BIOTECHNOLOGY

(CH 373 )

Chapter Two B

Industrial Microbiology

Instructor :
Dr Ato Fanyin-Martin
 Chapter Objectives

1. Introduction to Industrial Microbiology

2. Overview of microorganisms of industrial importance
3. Review of the fermentation processes
4. Overview of measurements and control of fermentation parameters
5. Isolation of industrially important microbial strains
6. Review of microbial production of industrial products
Introduction to Industrial Microbiology
• Industrial microbiology can be defined as the study of the large-scale and profit-
motivated production of microorganisms or their products for direct use or as
inputs in the manufacture of other goods.

• For instance, yeasts may be produced for the following purposes:

1. Direct consumption as food by humans
2. Animal feed
3. Active ingredients in bread-making
4. Ethanol products may be consumed in the form of alcoholic beverages or used in
the manufacture of perfumes, pharmaceuticals, etc.
Introduction to Industrial Microbiology
• Ethanol production from malt and fruit extracts by yeast action on a large scale
emerged as the first industrial microbiological process.
• Industrial microbiology extends the term fermentation to describe any process for the
production of microbial products by the mass culture of microorganisms.
• In industrial microbiology, fermentation refers to both aerobic and anaerobic
processes involving microbial cultivation on a large scale
• Industrial microbiology spans industries of pharmaceuticals, food and alcoholic
beverages
 Introduction to Industrial Microbiology

• The term fermentation is from the Latin verb “fervere”

meaning to boil, suggestive of the boiling appearance of
fermented products
• Typical of a microbial process, fermentation is influenced by strain
type, culture media and growth conditions.
• Manipulation of conditions and strains can improve product yield

• Different methods of fermentation, such as batch, fed-batch and

continuous, are employed to increase productivity

• Fermentation in a bioreactor ensures cell immobilization, increased

biomass concentration and increased biocatalyst concentration
which translates into higher productivity
Introduction to Industrial Microbiology
• Over millennia, enzymes have been used to produce beer, wine, cheese, and yoghurt, among
several other valuable products.
• A classic example is the production of ethanol and carbon dioxide from sugars by
enzymatic catabolism in yeast (using zymase)
• With an extensive variety of applications, enzymes have been employed in over 500
industrial processes
• E.g. Pharmaceuticals, food, leather, detergents, paper, cosmetics, fuel, feed, etc.
• Enzymatic actions are dependent on substrate, pH, inhibitors, temperature, etc. Thus,
process conditions must be optimized to ensure the maximum yield of products
Introduction to Industrial Microbiology

• Microorganisms, plants and animal tissues are potential sources of enzymes

• Although, microbial enzymes are the most preferred owing to specificity and accessibility

• Commercial enzymes include proteases, lipase, pectinases, amylases, cellulases, ligninases,

and milk clotting enzymes (rennet), among several others

• Enzyme immobilization can be used to ensure enzyme recovery thereby reducing the cost
of production significantly
History of Industrial Microbiology

• In 1837, Theodore Schwann et al. proposed that

yeast cells were responsible for alcoholic
fermentation (conversion of sugars to alcohol)

• In 1856, M. Bigo (an industrialist whose business

produced ethanol from the fermentation of beet
sugars in Lille) requested the assistance of Pasteur
to address an industrial challenge (low alcohol
yields and sourness)
Pasteur’s Contribution
• Pasteur found that alcohol-producing yeast had been replaced by lactic acid-
producing microbes
• Pasteur demonstrated that all fermentation processes were carried out by specific
yeasts and bacteria
• He studied wine diseases and developed pasteurization to preserve wine during
storage
• He studied fermentation for almost 20 years
• Discovered that some fermentative microbes were obligate anaerobes while
others were facultative anaerobes
Summary of the Chronological Development of Industrial Microbiology
Stage Period Main Products Strain Selection
1 Pre-1900 Alcohol and Vinegar Pure yeast cultures used at the Carlsberg brewery
(1886) Fermentations inoculated with ‘good’ vinegar

2 1900-1940 Baker’s yeast, glycerol, citric acid, Pure cultures used

lactic acid, and acetone/butanol

3 1940-date Penicillin, streptomycin, other Mutation and selection programmes essential

antibiotics, gibberellin, amino acids,
nucleotides, transformations, enzymes

4 1964-date Single-cell protein using hydrocarbon Genetic engineering of producer strains attempted
and other feedstocks

5 1982-date Production of heterologous proteins Introduction of foreign genes into microbial and
by microbial and animal cells animal cell hosts. In vitro recombinant DNA
techniques used in the improvement of stage 3
products
6 2000-date Use of “synthetic biology” to improve Synthetic biology used to develop existing and
established fermentations and develop novel fermentations
new bulk chemical processes
 Development of Industrial Microbiology – Stage
1
• Pre-1900 (beer was first brewed by ancient Egyptians, but large-scale breweries (using
wooden vats of 1500 barrel capacity) began in the early 1700s)

• Products confined to potable alcohol and vinegar using a batch culture method.
• Process control was attempted in 1757 (use of thermometers) and in 1801 (development of
primitive heat exchangers)

• By the mid-1800s, the role of yeasts in alcoholic fermentation had been demonstrated
independently by Cagniard-Latour, Schwann, and Kutzing but it was Pasteur who convinced
the scientific world of the obligatory role of yeasts in the process
Development of Industrial Microbiology – Stage
1
Development of Industrial Microbiology – Stage
1
• During the late 1800s, Hansen developed methods for isolating
and propagating single yeast cells to produce pure cultures and
established techniques for producing starter cultures at the
Carlsberg brewery.
• Vinegar was originally produced by leaving wine/beer in shallow
bowls or partially filled barrels to slowly oxidize to vinegar by
the development of a natural flora
• The need for air in the process led to the development of a
vinegar generator, which consisted of a vessel packed with an
inert material over which the wine/beer trickled
Development of Industrial Microbiology – Stage
1

• The vinegar generator may be considered as the first aerobic fermenter to be developed

• By the late 1800s to early 1900s, initial medium (substrate) was being pasteurized and
inoculated with 10% good vinegar to make it acidic (i.e. resistant to contamination) and
providing a good inoculum

• By the beginning of the 20th century, concepts of process control were well established
in both brewing and vinegar industries
Development of Industrial Microbiology – Stage
2
• Between 1900 and 1940
• Notable advances were the developments in the bakers’ yeast and solvent
fermentations
• The main new products were yeast biomass, glycerol, citric acid, lactic acid,
acetone, and butanol
 Development of Industrial Microbiology – Stage
2
• Production of Baker’s yeast is an aerobic process
• The rapid growth of yeast in the wort resulted in the depletion of oxygen which
in turn resulted in the production of ethanol at the expense of biomass formation
• This problem was addressed by restricting the initial wort concentration such that
the growth of cells was limited to the availability of carbon source rather than
oxygen
• Subsequent growth of culture was controlled by adding further wort in small
increments, thus the development of the fed-batch culture technique
• The fed-batch culture technique is widely employed in fermentation industries
to avoid conditions of oxygen limitations
• The aeration of these early yeast cultures was improved by the introduction of
air through sparging tubes which can be steam-cleaned
Development of Industrial Microbiology – Stage
2
• The development of acetone-butanol fermentation by Weizmann during WW1 led to
the establishment of the first aseptic fermentation
• The anaerobic butanol process was susceptible to contamination
• Aerobic bacteria in the early stage of fermentation
• Acid-producing anaerobic bacteria, once anaerobic conditions had been established
• Fermenters were vertical cylinders with hemispherical tops and bottoms made from
mild steel
• The use of huge fermenters (approx. 2000 hectoliters capacity) presented
problems with inoculum development and maintenance of aseptic conditions
during inoculation
• The use of steel fermenters made it possible for fermenters to be steam-sterilized
under pressure and designed to minimize possible contamination
Development of Industrial Microbiology – Stage
2
 Development of Industrial Microbiology – Stage
2

• Techniques developed for organic solvents production paved the way for aseptic aerobic
processes in 1940s
• However, solvent fermentation became uneconomic with the development of
competing processes based on petrochemical feedstocks and ceased to exist
• Rising cost of crude oil, attractiveness of environmentally friendly processes and
advances in metabolic engineering may lead to the resurrection of modern versions of
these old processes
Development of Industrial Microbiology – Stage
3
• Marked by the production of penicillin in submerged culture under aseptic
conditions in the 1940s
• Production of penicillin is an aerobic process that is very vulnerable to
contamination
• Problems of sparging a culture with large volumes of sterile air and mixing a
highly viscous broth had to be overcome
• Penicillin was synthesized in very small quantities by the initial isolates and this
resulted in the establishment of strain-improvement programs
• Process development was also aided by the introduction of pilot-plant facilities
• This enabled the testing of new techniques on a semi-production scale
• Development of a large-scale extraction process for the recovery of penicillin
was another major advance at this time
Development of Industrial Microbiology – Stage
3

