Pamantasan ng Lungsod ng Maynila

College of Engineering and Technology

Chemical Engineering Department

Basic Microbiology
and Biochemistry

Submitted by:

Crisnuel C. Ramirez

Submitted to:

Dr. Denvert Pangayao

September 4, 2019
 Pamantasan ng Lungsod ng Maynila
College of Engineering and Technology
Chemical Engineering Department

TABLE OF CONTENTS
Basic Microbiology 2

Classification of Microbes 6

Microbial Anatomy 9

Nutritional Requirements for Growth 23

Industrial Applications of Microorganisms 27

Basic Biochemistry 42

Lipids 43

Proteins and Amino Acids 48

Carbohydrates 54

Nucleic Acids and Nucleotides 61

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I. BASIC MICROBIOLOGY
Microbiology is the study of microorganisms, which are not only
microscopic and exist as single cells, but also those ultramicroscopic organisms
which are not cellular and hence cannot exist independently, e.g. viruses.
Microbiology deals with the study of functioning of cells, their diversity and
evolution, their interaction with environment, another living organisms and man.
Thus, it forms the basis for the complimentary development in cell biology,
biochemistry, molecular biology and genetics.

Microbiology has evolved as an important branch of science, and is

studied with respect to two major aspects:

• as a basic biological science

• as an applied biological science.

As a basic biological science, it provides a system to understand the nature

of life processes, the principles behind it, and the genetics which is involved in the
inheritance of traits to the next generation. Basic microbiology also consists of
various sub-sections as shown in Table 1.

Table 1 – Various Branches of Basic Microbiology

Branch Areas of coverage

Medical Study of pathogenic microorganisms that cause diseases, and
microbiology ways to eliminate
them.
Agricultural Study of plant diseases, understanding various beneficial
microbiology interactions with plant
systems, like soil fertility, crop-protection and increasing yield.
Environmental Study of relationship of microorganisms with its habitat;
microbiology pollution effect and its
impact on environment from the stand points of ecological
balance and health.
Food & Dairy Study of microorganisms that produce various food and dairy
microbiology products; study of
control of microorganisms in foods and transmission of food-
borne diseases.

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As an applied biological science, it deals with the study of useful

microorganisms as well as that of pathogenic organisms. The orientation of
applied microbiology is in several fields, and it uses the basic information and
techniques of different sub-divisions. Industrial microbiology is the study of
exploitation of the biochemical potential of microbes to produce various
products like antibiotics, vaccines, steroids, solvents, vitamins, leaching of
minerals, etc. It is also serving as a base for Genetic engineering which is an
upcoming branch of biotechnology and deals with improving the performance
of microorganisms.

HISTORIC BACKGROUND

One of the most important discoveries in the history of biology occurred in

the year 1665 with the help of studies using a relatively crude microscope. An
English scientist, Robert Hooke, reported small structural units called cell, by fruiting
structure of mold, and published a major work called Micrographie. But the first
person to see the microorganisms in some detail was Antoni van Leeuwenhoek
(1674) of Holland. He gave a reasonably detailed description of yeast cells in
brewing of beer; but did not realize that they were living organisms. It has taken
almost one and half centuries to realize the existence of the yeast cells which
were responsible for the production of beer. Hoogerheide (1977) noted that the
derailing of progress by at least a quarter century was mainly due to the then
powerful and scholarly chemists like Gay-Lussac, Berzelius, Wohler von Liebig,
Berthelot and Mitscherlich. Justis Liebig, in the year 1839, reported that all the living
activities, which were being reported till that time, were mainly due to chemical
and physical reactions in the medium.

Robert Hooke's writings awakened the learned Europe, and many other
scientists were quick to follow his lead. The discoveries by Hooke, Swammerdom
and other scientists showed that microscope was an important tool for unlocking
the secrets of the world of small creatures. Antoni van Leeuwenhoek, in the year
1674, was credited to be the first person ever to make a crude microscope. He
used to grind pieces of glass during his spare times into fine lenses by placing them
between two silver or brass plates riveted together and added an adjustable
device for holding tiny specimens and could magnify an object over 200 times.
Initially he used the lenses to inspect the quality of cloth and observed tiny
organisms which he called 'animal cules'. These studies opened the doors to a
completely new concept dealing with living things.

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However, microbiology as a subject did not develop until the 19th century,
despite the development of microscope, due to inefficient techniques for
studying microbes. It achieved significant importance in the second half of the
19th century with the theory of spontaneous generation. With the significant work
of Louis Pasteur, there was an explosion of discoveries in microbiology. Hence, the
period from 1857 to 1914 has been named as the 'golden age of microbiology'
(Table 2). During this period, there were many discoveries, which included the
study of pathogens and the role of immunity in the prevention and cure of
diseases, improved techniques for performing microscopy and culturing of
microorganisms. The need for a good microscope to have a vision of the
microorganisms was felt all the way.

MICROSCOPY

Microorganisms and their structural components are measured in smaller

units such as micrometers or microns (104 m), nanometers or millimicrons (109 m)
and angstroms (1010 m). The earliest report on the use of magnifying lens
(telescope) was that of Conrad Gesber in the year 1558. A Dutch spectacle
maker, Zaccharias Jonssen is credited with making the first compound
microscope, but in 1830, better microscopes were developed by Joseph Jackson
Lister. Various improvements were made on Lister's microscope that resulted in the
development of modern compound microscope. Table 3 shows some of the limits
of microscopic resolutions. Subsequent microscopic studies of live specimens
have revealed dramatic interactions between microbes.

