synthase, making acetyl-CoA available for con- ments (5.6—6.3 kb) which are present in free form
version to mevalonic acid, a key precursor of the in the mitochondria.
ergoline ring system. Increased amounts of ace- A frequently occurring problem in strain
tyl-CoA flow to fatty acid biosynthesis during preservation is the degeneration of high-yield-
activation of acetyl-CoA carboxylase, thus ex- ing strains. The proportion of unproductive var-
plaining the parallelism of lipid and alkaloid pro- iants after a single transfer was 3% in an ergo-
duction. Since the enzymes of the pentose phos- toxine-producing C. purpurea strain, but after 7
phate cycle are derepressed, a sufficient quantity passages it was 78%. Thus, strains must be care-
of NADPH, is produced, which is necessary for fully maintained in storage (mycelium fragments
lipid synthesis. or conidia in nutrient solution containing 10-
In submerged culture, synthesis of the alka- 20% glycerol can be stored in liquid nitrogen).
loids is increased considerably when asparagine Occasional reisolation of high-yielding variants
is used as a nitrogen source instead of ammo- may also be necessary in order to guarantee al-
nium salts. Measurements during the growth kaloid production.
284 / CHAPTER 14 / ERGOT ALKALOIDS

Yield increases have been achieved in genetic can be anticipated that these new genetic tech-
research primarily by use of mutation and selec- niques may lead not only to further increases in
tion. Conidia, mycelium fragments, or proto- yield but to the discovery of new structural var-
plasts have been treated with mutagens, such as iants of the ergot alkaloids.
ethyl methanesulfonate, nitrosoguanidine, ni-
trous acid, ethylenimine, or ultraviolet radiation.
REFERENCES
Such studies have led in every case to increased
yields: for example, a 160-fold increase in an er- Amici, A.M., A. Minghetti, T. Scotti, C. Spalla, and L.
gocristine producer. Mutagenesis has resulted Tognoli. 1969. Production of peptide ergot alkaloids in
submerged culture by three isolates of Claviceps pur-
primarily in changes in the amount of alkaloids purea. Appl. Microbiol. 18: 464-468.
produced rather than in the proportion of the Arcamone, F., E.B. Chain, A. Ferretti, A. Minghetti, P.
various products. However, in C. purpurea shifts Pennella, A. Tonolo, and L. Vero. 1961. Production of
a new lysergic acid derivative in submerged culture by
in the proportions of ergotamine to ergotoxine, a strain of Claviceps paspali. Proc. Roy. Soc. (London)
ergocornine/ergocryptine to ergocrystine, or er- Ser. B 155: 26-54.
Desai, J.D., H.C. Patel, and A.J. Desai. 1986. Alkaloid pro-
gotamine to ergosine. Due to the absence of the
duction during the cultivation with shaking of Clavi-
sexual phase in vitro, for a long time there was ceps sp.: Effects of asparagine. J. Ferment. Technol.
no means of performing genetic analysis or using 64:339-342.
genetic recombination for strain development, Floss, H.G. 1976. Biosynthesis of ergot alkaloids and re-
lated compounds. Tetrahedron 32: 873-912.
but protoplast fusion has overcome this diffi- Hofmann, A. 1964. Die Mutterkornalkaloide (The ergot
culty. Fusion of an ergocristine producer with an alkaloids). Enke Verlag, Stuttgart.
Hoffmann, A. and H. Tscherter. 1960. Isolierung von Ly-
ergocornine/ergocryptine producer resulted in
sergsaure-Alkaloiden aus der mexikanischen Zaub-
strains which appeared to be hybrids, as judged erdroge Ololiuqui (Rivea corymbosa). (Isolation of ly-
by their nutrient requirements (citrate or succi- sergic acid alkaloids from the Mexican magic drug
plant). Experientia 16: 414.
nate) and by the amount of alkaloids produced, Kelleher, W.J. 1970. Ergot alkaloid fermentations. Adv.
which ranged between the values of the parent Appl. Microbiol. 11: 211-244.
strains. Kobel, H. and J.J. Sanglier. 1986. Ergot alkaloids, pp. 569-
609. In: Rehm, H.J. and G. Reed (eds.). Biotechnology,
Experiments with mutasynthesis (see Sec- vol. 4.-VCH-Verlagsgesellschaft mbH, Weinheim.
tion 3.6) using C. purpurea show promise of ob- Kobel, H., E. Schreier, and J. Rutschmann. 1964. 6-Methyl
taining new pharmacologically useful alkaloids. 4°9-ergolen-8-carbonsaure, ein neues Ergolinderivat
aus Kulturen eines Stammes von Claviceps paspali. (6-
Auxotrophs of an ergocristine-producing strain
Methyl-4*°-ergolen-8-carboxylic acid, a new ergoline
(Phe-) and an ergocornine/ergocryptine-produc- derivative from cultures of strains of Claviceps paspali).
ing strain (Leu) produced the appropriate al- Helv. Chim. Acta 47: 1052-1064.
Kopp, B. and H.J. Rehm. 1984. Semicontinuous cultivation
kaloid analogs after the addition of phenylala- of immobilized Claviceps purpurea. Appl. Microbiol.
nine analogs (p-chlorophenylalanine, p- Biotechnol. 19: 141-145.
fluorophenylalanine) or leucine analogs (norleu- Kren, V., S. Chomatova, J. Bremek, P. Pilat, and Z. Re-
hacek. 1986. Effect of some broad-spectrum antibiotics
cine, norvaline). By feeding synthetic amino acid on the high-production strain Claviceps fusiformis W1.
analogs it has been possible to exchange both of Biotechnol. Letters 8:327-332.
the other amino acids of the tripeptide moiety. Kybal, J. and B. Sikyta. 1986. Renaissance of surface cul-
ture: Production of ergot alkaloids and spore forma-
For the development of a cloning system, tion. Acta Biotechnol. 6:245-351.
mitochondrial (mt) DNA from C. purpurea -has Kybal, J., E. Svoboda, K. Strnadova, and M. Kejzlar. 1981.
been used as a vector. Chromosomal DNA of C. Role of organic acid metabolism in the biosynthesis of
purpurea containing the gene for the phospho- peptide ergot alkaloids. Folia Microbiol. 26; 112-119.
Maier, W., D. Erge, and D. Gråger. 1981. Studies on the
ribosylanthranilate isomerase, has been cloned in cell-free biosynthesis of ergopeptines in Claviceps pur-
E. coli. This raises the possibility of using protein purea. Federation Europ. Microbiol. Soc. Microbiol.
Lett. 12: 143-146.
engineering to increase the action of the rate- Mantle, P.G. 1975. Industrial exploitation of ergot fungi,
limiting enzymes in the biosynthetic sequence. It pp. 281-300. In: Smith, J.E. and D.R. Berry (eds.), The
 REFERENCES / 285

filamentous fungi, vol. 1, Industrial mycology. Edward Rehacek, Z. and P. Sajdl. 1979. Changes in activity of
Arnold, London. Krebs and glyoxylate cycles during biosynthesis of
Puc, A., S. Milicic, M. Kremser, and H. Socic. 1987. Reg- agroclavine and elymoclavine. Biotechnol. Lett. 1: 53-
ulation of ergotoxine biosynthesis in Claviceps pur- 578
purea submerged fermentation. Appl. Microbiol. Bio- Robbers, J.E., W.W. Eggert, and H.G. Floss. 1978. Phys-
technol. 25:449-452. iological studies on ergot: Time factor influence on the
Rehacek, Z. 1980. Ergot alkaloids and their biosynthesis. inhibitory effect of phosphate and the induction effect
Adv. Biochem. Eng. 14: 33-60. of tryptophan on alkaloid production. Lloydia 41: 120-
129)
 Microbial
transformations

appears as a result of the enzyme reaction,

15.1 INTRODUCTION
the reaction product is normally optically ac-
Microorganisms have the ability to chemically tive.
modify a wide variety of organic compounds. Reaction conditions: Enzymatic reactions do
Such changes are called biological or microbial not cause destruction of sensitive substrates,
transformations, or more generally bioconver- due to the mild conditions of conversion. Sev-
sions. In these enzymatic reactions, the substrate eral reactions can be combined, either in one
may be metabolized, but in some cases the con- fermentation step using an organism with
version may take place without energy gain (co- suitable enzyme systems, or by step-wise
metabolism). In general, an industrial process can conversions using different microorganisms.
be implemented either by chemical synthesis or The reactions cause less environmental haz-
by bioconversion, but bioconversion is often ard, as they take place chiefly in water.
preferable for the following reasons:

¢ Substrate specificity: Only one specific reac- 15.2 TYPES OF BIOCONVERSION

tion step is normally catalyzed by an enzyme. REACTIONS
Site specificity (regiospecificity): If several The most important microbial transformation re-
functional groups of one type are present in actions are outlined in Table 15.1. Chemicallly,
the molecule, only one specific position may these transformations can be grouped under the
be affected. following categories: oxidation, reduction, hy-
Stereoselectivity: If a racemic mixture is used drolysis, condensation, isomerization, formation
as starting material, only one specific enan- of new C—C bonds, introduction of hetero func-
tiomer is converted. If a center of asymmetry tions. Oxidation reactions are particularly useful

286
 15.2 TYPES OF BIOCONVERSION REACTIONS / 287

Table 15.1 Bioconversion reactions of various types

Type of reaction Example Conversion
efficiency
(%)
OXIDATIONS
: COOH
Hydroxylation | 100
HC—NH2
|
CH, HO R
| B. subtilis |
Nate aed
shan es N :
H H

Tryptophan 5-Hydroxy-
tryptophan

Are Pseudomonas
Epoxidation Slgeverans 25
FVU VMS Se ee KØRES
SES SEA

1,7-Octadiene 7,8-Epoxy-1-octene

Dehydration of -CH-CH- 60

Fusarium 3 Øg
solani
———————

Glaucine Didehydro-
glaucine

Nocardia sp.
Oxidation of aliphatic side chains with (Ny Hg ————> CH;-COOH 80
the formation of aldehydes, ketones or
carboxyl functions
n- Dodecyl- Phenyl-
benzene acetic acid

Oxidative breakdown of alkyl side (CHa) CH KERES CH, COOH ?

chains ER SER
T16 OH
2-Hydroxy-
1-Phenyl- phenylacetic
dodecane acid

BS CH2-COOH
67-85

Phenylacetic
acid
288 / CHAPTER 15 / MICROBIAL TRANSFORMATIONS

Table 15.1 (continued)

Type of reaction Example Conversion
efficiency
(%)
Oxidative splitting of aromatic rings Corynebacterium 70

OC.
nov. sp. ATCC 15570

COOH
Napthalene Salicylic acid

Saeys
Oxidative splitting of substituents 100
Cunninghamella
(oxidative deamination, N-CH,- blakesleana
demethylation, O-CH,-demethylation) i CH, ATCC 9245
mm—
HCO. HCO
H,CO a] HO

10,11-Dimethoxy- lsoapocodeine
aporphine

Oxidation of heterofunctions (amino Ry

N
weer
R, N Streptomyces sp.
groups to nitro groups; formation of N-
oxides and sulfoxides) Bes oe NO2
H

2-Amino-4-alkyl- 2 Nitro-4-alkyl-
imidazole imidazole

R,= H; R,=H
-CH, H 50
“CH, H 25
36

REDUCTIONS
Saccharomyces
Reduction of carboxyl functions
DS) pana
H cerevisiae 50
CH20H
(9)

Benzaldehyde Benzyl alcohol

Reduction of heterofunctions NO2

Streptomyces
NH,
(particularly -NO,) Cl Cl aureofaciens Cl
re
Cl > Cl Cl
Cl Cl

Nitropenta- Pentachlor-
chlorbenzol aniline
 15.2 TYPES OF BIOCONVERSION REACTIONS / 289

Table 15.1 (continued)

Type of reaction Example Conversion
efficiency
(%)
ee =
ydration of carbon-carbon double
ee
e ?
R3 COO” clostridium R3 4H
bonds \ / La 1 | |
/ \ NS eB

R, R, H RR, COO

a,4 -unsaturated
carboxylic acids '

HYDROLYTIC REACTIONS
Hydration of carbon-carbon double | 55
bonds
CH; N(CH)), H,C OH
OH S. aureofaciens y
ae RA _ATCC 10762
; co
OH O OH O NH

Anhydrotetracycline Tetracycline

CH3 Mycobacterium CH,

Hydrolysis of carboxylic acid esters phlei

QsSR rer
O-C-(CH,),5- CH, OH
H3C -CH H;C—CH

CH, CH,
d,l-Menthy! laureate |-Menthol

Q
Hydrolysis of N-derivatives R-¢=NH>

Brevibacterium

R-COOH

CONDENSATIONS
S. griseus
Phosphorylation Streptomycin ——————®_ Streptomycin-P
290 / CHAPTER 15 / MICROBIAL TRANSFORMATIONS

Table 15.1 (continued)

Type of reaction |Example DA

(%)
Forn 16
N-Glycosidation O sg
ove E.coli ATCC 10798

Lee
H

6-Azauracil OH OH

6-Azauracil -
riboside

å idati Beauveria 60
O-Glycosidation R-O sulfurescens R=H 0
ATCC 7159
ce HOH,C
0)

CH,0 OH

R,=R, = REDE Methylgluco-

Cyclofenil pyranoside
derivative

N-Acylation 6-APS Kluyvera 63

citrophila
+ eee Ampicillin

D-Phenyl-
glycine methyl
ester

? = data unavailable or substances in mixture not isolated.

in industrial production. To a lesser extent, iso- of this procedure is to use a very large inoculum
merization, reduction, hydrolysis, and conden- and to add the concentrated substrate immedi-
sation also have industrial application. ately without allowing for a growth period.
Emulsifiers such as Tween or water-miscible sol-
15.3 PROCEDURES FOR vents with low toxicity (ethanol, acetone, di-
BIOTRANSFORMATION methyl formamide, dimethyl sulfoxide) may be
used to help solubilize poorly soluble com-
Spores, growing cultures, resting cells, enzymes, pounds. Steroid conversions, in which the low
immobilized cells, or immobilized enzyme solubility limits the amount of substrate which
systems can be used in the microbial conversion can be added, are commonly carried out at sub-
of organic compounds. strate concentrations between 0.1-10 g/l me-
In processes with growing cultures, the dium, although in some cases, up to 30 g/l can
strain used is cultivated in a suitable medium and be converted. Solvent concentrations between 5
a concentrated substrate solution is added after and 15 ml per liter of medium can be used. In
suitable growth of the culture (6-24 h). A variant some steroid transformations, the substrate is
 15.3 PROCEDURES FOR BIOTRANSFORMATION / 291

added and converted in fine crystalline form. and the cells can be used over and over again.
These so-called pseudo-crystalline fermentations Immobilized bacterial cells, which catalyze one-
can be carried out with relatively high concen- stage or multi-stage reactions, are presently used
trations of substrate (for example, 15-50 g/l with commercially in the production of aspartic acid,
progesterone). L-alanine, and malic acid.
For the biotransformation of lipophilic ma- Cell-free enzyme extracts, generally inolv-
terials it is possible to employ a polyphase sys- ing the use of immobilized enzymes (see Sec-
tem. The aqueous phase containing the cell ma- tion 11.11), are usually employed in biotrans-
terial or the enzyme is overlayed with a water- formation reactions when undesirable side
immiscible fluid phase in which the substrate has reactions or further breakdown of the reaction
been dissolved. The substrate passes slowly into products must be avoided or when the rate of
the aqueous phase and as the transformation re- the reaction is hindered by transport of substrate
action proceeds, the product passes back into the or product through the cell membrane. The use
solvent phase. In some cases, the actual trans- of carrier-bound enzymes also makes possible
formation occurs at the interface of the aqueous the development of continuous processes. We
and solvent phases. discussed in Chapter 11 the use of immobilized
Conversion time is related to the type of re- enzymes for several processes which could be
action, the substrate concentration, and the considered biotransformations, for example pen-
microorganism used. Oxidation and dehydration icillin acylase, glucoamylase, and glucose isom-
reactions using bacteria are often completed in a erase. A pilot-scale process has been developed
few hours; conversions with yeast and especially for the synthesis of amino acids by the reductive
fungi can take several days. Hydrolysis reactions amination of a-keto acids by amino acid dehy-
with most kinds of microorganisms can be ac- drogenases. This system permits the regeneration
complished in a few hours. of the required NADH cofactor.
Transformation reactions in large-scale The end products of transformation reactions
equipment are carried out under sterile condi- are usually extracellular and may occur in either
tions in aerated and stirred fermenters, the con- dissolved or suspended form. For further pro-
version process being monitored chromato- cessing, bacteria and yeasts are generally not sep-
graphically or spectroscopically. The process is arated, whereas fungal mycelium is usually re-
terminated when a maximal titer is reached. Ste- moved by filtration. In all cases, separated cell
rility is necessary because contamination can material must be washed repeatedly with water
suppress the desired reaction, induce the for- or organic solvents since a significant amount of
mation of faulty conversion products, or cause the reaction product can be adsorbed to the cells.
total substrate breakdown. Depending on the solubility of the product, re-
If enzyme induction by the added substrate covery is performed by precipitation as the cal-
is not necessary, resting cells may be used. This cium salt, by adsorption to ion exchangers, by
has the considerable advantage that growth in- extraction with appropriate solvents, or, for vol-
hibition by the substrate is eliminated. High cell atile substances, by direct distillation from the
densities, which promote increased productivity, medium.
may be used; at the same time, risk of contam-
ination is reduced. Since the transformation re- 15.4 APPLICATIONS OF
action occurs predominantly in the buffer solu- BIOCONVERSIONS
tion, the recovery of the product is relatively
easy. A number of transformation processes em- Although a vast array of biotransformations have
ploy immobilized cells, offering the advantage been described, only a few of these processes
that the process can be carried out continuously have found industrial application. Some pro-
292 / CHAPTER 15 / MICROBIAL TRANSFORMATIONS

cesses have insufficient yields and for others the formation processes is limited. Research is now
market is too limited. In the future, a wide range primarily directed at the optimization of the ex-
of applications is expected to arise in connection isting processes, primarily by use of immobilized
with new technology, such as the more cost-ef- cells or enzymes or by the optimization of the
fective processes using immobilized cells or en- reaction system, such as by the use of polyphase
zymes. Other improvements are expected with systems. Further optimization work is in the di-
the use of strains which have been genetically rection of finding better starting materials or re-
optimized for specific processes. ducing the degradative side reactions. Genetic
engineering research on the microorganisms
15.55 TRANSFORMATION OF STEROIDS used for steroid transformations is also under in-
AND STEROLS vestigation. Additionally, studies are under way
on the use of plant cell cultures for steroid trans-
Naturally occurring steroids have hormone formations.
properties. Examples are the adrenal cortex hor-
mones (glucocorticoids and mineral corticoids),
androgens, estrogens, and hormones active dur- Types of transformations
ing pregnancy, such as progesterone. All steroids
have the same basic structure, a cyclopen- Because the steroid molecule contains several
tanoperhydrophenanthrene. Figure 15.1 shows asymmetric centers, total synthesis is very dif-
the structure of the most important types. Estro- ficult. The original chemical process involved 31
gens, progesterone, and androgens are used separate reaction steps and yielded 1 g cortisone
therapeutically; derivatives of progesterone and acetate from 615 g deoxycholic acid. More recent
estrogens are also used as contraceptives. In ad- chemical processes are simpler and deoxycholic
dition, steroids are used as sedatives, in antitu- acid (from ox bile) is presently used as substrate
mor therapy, and as veterinary products. The in several production processes.
glucocorticoids are valuable compounds with Preliminary research on the 11la-hydroxyla-
wide therapeutic uses. Cortisone is especially tion of progesterone pointed to the possibility of
useful because of its anti-inflammatory action in the microbial introduction of oxygen into the ste-
such conditions as rheumatoid arthritis and skin roid nucleus in a site-specific and stereospecific
diseases. By altering the structure, specifically by manner without prior activation. These reactions
introducing a 1,2 double bond in ring A of the worked well, and cost-effective production of
cortisol or cortisone molecule to produce pred- cortisone became possible. The oxygen atom at
nisolone or prednisone, substances can be pro- C-11 is essential for the anti-inflammatory effect
duced with markedly increased anti-inflamma- of cortisone. In 1949, 1 g of cortisone cost $200
tory effect. Addition of fluorine and methyl to produce, but as a result of the introduction of
groups leads to the formation of compounds with the microbial process for the 11-a-hydroxylation
reduced mineral corticoid activity (Na? retention, of progesterone, the cost had decreased to under
K* excretion), such as 16a-hydroxy-9a-fluoro- $1 by 1979. Currently, almost all positions of the
prednisolone (triamcinolone) or 6a-methylpred- steroid molecule can be specifically hydroxylated
nisolone (medrol). By means of transformation, by different microorganisms and the number of
anabolic steroids with reduced androgenic effects transformation reactions is larger than the num-
have been developed, such as 1-methyl-A!-an- ber carried out by animal tissue (Figure 15.2). The
drostenolone. microbial hydroxylation reactions are carried out
At the present, the available steroids serve by highly specific monooxygenases; some ex-
most of the medical requirements quite well so amples are an 1la- or 116-hydroxylase, a 17a-
that further development of new steroid trans- hydroxylase and a 21-hydroxylase.
 15.5 TRANSFORMATION OF STEROIDS AND STEROLS / 293

Hormones of the adrenal cortex

Glucocorticoids
CH. OH CH,OH CHLOH
"ke: fe 2
c=0 c=0 C=O

HO OH HO

og

Cortisone Cortisol Corticosterone

CH,OH
|
Mineralcorticoids =e Cc=0
HO

oF

Aldosterone

Androgens
OH

oF

Testosterone
Estrogens
OH i OH
OH

HO HO HO
Estradiol-178 Estrone Estriol-3,16a, 178
CH
Gestagens ee

oF

Progesterone

Figure 15.1 Structure of several naturally occurring steroid hormones

294 / CHAPTER 15 / MICROBIAL TRANSFORMATIONS

21 CH, Table 15.2 Reaction types of microbial transformations

from steroids
A. Oxidations
Conversion of secondary alcohols to ketones
Introduction of primary hydroxyl groups into the
steroid side chain
Introduction of secondary hydroxyl groups into the
basic steroid framework
Introduction of tertiary hydroxyl groups into the
basic steroid framework
Dehydration of ring A in positions 1(2) and 4(5)
Aromatization of ring A
Oxidation of the methylene group to the keto group
58-Pregnane Splitting of the side chain of pregnane at C-17
during production of a ketone
Splitting of the side chain of pregnane at C-17 and
opening of ring D during production of
Microorganisms Animal tissue testololactone
Splitting of the steroid side chain during production
la Ta 15a la 15a
of a carboxyl group
18 7B 158 2a l6a
Splitting of the side chain uF pregnane at C-17
2a 9a l6a 26 l7a
during production of a secondary alcohol
28 108 168 6a 18
Production of epoxides
38 lla 17a 68 19
Decarboxylation of acids
Sa 118 18 Ta 21
58 12a 19 118 B. Reductions
68 128 21 12a
Reduction of ketones to secondary alcohols
14a
Reduction of aldehydes to primary alcohols
Hydration of double bonds in position 1(2) in ring A
Hydration of double bonds in position 4(5) in ring A
Figure 15.2 Steroid hydroxylation by microorganisms as and 5(6) in ring B
contrasted with the process in animal tissue Elimination of secondary alcohols
C. Hydrolysis
As outlined in Table 15.2, microorganisms Saponification of steroid esters
are capable of carrying out a wide variety of other D. Ester production
steroid transformation reactions besides hydrox- Acetylation
ylation.
(Sebek and Perlman, 1979)

Economically important transformations

of ring A is used in estrogen production. The
In recent decades, thousands of modified steroids conditions for several bioconversion reactions of
produced by a combination of chemical and mi- this type are given in Table 15.4 (examples 1-6).
crobial reaction steps have been tested for their Steroid transformations have thus far been con-
therapeutic effectiveness. A typical example of ducted as batch fermentations, but progress is
such a combined synthesis is the production of being made with the use of immobilized cells or
cortisone and its 1-dehydro-derivatives from enzymes. Advantages of the latter include re-
diosgenin via Reichstein’s Substance S (11- duced risk of contamination, simplified product
deoxycortisol), as shown in Figure 15.3. recovery, shorter conversion times, and increased
The microbial reaction steps listed in Table substrate concentrations. In several processes,
15.3 are of great economic significance. More- more than one biochemical step can be com-
over, progesterone transformation to a C,,-ste- bined. For instance, immobilized mycelium of
roid is used industrially in testosterone and es- Curvularia lunata or immobilized cells of Arthro-
trogen production and the microbial dehydration bacter simplex have been used to carry out the
 15.5 TRANSFORMATION OF STEROIDS AND STEROLS / 295

CH,OH
C=O

Diosgenin Reichstein’s Cortisol Prednisolone

substanceS (Hydrocortisone)
(11-Deoxycortisol)
D

Figure 15.3 Production of cortisol, cortisone and the 1-dehydro oS

compounds from diosgenin via Reichstein’s substance S (A, several
steps; B, 118-hydroxylation with Curvularia lunata; C, 1-dehydration
with Corynebacterium simplex; D, chemical oxidation; E, 1-dehydra-
tion with Corynebacterium simplex) Cortisone Prednisone

Table 15.3 Examples of commercial steroid processes

a |

Reaction Substrate Product Microorganism Manufacturer

1la-Hydroxylation Progesterone — 1la-Hydroxyprogesterone Rhizopus nigricans Upjohn Company
116-Hydroxylation Component S — Cortisol Curvularia lunata Pfizer Inc.,
Gist-Brocades

16a-Hydroxylation 9a-Fluorocortisol — 9a-Fluoro-16a- Streptomyces E. R. Squibb and Sons,

hydroxycortisol roseochromogenes Lederle Laboratories

1-Dehydrogenation Cortisol — Prednisolone Arthrobacter simplex Schering Corp.

Diendiol? — Triendiol? Septomyxa affinis Upjohn Company
1-Dehydration, Progesterone — 1-Dehydrotestololactone Cylindrocarpon E. R. Squibb and Sons
side chain splitting radicicola

Side chain splitting B-Sitosterol — Androstadiendione, Mycobacterium sp., G. D. Searle and Co.
and/or 9a-hydroxyandrostendione M. fortuitum Upjohn Company
(see Fig. 15.5 for structure) mutants

2 Diendiol = 116,21-dihydroxy-4,17(20)-pregnadiene-3-one
> Triendiol = 118,21-dihydroxy-1,4,17(20)-pregnatriene-3-one (precursors in the production of
6a-methylprednisolone).
(Sebek and Perlman, 1979)

two-step reaction from Reichstein’s component a system consisting of 25% (w/w) polyethylene
S to prednisolone. In several processes, fungal glycol (PEG) 8000 and 6 (w/w) dextran T40.
spores are being used directly to catalyze the
transformation. Since most steroid substrates are
Microbial breakdown of sterol side chains
not very soluble, transformation conditions have
been developed for some steroids in a solvent The growing demand for steroids caused a short-
system which is water-immiscible, e.g., for tes- age of steroid precursors for bioconversion, such
-tosterone with immobilized cells of Nocardia rho- as the compound diosgenin, which is obtained
dochrous. Since the organic solvent is often toxic from the Mexican yam root (Dioscorea composita)
to the cells or enzyme, an alternative is the use or the South African plant Testudinaria sylvatica.
of an aqueous two-phase system. For example, Intensive studies were conducted on the use of
the 1-dehydration of cortisol to prednisolone can low-cost sterols of animal origin, such as cho-
be carried out by cells of Arthrobacter simplex in lesterol, or of plant origin, such as (-sitosterol
296 / CHAPTER 15 / MICROBIAL TRANSFORMATIONS

Table 15.4 Conditions for operation of several steroid and sterol transformations
No. Substrate Product Yield Microorganism Medium Conditions used
(weight %)
1 Progesterone 1-Dehydrotestololactone 50 Cylindrocarpon a YP) Ney 78)
radicicola

2 Progesterone 1,4-Androstadiene-3,17- 85 Fusarium solani b 96 h, 25°C

dione

3 Progesterone 15a-Hydroxy-4-pregnene- 11 Streptomyces c IPN PAE

3,20-dione aureus

4 4-Androstene- 11la-Hydroxy-4- 25 Rhizopus arrhizus d 96 h, 28°C

3,17-dione androstene-3,17-dione

5 Progesterone 11-a-Hydroxy- "07 Aspergillus e 120 h, 28°C

progesterone ochraceus

6 Hydrocortisone Prednisolone 93 Arthrobacter 2 f 20h 2one

simplex Pseudo-
crystal
fermentation

a Cholesterol 1,4-Androstadiene-3,17- 90 Arthrobacter g 44 h, 30°C

dione simplex + chelating
agents
8 B-Sitosterol 9a-Hydroxy-4-androstene- Data Mycobacterium h 336 h, 30°C
Cholesterol 3,17-dione unavailable fortuitum mutant with
Stigmasterol block in
Campesterol breakdown of
steroid nucleus

Media in Table 15.4

a. 3 g Corn steep liquor (dry weight), 3 g NH,H,PO,, 2.5 g CaCO,, 2.2 g soy bean oil, 0.5 g progesterone, distilled
H,O to 11, pH 7.0.
b. 15 g Peptone, 6 ml corn steep liquor; 50 g glucose, distilled H,O to 11, pH 6.0; 0.25 g progesterone, added after
48 h.
(Cs 2.2 g Soy bean oil, 15 g soy meal, 10 g glucose, 2.5 g CaCO,, 0.25 g progesterone, distilled H,O to 1 1.
d. 20 g Peptone, 5 ml corn steep liquor, 50 g glucose, tap water to 1 1, pH 5.5-5.9; 0.25 g androstendione, added
after 27 h.
10 g peptone, 5 g yeast extract, 30 g sucrose, tap water to 1 liter; pH 6.5; 40 g progesterone dissolved in acetone
added to the growing culture after 24 h.
f. 5g Peptone, 5 g corn steep liquor, 5 g glucose; distilled H,O 1 1; pH 7.0; 1-50% finely ground hydrocortisone,
suspended in ethanol, added to a 24 hour old culture.
10 g Corn steep liquor, 2 g meat extract, 5 g glucose, 0.5 g K,HPO,; distilled H,O 1 1; pH 7.0; cholesterol (1 g)
added after 20 h (dispersed in water); 0.8 mM a,a'-dipyridyl (an iron chelating agent) in ethanol added after 26 h.
10 g Glycerol, 8.4 g Na,HPO,, 4.5 g KH,PO,, 2 g NH,Cl, trace elements; distilled H,O 1 1; 1 g soy meal and 10 g
sitosterol are added.
(Sebek and Perlman, 1979)

and stigmasterol (from soy beans) or campesterol the substrate led only to strains carrying out a
(produced in great amounts as a byproduct of total breakdown of sterol to CO, and H,O. The
paper manufacture). breakdown of the side chain to yield a C-17 ke-
The objective of these studies was the selec- tocompound, shown in Figure 15.4, involves a
tive removal of the aliphatic side chain without mechanism which is similar to that of the B-ox-
further breakdown of the steroid nucleus. How- idation of fatty acids. A C-1(2)-dehydration and
ever, a screening procedure with cholesterol as 9a-hydroxylation are mandatory for further
 15.5 TRANSFORMATION OF STEROIDS AND STEROLS / 297

COOH
um | ~COOH O
It

Seley
C27 C 24 €:22 Cry,

Figure 15.4 Side chain break- + CH;CH zCOOH + CH;COOH + CH; CH; COOH
down of sterols Propionic acid Acetic acid Propionic acid

LÅ

breakdown of the steroid ring. As shown in tion or 9a-hydroxylation, such as compounds

Figure 15.5, the breakdown product 3-hydroxy- which chelate Fe?” or Cu”, bivalent ions
9,10-secoandrostatriene-9,17-dione is produced which replace Fe?*, or substances which block
from cholesterol via an opening of the B ring, sulfhydryl functions, such as Ni”, Co?*, and
with the producticn of two useful intermediate lagen
products, androstendione and androstadien- ¢ Mutants with inactive C-1(2)-dehydrogenase
dione. In order to modify the steroid nucleus and/or 9a-hydroxylase can be used. Myco-
without breaking any of the rings, methods for bacterium mutants which transform choles-
selectively blocking attack on the ring are terol, stigmasterol, and sitosterol into the
needed. Several methods are available to selec- main product androstenedione and the sec-
tively block the breakdown of the steroid nu- ondary product androstadiendione have been
cleus: isolated. With these mutants, there is no fur-
ther breakdown of these latter compounds.
¢ The breakdown reaction can be blocked by
chemical modification of the substrate. Some of the processes just mentioned are
«+ The conversion can take place in the presence used commercially (see Table 15.4, examples 7,
of inhibitors which prevent C-1(2)-dehydra- 8).