• Technology established for the penicillin fermentation provided the basis for the
development of a wide range of new processes including other antibiotics,
vitamins, gibberellin, amino acids, enzymes, and steroid transformations

• From the 1960s onward, microbial products were screened for activities other
than simply antimicrobial properties and screens became more and more
sophisticated
• Screens have given rise to those operating today utilizing miniaturized culture
systems, robotic automation, and elegant assays
Development of Industrial Microbiology – Stage
3
• One catabolic product gained significance in the mid-1970s
• Brazil and the United States of America initiated programs for the manufacturing of
ethanol as a motor fuel in 1975 and 1978 respectively
• Attempt by both countries to lessen their dependency on imported petroleum by using
fermentation processes to convert carbohydrates to ethanol (bioethanol)
• Brazilian process being based on sugarcane and the American process based on
maize starch.
• Political and social issues associated with the diversion of land from food production to
fuel are obvious and thus the development of processes using cellulose and lignin
feedstocks rather than sugar and starch was the next stage in this saga.
• Largest mechanically stirred fermentation vessels developed during stage 3 were in the
range of 80,000 to 150,000 dm3
Development of Industrial Microbiology – Stage
4
• In the early 1960s, several multinational companies investigated the production
of microbial biomass as a source of feed protein
• The relatively low selling price of microbial biomass necessitated its production
in much larger quantities than other fermentation products in order for the
process to be profitable
• Also, hydrocarbons were considered as the potential carbon sources that would
result in increased oxygen demands and high heat outputs by these fermentations
• Thus, the development of the pressure jet and pressure cycle fermenters that
eliminated the need for mechanical stirring
• Another feature of these potential processes was that they would have to be operated
continuously if they were to be economic
• The technique of growing an organism continuously by adding fresh medium to
the vessel and removing culture fluid
Development of Industrial Microbiology – Stage
4
• Several companies persevered in the biomass field and a few processes came to
fruition
• Most long-lived was the ICI Pruteen animal feed process, which utilized a
continuous 3,000,000-dm3 pressure cycle fermenter for the culture of
Methylophilus methylotrophus with methanol as carbon source
• Although the Pruteen process was a technological triumph, it became an
economic failure because the product was out-priced by soybean and
fishmeal.
• In 1989, the plant was demolished, marking the end of a short, but very
exciting, era in the fermentation industry
Development of Industrial Microbiology – Stage
4

• The operation of an extremely large continuous fermenter for time periods in

excess of 100 days presented a considerable aseptic operation problem
• The aseptic operation of fermenters of this type was achieved as a result of:
• High standards of fermenter construction
• Continuous sterilization of feed streams
• Utilization of computer systems to control the sterilization and operation
cycles, thus minimizing the possibility of human error.
Development of Industrial Microbiology – Stage
5
• The establishment of very high-value, low-volume products; a stage often referred
as “new biotechnology.”
• In vitro recombinant DNA technology enabled the expression of human and
mammalian genes in cultured animal cells and microorganisms
• Enabling the development of relatively large-scale fermentation processes for
the production of human proteins, which could then be used therapeutically
• The exploitation of genetic engineering coincided approximately with the
production of monoclonal antibodies (mAbs) as therapeutic agents
• Initial use of mAbs was limited to analytical applications, they are now well-
established as therapeutic biopharmaceuticals
• Produced in fermentation systems employing both mammalian cells and
microorganisms as production agents
Development of Industrial Microbiology – Stage
5
• In 1982, recombinant human insulin became the first heterologous protein to be
approved for medical use.
• Eight more products were approved in the 1980s comprising:
• Two approvals for human growth hormone
• Two for interferons,
• One monoclonal antibody
• One recombinant vaccine for hepatitis B
• One for tissue plasminogen activator
• One for erythropoietin
Development of Industrial Microbiology – Stage
5
• The pharmaceutical industry was very active in developing “conventional” microbial
processes which resulted in a number of new microbial products in the late 1980s and
early 1990s
• Four secondary metabolites were launched in the 1980s:
• Cyclosporine, an immunoregulant used to control the rejection of transplanted
organs
• Imipenem, a modifed carbapenem, which had the widest antimicrobial
spectrum of any antibiotic
• Lovastatin, a drug used for reducing cholesterol levels
• Ivermectin, an antiparasitic drug which has been used to prevent “African River
Blindness” as well as in veterinary practice.
Development of Industrial Microbiology – Stage
5

• By the end of 2012, approximately 220 biopharmaceuticals had been approved

• 31% being produced in E. coli,
• 15% in yeast
• 43% in mammalian cells (mainly Chinese Hamster Ovarian cell lines, CHO)
• 11% being produced by hybridoma cells, insect cells, and transgenic animals
and plants.
• Whereas the approved products in the 1980s were predominantly hormones and
cytokines, the approvals over the 2005–12 period were dominated by antibody-
based products and engineered proteins (i.e. proteins modified post-synthesis).
Development of Industrial Microbiology – Stage
5

• These modifications include:

• Antibody-drug conjugates in which, for example, anticancer drugs are linked to
monoclonal antibodies which bind to the tumor, thus directing the drug to its
target.
• Modifications which extend the half-life of the therapeutic protein in the body.
• Modifications which alter the pharmacokinetic properties of the therapeutic
protein.

Absorption, Distribution, Metabolism,

Excretion
 Development of Industrial Microbiology – Stage
5

• Recombinant proteins are classified as biologicals, not as drugs, and thus come
under the same regulatory authorities as do vaccines.
• The major difference between the approval of drugs and biologicals is that the
production process for a biological must be precisely specified and carried out in
a facility that has been inspected and licensed by the regulatory authority
• For drugs, only major changes require approval prior to implementation.
• These stringent containment and regulatory requirements results in the extremely
high cost of developing recombinant protein processes
• The cost of building a 3000 dm3 scale facility for biologics is estimated to be
equivalent to building a 200,000 dm3 scale facility for an antibiotic.
Development of Industrial Microbiology – Stage
6

• Advances in genomics, proteomics, and metabolic flux analysis are the basis of
the sixth stage in the progress of the industry
• The sequencing of the complete genomes of a wide range of organisms and
the development of computerized systems to store and access the data has
enabled the comparison of genomes and the visualization of gene expression
in terms of both mRNA (transcriptomes) and protein profiles (proteomes)
• The adoption of a systems biology approach (functioning of the whole
organism) by fermentation scientists has enabled them to build upon
established fermentation processes and take them to a further level
Development of Industrial Microbiology – Stage
6

• The goal of synthetic biology (application of systems biology to biotechnology)

is to maximize the yield of the desired product while minimizing that of
unwanted or unnecessary metabolites.
• This has always been the goal of the fermentation scientist, but the tools of
synthetic biology make this goal a reality.
• An exciting application of the approach is in the production of bulk chemicals
and feedstocks for the chemical industry, in competition with the
petrochemicals.
 Biotechnology Timeline
1750 BC The Sumerians brew beer.
500 BC Chinese use moldy soybean curds as an antibiotic to treat boils
1590 Janssen invents the microscope

1675 Leeuwenhoek discovers cells

(bacteria, red blood cells)
1830 Proteins are discovered
1833 The first enzymes are isolated
1855 The Escherichia coli bacterium is discovered
 Biotechnology Timeline

1859 Charles Darwin publishes On the Origin of Species

1864 Louis Pasteur shows all living things are produced by other
living things

1865 The age of genetics begins

1902 Walter Sutton coins the term ‘gene’ - proposed that

chromosomes carry genes
1910 Chromosomal theory of inheritance proved

1928 Fleming discovers antibiotic

properties of certain molds

1941 George Beadle and Edward Tatum propose that one

gene makes one protein

1949 Sickle cell anaemia demonstrated to be molecular

disease
 1952 The ‘Waring Blender’ experiment

1953 The double helix is unravelled

1967 The genetic code is cracked

1973 Recombinant DNA technology begins

1975 First international conference on recombinant DNA technology

1975 DNA sequencing discovered

1975 Monoclonal antibody technology introduced

1978 Genentech Inc. established

1978 Genentech use genetic engineering to produce human insulin in

[Link] - 1980

1978 Kary Mullis discovers PCR

 1989 The Human Genome Project begins

1990 First use of gene therapy

1990 First product of recombinant DNA technology introduced into

US food chain

1993 FDA announces that transgenic food is safe

1994 The FLAVR SAVR tomato - first genetically

engineered whole food
1996 First mammal cloned from adult cells

1990s First conviction using genetic fingerprinting

1996 Development of Affymetrix GeneChip

1997 First artificial chromosome

 1998 Human embryonic stem cells grown

1999 Celera announces completion of

Drosophilia genome sequence

2000 90% of Human Genome sequence

published on web

2001 Human genome project complete

Overview of Microorganisms of Industrial
Importance

Yeasts (Saccharomyces Moulds (Aspergillus

cerevisiae) niger)

Bacteria (Escherichia Actinomycetes (Streptomyces

coli) griseus)
Industrial Microorganisms

• Microorganisms are used extensively to provide a vast range of products and services.