Table 2 – Important Discoveries in the Golden Age of Microbiology

Year of discovery Name of the scientist Discovery

1857 Louis Pasteur Fermentation
1861 Louis Pasteur Disproved spontaneous
generation
1864 Louis Pasteur Pasteurization
1867 Joseph Lister A septic surgery
1870 Ernst Karl Abbe Condenser and Oil
immersion objective
1876 Robert Koch Germ theory of diseases

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1879 Neisser Neisseria gonorrhoeae

(organism)
1880 Louis Pasteur Immunization technique
1881 Robert Koch Pure cultures
1882 Robert Koch Microbacterium
tuberculae
1882 Walter Hess Agar (solid) media
1883 Robert Koch Vibro cholera
1884 Elic Metchnikoff Phagocytosis
1884 Christian Gram Gram staining procedure
1884 Escherich Escherichia coli
1887 Petri Petri dish
1890 Von Bering Diphtheria antitoxin
1890 Paul Ehrlich Theory of immunity
1892 Winogradsky Sulphur cycle
1898 Shiga Shigella dysenteriae
1910 Paul Ehrlich Syphilis

Table 3 – Limits of Microscopic Resolutions

Size range of specimen Type of specimen Operation range of

microscope
1 cm Unaided eye
1 mm Unaided eye
0.1 mm Most unicellular Light microscope
organisms,
algae, fungi, protozoa
100 —10 pm Most eukaryotic cells Light microscope
10—1 Most bacteria, Light
mitochondria microscope/Electron
microscope
I—100 nm Mycoplasma Light
microscope/Electron
microscope

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100—10 nm Viruses, ribosomes Light

microscope/Electron
microscope
10—1 nm Proteins, lipids Electron microscope
1_0.1 nm Small molecule Electron microscope
0.1 nm Atoms Electron microscope

A. CLASSIFICATION OF MICROBES
Taxonomy is the subject of classification biological entities. Earlier, all living
organisms were classified under two main kingdoms, namely, Plantae and
Animalia – by Swedish botanist Carolus Linneaus, in the 18th century, in his Systema
Nature. Later in 1886, German naturalist Earnst H. Haeckel separated
microorganisms from plant and animal kingdoms, and placed them under
Protista kingdom which included microorganisms, fungi, protozoa, and
microscopic algae. Many taxonomists opposed this, and there have constant
endeavors to revise this system of classification.

Finally, in 1969, Robert H. Whittaker proposed a five-kingdom classification

which received wide acceptance. According to this system, the five kingdoms
are:

➢ Monera, which include true bacteria and blue green algae.

➢ Protista, which include eukaryotes such as protozoa which are unicellular,
contain chlorophyll (plant like) and possess movement (animal like).
➢ Fungi, which include non-green eukaryotic organisms like slime molds,
bread molds, sac fungi. The composition of cell wall differed from that of
plants and had adjacent cells without separation (coenocytic condition),
and hence not considered as true multicellular organisms.
➢ Plantae, which include algae, all mosses, ferns, conifers, and flowering
plants.
➢ Animalia, sponges, worms, insects, and vertebrates.

The taxonomical classification of all the microorganisms is shown in Figure

1. The entire taxonomical classification of Figure 1 can be simplified to a form
shown in Figure 2.

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Figure 1 – Taxonomical Classification of Microogranisms

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Figure 2 – Simplified Classification of Universal Ancestor

Microbial Diversity

Evolutionary roots of cells form a basis for understanding the microbial

diversity (which requires an appreciation of the evolutionary roots of cells). The
evolution has shaped all life on earth which is responsible for structural and
functional diversity in cells. Microbial diversity can be seen in terms of variations in
cell size, morphology, metabolism, adaptation, growth, etc. Several evolutionary
branches occur in bacteria, as some of them are pathogenic, while many others
are found in soil, water, and animal digestive tract; some of them use light as
energy source, while others use organic and inorganic chemicals as fuel for
cellular metabolism. They are observed in oxic and anoxic environments. They
also thrive under extreme environmental conditions like hot springs, salty water
bodies, highly acidic or alkaline soils.

Microbial Nomenclature

In microbiology, the basic taxonomic unit is species which can be defined

as a collection of similar strains. The species concept gives a formal taxonomic
identity to the selected strains. Groups of species are collected into genera. They

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share major properties but differ by the presence or absence of a few

characteristics. Groups of genera are classified into families, families into orders,
and orders into division.

Following the binomial system of nomenclature, microorganisms are given

genus and species name, which are either Latin or Greek derivations of some
descriptive property appropriate for that species. For example, Bacillus subtilus or
Bacillus meaning 'rod shape', which indicates its slender nature; B. cereus
meaning 'waxen'; B. stearothermophilus meaning 'heat loving'; and B.
acidoceldarius meaning 'acid-thermal'. They are written in italics with the genus
name starting with capital letter and the species name with a small letter. The
rules for bacterial nomenclature are described in a publication called "The
International Code of Nomenclature of Bacteria" which spells the rules for naming
newly isolated organisms as new genera or new species (Lapage, et al, 1975).