AN
HO HO O

Cholesterol 36 -Hydroxy-5- Androstendione

androstene-17-one

ae]
Figure 15.5 Biotransforma- :
2
OG SO pres
HO
tion of cholesterol to androsta-
diendione and androstatrien- Androstadiendione 9a -Hydroxy- 3-Hydroxy-9,10-secoandrosta-
dione by mycobacteria androstadiendione 1,3,5(10)triene-9,17-dione
298 / CHAPTER 15 / MICROBIAL TRANSFORMATIONS

commercially significant bioconversions among

15.6 TRANSFORMATION OF the many known reactions are discussed here.
NONSTEROID COMPOUNDS
In addition to the steroid transformations, mi- L-Ascorbic acid (Vitamin C)
crobial transformations of alkanes, of alicyclic,
The established process for producing L-ascorbic
aromatic, and heterocyclic compounds, of ter-
acid is the so-called Reichstein-Griissner synthe-
penes, and of alkaloids have been described. In
sis, shown in Figure 15.6. This process consists
some cases, microbial transformations are diffi-
of several chemical steps and one microbial con-
cult to control. For example, in experiments with
version. L-ascorbic acid is used in vitamin prep-
gibberellins, carotenoids, and camphor, break-
aration or as an antioxidant in food manufacture
down reactions predominate and there is only
and world production by this process is about
slight accumulation of utilizable intermediary
40,000 tons per year. The oxidation stage from
products; attempts to isolate mutants blocked in D-sorbitol to L-sorbose is carried out by Aceto-
the breakdown reactions have been unsuccessful.
bacter suboxydans in a submerged process at 30—
In other cases, as in alkaloid biotransformations, 35°C with vigorous stirring and aeration. Sor-
reaction rates have been too low for commercial bitol is added at an initial concentration of 20%
processes. However, some transformation reac- to a nutrient solution consisting of 0.5% yeast
tions are economically important; some of these extract or corn steep liquor, and CaCO,. A quan-
have already been covered earlier (e.g., gluconic titative conversion is completed after about 24
acid, kojic acid, in Chapter 8; manufacture of L- hours; higher sorbitol concentrations prolong the
amino acids through racemic separation or hy- conversion time. Today this process is carried out
dantoin cleavage, in Chapter 9; penicillin acylase, continuously in two stages; there are even some
glucose isomerase, in Chapter 11). A few more installations in which polyacrylamide-immobi-

CH,OH CH,0H
| |
Boece! Catalytic et Sorbitol dehydrogenase
HO-C-H reduction HO=C=H Acetobacter suboxydans, A. xylinum
| I
HEÆC-OH H-C-OH
| 5 | CH,OH
HO=C=H HOsCsiH |
| | NAD NADH, c=0
CHO CH,OH |
HO=C=H
|
D-Glucose D-Sorbitol BEC oH
HOSCEH
|
CH,OH

ioe COONa COOH L-Sorbose

|
C=O : C-ONa C=0 10)
i (eg) Acid i | Chemical
ree treatment (EO Na 5 MOGs H oxidation

HG H-C-OH / H=C=OH
| | | H20
HO-C-H HO-C-H 2H HO-C-H
| a 5
CH,OH CH,OH CH,OH Figure 15.6 Microbial dehy-
dration of D-sorbitol to L-sor-
L-Ascorbic acid Sodium salt/Enol 2-Keto-L- bose in the production of L-as-
form/2-Keto-L- gulonic acid corbic acid (Reichstein-
gulonic acid Griissner synthesis)
 15.6 TRANSFORMATION OF NONSTEROID COMPOUNDS / 299

lized cells are used. In the overall process ap-

Dihydroxyacetone from glycerol
proximately 1 kg of L-ascorbic acid is produced
from 2 kg of glucose. The microbial conversion of glycerol to dihy-
In addition to the Reichstein-Grussner syn- droxyacetone (used in suntan lotions and cos-
thesis shown in Figure 15.6, a two-step fermen- metics), is of some significance (Figure 15.8). Var-
tation process has been developed and carried to ious acetic acid bacteria convert 10% glycerol in
commercial production. As shown in Figure 15.7, a suitable nutrient solution (0.5% yeast extract,
the first step involves the oxidation of glucose by 0.5% KH,PO,, 2% CaCO,) at 28°C and at a pH
an Erwinia species to 2,5-diketo-D-gluconic acid below 6.0. Conversion time is 72-96 hours but
(2,5-DKG), via D-gluconic acid and 2-keto-D- can be reduced to 33 hours with Gluconobacter
gluconic acid. During a 26 hour incubation, 328 melanogenus IFO 3293 by using O,-enriched air
8/1 calcium 2,5-DKG is formed with a 94% ef- (oxygen partial pressure 0.05 bar). Growth is not
ficiency. The second step, a reduction of 2,5-DKG affected, but the amount of dihydroxyacetone
to 2-keto-1-gulonic acid, is catalyzed by a Co- produced is raised from 12.2 g/g biomass in nor-
rynebacterium sp. In this process, after the Co- mally aerated cultures to 35.8 g/g in cultures
rynebacterium has grown for 16 hours, it is fed with improved oxygen supply.
with a sterilized Erwinia culture. After 66 hours
incubation, 106 g of calcium 2-keto-L-gulonate Prostaglandins
is formed, with an efficiency of 92%. The latter
product is easily transformed chemically into L- Prostaglandins are unsaturated C,, fatty acids
ascorbic acid and the overall balance, based on that function as tissue hormones. They are of
glucose consumed, is 86%. increasing medical significance because of their
Scientists at the Genentech Company have varied physiological activities. Currently mar-
succeeded in cloning and expressing the gene for keted are PGE, as a contraceptive, PEG, for the
the 2,5-DKG reductase of Corynebacterium into alleviation of pain of childbirth, PEG, for the
Erwinia herbicola, opening up the possibility of a treatment of congenital heart failure, and a
single-step process from glucose to 2-keto-L-gu- methyl derivative of PEG, for the treatment of
lonic acid. However, the hybrid strain produced digestive diseases. The prostaglandins PGE,,
exhibited a low tolerance to high concentrations PGE,, PGF,, and PGF, can be produced from un-
of glucose and a yield of only 0.6 g/l of 2-keto- saturated fatty acids (particularly arachidonic
L-gulonic acid was obtained from 20 g/1 glucose acid) (Figure 15.9) by microbial transformation
after 57 hours incubation. with fungi. The isolation of compounds with im-

CHO CCDE ewe

|
H-C—OH : C=0 Coryne- C=0

Hoc —H |EMS. i by bactenum sp. HOCH

| | ———“o |
H—C— OH pes OB a cag

H=G Or GER HO-C —H

CH20H CH-OH CH 20H

D-Glucose 2,5-Diketo- 2-Keto-

D-gluconic acid L-gulonic acid

Figure 15.7 Two-stage fermentation for the production of 2-keto-L-gulonic acid from D-glucose
300 / CHAPTER 15 / MICROBIAL TRANSFORMATIONS

CH, OH Acetobacter CH,OH duction of new and improved antibiotics by the

suboxydans (97 °/o) | microbial transformation of existing compounds.
H-C —OH ——~>_ C=0
A. xylinum (100 °/o) | The objective here is the development of mod-
CH, OH CH,OH ified antibiotics with improved effectiveness, re-
duced toxicity, wider antimicrobial spectrum, im-
Glycerol Dihydroxyacetone proved oral absorption, decreased development
of resistance, or lower allergic effects. In most
Figure 15.8 Conversion of glycerol to dihydroxyacetone cases, any transformation step causes a partial or
complete inactivation of the antibiotic. Thus,
many biotransformations must be attempted and
their products assayed to find those rare cases
where the modified product has desirable im-
proved properties.
Arachidonic acid
Several typical examples of the many possi-

(9)
\ i
|
a a COOH
ble reactions are given here.

Indirect transformation The addition of inhibi-

tors or modified precursors to the medium may
result in the synthesis of altered antibiotics via
HO OH controlled biosynthesis. For instance, in the
Prostaglandin E,
presence of cis-4-methylproline, Streptomyces
parvulus produces two new actinomycins (K,.
KN and K,.) in which proline is replaced by this pro-
line analog. New compounds have been found
when mutants blocked in the synthesis of a par-
HO OH ticular antibiotic were used. For instance, ribos-
tamycin.(Figure 15.10) accumulates as an inter-
Prostaglandin E, mediate product of neomycin biosynthesis with
a mutant of the neomycin producer S. fradiae. A
HO few improved antibiotics have been isolated after
pe AAN COOH
OR
mutational synthesis (see Section 3.6), of which
Fa A Y only 5-epi-sisomicin has proved of sufficient util-
HO OH ity to undergo clinical trials. Several genetic tech-
niques, mutagenesis, intra- and interspecific re-
Prostaglandin Fog
combination by protoplast fusion, and
recombinant DNA technology, have been used
Figure 15.9 Biotransformation of arachidonic acid to
prostaglandins to isolate strains capable of producing modified
versions of rifamycins, aminoglycosides, and ty-
losin.
proved effectiveness can be expected in light of
new reports on successful biotransformations of
Direct transformation Acylation reactions have
this molecule.
been described for various antibiotics. Inactive
compounds develop in most cases but in the case
15.7 TRANSFORMATION OF of lankacidin-C-14-butyrate, a bioconversion
ANTIBIOTICS
product formed from lankacidin C and methyl-
In addition to screening procedures, studies in butyrate by Bacillus megaterium IFO 12108, im-
recent years have also been directed at the pro- proved antimicrobial activity with lower toxicity
 15.8 TRANSFORMATION OF PESTICIDES / 301

CH
HO 29
HO
HON it
CHR 26
0 HON
ocr 0/7 R !
R;

O OH Mee 2 HON

re,
H

RO OH ) Cc HO NH,
OG

O
HO R,
Rees =i Ribostamycin
ON >
Rats ee
H ©
Neomycin B
dar (P) star
Ae \ NH2
CH,NH, Gentamicin R, Ro R3z Ry Rs Re Ry

Figure 15.10 Ribostamycin formation by a mutant of Gentamicin A H OH OH OH NHCH3 H On

Streptomyces fradiae blocked in neomycin production Gentamicin A> H OH OH OH OH OH 4H
Gentamicin Xz H OH OH OH NHCH3 OH CH3
GentamicinC,, H NH, H H NHCH3; OH CH3
Gentamicin Co NHCH3 OH CH3
GentamicinC, | CH3 NHCH3 H H NHCH3 OH CH
was obtained. Deacylation reactions have been Figure 15.11 Structure of gentamicins with indications
described in particular for the macrolide anti- of the site of action of the inactivation enzymes
biotics. The biologically less active deacylated
products can then be used for the production of
semisynthetic compounds. Phosphorylation via
Hydroxylation is another frequent microbial
different phosphotransferases and adenylylation
transformation reaction in antibiotics (example in
via adenyltransferases take place chiefly with
Figure 15.13).
aminoglycosides and lead to inactivation. Acet-
ylation (Ac), phosphorylation (P), and adenylyl-
ation (Ad) are reactions that are primarily re- 15.8 TRANSFORMATION OF
sponsible for the development of bacterial PESTICIDES
resistance to aminoglycosides. The example of
Until now, we have been discussing microbial
gentamicin in Figure 15.11 shows the target at
transformations that lead to the production of
which the three modifying enzymes act. commercially useful compounds. We now
Hydrolysis reactions are especially significant change our perspective and discuss some trans-
in B-lactam antibiotics (Figure 15.12). Hydrolysis formations that cause the destruction of a major
involves the splitting of the lactone ring of pen- class of commercially useful compounds, the pes-
icillins and cephalosporins by 6-lactamases, lead- ticides. Agents for plant disease and pest control
ing to inactivation of the antibiotics. Bacterial re- are necessary for the survival of the world’s pop-
sistance to B-lactam antibiotics is mainly caused ulation. Presently one-third of the world’s har-
-" by this reaction. On the other hand, the enzy- vest is lost because of damaging organisms. It is
matic splitting of penicillin by penicillin acylase estimated that another one-third would be lost
into 6-aminopenicillanic acid (Section 11.9) is of without the use of chemical control agents. Con-
great economic significance. tagious diseases such as malaria, chagas disease,
302 / CHAPTER 15 / MICROBIAL TRANSFORMATIONS

GR OS
ER
N S

COOH
PenicillinG

Penicillin Penicillinase
acylase (6 -Lactamase)

H
Gir H.N S Crs S
Il nr
ce =| 0 ] =
eas COOH 07 on N COOH
Phenylacetic acid 6-Aminopenicillanic acid Benzylpenicilloic acid

Chemical
reacylation

Semisynthetic Figure 15.12 Microbial transformation of

penicillin penicillin G

typhus, cholera, and spotted fever have been re- the enzymes involved in these transformations
duced in severity and in some regions completely must be induced and different organisms are fre-
eliminated through the control of disease-car- quently involved in the breakdown of a com-
rying insect vectors. High stability (persistence) pound. Depending on chemical structure, some
of the compounds used is vital for these vector- compounds cannot be easily converted; thus per-
control programs, but this stability has a negative sistence times in the soil range from a few days
effect on the environment. to several years.
This problem of environmental persistence is Removal of xenobiotics from ecosystems can
apparent with chlorinated hydrocarbon insecti- be accomplished through various mechanisms.
cides such as DDT, lindane, and dieldrin. A re-
markable success in infectious disease control can Metabolism Xenobiotics can serve as substrates
be attributed to DDT, but due to its resistance to for microbial growth and energy production.
decomposition, the compound accumulates in Complete breakdown of some substances to CO,
microorganisms and thus enters into the food and H,O has been described. Figure 15.14 shows
chain. This development, along with the re- the example of the herbicide dalapon, a chlori-
stricted use of DDT since the early 1970’s, has nated fatty acid, which is converted by Arthro-
led to research for new control methods and tox- bacter sp. into pyruvate after oxidative dehalo-
icologically and environmentally safe prepara- genation.
tions.
In this context, microbial transformation is of Cometabolism In cometabolism, microorga-
interest not for the production of new active nisms do not obtain energy from the transfor-
agents, but for the greatest possible detoxification mation reaction and require another substrate for
of the environment. This involves enzymatic growth. Hence, cometabolism normally causes
conversions of so-called xenobiotics, substrates mere modification of molecules, which may re-
which do not normally occur in nature, such as sult in either a decrease or an increase in toxicity.
halogenated hydrocarbons, aromatic nitro-com- A further breakdown can be achieved through
pounds, and sulfonic acid derivatives. Many of the combined action of different organisms.
'
 15.8 TRANSFORMATION OF PESTICIDES / 303

Table 15.5 Cometabolism of pesticides

Substrate Conversion Microorganism
product
Chlorbenzilate 4,4'- Rhodotorula
CH, Narbomycin
(ethyl-4,4'-di- Dichlorbenzo- gracilis
0 O chlorobenzilate) | phenone

N(CH3)2 Chloroneb 2,5-Dichlor-4- Fusarium sp.

(1,4-dichloro-2,5- methoxyphenol
dimethoxy-
CH, benzene)

DDT p,p’-Dichlor- Aerobacter

diphenylmethane aerogenes
p,p’-Dichlorodi- _ p-Chlorpheny]- Hydrogenomonas
Streptomyces sp.
phenylmethane acetate sp.
2,4,5-Trichloro- 3,5-Dichlor- Brevibacterium
phenoxyacetate catechol sp.
3,5-Dichlor- 3,5-Dichlor-2- Achromobacter
catechol hydroxymuconic- sp.
acid
semialdehyde
(Bollag, 1974)
Picromycin
Cl
| CH= C ='COOH
CC COOH I
| = Cl
Cl

Dalapon 2-Chloroacrylate
Figure 15.13 Hydroxylation of narbomycin (2,2-Dichloropropionic acid)

Some examples of cometabolism are given in Ta-

ble:45:5.
Dehalogenation or oxidative dehalogenation
reactions (Figure 15.15) are important cometab-
olism reactions which may make pesticide mol- CHz—
Q
C— COOH
|cH,—COH—COOH
|
i

ecules accessible for further breakdown. Some

Cl
compounds such as chlordecone, a hexachloro-
cyclopentadiene derivative with excellent insec-
Pyruvic acid 2-Chloro-2-hydroxy-
ticide and acaricide effects, are not easily attacked
propionate |
by microorganisms because of their complicated
structure and high degree of halogenation.
Figure 15.14 Microbial breakdown of dalapon

Conjugate formation Linkage of xenobiotics or

decomposition products with naturally occurring toxic compound can be released again at any
compounds such as amino acids or carbohydrates time. Figure 15.16 shows the conversion of a di-
results only in a temporary detoxification; the thiocarbamate fungicide.
304 / CHAPTER 15 / MICROBIAL TRANSFORMATIONS

Reductive dehalogenation absorb DDT and concentrate it by a factor of 100.

These microorganisms are eaten by marine ani-
mals and the DDT is then stored in fat tissue,

co chee, ss
Aerobacter
aerogenes
or CHCHCl,
leading to an even greater concentration factor.
The end result is an accumulation of the com-
pound to high levels as it passes up the food

al
ist io
Cl
chain.
Intensive research of recent years has shown
a whole series of aerobic and anaerobic biode-
DDT TDE
gradative reactions that can occur with environ-
mentally significant compounds. Further prog-
Dehydrodehalogenation
ress can be anticipated through the following:
e The search for microorganisms capable of

loa Trichoderma

CHCCl, §——————$
viride
len C=CCL
breaking down compounds of interest, by use
of strong selective pressure in the chemostat
for enrichment culture. If particular enzyme
systems are being sought, special genetic
probes can be used to quickly screen and
Cl cl identify a wide variety of natural isolates.
DDT DDE
«+ The use of recombinant DNA technology to
Figure 15.15 Transformation of the chlorinated hydro- construct microorganisms that contain the
carbon DDT (DDT=2,2-Bis-[4-chlor-pheny]]-1,1,1-tri- -complete biochemical sequence for the break-
chlor-ethane; TDE=2,2-Bis-[4-chlor-pheny]]-1,1-dichlor- down of a particular chemical. It should be
ethane; DDE=1,1-dichlor-2,2-Bis-[4-chlor-phenyl]-ethyl-
ene) possible to combine in one organism that pro-
cesses that occur now only in co-metabolizing
organisms. For example, a hybrid Pseudo-
Hansenula
anomala
CH,
S
monas has been constructed capable of break-
Saccharomyces
cerevisiae
eae (CHG —COOH
. ing down chlorosalicylic acid.
CH, Ss NH
CH
E.coli
A illus ¢ Immobilized cells should tolerate higher con-
ay Riga @
N—C—SNa centrations of xenobiotics and should also
5 il
CH, carry out the desired biochemical reactions
CH,
: N-C-S~(CH)3C-
(CH,)5 C -COOH
more rapidly. This approach has already been
O TEEN Bee TT used to develop a continuous process for the
CHEERS 0
ad biodegradation of phenolic compounds.
@ sodium-dimethyl-dithiocarbamidate
Ø 4-(Dimethylthiocarbamyithio)-a-aminobutyric
acid
REFERENCES
@® Corresponding keto acid
Anderson, S., C. Marks, R. Lazarus, J. Miller, K. Stafford,
Figure 15.16 Transformation of a thiocarbamate fungi- J. Seymour, D. Light, W. Rastetter, and D. Estell. 1985.
cide Production of 2-keto-1-gulonate, an intermediate in L-
ascorbate synthesis, by a genetically modified Erwinia
herbicola. Science 230: 144-149.
Bettman, H. and H.J. Rehm. 1985. Continuous degradation
Accumulation of xenobiotics When microorga- of phenol(s) by Pseudomonas putida P8 entrapped in
nisms absorb xenobiotics, only temporary detox- polyacrylamide hydrazide. Appl. Microbiol. Biotech-
nol. 22; 389-393.
ification of the environment occurs. It has been Bollag, J.M. 1982. Microbial transformation of pesticides.
found that marine microorganisms and plankton Adv. Appl. Microbiol. 18:75-130.
 REFERENCES / 305
Fukui, S., S.A. Ahmed, T. Omata, and A. Tanaka. 1980. Rehm, H.J. and G. Reed. 1984. Biotechnology, Volume 6a.
Bioconversion of lipophilic compounds in non- Biotransformations. VCH Publishers, Deerfield Beach,
aqueous solvent. Effect of gel hydrophobicity on di- FE
verse conversions of testosterone by gel-entrapped No- Rubio, M.A., K.H. Engesser, and H.J. Knackmuss. 1986.
cardia rhodochrous cells. Europ. J. Appl. Microbiol. Bio- Microbial metabolism of chlorosalicylates: accelerated
technol. 10: 289-301. evolution by natural genetic exchange. Arch. Micro-
Kaul, R. and B. Mattiasson. 1986. Extractive bioconversion biol. 145: 116-122.
in aqueous two-phase systems. Production of predni- Sariaslani, F.S. and J.P.N. Rosazza. 1984. Biocatalysis in
solone from hydrocortisone using Arthrobacter simplex
natural products chemistry. Enzyme Microb. Technol.
as catalyst. Appl. Microbiol. Biotechnol. 24: 259-265.
Kieslich, K. 1978. Microbial transformations—type reac-
6: 242-253.
Sebek, O.K. 1974. Microbial conversion of antibiotics.
tions, pp. 57-85. In: Hutter, R., T. Leisinger, J. Nuesch,
and W. Wehrli (eds.), Antibiotics and other secondary Lloydia 37: 115-133.
metabolites. Federation Europ. Microbiol. Soc. Symp. Sebek, O.K. and D. Perlman. 1979. Microbial transfor-
No. 5. Academic Press, London. mation of steroids and sterols, pp. 483-496. In: Pep-
Leuenberger, H.G.W. 1978. Microbial transformations— pler, H.J. and D. Perlman (eds.), Microbial technology,
some applications in natural product chemistry, pp. vol. 1. Academic Press, New York.
87-100. In: Hitter, R., T. Leisinger, J. Nuesch, and W. Sonoyama, T., H. Tani, K. Matsuda, B. Kageyama, M. Tan-
Wehrli (eds.), Antibiotics and other secondary metab- imoto, K. Kobayashi, S. Yagi, H. Kyotani, and K. Mit-
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Academic Press, London. from D-glucose by two-stage fermentation. Appl. En-
Mazumder, T.K., K. Sonomoto, A. Tanaka, and S. Fukui. viron. Microbiol. 43: 1064-1069.
1985. Sequential conversion of cortexolone to pred- Wichmann, R., C. Wandrey, A.F. Biickmann, and R.M.
nisolone by immobilized mycelia of Curvularia lunata Kula. 1981. Continuous enzymatic transformation in
and immobilized cells of Arthrobacter simplex. Appl. an enzyme membrane reactor with simultaneous
Microbiol. Biotechnol. 21: 154-161. NAD(H) regeneration. Biotechnol. Bioeng. 23: 2789-
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ogy, Volume 3. Pergamon Press, Oxford.
 Single-cell
protein (SCP)

amino acids. In many countries, however, there

16.1 INTRODUCTION
are psychological barriers to the use of microor-
The term ”single-cell protein” (SCP) was coined ganisms as a major food source. The following
at Massachusetts Institute of Technology in 1966 points must also be considered with regard to
and is used today to refer to microbial biomass safety:
used as food and feed additives. Either the iso-
lated cell protein or the total cell material may + The high nucleic acid content (4—6% in algae,
be called SCP. 10-16% in bacteria, 6-10% in yeast, and 2.5-
The human food chain is diagrammed in Fig- 6% in fungi) can be hazardous to health.
ure 16.1. Animals and plants have always pro- Toxic or carcinogenic substances adsorbed
vided the main food sources, but microorganisms from the microbial growth substrate (odd-
are also eaten in small quantities in such products numbered and branched-chain hydrocar-
as cheese, vinegar, mushrooms, koji, yeast, and bons, polycyclic aromatic compounds) may
blue-green algae (the species Spirulina is eaten be present. There is also the possibility that
in parts of Africa). microorganisms may produce highly toxic
In view of the insufficient world food supply substances (aflatoxins are produced by some
and the high protein content of microbial cells fungi, for example.).
(Table 16.1), the use of biomass produced in the Slow digestion of microbial cells in the diges-
fermenter would be an ideal supplement to con- tive tract may cause indigestion and allergic
ventional food supply. Single-cell protein is of reactions.
great nutritional value because of its high pro-
tein, vitamin, and lipid content and the general The following substrates are presently being
presence of a complete array of all essential studied for SCP production: alkanes, methane,

306
 16.2 PRODUCTION OF SINGLE-CELL PROTEIN FROM ALKANES 72307

Sore price structure is more critical than in more in-

ee
[rens Jr] [Pane]
dustrialized countries.

16.2 PRODUCTION OF SINGLE-CELL

PROTEIN FROM ALKANES
The use of longer-chained alkanes will be dis-
Hydrocarbon Solar energy
Carbon dioxide Carbon dioxide
cussed in this section, and gaseous methane, the
Energy simplest alkane, will be discussed in the next sec-
tion. Alkanes can be catabolized by many yeasts
Figure 16.1 Food chain of humans and by some fungi (for instance, fungi of the or-
ders Mucorales and Moniliales) and by some bac-
teria (partially through cooxidation). The follow-
methanol, cellulose, carbohydrates, and waste ing yeast species have been intensively studied
materials. for SCP production: Candida tropicalis, Candida
Cost of production will also be a deciding oleophila, and Saccharomycopsis lipolytica.
factor in determining the ultimate place of SCP The disadvantage of the use of alkanes is that
in the human or animal diet. It is clear that SCP they are not easily soluble. During growth in bio-
reactors with impellers or in airlift fermenters,
must compete with natural protein sources such
large alkane drops are formed which are 1-100
as soy meal or fish meal. The production costs
um in size and which remain suspended. Con-
will be strongly influenced by the nature of the
sidering the low water solubility of alkanes (sol-
raw starting material used. If a starting material
ubility 10-*-10~° v/v), the observed high growth
is used that must otherwise be disposed of as a
rates of microorganisms on alkanes cannot be
waste product, for instance, sulfite-waste liquor
explained merely by transport of alkane dis-
from the paper industry, then the raw material solved in water; other mechanisms of uptake
cost is actually negative. However, if a useful must be present. It seems likely that the cells
material such as natural gas is used, the cost of form emulsifying substances which convert the
the starting material will play a major role in the insoluble alkanes into droplets of 0.01-0.5 um.
final cost of the SCP. Another factor is that the Alkane molecules can then reach the cytoplasmic
price of SCP varies depending on how it is used. membrane through the cell wall via passive dif-
For instance, SCP for humans is about 10 times fusion. Cells growing on alkanes are enriched in
more expensive than SCP for animal feed be- lipids and it seems likely that these lipids play a
cause a more highly refined product must be role in the transfer of alkanes through the cell
used. Further, in less industrialized countries the membrane.