• They have proved to be particularly useful because of the ease of their mass cultivation, speed
of growth, use of cheap substrates (which in many cases are wastes) and the diversity of
potential products.

• Their ability to readily undergo genetic manipulation has also opened up almost limitless
further possibilities for new products and services from the fermentation industries.
Products and Services of Industrial
Microorganisms
• Microbial products may be:
• Microbial biomass: microbial cells (living or dead) and
components of microbial cells
• Microbial metabolites
• Intracellular or extracellular enzymes
• Chemicals produced by the microbes utilizing the medium
constituents or the provided substrate
• Modified compound that has been microbiologically
transformed
• Recombinant products through the DNA recombinant
technology
• Microbial products can be broadly categorized into the following
services:
• Metabolic production
• Biotransformation
• Production of biofuels
• Treatment of organic and industrial wastes
• Recovery of metals
• Production of microbial biomass (microbial protein or single cell protein)
for food and feed
• Production of biocontrol agents
• Fermentation of food products.
Development of Industrial
Microorganisms
• Traditional fermentations were originally performed
(and still are in many cases) by a mixture of wild
microorganisms emanating from the raw materials or
the local environment.
• For example, some food and alcoholic beverage
fermentations.
• Initial attempts to improve the microorganisms
involved occurred a little more than 120 years ago
when they were first isolated from these processes as
pure cultures from which the most useful strains
Development of Industrial
Microorganisms
• The specific microorganisms employed were often isolated from
the natural environment which involved the random screening
of many isolates.
• Alternatively, suitable microorganisms were acquired from culture
collections.
• Most of these microorganisms, irrespective of their origins, were
subsequently modified by conventional strain improvement
strategies, using mutagenesis or breeding programmes, to improve
their properties for industrial use.
• Several processes developed in the past 20 years have involved
recombinant microorganisms and genetic engineering
technologies have increasingly been used to improve established
 Regulation of Industrial
Microorganism Usage
• Regulatory considerations are of major importance when choosing
microorganisms for industrial use.
• Fermentation industries often prefer to use established
Generally Regarded As Safe (GRAS) microorganisms,
particularly for manufacturing food products and ingredients.
• This is because requirements for process and product
approval using a “new” microbe are more stringent and
associated costs are much higher.
• Where pathogens and some GMMs are used as the producer
organism, additional safety measures must be taken.
• Special containment facilities are employed and it may
be possible to use modified (“crippled”) strains that
 Advantages of Using Microorganisms for
Industrial Processes
• Although plants and animals as well as their cell cultures are
used in biotechnology, microorganisms are the preferred choice
due to the following advantages:
• Microorganisms grow rapidly in comparison with plants and
animals.
• Generation time (the time for an organism to mature and reproduce) is about
12 years in man, about 24 months in cattle, 18 months in pigs, and 6 months
in chicken, but approx. 20 minutes in the bacterium, E. coli.
• Consequently, biotechnological products which can be obtained from
microorganisms in a matter of days may take many months in animals or
plants.
• The space requirement for growing microorganisms is small.
• A 100,000-liter fermenter can be housed in about 100 square yards of space,
Advantages of Using Microorganisms for
Industrial Processes

• Microorganisms are not subject to the problems of the

changes of weather which may affect agricultural
production, especially among plants.

• Microorganisms are not affected by diseases of

plants and animals, although they do have their
peculiar scourges in the form of phages and
contaminants, but there are procedures to contain them.
 Commonly Employed Microorganisms

• The major organisms used in industrial microbiology are

certain prokaryotes
(Streptomyces and E. coli) and fungi (yeasts and
moulds).
• Classical genetic methods/tools are employed by industrial
microobiologists to select high-yielding microbial variants,
with the goal being to increase the yield of the product to
the point that an economically feasible process is possible.
• Thus, the behaviour of the actual production strain may be
far removed from that of the original wild-type strain.
 Escherich
ia coli
• Escherichia coli is a well-known prokaryotic bacterium that is
widely used as a
model organism for a variety of applications due to its
adaptability.
• E. coli is more understood than other living species because of its
minimal dietary requirements, rapid growth rate, and, most
critically, well-established genetics.
• E. coli cells divide once every 20–30 min on average, allowing
them to adapt to their surroundings quickly.
• It is a good model organism for industrial applications because of
its ability to grow quickly on low-cost media and the
availability of molecular tools to perform genetic
Escherich
ia coli

• Vaccine development, bioremediation, biofuel generation,

and immobilized enzymes have all exploited modified E.
coli cells.

• Furthermore, because E. coli reproduce primarily asexually,

alterations to the genome are preserved, and the effects
exhibited in these mutants are repeatable.
• Because of these characteristics, E. coli is an excellent
model organism for industrial microbiology applications.
Actinomyc
etes
• They have branching filamentous hyphae which resemble the
mycelia of fungi.
• They are unrelated to fungi and regarded as bacteria for the
following reasons:
• First, they have peptidoglycan in their cell walls
• Second, they are about 1.0 µ in diameter, whereas fungi are at least
twice that size in diameter.
• As a group, the actinomycetes are unsurpassed in their
ability to produce secondary metabolites of industrial
importance, especially as pharmaceuticals.
• The best-known genus is Streptomyces, from which many
antibiotics as well as non- anti-microbial drugs have been
Fungi
• Fungi are members of the Eukarya which are commonly used in
industrial production.
• The following are currently used in industrial microbiology:
• Rhizopus and Mucor are used for producing various
enzymes.
• Yeasts (Saccharomyces cerevisiae) are used for the
production of ethanol and alcoholic beverages.
• Claviceps purperea is used for the production of ergot
alkaloids.
Objective 2

To isolate, screen and identify fungal strains from fruit waste to be used
for the production of cellulolytic enzymes.

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 Methods
• Two methods were used in obtaining desired fungal strains.
1. Taking decomposing material from the Blue Skies compost site and isolating
organisms
2. Taking fresh fruit waste samples, undertaking controlled decomposition and
isolating organisms from the decomposed material
• Controlled decomposition was carried out in 3 environments for 5 days
• Incubator at 30 ± 1℃
Mango
• Cupboard at 25 ± Pineapple 2℃
pawpaw Blend

• Soil environment 28℃ (samples were put in a Ziploc bag before being buried in
the soil)
• Swabs from the fresh samples wereCompost
Fruit waste from the factory sent to the
also taken and incubated for fungal isolates
Sampling
• An
compost siteantibacterial agent, chloromphenical was added to eliminate bacteria

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Identification of fungal isolates
Cultural and morphological characteristics such as:
• colony (mycelia)
• colour,
• surface texture,
• pattern
• exuded pigment
• spore shape
• hyphal shape
• of the various fungal isolates were carefully observed with the aid of a
magnifying lens on MEA identified using a standard identification key
(Watanabe, 2002; Gaddeyya et al., 2012).

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Results
Fungal isolates – Compost site
• Samples from the compost site produced three different organisms:
• Comp1, Comp 2 and Comp 3

Comp 1 Comp 2 Comp 3

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 Fungal isolates – Controlled
decomposition
• All samples yielded fungal isolates • Preliminary macroscopic
• A total of 108 isolates were identified examination of cultural
• 61 fungal isolates were obtained from the characteristics of isolates revealed
fresh samples that some isolates were similar to
• 29 from the soil others from the same source.
• 13 from the incubator • The numbers were thus reduced to
• 5 from the cupboard 64 isolates.

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 Preliminary Assessment of Organisms

• Preliminary assessment of organisms was done to investigate their hydrolytic

abilities
• The organisms were cultivated on Pineapple peels and those with lower
hydrolytic potential were eliminated.
• After further assessment seven organisms were obtained:
• Comp 1, Comp 2, Comp 3, 5IPa2d, Bl1E, Pa1E, Pi3f

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Comp 1

• Comp 1 had a pale green pigmentation

A B

• Observation under the microscope showed:

conidiophores hyaline, inflated clavately at the
apex forming nodded vesicles: conidial heads
dark bluish green.

• Using literature as guide (Gaddeyya et al., 2012, Gilman, 2001,

Nagamani et al., 2006, Watanabe, 2002), Comp1 was identified
as Aspergillus fumigatus.

C D
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 Comp2
A B
• Comp 2 had black homogenous pigmentation

• Observation under the microscope showed

conidiophore, erect, thick-walled, inflated at the
apex forming globose vesicles and conidia
phialosporous, black in mass
C D

• Using literature as guide (Gaddeyya et al., 2012, Gilman,

2001, Nagamani et al., 2006, Watanabe, 2002),

• Comp 2 was identified as Aspergillus niger.