There are many ways of grouping (classification) of prokaryotes. The

characteristics of taxonomic approach include morphology, Gram reaction ,
nutrition, cell wall chemistry, pigments, storage products, ability to use various
substrates, fermentation product, gaseous needs, temperature, pH requirement,
tolerances, sensitivity, pathogenicity, symbiotic relationships, immunological
characteristics and habitat.

Molecular taxonomy includes guanine and cytosine ratio (GC ratio) in

organisms, the extent of DNA: DNA hybridization, amino acid sequencing and
protein analysis. Bergey's Manual of Systematic Bacteriology is a compendium of
classical and molecular information of all recognized species of prokaryotes and
contains a number of dichotomous keys for identification.

B. MICROBIAL ANATOMY
Cells are composed of small molecules as well as macromolecules, and are
made up of four basic elements, viz. carbon, oxygen, hydrogen and nitrogen.
Apart from these elements, phosphorous, iron, sulfur, zinc, manganese, copper,
molybdenum and cobalt are also present. Water accounts for about 90% of the
weight of the cells. Of the macromolecules, proteins are most abundant by
weight (55%). The chemical compositions of a prokaryotic cell are given in Table
4. The dry weight of an actively growing E. coli cell ≈ 2.8 x 10-13 g.

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Table 4 – Chemical Composition of a Prokaryotic Cell

Molecule Percent of dry Molecules per Different kinds

weight cell
Total 96 24,610,000 ~2500
macromolecules
➢ Protein 55 2,350,000 ~1850
➢ Polysaccharide 5 4,300 2
➢ Lipid 9.1 22,000,000 4
➢ DNA 3.1 2.1 1
➢ RNA 20.5 255,500 ~600
Total Monomers 3.5 ~350
➢ Amino acids 0.5 ~100
and precursors
➢ Sugars and 2 ~50
precursors
➢ Nucleotides 0.5 ~200
and precursors
Inorganic ions 1 18
Total (%) 100
Most prokaryotes require an organic compound of some sort as their source
of carbon. After carbon, the most abundant element in cells is nitrogen (12% by
dry weight), which is a major constituent of proteins and nucleic acids. Nitrogen
is found in organic and inorganic forms, and is mostly assimilated as ammonia
(NH3), nitrates (N03) or N2.

Prokaryotic Cell

The word prokaryote means primitive nucleus in Greek, and has the following
features:

➢ the genetic material is not enclosed within membrane

➢ they lack membrane-bound organelles
➢ cell walls contain complex polysaccharide peptidoglycon
➢ they have a simple method of reproduction.

The prokaryotes include a vast heterogeneous group of very small

unicellular organisms which comprise eubacteria and archaeobacteria. Most

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prokaryotes range from 0.5 to 3gm in diameter and 2—8gm in length. They have
a volume of 10 12 ml per cell and may weigh approximately 10-12 g per cell. They
are variously shaped like cocci (meaning berry), spherical to oval, which may be
attached in a chain pattern, streptococci, or may be found as tetrads, or as a
bunch of grapes (groups of eight sarcinae), or may be rod-shaped bacillus
(meaning little staffs) or may be spiral. Curved rods are called vibrios and the
helical-shaped ones are called spiralla. Usually, the shapes are determined by
genetic factors, and most of them maintain single shape (monomorphic), but a
few undergo alteration, e.g. corny bacterium, rhizobium.

External Appendages

These appendages are present external to the cell wall, and include
glycocalyx, flagella, axial filaments, fimbriae and pili. They are described in the
following paragraphs.

Glycocalyx is a viscous gelatinous polymer composed of polysaccharide which

may be loosely attached (slime layer) or firmly attached (capsule).

Flagella (meaning whip) is a long filamentous appendage which helps in

movement and is variously arranged, viz. monotrichous (single polar flagellum),
amphitrichous (two or more at one end of cell), or peritrichous (around the cell).
It comprises of three basic parts:

1. Long outermost filament made of spherical protein flagellin arranged as

helix around a hollow core.
2. A hook which connects the external filament and basal body.
3. The basal body which anchors flagellum to cell wall and plasma
membrane.

Prokaryotic flagellum is simple compared to eukaryotes. It is semi-rigid and helps

in movement of cell similar to the shaft of an electric motor. The mobility enables
the bacterium to move towards favorable environment.

Axial filaments are composed of fibril bundles, which arise at the end of the cell
and beneath the outer sheath. They are mainly present in spirochetes, and the
rotation of these filaments produces spiral motion similar to corkscrew moving
through a cork.

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Fimbriae are usually present in gram -ve bacteria. These are short and thin
appendages, mainly useful for attachment rather than for motility. Fimbriae occur
at poles of bacteria or may be present around the cell. These appendages may
be few to several hundreds and are useful for colonization. Pili are usually longer
than fimbriae (1—2 per cell), and are made up of special proteins called pilin. Pili
help bacterial cells for transfer of DNA from one cell to another, and hence they
are occasionally referred to as sex pili.

Cell Wall

The bacterial cell wall is complex, semi-rigid and is responsible for

characteristic shape. It protects cell organelles from external environment and
helps in anchorage. It is credited for its taxonomic value for classification of major
types of bacteria.

The cell wall is composed of a macromolecular network called

peptidoglycan (murein) which consists of a repeated disaccharide (N-acetyl
glucosamine (NAG) and N-acetyl muramic acid (NAM)) attached by
polypeptides to form a lattice (Fig. 3). Alternating NAM and NAG molecules are
linked in rows of 10—65 sugars to form a carbohydrate backbone (glycon portion
of peptidoglycan). The adjacent rows are linked by polypeptides (peptide
portion of peptidoglycan). Although the structure of polypeptide link varies, it
always includes tetrapeptide side chains which consist of four amino acids
attached to NAMs in the backbone. The amino acids occur in alternating
patterns off) and L forms.