Table 16.1 Composition of crude single-cell protein (percent)

Alkane Methanol Protein Fungus Alga Soy Milk
yeast bacterium isolate meal powder
Raw protein 60.0 83 80 42 70 45.0 34.0
Fat 9.0 7.4 8-10 13 5 1.8 1.0
Nucleic acid 5.0 15 1-2 9.7 4
Mineral salts 6.0 8.6 8-12 6.6 7 6.0 8.0
Amino acids 54 65 40
Moisture 4.5 2.8 4.0 13.0 6 12.0 5.0
The values for soy meal and milk powder are given for comparison.
308 / CHAPTER 16 / SINGLE-CELL PROTEIN (SCP)

C,. Internal oxidation refers to subterminal oxi-

Catabolism of longer-chained alkanes
dation which does not take place in position C,.
The first step in the utilization of alkanes is the Organisms show specificity in which carbon
introduction of molecular oxygen into the mol- atom is attacked in the subterminal oxidation
ecule. There are two pathways for oxygen intro- process. Further catabolism of the secondary al-
duction: terminal oxidation and subterminal ox- cohol is variable. a-Oxidation with subsequent
idation (Figure 16.2). In terminal oxidation, the decarboxylation and B-oxidation has been found
corresponding monocarboxylic acid is produced in Candida. It is also possible for the molecule to
via the intermediate stages of the primary alcohol be converted to an ester which is then split with
and aldehyde. After this terminal oxidation, an esterase. The acid and the alcohol, which are
breakdown generally proceeds to acetyl-CoA products of the esterase action, are then metab-
units by means of 6-oxidation. Terminal oxida- olized further.
tion is the chief pathway of metabolism for bac-
teria and yeast. In some cases, terminal oxidation
Large-scale processes using yeast
at both ends of the molecule occurs by means of
w-oxidation, leading to the production of the cor- Two petroleum products have been used as start-
responding dicarboxylic acid, which is further ing material:
broken down into acetate units and succinate by
e Gas oil, also known as fuel oil or diesel oil,
means of 6-oxidation. The enzymatic mecha-
is a fraction derived from crude oil and con-
nisms of terminal oxidation are not yet fully
tains 10-25% C,;-C,, alkanes.
understood.
¢ C,.-C,; alkanes or C,,;-C,, alkanes are sepa-
In subterminal oxidation, the appropriate
rated from gas oil with molecular sieves.
ketone is first produced via a secondary alcohol.
This can either happen in the C, position or in British Petroleum Co. has developed pro-
the interior of the molecule, e.g. at C,, C,, C; or cesses using both of these substrates.

Gai (CH), ae CH,—CH,

at an Mao
Q
CHz=(CH)E= Choe COOH
Leven ann CH+(CH,),- C — CH;
em,” &-Oxidation

HOOC-(CH,),—CH-COOH 0
CHER == C —COOH

B-Oxidation B-Oxidation Decarboxylation

Acetate Acetate + Succinate CH+(CH,),— COOH + CO,

B-Oxidation

Acetate + Propionate

Figure 16.2 Alkane oxidation in Candida

 16.4 METHANOL FERMENTATIONS / 309

Gas-oil process Candida tropicalis was tested in lated, and thus far only relatively few methane-
the gas-oil process in a nonsterile, continuous utilizing bacteria have been identified. Among
system (16,000 tons/year) in Cap Lavera, France … the bacteria are Methylomonas methanica, Meth-
from 1973-1975. In Grangemouth (U.K.), Sac- ylococcus capsulatus, Methylovibrio soehngenii,
charomycopsis lipolytica (previously called Can- Methanomonas margaritae, and some unclassified
dida lipolytica) was grown aseptically with n-al- organisms.
kanes in a 300 må bioreactor in a system which The enzyme methane oxygenase oxidizes
produced 4000 tons SCP/year over a period of methane to methanol, which is further channeled
several years. A complete factory of 100,000 into the primary metabolism (see Section 16.4).
tons/year with three 1000 m? fermenters was
built in Sardinia for the alkane procedure, but
CH, + O, + XH, > CH;0H + H,0 + X
was subsequently not put into operation for po-
litical reasons.
The gas-oil fermentation was run in an airlift Methanol accumulates as a result of the oxidation
bioreactor with an increased aeration rate. Since process and inhibits the growth of bacteria. Pri-
alkanes make up only a small proportion of gas mary metabolites, such as amino acids, sugars,
oil, insoluble gas oil had to be fed repeatedly to or acetate, can also inhibit growth and methane
the growth medium. This resulted in poor mix- oxidation.
ing, poor oxygen transfer, and low yields. Using Methylococcus capsulatus, 0.4 g/1 dry
weight was obtained with a yield of 1.00-1.03 g
Alkane process In comparison to glucose, Can- dry weight/g methane.
dida tropicalis grows much more poorly on al- Since methanol is much easier to handle and
kanes (Um. = 0.28 as compared with 0.62 on can be produced chemically from methane,
glucose), but the yield is better (0.98 g cells/g methanol is the preferred starting material for all
alkane versus 0.51 g cells/g glucose) and the oxy- systems using C, substrate (see below).
gen uptake rate is similar (14 uM O,/g-h on al-
kane versus 11 uM O,/g°h on glucose).
16.4 METHANOL FERMENTATIONS
Methanol was at one time the most important
16.3 BACTERIA WHICH UTILIZE
substrate for single-cell protein production and
METHANE
extensive research on methanol-utilizing orga-
There is an excess of methane, the chief com- nisms was carried out. Although cost factors cur-
ponent of natural gas, in some parts of the world, rently rule out methanol as a starting material,
making this a desirable energy source for SCP it still has many advantages as a substrate for
production. Methane can be obtained as a very SCP production and changing economics could
pure gas. However, in contrast to higher hydro- easily bring it back.
carbons, methane cannot easily be liquefied, Methanol may be obtained from synthesis
making long-distance transport difficult and ex- gas, natural gas, methane, oil, or coal. Wood
pensive. Also, considerable security measures could theoretically also be used as a starting ma-
must be taken when handling methane, due to terial for methanol production. Bacteria, yeasts,
the risk of explosion. and fungi may all be considered for the produc-
Methane-oxidizing bacteria are classified tion of SCP from methanol (Table 16.2). Besides
among the obligate methylotrophs. This group the obligate methylotrophic bacteria which only
grows only on C, substrates (methane, methanol, grow on C, compounds, facultative methylo-
methylamine, formaldehyde, or formate). Yeasts trophic bacteria, yeasts, and fungi which metab-
which assimilate methane have not yet been iso- olize longer-chained hydrocarbons as well are
310 / CHAPTER 16 / SINGLE-CELL PROTEIN (SCP)

Table 16.2 Microorganisms that grow on methanol Yeasts oxidize methanol by means of a non-
1. Obligate methylotrophic bacteria specific, FAD-containing, inducible methanol ox-
Methylobacter Methylocystis idase. Methanol can also be oxidized by means
Methylococcus Methylosinus of H,O, with the peroxidase activity of an in-
Methylomonas
ducible catalase, the hydrogen peroxide being
2. Facultative methylotrophic organisms produced by methanol oxidase. Methanol oxi-
a. Bacteria dation to formaldehyde results in no energy gain
Arthrobacter Protaminobacter for the yeast.
Bacillus Pseudomonas
The next step is formaldehyde oxidation,
Hyphomicrobium Rhodopseudomonas
Klebsiella Streptomyces which in bacteria can be carried out by several
Micrococcus Vibrio enzymes:
b. Yeast
e Conversion of formaldehyde to formate with
Candida boidinii Pichia haplophila
Pichia lindnerii
reduced glutathione (GSH) by means of an
Candida parapsilosis
Hansenula capsulata Pichia pastoris NAD-dependent formaldehyde dehydrogen-
Hansenula henricii Torulopsis glabrata ase, a reaction which also occurs in yeast.
Hansenula minuta Torulopsis methanolovescens
Hansenula nonfermentans Torulopsis methanosorbosa
Hansenula wickerhamii Torulopsis molischiana GSH
HCHO + NAD+ H,O0 —> HCOOH + NADH,
Torulopsis memodendra

c. Fungi
Gliocladium delinquescens e A dichlorophenol-indophenol (DCPIP)-de-
Paecilomyces varioti
Trichoderma lignorum pendent formaldehyde dehydrogenase.
e An unspecific methanol dehydrogenase.
(Sahm, 1979)
Two to three ATP per mole of substrate are
obtained at this oxidation level.
included in this list. In contrast to many bacteria, The last step, the oxidation of formate, is
yeasts are unable to use any C, compounds other common: to all the methanol-utilizing microor-
than methanol. ganisms. Formate oxidation involves a NAD-de-
In methanol fermentation for SCP produc- pendent formate dehydrogenase and yields 3
tion, bacteria rather than yeasts are employed in ATP per mole of substrate:
essentially all existing production processes for
the following reasons: rapid growth (Table 16.3),
higher protein content, better yields, and simpler HCOOH + NAD — CO, + NADH,

culture medium requirements.

Table 16.3 Maximal specific growth rates (4) of

Methanol oxidation various methanol utilizers

Microorganism KEANE)
Methanol is oxidized to CO, by bacteria via the
following intermediate steps: Pseudomonas rosea (ICI) 0.38—0.50
Protaminobacter ruber 0.10
Pseudomonas extorquens 0.18
CH,0H — HCHO — HCOOH — CO, Methylomonas methanolica 0.53
Pseudomonas B45 0.198
Kloeckera sp. 2201 0.075
Hansenula polymorpha 0.22
The first step to formaldehyde requires an in- Torulopsis glabrata 0.11
ducible nonspecific methanol dehydrogenase. (Braunegg, 1975)
 16.4 METHANOL FERMENTATIONS / 311

In some obligate methanol-utilizing bacteria (for CH,0H—— HCHO

example, Methylomonas M 15), formaldehyde de-
hydrogenase and formate dehydrogenase are not
found. Instead, these organisms oxidize formal- Ri bulose-5-P 3-Keto-6P-Hexulose
dehyde by means of the phosphogluconate path-
CO,
way (Figure 16.3).
NAD(P)H,

Carbon assimilation by methanol- Fructose-6-P

oxidizing organisms
NAD(P) i
Bacteria growing on methanol must produce C,
molecules which then feed into primary metab-
olism as pyruvate. Three distinct pathways for 6-P-Gluconic acid Glucose-6-P
one-carbon assimilation have been recognized:
. CO, may be taken up in photosynthetic or-
ganisms via the ribulose diphosphate (Cal- | NAD(P)H, NAD (P)

vin) cycle. Assimilation

+ Formaldehyde may be condensed with ri-

Figure 16.3 Phosphogluconate pathway
bulose-5-P in the ribulose monophosphate
cycle (Quayle cycle). The enzyme hexulose
phosphate synthase (HUPS) carries out this regenerated in the ribulose monophosphate
reaction (Figure 16.4). After isomerization of cycle through transketolase and transaldolase
3-keto-6-P-hexulose to fructose-6-P, either reactions. HUPS, the key enzyme of the cy-
dihydroxyacetone-P is produced through gly- cle, is partly constitutive, partly inducible by
colysis or pyruvate is produced through the methanol, and is repressed by formaldehyde.
Entner-Doudoroff pathway. Ribulose-5-P is e In the serine pathway, condensation of for-

3 Ribulose-5-P 3 3Keto-6P-Hexulose Fructose-6-P

Glucose-6-P
3 Fructose-6-P
NADP
Fructose-1,6di-P
NADPH2
3 Pentose-5P 2-Fructose-6
-P
6-P-Gluconate
Transaldolase

Transketolase —— Dihydroxyacetone-P SEE

Reactions Glyceraldehyde-3-P

Figure 16.4 Ribulose monophosphate (Quayle) cycle

312 / CHAPTER 16 / SINGLE-CELL PROTEIN (SCP)

Methanol Formaldehyde A pathway very similar to the ribulose mon-

ophosphate cycle, the dihydroxyacetone cycle,
Glycine L-Serine has been found in yeasts (Figure 16.6).
Glyoxylate Table 16.4 shows theoretical yield coefficients
Acetyl-CoA for bacteria and yeasts that use the various one-
Malyl-CoA Hydroxypyruvate
carbon pathways. It is clear from this table that
bacteria using the ribulose monophosphate path-
ADP+P. NADH,
way are the best producers; therefore, all com-
ATP CoA NAD
mercial processes are conducted with this group.
Malate Glycerate

NAD ATP:
Production processes
NADH3 ADP
Imperial Chemical Industries (ICI) was the first
Oxalacetate 2-P-Glycerate
company to develop a continuous methanol fer-
mentation for the commercial production of SCP.
CO,
They studied the effect of O,, CO,, and methanol
Phospho-
enolpyruvate
concentration on productivity and the effect of
the pressure differential between the bottom and
Figure 16.5 Formaldehyde fixation via the serine path- the surface of the bioreactor. The ‘ICI Pressure
way Cycle Fermenter’’, a combination of air lift and
loop reactor, is illustrated in Figure 16.7. This 37
maldehyde and glycine takes place through må, 30 m-high pilot fermenter consists of 3 units:
the action of serine transhydroxymethylase air lift column (I), down-flow tube with heat re-
(Figure 16.5). moval (II), and gas release space (III). The pilot

Xu-5P

GAP DHAP GAP

Biosynthesis

Figure 16.6 Formaldehyde

Transketolase and transaldoase reactions fixation in yeast via the dihy-
droxyacetone pathway
 16.4 METHANOL FERMENTATIONS / 313

Table 16.4 Yield coefficients of microorganisms fermenter and the cells are spray-dried. Based on
growing on methanol
the results of this pilot study, ICI invested £40
Microorganism Pathway Y, (g cell million in 1979 to install a continuous culture
dry weight/
g methanol)
system with a capacity of 50,000-70,000 tons/
year, which began operation in 1980. In this fer-
a. Bacteria
menter, which had a volume of 1000 m3, culti-
Pseudomonas C RMP 0.54
P. methylotrophus RMP 0.53 vation of the inoculum could be carried out right
Methylomonas RMP 0.49 in the fermenter.
methalonica Although the original chemostat process was
Pseudomonas AM 1 SER 0.30
Pseudomonas M 27 SER ; 0.41 methanol-limited, it was later operated as a ni-
Pseudomonas rosea SER 0.41 trogen-limited system. With the bacterial strain
b. Yeasts originally used, NH,-assimilation occurred via
Candida boidinii DA 0.32
two inefficient enzyme systems:
Hansenula polymorpha DA 0.38
RMP Ribulose monophosphate cycle, SER Serine Glutamine-Ketoacid Transaminase (GOGAT)
pathway, DA Dihydroxyactone cycle a -Ketoglurate + NAD(P)H + Glutamine — 2 Glutamate
(Sahm, 1979) + NAD(P)
Glutamine synthetase
Glutamate + NH; + ATP — Glutamine + ADP + P;

plant system, which had a capacity of 1000 tons/

The more effective glutamate dehydrogenase
year, operated at pH 6.5-6.9 and 34-37°C. The
system from Escherichia coli was therefore trans-
organism used for industrial SCP production is
ferred to a GOGAT- mutant of Pseudomonas
the methanol oxidizer Pseudomonas methylotro-
methylotrophus by means of genetic engineering,
phus, which was isolated by ICI and then sig-
and such strains, which presumably grow more
nificantly improved through genetic and phys-
rapidly due to improved nitrogen assimilation,
iological development (Table 16.5).
were placed in commercial production. However,
In the product recovery process, partial cell
the yield increase amounted to only 3%.
lysis is first achieved via heat and acid treatment
Because of difficulties in removing the cells
and the nutrient solution is then clarified by de-
from the liquid, the completely automatic large-
canting. The water is then recycled back into the
scale process for SCP was never placed in full
production due to economic reasons. For in-
) stance, in 1984 the price of soy meal was around
mm
“——______ Nutrient solution surface
Waste gas) $125-190 per ton, whereas the SCP from ICI
= Product removal
(going under the trade name Pruteen) was being
ua Sieve plate
sold at $600 per ton!

Table 16.5 Results of the optimization of the ICI

fermentation process for growing Pseudomonas
methylotrophus on methanol
Heat Raw protein 83% — 85%
exchanger
Pure protein 59% — 64%
Cell dry weight 4 — 30g/1
Nutrient = Aeration
solution >
[te 0.38h? > 05h?
xe — 0.5 g/g

Figure 16.7 ICI pressure cycle fermenter (Gow et al., 1975)

314 / CHAPTER 16 / SINGLE-CELL PROTEIN (SCP)

16.5 SINGLE-CELL PROTEIN FROM Cellulase is an enzyme complex consisting of

at least three enzymes:
WOOD
e Endo-@-1,4-glucanase, also called endocel-
At the present time cellulose from natural sources
and waste wood is still an attractive starting ma- lulase, carboxymethyl cellulase (CMCase), or
terial for single-cell protein production as well as C, cellulase.
a potential source for production of fermentation e Exo-@-1,4-glucanase, also called cellobiohy-
ethanol. Although there is an abundance of cel- drolase, avicelase, or C, cellulase. C, cellulase
lulose on earth, it is usually mixed with sub- comprises 80% of the cellulase complex dur-
stances such as lignin, hemicellulose, starch, pro- ing fermentation with T. viride.
tein, and salts (Table 16.6). Therefore, cellulose + $-1,4-glucosidase, or cellobiase. The extra-
sources must be pretreated physically and chem- cellular concentration of these cellobiose-
ically in order to break down the cellulose into splitting enzymes is low, with the bulk of the
fermentable sugars. Pretreatment may either be enzyme, in T. viride, being intracellular.
enzymatic (cellulases) or chemical (acid hydrol-
ysis). We discuss here the enzymatic processes
Current status of cellulase research
for cellulose hydrolysis. Great advances are
being made in the breakdown of lignin, and lig- Enzyme production has been optimized but cel-
ninase enzymes can now be produced by sub- lulase yields are still low. The effects on Tri-
merged fermentation. choderma viride of pH control, inoculum age, and
culture medium additives have been tested in a
basal medium containing KH,PO, 0.2%,
Physiology (NH,),SO, 0.14%, urea 0.03%, MgSO,°7 H,O
Only extracellular cellulases can be used com- 0.03%, CaCl, 0.03%, FeSO,°7 H,0'5.0 mg/L
mercially. Such cellulases are excreted by both MnSO,°H,O 1.6 mg/l, ZnSO, 1.4 mg/l and
bacteria (Cellulomonas and actinomycetes) and CoCl, 2.0 mg/l. Maximal yields were obtained
fungi (Trichoderma, Penicillium, Thermoascus, at 30°C after 140 hours’ growth. As seen in Fig-
Sporotrichum, and Humicola). For pilot plant stud- ure 16.8, yields are dependent on the cellulose
ies, the following fungi have been used: Tri- concentration. Purification of the enzyme was
choderma reesei (T. viride), T. koningii, and Spo- carried out using ion exchangers.
rotrichum pulverulentum. Based on pilot plant experiments, computa-
tions can be made for the production of cellulase
and for sugar production from cellulose with cel-
lulases. Production costs have been quoted at
$0.011/1 for crude culture broth and $0.11/1 for
Table 16.6 Composition of cellulosic substrates purified enzyme solution. Depending on the pro-
cess and the extent of cellulose breakdown, the
Cellulose Hemi- Lignin
% cellulose % production cost to make fermentable sugar from
%
cellulose ranges from $0.037-0.489/kg sugar.
Wood (Angiosperms) 40-55 24-40 18-25 The higher price corresponds to the world market
Wood 45-50 25-35 25-35 price of sucrose in 1980. Obviously, it would not
(Gymnosperms) be feasible to use the higher-priced sugar equiv-
Grasses 25-40 25-50 10-30
Leaves 15-20 80-85 — alents as substrate for the production of SCP
Newspaper 40-55 25-40 18-30 which itself entails a considerable production
Waste from paper 60-80 20-30 2-10 cost. Before SCP can be economically produced
manufacture
from cellulose, cellulase yields must show a dras-
 16.7 SCP FROM SEWAGE / 315

lution rate of 0.1 h. Mineral salts (Na-, K-, Mg-,

Mn-, Cu-, Ca-, Co-ions) and biotin are added,
with ammonium ions serving as the nitrogen
source. The pH is regulated at 6.0 and the in-
cubation temperature is 30°C. From 1 kg of glu-
cose 1 kg wet weight of fungal mycelium is ob-
tained, with a protein content of 136 g. The yield
is 3—3.5 kg/m%h. The culture taken directly
from the fermenter is heated to 64°C to inactivate
proteases and to activate endgenous RNases. Af-
on
hae
Ww
D
N(=)
oO
OD
oO
ter 30 min heating, the ribosomal RNA content
is reduced from 9 to 1.1% and the breakdown
products (principally 5’ nucleotides) diffuse from
Enzyme
(units/ml)
concentration
ro) the cells. The dried protein is eventually formed
into structures that resemble pork, chicken, or
beef meat and an appropriate artificial flavor
AO RER GO LU. = 140 added. For test marketing, ICI has used fermen-
Fermentation time (hr) ters of 2 X 1.3 m? size, with scale-up in pilot size
of 30 m°. The final product is intended to be
Figure 16.8 Effect of cellulose concentration on cellulase produced in the same fermenters that ICI used
production in Trichoderma viride QM 9414. Squares, for Pruteen production (see Section 16.4).
0.94% cellulose; circles, 2.55% cellulose; triangles, 5.04%
cellulose (Nystrom and DiLuca, 1978)
16.7 SCP FROM SEWAGE
tic increase, less expensive pre-saccharification Domestic sewage is at present not suitable for
wood treatment methods must be developed, large-scale SCP production but may be more im-
and energy-saving measures must be imple- portant for methane production in the future
mented throughout the process. (Section 17.5). Thus far, industrial waste waters
An alternate possibility is the direct fermen- from cellulose processing, coffee production,
tation of pretreated lignocellulose-containing starch production, and food processing have
materials with mixed cultures. The resulting bio- been used for SCP production. Sulfite waste li-
mass and the unmetabolized substrate can be quors from paper and cellulose production have
used directly as animal feed. also been extensively used to produce SCP for
the following reasons: Consistent availability of
large quantities, low investment and production
16.6 SCP FROM CARBOHYDRATES
costs, and availability of organisms capable of
Large-scale cultivation of yeasts on molasses is high growth rates on the substrate.
widely used in the manufacture of baker's yeast, Appropriate microorganisms for growth on
and in smaller-scale for food yeast and for the sulfite waste liquor are Candida utilis, C. tropi-
production of wine yeasts. In addition, several calis, Chaetomium cellulolyticum, and Paecilo-
large-scale yeast processes employ whey (whose myces varioti.
principal sugar is lactose) as a starting material. A continuous process was used in a Finnish
A promising product is a mycoprotein which plant with two 360 m? fermenters. The SCP
was introduced on the market in England in yields were 2.7-2.8 kg/m°-h using Paecilomyces
1986. The fungus Fusarium graminearum is grown at dilution rates of 0.2 h”. With a solution con-
completely continuously on glucose with a di- taining 32 g/l reducing sugar, 55% of the sugar
316 / CHAPTER 16 / SINGLE-CELL PROTEIN (SCP)

was converted into biomass. This installation is Leisola, M., V. Thanei-Wyss, and A. Fiechter. 1985. Strat-
egies for production of high ligninase activities by Pha-
no longer in operation. nerochaete chrysosporium. J. Biotechnol. 3: 97-107.
In Czechoslovakia a SCP factory producing Litchfield, J.H. 1985. Bacterial biomass. pp. 463-481. In:
25,000 tons per year is operating using the ef- Moo-Young, M. (ed.), Comprehensive Biotechnology,
Volume 3, Pergamon Press, Oxford.
fluent from a paper-manufacturing facility. The Magee, RJ. and N. Kosaric. 1985. Bioconversion of hem-
so-called System Paskov uses Candida utilis in 3 icellulosics. Adv. Biochem. Eng./Biotechnol. 32: 61-
x 800 m° fermenters, operated continuously 93:
with a capacity of 3 X 1.5 tons/h. Product re- Nystrom, J.M. and A.L. Allen. 1976. Pilot scale investi-
gations and economics of cellulase production. Bio-
covery involves two concentration steps in a sep- technol. Bioeng. Symp. 6: 55—74.
arator (to 18% and 25%), followed by drying. In Nystrom, J.M. and P.H. DiLuca. 1978. Enhanced produc-
this installation, SCP production is an ancillary tion of Trichoderma cellulase on high levels of cellulose
in submerged cultures. Proc. Bioconversion Symp. IIT
result of waste-water stabilization and purifica- Delhi, pp. 293-304.
tion. Oura, E. 1983. Biomass from carbohydrates. pp. 3-41. In:
Rehm, H.J. and G. Reed (editors), Biotechnology, Vol-
ume 3, VCH Publishers, Deerfield Beach, FL.
REFERENCES Rehm, H.J. and I. Reiff. 1981. Mechanisms and occurrence
of microbial oxidation of long-chain alkanes. Adv.
Braunegg, G. 1975. Methanol—eine neue, billige Kohlen- Biochem. Eng. 19: 175-215.
stoffquelle in der Fermentationstechnik (Methanol—a Romantschuk, H. and M. Lehtomaki. 1978. Operational
new inexpensive carbon source for use in large-scale experiences of first full scale Pekilo SCP-mill appli-
fermentation), pp. 218-234. 1. Arbeitstagung Biotech- cation. Proc. Biochem. 13: 16-17,29.
nologie in Osterreich. Sahm, H. 1979. Production of SCP by methanol utilizing
Busche, R.M. 1985. The business of biomass. Biotechnol.
microorganisms. Int. Microbiol. and Food Ind. Congr.,
Progress 1: 165-180.
Paris.
Fukui, S. and A. Tanaka. 1981. Metabolism of alkanes by
Smith, A.J. and D.S. Hoare. 1977. Specialist phototrophs,
yeasts. Adv. Biochem. Eng. 19: 217-237.
Gow, J.S., J.D. Littlehailes, S.R.L. Smith, and R.B. Walter. lithotrophs and methylotrophs: a unity among a di-
1975. SCP-production from methanol: Bacteria, pp. versity of procaryotes? Bacteriol. Rev. 41: 419-448.
370-384. In: Tannenbaum, S.R. and D.I.C. Wang Solomons, G.L. 1985. Production of biomass by filamen-
(eds.), Single cell protein, vol. II. MIT Press, Cam- tous fungi. pp. 483-505. In: Moo-Young, M. (editor),
bridge, MA. Comprehensive Biotechnology, Volume 3, Pergamon
Harwood, J.H. and S.J. Pirt. 1972. Quantitative aspects of Press, Oxford.
growth of the methane oxidizing bacterium Methylo- Tanaka, M. and Matsuno, R. 1985. Conversion of ligno-
coccus capsulatus on methane in shake flasks and con- cellulosic materials to single-cell protein (SCP): recent
tinuous chemostat culture. J. Appl. Bacteriol. 35: 597- developments and problems. Enzyme Microb. Tech-
607. nol. 7: 197-206.
 17
Newer |
approaches to
sewage
treatment

nisms, environmental pollution due to odor

17.1 INTRODUCTION
emission, and fog formation.
In urbanized countries, vast amounts of indus- The developments in sewage treatment over
trial and domestic sewage are treated biologi- the past century have been carried out primarily
cally. At present, sewage is treated primarily by in an empirical fashion, but aerobic and anaer-
the classic, aerobic, activated sludge process with obic sewage treatment processes are being in-
surface aeration (Figure 17.1) or with oxygen creasingly studied scientifically, since it is felt
supplied by forced air. In another type of aerobic that optimization will lead to the greatest success
treatment process (the trickling filter), a bed of in improving these processes. Although studies
stones or sand is used and the waste water is on aerobic processes have been primarily di-
allowed to trickle down over the supporting me- rected to an understanding of carbon metabo-
dium, the organisms growing attached to the sur- lism, in the future it is anticipated that research
faces of the filter bed and oxidizing the organic will be directed at sulfur, nitrogen, and phos-
compounds in the waste material. In another pro- phate metabolism, since elimination of these ele-
cess, anaerobic digestion is used (Figure 17.2), ments from sewage will become increasingly im-
occasionally directly on raw sewage or more fre- portant. Further, accumulations of heavy metals
quently on the solid material (sludge) obtained in the stabilized sludge are being increasingly
from sedimentation in the aerobic treatment pro- recognized to be of serious concern.
cesses. A further critical problem is the phenomenon
Aerobic systems have the following disad- of bulking in sludge, which inhibits the settling
vantages: open construction which restricts pro- process in the clarification basins. Bulking is due
cess control, uncontrolled populations of orga- to the development of filamentous microorga-

O17
318 / CHAPTER 17 / NEWER APPROACHES TO SEWAGE TREATMENT
; Air
Primary Secondary
sedimentation ! sedimentation
Raw basin basin
sewage

Sludge recycle

Aeration Sewage

Figure 17.1 Activated sludge process:

above, aeration basin; below, oxidation
S = Sedimentation basin | Exit ditch

nisms that do not settle well. Although bulking markable decrease in organic material in indus-
can be controlled in some cases by introduction trial effluents. This has resulted from extensive
of flotation processes, in domestic sewage this application of research and development efforts
technical solution is too expensive. Therefore, a and considerable expenditures for new treatment
microbiological solution to the bulking problem installations.
must be found so that a satisfactory clear effluent In this chapter four new processes will be
can be delivered from the final settling basin. discussed which have the potential for increasing
One of the most encouraging developments the efficiency of sewage treatment.
over the past several decades has been the re-
17.2 STARTER CULTURES FOR
TREATMENT PROCESSES
Gas removal
Conventional sewage treatment involves the use
of microorganisms which develop naturally
within the sewage treatment system, no attempt
being made to optimize the organisms involved.
An approach which may have some potential for
increasing the efficiency of the sewage treatment
process is to inoculate the system wih microor-
ganisms which have been specially selected for
Recycle the particular sewage-treatment process. In anal-
ogy with their use in food fermentations, such
organisms might be called ‘starter cultures”. Al-
though starter cultures might find use in the
treatment of domestic sewage, it seems more
likely that they will find use in the treatment of
special or unusual industrial wastes or in the
treatment of accidental spills of industrial chem-
Figure 17.2 Sludge digester icals. Such wastes often cannot be channeled into
 17.3 AEROBIC SEWAGE TREATMENT / 319

ordinary treatment plants because their toxicity dividual compounds are underway, but practical
and lack of biodegradability cause significant applications are not yet widespread.
damage to the nonadapted organisms. Another
way in which such starter cultures might be used
is in shortening the start-up time that is generally 17.3 AEROBIC SEWAGE TREATMENT—
required after a sewage treatment plant is shut AIRLIFT PROCESS
down for one or another reason. Before the sys- A disadvantage of customary aerobic sewage
tem can become fully operative once again, the treatment by the activated sludge system is the
optimal bacterial culture mixture must be re-es- low efficiency of oxygen transfer and the large
tablished; this usually requires aweek-long en- amount of space required by ‘the installation. Be-
richment process. A starter culture could be ex- cause these installations are open to the atmos-
pected to shorten this start-up time. In the United phere, another problem is the odor which they
States, starter cultures resembling those of the emit. Tower reactors have been developed in
dairy industry have been developed and are suit- which aeration and oxygen utilization are im-
able for a variety of special applications in sew- proved and the efficiency of the overall process
age treatment, such as tank cleaning, pipeline increased. Both tube reactors and airlift fermen-
cleaning, start-up of city purification plants, and ters have been used. These installations are fa-
breakdown of special substances contained in vorable economically because of 30% less space
sewage. required, 20% lower investment costs, and 20%
Bacteria from cold habitats have been isolated less energy costs.
which degrade alkanes and aromatic compounds The British chemical company ICI uses a tu-
at 0-15°C in saline habitats; these could be useful bular loop reactor which is embedded 100 meters
in the degradation of oil spills in the ocean. into the ground. Two German companies, Bayer
Mixed cultures which metabolize DDT, poly- (Biotower) and Uhde/Hoechst (Bio-high Reac-
chlorinated diphenols, and phenols or which tor) have constructed bioreactors 30 m in height.
possess high protease, lipase, or cellulase activity In these systems, the circular settling basins are
are also on the market. If special operating con- located around the top rim of the bioreactor (Fig-
ditions are desired, such as growth at pH<5.0 ure 17.3). The dimensions of this type of acti-
or = 9.0, these requirements can also be met with vated sludge fermenter allow a considerably bet-
selected enrichment cultures. ter oxygen-transfer efficiency. Table 17.1 shows
A patented process has been developed with performance data from various systems of the
a strain of Pseudomonas putida containing plas-
mids which code for the breakdown of octane, Gas out
xylene, metaxylene, and camphor. Starter cul-
Process regulator
tures have also been used to deodorize animal
excrements.
In 1978, the starter-culture industry grossed
$2-4 million in the United States, but the poten- Tower-shaped Clarified
tial total value is estimated at $200 million. reaction vessel effluent
Twenty manufacturers produce single strains or
mixed cultures under sterile conditions in vessels
up to 10 m/ capacity. The culture conditions used
are determined by how the cultures are to be
used, in order to ensure that the cells are fully Gas sparger
induced. Research in this field is still in its infancy
and scientific studies on the breakdown of in- Figure 17.3 Bayer Biotower
320 / CHAPTER 17 / NEWER APPROACHES TO SEWAGE TREATMENT

Table 17.1 Data from an activated sludge system

(Uhde/Hoechst)
20mm
Water depth (m) 4 8 20
O,-Saturation 13 iy 28
(g 0,/m?)
Oxygen yield (%) 8-15 15 -30 40-60

|
Air required 42-23 23——11 9—6
(Nm? /kg O,)
Energy-specific O, 2- 2.5 25-13 3-39
input (kg O,/kWh)
Bioreactor diameter 18 13 8
(m)
Reactor surface area 250 125 50
for 1000 m3
volume (m7?)