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Comp 3
• Comp 3 had white pigmentation with spongy
mycelia
• Observation under the microscope showed
sporangiophores erect, simple rhizoidal,
connected directly to sporangiophores, bearing
sporangia terminally.
• Sporangiospores subglobose to subellipsoidal,
with bluish stripes
• Using literature (Gaddeyya et al., 2012, Gilman, 2001,

Nagamani et al., 2006, Watanabe, 2002) as guide,

• Comp 3 was identified as Rhizopus sp
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BL1E
• BL1E had a thick mycelium with its outer layer
pigmented and the inner layer being hyaline.
• The reverse of the mat was brownish.
• Observation under the microscope reveals:
• Conidial anastomosis tubes which are special
hyphae and conidophore as well as ascospore
cells which are typical of Neurospora sp (Frederick
et al., 1969, Ho, 1986, Perkins et al., 1976).

• BL1E was thus identified as Neurospora sp.

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 • 5IPa2d had greyish-white pigmentation with
5IPa2d spongy mycelia on the obverse and cream to
beige coloration with a pink peach on the
reverse.
• Observation under the microscope showed
sporangiophores erect, simple rhizoidal,
connected directly to sporangiophores,
columellae globose.
• Sporangiospores subglobose to subellipsoidal,
with bluish stripes (lines).
• Using literature (Gaddeyya et al., 2012, Gilman, 2001,

Nagamani et al., 2006, Watanabe, 2002) as guide,

• 5IPa2d was identified as Rhizopus sp.

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Pa1E • Pa1E had blue - green pigmentation with a white
edge hem (obverse) and cream to beige coloration
(reverse).
• Clear exudate droplets were observed
• Observation under the microscope revealed
conidiophores hyaline, erect, branched
penicillately at the apexes, verticillate phialides
on each metula, and rather aggregated, compact,
forming cylindrical columnar grayish green
conidial head.
• Using literature Pa1E was identified to be
Penicillium sp. (Demjanová et al., 2021, Watanabe, 2002, Zain et
al., 2009).
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Pi3f
• Pi3f was observed to have black homogenous
pigmentation on MEA.
• Observation under the microscope showed
conidiophore, erect, thick-walled, inflated at the
apex forming globose vesicles, and Conidia
phialosporous, black in mass.
• Using literature as guide (Gaddeyya et al., 2012, Gilman,

2001, Nagamani et al., 2006, Watanabe, 2002) ,

• Pi3f was identified as Aspergillus niger.

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Table 3 Fungal isolates obtained from fruit peels

Isolate Substrate Isolated from Identified Organism

Comp 1 Compost site Aspergillus fumigatus
Comp 2 Compost Site Aspergillus niger
Comp 3 Compost Site Rhizopus sp.
BL1e Fresh Blended peels Neurospora sp.
5IPa2d
Pawpaw Incubator decomposed Rhizopus sp.
Pa1E Fresh pawpaw Penicillium sp.
Pi3f Fresh pineapple Aspergillus niger.

[Link]
10/27/2025 69
Isolation of Suitable
Microorganisms
• The isolation of a suitable industrial microorganism from the
environment can be divided into two types, “shotgun” and
objective approaches.
• In the shotgun approach, samples of free-living microorganisms,
biofilms or other microbial communities are collected from animal
and plant material, soil, sewage, water and waste streams,
particularly from unusual man-made and natural habitats.
• These isolates are then screened for desirable traits.
• The alternative is to take a more objective approach by sampling
from specific sites where organisms with the desired
characteristics are considered to be likely components of the
natural microflora.
• For example, when attempting to isolate an organism that can degrade or
Culture
Development
• Once the samples have been collected, a major problem is
deciding on the growth media and cultivation conditions that
should be used to isolate the target microorganism(s).
• An initial step is often to kill or repress the proliferation of
common organisms and encourage the growth of rare ones (i.e.
selective growth).
• Enrichment cultures may then be performed in batch culture,
or often more suitably in continuous systems.
• This encourages the growth of those organisms with the
desired traits and increases the quantity of these target
organisms, prior to isolation and screening.
• However, this mode of selection is suitable only for cases where
Culture
Development
• Subsequent isolation as pure cultures on solid growth
media involves choosing or developing the appropriate
selective media and growth conditions.
• Once isolated as pure cultures, each must be
screened for the desired property, production of a
specific enzyme, inhibitory compound, etc.
• However, at this stage the level of activity or
concentration of the target product per se is not of
major concern, as strain development can normally be
employed to vastly improve performance.
• Selected isolates must also be screened for other
Culture
Development

• These isolation and screening procedures are more

easily applied to the search for a single microorganism.
• However, it is much more difficult to isolate consortia
which together have the ability/characteristic that is
sought and whose composition may vary with time.
• Such groups can be more efficient, particularly where
the ability to degrade a complex recalcitrant
compound is involved.
Culture
Collections
• Microbial culture collections provide a rich source of
microorganisms that are of past, present and potential future
interest.
• There are almost 500 culture collections around the world; most
of these are small, specialized collections that supply cultures or
other related services only by special agreement.
• Others, notably national collections, publish catalogues listing the
organisms held and provide extensive services for industrial and
academic organisations.
• In the UK for example, the National Culture Collection
(UKNCC) is made up of several collections. They are housed in
separate institutions and tend to specialize in bacteria, yeasts,
filamentous fungi or algae of either industrial or medical
Culture
Collections
• The prime functions of a culture collection are to maintain the
existing collection, to continue to collect new strains and to
provide pure, authenticated culture samples of each organism.
• Problems of culture maintenance have been aided by the
development and use of cryopreservation and freeze-drying
(lyophilization) techniques, along with miniaturized storage
methods.
• One convenient method involves the adsorption of cells to glass beads (2 mm diameter)
that may be placed in frozen storage, from which individual beads may be removed
without thawing the whole sample.
• Use of microorganisms selected from a culture collection provides
significant cost savings compared with environmental isolation
and has the advantage that some characterization of the
Characteristics of
Industrial Strains
Microorganisms should ideally exhibit the following properties to be considered
suitable for industrial application:
1. Large-scale cultivation. First and foremost, the organism must be capable of
growth and product formation in large-scale culture.
2. Reproductive structures for propagation. It should produce spores (if
fungi or yeast) or some other reproductive cell form so that it can be easily
inoculated into large vessels used to grow product-producing
microorganisms on an industrial scale.
3. Rapid growth and production of desired products over a short period.
A slow-growing organism, regardless of its efficiency in terms of the
production of the target material, could be a liability. In the first place, the
slow rate of growth exposes it to a greater risk of contamination in
comparison to other equally effective producers which are faster growers.
Second, the rate of the turnover of the production of the desired material is
Characteristics of Industrial
Strains
4. Genetic stability. The organism should have reasonable genetic and
physiological stability. An organism which mutates easily is an expensive risk.
It could produce undesired products if a mutation occurred unobserved. The
result could be reduced yield of the expected material, production of an
entirely different product, or even a toxic material. None of these situations is
a help towards achieving the goal of the industry, which is the maximization of
profits through the production of goods with predictable properties to which
the consumer is accustomed.
5. Utilization of a wide range of low-cost and readily available carbon
sources. It must also be able to grow in a liquid culture medium obtainable in
bulk quantities at a low price. Many industrial microbiological processes use
waste carbon from other industries as major or supplemental ingredients for
large-scale culture media. These include corn steep liquor (a product of the
corn wet-milling industry that is rich in nitrogen and growth factors) and whey
(a waste liquid of the dairy industry containing lactose and minerals).
Characteristics of Industrial
Strains
7. Amenability to genetic manipulation. The organism should be fairly
easily amenable to genetic manipulation to enable the establishment of
strains with more acceptable properties. Increased yields are often
obtained using mutation and classical genetic selection techniques. A
genetically stable and easily engineered microorganism is thus a clear
advantage for an industrial process.
8. Safety, non-pathogenicity and should not produce toxic agents,
unless this is the target product. Its end products should not include
toxic and other undesirable materials, especially if these end products are
for internal consumption. An industrial microorganism should not be
pathogenic, especially to humans or economically important animals or
plants.
9. Competing advantage. Wherever possible, organisms which have
physiological requirements which protect them against competition from
contaminants should be used. An organism with optimum productivity at
Characteristics of
Industrial Strains
[Link] harvesting from the fermentation as well as ready breakage,
if the target product is intracellular. The organism should lend itself to
a suitable method of product harvest at the end of the fermentation. If, for
example, a yeast and a bacterium were equally suitable for manufacturing
a certain product, it would be better to use the yeast if the most
appropriate recovery method was centrifugation. This is due to the
bacterial diameter of approximately 1 µm, while yeasts are approximately
5 µm. Assuming their densities are the same, yeasts would sediment 25
times more rapidly than bacteria. The faster sedimentation would result in
less expenditure in terms of power, personnel supervision, etc. which
could translate to higher profit.
[Link] to contamination. The organism should be reasonably
resistant to predators such as Bdellovibrio spp. or bacteriophages. It
should therefore be part of the fundamental research of an industrial
establishment using a phage-susceptible organism to attempt to produce
phage-resistant, yet high-yielding, strains of the organism.
Problems Associated with Industrial
Microbial Processes
1. Finding the least expensive medium in which to grow the microbe to
maximize yield and profits.
• Often this is a waste product from another industrial process, such as
corn-steep liquor, sugar processing wastes or whey.
2. Maintaining strain purity and developing better strains for improving
the yield.
• A single mutation may decrease the yield by a significant percentage or
result in undesirable substances being produced. The industrial research
laboratories constantly seek better strains for the production of their
product.
3. Preventing contamination by other microbes and by viruses (phage)
that live on the microbe involved.
• The media must be sterilized before being inoculated with the desired
organism and purity must be maintained throughout the production
process. A small quantity of a contaminant may produce an enzyme that
Problems Associated with Industrial
Microbial Processes
4. Develop rapid and efficient methods for purification of the desired
produce in a stable form that is safe to use.
• The products of many fermentations are often unstable in impure form or
subject to unwanted modifications if they are not purified quickly. The final
growth mixture may contain dangerous substances from which the desired
product must be separated. As every step in the purification results in a loss of
the product, the search for more efficient purification procedures is never-
ending.
5. Always striving to improve yield by modifying the strain, nutrients or
environmental conditions.
• As product yields are sensitive to subtle modifications in the nutrient and the
environmental conditions, these are constantly monitored. For example, the pH,
oxygen content, nitrogen/phosphorous ratio etc. may be adjusted during the
production process.
6. Safe and inexpensive disposal of the massive quantities of waste products
Selection and Screening of Industrial
Microorganisms