Certain primitive group of prokaryotes does not possess at all or possesses a vet"
little cell wall material, e.g. mycoplasma which are smallest bacteria. They possess
unique plasma membrane having lipids known as sterols, which help in prevention
of osmotic lysis. Archaebacteria lack or have unusual cell walls of polysaccharides
and proteins without peptidoglycan or contain pseudomurein which is similar to
peptidoglycan. Pseudomurein is made up of IV-acetyl alosaminuronic acid
instead of NAM and lacks D-amino acids. Gram +ve bacterial cell wall consists of
thick, rigid structure due to several peptidoglycan layers, and contains teichoic
acids (such as glycerol or ribitol and phosphate). Teichoic acid is mainly helpful in
the movement of cations across the membrane, prevents cell lysis, and provides
antigenic specificity. There are two classes of teichoic acids:

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1. lipoteichoic acid, which extends over the peptidoglycan layer and links
to the plasma membrane.
2. wall teichoic acid, which links to the peptidoglycan layer.

Figure 3 – Macromolecular Network of Peptidoglycan

Gram -ve cell wall contains a few layers of peptidoglycan and outer
membrane (which is made up of lipoproteins, lipo- saccharide and
phospholipids). The peptidoglycan is bonded to lipoproteins in outer membrane
and is in the periplasmic space (in between outer membrane and plasma
membrane). The periplasmic space contains a high concentration of
degradative enzymes and transport proteins. Absence ofteichoic acid and fewer
layers of peptidoglycan make the cell more susceptible to mechanical
breakage. The negative charge on the outer membrane acts as a defence
mechanism from the evading phagocytes.

Cytoplasmic Membrane

It is a thin phospholipid bilayer surrounding the cell (about 8nm thick) acting
as a barrier from external environment. It is a highly selective membrane enabling
the cell to concentrate specific metabolites and excrete waste. It primarily
consists of phospholipids and proteins and lacks sterols; and hence it is less rigid

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than eukaryotic organisms. The membrane has phospholipid bilayer and proteins
embedded in it with hydrophobic region towards inside. Hydrophilic region is
exposed to the external environment. The overall structure of the cytoplasmic
membrane is stabilized by hydrogen bonds and hydrophobic interactions, cations
like Ca2 and Mg ionic bonds with negatively charged phospholipids.

The outer surface of the membrane faces the environment. In certain

bacteria, it makes contact with a variety of proteins that serve to bind substrates,
or process large molecules for transport into cell. The inner side of the membrane
faces the cytoplasm and interacts with proteins involved in energy yielding
reactions. Although the cytoplasmic membrane appears rigid, it is fluid in nature,
and allows phospholipid and protein molecules to have freedom to move about
the membrane. The membrane has selective permeability and prevents
destruction by antimicrobial agents. It permits active and passive transport of
nutrients.

Cytoplasm

It is the polyphasic, colloidal, living system of cell, which is thick,

semitransparent and elastic in nature, and has higher water content. It contains
hydrophilic components (protein particles, carbohydrates and salts) and
hydrophobic components (globules of lipid or fats). The physical properties of
cytoplasm are

➢ sol-gel phase reversal during metabolic activities

➢ molecules form large aggregates due to cohesive forces
➢ absorption and excretion
➢ cytoplasm shows resistance towards flow, which makes the particles to be
in a state of suspension rather than settling down due to gravitational pull
➢ it exhibits Brownian movement.

Nuclear Area

Prokaryotes do not have nuclear envelope and do not show cytoplasmic

organelles like endoplasmic reticulum, Golgi complex, mitochondria and
lysosomes. The bacterial cell shows single chromosome in cytoplasm which
consists of single, circular, supercoiled naked DNA (i.e. not associated with
histones); this closed loop of DNA contains hereditary information of cell, and this
region is referred to as nucleoid.

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Smaller molecules of DNA exist apart from chromosome loops called as

plasmids, contain few genes which are not essential for growth and metabolism,
but confer special adaptive methods for better survival e.g. resistance factor (R-
factor) for drugs or extreme environmental conditions. Plasmids replicate
independent of cell DNA, and transfer from one cell to another by the
recombination process

Ribosomes are usually present in cytoplasm and are more active in protein-
synthesizing cells. They contain RNA and protein and are essential machinery for
protein synthesis. Prokaryotic ribosome (70S) comprises two subunits 50s and 30s.
50s subunit is spherical to dome-shaped in structure, and 30s subunit is ellipsoid to
prolate in structure.