Table 17.2 Parameters for a Bayer Biotower in

continuous operation
Process Parameters

Pretreatment Screening, neutralization,

primary sedimentation
Bioreactors 4 parallel, rubber-lined tanks
(26 X 30 m)
with 13,600 m3 total volume
Aeration 22,000 m3/h
Sewage Figure 17.4 Bayer injector (Zlokarnik, 1985)
From industry 90,000 m3/d with 95 tons/d
BOD
From domestic 70,000 m3/d with 14 tons/d
sources BOD United States and the Federal Republic of Ger-
Residence time 14.5h many. The objective has been to increase the ef-
Treatment capacity 105 tons/d BOD
Oxygen requirements 120 tons/d O, ficiency of oxygen transfer, since the loading ca-
pacity of a conventional purification plant and
the size of the microbial population are limited
Uhde/Hoechst type and Table 17.2 shows data by the low solubility of dissolved oxygen.
for the Bayer system. Pure oxygen has 4.8 times the partial pressure
The Hoechst system has baffles to facilitate of oxygen from the air, so that aeration with pure
mixing. In the Bayer Biotower process, two-com- oxygen markedly increases the oxygen content
ponent nozzles have been developed as injectors of the activated sludge system. Dissolved oxygen
by means of which the kinetic energy of the liq- in such a system is 90-95% utilized, and the off
uid stream is utilized to disperse air in small gas gas (1% of that from conventional aeration) can
bubbles in a manner similar to a water spray subsequently be treated chemically or thermally
pump (Figure 17.4). These injectors are installed in the closed system in order to prevent unpleas-
in groups of four (injector clusters) above the bot- ant odors.
tom of the tower reactor (1 injector/1-2 m? bot- High loading capacity in the oxygen process
tom surface).
allows smaller aeration tanks and smaller settling
basins. Due to the smaller amount of sludge pro-
17.4 AERATION WITH PURE OXYGEN
duced, the costs of facilities for further treatment
Aeration of activated sludge treatment facilities are also reduced. On the negative side is the
with pure oxygen has been tried in both the higher capital investment, the more complicated
'
 17.5 METHANE PRODUCTION / 321

installation, the necessity for careful controlof flows through this second treatment stage to re-
the process parameters, and the need for highly move material which is not easily broken down
trained personnel. (Figure 17.6). After a third treatment stage, the
In both domestic and industrial installations clarified waste water proceeds to the receiving
systems are in use that employ pure oxygen. stream. Table 17.3 gives performance character-
These systems are especially used in the paper, istics using the Unox process.
chemical, and food industries.
The Unox system is one such example. The
17.5 METHANE PRODUCTION
first aeration stage consists of 2 channels, each
of which is connected in a cascade arrangement Methane, or natural gas, is a microbial product
to three bioreactors (Figure 17.5) with capacities of the anaerobic decomposition of organic mat-
of 1500, 750 and 730 må. In the first bioreactor ter. Methanogenesis is a widely used process in
(activated sludge basin), pure oxygen from an air- organic waste disposal, primarily because the
separation plant is forced in via injectors. In this methane gas, being insoluble, is readily removed
stage, 90-93% of the organic, decomposable ma- from the treatment system. Since it is a fuel, the
terial is eliminated. The CO, produced is col- methane produced in the treatment system can
lected along with the residual oxygen and treated also be used as a source of energy. In some ag-
at 1000°C. In the second treatment stage (settling ricultural situations, the methane formed is a sig-
basin), the accumulating sludge is pumped back nificant energy source (biogas) and the main goal
into the activated sludge basin. The waste water of the treatment process is the maximization of

Oxygen

Figure 17.5 Diagram of an activated sludge system using pure oxygen

322 / CHAPTER 17 / NEWER APPROACHES TO SEWAGE TREATMENT

Table 17.3 Data from a Unox purification plant with

| pure oxygen
e
Amount of sewage 20,000 m3/day
| Chemical oxygen demand 61 tons/day
1000 Raw material
(COD)-breakdown
| capacity
Biological oxygen demand 34 tons/day
(BOD);-breakdown
capacity
Oxygen input
Activation stage 1 51 tons/day
Activation stage 2 7 tons/day
= | Volume
E VEX Activation stage 1 6200 m°
S 5007 Activation stage 2 3200 m°
& Exit; ee Intermediate purification: 4400 m°
sko) J Final purification 4800 m?
Time required
Activation stage 1 7.5h
| @ Activation stage 2 3.8h
Intermediate purification: 5.3h
Final purification 5.8h
Breakdown activity
| Exit; stage 2 Activation stage 1 max. 80% COD
. oe
max. 93% BOD,
aa a Activation stage 2 max. 7% COD, total 87%
1 2 3 4 5 6 7 8 max. 5% BOD,, total 98%
Sludge load
Day
Activation stage 1 0.7 kg BOD. /kg dry
weight:d
Figure 17.6 Methanol breakdown in a two-stage puri- Activation stage 2 0.2 kg BOD, /kg dry
fication plant using pure oxygen (Mack, 1973) weight-d
Space requirement
Activation stage 1 5.6 kg BOD,/m*d
methane yields. Anaerobic installations for sew- Activation stage 2 0.8 kg BOD,/m?-d
age treatment are called digestors, because the Excess sludge 25% of the BOD,
primary process taking place is the digestion of load
the organic material to soluble and gaseous con- (Lemke and Mack, 1978)
stituents. In most waste treatment systems, how-
ever, even though the methane may be used as
a fuel, the main goal has not been the production
ergy expenditure for aeration is not required, sav-
of methane as an energy source but rather the
ing considerably on operating costs. The organic
stabilization of the sludge so that it can be safely
waste is converted almost quantitatively (>90%)
disposed of in the environment. In such systems,
to methane and CO,. Biomass formation is low,
little attention has been given to the optimization
of the methane production process. In one study so that the cost of sludge disposal is lower. Also,
of 275 sewage treatment systems in western Eu- odor problems are decreased since the process is
rope, only a small fraction of the methane pro- carried out in closed reactors. Finally, the meth-
duced was distributed to other users, although ane produced can be utilized as an energy source.
75% of the gas produced was used as an energy However, the anaerobic process is not suit-
source within the treatment plants themselves. able for the more dilute liquid wastes that are
In comparison to aerobic processes, anaerobic primarily treated by the aerobic activated sludge
process have some significant advantages. En- processes.
 17.5 METHANE PRODUCTION / 323

The methanogenic bacteria constitute a The third group of organisms are the ex-
unique group of organisms which are the final tremely anaerobic methanogens. As noted, only
and key link in the breakdown of organic matter a limited range of substrates are used by these
in the anaerobic food chain. The methanogenic bacteria. Most methanogens will use H, as en-
bacteria are able to utilize only a restricted group ergy source and CO, as carbon source and elec-
of substrates for the production of methane, in- tron acceptor. A few species of morphologically
cluding: acetate, methanol, formate, and H, + diverse groups (cocci, sarcinae, and spirillae) also
CO,. In waste disposal systems, about 75% of metabolize formic acid, methanol, or methylam-
the methane is derived from acetate and most of ine. The most important organic substrate of
the rest from H, + CO,. The starting materials methanogens is acetic acid, which is fermented
of the anaerobic decomposition process are com- in a reaction that is only weakly favorable en-
plex polymeric materials such as cellulose, starch, ergetically:
fats, and proteins, none of which the methano- CH,COO- + H* — CH, + CO,
genic bacteria are able to use. The methanogenic AG > 37k]
bacteria are consequently dependent upon other Because of this, these bacteria grow very
anaerobic fermentative organisms for the initial slowly and are the most critical organisms in the
breakdown of the substrates and the production mixed culture of the anaerobic digestor.
of acetate and H, + CO... Because of the low biomass formed and the
Three groups of microorganisms participate low energy yield, the rate of the digestion process
in the anaerobic process. The first group breaks is low, the average residence time in the digestor
down the original organic material (starches, fats, being greater than 20 days. Immobilization of
proteins) into organic acids (propionic acid, bu- microorganisms in a fixed-bed reactor can be
tyric acid, acetic acid, lactic acid, and valeric acid), used to increase greatly the biomass concentra-
alcohol, H,, and CO,. Since one of the constit- tion. Solutions with very high organic loads
uents of the initial load, lignin, is not broken
(chemical oxygen demand >3 g O, consump-
down anaerobically, any lignocelluloses in the tion/l) can be treated with such fixed-bed reac-
starting material are metabolized slowly. The tors within a few hours. Some studies have been
group of bacteria involved in this initial break-
carried out using porous sintered glass as carrier
down of organic matter include obligate anaer-
material for fixed-bed reactors. In a 1000 I reactor
obes such as the clostridia and facultative an-
the biochemical oxygen demand (BOD) was re-
aerobics such as the streptococci and enteric
duced from 6.4 kg/m: to 1.3 kg/m: in 4.8 hours.
bacteria.
In another installation for treating high-concen-
The second group of bacteria convert the
tration wastes from a fermentation plant, a two-
longer-chain fatty acids (for instance, propionic
stage process was used, the first reactor being
and butyric acids) and alcohols to acetic acid, H,,
used for acid production, the second for meth-
and CO,. This conversion is endergonic at pH 7
anogenesis. Both reactors were 380 m? in volume
and can only occur if coupled with exergonic re-
and sand was used as the carrier. Waste volumes
actions. Thus, the reaction only occurs in mixed
of 150-200 m3/h were treated within 1—1.5 hours
cultures. For example, ethanol is converted to
acetic acid and H, by one bacterium and in a with a removal efficiency of 30 kg COD/m>-day.
second reaction a methane bacterium utilizes the In the United States, some studies have been
H,, thus pulling the reaction: done to produce methane from the biomass of
the water hyacinth, a plant which causes con-
CH,CH,OH + H,O — siderable problems by excess growth in canals
CH,COO- + H* + 2H, and streams in the warmer parts of the country.
4H, + CO, — CH, + 2H,O Under optimal conditions, about 2 tons dry mass
324 / CHAPTER 17 / NEWER APPROACHES TO SEWAGE TREATMENT

per day and per hectar could be treated, yielding REFERENCES

theoretically 800 m° biogas.
When optimizing methanogenesis, it is im- Aivasidis, A., H.J. Brandt, A. Joeris, J. Kriese, R. Pick, and
C. Wandrey. 1986. Recent developments in process
portant to note that the rate is affected by the and reactor design for anaerobic waste water treat-
nature of the substrate as well as by the tem- ment. Biotech Forum 3:141-150.
Barnard, G.W. and D.O. Hall. 1983. Energy from renew-
perature (Table 17.4). able resources, pp. 592-625. In: Rehm, H.J. and G.
With mixed sludges, as much as 600 | biogas Reed (eds.). Biotechnology 3. Verlag Chemie, Wein-
per kg dry organic matter have been produced. heim.
Bishop, P.L. and N. E. Kinner. 1986. Aerobic fixed-film
The gas consists of about 60-70% methane, processes, pp. 113-176. In: Rehm, H.J. and G. Reed
about 25-30% CO,, and the rest H, and N,. De- (eds.). Biotechnology 8. Verlag Chemie, Weinheim.
Chakrabarty, A. 1974. US Patent 3,813,316.
veloping countries represent the most promising Dohne, E. 1985. Biogasverwertung, Biogasmotoren und
areas for further development of biogas produc- Wirtschaftlichkeit. (Economic aspects of biogas utili-
tion, since energy is often in short supply and zation and biogas motors.) GIT Fachz. Lab 12/
85:1241-1254.
waste disposal can often not be done by the elab- Holt, F. 1986. Environmental management: is there a busi-
orate processes used in developed countries. In ness? World Biotech Report 1: 89-99. Online, London.
Kandler, O. 1979. Optimierungsméglichkeiten von Bio-
such cases, the efficiency of the process is less gasprozessen unter Einsatz thermophiler Mikroorga-
critical than the energy produced; biogas pro- nismen (Optimization of the biogas process using ther-
duction in developing countries can often be ac- mophilic microorganisms), pp. 19-31. BMFT (ed.),
Biokonversion. 2nd BMFT-Statusseminar “Bioverfah-
complished in simple installations with little re- renstechnik’’.
quirement for complicated engineering skills. Lemke, J.R. and K. Mack. 1978. Biologische Abwasserrei-
nigung mit technisch reinem Sauerstoff in der Klar-
Biogas can also be used as a motor fuel in those anlage BAYER AG, Werk Wuppertal-Elberfeld (Bio-
regions where petroleum sources are expensive logical sewage treatment in activated sludge plants
or in short supply. using pure oxygen). Chemie-Technik 7: 193-196.
Mack, K. 1973. Biologische Reinigung von Abwasser mit
Sauerstoff (Biological treatment of sewage with oxy-
gen). Wasser, Luft, und Betrieb 17: 108-111.
Table 17.4 Biogas production in relation to Magruder, G.C. and J.L. Gaddy. 1981. Production of farm
temperature energy from biomass, pp. 269-274. In: Moo-Young,
M. (ed.), Advances in biotechnology, vol. II. Pergamon
Substrate Temper- % Dry Time 1 Methane/ Press, Toronto.
ature weight h g dry Muller, H.G. 1984. Der Biohochreaktor—eine platz- und
Xe in input weight energiesparende biologische Abwasserreinigungsan-
lage. (The vertical bioreactor—a space- and energy-sav-
Sludge 35-40 21 23 0.488+ ing sewage-treatment installation.) GIT-Suppl. 4:16-
50 AS 9 0.488* 255
60 2.1 12/5 0.488+ Nyns, E.J. 1986. Biomethanation processes, pp. 207-267.
Sludge 30 3 33-50 0.371 In: Rehm, H.J. and G. Reed (eds.). Biotechnology 8.
50 3 20 0.368 Verlag Chemie, Weinheim.
Ohta, T. and M. Ikeda. 1981. Rapid microbial deodori-
Waste and 30 3 30 0.288* zation of agricultural and animal wastes, pp. 621-626.
sludge 50 3 10 0.369* In: Moo-Young, M. (ed.) Advances in Biotechnology
60 3 4 0.455* II. Pergamon Press, Toronto.
Reimann, H., M. Morper, and C.H. Gregor. 1982. Reini-
55 2 3 0.16 gung industrieller Abwåsser unter Einsatz von tech-
9 0.19 nischem Sauerstoff. (Purification of industrial waste
water with oxygen.) Chemie Technik 11:27-32.
Cow 55 6 3 0.16
Sahm, H. 1984. Anaerobic wastewater treatment. Adv.
manure 9 0.19
Biochem. Eng./Biotechnol. 29:83-115.
55 10 3 0.10 Shailubhai, K. 1986. Treatment of petroleum industry oil
9 0.19 sludge in soil. Reviews. TIBTECH, August 202-206.
Shieh, W.K. and J.D. Keenan. 1986. Fluidized bed biofilm
*= Biogas reactor for wastewater treatment. Adv. Biochem. Eng./
(Kandler, 1979) Biotechnol. 33:131-169.
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management. World Biotech Report 1986 1:75—79. On- heim.
line, London. å Uhde/Hoechst. Bio-Hochreaktor. No date.
Sojka, S.A. 1986. Genetic engineering for dehalogenatio Zlokarnik, M. 1985. Tower-shaped reactors for aerobic bi-
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 Leaching

metal sulfides or other ores. The metals are con-

18.1 INTRODUCTION
verted to water-soluble metal sulfates with the
Microbial leaching is the process by which metals aid of biochemical oxidation processes.
are dissolved from ore-bearing rocks using mi- In commercial applications, copper and ura-
croorganisms. At present a number of ores can- nium have been widely produced through the
not be economically processed with chemical use of microorganisms. However, there have
methods due to their low metal content. In ad- been difficulties in extrapolating the results from
dition, large quantities of low-grade ores are pro- laboratory and pilot-plant studies into practical
duced during the separation of higher-grade ores field conditions. In addition, problems may arise
and are generally discarded in waste heaps. when the large-scale leaching process of a waste
Throughout the world there are vast quantities dump is improperly managed. Leach fluids con-
of such low-grade copper ores that cannot be taining large quantities of metals and having ex-
profitably purified by conventional chemical tremely low pH values (pH 3) can seep from such
methods, but that could be processed by micro- dumps into nearby natural water supplies and
bial leaching. There are also significant quantities ground waters, causing enormous and lasting
of nickel, lead, and zinc ores which could be damage.
leached.
Leaching was first discovered as a process oc-
18.2 ORGANISMS FOR LEACHING
curring in pumps and pipelines installed in mine
pits containing acidic water. It was subsequently The two most commonly used organisms in mi-
developed for the recovery of metal from low- crobial leaching are Thiobacillus thiooxidans and
grade ores. For many metals, there are now Thiobacillus ferrooxidans. A number of others
leaching methods which permit extraction from may also be used including: Thiobacillus concre-

326
 18.4 COMMERCIAL PROCESSES / 327

tivorus, Pseudomonas fluorescens, P. putida, Achro- lowing equation describes the initial oxidation of
mobacter, Bacillus licheniformis, B. cereus, B. lu- pyrite by ferric ions:
teus, B. polymyxa, B. megaterium, and several
thermophilic bacteria including Thiobacillus ther-
FeS, + Fe,(SO,), —> 3 FeSO, +2 S° [4]
mophilica, Thermothrix thioparus, Thiobacillus
TH1, and Sulfolobus acidocaldarius. The hetero-
trophic organisms listed have not as yet actually The sulfur which is formed via this process is
been used, but it seems likely that processes will reoxidized as shown in equation 2.
be developed by which metals are extracted from Examination of leaching dumps always
ores with microbially produced organic acids via shows the presence of mixtures of T. thiooxidans
chelate and salt formation. Because of their more and T. ferrooxidans. In pilot-plant reactors (50 li-
rapid growth rate, the thermophilic bacteria may ter), leaching can be performed continuously in
significantly accelerate the leaching process. a cascade series with recycling of the cells and
leachate.
18.3 CHEMISTRY OF MICROBIAL Yields such as those in other areas of micro-
LEACHING biology can be attained in the laboratory under
optimal conditions (temperature control, O, and
Thiobacillus ferrooxidans is the organism that has CO, adjustment, maintenance of pH around 2-
been most extensively studied. It is a Gram-neg- 3 and Eh around —300 mV) with very finely
ative rod-shaped bacterium which is 0.5—0.8 ground ores in a tower (percolator), or better yet
um X1.0-2.0 um in size. An autotrophic aerobe, in fermenters under optimal conditions. How-
it can obtain carbon for biosynthesis solely from ever, in field experiments, these conditions and
CO, fixation, and obtains its energy from the ox- yields cannot be realized due to the high cost.
idation of Fe?" to Fe?" or from the oxidation of
elemental sulfur and reduced sulfur compounds
to sulfate.
18.4 COMMERCIAL PROCESSES
Three methods have practical application (Figure
4 FeSO, +2 H,SO, +O, > 2Fe,(SO,),+2H,O [1]
18.1):
2S°+3 O,+2H,O >2H,S0, [2]
e Slope leaching. Finely ground ores (up to
2 FeS, +7 0, + 2H,O — 2 FeSO, + 2H,SO, [3] 100,00 tons) are dumped in large piles down
a mountainside and continuously sprinkled
with water containing Thiobacillus. The water
The oxidation of insoluble sulfur to sulfuric acid, is collected at the bottom and reused after
which is also performed by Thiobacillus thiooxi- metal extraction and possible regeneration of
dans, occurs in the periplasmic space. According the bacteria in an oxidation pool.
to equation 3, iron is dissolved through ”direct + Heap leaching. The ore is arranged in large
bacterial leaching”. heaps and treated as in slope leaching.
In addition to this leaching process performed e In-situ leaching. Water containing Thioba-
only by microorganisms, there is another pro- cillus is pumped through drilled passages to
cess, “indirect, bacterially supported leaching” unextracted ore which remains in its original
which takes place slowly in the absence of mi- location in the earth. In most cases, the
crobes. The oxidation of pyrite can be used as an permeability of the rock must be first in-
example. Pyrite is a common rock mineral that creased by subsurface blasting of the rock.
is found in association with many ores. The fol- The acidic water seeps through the rock and
328 / CHAPTER 18 / LEACHING

CuS
+ 20,— CuSO, [6]

Copper leaching plants have been in wide use

Input
throughout the world for many years, generally
Product operated as simple heap leaching processes but
recovery
sometimes as combinations of heap and in-situ
leaching. The leaching solution (sulfate /Fe?" so-
lution) carries the microbial nutrients in and the
dissolved copper out. The solution is sprinkled
over the heap and percolates through the rock
pile to the lower level where the copper-rich liq-
uid is collected. The copper-containing solution
(up to 0.6g/1) is removed, the copper is precip-
itated, and the water is reused after readjusting
the pH to 2.
Countries in which microbial leaching of cop-
per has been widely used include the United
States, Australia, Canada, Mexico, South Africa,
Portugal, Spain, and Japan. About 5% of the
world copper production is obtained via micro-
bial leaching. A single installation in the United
States has produced up to 200 tons of copper per
day.

Figure 18.1 Diagram of (a) slope, (b) heap, and (c) in-
situ leaching Uranium leaching
Although less uranium than copper is obtained
collects in the bottommost cavity from which by microbial leaching, the uranium process is
it is pumped, the minerals extracted, and the more significant economically. Because a thou-
water reused after regeneration of bacteria. sand tons of uranium ore must be handled to
obtain one ton of uranium, in-situ microbial
Copper leaching leaching is gaining greater acceptance, since it
eliminates the expense of moving such vast
If chalcocite, chalcopyrite, or covellite are used amounts of material.
for the production of copper, several metals are In the uranium leaching process, insoluble te-
usually found together. For example, chalcopy- travalent uranium is oxidized with a hot H,SO,/
rite contains 26% copper, 25.9% iron, 2.5% zinc, Fe?" solution to soluble hexavalent uranium sul-
and 33% sulphur. Chalcopyrite is oxidized as fol- fate.
lows: É

2 CuFeS, +8 1/2 O, k H,SO, me UO, + Fe,(SO,); —> UO,SO, + 2 FeSO, [7]

2 CuSO, EJE Fe,(SO4)3 ot H,O [5]

This is an indirect leaching process since the mi-

Covellite is oxidized to copper sulfate: crobial attack is not on the uranium ore directly
 REFERENCES / 329

but on the iron oxidant. Ferric sulfate and sulfuric deeply into the ore. In such situations the heap
acid can be produced by T. ferrooxidans from the system is often still used commercially for leach-
pyrite within the uranium ore. ing of uranium.
Areas where uranium leaching has been car-
ried out include the United States, Canada, and
2 FeS, +H,O+7.5 0, > Fe,(SO,), + H,SO, [8] South Africa.