• In industrial microbial processes, the aim is primarily to

improve/optimize the particular characteristics sought in an organism.
• Advances have been achieved in this area by using screening and
selection techniques to obtain better organisms.
• In a selection system, all rare or novel strains grow while the
rest do not.
• In a screening system, all strains grow but certain strains or cultures
are chosen because they show the desired qualities required by the
industry in question.
Strain
Improvement
• Genetic manipulations are used to produce microorganisms with
new and desirable characteristics.
• The classical methods of microbial genetics play a vital role in
the development of cultures for industrial microbiology.
• Some strain improvement techniques commonly employed
include:
1. Mutation
2. Protoplast and cell fusion
3. Genetic engineering (recombinant DNA technology)
Mutati
on
• Once a promising culture is found, a variety of techniques can be used
for culture improvement, including chemical mutagenesis and
ultraviolet light.
• However, such methods normally lead only to the loss of undesirable
traits or increased production due to loss of control functions.
• It has rarely led to the appearance of a new function or property.
• Thus, an organism with a desired feature will be selected from the
natural environment, propagated and subjected to a mutational
programme, then screened to select the best progeny.
• As an example, the first cultures of Penicillium notatum, which could
be grown only under static conditions, yielded low concentrations of
penicillin.
• In 1943 a strain of P. chrysogenum was isolated – strain NRRL 1951 –
which was further improved through mutation (using X-ray treatment, UV
and mustard gas, the yield was increased from 120 IU to 2,580 IU).
Protoplast
Fusion
• Protoplast fusion is now widely used with yeasts and moulds.
• Most of these microorganisms are asexual or of a single mating type, which decreases the
chance of random mutations that could lead to strain degeneration.
• To carry out genetic studies with these microorganisms, protoplasts
are prepared by growing the cells in an isotonic solution while
treating them with enzymes, including chitinase, cellulase and ß-
galacturonidase.
• The protoplasts are then regenerated using osmotic stabilizers such
as sucrose.
• If fusion occurs to form hybrids, desired recombinants are identified using selective plating
techniques.
• After regeneration of the cell wall, the new protoplasm fusion product can be used in further
studies.
• A major advantage of the protoplast fusion technique is that
 Protoplast
Fusion
• One of the most exciting and commercially rewarding areas of
biotechnology involves a form of mammalian cell fusion to the
formation of monoclonal antibodies.
• In 1975, pure monoclonal antibodies were produced from the
fusion product of (hybridoma) of ß-lymphocytes and myeloma
tumour cells.
• The monoclonal antibody technique changes antibody-secreting
cells (with a limited life span) to cells that are capable of
continuous growth (immortalisation) while maintaining their
specific antibody-secreting potential.
• Monoclonal antibodies are now widely applied in many diagnostic
techniques which require a high degree of specificity.
• Specific monoclonal antibodies have been combined into diagnostic kits in health care,
Protoplast
Fusion

The formation of antibody-producing hybridomas by fusion techniques.

Genetic
Engineering
• Recombinant DNA technology or genetic engineering offers
unlimited opportunities for creating new combinations of
genes.
• These techniques allow the splicing of DNA molecules of quite
diverse origin and when combined with the techniques of
genetic transformation, facilitate the introduction of foreign
DNA into other organisms.
• DNA can be isolated from plants, animals or microorganisms
(the donors), and fragmented into groups of one or more genes.
• Such fragments can then be coupled to another piece of DNA
(the vector) and then passed into the host or recipient cell,
becoming part of the genetic complement of the new host.
• The host cell can then be propagated in mass to form novel
Strategies Involved in Genetic
Engineering

STRATEGY METHOD
Formation of DNA Extracted DNA can be cut into smaller fragments by
fragment specific enzymes -
restriction endonucleases found in many species of
bacteria.
Splicing of DNA into The small sequences of DNA can be joined or spliced
vectors into the vector DNA
molecules by an enzyme DNA ligase, creating an
artificial DNA molecule.
Introduction of The vectors are either viruses or plasmids and are
vectors into replicons and can exist
host cells in an extra-chromosomal state; they can be
transferred normally by transduction or
transformation.
Selection of newly Selection and ultimate characterization of the
 Recombinant DNA
Technology

Recombinant DNA: The technique of recombining genes from one species with those of another.
Genetic
Engineering

• Short lengths of chemically synthesized DNA sequences can

be inserted into recipient microorganisms by the process of
site-directed mutagenesis.
• This can create small genetic alterations leading to a change of
one or several amino acids in a target protein.
• Such minor amino acid changes have been found to result in the
development of new products
• Development of thermostable enzymes and enzymes that can catalyze desired reactions.
• Enzymes and bioactive peptides with markedly different
characteristics (stability, kinetics, activities) can be created.
• This approach may lead to improved transformation of recalcitrant materials or even
the degradation of materials that have previously not been amenable to biological
processing.
Preservation of
Microorganisms

• Once a microorganism or virus has been selected or

created to serve a specific purpose, it must be
preserved in its original form for further use and study.
• Periodic transfers of cultures have been used in the past
although this can lead to mutations and phenotypic
changes in microorganisms.
• To avoid these problems, a variety of cultural
preservation techniques may be used to maintain
desired cultural characteristics.
Microbial Culture Preservation
Methods
METHOD COMMENT
Periodic Variables of periodic transfer to new media include frequency, medium used
transfer and holding temperature. This can lead to increased mutation rates and
production of variants.
Mineral oil A stock culture is grown on a slant and covered with sterilized mineral oil;
slant the slant can be stored at refrigerator
temperature
Minimal Washed cultures are stored under refrigeration; these cultures can be viable
medium, for 3-5 months or longer.
distilled
water, or
water agar
Freezing in Not reliable, can result in damage to microbial structures, with some
growth microorganisms, however this can be a useful
medium means of culture maintenance.
Drying Cultures are dried on sterile soil (solid stocks), on sterile filter paper disks, or
in gelatin drops; these can be stored in a
desiccator at refrigeration temperature or frozen to improve viability.
Freeze-drying Water is removed by sublimation, in the presence of a cryoprotective agent;
sealing in an ampoule can lead to long-term
viability, with 30 years having been reported.
Ultra- Liquid nitrogen at -196˚C is used, and cultures of fastidious microorganisms
Culture Maintenance
Media

• These media are used for the storage and subculturing of key
industrial strains.
• They are designed to retain good cell viability and minimize
the possible development of genetic variation.
• In particular, they must reduce the production of toxic
metabolites that can have strain- destabilizing effects.
• If strains are naturally unstable, they should be maintained
on media selected for the specific characteristic that must be
retained.
Screening of
Microorganisms

• In industrial microbiology, microorganisms hold the key to the

success or failure of a fermentation process.
• It is therefore important to select the most suitable
microorganisms to carry out the desired industrial process.
• The most important factor for the success of any fermentation
industry is a production strain.
• Screening may be defined as the use of highly selective
procedures to allow the detection and isolation of only those
microorganisms of interest from among a large microbial
population.
• Thus, to be effective, screening allows the discarding of many
valueless microorganisms, while at the same time allowing
 Screening of
Microorganisms
• The screening programmes generally consist of:
1. Primary screening
2. Secondary screening

• Primary screening: It is carried out just to detect and isolate

the species from mixed population that possess desired
capacity.

• It is generally carried out by crowded plate technique.