The prokaryotic cytoplasm has many inclusions, commonly called as

inclusion bodies, which store nutrients for starvation periods. Some of these are as
follows:

➢ Metachromatic granules: These are large inclusions, stained with a dye like
methylene blue. They are also known as volutin granules. They are reservoirs
of inorganic phosphate, and used in the synthesis of ATP
➢ Polysaccharide granules: They are reservoirs of glycogen and starch
➢ Lipid inclusions/granules: They are reservoirs of mainly polymer poly B-
hydroxy butyric acid
➢ Sulfur granules: They are reservoirs of elemental sulfur
➢ Carboxysomes: They are polyhedral, hexagonal inclusions, and mostly
contain enzyme-ribulose, 5- diphosphate carboxylase.
➢ Gas vacuoles: These are hollow cavities in the cytoplasm, useful in
maintenance of buoyancy at depth in water, and allow cells to float up
and down in water
➢ Magnetosomes: They are intracellular crystal particles of iron mineral
magnetite Fe,04 responsible for magnetotaxis in cell

Endospores

Mostly Grams +ve bacteria produce highly resistant special structures

called endospores (mostly by members of genera Bacillus and Clostridium). These
organisms grow, mature and reproduce for several hours as vegetative cells.
Endospores contain a little amount of water and exhibit very few reactions. They
have a large amount of dipicolinic acid which stabilizes the proteins. The spore

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formation begins under unfavorable conditions. The bacterial chromosome

replicates, a small amount of cytoplasm gathers with it, and the membrane grows
to seal off the developing spore; then thick layers of peptidoglycan coat it and
vegetative cell degenerates. On return of favourable conditions, protective
layers break down, and spores germinate to vegetative cells. Thick layers of
peptidoglycan form and coat it and the vegetative cell disintegrates. As a result,
the spore is freed.

Eukaryotic Cell

The word eukaryote means "true nucleus". Eukaryotes consist of three main
components:

➢ cell membrane
➢ cytoplasm
➢ nucleus

Eukaryotic cells are larger and more complex than the prokaryotic cells. They
have the genetic material enclosed in a specialized membrane. They contain
distinct structures called cell organelles within cytoplasm (Fig. 5). Typical
eukaryotic cells range from 2 to 200 um in diameter

Cell Membrane

The general structure of most biological membranes consists of a

phospholipid bilayer; but the major difference in the chemical composition of
membranes of eukaryotes as compared to that of prokaryotes is that the
eukaryotic membranes have sterols. Five to twenty-five percent of the sterols
constitute the total lipids of eukaryotes. Sterols are rigid and planar molecules
which stabilize membrane structure. They are responsible for providing rigidity to
the membrane, which is essential to endure against physical stress in the absence
of cell wall. However, sterols can be easily destabilized by certain groups of
antibiotics like polyenes (which include filipin, nystatin, candicidin). Hence,
absence of sterols in the membrane of prokaryotes protects them.

The membrane thickness is generally of the order of 70-100 A and the

thickness of the lipid layer is 30A, while that of the protein layer is 25 A. The protein
part is responsible for wettability and flexibility. The important lipids in the

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phospholipid membrane are phospholipids, sphingolipids, and cholesterol

glyophosphaticles.

Figure 4 – A Typical Eukaryotic Cell

Cytoplasm

The cytoplasm is a complex, colloidal, viscous, semitransparent, jelly-like

substance and it is pervaded by a complex endomembrane system. The
endomembrane system consists of endoplasmic reticulum, Golgi complex and
nuclear envelope. This system is involved in secretion, processing, package, and
transport of substances within the cell. The cytoplasm present outside the
endomembrane system is called ground cytoplasm or cytoplasmic matrix proper.
The cytoplasm of eukaryotes includes a number of organelles which are absent
in prokaryotes, such as endoplasmic reticulum, Golgi complex, mitochondria,
lysosomes, peroxysomes, glyoxysomes, microtubules and true nucleus

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➢ Endoplasmic Reticulum

Endoplasmic reticulum gives mechanical support to the cytoplasm and

acts as a circulatory system. It is an extensive complex of vesicles and tubules of
vacuolar nature, and it connects plasma and nuclear membrane. It is of two
types:

a) rough endoplasmic reticulum (associated with ribosomes

b) smooth endoplasmic reticulum (without ribosomes).

Rough endoplasmic reticulum present in cells which actively synthesize proteins

and represents an intricate arrangement of membrane-bound spaces in the form
of channels with distinct inside and outside paths. Thus, the division of cytoplasm
into compartments allows no interference of different metabolic reactions.
Numerous enzymatic proteins are associated with endoplasmic reticulum
membrane which provides increased surface area for biochemical reactions.
Ionic gradients and electrical potentials help in conduction of impulses within the
cell

➢ Golgi Complex

The main function of Golgi complex is storage and absorption; it stores

mainly proteins, lipids, hormones, enzymes, vitamins, gums, mucilages, and
hemicellulose. Iron, silver and copper accumulate in Golgi membrane. Golgi
complex participates in recycling of plasma membrane, helps to concentrate
secretory materials and packs them into secretory residues, and mainly secretes
proteins, lipids, pectin, cellulose steroids, etc.

Several units of Golgi complex are scattered in cytoplasm and each unit is
called dictyosome. Each dictyosome consists of cisternae, clusters of tubules,
vesicles and large vacuoles. Cisternae or lamellae are flattened fluid-filled sacs,
curved long ( 20-30 nm wide), arranged parallelly, bound to a single membrane
and are connected to smooth endoplasmic reticulum. Vesicles are small drop like
structures of about 60 nm diameter, associated with convex forming face of
cisternae. Golgi vesicles form cell plate during cytokinesis and play an important
role in metabolic activities of cell. High concentration of enzymes like acid
phosphatase are transferred from Golgi complex to lysosomes. Vacuoles are
rounded sacs associated with maturing face of cisternae

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➢ Lysosomes

They are single membrane bound, spherical structures (0.4um) containing

hydrolytic enzymes. They enclose fine granular fluid, rich in digestive enzymes and
originate from tubules of endoplasmic reticulum or from cisternae of Golgi
complex. They release, due to external forces like freezing, thawing, blending or
UV radiations, acid phosphates, ribonuclease, cathepsin, collagenase α-
glucosidase, β-glucosidase, α-mannosidase, sulphatase, etc.