REFERENCES
The pyrite reaction is used for the initial pro-
duction of the Fe?" leach solution. Pilot plants Atkins, A.S., F.D. Pooley, and C.C. Townsley. 1986. Com-
parative mineral sulfide leaching in shake flasks, per-
operate with surface reactors similar to the trick- colation columns, and pachuca reactors using Thioba-
ling filters used in sewage. cillus ferrooxidans. Process Biochemistry Febr. 3-10.
Optimal uranium leaching conditions are pH Bosecker, K. 1987. Microbial leaching, pp. 551-559. In:
Prave, P. (ed.). Handbook of Biotechnology. Olden-
1.5-3.5, 35°C and 0.2% CO, in the incoming air. bourg Publishers, Munich.
Some thermophilic strains are known which Ebner, H.G. 1977. Metal extraction from industrial waste
have a temperature optimum of 45-50°C. with Thiobacilli, pp. 217-222. In: Schwartz, W. (ed.),
Conference on Bacterial Leaching. Verlag Chemie,
In commercial processes, the dissolved ura- Weinheim.
nium is extracted from the leach liquor with or- Kelly, D.P., P.R. Norris, and C.L. Brierley. 1979. Micro-
ganic solvents such as tributylphosphate and the biological methods for the extraction and recovery of
metals, pp. 263-308. In: Bull, A.T., D.C. Ellwood, and
uranium is subsequently precipitated from the C. Ratledge (eds.), Microbial technology: Current state,
organic phase. Adsorption of the uranyl ions future prospects. Cambridge University Press, Cam-
with ion exchangers is another possibility. The bridge.
Olsen, G.J. and R.M. Kelly. 1986. Microbiological metal
organic solvents which remain in the water sys- transformations: Biotechnological applications and po-
tem after extraction may be toxic and hence cause tential. Biotechn. Progress 2:1—-15.
problems when the microbiological system is Sanmugasunderam, V., D.W. Duncan, and R.M.R. Bran-
ion. 1981. Novel reactor configuration for small scale
reused. microbiological leaching, pp. 595-600. In: Moo-Young,
In-situ leaching has the disadvantage that the M. (ed.), Advances in biotechnology, vol. I. Pergamon
Press, Toronto.
permeability of the rock may be low and the
Torma, A.E. 1977. The role of Thiobacillus ferrooxidans in
drilled passages may not always allow an ade- hydrometallurgical processes. Adv. Biochem. Eng. 6:
quate supply of nutrients and oxygen to enter 1-37.
 Extracellular
polysaccharides

Polysaccharides are used commercially to pro- charides vary greatly in such rheological prop-
duce gels, and to thicken and stabilize foods, erties as pseudoplasticity, thixotrophy, and vis-
medicines, and industrial products. They are also coelasticity.
used as polymers in the fluids used to force oil Table 19.1 shows the most important micro-
to the surface in the tertiary oil recovery process. bial exopolysaccharides produced commercially.
Although polysaccharides of plant origin (such In this list, alginate has thus far been produced
as starch, alginate, or agar) have been used for commercially only from seaweed, but in the fu-
many years, microbial polysaccharides have be- ture Azotobacter may be used. Alginate and xan-
come widely used over the past several decades. than are anionic polysaccharides composed of
Both intracellular polymers such as polybutyric uronic acid residues.
acid and poly-6-hydroxybutyric acid/polyhy- The biosynthesis of heteropolysaccharides
droxyvaleric acid copolymer, and extracellular is comparable to that of bacterial cell wall com-
polysaccharides are produced commercially. ponents. Proceeding from glucose, the appro-
Around 20 different microbial polysaccharides priate sugar nucleotide is produced via glucose
with market potential have been described, but phosphate, followed by transformation of the
the largest part of the market is held by xanthan, glucose into another sugar. At the sugar nucleo-
with production of about 10,000 tons per year. tide level, further transformations can take place,
However, microbial polysaccharides are really such as UDP-mannose — UDP-mannuronic acid.
only a small part of the polysaccharide market The transport of monosaccharides through the
as shown by the fact that xanthan holds only membrane and out of the cell takes place after
about 4% of the market. An important aspect of coupling to a C-55 isoprenoid alcohol phosphate.
a polysaccharide, if it is to have market potential, Relatively little is known about the manner in
is its rheological properties. Microbial polysac- which the polysaccharide polymerase on the out-

330
 EXTRACELLULAR POLYSACCHARIDES / 331
Table 19.1 Microbial polysaccharides with commercial
uses which degrade these polymers and hence reduce
the viscosity of the final product.
Poly- Structure Organism
saccha- Various factors regulate the rate of exopoly-
ride saccharide biosynthesis. Molecular oxygen
Xanthan _ Gle 1 Bog Gle Xanthomonas
(>90% saturation) is necessary not only for pri-
3 campestris mary energy metabolism but also for the oxi-
tie dation of sugar to the corresponding alcohol and
Mann 6-OAc for reoxidizing reduced pyridine nucleotides. A
2
Me carbon/nitrogen ratio of around 10:1 is generally
GlcA most favorable for optimal yield. For some poly-
4 saccharide-forming organisms, Xanthomonas,
418 Pseudomonas, Azotobacter, and Aureobasidium,
Mann 4,6-OPyruvate
growth and product formation can be described
Alginate _ 4p _MannA 1 £4pD Pseudomonas
quantitatively by the Leudeking-Piret equation:
~MannA 1: aeruginosa,
"a Azotobacter
E al ee vinelandii

Curdlan _ Gig 1 $3 Gic — Alcaligenes Especially in large-scale fermentations, the

marked increase in viscosity which occurs as the
Sclero "| Gic 1 > 3 Cle KS Sclerotium polysaccharide concentration increases causes
glucan 6 glucanicum, the stirring rate to decrease, resulting in a slowing
ME S. delphinii, of the mixing time. This leads to a reduction in
Gle S. rolfsii
mass transfer efficiency and hence to the for-
mation of a polymer of heterogeneous quality.
Pullulan _, 6 (Gle 1 Gere Aureobasidium
4GIc)1—> pullulans Isolation of the polymer from the fermenta-
tion broth involves precipitation by treatment
Dextran Glc 1 * 6 Glc Acetobacter
with an organic solvent, a salt, or an acid. The
several 1 > 2,1 —3 and sp., producing bacteria become entrapped in the pre-
1 — 4 compounds Leuconostoc cipitating polymer and are carried out of the
mesenteroides,
broth also.
Leuconostoc
dextranicum,
Streptococcus
mutans Xanthan
Glc=Glucose, Mann=Mannose, GlcA=Glucuronic Xanthan was the first heteropolysaccharide to be
acid, MannA=Mannuronic acid, GulA=Guluronic acid,
Ac= Acetate commercially available. It is produced by Xan-
thomonas campestris, and was developed in the
late 1950’s and marketed in 1964. The molecular
side of the cell membrane acts to bring about weight of this polymer is around 2-15 X 10°
chain lengthening and termination and the in- Daltons. As shown in Figure 19.1, the basic re-
‘fluence on the development of secondary and peating unit of xanthan consists of a pentasac-
tertiary structure. An additional factor influenc- charide containing glucose, mannose, glucuronic
ing the structure of the resulting polysaccharide acid, acetate, and pyruvate. The number of py-
is degradation by microbial enzymes. It is known ruvate units in the molecule and the molecular
for alginate and pullulan, for instance, that at the weight determines the viscosity of the resulting
end of the fermentation enzymes are excreted xanthan gum.
332 / CHAPTER 19 / EXTRACELLULAR POLYSACCHARIDES
Table 19.2 Influence of limiting nutrient and dilution
rate on yield of xanthan
Limitation Medium Dilution rate Yield
(h”) (%)
Nitrogen Complex 0.023 71
mdCHP Nitrogen Complex 0.054 82
Sulfur Synthetic 0.035 60
OH Sulfur Synthetic 0.050 32
HO Phosphate Synthetic 0.030 40
COO°M® 9g/ Magnesium Synthetilc 0.050 28
0.
Yield based on added glucose
H
Data of Sutherland (1983)
COO®M®0 CHa M°=Na,K, 1/2 Ca
\—~
C fo 9 OH
a
CH, AG OH
(9) Alginate

Figure 19.1 Repeating unit of a xanthan gum Pseudomonas aeruginosa and Azotobacter vinelan-
dii produce alginate from two uronic acids, man-
nuronic acid and guluronic acid, in proportions
For commercial production, the nutrient so- of 4:1 to 20:1. Sodium alginate is a commonly
lution consists of 4-5% carbohydrate (glucose, used agent for the immobilization of microor-
sucrose, corn starch hydrolysate), 0.05-0.1% ni- ganisms (see Chapter 11) and also finds use as
trogen (yeast extract, peptone, ammonium ni- an ion-exchange agent.
trate, or urea) and salts, with the pH controlled
at 7.0. During the 2-day batch culture, an in- Curdlan
crease in viscosity begins during the log phase
Alcaligenes faecalis var. myxogenes produces two
and continues into the resting phase. Large-scale
B-1,3 glucans, curdlan and succinoglucan. Suc-
fermentations can have an apparent viscosity up
cinoglucan contains glucose, about 10% succinic
to 20-30,000 centipoise. However, viscosities
acid, and galactose, whereas curdlan is a simple
greater than 10,000 cP may cause technical prob-
glucose polymer. Yields of curdlan of 40 g/l are
lems. The efficiency of production of xanthan in
obtained on a defined medium containing 8%
weight per weight of carbohydrate used is 70-
glucose in the course of the 80-hour fermenta-
80% and the yield is 25-30 g/l. Product recovery
tion. Because curdlan is water insoluble, the vis-
is carried out by precipitation of the polysaccha-
cosity does not increase during the fermentation;
ride with isopropanol or methanol, a step which
it only begins to form a gel at temperatures above
also kills the culture. The precipitated xanthan is
54°C. There is an increase in viscosity with suc-
then dried and ground. Production in continous cinoglucan, however, so it must be produced
fermentation has been successfully tested; the ef- from a 4% glucose medium, with an efficiency
fect of the limiting nutrient and the dilution rate of production of 35%.
on xanthan yield is shown in Table 19.2.
In one actual installation capable of produc-
ing 2000 tons of xanthan per year (40 hour fer- Scleroglucan
mentation time, yield of 0.7 kg xanthan/kg glu- Sclerotium glucanicum, S. delphinii, S. rolfsii and
cose, 2.5% xanthan final concentration), Helotium sp. produce scleroglucan, a polysac-
calculations showed that the production cost per charide with glucose units connected primarily
m? fermenter volume was markedly lower with in B-1,3- with occasional 6-6,1-glycosidic bonds.
the continuous process. The organisms grow on hydrolyzed lignocellu-
 EXTRACELLULAR POLYSACCHARIDES / 333

lose substrates. Scleroglucan is used in food pro- _

duction and is under development for use in ter- 5 $$ S E

€
tiary petroleum recovery. +150 g =
o
lo 5 ° 20 9
Figure 19.2 shows the kinetics of the pro- a

duction of scleroglucan with Sclerotium rolfsii 34 5= — 154-30 3

WE = =
growing on glucose under nitrate limitation. Pro- E =
= S= bey iSan
duction (fermentation, purification and possibly DS

=
r100—

2
c
ie)
5
S
enzymatic splitting) of scleroglucans has been SA927 eg3 Stores
3 10+205
shown to be an economically feasible process,
is) oO a—as f= E
€ =:
o 3 8
provided the conditions of production are optim-
fi BR
oO 9 ©

ized. 1) 4 205+10 2
Å 2
oO
a
°

0 sBeet, s s

Pullulan ey £ "Aa a

20 40 60 80
Pullulan, a glucan with a-1,4- and a few a-1,6- Fermentation time (hr)

glycosidic bonds, is produced by the fungus Au-

reobasidium pullulans from a 5% glucose solution Figure 19.3 Pullulan production by Aureobasidium pul-
lulans in a medium with 140mM glucose and 4.5 mM
with 70% efficiency within 5 days. The enzyme ammonium sulfate. O——O cell dry weight (mg/ml);
pullulanase, produced by bacteria, was discussed O— O extracellular glucose (uM/ml); 4 — 4 pullulan
in Section 11.2. The physiology of pullulan for- (uM anhydroglucose/ml); ™ — mø C-accumulation rate as
a measure of glucose assimilation rate (uM/mg cells-h);
mation has been intensively studied; Figure 19.3 4 — 4 pullulan formation rate (uM anhydroglucose/mg
shows the fermentation kinetics of pullulan pro- cells:h) (Catley, 1973).
duction.

Dextran
—
a
Se Dextrans are important as blood plasma exten-
2 a
ders and are also used in food production. They
14 4 38 >
S =O oe
are a complex group with molecular weights
12 +1200 VV from 15,000-500,000. Most occur as glucans with
= ° S 1,6-glycosidic bonds, but some dextrans also
2
2 10
ri oa e have 1,2-, 1,3-, or 1,4-glycosidic bonds.
5a f The polysaccharide dextran is produced by
8 + 800
2© a the enzyme dextransucrase, a transglucosidase
© 6 4 which acts on sucrose (glucose-fructoside), po-
= rN
io) lymerizing the glucose units into dextran and lib-
a
4 + 400
e e
erating free fructose. This enzyme is produced
by many strains of the lactic acid bacterium Leu-
Zz 4 Va conostoc mesenteroides. In contrast to other exo-
polysaccharides, dextran is produced extracel-
h—— 2-4 : ; = re
24 48 72 96 120 lularly in the medium. Commercial dextran
Fermentation time (hr) production can be carried out in a batch process
with L. mesenteroides with a medium containing
Figure 19.2 Scleroglucan production by Sclerotium rolfsii inorganic phosphate and an organic nitrogen
ATCC 15206 during nitrate limitation. e——e biomass;
a — ascleroglucan; O — O viscosity (Griffith and Com-
source. The crude dextran (MW 500,000) ob-
pere, 1978). tained through alcohol precipitation is treated
334 / CHAPTER 19 / EXTRACELLULAR POLYSACCHARIDES

with acid for hydrolysis. The desired dextrans and D.C. Ellwood (eds.), Microbial polysaccharides
and polysaccharases. Academic Press, London.
(MW 40,000 to 60,000) are subsequently frac- Compere, A.L. and W.L. Griffith. 1981. Scleroglucan bio-
tionated by ethanol precipitation and dried. The polymer production, properties, and economics, pp.
batch process has been further developed into a 441-446. In: Moo-Young, M. (ed.), Advances in bio-
strictly enzymatic process, in which the cells are technology, vol. III. Pergamon Press, Toronto.
Griffith, W.L. and A.L. Compere. 1978. Production of a
separated from their medium, which now con- high viscosity glucan by Sclerotium rolfsii ATCC 15206.
tains the enzyme, after fermentation. The extra- Dev. Ind. Microbiol. 19: 609-617.
cellular dextransucrase is able to carry out the Jeanes, A.R. 1978. Dextran bibliography. U.S. Dept. Agric.,
Res. Serv. Misc. Publ. 1355.
transformation of sucrose into dextran in the cell- Margaritis, A. and G.W. Pace. 1985. Microbial polysac-
free nutrient solution at pH 5.0—5.2 and a tem- charides. pp. 1005-1044. In: Moo-Young, M. (editor),
perature of 25-30°C. The fructose remaining in Comprehensive Biotechnology, Volume 3, Pergamon
the solution can be recovered. Press, Oxford.
Mian, F.A., T.R. Jarman, and R.C. Righelato. 1978. Bio-
synthesis of exopolysaccharide by Pseudomonas aeru-
REFERENCES ginosa. J. Bacteriol. 134: 418-422.
Sutherland, J.W. 1983. Extracellular polysaccharides. pp.
Catley, B.J. 1973. The rate of elaboration of the extracel- 531-574. In: Rehm, H.J. and G. Reed (editors). Bio-
lular polysaccharide, pullulan, during growth of Pul- technology, Volume 3, VCH Publishers, Deerfield
lularia pullulans. J. Gen. Microbiol. 78: 33-38. Beach, FL.
Catley, B.J. 1979. Pullulan synthesis by Aureobasidium pul- Uttley, N.L. 1986. Polyhydroxybutyrate: a commercial
lulans, pp. 67-84. In: Berkeley, R.C.W., G.W. Gooday, challenge. World Biotech Report 1: 171-177.
 20
Other
fermentation
processes and
future
prospects

In this chapter we discuss several fermentations gibberellins are also active, e.g. GA, from Spha-
that do not fit into the categories established in celoma manihoticola. Plants produce only a few
the previous chapters, including several which gibberellins (GA,, GA,, GA,, and GA,), and the
are not now of commercial use but may become majority of the rest are produced by fungi. Bio-
significant in the future. synthesis takes place via isoprenoid units.
Fusarium moniliforme, the imperfect stage of
Gibberellins the fungus Gibberella fujikuroi, is used for com-
mercial production. This fungus produces the
The gibberellins are one class among the five antibiotic bikaverin simultaneously with its pro-
known classes of phytohormones. Gibberellins duction of gibberellins. Six physiologically dis-
are used as growth hormones and have found tinct phases have been characterized for the pro-
application with barley to improve grain quality duction process (Figure 20.2).
in malt production. Sales of plant growth regu-
lators are estimated at $40 million for 1980 in 1. Lag phase
American agriculture, with gross world sales at 2. Growth phase (24-36 h) without nitrogen
$118 million. limitation, low production of gibberellin
The gibberellins are derived from gibberellan Glycine limitation, little cell reproduction,
(Figure 20.1) and are given arbitrary consecutive slow gibberellin production
numbers according to when they were discov- Glycine absent, remaining glucose catabol-
ered. From the first gibberellin discovery in 1938, ized, strong gibberellin production
many more have been described; by 1975 the Glucose content zero, slow accumulation of
number was up to GÅ,5. The most important are gibberellins
GA, (also called gibberellic acid) and GA,. Other Lysis of cells, increase in pH

335
 336 / CHAPTER 20 / OTHER FERMENTATION PROCESSES AND FUTURE PROSPECTS

CoQ eee.
tures as well as submerged fed-batch cultures.
Continuous processes have been described, but
OH have not become widely used. Table 20.1 shows
V that in continous culture, gibberellins are formed
He Coon er
only at very low dilution rates.
I I]

Figure 20.1 Structure of gibberellan (I) and gibberellic

acid (II) Zearalenone

Zearalenone (Figure 20.3) and its derivatives

have estrogenic activity. The various zearalenone
a \ \
derivatives differ by the presence of a keto or
(g/l) \
hydroxyl group on the 6’ carbon and by the pres-
Dry
weight
»
+ 200
ence or absence of a double bond in the mac-
rolide lactone ring. These substances are formed
by Gibberella zea, the sexual stage of the fungus
+ 100
(mg/l) Fusarium roseum graminearum. Production scale
Gibberellins

today is carried out in a submerged process in

aj 80 m? fermenters. The yields at 32°C over a 21-
ean osvo day incubation period are 30 g/l. Zearalenones
2 oO
ae
oS
Re
ce
io)
are used as anabolic growth-stimulating com-
=
pounds in swine and sheep production.
g
£sd z
= 5
role

2 DE Table 20.1 Gibberellin formation by Gibberella fujikuroi

=(3) 5%
© 005
Cores
Qa x
under glycine limitation
Q no
2 =2 Dilution rate 0.12 0.09 0.07 0.05 0.02
hh”
re)
co)
Q
n Mycelium dry 7.6. 116 14.4 15:65 2255
weight g/l
Fermentation time (hr)
Glycine 1250/90 0:49 OFS LOL?
content g/l
Figure 20.2 Batch fermentation with glycine limitation Glucose 100 92 81 63 53
for the production of gibberellins by Gibberella fujikuroi. content g/l
Upper graph: 4 — 4 pH; e — e glycine; O — O myce- Gibberellin 0 0 0 0.45 2.05
lium dry weight; X—-—-— X gibberellin concentration;
Lower graph: — — — specific gibberellin formation rate;
mg/g
weight
dry
specific growth rate (Bu’Lock et al., 1974)
(Bu’Lock et al., 1974)

A low nitrogen content (<0.2%) and a mix-

ture of different carbon sources are two important
conditions for gibberellin production. Yields in
production-scale fermenters after 120-150 hours
are 1-2 g/l of total gibberellin at 25°C, with the
aeration rate at 0.5 vvm. In addition to direct
fermentation in submerged culture, some studies
have been carried out on the use of surface cul- Figure 20.3 Zearalenone
 OTHER FERMENTATION PROCESSES AND FUTURE PROSPECTS / 337

Table 20.2 Protease inhibitors

Triglycerides and fatty acids
Enzyme Enzyme Microorganism Appli-
Fats and oils derived from plant sources find wide inhibitor cation
use in foods and for industrial applications. Tech- Pepstatins Acid Streptomyces Ulcers
niques have been developed for the microbial proteases testaceus,
production of fats and oils, but such processes e.g. pepsin S. argenteolus
have not been cost-effective for the most signif- Antipain Papain S. yokosu- Reduces
icant materials. The fats and oils for food uses kanensis fever
S. michiga-
mostly contain the longer-chain fatty acids (C,,- nensis
cs), but uses for those with other chain lengths
Chemostatin Chymo- S. hygrosco- Reduces
also exist. Because of the high prices for the fats trypsin picus fever
with shorter-chain fatty acids (C, and C,) and S. lavendulae
for a few of the longer-chain materials, consid- Elastatinal Elastase S. griseo- Pancrea-
erable research is underway to produce glycer- ruber titis
ides with such fatty acids. The proportion of ster- Leupeptin Trypsin Reduces
ols produced in such fermentations must be low Plasmin fever
(<0.5%) and the fatty acid contents of the cells Bestatin Amino S. olivoreticuli Inhibits
must be greater than 40%. Only yeasts and fi- peptidases cancer
lamentous fungi apppear suitable for such pro- Subtilisin Subtilisin — S. albogriseolus
cesses. In England a process has been developed inhibitor
using species of the fungus genus Mucor. Using
glucose as the starting material, an oil containing
7% y-linolenic acid has been produced in 220 m? pressives. Table 20.2 gives examples of several
fermenters. y-Linolenic acid, a C,, unsaturated microbially produced protease inhibitors.
fatty acid with three double bonds (C,, C,, and Figure 20.4 shows the peptide structure of the
C,,) is a starting material in prostaglandin bio- substance antipain, an inhibitor of papain. In
synthesis and is also used in dietary foods. contrast to the relatively simple structure of an-
Another approach to the production of de- tipain, the inhibitor of the enzyme subtilisin con-
sired oils is by the use of specific lipases which tains 113 amino acids.
will catalyze the exchange of a fatty acid ester of A second group of substances that have been
lower value for one of higher value. This ap- isolated are inhibitors of amylase and other gly-
proach is currently undergoing research for large- coside hydrolases. These substances are impor-
scale production. tant in medicine because they inhibit the break-
down of carbohydrates in the digestive tract.
Enzyme inhibitors Included here are inhibitors of a-glucosidases, B-
glucosidases, cellulases, and dextranases. a-Glu-
It has been known for a long time that animals
and plants produce enzyme inhibitors. Micro-
organisms have been studied for enzyme-inhib- HOOC-CH -NH- CO-NH-CH- oe COSNAF ER-CHO
itor production in Japan since 1965 and world- |
Chie
|
(GRE) CH (CH,)3
wide beginning more recently. By 1974, 50 | ZEN |
va CH, CH, Ne
microbial enzyme inhibitors were known. Ini-
were sought, which C=NH C=NH
tially, protease inhibitors
| |
were usually peptides themselves and were de- NH, NH,
veloped for varying applications, including pos-
sible use as antitumor agents or as immunosup- Figure 20.4 Antipain
338 / CHAPTER 20 / OTHER FERMENTATION PROCESSES AND FUTURE PROSPECTS

cosidase inhibitors are widely distributed (Table CHaICHACHs CH: CHz Cherie cit Eb:CHCH (Cele CH3
20.3). Chemically, such inhibitors are pseudo-ol- O Cae
|
igosaccharides, monosaccharides, cyclopeptides, co 4
I
and polypeptides. Figure 20.5 shows the general CH-NH-CO-CH3
formula of a group of a-glucosidase inhibitors |
CHsCO-NH2
which contain a central molecule of an unsatu-
rated cyclitol plus 4,6-dideoxy-4-amino-D-glu- Figure 20.6 Esterastin
copyranose with a-1,4-glucopyranose. This

group of acarboses are produced by species of

Table 20.3 a-Glucosidase inhibitors produced by the genus Actinoplanes using a medium contain-
microorganisms ing maltose and yeast extract. For purification,
Microorganism Type of Specificity the products are complexed to an ion-exchange
compound resin and then eluted for further purification.
Actinomycetes Proteins 1,2 plus Nojirimycin (see Figure 13.19) is an example
Pseudo- microbial a- of a substance which has anti-6-glucosidase ac-
oligosaccharides amylases
Streptomyces Pseudo- 23 tivity.
flavochromogenes __ oligosaccharides Enzyme inhibitors of adrenalin biosynthesis
S. diastaticus Pseudo- 3 plus a- (dopastin, fusaric acid, isoflavone, phenolpico-
oligosaccharides amylases,
microbial a- linic acid, oudenone, and oosponol) have been
amylases intensively studied because of their blood-pres-
S. fradiae Acid polypeptides Human a- sure regulating effect. The structure of esterastin,
amylases
Bacilli, Nojirimycin, 2,3 plus 6- an esterase inhibitor (Figure 20.6) is unusual be-
S. lavendulae 1-deoxynojiri- glucosidases cause it contains a 6-lactone ring. 6-Lactamase
mycin from plants inhibitors such as clavulanic acid are valuable
and micro-
organisms because they overcome bacterial penicillin re-
S. calidus Glycopeptide 1 plus sistance. Because antibiotics which are active
intestinal against microbial cell wall synthesis are generally
maltase and
sucrase less toxic to humans, penicillinase inhibitors are
S. violaceoruber Polypeptide 1 being sought for possible use in penicillin ther-
Aspergillus niger Glycopeptide Aspergillus apy. Another area of application for enzyme in-
glucoamylase
Cladosporium Glycopeptide hibitors concerns the inhibitors of chitin synthe-
herbarum sis, such as the polyoxins, which have found
Cladosporium Acid glycopeptide Human a- limited use as insecticides.
cladosporioides amylases
1 = pancreatic amylase; 2 = intestinal amylase and
disaccharase; 3 = Rhizopus glucoamylase. Microbial insecticides
(Miller, 1986)

BLESS
The production of insecticides from microbiolog-
ical sources is of considerable interest. The em-
phasis here is not on the production of insecti-
cide-active chemicals, but on the use of microbes
-OH
which are themselves insect pathogens. In such
Onl Only On OH
applications, the living microorganism must be
dispersed into the environment. Five specific re-
Figure 20.5 a-Glucosidase inhibitor complex. quirements must be met before living prepara-
(m+n=1-8) tions can be used as insecticides:
 OTHER FERMENTATION PROCESSES AND FUTURE PROSPECTS [1339

1. Large-scale production must be feasible Table 20.4 Fungi pathogenic against insects
2. Viability must be maintained Fungus Production Status Countries
3. The organism must not be toxic or pathogenic technique
to other animals and plants Aschersonia Submerged Pilot plant USSR,
4. The organism must be less expensive than sp. Holland, UK
chemical agents. Beauveria Submerged/ Approved for USSR
bassiana 2 stage market
5. No development of resistance by the target Semi-solid Small scale USA
insects. medium /
2 stage
Bacteria, viruses, fungi, or protozoa can be Conidiobolus Submerged Pilot plant France, UK,
obscurus ; USA
used as insecticides. They are produced com- Culcinomyces Submerged Small scale Australia
mercially either in large-scale fermentation or in clavosporus
insect hosts. Entomophthora In insects Small scale USA
grylli
Five protozoa, (Nosema locustae, N. algerae, N. Erynia Submerged Small scale UK
pyrausta, Vairimorpha necatrix, and Mattesia tro- neoaphidis In insects
godermae) have been tested thus far, but have not Hirsutella Semi-solid Pilot plant USA
thompsonii
yet found practical application. Lagenidium Submerged Small scale USA
Over 400 fungi are known which attack in- giganteum
sects. They are relatively nonspecific in host Metarhizium Semi-solid Approved for Brazil
anisopliae market
range but are also less effective than other agents. Nomurea Solid Small scale USA
Those fungi currently under study are summa- rileyi
rized in Table 20.4. Many of these fungi can be Verticillium Submerged Approved for UK
lecanit market
cultured well in open dishes in semi-solid me- Zoophthora Submerged Laboratory USA
dium such as rice seeds. Within 15-20 days at radicans studies
26-29°C, about 2 X 10° conidia per gram are (Quinlan and Lisanski, 1983)
produced.
Over 650 insect viruses have been described.
Such viruses offer the best possibilities for prac- sibility. The active ingredient of B. thuringiensis
tical use as insecticides and several viruses are is a polypeptide toxin (y-endotoxin) which is pro-
already being marketed. One disadvantage of vi- duced during the bacterial sporulation process.
ruses for large-scale production is that they must The cultivation procedure is therefore designed
be cultured on living insects, making scale-up to obtained a high percentage of sporulation and
difficult. toxin accumulation. The fermentation is carried
Three bacteria (Bacillus popilliae, B. moritae, out in a medium containing starch, corn-steep
and B. thuringiensis) are commercially produced. liquor, casein, and yeast extract. Sporulation oc-
The first of these can only be cultivated in living curs after 25-30 hours and the y-endotoxin ac-
insects but the latter two can be grown in sub- cumulates as parasporal crystalline inclusions
merged culture. B. thuringiensis is widely culti- that amount to around 30% of the cell mass. This
vated in North America and Europe, with toxin is quite selective in its action and is ad-
amounts of 2300 tons per year, and is also pro- ministered by being incorporated into a suitable
. duced in the USSR and China. About 40 different insect food. Another toxin, the 6-exotoxin, is a
insects that cause agricultural or forest diseases nucleoside which is produced during the growth
are treated with B. thuringiensis. In addition, con- phase. It is a nonspecific toxin which rapidly kills
trol of certain insects which are carriers of human caterpillars. Between 10-15 minutes after inges-
diseases (for example, Anopheles sp., malaria; Si- tion of the B-exotoxin, the animals quite feeding
mulium damnosum; river blindness) is also a pos- and die within 3-5 days.
340 / CHAPTER 20 / OTHER FERMENTATION PROCESSES AND FUTURE PROSPECTS

bonyl compounds, and 38 aliphatic and aromatic

Cyclosporin A
alcohols. The aroma components and the per-
The cyclosporins are a group of oligopeptides centage distribution vary depending on the strain
produced by the fungus Tolypocladium inflatum and substrate solutions.
which have antifungal, anti-inflammatory, and In addition to the flavor enhancers already
immunosuppressive properties. The immuno- discussed (sodium glutamate, Chapter 9, and 5’-
suppressive effect is due to the inhibition of the nucleotides, Chapter 10) a number of specific fla-
activation of the T cell response. Cyclosporin A voring agents can be produced by fermentation.
is an eleven-membered cyclic peptide which is An example is the production of complex fla-
used because of its immunosuppressive proper- voring ingredients suitable for cheese produc-
ties to prevent rejection reactions during organ
tion. The most important flavoring substances in
transplantation. The cyclosporins are produced
cheese are methyl] ketones (2-heptanone, 2-non-
in a 15-day submerged fermentation from a me-
anone, 2-pentanone) and secondary alcohols
dium containing 4% glucose, 1% peptone, and
with 5-11 carbon atoms. Such flavoring sub-
mineral salts.
stances can also be produced in separate fer-
mentation processes and then used as supple-
Biochips ments to increase the flavor of prepared foods
A quite different application of biotechnology in- such as salad dressings and cheese pastries. As
volves the combination of electronics, biochem- an example, the fermentation of Penicillium ro-
istry, and genetics in the development of the con- queforti, the fungus involved in Roquefort cheese
cept of the biocomputer. Microprocessors of the production, can be described. Milk, salt, and milk
future may consist of chips in which monolayers fat are added for the fermentation which is car-
of protein are the functional material. Studies ried out at 24-26°C for three days. During this
with polylysine have given some success. These period, the milk fat is converted by the fungal
chips have over 100,000-fold more switches than lipases into methyl ketones and secondary al-
conventional microprocessors. The appropriate cohols. _
proteins may be produced from microorganisms
economically by the use of genetic engineering
Lipases i:
techniques. Fats LÆSES Fatty acids ———> 8 -Keto acids
—— Methylketones ——— > Secondary alcohols