• A natural source such as soil is diluted to provide cell
concentration such that amount spread, applied in some manner to
the surface of agar plate which will yield colonies not touching
Screening of
Microorganisms

The following are some examples of primary

screening:
i) Screening of antibiotic producing organisms;
ii) Screening of organic acid and amine producing
organisms; and
iii) Screening of organisms producing vitamins, amino
acids and growth factors extracellularly
Screening of
Microorganisms
• Secondary screening allows the further sorting of
those microorganisms that have real value for
industrial purposes and discarding valueless
microorganisms.
• Thus, the secondary screening allows determination of the
most efficient and suitable organism to be used for
commercial production of the fermentation product from
different potential strain as detected and isolated by
primary screening.
• There are two types of secondary screening techniques:
• a) Qualitative secondary screening
 Secondary screening

• The qualitative secondary screening tells us the spectrum or range

of microorganisms which are sensitive to newly discovered antibiotic,
but quantitative secondary screening tells us yield of antibiotic
which can be expected when microorganisms is grown in various
different media.
• Secondary screening should yield the type of information that is
needed in order to evaluate the potential microorganisms for
industrial use
• Yield and spectrum; Side effects in humans; Molecular structure; Nutritional
requirements of the culture; Genetic stability; Pathogenicity
Fermentation
Processes
Review of Fermentation
Processes
• Generally, an established fermentation process may be divided
into six (6) parts:
1. The formulation of media to be used in culturing the
process organism during the development of the inoculum
and in the production fermenter.
• The production medium is almost always complex and is devised to maximize
yields of the enzyme/product
2. The sterilization of the medium, fermenters, and ancillary
equipment.
3. The production of an active, pure culture in sufficient quantity
to inoculate the production vessel.
4. The organism’s growth in the production fermenter under
optimum conditions for product formation.
Schematic Representation of a Typical
Fermentation Process
Media for Industrial
Fermentation
• The medium composition is as critical to product yields as high-
producing strains of microorganisms. The medium not only
provides the nutrients needed for microbial growth but also for
metabolite production.
• All microorganisms require water, sources of energy,
carbon, nitrogen, mineral elements, and possibly vitamins
plus oxygen if aerobic.
• On a small scale it is relatively simple to devise a medium
containing pure compounds, but the resulting medium, although
supporting satisfactory growth, may be unsuitable for use in a
large-scale process.
• The various media for bioreactors may be grouped into two
broad categories:
Media for Industrial
Fermentation
• A synthetic or chemically defined medium is desirable for
various studies, but product yields from such media are
generally low.
• Foaming is not a problem with such media.
• The complex media contain undefined constituents like soybean
meal, molasses, corn steep liquor, etc., and give much higher
yields of metabolites.
• Carbon sources can be simple, e.g., sugar, alcohol, etc., or
complex carbohydrates, proteins, molasses, potatoes, sweet
potatoes, etc.
• In many processes, precursors need to be provided, e.g.,
phenylacetic acid for penicillin G, inorganic cobalt for vit. B12.
Media for Industrial
Fermentation
• On a large scale one must normally use sources of nutrients
to create a medium which will meet as many as possible of
the following criteria:
1. It will produce the maximum yield of product or biomass per
gram of substrate used.
2. It will produce the maximum concentration of product or
biomass.
3. It will permit the maximum rate of product formation.
4. There will be a minimum yield of undesired products.
5. It will be of a consistent quality and be readily available
throughout the year.
6. It will cause minimal problems during media making and
sterilization.
 Media for Industrial
Fermentation

• The following carbon and nitrogen sources tend to meet most

of the above criteria for production media and are cheap
substrates:
• Carbon sources: Cane molasses, beet molasses, cereal grains,
starch, glucose, sucrose, and lactose
• Nitrogen sources: Ammonium salts, urea, nitrates, corn steep
liquor, soya bean meal, slaughter- house waste, and fermentation
residues
• For much fermentation, e.g., antibiotic production, a medium
suited for rapid cell growth is unsuitable for product formation.
• In such cases, specialized media for production have to be devised.
 Types of Industrial
Fermentation Processes
• A fermentation process is a biological process and, therefore, has
requirements of sterility and the use of cellular enzymatic
reactions
• Some of the most important types of fermentation are as follows:
1. Submerged fermentation
2. Solid-state fermentation
3. Anaerobic fermentation
4. Aerobic fermentation
5. Immobilized cell/enzyme bioreaction
 Submerged or Liquid
Fermentation (SmF/LF)

• SmF can be defined as “the growth of microorganisms (mainly

bacteria and yeasts) on substrates in excess/free moisture (i.e.
liquid that will drain freely by gravity from solid materials).
• This type of fermentation can be categorized into three (3) forms:
1. Batch fermentation
2. Continuous fermentation
3. Fed-batch fermentation
 Submerged Fermentation – Batch
Fermentation Process

• A closed-loop system where yeasts or bacteria grow in a

culture media and an additional material is not added is
called batch culture.
• Growth in a batch culture undergoes some series of
steps:
[Link] initial phase seems to record no growth, termed the
lag phase (considered as a time of adaptation)
• In a commercial process, the length of the lag phase
should be reduced as much as possible by using a
suitable inoculum and cultural conditions
 Submerged Fermentation – Batch
Fermentation Process

1.A gradual increase that builds up into a constant,

maximum growth rate often referred to as the logarithmic
phase or exponential phase
[Link] is terminated by progressive decrement in the
rate of growth until the stationary phase is reached.
• This is because of the limitation of one or more of the
essential nutrients and /or the production of inhibitory
substances.
Schematic Representation of
Batch Fermentation
Description of Batch Fermentation
Process
• A fermenter is filled with the prepared mash of raw materials to
be fermented.
• The temperature and pH for microbial fermentation are properly
adjusted, and occasionally nutritive supplements are added to
the prepared mash.
• The mash is steam-sterilized in a pure culture process.
• The inoculum of a pure culture is added to the fermenter, from a
separate pure culture vessel.
• Fermentation proceeds, and after the proper time the contents of
the fermenter are taken out for further processing.
• The fermenter is cleaned and the process is repeated. Thus,
each fermentation is a discontinuous process divided into
 Submerged Ferment. – Continuous
Fermentation Process

• In continuous fermentation, the substrate is added to the

fermenter continuously at a fixed rate.
• This maintains the organisms in the logarithmic growth phase.
• Exponential growth in batch culture may be prolonged by the addition of fresh medium to
the vessel.
• The fermentation products are taken out continuously.
• The fermenter used for continuous fermentation is also referred
to as chemostat.
Submerged Ferment. – Continuous
Fermentation Process

• In a substrate-limited system, the medium is designed such that

growth is limited by some component of the medium and not
toxins. Thus, growth will proceed until additional substrate is
exhausted
• An overflow device can be fitted to the fermenter such that the
added medium displaces an equal volume of culture from the
vessel, so that continuous production of cells can be achieved
• The feeding of culture medium continuously to such a culture at a
suitable rate could result in the creation of a steady state (i.e. the
formation of new biomass is balanced by the loss of cells from the
vessel).
Schematic Representation of
Continuous Fermentation
 Submerged Ferment. – Fed-Batch
Fermentation Process
• When a batch culture is subsequently fed with fresh nutrient
medium without removing the growing microbial culture, it is
called fed-batch culture.
• Fed-batch culture allows one to supplement the medium with such
nutrients that are depleted or that may be needed for the terminal
stages of the culture (for example, the production of secondary
metabolites).
• Therefore, the volume of a fed-batch culture increases with time.
• Fed-batch cultures achieve higher cell densities than batch
cultures.
• It is used when high substrate concentration causes growth
inhibition.
 Submerged Ferment. – Fed-Batch
Fermentation Process
• A fed-batch culture is established initially in batch mode and is then fed
according to one of the following feed strategies:
[Link] same medium used to establish the batch culture is added,
resulting in an increase in volume.
2.A solution of the limiting substrate at the same concentration
as that in the initial medium is added, resulting in an increase
in volume.
3.A concentrated solution of the limiting substrate is added at a
rate less than in
(1) and (2), resulting in an increase in volume.
4. A very concentrated solution of the limiting substrate is added
at a rate less than in (1), (2), and (3), resulting in an insignificant
increase in volume.
• Fed-batch systems employing strategies (1) and (2) are described as
variable volume, whereas a system employing strategy (4) is described
Schematic Representation of Fed-
Batch Fermentation
Solid State
Fermentation
• This process can be defined as “the growth of
microorganisms (mainly fungi) on moist solid materials in
the absence of free-flowing water”.
• The fungi used for solid-state fermentations are usually obligate aerobes.
• This method allows greater surface area for growth.
• The production of the desirable substance and the recovery is
generally easier and more satisfactory.
• In such fermentations, microbial growth and product
formation occur at the surface of solid substrates.
• Examples of such fermentations are mushroom cultivation, mould-
ripened cheeses, starter cultures, etc.
• More recently, this approach has been used for the production of
extracellular enzymes, certain valuable chemicals, fungal toxins, and
Solid State
Fermentation
• According to the physical state, solid-state fermentations are
divided into two groups:
[Link] moisture solids fermentation without or with
occasional/continuous agitation

Stirred drum bioreactors with

Tray bioreactor with
occasional and
occasional agitation
continuous agitation
Solid State
Fermentation
2. Suspended solids fermentation in packed columns, through
which liquid is circulated.