Lysosomes digest larger food molecules which enter the cell (heterophagy) or
digest stored food materials within the cell (autophagy); they also digest
damaged or ageing cells by releasing hydrolytic enzymes (autolysis). They even
digest the extracellular materials by releasing hydrolytic enzymes outside the cell

➢ Ribosomes

They are ribonucleoprotein particles of cytoplasm associated with protein

synthesis. They are basophilic granules (mostly present on the endoplasmic
reticulum) with a spheroidal structure of about 23 nm and consist of two unequal
subunits. Their size is measured by the speed with which they sediment in
centrifuge and is given by Swedberg unit.

Ribosomes occur in all organisms including PPLO (Pleuro Pneumonia like

organisms-mycoplasma). The eukaryotic 80s ribosome consists of 60s and 40s
subunits, and they assemble into ribosome only during protein synthesis. Ribosome
provides the surface area for the synthesis of protein, and guide multiple
interactions involved in protein synthesis.

➢ Mitochondria

They are granular or filamentous cytoplasm organelles and are found in the
form of minute globules, vesicles, threads and rods. Their number is more in
actively metabolizing cell and is about 7 Hm length and 0.5 um in diameter. It is
surrounded by two membranes:

(a) outer membrane (6 nm thick, smooth and lipoproteinaceous) and (b)

inner membrane (6 nm thick, lipoproteinaceous and projects into mitochondrial
matrix in the form of fine finger-like foldings called 'mitochondrial crests' or
'cristac'). This increases the surface area for biochemical reactions. The inner

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surface of cristac contains oxysomes or F1 particles which play an important role

in electron transport and hydrogen transfer systems

Mitochondria is made up of proteins (65-75%), lipids (25-30%), RNA, DNA,

etc.; the proteins constitute 70% isoenzymes and 30 % structural proteins, whereas
lipids constitute 90% phospholipids and 10% cholesterol. Due to the presence of
DNA and RNA, mitochondria are regarded as self-duplicating organelles. The F1
particle is about 8.5 nm long and has basal piece, stalk piece, and head piece.
The head piece consists of a special enzyme called ATP synthetase which
catalyzes synthesis of ATP from ADP and Pi

Mitochondria are involved in the oxidation of food materials, and the so-
obtained potential energy is converted into kinetic energy. They are the centers
for production of ATP, and hence they are called as 'powerhouses' of the cell

➢ Plastids

Plastids are special cytoplasmic organelles present mostly in eucaryotic

plant cells, and are classified into the following

(a) Leucoplasts
(b) Chloroplasts
(c) Chromoplasts

(a) Leucoplasts: They are colorless plastids, which lack ribosomes and thylakoids,
and their main function is storage. Depending on the type of food stored, they
are further classified into:

⚫ AMYLOPLASTS, which store starch

⚫ ELAIOPLASTS, which store oils and fats (also called as oleosomes)
⚫ PROTEOPLASTS, which store protein

(b) Chloroplast: They are green plastids and are mainly present in photosynthetic
organisms. A chloroplast mainly shows three main components:

⚫ Outer envelope containing two membranes, each of 4-6 nm thickness,

lipoproteinaceous materials and a small amount of xanthophylls.
⚫ Stroma' is a heterogeneous proteinaceous matrix, colourless, gel-like with
70s ribosomes, RNA, highly twisted circular DNA, starch grains, granules and
enzymes of Calvin cycle.

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Chloroplast is a self-duplicating organelle (like mitochondria) due to the presence

of DNA and RNA. It is the seat of photosynthesis which is two phased; light phase
takes place in grana and dark phase in stroma.

(c) Chromoplast: It is a colored plastid (other than green), usually containing

yellow (xanthophyll) or orange (carotene) pigments. It is variously shaped as
needle of rhomboid. Chromoplast of red algae contains phycocyanin,
phycoerythrin (phycobilins), while those of brown algae contain fucoxanthin (a
type of xanthophyll).

➢ Peroxisomes

They are small, oval-shaped, membrane-bound structures of cytoplasm

containing oxidizing enzymes (like peroxidases, catalases or oxidases) in fine
granular matrix. They carry out photorespiration and protect the cell from toxic
effects of hydrogen peroxide, which is produced as a byproduct within the cell.
They are involved in "-oxidation of fatty acids and initial steps of phospholipid
synthesis.

➢ Glyoxysomes

They are small, single membrane-bound and oval-shaped (0.6 pm),

enclosing an amorphous protein matrix containing enzymes of glyoxylate cycle.
They are involved in degradation of fats to carbohydrates via glyoxylate cycle.

➢ Spherosomes

They are small (2.5 pm), single membrane-bound organelle associated with
synthesis and storage of lipids.

➢ Cytoskeleton

The cytoplasmic matrix proper shows cytoskeleton formed by the following:

a. Microtubules: They are 25 μm in diameter and several microns long. They

occur freely in most eukaryotic cells, made up of special protein called
tubulin. They provide mechanical support to cytoplasm, play an
important role in cell division and are associated with intracellular
transport. During cell division, they form spindle fibers and help in
contraction and movement of chromosomes.