Flavoring substances
After growth, the sterilized culture broth contains
We discussed the production of flavor-enhancers
4% fat, 5% protein, 8% carbohydrates, 5% salts,
in Chapters 9 and 10. Substances with inherent
flavor properties are also produced by microor- and 78% water; this final fermentation product
ganisms and are of great significance in the man- has 6-20 times more cheese aroma than does the
ufacture of food and drink. Moreover, microbial cheese itself.
enzymes used in food can alter the substrate and A number of specific microbial products that
thus produce flavoring substances. Intensive have distinctive odors are summarized in Table
studies are being conducted on the use of mi- 20.5. In addition, biotransformation of terpenes
crobes in food production and on the genetics to terpenols have been used to produce flavor
and biochemistry of aroma production. Among ingredients. For instance, Penicillium digitatum is
various alcoholic drinks alone, 400 different sub- able to convert the terpene R(+)limonene to a-
stances have been identified; of these 118 were terpineol, a substance with a lilac odor that is
esters, 80 aliphatic and aromatic acids, 41 car- used in perfumes.
 REFERENCES / 341
Table 20.5 Microbial compounds with specific odors Baldwin, B. 1986. Commercialisation of microbially pro-
duced pesticides, pp. 39-49. World Biotech Report 1.
Compound Structure Type of odor .
Online, London.
Diacetyl CH3;-CO—CO-—CH; _ Butter flavor Bu’Lock, J.D., R.W. Detroy, Z. Hostalek, and A. Munim-
(buttermilk) Al-Shakarchi. 1974. Regulation of secondary biosyn-
thesis in Gibberella fujikuroi. Trans. Br. Mycol. Soc.
Acetaldehyde CH3;—CHO Oranges and 62:377-389.
yogurt Gatfield, I.L. 1988. Die enzymatische Bildung von Aro-
mastoffen. (Enzymatic formation of perfume ingredi-
-Decalactone Peach ents.) In: Crueger, W. P. Prave, M. Schlingmann, R.
CH;—(CH,);— CH ——— CH, Thauer, F. Wagner (eds.). Jahrbuch Biotechnologie
| | 1988/89. Hanser Verlag, Miinchen.
O CH Hidy, P.H., R.S. Baldwin, R.L. Greasham, C.L. Keith, and
RS Co å J.R. McMullen. 1977. Zearalenone and some deriva-
|| tives: Production and biological activities. Adv. Appl.
O Microbiol. 22:59-82.
Esters Fruit Khachatourians, G.G. 1986. Production and use of bio-
Pyrazine Roast and nut logical pest control agents. TIBTECH May, 120-124.
Krieg, A. 1986. Bacillus thuringiensis, Tagungsbericht.
Forum Mikrobiol. 6/86:334-336.
Kumar, P.K.R. and B.K. Lonsane. 1987. Potential of fed-
Biofilters batch culture in solid state fermentation for production
of gibberellic acid. Biotech. Letters 9: 179-182.
Biofilters have been used since about 1970 for Luthy, P. and M.G. Wolfersberger. 1986. The delta-endo-
toxin of Bacillus thuringiensis. Swiss Biotech 4:11-14.
the removal of odors from air streams arising Muller, L. 1986. Microbial glycosidase inhibitors, pp. 531-
from sewage treatment plants, animal feed lots, 567. In; Rehm, H.J. and G. Reed (eds.). Biotechnology
paper mills, tobacco, chocolate and coffee roast- 4. VCH Verlag, Weinheim.
Quinlan, R.J. and S.G. Lisanski. 1983. Microbial insecti-
ing plants. Highly odoriferous off-gasses are pur-
sides, pp. 233-254. In: Rehm, H.J. and G. Reed (eds.).
ified and moistened by passing through beds Biotechnology 3. VCH Verlag, Weinheim.
containing compost, peat, pine needles, moss, or Rowe, G.E. and A. Margaritis. 1987. Bioprocess devel-
plastic bodies. Microorganisms growing on the opments in the production of bioinsecticides by Bacillus
thuringiensis. CRC Critical Rev. Biotechnol. 6:87 ff.
beds are responsible for the removal of much of Sinden, K.W. 1987. The production of lipids by fermen-
the odor. During the time which the gas passes tation within the EEC. Enzyme Microb. Technol.
through the bed (generally only a few seconds), 9:124-125.
the odors are absorbed and metabolized to bio- Tholander, P.J. 1987. Biologische Verfahren zur Beseiti-
gung von Geruchsquellen im grosstechnischen Be-
mass and CO. Biofilters themselves only have a reich. (Biological technique for the removal of odors in
compost-like odor. Biofilters may also be used in large-scale installations.) BioEng. 1:54—-55.
the future to purify the air from large-scale fer- Umezawa, H., T. Takita, and T. Shiba (eds.). 1978. Bioac-
tive peptides produced by microorganisms. John Wiley
mentation installations. & Sons, New York.
Vogel, R. 1984. Nattrliche Enzym-Inhibitoren. (Natural
REFERENCES enzyme inhibitors.) Thieme Verlag, Stuttgart.
Wink, J., A. Lotz, and P. Prave. 1987. Biotechnologisch
Anke, T. 1986. Further secondary products of biotech- hergestellte Aromastoffe. (Use of biotechnology for the
nological interest, pp. 611-628. In: Rehm, H.J. and manufacture of perfume ingredients.) Biotech-Forum
Reed (eds.). Biotechnology 4. VCH Verlag, Weinheim. 4:235-238.
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 Index

Absidia, 215 Actinomyces levoris, 52 L-Alanine, 152-56, 291

Absolute filter, 112 Actinomycete, 230 D,L-Alanine, 151-54, 156
Acarbose, 120, 338 recombination, 31-32 Alanine racemase, 244
Acetaldehyde, 131, 341 Actinomycin, 37-39, 52, 230-31, 244- Alarm system, 107
Acetaldehyde dehydrogenase, 144 45, 263, 300 Albomycin, 247
Acetamidocinnamate amidohydrolase, Actinoplanes, 6, 338 Albumin, serum, 49, 55
155 Actinoplanes missouriensis, 200 Alcaligenes, 206, 209, 331
Acetic acid, 48, 61, 129, 134 Actinorhodin, 32, 48, 51-52, 230 Alcaligenes faecalis, 332
amino acid production from, 154 Activated charcoal, 214 Alcohol dehydrogenase, 125, 144, 189
biosynthesis, 129-30, 144—45 Activated sludge basin, 321 Alcohol oxidase, 216
as carbon source, 74, 92 Activated sludge process, 317-20 Alginate, 330-32
glutamic acid production from, 159, N-Acylation reaction, 290 Aliphatic antibiotic, 230
161, 163-64 Acyltransferase, 38 Alkaline phosphatase, 180-81
lysine production from, 168-69 Adaptive repair, 14-15, 17 Alkaline protease, 203-4
methane production from, 323 Adenine, 28, 176 - Alkaloid, 39
production: Adenine nucleotide, 186-87 ergot, see Ergot alkaloid
product recovery, 147 Adenosine, 176, 186-87 indole, 275
submerged process, 144-47 Adenosine deaminase, 186 n-Alkane, 69
surface process, 143-45 S-Adenosylhomocysteine, 187 amino acid production from, 154
riboflavin production from, 225 S-Adenosylhomocysteine hydrolase, as carbon source, 61, 92
Acetobacter, 144, 234, 331 187 catabolism, 308
Acetobacter aceti, 144 S-Adenosylmethionine, 187 citric acid production from, 137,
Acetobacter pasteurianus, 144 Adenyl cyclase, 186-87 141-42
Acetobacter peroxidans, 144 Adenyl deaminase, 180 glutamic acid production from, 164
Acetobacter suboxydans, 135, 143, 298 Adenylsuccinate lyase, 176, 178 lysine production from, 168-69
Acetone, 48, 52, 61 Adenylsuccinate synthetase, 178 organic acid production from, 135
biosynthesis, 129-30 Adriamycin, 51, 230-31, 267 riboflavin production from, 225
production, 130-31 Adsorption chromatography, 120 single-cell protein production from,
Acetylation reaction, 294
Aeration, 85, 105 306-9
Acetyl-CoA carboxylase, 161, 263-64, vitamin B,, production from, 223
Aeration basin, 318
283
Aeration rate, 88 Alkylating agent, 17
3-Acetylpyridine, 28
Aerobacter, 135, 209 O%-Alkylguanine, 17
Acholeplasma laidlawii, 7
Aerobacter aerogenes, 92, 164, 198, O4-Alkylthymine, 17
Achromobacter, 207, 303, 327
Achromobacter lipolyticum, 208 200, 303 Allosteric effect, 35
Achromobacter liquidum, 170 Affinity chromatography, 120 Allosteric site, 35, 37
Achromobacter obae, 155, 165 Agar plug method, 28 Alternaria solani, 269
Agriculture, 54-55 Aluminum oxide, 214
Acidophile, 6
Acid protease, 203, 205 Agrobacterium, 44, 198 Amberlite, 120
Aclacinomycin, 231 Agrobacterium radiobacter, 234 Amidinotransferase, 38
Aconitase, 136 Agrobacterium tumefaciens, 54-55 Amikacin, 251
cis-Aconitic acid decarboxylase, 148 Agroclavine, 275, 278, 280-81 Amino acid, 2, 10, 27, 48, 51, 71, 105,
Acrasiomycete, 50 AICA, 185 112, 150-73, 298
Acremonium chrysogenum, 240 AICAR, 184-85 biosynthesis, 36, 157
Acridine orange, 16, 18 AICARP, 177, 185 production, 151-56, 215
Acriflavine, 18 AICARP formyltransferase, 185 chemical synthesis, 153
Actilyse, see Tissue plasminogen Air, sterilization, 100-3 process control, 157-58
activator Airlift process, 76, 319-20 product recovery, 158

343
344 / INDEX
from protein hydrolysate, 153 Ansamycin, 230, 262 Aspartic semialdehyde dehydrogenase,
strains, 156—57 Anthracene synthase, 263 166
racemic mixture, 153 Anthracycline, 5, 230-31, 267 Aspartokinase, 35, 166-68
uses, 151—52 Anthranilate synthase, 172-73 Aspergillus, 29, 33, 136, 156, 192-93,
Amino acid antibiotic, 230, 232, 243- Anthranilic acid, 170-71 206-7, 233, 274
49 Antibiotic, 1-2, 6, 51, 69, 71, 112-13, Aspergillus awamori, 49, 195
Amino acid dehydrogenase, 153, 291 121 Aspergillus clavatus, 135
Amino acylase, 153, 214 biosynthesis, controlled, 300 Aspergillus flavus, 148, 203
Aminoadipic acid pathway, 165 classification, 230 Aspergillus fumigatus, 34
p-Aminobenzoic acid, 28 economic significance, 232 Aspergillus itacontcus, 148
a-Aminobutyric acid, 28 hybrid, 52-53 Aspergillus nidulans, 29, 31, 34, 66,
DL-a-Aminocaprolactam, 165 increases in production, 10 234
L-Aminocaprolactam hydrolase, 155 microbial groups producing, 230 Aspergillus niger, 49, 66, 85, 89, 139—
D-Aminocaprolactam racemase, 155, microbial transformation, 300-3 41, 143, 192, 195-98, 203, 206-8,
165 modified, 51-53 210, 338
7-Aminocephalosporanic acid, 210, mutational synthesis, 300 Aspergillus ochraceus, 208, 296
241 in producer strain, 230 Aspergillus oryzae, 49, 148, 153, 180,
S-(6-Aminoethyl)-L-cysteine, 154, 165, production: 192, 195, 198, 203-4, 210
169 strains, 10, 51 Aspergillus rugulosus, 34
Aminoglycoside antibiotic, 39, 71, 105, levels, 25, 28-29, 32 Aspergillus sojae, 203-4
230, 232, 250-51, 301 purification, 120 Aspergillus terreus, 148-49
biosynthesis, 253-55 research on, 232-33 Aspergillus wentit, 135, 206
mode of action, 251-53 screening for, 6—7 Astaxanthine, 226
production: uses, 231-33 Asymmetric control, 178
methods, 255-56 Antibiotic resistance, 21, 27, 44, 46, ATC oxygenase, 265
strains, 253-55 48, 231, 233, 301 ATP, 35, 40, 176, 186, 216
structure, 253 Antifoam agent, 76-77, 88, 115, 141 Atrial natriuretic factor, 55
6-Aminopenicillanic acid, 208-10, Antimetabolite resistance, 27-28, 37, Attenuation, 36
234-36, 301 165 Aureobasidium, 331
p-Aminophenylalanine, 154, 171 Antimycin A,, 52 Aureobasidium pullulans, 331, 333
2-Aminopurine, 18 Antipain, 337 Autolysis, 118
p-Aminotyrosine, 154 Antithrombin IIL, 55 Autoregulation, 40-41
Ammonia, 163, 168 _ Antitumor agent, 7, 51, 203, 231, 249, Auxotrophic mutant, 27-29, 32, 36,
gaseous, 62 267, 271 156, 165
Ammonium salt, 62 AP endonuclease, 13 Avermectin, 261
Amoxycillin, 243 Apramycin, 52, 252 Avidin, 42
5'-AMP, 176, 178, 180 6-D-Arabinofuranosyladenine, 176 Avilamycin, 250
AMP deaminase, 178, 180 Arabinosyladenine, 187 8-Azaguanine, 175, 182, 185-86
Amphibolic pathway, 71 Arachidonic acid, 300 4-Azaleucine, 28
Amphomycin, 247 L-Arginine, 28, 152, 154 Azaserine, 186, 243
Amphotericin B, 39, 260 Arginine hydroxamate, 154 6-Azauridine, 176
Ampullariella regularis, 268 Aroma component, 340-41 8-Azaxanthine, 28, 186
Amylase, 7, 105, 112, 137, 189-98 Aromatic antibiotic, 230, 268-69 Azide, 171
saccharogenic, 191-92 Arrhenius equation, 96-97 Azotobacter, 330-31
starch-liquefying, 191-92 Arthrobacter, 158, 192, 200, 223, 302, Azotobacter vinelandii, 331-32
a-Amylase, 127, 139, 191-98, 211-12, 310 Azthreonam, 151, 243
214 Arthrobacter hyalinus, 223 Azureomycin, 7
bacterial, 192-96 Arthrobacter paraffineus, 135, 154, 164,
fungal, 195 187 Bacillus, 6, 33, 48, 182, 193, 198, 210,
uses, 193 Arthrobacter simplex, 294-96 230-31, 245, 310
B-Amylase, 191, 195, 198, 214 Artificial sweetener, 2, 151 Bacillus acidocaldarius, 192
Amylase inhibitor, 337 Arylamine synthetase, 38 Bacillus amyloliquefaciens, 54, 191-92,
Amyloglucosidase, 189 Aschersonia, 339 203
Anaerobe, 6 Ascorbic acid, 216, 219, 298-99 Bacillus amylosolvens, 194
Anaerobic digestion, 317, 322-24 Ascorbic acid oxidase, 216 Bacillus brevis, 39, 247
Anaplerotic reaction, 136 Ashbya gossypii, 223-25 Bacillus caldolyticus, 192, 212
Androgen, 292 Asparaginase, 189, 203, 283 Bacillus cereus, 116, 191, 195, 204, 327
1,4-Androstadiene-3,17-dione, 295-97 L-Asparagine, 152-53 Bacillus circulans, 53, 252, 255-56
Androstatriendione, 297 Aspartame, 2, 151, 156 Bacillus coagulans, 191-92, 200-1
Androstendione, 297 Aspartase, 155, 214 Bacillus firmus, 203
Animal feed, 220, 231-32, 246, 250 Aspartate decarboxylase, 155 Bacillus licheniformis, 31, 39, 185, 192,
lysine content, 151 Aspartate phenylalanine transaminase, 203-4, 246-49, 327
Animal growth promoter, 231-32 155 Bacillus luteus, 327
Ansa chain, 262 L-Aspartic acid, 150-52, 154-56, 191, Bacillus megaterium, 153, 185, 203-4,
Ansamitocin, 262 291 221, 300, 327
 INDEX / 345
Bacillus mesentericus, 206 stirred, 76-78, 80, 88 N-Carbamoy]l-L-tryptophan hydrolase,
Bacillus moritae, 339 Biosensor, 106 170
Bacillus natto, 192 Biosynthetic pathway: Carbapenem, 243
Bacillus polymyxa, 52, 192, 195, 198, branched, 35, 37 Carbohydrate, single-cell protein
206, 247, 327 unbranched, 35 production from, 307, 315
Bacillus popilliae, 339 Biotin, 42, 159, 161, 163, 168 Carbohydrate antibiotic, 230, 249-56
Bacillus pumilus, 31, 185, 203, 222 Biotransformation, see Microbial Carbomycin A, 257
Bacillus sphaericus, 155 transformation 1-Carbon assimilation, 311
Bacillus stearothermophilus, 96, 98, Bisulfite, 21 Carbon catabolite repression, 38
158, 192 Blade-wheel reactor, 76 Carbon dioxide, 86
Bacillus subtilis, 7, 29, 31, 34, 45, 47- Blakeslea trispora, 225, 227-28 effect on fermentation, 92-93
48, 170-71, 178, 181-86, 191-94, Blasticidin S, 232, 267 measurement, 106
198, 204, 206 Bleomycin, 230-31, 249 Carbon dioxide electrode, 106-7
Bacillus subtilis-ituriensiens, 247 Bluensomycin, 252 Carbon source, 38, 60-61
Bacillus thuringiensis, 55, 339 Bordetella, 209 Carboxymethyl cellulose, 214
Bacilysin, 245 Botrytis, 269 8-Carotene, 219, 225
Bacitracin, 39, 120, 230, 246-49 Bovine growth factor, 55 biosynthesis, 226-27
Bacitracin synthetase, 247-48 Brevibacterium, 33, 48, 51, 116, 155, economic significance, 226
Bacteria, recombination, 30-31 158-59, 164-65, 182, 303 occurrence, 226
Bacteriocin, 44 Brevibacterium ammoniagenes, 182-84, production, 227-28
Bacto-soytone, 200 186-87, 215 structure, 227
Baffle, 76, 83 Brevibacterium divaricatum, 163-64 Cartridge filter system, 102
Baffle centrifuge, 116 Brevibacterium flavum, 154, 156, 163- Casein hydrolysate, 200
Bafilomycin, 261 64, 167, 169, 171, 185 Cassava, 125-27
Bagasse, 127 Brevibacterium imperiale, 200 Cassette filtration system, 114-16
Baker’s yeast, 95 Brevibacterium lactofermentum, 34, 154, Catabolite activator protein, 38
Ball mill, 118 156-57, 165 Catabolite repression, 38-39, 59, 67,
Base analog, 16, 18 Brevibacterium liquifaciens, 186 194, 200
Base excision repair, 13 5-Bromouracil, 16, 18-19 Catalase, 189, 216, 310
Base substitution, 11 Bubble column, 88 CDP-choline, 176, 187
Basic research, 53-54 Bulking, sludge, 317-18 Cell concentration, 66, 68-69
Batch fermentation, 65-67, 70, 103 Butadiene, 129 Cell formation rate, 69
Batch sterilization, 98-99 2,3-Butandiol, 61 Cell-free extract, 291
Bayer Biotower, 319-20 Butanol, 48, 51, 61 Cell stabilization, 210-17
Beauveria bassiana, 339 Cellulase, 32, 314-15, 319
biosynthesis, 129-30
Beer, 70 Cellulase inhibitor, 337-38
production, 130-31
Bentonite, 214 Cellulomonas, 209, 314
Butirosin, 39, 53, 252, 255-56
Benzoic acid, 164 Cellulose:
Butyribacterium rettgeri, 221
Benzoquinone antibiotic, 230-31 as carbon source, 61
Butyric acid, 48, 129-30
Benzylalcohol dehydrogenase, 212 decomposition, 69
Butyryl phosphoadenosine, 41
Benzylpenicillin, 234, 236 organic feedstocks from, 124
Benzylthiocyanate, 265 single-cell protein production from,
Calcium phosphate gel, 214
Bestatin, 337 307
Calvin cycle, 311 Cellulose triacetate, 215
Betaine, 222
Campesterol, 296 Centrifugation, 115-17
B-rays, 16
Camphor degradation, 44, 319 Cephalosporin, 33, 38, 51, 234
Bgll, 21
Canavanine, 28 assay, 216
Bialophos, 245-46
Candicidin, 37, 39-40, 230, 260-61 biosynthesis, 241-42
Bikaverin, 335
Bingham-plastic solution, 84-85 Candida, 33, 135, 192, 206-7, 308 production:
Biochemical assay, 216-17
Candida boidinii, 116, 186, 310, 313 methods, 242
Biochemistry, antibiotics as tools in, Candida cylindraceae, 208 strains, 242
231-32 Candida flareri, 223 regulation, 241-42
Biochip, 340 Candida guilliermondii, 135 semisynthetic, 156, 210, 240-41
Biocomputer, 340 Candida lipolytica, see structure, 240
Bioconversion, see Microbial Saccharomycopsis lipolytica Cephalosporin C, 31, 39, 52, 210, 230,
transformation Candida oleophila, 307 240-42
Biofilter, 341 Candida parapsilosis, 310 Cephalosporin N, 52
Biogas, 321-23 Candida periculosa, 165 Cephalosporin P, 52
Biogel, 119 Candida tropicalis, 34, 307, 309, 315 Cephalosporin acylase, 210
Biomass concentration, 66 Candida utilis, 92, 170, 179, 315-16 Cephalosporinase, 216
Bioreactor, see also Fermenter Candidin, 260 Cephalosporium, 33, 192, 208
construction types, 74-76 Candihexin, 38-39 Cephalosporium acremonium, 29-33,
homogeneously mixed, 68 Capillary bundle filtration, 114-16 49-52, 234, 240-42
for immobilized enzymes or cells, Capreomycin, 94, 246-47 Cephamycin, 37, 39, 240-41
78-81 Caramelization, 99 Chaetomium cellulolyticum, 315
346 / INDEX