Packed-bed
bioreactor
 Solid State
Fermentation

• Solid-state fermentation on a large scale uses stationary or

rotary trays.
• Temperature and humidity-controlled air is circulated through
the stacked solids.
• Less frequently, rotary drum-type fermenters have been used.
• The pH, temperature of incubation, aeration etc., are all
important factors in fermentations and these have to be
optimized for each type of fermentation.
 Solid State
Fermentation

• The composition of the culture medium plays a major

role in the fermentation process and will determine to a
very great extent the level of the end product.
• For example, a culture medium containing sucrose enables better
production of citric acid by A. niger than any other carbohydrate.
• Feedstocks such as bagasse, rice straw, cassava starch
processing residue, wheat bran, potato pulp, etc. have
been used to grow industrial strains
• Emphasis is generally placed on the use of cheap raw materials so that
the cost of production is low.
Solid State
Feedstocks
• Traditional substrates are several agricultural products,
rice, wheat, maize, soybean, etc.

Cassava starch processing residue

Rice straw

Corn stover Soybean residue

Solid State
Fermentation
• The substrate provides a rich and complex source of nutrients,
which may or may not need to be supplemented.
• Such substrates selectively support mycelial organisms, which can grow at high
nutrient concentrations and produce a variety of extracellular enzymes
• For example, a large number of filamentous fungi, and a few bacteria (Actinomycetes and
one strain of Bacillus).

Fungal growth under SSF

Advantages of SSF over
SmF
[Link] yields: Comparative studies between SmF and SSF
claim higher product yields with SSF than with the
corresponding SMF
[Link] contamination: The low availability of water reduces
the possibility of contamination by bacteria and yeast. This
allows working in aseptic conditions in some cases.
[Link] growth: Similar environmental conditions to those of
the natural habitats for fungi, which constitute the main group of
microorganisms used in SSF.
[Link] aeration: Higher levels of aeration, especially
adequate in those processes that demand an intensive oxidative
metabolism.
Advantages of SSF
over SmF
[Link] media are often quite simple. The substrate
usually provides all the nutrients necessary for growth.
[Link] design reactors, with few spatial requirements
can be used due to the concentrated nature of the
substrates.
[Link] energy requirements (in some cases autoclaving
or vapour treatment, mechanical agitation and aeration
are not necessary).
[Link] volumes of polluting effluents are produced in
solid-state fermentation (SSF). Due to the high concentration
of materials in SSF, fewer dissolvents are required for
Advantages of SSF over
SmF
9. The low moisture availability in SSF may stimulate the
production of certain compounds that may not be produced
or may only be poorly produced in submerged liquid
fermentation (SLF or SmF).
[Link] some cases, the properties of the products obtained in
SSF may differ slightly
from those produced in SLF. For instance, they may exhibit
more thermo-tolerance.
[Link] SSF, due to the concentrated nature of the substrate,
smaller reactors can be used
to hold the same amount of substrate as in SmF.
Disadvantages of SSF
over SmF
1. Only microorganisms that can grow at low moisture levels can
be used.
2. Microbial biomass determination is very difficult.
3. Mass transfer is limited due to the low rate of diffusion.
4. The solid nature of the substrate causes problems in the
monitoring of the process parameters (pH, moisture content,
and substrate, oxygen and biomass concentration).
5. Agitation may be very difficult. For this reason, static
conditions are preferred.
6. Possibility of contamination by undesirable fungi.
7. The removal of metabolic heat generated during growth may
be very difficult.
8. Cultivation times are longer than in SmF, especially for
Anaerobic
Fermentation
• Basically a fermenter designed to operate under
microaerophilic or anaerobic conditions will be the same as
that designed to operate under aerobic conditions, except
that arrangements for intense agitation and aeration are
unnecessary
• Many anaerobic fermentations do, however, require mild
aeration for the initial growth phase, and sufficient agitation
for mixing and maintenance of temperature. In anaerobic
fermentation, a provision for aeration is usually not needed.
• But in some cases, aeration may be needed initially for
inoculum build up. In most cases, a mixing device is also
unnecessary; while in some cases initial mixing of the
Anaerobic
Fermentation
• The air present in the headspace of the fermenter should be
replaced by CO2, H2, N2 or a suitable mixture of these; this is
particularly important for obligate anaerobes like Clostridium.
• The fermentation usually liberates CO2 and H2, which are
collected and used.
• E.g., CO2 for making dry ice and methanol, and for bubbling into freshly inoculated
fermenters.
• In case of acetogens and other gas utilizing bacteria, O2 free
sterile CO2 or other gases are bubbled through the medium.
• Acetogens have been cultured in 400 L-fermenters by bubbling sterile CO2 and 3kg
cells have been harvested in each run.
• Recovery of products from anaerobic fermenters does not
require anaerobic conditions.
 FERMENTATION PATHWAY

Complex polymers Hydrolysis

Sugars, Amino acids,
Glycerol Acidogenesis
VFA,
ethanol,H2, Acetogenesis
Acetic
acid,CO2, H2 Methanogenesis

Methane, CO2

10/27/2025 132
 • Inoculum - Halton Recycling AD. (New Market, Ontario,
Canada)
Influent Influent
• Substrate - Food waste (Columbia University cafeteria, New
York, NY)

 Two lab-scale fermenters, with operational volumes of 6.0L

Sequencing Step- Feed
batch reactor Reactor
were set up for anaerobic operation
 The fermenters were operated at three different hydraulic
retention times (HRT) of 2 days, 4 days and 8 days HRT and
fed an influent of 12800±351 mg COD/L.
 The SBR was fed the desired volume at one shot to achieve
the required HRT
 Step feeding in the SFR was achieved by automated addition
of required volume in three aliquots
 Samples were taken for analyses at the end of each cycle and
the effluent pumped out once
 The temperature in the fermenters was maintained at 37±1 oC
 The pH was controlled at 7.0 ±0.2
Monitored
Parameters
• Total and soluble COD (HACH Method 8000)
• Total VFA (HACH Method 8196)
• Biogas flowrate, online monitoring using Omega FMA 1604A
• VFA speciation with a Thermo Scientific Dionex™ ICS-2100 Ion
Chromatography System using an AS 11HC column
• Biogas composition with a Gas Chromatograph SRI 8610C equipped
with a Heyesep D column
• Total suspended solids (TSS) and volatile suspended solids (VSS) (US
EPA Method 160.2)

[Link]

10/27/2025 134
Objective 5

To investigate process routes involved in the

anaerobic fermentation of food waste.

[Link]

10/27/2025 135
Methods
• Biomass samples taken from the food waste reactors were stored in a freezer at -80°C for
molecular analysis.
• DNA was extracted from the samples using Qiacube (Qiagen, CA).
• Preparation and sequencing of shotgun libraries were done using Illumina MiSeq kits (Illumina,
CA).
• Reads were aligned against the National Center for Biotechnology Information’s (NCBI) protein
database.
• Conversion of protein alignments to taxonomic and functional profiles was done using R software.
• A survey was done for prevailing literature on metabolic pathways for the processing of organic
carbon targeting acidogenic and acetogenic enzymes.
• This database was employed in the creation of an expanded AD model.
[Link]

10/27/2025 136
sults

Comparison of microbial community profiles in SBR and SFR at 8d, 4d, and 2d HRT
 Reads per million (RPM)

assigned to key genera (RPM

≥ 50,000 in at least 1 sample)

in SBR and SFR food waste

digesters at 2, 4, and 8d HRT

Genera contributing significantly (RPM ≥ 50,000) to overall coding potential

(total protein coding regions, CDS) in SBR and SFR at 8-, 4-, and 2-d HRT

[Link]

10/27/2025 138
Fermentation Pathway

10/27/2025 139
Aerobic
Fermentation
• A number of industrial processes, although called 'fermentations',
are carried on by microorganisms under aerobic conditions.
• In older aerobic processes it was necessary to furnish a large surface
area by exposing fermentation media to air.
• n modern fermentation processes aerobic conditions are maintained in a
closed fermenter with submerged cultures.
• The contents of the fermenter are agitated with an impeller and aerated
by forcing sterilized air
• The main feature of aerobic fermentation is the provision for adequate
aeration; in some cases, the amount of air needed per hour is about 60-
times the medium volume.
• Therefore, bioreactors used for aerobic fermentation have a provision for
adequate supply of sterile air, which is generally sparged into the
Aerobic
Fermentation
• In addition, these fermenters may have a
mechanism for stirring and mixing of the medium
and cells. Aerobic fermenters may be either of the:
i. Stirred tank type in which mechanical motor
driven stirrers are provided
[Link] lift type in which no mechanical stirrers are used
and the agitation is achieved by the air bubbles
generated by the air supply.
• Generally, these bioreactors are of closed or batch
type, but continuous flow reactors are also used,
such reactors provide a continuous source of cells
Immobilized Cell
Bioreactors