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b. Microfilament: It is thin (5—7nm wide), just beneath the cell membrane,

made up of contractile proteins (like actin and myocin), and helps in
cytoplasmic streaming.
c. Intermediate filaments: They are intermediate in size between
microtubules and microfilaments (10 nm thick), form a cage around the
nucleus, and connect the nucleus with the cell surface. They play an
important role in shaping a cell compartmentation within the cell.
➢ Vacuole

It is a cavity of cytoplasm filled with a watery fluid called cell sap and
surrounded by a membrane called tonoplast. The cell sap contains 98% water
and 2% secretory products, stored products, excretory products, which include
organic materials like carbohydrates, proteins, alkaloids, glucosides, tannins, etc.
inorganic substances such as sulphates, phosphates, nitrates, carbonates,
oxalates, etc., and sometimes pigments also. Vacuoles regulate the process of
osmosis, maintain turbidity of cells, and store water, food and byproducts (toxic
sometimes) of metabolism.

Nucleus

It is a highly specialized structure of protoplasm. It is the dynamic center of

the cell, and the site of storage and replication of hereditary materials. The
position and number of nuclei vary from organism to organism.
For example:

a. Acetabularia is located at the basal region. Meristematic cells are

centrally located. Many cells are peripherally located.
b. Most of the cells have single nucleus called as uninucleate. Ascogenous
hyphal cells and secondary mycellium of Basidiomyceles are binucleate.
Rhizopus is multinucleate.

Nucleus shows four main parts:

1. nuclear envelope
2. nucleoplasm
3. chromatin
4. nucleolus

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1. Nuclear envelope: It consists of two concentric membranes, called outer

and inner membranes, with perinuclear space in between. The outer
surface is rough and stuffed with ribosomes, and the inner membrane is
smooth and a layer of fibrous protein called nuclear lamina is attached to
it. The nuclear lamina helps in disassemblage and reassemblage of nuclear
envelope during cell division. Nuclear envelope has a number of pores
where the two membranes fuse together; the pore is not a wide open
channel but plugged with protein to form annulus which allows transport
between nucleoplasm and cytoplasm.
2. Nucleoplasm: It is a semi-fluid substance in which glycoproteins,
ribonucleoproteins, and hydrolytic enzymes are suspended, and has
dispersed chromatin.
3. Chromatin: It is a viscous gelatinous substance, made out of DNA, RNA,
histone proteins and non-histone proteins. It is formed by repeating units
called nucleosome, which is nothing but tightly coiled DNA along with
histone proteins.
4. Nucleolus: It has deeply stained, prominent body consisting of central
fibrillar (made up of 5—10 nm fibrals) and peripheral granular zone
(granules of 10—20 nm in diameter which are precursors of ribosomes). It is
composed of RNA, DNA and proteins. It disappears during prophase and
reappears at telophase stage of cell division.

Centrosome

They are present in lower plants, algae and fungi. They are minute inclusions near
nucleus and have deeply stained granules called centriole, which is embedded
in a mass of hyaline substance (called centrosphere). Centrosome is involved in
formation of bipolar spindle during cell division, and also in the formation of cilia.

C. NUTRITIONAL REQUIREMENTS FOR GROWTH

Nutritional studies revealed that organisms can assimilate various organic
compounds, and use them to make new cell wall material, which may be amino
acids, fatty acids, organic acids, sugars, nitrogen bases and aromatic
compounds. Other macronutrients that are required are:

➢ Phosphorous, which is required by the cell during nucleic acid synthesis and
phospholipids

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➢ Sulfur, which has a structural role in amino acids like methionine, cysteine,
and vitamins like thiamine, biotin, lipoic acid and co-enzyme A
➢ Potassium, which is involved in the protein synthesis and for proper
functioning of enzymes
➢ Magnesium, which stabilizes ribosomes, cell membrane, nucleic acid and
is required for the activity of enzymes
➢ Calcium, which is essential for growth, stabilizes cell walls and is responsible
for the stability of endospores.

Table 5 – Macronutrients in Nature and Culture Media

Element Usual form of nutrient Chemical form supplied in

found in the environment culture media
Carbon (C) CO2 organic compounds Glucose, malate, acetate,
pyruvate, etc. or
in the form of yeast extract,
peptone, etc.
Hydrogen (H) H2O organic compounds H2O organic compounds
Oxygen (O) H2O, O2 organic H2O, O2 organic compounds
compounds
Nitrogen (N) NH3, NO3, N2 organic Inorganic: NH4Cl, (NH4)2SO4,
nitrogen compounds KNO3, N2
Organic: Amino acids, nitrogen
bases of nucleotides, many
other N-containing organic
compounds
Phosphorus (P) PO4 3- KH2PO4, Na2HPO4
Sulphur (S) H2S, SO4 , organic sulfur Na2SO4,
2- Na2S2O3, Na2S,
compounds, metal sulfides cysteine, or other organic sulfur
(FeS, CuS, ZnS, NiS, and so compounds
on)
Potassium (K) K+ in solution or as various K KCl, KH2PO4
salts
Magnesium Mg2+ in solution or as MgCl2, MgSO4
(Mg) various Mg salts
Sodium (Na) Na+ in solution or as NaCl, NaCl
or other Na salts

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Calcium (Ca) Ca2+ in solution or as CaCl2

CaSO4, or other Ca salts
Iron (Fe) Fe2+ or Fe3+ in solution or as FeCl3, FeSO4, various chelated
FeS, Fe(OH)3, or many Fe iron solutions (Fe3+ EDTA, Fe3+
salts citrate, and so on)

Various micronutrients are also needed by living organisms and are

tabulated in Table 6.