Chalcopyrite, 328 isolation of DNA sequences, 41-47 Cyclic AMP, 38, 40, 176, 186-87
Chanoclavin, 275, 278, 281 in acrasiomycetes, 50 Cyclic peptide antibiotic, 245-47
Chanoclavin-I-cyclase, 38, 282 Clostridium, 52 Cycloalkane antibiotic, 230
Chelate-forming antibiotic, 230, 247— Clostridium acetobutylicum, 129-30, Cycloheximide, 38-39, 52, 230, 232
49 223 Cyclol-synthetase complex, 279
Chemical modification of products, 4 Clostridium butylicum, 129 Cyclonic reactor, 80
Chemical sterilization, 98 5'-CMP, 180 Cycloserine, 230, 243-44
Chemostat, 68 Cobalamin, see Vitamin B,, Cyclosporin A, 340
Chemostatin, 337 Coenzyme A, 187 Cylindrocarpon radicicola, 295-96
Chitinase, 32, 268 Coenzyme B,,, 220 L-Cysteine, 151-53, 155-56, 163
Chitin synthase, 268 Colistin, 29 Cysteine desulfhydrase, 155
Chloramphenicol, 32, 38-39, 44, 229— Colony hybridization, 47 L-Cystine, 153
30, 268 Column chromatography, 118-19
Chlorbenzilate, 303 Combination electrode, 106 Dactinomycin, 244
a-Chlorcaprolactam, 154, 169 Cometabolism, 286, 302-3 DAHP synthetase, 172-73
Chlordecone, 303 Complementary DNA, 42-43 Dalapon, 302-3
Chlorobenzoate degradation, 44 Computer, 107-8 Dapiramine, 268
Chloroneb, 303 Comutation, 20 Data:
p-Chlorophenylalanine, 284 Condensation reaction, 286, 289-90 acquisition, 107
Chlorsalicylic acid, 304 Conidiobolus obscurus, 339 analysis, 107-8
Chlortetracycline, 10, 32, 37, 39, 51- Coniothrium hellbori, 85 Daunomycin, 51, 231, 267
52, 231, 262-67 Conjugation, 30-31, 34 Daunorubicin, see Daunomycin
Cholesterol, 216, 295-97 Contact dryer, 121 DDT, 302-4, 319
Cholesterol oxidase, 189, 216 Continuous fermentation, 2, 67—70, Death phase, 65, 67
Chorismate mutase, 172 72=73, 103 Death rate, 96-97
Chromatography, 118-21 Continuous gas phase, 76 y-Decalactone, 341
Chromobacter violaceum, 234 Continuous sterilization, 99-102 Decane degradation, 44
Chromomycin A,, 231 Convection dryer, 121 Decanter, 116-17
Chromopeptide antibiotic, 230-31, 244 Cooling system, 73 Decoyinine, 154, 186
Chromosome mutation, 11, 16 Copper leaching, 326, 328 Dehalogenation reaction, 303-4
Chymase, see Rennin Co-repressor, 36, 40 1-Dehydration, 295
Chymosin, see Rennin Coriolus, 206 Dehydration reaction, 287, 291
Cider, 144 Corn starch, 125-27, 197 C-1(2)-Dehydrogenase, 297
Citrate synthase, 136, 283 Corn steep liquor, 62, 131, 200, 225 1-Dehydrogenation, 295
Citric acid, 71, 76, 112, 134-35, 148 Cortisol, 292, 295 3,4-Dehydroproline, 28
biosynthesis, 136-37 Cortisone, 292, 294-95 1-Dehydrotestololactone, 295-96
production: Corynebacterium, 33, 48, 51, 135, 165, Deletion, 11, 15, 17-18, 20-21
media, 137-38 182, 206, 209, 223, 299 6-Demethy] tetracycline, 52
process, 138-39 Corynebacterium acetoacidophilum, 154 5-Deoxybutirosamine, 53
product recovery, 142 Corynebacterium alkanolyticum, 164 Deoxycholic acid, 292
strains, 135-36 Corynebacterium glutamicum, 103, 150, Deoxycortisol, 294-95
submerged process, 138, 140-42 154, 156, 158-59, 162-73, 183 2-Deoxystreptamine, 253, 255
surface process, 138-40 Corynebacterium guanofaciens, 185 2-Deoxystreptidine, 53
yield, 137, 140 Corynebacterium hydrocarboclastus, 164 Depsipeptide antibiotic, 230, 244-45
uses, 135 Corynebacterium murisepticum, 186 Depth filter, 101-2, 112
Citrinaxanthine, 226 Corynebacterium petrophilum, 182, 185 Deregulated mutant, 59
Citrobacter, 216 Corynebacterium simplex, 295 Design criteria, 96
Citromyces pfefferianus, 135 Cosmid, 44, 51 Destomycin, 252
Cladosporium cladosporioides, 338 Cost effectiveness, 9, 72, 115 Dextran, 2, 331, 333-34
Cladosporium herbarum, 338 Cosynthesis, 52 Dextranase inhibitor, 337-38
Claviceps, 29, 33, 277 Covellite, 328 Dextransucrase, 333
Claviceps fusiformis, 275, 280-81 Critical oxygen concentration, 89-90, Dextrin, 61, 191, 195
Claviceps gigantea, 275 94 Diacetyl, 341
Claviceps paspali, 275, 280 Cross-flow filtration, 114, 116 Dialysis, 81
Claviceps purpurea, 274-75, 279-80, Crossing over, 29-30 Diaminobutyric acid, 246
283-84 Cryptococcus laurentii, 155, 165 Diaminohydroxyazelain acid, 246
developmental cycle, 275-76 Crystallization, 112, 121 meso-Diaminopimelate decarboxylase,
Clavine, 274, 276 Culcinomyces clavosporus, 339 166
Clavulanic acid, 234, 243, 338 Culture media, 59-63 Diaminopimelate epimerase, 166
Clindamycin, 250 optimally balanced, 59 Diaminopimelic acid pathway, 164-66
Cloning, 4, 41, 47-50, 284 sterilization, 60, 96-103, 105 Diaminopropionic acid, 246
in eucaryotes, 49 Curamycin, 250 2,6-Diaminopurine, 28
in filamentous fungi, 49 Curdlan, 331-32 Diatomaceous earth, 113
in gram-negative bacteria, 47-48 Curvularia lunata, 294-95 Diauxy, 67
in gram-positive bacteria, 48-49 Cyanocobalamin, 220 Diazomethane, 16, 18
 INDEX / 347
Dibekacin, 251 Dynamic method, determination of Ergosterol, 219
Dicarboxylic acid cycle, 283 oxygen uptake rate, 90-91 Ergot, 274
3,5-Dichlorcatechol, 303 Dynamic viscosity, 84 Ergot alkaloid, 38, 40, 274-84
p-p'-Dichlorodiphenylmethane, 303 biosynthesis, 277-79
Di-(2-chloroethyl)-sulfide, 16-17 Edein A, 246 occurrence, 274
2,4-Dichlorophenoxyacetic acid Effector, 35, 40 production:
degradation, 44 Ejector aerator, 88 chemical synthesis, 279
Dictyostelium discoideum, 50 Elastatinal, 337 extraction from sclerotia, 279-80
2,4-Dideoxystreptamine, 53 Electrofusion, 32-33 fermentation, 280-81
Dieldrin, 302 Elymoclavine, 275, 278, 281 regulation, 281-83
Diepoxybutane, 16-17 Embden-Meyerhof-Parnas pathway, semicontinuous with immobilized
Diethylsulfate, 16-17 136, 159 mycelium, 281
Dihydrodipicolinate reductase, 166 Emericellopsis, 29, 240 strains, 283-84
Dihydrodipicolinate synthase, 165-67 Emericellopsis salmosynnemata, 31 structure, 275-77
Dihydroelymoclavine, 275 Emericellopsis terricola, 31 uses, 274-75
Dihydroergotamine, 275 Endoenzyme, 191 Ergotamine, 274-75, 277, 279, 281,
Dihydropenicillin F, 209 Endo-6-1,4-glucanase, 314 284
Dihydrostreptomycin, 29, 251-52 Endomyces, 85-86 Ergotism, 274
Dihydroxyacetone, from glycerol, 299- Endomycopsis, 143 Ergotoxine, 275, 281, 284
300 Endonuclease, 180-81 Erwinia, 198, 209, 299
Dihydroxyacetone cycle, 312-13 Endothia, 206 Erwinia aroideae, 203
L-Dihydroxy phenylalanine, 156 Endothia parasitica, 206 Erwinia carotovora, 203
2,5-Diketo-D-gluconic acid, 299 End product, 35 Erwinia herbicola, 155, 299
Dilatant solution, 84 End product regulation, 38 Erynia neoaphidis, 339
Dimethylallylpyrophosphate, 278 Enduracidin, 232, 247 Erythromycin, 10, 32, 51, 53, 92, 108,
4-Dimethylallyl tryptophan, 278 Energy charge, 35 230, 256-59
4-Dimethylallyl tryptophan synthase, Energy metabolism, 70-71 Escherichia, 192, 209
38, 40, 282 Enrichment procedure, 6 Escherichia coli, 2, 35-36, 44-48, 51,
Dioscorea composita, 295 Enterobacter cloacae, 171 96, 154-56, 164-65, 170, 172-73,
Diosgenin, 294-95 Entner-Doudoroff pathway, 311 185-86, 203, 206
Diploidization, 51 Entomophthora, 206 concentration, 115
Disintegration of microorganisms, Entomophthora grylli, 339 conjugation, 31
117-20 Environmental protection, 55 critical oxygen concentration, 90
Distiller’s solubles, 200, 225 Enzyme, 105, 112 DNA repair, 14-15
DNA: breakdown, 35 filtration, 116
alkylation, 11-15, 17-18 carrier-bound, 211, 213-14, 291 foreign protein production, 42
constitutive, 36-37 growth rate, 66
crosslinks, 11-12, 15-17
cross-linked, 211-13 lysine biosynthesis, 165-67
deamination, 11-13, 17, 21
encapsulated, 204, 211, 214-15 mutagenesis, 17-20
double-strand breaks, 11, 16
immobilized, see Immobilized oxygen requirement, 92
foreign:
enzyme penicillin acylase, 103, 209
incorporation into plasmid, 45
modification, 35 protoplast, 33
insertion into host, 45
plate assay, 28 specific oxygen requirement, 89
isolation of sequences for cloning,
purification, 121 Escherichia freudii, 200
41-47
regulation of activity, 35 Escherichia intermedia, 200
nonreplicating, 16-17 regulation of synthesis, 36 Essential amino acid, 151, 164
repair mechanisms, 11-15 stabilization, 210-17 Esterase inhibitor, 338
single-strand breaks, 11, 16 uses, 189-90 Esterastin, 338
synthetic, 42 Enzyme electrode, 216 Estrogen, 292
DNA endonuclease, 12 Enzyme inhibitor, 337-38 Ethanol, 112
DNA exonuclease V, 14 Enzyme/thermistor, 216 acetic acid production from, 144-46
DNA gyrase, 269 Epidermal growth factor, 55 assay, 216
DNA polymerase, 11-13, 18 Epidermophyton floccosum, 208 biosynthesis, 125-26
L-DOPA, 155-56 2-Epistreptamine, 53 as carbon source, 61, 74, 92
Dopastin, 338 Epithienamycin, 234, 243 from cellulose, 61
Doubling time, 69 Epoxidation reaction, 287 citric acid production from, 137
Dowex, 120 Eremothecium ashbyti, 223-24 glutamic acid production from, 164
" Downstream processing, see Product, Ergoblasine, 274 lysine production from, 168-69
recovery Ergocornine, 275-77, 280-81, 284 production, 2, 70, 124-28, 216
Doxorubicin, see Adriamycin Ergocristine, 275-77, 280-82, 284 from starch, 61
Dry ice, 131 Ergocryptine, 275-77, 280-82, 284 vitamin B,, production from, 223
Drying of product, 112, 121 Ergometrine, 275-76, 278, 280-81 Ethidium bromide, 21
Dry malt extract, 60 Ergopeptam, 276, 279 Ethionine, 28, 154
Dunaliella salina, 132, 225 Ergopeptin, 276-79 Ethylmethanesulfonate, 16-17
Duplication, 11, 20 Ergosine, 275, 277, 279-81, 284 Eucaryote:
348 / INDEX
cloning in, 49 p-Fluorophenylalanine, 28, 30, 154, Gas:
genetic organization, 42 171, 284 distribution:
Everninomicin, 230, 250-51 B-Fluoropyruvate, 154, 169 continuous gas phase, 76
Excision repair, 12-15, 17 Fluorotryptophan, 171 by pressurized air, 76
Exoenzyme, 48-49 5-Fluorouracil, 28 by pump, 76
Exo-6-1,4-glucanase, 314 Foam separator, 76-78 by stirring, 74-76
Exon, 42, 53 Folic acid, 219 sterilization, 96-103
Exonuclease, 180-81 Food, lysine content, 151 Gaseous phase, 81
Expandase reaction, 241 Food additive, 2 Gas exchange, 86-92
Expression vector, 44-45 Food chain, human, 306-7 Gas oil, 308-9
Extracellular polysaccharide, 330-34 Food industry, amino acids used in, Geldanamycin, 262
Extraction, 121 151 Gel enclosure, of enzymes, 214-15
Food preservative, 231 Gel filtration, 119
Factor A, 38, 40, 253, 255 Foreign protein: Gene amplification, 50-51
Factor VIII, 2, 54-55 glycosylation, 47, 49 Gene bank, 41-42, 44
Factor IX, 55 proteolysis, 48 Gene cloning, see Cloning
FAD, 176, 187 secretion, 47-48 Gene dose, 9
FAICARP, 177, 185 solubility, 48 Gene manipulation, 41
Fats, as carbon source, 61 Formaldehyde, fixation, 311-12 Gene mutation, see Point mutation
Fatty acid, 161, 337 Formaldehyde dehydrogenase, 310 Gene probe, 42, 54
Fed-batch fermentation process, 67 Formate, methane production from, Generalized transduction, 30-31
Feedback inhibition, 35 323 Gene technology, 41-50
elimination, 36-37 Formate dehydrogenase, 310 applications, 53-55
Fermentation process, 64-108 N-Formimidoylthienamycin, 243 Gene therapy, 54
classification, 70-71 Fortimicin, 251-52, 255-56 Genetic change, 5
conditions, 72-73 Fosformycin, 38, 230 Genetic engineering, 1, 41
control, 107 Fradicin, 52 risks, 50
models, 108 Frameshift mutation, 11, 15-19 Genetic recombination, see
optimization, 107 Frings bioreactor, 88 Recombination
stages, 103-6 Fritted disc aerator, 88, 95 Genome fragment, 41-42
Fermenter, 74-81, see also Bioreactor Frone, see Interferon beta Genome mutation, 11
layout for installation, 78-79 Froude’s number, 83 Gentamicin, 32, 34, 232, 252, 254-56,
size, 105 Frozen culture, 104 301
Fermenter preculture, 103, 105 Fructose, 198-200 Geotrichum, 207
Ferrimycin, 247 Fructose syrup, 201-3 Geotrichum candidum, 66, 207-8
Ferrioxamine, 247 Fumagillin, 95-96 Gibberella fujikuroi, 335-36
Fertility factor, 31, 44 Fumarase, 215 Gibberella zea, 336
Festuclavine, 275 Fumaric acid, 135, 154 Gibberellan, 335-36
F factor, 31 Fungi: Gibberellic acid, 61, 335-36
Fibrous polymer, immobilization of antibiotics produced by, 230 Gibberellin, 39, 335-36
enzymes, 215 imperfect, 30 Glass fiber filter cartridge, 102
Filamentous fungi, cloning in, 49 parasexual cycle, 29-31 Glucoamylase, 85-86, 127, 190-91,
Filipin, 260 recombination, 31 195-98, 214-15, 291
Filter cake, 113 sexual cycle, 29-31 Glucocorticoid, 292
Filter cartridge, 102 Fungicidin, 38 Gluconic acid, 70, 134, 142-43, 298
Filter centrifuge, 116 Fungimycin, 260 Gluconobacter, 144, 234
Filter press, 113-14 Fusaric acid, 338 Gluconobacter melanogenus, 299
Filter sterilization, 98, 101 Fusarium, 303 Gluconobacter oxydans, 144
Filtration of product, 112-15 Fusarium avanaceum, 66 Gluconolactone, 142-43
Filtration enrichment, 28 Fusarium graminearum, 66-67, 315 Glucose:
Fixed-bed reactor, 80, 323 Fusarium moniliforme, 335 assay, 216
Flambamycin, 250 Fusarium roseum graminearum, 336 as carbon source, 60, 66, 74, 92
Flavobacterium, 209, 225 Fusarium semitectum, 208 catabolite repression by, 38-39
Flavobacterium aminogenes, 170-71 Fusarium solani, 296 conversion to amino acids, 154, 157 ,
Flavobacterium ammoniagenes, 156 Fusidic acid, 230, 270 160, 162
Flavobacterium arborescens, 200 Fusidium coccineum, 270 conversion to ethanol, 125-26
Flavomycin, 95, 112 Fusion protein, 45 conversion to fructose, 191
Flavor enhancer, 2, 151, 175, 340 conversion to gluconic acid, 143
Flavoring compound, 340-41 Galactose, 216 enzyme production from, 197
Flexibacter, 234 Galactose oxidase, 189, 216 isomerization, 199
Flocculation, 112 a-Galactosidase, 215 kojic acid production from, 148
Flotation, 112 B-Galactosidase, see Lactase lactic acid production from, 147
Flow rate, 68-70 Gamma rays, 16 lysine production from, 169
Fluidized-bed reactor, 80 Gas absorption rate, 89 organic acid production from, 134-
9a-Fluoro-16a-hydroxycortisol, 295 Gas bubble, 86-88 35
 INDEX / 349
single-cell protein production from, Guanylic acid, see 5'-GMP 9a-Hydroxy-4-androstene-3,17-dione,
315 295-96
sweetness, 199 Haemophilus, 269 11a-Hydroxy-4-androstene-3,17-dione,
tryptophan production from, 171 Hallucinogen, 275 296
Glucose isomerase, 70, 189-90, 198- Halomycin, 262 2-Hydroxybutirosin, 53
200, 211, 214-15, 291, 298 Halophile, 6, 132 N‘-Hydroxycytidine triphosphate, 21—
Bacillus, 200-1 Hamycin, 260 22
immobilization, 201-3 Hansenula anomala, 170-71 N‘-Hydroxydeoxycytidine triphosate,
Streptomyces, 201 Hansenula capsulata, 310 21
Glucose oxidase, 142-43, 189, 214, Hansenula henricii, 310 a-Hydroxyethyllysergamide, 275, 278,
216-17 Hansenula minuta, 310 280
Glucose-6-phosphate dehydrogenase, Hansenula nonfermentans, 310 16a-Hydroxy-9a-fluoroprednisolone,
40 Hansenula polymorpha, 29, 190, 225, 292
Glucose syrup, 61, 198-99 310, 313 D-Hydroxyisocaproate dehydrogenase,
B-Glucosidase, 314 Hansenula wickerhamii, 310 155
Glucosidase inhibitors, 337-38 Haploidization, 30 Hydroxylamine, 17
L-Glutamic acid, 61, 103, 105, 135, Heat exchanger, 100-1 Hydroxylase, 292, 295, 297
150-52, 154, 156, 158, 175 Heat loss, 73 Hydroxylation reaction, 287
biosynthesis, 159-61 Heat of combustion, 74 in steroid process, 292-295, 297
production: Heat production, 73-74 6-Hydroxynorleucine, 28
conditions, 161-63 Heat sterilization, 97-99, 101 15a-Hydroxy-4-pregnene-3,20-dione,
effect of permeability, 159-61 Heat transfer coefficient, 100 296
processes, 163-64 Helotium, 332 11la-Hydroxyprogesterone, 295-96
purification, 121-22 Henry’s law, 86 2-Hydroxysagamicin, 53
strains, 158-59 Hepatitis B vaccine, 54-55 3-Hydroxy-9,10-secoandrostatriene-
Glutamic acid dehydrogenase, 39, 69, Herofor, see Interferon alpha 2 9,17-dione, 297
158-60, 313 Heterocyclic antibiotic, 230, 232 Hygromycin B, 231, 252
L-Glutamine, 152, 154 Heterokaryon, 23, 29-30, 33-34 Hygromycin B phosphotransferase, 50
Glutamine synthetase, 35, 39, 158, Heterologous protein, see Foreign Hyperinduced strain, 51
160, 283 protein Hyphomicrobium, 310
Glutaraldehyde, 212-13 Hexacyanoferrate, 137 Hypoxanthine, 183-84
Glyceraldehyde phosphate Hexamethylene diisocyanate, 213 Hypoxanthine DNA glycosylase, 13
dehydrogenase, 132 Hexane degradation, 44
Glycerol, 131-32, 216 Hexokinase, 189, 216 ICI Pressure Cycle Fermenter, 312-13
as carbon source, 39, 92 Idiolite, 38
Hexulose phosphate synthase, 311
deficiency, 161 Hfr strain, 31
Idiophase, 38-39, 69-71, 140
dihydroxyacetone from, 299-300 Immobilized cells, 2, 78-81, 126, 153,
High-fructose syrup, 2, 199, 215
Glycine, 151-53 201, 211, 2137, 291
Hindill, 43
Glycolipid antibiotic, 230 analytical biochemical uses, 216-17
Hirsutella thompsonii, 339
Glycolysis, 125, 311 L-Histidine, 28, 36, 151-52, 154, 156-
industrial uses, 215-16
Glycopeptide antibiotic, 231, 250 Immobilized enzyme, 2, 78-81, 153,
57
Glycosidation reaction, 290 200-1, 211-14, 291
Homocitrate synthase, 236
Glycoside antibiotic, 230-31, 249-50 analytical biochemical uses, 216-17
Homogenizer, 118-20
1,6-Glycoside-splitting enzyme, 197- industrial uses, 215-16
L-Homoserine, 168
98 5'-IMP, 175-76
Homoserine dehydrogenase, 167-68 biosynthesis, 176-78
Glyoxylate cycle, 137, 159-60, 283
Hormone, 292 production:
Glyphosate, 151
Hot spot, 24 direct fermentation, 181-84
5'-GMP, 175-76
Human gonadotropin, 55 RNA hydrolysis, 179-81
biosynthesis, 176-78
Human growth hormone, 2, 10, 54-55 regulation, 178
production:
direct fermentation, 184-86 Humicola, 314 IMP dehydrogenase, 178, 182, 185-86
RNA hydrolysis, 179-81 Humulin, see Insulin Impeller, see Stirrer
regulation, 178 Hybrid antibiotic, 52-53 Inactivation factor, 96
GMP reductase, 178, 185-86 Hybridization, 32, 34 Inclusion body, 48, 111
GMP synthetase, 178, 184-86 Hybridoma technique, 2, 54 Indole, 170, 172
Gonatobotrys, 143 Hybrimycin, 53 Indole alkaloid, 275
_ Grain, ethanol production from, 126 Hydantoin, 153-56 Indolizomycin, 52
Grain spirits, 144 Hydantoinase, 153-56, 171 Indolmycin, 39
Gramicidins, 38-39, 246-47 Hydration reaction, 289 Induction, 36, 38
Grass, 314 Hydrocortisone, 296 Injection port, 78
Griseofulvin, 31, 52, 112, 230, 269 Hydrogenomonas, 29, 303 Inoculation, 104
Growth curve, 65, 107-8 Hydrol molasses, 60 Inoculum:
Growth kinetics, 64-74 Hydrolysis reaction, 286, 289, 291, build-up, 103-5
Guanine, 28 294 concentration, 65
Guanosine, 184-85 Hydroxocobalamin, 220 physiological condition, 65
350 / INDEX
preservation, 103—4 Kasugamycin, 28, 32, 232, 251-52, Ligninase, 314
size, 105 254-55 Limonene, 340
Inosine, 176, 181—84 a-Ketoacid, 153 Lincomycin, 114, 250
Inosinic acid, see 5'-IMP Ketogluconic acid, 135 Lindane, 302
Insecticide, microbial, 338—39 a-Ketoglutarate, 134-35, 159 Linear peptide antibiotic, 245-47
Insect virus, 339 a-Ketoglutarate dehydrogenase, 158- Linker-adaptor method, directed
Insertion, 18 59 mutagenesis, 21
site-specific, 21 2-Keto-1-gulonic acid, 299 y-Linolenic acid, 337
Insertion vector, 44 Ketozyme, 200 Lipase, 7, 189, 207-8, 214, 319, 337
In situ leaching, 327-29 Killer factor, 51 Lipomyces kononkoae, 34
In vitro recombination, 41 Klebsiella, 223, 310 Liposome, 32
Instrumentation, 106-7 Klebsiella aerogenes, 89, 155 Liquid phase, 81
Insulin, 10, 42-43, 45, 54-55, 119 Klebsiella pneumoniae, 116, 221 Lividomycin, 252
Interferon, 2, 10, 49, 54-55, 119, 121 Kloekera, 310 Log phase, 65-67
Interleukin-2, 49, 55 Kluyveromyces, 33 Loop reactor, 76
INTERMIG stirrer, 78 Kluyveromyces fragilis, 34, 125, 210 LSD, 275
Intron, 42, 53 Kluyveromyces lactis, 34, 210 Lycopene, 225-26
Intron A, see Interferon alpha 2 Kojic acid, 148, 298 Lymphokine, 55
Inversion, 11, 16 Lyophilization:
Invertase, 214-17 B-Lactam antibiotic, 34, 49, 52, 230, product, 121
Invertase inhibitor, 120 233-43, 301-2 strain, 104
Ion exchange chromatography, 120 B-Lactamase, 241, 301 Lysergic acid, 274-76, 278, 281
Ion exchanger, as enzyme carrier, 8-Lactamase inhibitor, 7, 243 A®?-Lysergic acid, 278, 280-81
213-14 Lactase, 210, 215-16 Lysergic acid alkaloid, 276
Ionizing radiation, 15-16 Lactate dehydrogenase, 147 Lysergic acid diethylamide, 275
B-Ionone, 228 Lactic acid, 61, 71, 134, 147 Lysergylalanine, 278
Ionophore, 270 Lactivicin, 7 L-Lysine, 150-52, 154-56, 158, 168,
Iron: Lactobacillus, 33, 192, 198, 206 242
in citric acid fermentation, 138 Lactobacillus bulgaricus, 147, 205 biosynthesis, 165-68
in riboflavin biosynthesis, 224 Lactobacillus casei, 116, 155 production:
Irpex, 206 Lactobacillus confusus, 155 from a-amino caprolactam, 165
Irumamycin, 261 Lactobacillus delbreuckii, 147 conditions, 168-69
IS-element, 19-20 Lactobacillus helveticus, 205 direct fermentation, 165-69
Isoamylase, 191, 197-98 Lactobacillus leichmannii, 147 strains, 165
Isocitrate dehydrogenase, 35, 136, Lactobacillus pentosus, 147 via diaminopimelic acid, 164-65
159-60 Lactone synthase, 258 regulation, 165-68
Isocitrate lyase, 137, 158-60 Lactose, 61, 135, 197, 216 Lysobacter, 6
Isocitric acid, 142 Lagenidium giganteum, 339 Lysozyme, 32
Isoelectric focusing, 120-21 Lag phase, 65
Isoenzyme, 35 Laminar flow, 82 Macarbomycin, 232, 250
Isoflavone, 338 Lankacidin C, 300 Macrocyclic lactone antibiotic, 256-62
L-Isoleucine, 28, 152-54, 156 Lard oil, 141 Macrolide antibiotic, 52, 230, 232,
Isomerase, 189 Lasalocide, 231, 270 256-59, 301
Isomerization, 286 Leaching, microbial, 76, 326-29 atypical, 261-62
Isoniazid, 28, 228 chemistry, 327 Macrotetrolide antibiotic, 230, 232,
Isooctanoic acid, 246 commercial processes, 327-29 256, 261
Isopenicillin, 241 organisms, 326-27 Maillard reaction, 60-61, 99
Isopenicillin N synthetase, 242 Lead leaching, 326 Malate synthase, 137
Isopentenylpyrophosphate, 278 Leaves, composition, 314 Malic acid, 74, 134-35, 215, 291
Isoproduction curve, 108 Lethal mutation, 24 Malic enzyme, 159-60
Isopropanol, 48, 61, 129 L-Leucine, 28, 94, 152, 154-55, 167 Malt, 144
biosynthesis, 129-30 Leucine dehydrogenase, 155 Malt extract, 60, 200
as carbon source, 74 Leucomycin, 52, 256-57 Maltose, 197
vitamin B,, production from, 223 Leuconostoc, 198 Maltose syrup, 191
G-Isopropylmalate dehydrogenase, 49 Leuconostoc brevis, 135 Manihot esculenta, 126
Isotime curve, 108 Leuconostoc dextranicum, 331 Manioc, 126
Istamycin, 52, 252 Leuconostoc mesenteroides, 331, 333 Mannosido-streptomycin, 52
Itaconic acid, 71, 134, 148-49 Leudeking-Piret equation, 331 Maridomycin, 257
Itatartaric acid, 148 Leukaemomycin, 41 Marker inactivation technique, 46-47
Iturin A, 247 Leupeptin, 337 Mass spectrometer, 106
Levorin, 52, 260 Mass transfer, 86-92
Josamycin, 257 Levoristatin, 52 Master strain, 104
Lewatit, 120 Mattesia trogodermae, 339
Kanamycin, 39, 114, 251-53 L factor, 41 Maximal productivity, 72
Karyogamy, 29 Licheniformin, 245 Maximum specific growth rate, 66, 69
 INDEX / 351
Mechanical foam separator, 77-78 O*-Methylguanine DNA methyl Mikamycin, 232, 245
Media, see Culture media transferase, 15 Milbemycin, 261
Medicine: Methyl ketone, 340 Mildiomycin, 232, 267
amino acids used in, 151 y-Methyl-L-lysine, 154, 169 Millet, 127
diagnostics techniques, 54 Methylmethanesulfonate, 16-17 Mineral corticoid, 292
nucleotides and nucleosides used in, a-Methylmethionine, 28 Mithramycin, 231
175 6'-N-Methylneamine, 53 Mitomycin, 14, 39, 230-31, 270-71
Medrol, see 6a-Methylprednisolone N-Methyl-N'-nitro-N- Mitotic recombination, 23
Meiosis, 29 nitrosoguanidine, 16-18, 20 Mixed culture, 97
Membrane/enzyme reactor, 81 N-Methyl-N-nitrosourea, 16-17 Mixing, 81-86
Membrane filtration: Methylobacter, 310 Mixing time, 82, 94
cross-flow, 114 Methylobacter acidophilus, 179 Mocimycin, 232
static, 114 Methylococcus, 310 Moenomycin, 230, 232, 250
6-Mercaptoguanine, 182, 186 Methylococcus capsulatus, 309 Molasses: ,
6-Mercaptopurine, 175, 182—83 6-Methyloctanoic acid, 246 acetone/butanol production from,
Merozygote, 30 Methylocystis, 310 129, 131
Metabolite: Methylomonas, 310-11 as carbon source, 60
excess production, 36—41 Methylomonas methalonica, 313 citric acid production from, 137-39
primary, 5—6, 36-37 Methylomonas methanica, 309 ethanol production from, 126-28
production, 69 Methylomonas methanolica, 310 glutamic acid production from, 162,
regulation, 37-41 Methylomonas methylovora, 164 164
screening for new, 6 Methylosinus, 310 lysine production from, 168
secondary, 5-6, 37-41 Methylotroph, 309-10 single-cell protein production from,
modified, 51-53 Methylovibrio soehngenii, 309 315
Metalloprotease, 120 6a-Methylprednisolone, 292 tryptophan production from, 171
Metarhizium anisopliae, 339 cis-4-Methylproline, 300 in yeast production, 179
Metaxylene degradation, 319 6-Methylthiopurine, 182 Molecular biology, antibiotics as tools
Methane: O-Methyltransferase, 48 in, 231-32
as carbon source, 74, 92 4-Methyltryptophan, 171 Molecular sieve chromatography, 119
production from sewage, 321-23 5-Methyltryptophan, 28, 154, 171 Monensin, 230-31, 270
single-cell protein production from, 6-Methyltryptophan, 28 Moniliales, 307
306-7, 309 Microbacterium, 158, 182, 186 Monoamine oxidase, 216
Methane oxygenase, 309 Microbacterium ammoniaphilum, 154 Monobactam, 234, 243
Methanobacillus omelianski, 223 Microbial growth, oxygen transfer rate Monoclonal antibody, 2, 54
Methanobacterium soehngenii, 223 from, 91-92 Monod equation, 66-68
Methanogen, 323-24 Microbial transformation, 4, 219, 286 Mucor, 33, 192, 206-7, 337
Methanol: antibiotics, 300-3 Mucor hiemalis, 66
amino acid production from, 153 applications, 291-92 Mucor miehei, 206
as carbon source, 61, 74, 92 nonsteroid compounds, 298-300 Mucor piriformis, 135
citric acid production from, 137 pesticides, 301-4 Mucor pusillus, 206
fermentation, 310 procedures, 290-91 Mucorales, 307
glutamic acid production from, 164 steroids and sterols, 292-97 Multi-purpose cloning site, 44
methane production from, 323 types, 286-90 Multivalent inhibition, 35
oxidation, 310-11 Microbispora rosea, 200 Muramidase, 189
single-cell protein production from, Microcapsule, 215 Mustard gas, see Di-(2-chloroethyl)-
307, 309-13 Micrococcus, 192, 209, 310 sulfide
vitamin B,, production from, 223 Micrococcus glutamicus, see Mutagen, 11, 15
Methanol dehydrogenase, 310 Corynebacterium glutamicum Mutagenesis:
Methanol oxidase, 310 Micrococcus luteus, 13 with chemical agents, 16-18
Methanomonas margaritae, 309 Microellobospora flavea, 200 direct, 17
Methanosarcina barkeri, 223 Microfiltration, 114 directed, 20-22
Methionine, 28, 38, 151-54, 156, 158, Micromonospora, 33, 84, 221 optimization, 24-25
167-68 Micromonospora carbonaceae, 250 radiation, 15-16
Methionine hydroxamate, 154 Micromonospora chalcea, 32 sequential, 20
Methionine sulfoxide, 182, 185-86 Micromonospora coerula, 200 site-directed, 10
7-Methoxycephalosporin, 234 Micromonospora echinospora, 32, 252 treatment conditions, 24
8-Methoxypsoralen, 15-16 Micromonospora grisea, 252 Mutamicin, 53
3-Methyladenine DNA glycosylase II, Micromonospora inyoensis, 24-25, 53, Mutarotase, 216-17
15 252 Mutase, 81
1-Methyl-6!-androstenolone, 292 Micromonospora olivoasterospora, 252 Mutasynthesis, 52-53, 284