• Bioreactors of this type are based on immobilized

cells.
• Cell immobilization is advantageous when:
• The enzymes of interest are intracellular
• Extracted enzymes are unstable
• The cells do not have interfering enzymes or such enzymes are
easily inactivated/removed
• The products are low molecular weight compounds released into
the medium.
Immobilized Cell
Bioreactors

• Under these conditions, immobilized cells offer the following

advantages over enzyme immobilization:
i. enzyme purification is not needed
ii. high activity of even unstable enzymes
[Link] operational stability
iv. lower cost
v. Possibility of application in multistep enzyme reactions.
vi. In addition, immobilization permits continuous operation of bioreactor,
which reduces the reactor volume and, consequently, pollution
problems.
• Obviously, immobilized cells are used for such biotransformation of
compounds, which require action of a single enzyme.
Immobilized Cell
Bioreactors
• Cell immobilization may be achieved in one of the following ways:
1. Cells may be directly bound to water insoluble carriers, e.g.,
cellulose, dextran, ion exchange resins, porous glass, brick, sand,
etc., by adsorption, ionic bonds or covalent bonds.
2. They can be cross linked to bi- or multi-functional reagents, e.g.,
glutaraldehyde, etc.
3. Polymer matrices may be used for entrapping cells; such matrices are
polyacrylamide cell, carrageenan (a polysaccharide isolated from a
seaweed), calcium alginate (alginate is extracted from seaweed), poly
glycol oligomers, etc. Out of these approaches, calcium alginate
immobilization is the most commonly used since it can be used for
even very sensitive cells. Cell immobilization has been used for
commercial production of amino acids, organic acids, etc.
Ferme
nter
• A fermenter (or fermentor) is a vessel for the growth of
microorganisms which, while not permitting contamination,
enables the provision of conditions necessary for the maximal
production of the desired products.
• In other words, the fermenter ideally should make it possible to
provide the organism growing with optimal pH, temperature,
oxygen, and other environmental conditions.
• Fermenters are therefore also known as bioreactors
• Fermentations may be liquid, also known as submerged, or
solid state, also known as surface.
• Most fermentations used in the industry are submerged processes,
because the fermenters used in submerged fermentation save
Bioreact
or

• An apparatus in which a biological

reaction or process is carried out,
especially on an industrial scale.
• It may be any manufactured or
engineered device or system that
supports a biologically active
environment.
• In one case, a bioreactor is a
vessel in which a chemical
process is carried out which
involves organisms or
biochemically active substances
Bioreac
tor
Bioreac
tors
Bioreac
tors

• Bioreactors are extensively used for

food processing, fermentation, waste
treatment, etc.
• On the basis of the agent used,
bioreactors are grouped into two
broad classes:
• those based on living cells and
• those employing enzymes
Bioreac
tors
• For process requirements, they are of the following types:
• aerobic
• anaerobic
• solid state and
• immobilized cell bioreactors.
• All bioreactors deal with heterogeneous systems having two or
more phases, e.g., liquid, gas, solid.
• Therefore, optimal conditions for fermentation necessitate
efficient transfer of mass, heat and momentum from one phase
to the other
 Key Components of a
Bioreactor

• A bioreactor should provide for the following:

1. Agitation (for mixing of cells and medium)
2. Aeration (aerobic fermenters; for O2 supply)
3. Regulation of factors like temperature, pH, pressure, aeration, nutrient
feeding, liquid level, etc.
4. Sterilization and maintenance of sterility, and
5. Withdrawal of cells/medium (for continuous fermenters).
• **Modern fermenters are usually integrated with
computers for efficient process monitoring, data
acquisition, etc.
Dimensions of
Fermenters/Bioreactors
• Depending on the purpose, fermenters can be as small as 1 liter
or up to about 20 liters in laboratory-scale fermenters and range
from 100,000 liters to 500,000 liters, or occasionally even more,
for factory or production fermenters.
• Fermenters of up to 1.2 million litres have been used.
• Between these extremes, pilot fermenters are found. It should be
noted that while fermenter size is measured by the total volume,
only about 75% of the volume is usually utilized for actual
fermentation, the rest being left for foam and exhaust gases.
• Generally, 20-25% of fermenter volume is left unfilled with medium as "head space" to
allow for splashing, foaming and aeration.
Types of
Fermenters

• Several types of fermenters are known which may be grouped

in several ways: shape or configuration, whether aerated or
anaerobic, and whether they are batch or continuous.
• The fermenter design varies greatly depending on the type of fermentation for which
it is used.
• The most commonly used type of fermenter is the Aerated
Stirred Tank Batch Fermenter.
• Unless specifically qualified, the word fermenter usually refers to the Aerated Stirred
Tank Batch Fermenter.
Components of the Aerated Stirred-Tank Batch
Fermenter
• A typical fermenter of this type is an upright closed cylindrical tank fitted with:
• Four or more baffles attached to the side of the wall to obstruct radial swirl and convert it to axial mixing
• A water jacket or coil for heating and/or cooling
• A device for forcible aeration (known as a sparger)
• A mechanical agitator usually carrying a pair or more impellers
• Means for introducing organisms and nutrients and for taking samples, and outlets for exhaust gases.

• Modern fermenters are highly automated and usually have means of continuously
monitoring, controlling or recording pH, oxidation-reduction potential, dissolved
oxygen, effluent O2 and CO2, and chemical components of the fermentation broth.

• Nevertheless, the fermenter need not have all these gadgets, and many automated
activities can also be operated manually.
Components of the Aerated Stirred-Tank Batch
Fermenter
Anaerobic Batch
Fermenters
• Some processes do not require the high levels of oxygen needed in aerobic
fermentation.
• Indeed, some such as clostridial fermentations do not require oxygen at all.
• These are collectively referred to as ‘anaerobic’ fermentations although in strict terms
some may be micro-aerophilic.
• Anaerobic fermenters, whether strict or micro-aerophilic (requiring small amounts of oxygen), are not commonly used in
industry.
• When they do (require oxygen), they are essentially the same as the typical
(aerobic) fermenter.
Features of the Anaerobic Batch
Fermenters
i. Vigorous aeration through air sparging is absent, as oxygen is not required.
ii. If present, agitation is aimed only at achieving an even distribution of organisms,
nutrients, and temperature, but not for aeration.
• In some cases, agitation may be essential only initially; the emergence of CO2 and H2 in anaerobic fermenters may
stir the medium.
iii. The medium is introduced into the fermenter while hot to prevent the absorption of
gases; and usually, it is introduced at the bottom of the fermenter.
iv. The fermenter itself is filled as much as possible, in order to avoid an airspace which
would introduce oxygen
Solid-state
Fermenters
• In solid-state or solid substrate fermenters, rice bran or similar solids are
used.
• Molasses may be added and a nutrient solution of ammonium and phosphate may be introduced.
• It is used mainly in Japan for enzyme production, and has been used for
citric acid production.
• Fungal bioinsecticides are also cultivated as surface cultures. Certain
mushrooms are also grown in tray fermenters

Solid-state (Surface) Tray

Solid-state
Fermenters

• In the surface fermenter, a series of shallow trays no more than about 7 cm in depth is
used, the solid medium not being more than about 5 cm so that air can penetrate into the
solid medium.

• Humid air is blown into the chamber containing the trays.

• The incoming air and the outgoing air may be filtered, especially when fungi are used to
save the dissemination of the spores in the atmosphere.

• In some fermentations, some form of temperature control is imposed through blowing

cold air into the fermenter and also by cooling the room where the fermenter is located
Fermenter
Configurations
• Batch fermenters – Stirred Tank Batch Fermenters

• Continuous stirred tank fermenters - Essentially similar to that of the

batch fermenter. It differs by the provision of the inlet of medium and the outlet of
broth.

• Tubular fermenters: Continuous unstirred fermenters in which the reactants

move in a general direction. Reactants enter at one end and leave from the other and no
attempt is made to mix them.

• Due to the absence of mixing, there is a gradual fall in the substrate concentration
between the entry point and the outlet while there is an increase in the product in the
same direction.
Fermenter
Configurations

• The fluidized bed fermenter: It is intermediate in nature between

the stirred tank and the tubular fermenter.

• The microorganisms which are in a fluidized bed fermenter are kept in

suspension by a medium flow rate whose force just balances the gravitational
force.

• The tower fermenter for the brewing of beer and production of vinegar is an
example of a fluidized bed fermenter.
Fermenter
Configurations
Overview of Measurements and Control of
Fermentation Parameters
 Thanks for your
attention!
• Thank you for your
attention!!!