6'-N-Methylbutirosin, 53 Micromonospora purpurea, 32, 34, 252 Mutation, 10, 104
Methylcobalamin, 220 Micromonospora sagamiensis, 53, 252 induced, 10-11
Methylenomycin, 31-32, 44, 48, 51 Micronomicin, 252 multiple, 26
Methylergoblasine, 274 Midekamycin, 257 phenotypic expression, 22-24
N-Methyl-N-glucosamine, 253 MIG stirrer, 78 recessive, 22-24
352 / INDEX
during SOS repair, 14—15 Notatin, 143 Paecilomyces, 240
spontaneous, 10-11 Novobiocin, 39, 52, 85, 89, 95, 230, Paecilomyces divaricatum, 135
Mutation and selection program, 26 269 Paecilomyces varioti, 315
Mutation rate, 10-11 Nuclease P,, 179-80 Palm oil, 61
Mutator gene, 19 Nuclease $1, 21, 43 Pantothenic acid, 219
Mycelianamide, 52 Nucleic acid, 112 Paper manufacturing waste, 314-16
Mycelium, 71, 84-85, 88, 114, 140-42 Nucleosidase, 182, 184-85 n-Paraffin, 74
Mycobacterium, 295, 297 Nucleoside, 5, 175-76 Parasexual cycle, fungi, 29-31
Mycobacterium fortuitum, 295-96 Nucleoside antibiotic, 187, 230, 232, Paromomycin, 252
Mycobacterium phlei, 225 267-68 Particle-bound system, 80
Mycobacterium smegmatis, 32, 34, 223 Nucleotidase, 180-84 Parvulin, 247
Mycobacterium tuberculosis, 244 Nucleotide, 27, 48, 175 Patent applications, 2
Mycoheptin, 260 biosynthesis, 176-78 Patulin, 31
Myeloma, 54 production: Pectate lyase, 206-7
Myocandida riboflavina, 223 direct fermentation, 175, 178-79, Pectin, 206-7
Myxobacteria, 6 181-86 Pectinase, 189-90, 206-7
RNA hydrolysis, 175, 178-81 Pectinesterase, 206
NAD, 7, 120, 176, 187 regulation, 178 Pectin lyase, 206-7
NADH,, 106 structure, 176-78 Pediococcus, 198
NADP, 187 uses, 175-76 PEG fusion, 32
NADPH, 187 Nucleotide analog, 21-22 Penicillin, 1, 10, 30, 37-39, 67, 69, 82,
Nalidixic acid, 29 Nucleotide-excision repair, 12, 16 85, 92, 95, 105, 112, 116, 121, 229-
Naphthalene degradation, 44 Nystatin, 29, 85, 95-96, 260 30, 233-34
Naphthoquinone antibiotic, 230 biosynthesis, 236
Narbomycin, 303 Octane degradation, 44, 319 biosynthetic, 235
Nebramycin, 255 Odor removal, 341 glutamic acid excretion and, 161
Neocarzinostatin, 231, 247 Oils, as carbon source, 61 microbial transformation, 301-2
Neomycin, 32, 37, 39, 52-53, 114, Oil spill degradation, 319 natural, 234
252, 254-55, 300-1 Oleandomycin, 52, 256-57 production:
Neplanosin A, 268 Oleic acid deficiency, 159, 161 methods, 238-40
Netilmicin, 251 Oligonucleotide mutagenesis, 22 strains, 236-38
Neurospora, 192 Olivanic acid, 234, 243 regulation, 236
Neurospora crassa, 44, 49 Olive oil, 61 semisynthetic, 156, 208, 235
Neurospora sitophila, 66 structure, 234-36
Oosponol, 338
Neutral protease, 203-5 Operating cost, 72 Penicillin B, 143
Newspaper, composition, 314 Optisweet, 200
Penicillin G, 31, 114, 208-9, 235, 238-
Newton’s law, 84 40
Oraspor, 242
Nickel leaching, 326
Ore, leaching, 326-29
splitting with carrier-bound
Nicotinic acid, 28 penicillin acylase, 210
Organic acid, 134-49
Nifirimycin, 338
Organic feedstock, 124-32
splitting with whole bacteria, 209-
Nikkomycin, 94, 268 10
Organic solvent, 69
Nisin, 231, 247 Penicillin K, 209
Origin of replication, 45, 48
Nitrogen source, 62-63 Penicillin N, 31, 240-41
L-Ornithine, 152, 154
catabolite repression, 39 Penicillin O, 31, 235
Orotic acid, 176, 187
Nitrous acid, 16-18, 24-25 Penicillin V, 31, 208-9, 235, 238-40
Orthosomycin, 230, 250-51
Nocardia, 6, 33, 198, 209, 223 Penicillin acylase, 51, 103, 105, 189,
Oudenone, 338
Nocardia asteroides, 200 208, 291, 298, 301
Oxalic acid, 142
Nocardia dassonvillei, 200 carrier-bound, 210
Nocardia erythropolis, 32 Oxaloacetate carboxylase, 159-60
classification, 208-9
Nocardia gardneri, 223 Oxidation, incomplete, 144
Escherichia coli, 209
Nocardia lactamdurans, 234, 241 Oxidation ditch, 318 Penicillinase, 45
Nocardia mediterranei, 32, 41, 262 Oxidation reaction, 286-88, 291, 294, Penicillinase inhibitor, 338
Nocardia orientalis, 34 308 Penicillin-binding protein, 234
Nocardia rhodochrous, 295 Oxidoreductase, 38, 189 Penicillin resistance, 7
Nocardia uniformis, 234 Oxygen, 86 Penicillin-selection procedure, 28
Nocardicin, 243 measurement, 106 Penicillium, 33, 143, 192, 206=7, 274,
Nocardin, 234 requirement, 92 314
Nojirimycin, 230, 249-50 as substrate, 88-89 Penicillium chrysogenum, 29-31, 34,
Nomurea rileyi, 339 transfer, 86-88 37, 49, 66, 69, 82, 85, 89-90, 92,
Norleucine, 28, 284 Oxygen electrode, 106-7 114, 208, 234, 236-39
Northern blotting technique, 47 Oxygen transfer rate, 87, 91, 95-96 Penicillium citrinum, 34, 135, 179-80
Norvaline, 284 Oxygen uptake rate, 89 Penicillium digitatum, 340
Nosema algerae, 339 determination, 89-92, 216 Penicillium griseofuluum, 52, 269
Nosema locustae, 339 Oxytetracycline, 10, 32, 51, 71, 263, Penicillium luteum, 135
Nosema pyrausta, 339 266 Penicillium luteum-purpurogenum, 143
 INDEX / 353
Penicillium notatum, 229, 233, 236 Pichia haplophila, 310 Propionibacterium freudenreichii, 34,
Penicillium patulum, 31, 269 Pichia lindnerii, 310 221
Penicillium roqueforti, 207-8, 340 Pichia miso, 225 Propionibacterium shermanii, 221
Pentose phosphate cycle, 40, 136, Pichia pastoris, 310 Propionic acid, 135
159-60, 265, 283 Pilot plant, 86 Prostaglandin, 299—300, 337
Pepstatin, 337 : Pimaricin, 231, 260 Protaminobacter, 310
Peptide antibiotic, 6, 48, 230-32, 243- Plant pathology, antibiotics for, 231- Protaminobacter ruber, 221, 223, 310
49 32 Protease, 7, 105, 112, 189—90, 197,
Peptone, 62-63, 200, 225 Plasmids, 21, 31, 42-45, 47-49, 54- 203-5, 212, 319
Pericularia oryzae, 204 55, 156, 165, 283 Protease inhibitor, 337
Permeability, 159-61 Plasmolysis, 118 Protein:
Permeability gene, 37 Plate separator, 116-18 excretion, 194
Pesticide, microbial transformation, Plug flow reactor, 68 foreign, see Foreign protein
301-4 Podospora anserina, 49 purification, 120
pH measurement, 106, 216 Point mutation, 11, 17, 21-22 Protein engineering, 54
Phage: Polyacrylamide gel, 214-15 Proteolysis, 48
contamination by, 158 Polybutyric acid, 330 Proteus, 192, 209, 269
filtration, 103 Polychlorinated diphenol, 319 Proteus rettgeri, 170, 172
Phage f1, 48 Polyene macrolide antibiotic, 230, 256, Protoplast:
Phage fd, 48 258-61 fusion, 1, 4, 10, 32-34, 156, 165,
Phage A, 31, 44, 48 Polyether antibiotic, 230, 270 284
Phage M13, 22, 48 Polygalacturonase, 206-7 transfection, 34
Phage Mu, 19-20 Polyglycyl enzyme, 212 transformation, 32, 34
Phage NPO2, 48 Polyhydroxybutyric acid, 70, 330 Protropin, see Human growth
Phage P1, 30-31 Polyhydroxyvaleric acid, 330 hormone
Phage p11, 48 Polylysine, 340 Providencia alcalifaciens, 33
Phage PBS1, 31 Polymyxin B, 52, 246-47 PRPP, 177
Phage $105, 48 Polynucleotide ligase, 12, 21 PRPP amidotransferase, 178, 181-85
Phage C31, 48 Polyoxin, 230, 232, 267, 338 Pseudo-crystalline fermentation, 291
Phage PS20, 156 Polysaccharide: Pseudomonas, 31, 44, 48, 143, 154,
Phage SP10, 31 biosynthesis, 330-31 192, 195, 206-10, 223, 231, 246,
Phenol degradation, 319 extracellular, 330-34 304, 310, 313, 331
Phenolpicolinic acid, 338 Polytyrosyl enzyme, 212 Pseudomonas acidophila, 234
Phenotypic expression, 22-24 Pseudomonas aeruginosa, 29, 204, 243,
Postreplicative recombination repair,
Phenoxazine synthase, 244 331-32
13-14
Phenoxazinone synthetase, 38
Potato, 125, 127, 139, 144 Pseudomonas amyloderamosa, 198
Phenylacetate-activating enzyme, 38 Pseudomonas aureofaciens, 223
Power consumption per volume, 94-
Phenylacetic acid, 208-10 Pseudomonas dacunhae, 155-56
95
L-Phenylalanine, 28, 94, 151-57, 171- Pseudomonas denitrificans, 221
Power number, 82-83
72 Pseudomonas extorquens, 310
Power requirement, aerated bioreator,
Phenylalanine ammonia lyase, 155 Pseudomonas fluorescens, 74, 327
85
Phenylalanine dehydrogenase, 155 Pseudomonas fragi, 208
Precipitation, 112, 121
Phenylalanine hydroxamate, 171 Pseudomonas mesoacidophila, 234
Prednisolone, 292, 295-96
Phosphatase, 7, 39 Pseudomonas methylotrophus, 313
Prednisone, 292
Phosphate-deregulated mutant, 40 Pseudomonas ovalis, 90, 223
Premutational lesion, 11, 17
Phosphate, inorganic, 39-40 Pseudomonas putida, 172, 319, 327
Phosphate regulation, 39-40, 59, 241,
Prephenate dehydratase, 172 Pseudomonas rosea, 310, 313
265, 282-83 Prephenate dehydrogenase, 172 Pseudomonas saccharophila, 198
Pressure, in fermenter, 106 Pseudoplastic solution, 84-85
Phosphoenolpyruvate carboxykinase,
136 Pressure cycle fermenter, 76 Psicofuranine, 28, 186
Phosphoenolpyruvate carboxylase, Procaryote, genetic organization, 42 Psoralen derivative, 15-16
263-64 Product: Psychrotroph, 6
Phosphogluconate pathway, 311 concentration, 111, 113 Pullulan, 331, 333
Phosphoglycolipid antibiotic, 232, 250 purification, 111 Pullulanase, 191, 197-98
Phosphonomycin, 229 rate of formation, 69, 71-73 Pullularia, 143
Phosphoribosylanthranilate isomerase, recovery, 9, 59, 64, 111-22 Pullularia pullulans, 198
284 yield, 59 Pump, gas distribution, 76
’ 5'-Phosphoribosylpyrophosphate, 176— Production fermentation, 103, 105-6 Purine analog, 175, 178
77 Productivity, 72-73 Purine arabinoside, 187
Phosphorylation reaction, 289 Proflavine, 18 Purine base, conversion to purine
Photolyase, 12 Progesterone, 292, 296 nucleotide, 184
Photoreactivation, 11-12, 15 L-Proline, 28, 152, 154, 156-57 Puromycin, 38-39, 268
Phytohormone, 335 1,2-Propanediol, 223 Pyrazine, 341
Pichia farinosa, 132 Propeller stirrer, 83 Pyridoxal, 219
Pichia guilliermondii, 225 Propionibacterium, 135 Pyrimidine dimer, 11-12, 14-15
354 / INDEX
Pyrimidine-psoralen adduct, 16 Rhizopus javanicus, 195 Scleroglucan, 331-33
Pyrite, 327, 329 Rhizopus nigricans, 295 Sclerotium, 206
Pyrithiamine, 28 Rhizopus niveus, 195 Sclerotium delphinii, 331-32
Pyroclavine, 275 Rhodococcus, 155 Sclerotium glucanicum, 331-32
Pyrrolnitrin, 229 Rhodopseudomonas, 310 Sclerotium rolfsii, 331-33
Pyruvate carboxylase, 136 Rhodopseudomonas protamicus, 221 Scopulariopsis, 143
Pyruvate dehydrogenase, 212 Rhodopseudomonas spheroides, 221, 223 SCP, see Single-cell protein
Rhodotorula glutinis, 92, 155 Screening, 4-6, 26-27
Quale cycle, 311 Rhodotorula gracilis, 303 test systems, 7
Quebemycin, 232, 250 Riboflavin, 112, 219 transformed cells, 45-47
Quinone antibiotic, 230 biosynthesis, 224-25 Sedimentation rate, 115
economic significance, 222-23 Seldomycin, 252
Radiation sterilization, 98 occurrence, 222-23 Selection of mutants, 25-29
Raffinose, 215 production, 222-23, 225 Selective marker, 20
Random screening, 26-27 structure, 223-24 Sensor, 106
recA-dependent repair, 12-13, 16 Ribostamycin, 252, 300-1 Sephadex, 119
Recombinant clone, 45-47 Ribulose diphosphate cycle, 311 Septomyxa affinis, 295
Recombinant DNA, 10, 41, 156 Ribulose monophosphate cycle, 311, Sequence vector, 44
Recombination, 29-34, 156 313 Sequential induction, 36
in actinomycetes, 31-32 Rice, 127 L-Serine, 61, 152-56, 172
in bacteria, 30-31 Rifampicin, 262 Serine hydroxymethyl transferase, 155
in fungi, 31 Rifamycin, 32, 41, 51, 92, 230, 262 Serine pathway, 311-13
parasexual, 29-30 Rimocidin, 263 Serine transhydroxymethylase, 312
protoplast fusion, 32-34 Ristomycin, 38 Serratia, 51, 192, 198, 206
sexual, 29 RNA: Serratia marcescens, 135, 154, 156-57,
Recombination enzyme, 11 content of different microorganisms, 203
Recombination repair, 17 179 Settling basin, 319, 321
Recombivax HB, see Hepatitis B hydrolysis, 178-81 Set-up time, 72
vaccine isolation, 179 Sewage, single-cell protein production
Recovery loss, 121-22 processing, 42 from, 315-16
Reduction reaction, 286, 288-89, 294 RNA polymerase, 38 Sewage treatment, 215, 317-24
Refrigerated culture, 104 R-plasmid, 44 aeration with pure oxygen, 320-22
Regulation, 34-41 Rubradirin, 32 airlift process, 319-20
altered, 27 methane production, 321-23
energy charge, 35 Saccharomyces, 29, 33, 128, 182, 216, starter culture, 318-19
enzyme activity, 35 219, 225 Sexual cycle, fungi, 29-31
enzyme synthesis, 36 Saccharomyces carlsbergensis, 187 Shake culture, 104
Regulatory gene, 37 Saccharomyces cerevisiae, 29, 34-35, Shear rate, 84-85
Regulatory mutant, 156 44, 47, 49-51, 89-92, 125-26,-165, Shear stress, 84-85
Reichstein’s Substance S, see 179, 216 Shotgun experiment, 41
Diosgenin Saccharomyces diastaticus, 34 Shuttle vector, 45
Reliability, 74 Saccharomyces fibuligera, 34 Sideramine, 247
Rennet, 205 Saccharomyces rouxti, 132 Siderochrome, 247-49
Rennin, microbial, 49, 189-90, 205-6 Saccharomycopsis lipolytica, 135, 141- Sideromycin, 247
Repair system, 24 42, 165, 307, 309 Sieve centrifuge, 116
Replica plating, 27, 47 Saccharopolyspora hirsuta, 252 Signal peptide, 194
Repression, 36 Sagamicin, 53, 252 Single-cell protein (SCP), 2, 61, 69-73,
Resistance gene, 37 Salicylic acid degradation, 44 76, 105, 112, 121, 306
Resistant mutant, isolation, 27 Salinomycin, 231, 270 from alkanes, 306-9
Respiration rate, 89 Salmonella, 209, 268 from carbohydrate, 307, 315
Resting cells, microbial transformation Salmonella typhimurium, 19, 29, 36, composition, 307
by, 291 186-87 from methane, 306-7, 309
Restriction endonuclease, 20-21, 41- Sampling device, 78 from methanol, 307, 309-13
42 SAMP, 177 from sewage, 315-16
Reverse mutant, 70 SAMP synthetase, 182-83, 185 from wood, 314-15
Reverse osmosis, 114 Sannamycin, 252 Siomycin, 39, 232, 247
Reverse transcriptase, 42-43 Sarcina, 209 Sisomicin, 25, 53, 92-93, 112, 252,
Reversion, 32 Sarcina lutea, 187, 249 255-56
Reynold’s number, 82-83 Scale up, 92, 107 5-epi-Sisomicin, 251, 300
Rhizobium, 274 with constant oxygen transfer rate, B-Sitosterol, 295-97
Rhizopus, 135, 192, 206-7 95-96 Sludge digester, 318
Rhizopus arrhizus, 296 with constant power per volume, Somatostatin, 10, 42, 45-46
Rhizopus delemar, 195, 208 94-95 Somatotropin, 49
Rhizopus formosaensis, 195, 197 parameters, 94 D-Sorbitol, 298
Rhizopus japanicus, 195 significance, 92-94 L-Sorbose, 298
 INDEX / 355
SOS repair, 13-14, 16-17 development, 9-55 Streptomyces flavogriseus, 234
Southern blotting technique, 47 isolation, 6 Streptomyces flavopersicus, 252
Soy bean oil, 61, 225 optimization, 50-51 Streptomyces flavus, 263
Soy meal, 63, 201, 255, 307 preservation, 103-4 Streptomyces floridae, 247
Soy protein, 168 purification, 6 Streptomyces fradiae, 32, 51-53, 114,
Specialized transduction, 31 for screening, 6-7 203, 232, 252, 257, 300-1, 338
Specific growth rate, 66-67 Streptamine, 53 Streptomyces fulvissimus, 244
Specific oxygen requirement, 89 Streptavidin, 42 Streptomyces fungicidicus, 232, 247
Spectinomycin, 252 Streptidine, 253 Streptomyces galilaeus, 231
Sphaceloma manihoticola, 335 Streptococcus, 33, 198, 206 Streptomyces garyphalus, 244
Spiramycin, 257 Streptococcus cremoris, 205, 247 Streptomyces geysiriensis, 250
Spirulina, 306 Streptococcus lactis, 34, 205 Streptomyces ghanaensis, 250
Sporaricin, 252 Streptococcus mutans, 331 Streptomyces glaucescens, 32, 51
Spore, heat resistance, 98 Streptococcus thermophilus, 205 Streptomyces griseochromogenes, 232,
Spore crop, 104, 138-39 Streptokinase, 189 267
Spore suspension, 138-39 Streptomutin A, 53 Streptomyces griseoflavus, 32, 269
Sporotrichum, 314 Streptomyces, 5, 33-34, 52, 85, 98, 182, Streptomyces griseoruber, 337
Sporotrichum pulverulentum, 314 195, 198, 206, 230, 310 Streptomyces griseus, 31-32, 34, 37,
Sporulation, 185 Streptomyces achromogenes, 32 39-41, 52-53, 85, 89, 185, 203-4,
Staphylococcus, 198, 207 Streptomyces acrimycini, 32 231-32, 252-55, 260, 269
Staphylococcus aureus, 48, 208, 243 Streptomyces alboflavus, 263 Streptomyces hachijoensis, 260
Staphylococcus carnosus, 34 Streptomyces albogriseolus, 337 Streptomyces halstedii, 257
Starch: Streptomyces alboniger, 268 Streptomyces hofunensis, 252
acetone/butanol production from, Streptomyces albus, 201, 270 Streptomyces humidus, 252
129 Streptomyces ambofaciens, 257 Streptomyces hygroscopicus, 232, 245-
as carbon source, 61, 100 Streptomyces antibioticus, 37, 39, 52, 46, 250, 252, 257, 337
citric acid production from, 137-39 231, 244-45, 257, 263 Streptomyces kagawaensis, 232
as enzyme carrier, 214 Streptomyces argenteolus, 337 Streptomyces kanamyceticus, 39, 114,
ethanol production from, 129 Streptomyces argillaceus, 231 252
glutamic acid production from, 162 Streptomyces atroolivaceus, 231 Streptomyces kasugaensis, 232, 252
hydrolysis by amylase, 191-93 Streptomyces aureofaciens, 29, 31-32, Streptomyces lasaleinsis, 270
lysine production from, 168 39, 52, 262-63, 265-67 Streptomyces lavendulae, 208, 244, 250,
organic feedstocks from, 124 Streptomyces aureus, 179-81, 247, 263, 337-38
tryptophan production from, 171 296 Streptomyces levoris, 260
Starch hydrolysate, 198 Streptomyces avermitilis, 261 Streptomyces lincolnensis, 114, 250
Starch saccharification residue, 60 Streptomyces avidini, 42 Streptomyces lipmanii, 37, 241
Starter culture, 69, 121, 205 Streptomyces azureus, 232 Streptomyces lividans, 34, 48-49
sewage treatment, 318-19 Streptomyces bambergiensis, 232, 250 Streptomyces lividus, 252
Static filtration, 114 Streptomyces bikiniensis, 32, 40, 253 Streptomyces lusitanus, 263
Stationary phase, 65, 67 Streptomyces bluensis, 252 Streptomyces michiganensis, 337
Steady state conditions, 68 Streptomyces cacaoi, 232, 267 Streptomyces mitakaensis, 232, 245
Sterility, maintenance, 78-79 Streptomyces caespitosus, 231, 270 Streptomyces mycarofaciens, 257
Sterilization: Streptomyces calidus, 338 Streptomyces narbonensis, 257
air, 100-3 Streptomyces californicus, 263 Streptomyces natalensis, 260
culture media, 96-103, 105 Streptomyces canus, 247 Streptomyces netropsis, 260
gas, 96-103 Streptomyces capreolus, 247 Streptomyces niveus, 85, 89-90, 269
Steroid: Streptomyces carzinostaticus, 231 Streptomyces nodosus, 39, 260
structure, 292-93 Streptomyces cattleya, 234, 243 Streptomyces nojiriensis, 250
transformations, 85, 105, 290-97 Streptomyces cellulosae, 263 Streptomyces noursei, 85, 260
economically important, 294-95 Streptomyces chrestomyceticus, 225 Streptomyces olivaceus, 29, 31-32, 201,
side chain breakdown, 295-97 Streptomyces chrysomallus, 244 221, 234
types, 292-94 Streptomyces cinnamonensis, 270 Streptomyces olivochromogenes, 257
uses, 292 Streptomyces clavuligerus, 34, 234, 241, Streptomyces olivoreticuli, 337
Steroid antibiotic, 230, 270 243 Streptomyces omiyaensis, 268
Steroid cephalosporin, 240 Streptomyces coelicolor, 18, 20, 31-34, Streptomyces orchidaceus, 244
Stigmasterol, 296-97 48, 51-52, 260, 266 Streptomyces orientalis, 39, 250
_ Stirred bioreactor, 76-78, 80, 88 Streptomyces curacoi, 250 Streptomyces parvulus, 34, 247, 300
Stirrer: Streptomyces diastaticus, 338 Streptomyces parvus, 263
geometry, 95 Streptomyces ederensis, 250 Streptomyces peucetius, 51, 231
speed, 94, 106 Streptomyces erythreus, 32, 51, 53, Streptomyces phaeochromogenes, 200,
types, 77-78 257-59 232, 268
Stirring, 73-76, 81-86, 106 Streptomyces favochromogenes, 338 Streptomyces pilosus, 247
Stoke’s law, 115 Streptomyces feofaciens, 263 Streptomyces platensis, 263
Strain: Streptomyces filipinensis, 260 Streptomyces plicatus, 231
degeneration, 283 Streptomyces flaveolus, 232, 261, 263 Streptomyces primprina, 260
356 / INDEX
Streptomyces pseammoticus, 52 Sugar beet, 60, 125-127, 129, 137, Thiotemplate mechanism, 247-48
Streptomyces ramocissimus, 232 139, 162, 164, 168 Three-phase system, 81
Streptomyces rectus, 203 Sugar cane, 60, 125-127, 129, 137, L-Threonine, 28, 151-52, 154, 156-58,
Streptomyces reticuli, 31 162, 168 167-68
Streptomyces ribosidificus, 252 Sulfaguanidine, 154, 171 Thymine dimer, 12
Streptomyces rimofaciens, 252 Sulfite method, determination of Tissue plasminogen activator, 49, 54—
Streptomyces rimosus, 31—32, 71, 252, oxygen uptake rate, 90-91 55
263, 265—66 Sulfite waste liquor, 61, 127, 147, 179, Tobramycin, 52, 252-54
Streptomyces roseochromogenes, 244, 8077315 Tocopherol, 219
250, 295 Sulfolobus acidocaldarius, 327 Toluene degradation, 44
Streptomyces rubiginosus, 200 Sulfonamide, 28 Tolypocladium inflatum, 340
Streptomyces sannanensis, 252 Superoxide dismutase, 55 Torula cremoris, 210
Streptomyces sayamaensis, 263 Suppressor mutation, 24, 27, 37 Torulopsis, 206-7
Streptomyces scabies, 32 extragenic, 24 Torulopsis glabrata, 310
Streptomyces sioyaensis, 232, 247 intragenic, 24 Torulopsis magnoliae, 132
Streptomyces spectabilis, 252 Surface aerator, 76 Torulopsis memodendra, 310
Streptomyces spheroides, 269 Surface reactor, .76 Torulopsis methanolovescens, 310
Streptomyces tateyamensis, 232, 247 Survival factor, 96 Torulopsis methanosorbosa, 310
Streptomyces tenebrarius, 52, 252 Sweetzyme, 200 Torulopsis molischiana, 310
Streptomyces tenjimariensis, 52, 252 Swine growth factor, 55 Torulopsis utilis, 90
Streptomyces testaceus, 337 Synthetase I, 38 Toxic waste, 55
Streptomyces vendargensis, 263 Synthetase II, 38 Trace element, in citric acid
Streptomyces venezuelae, 32, 268 Synthetic rubber, 128-29 production, 138
Streptomyces verticillus, 231, 249 Transduction, 30-31, 45
Streptomyces violaceoruber, 48, 338 T4 ligase, 43 Transfection, protoplast, 34
Streptomyces virginiae, 40-41, 232, 245 Takadiastase, 189 Transformation, 30-31, 45
Streptomyces viridans, 232 Taka-Sweet, 200 microbial, see Microbial
Streptomyces viridifaciens, 37, 262-63 Tannin aminohexyl cellulose, 214 transformation
Streptomyces viridochromogenes, 250 Tapioca flour, 126 protoplast, 32, 34
Streptomyces viridoflavus, 260 Temperature: Transient range of mixing speed, 83
Streptomyces wadayamensis, 34 constant, 73 Transition, 11, 15, 17-19, 21
Streptomyces yokosukanensis, 337 fermentation, 105-6 Translocation, 11, 16
Streptomycin, 10, 32, 38-40, 52-53, Terpenol, 340 Transmethylase, 38
85, 95, 112, 230-31, 251-55 a-Terpineol, 340
Transposon, 11, 19-21
Streptomycin phosphotransferase, 40 Testosterone, 295
Transposon Tn1, 21
Streptose, 253 Testudinaria sylvatica, 295
Transposon Tn3, 21
Streptothricin, 230 Tetracycline, 32, 39, 51-52, 230, 262-
Transposon Tn5, 21
Streptovaricin, 262 63
Transversion, 11, 15, 17, 19
Streptoverticillium kitasataensis, 52, 257 biosynthesis, 263-65
Triamcinolone, see 16a-Hydroxy-9a-
Streptoverticillium mycoheptinicum, 260 mode of action, 263
fluoroprednisolone
Streptoverticillium rimofaciens, 232, 267 production:
1,2,4-Triazol-3-alanine, 28
Structural gene, 37 methods, 266-67
Substitution vector, 44 Tricarboxylic acid cycle, 134, 136, 159
strains, 263, 265-66
Substrate, 59-63: regulation, 263-65 2,4,5-Trichlorophenoxyacetate, 303
Trichoderma, 314
Substrate concentration, 66-69, 72-73 structure, 262-63
Trichoderma koningii, 314
Substrate limitation, 69 Tetranactin, 230, 232, 261
Subtilisin, 54, 203 Thermoactinomyces, 107-8 Trichoderma reesei, 314
Subtilisin inhibitor, 337 Thermoactinomyces vulgaris, 31-32 Trichoderma viride, 135, 197, 315
Subunit vaccine, 54 Thermoascus, 314 Trichomycin, 260
Succinic acid, 148 Thermomonospora, 34, 192, 195 Trichophyton mentagrophytes, 208
Succinoglucan, 332 Thermomonospora curvata, 192 Trichosporon brassica, 216
Succinyldiaminopimelate Thermophile, 6, 124, 195, 327, 329 Trickle-film reactor, 76
aminotransferase, 166 Thermothrix thioparus, 327 Trickling filter, 215, 317
Succinyldiaminopimelate Thiamine, 28, 219 Trickling generator, 143-45, 147, 215
desuccinylase, 166 2-Thiazolalanine, 28, 154 Triendiol, 295
Succinylketoaminopimelate synthase, Thienamycin, 234, 243 5,5,5-Trifluoroleucine, 28
166 Thienylalanine, 28, 171 Triglyceride production, 337
Sucrose, 197, 199 Thiobacillus, 327 Trisporic acid, 227-28
acetone/butanol production from, Thiobacillus concretivorus, 326-27 Trophophase, 38-40, 69-71
129 Thiobacillus ferrooxidans, 326, 329 Tryptophan, 28, 36, 38, 151-56, 158,
amino acid production from, 154 Thiobacillus thermophilica, 327 169-71
assay, 217 Thiobacillus thiooxidans, 326-27 biosynthesis, 171-73
as carbon source, 60 6-Thioguanine, 182 production:
citric acid production from, 137-38 Thiopeptin, 232, 247 direct fermentation, 172-73
Sugar antibiotic, 230, 249-50 Thiostrepton, 231-32, 247 enzymatic, 170
 INDEX / 357
transformation of precursors, 170- L-Valine, 28, 94, 152-54, 156 composition, 314
71 Valinomycin, 230, 244-45 ethanol production from, 126-27
Tryptophanase, 155, 170, 172 Vancomycin, 34, 39, 230, 249-50 organic feedstocks from, 124
D,L-Tryptophan hydantoin, 172 Vector, 42-45 single-cell protein production from,
L-Tryptophan hydantoin hydrolase, Vengacide, 263 314-15
170 Venturicidin, 261
D-Tryptophan hydantoin racemase, Verdamicin, 252 Xanthan, 2, 61, 330-32
170 Verticillium agaricinum, 66 Xanthine, 184
Tryptophan hydroxamate, 171 Verticillium lecanii, 339 Xanthomonas, 209, 331
Tryptophan synthetase, 35, 170, 172 Veterinary medicine, antibiotics in, Xanthomonas campestris, 331
Tubular loop reactor, 88 231-32 Xanthomonas oryzae, 231, 249
Tumor necrosis factor, 55 Vibrio, 143, 310 Xanthophyil, 226-27
Turbidostat, 68 Vinegar, 76-77, 143-47, 215 Xanthylic add, see 5'-XMP
Turbine-type stirrer, 77, 87 Viomycin, 246-47 Xenobioiic, 302
Turbulent flow, 82-83 Virginiamycin, 38, 41, 232, 245 accumulation, 304
Tylosin, 38, 51-52, 231-32, 256-58 Viscoelastic solution, 83-84 cometabolism, 302-3
Tyrocidin, 246-47 Viscosity, 83-85 conjugate formation, 303-4
Tyrosinase, 155 Viscous solution, 83 metabolism, 302-3
L-Tyrosine, 28, 152-57, 171-72 Vitamin, 71, 112, 219-28 5'-XMP, 176, 184
Tyrosine hydroxamate, 154, 171 Vitamin A, 226 X-rays, 16
Tyrothricin, 247 Vitamin B,, see Riboflavin Xylan, 200
Vitamin B,,, 34, 52, 61, 112, 219 Xylene degradation, 44, 319
Uhde/Hoechst Bio-high Reactor, 319— biosynthesis, 221 Xylose, 200
20 economic significance, 219-20
occurrence, 219-20
Ultrafiltration, 114 a
Arey
production, 221-23
Ultrafiltration membrane, 81 Yq, 92
structure, 220
Ultrasonic disintegration, 118 YCp vector, 49
Ultraviolet radiation, 11-12, 15 Yeast
Vitamin C, see L-Ascorbic acid doning in, 47
long-wavelength, 15-16 Volumetric oxygen transfer coefficient,
short-wavelength, 15 commerdal production, 179
87, 90 Yeast extract, 62, 200-1, 225
Undecylprodigiosin, 48, 51 Volumetric oxygen transfer rate, 91
Unox system, 321-22 YEp vector, 49
Wash-out point, 68
Uracil, 28 Yield, 121-22
Waste-water treatment, 70, 76, 317-24
Uracil DNA glycosylase, 13 Yield coeffident, 73-74
Water jet pump, 76
Uranium leaching, 326, 328-39 Weissenberg effect, 84 Yield constant, 68
Urea, 62, 163, 168 Wellferon, see Interferon alpha Yip vector, 49
Urease, 163, 168 Western blotting technique, 47 YRp vector, 49
Urocanic acid, 151 Wheat bran, 139, 200
Urokinase, 2 Whey, 61, 126, 144, 147, 215 Zearalenone, 336
Wild strain, 9 Zeaxanthin, 225
Vaccine, 10, 54 Wine, vinegar production from, 144 Zoophthora radicans, 339
Vacuum drum filter, 113 Wood: Zorbamyan, 32
Vairimorpha necatrix, 339 acetone/butanol production from, Zorbonomyan, 32
Validamycin, 232, 251-52 129 Zymomonas mobilis, 125-26, 128
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