Table 6 – Micronutrients Required by Cells

Element Cellular function

Chromium (Cr) Required by mammals for glucose metabolism; no known
microbial requirement
Cobalt (Co) Vitamin B 12; transcarboxylase (propionic acid bacteria)
Copper (Cu) Certain proteins, notably those involved in respiration (for
example, cytochrome
c oxidase), or in photosynthesis (for example, plastocyanin)
some superoxide dismutases
Manganese (Mn) Activator of many co-enzymes; present in certain
superoxide dismutases and in water-splitting
enzyme of photosystem Il in oxygenic phototrophus.
Molybdenum Present in various flavin-containing enzymes; also in
(MO) molybdenum nitrogenase, nitrate reductase,
sulphite oxidase, DMSO-TMAO reductases, some formate
dehydrogenase, oxotransferases
Nickel (Ni) Most hydrogenases; co-enzyme F of methanogens; carbon
monoxide dehydrogenase; urease
Selenium (Se) Formate dehydrogenase: some hydrogenases; the amino
acid selenocysteine
Tungsten (W) Some formate dehydrogenases; oxotransferases of
hyperthermophiles (for example,
aldehyde: ferridoxin oxidoreductase of Pymcoccus
furiosus)

Vanadium (V) Vanadium nitrogenase; bromoperoxidase

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Zinc (Zn) Present in the enzymes carbonic anhydrase, alcohol

dehydrogenase, RNA and DNA
polymerases, and many DNA-binding proteins
Iron (Fe) Cytochromes, catalases, peroxidases, iron—sulfur proteins
(for example, ferredoxin), oxygenases, all nitrogenases

Growth factors

They are the organic compounds required in small amounts for growth and
metabolism, and these include vitamins, amino acids, purines and pyramidines.
Vitamins are not only needed for growth, but also play an important role as co-
enzymes. Vitamins and their functions are tabulated in Table 7. Most of the
vitamins are synthesized by organisms themselves, but some of them depend on
external sources.

Table 7 – Vitamins and their Functions

Vitamin Function
P-Aminobenzoic acid Precursor of folic acid
Folic acid One-carbon metabolism; methyl group transfer
Biotin Fatty acid biosynthesis; ß-decarboxylations; C02
fixation
Cobalamin (B 12) Reduction of and transfer of single-carbon fragments;
synthesis of deoxyribose
Lipoic acid Transfer of acyl groups in decarboxylation of pyruvate
and a-ketoglutarate
Nicotinic acid (niacin) Precursor of NAD+; electron transfer in oxidation-
reduction reactions
Pantothenic acid Precursor of co-enzyme A; activation of acetyl and
other acyl derivatives
Riboflavin Precursor of FMN, FAD in flavoproteins involved in
electron transport
Thiamine α-Decarboxylations ; transketolase
Vitamins (B6) Amino acid and keto acid transformations
(pyridoxal-
pyridoxamine group)

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Vitamin K group; Electron transport; synthesis of sphingolipids

quinones
Vitamin K group; Iron-binding compounds; solubilization of iron and
quinones transport into cell
Hydroxamates Required by certain methanogens; plays a role in
methanogenesis

D. INDUSTRIAL APPLICATIONS OF MICROORGANISMS

Microorganisms have been identified and exploited for more than a
century. The Babylonians and Sumerians used yeast to prepare alcohol. There is
a great history beyond fermentation processes, which explains the applications
of microbial processes that resulted in the production of food and beverages. In
the mid-nineteenth century, Louis Pasteur understood the role of microorganisms
in fermented food, wine, alcohols, beverages, cheese, milk, yoghurt and other
dairy products, fuels, and fine chemical industries. He identified many microbial
processes and discovered the first principal role of fermentation, which was that
microbes required substrate to produce primary and secondary metabolites, and
end products.

In the new millennium, extensive application of bioprocesses has created

an environment for many engineers to expand the field of biotechnology. One of
the useful applications of biotechnology is the use of microorganisms to produce
alcohols and acetone, which are used in the industrial processes. The knowledge
related to industrial microbiology has been revolutionized by the ability of
genetically engineered cells to make many new products. Genetic engineering
and gene mounting have been developed in the enhancement of industrial
fermentation. Consequently, biotechnology is a new approach to making
commercial products by using living organisms. Furthermore, knowledge of
bioprocesses has been developed to deliver fine-quality products.

Application of biological sciences in industrial processes is known as

bioprocessing. Nowadays most biological and pharmaceutical products are
produced in well-defined industrial bioprocesses. For instance, bacteria can
produce most amino acids that can be used in food and medicine. There are
hundreds of microbial and fungal products purely available in the biotechnology
market. Microbial production of amino acids can be used to produce L-isomers;

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chemical production results in both D- and L-isomers. Lysine and glutamic acid
are produced by Corynebacterium glutamicum. Another food additive is citric
acid, which is produced by Aspergillus niger. Table 8 summarises several
widespread applications of industrial microbiology to deliver a variety of products
in applied industries. The growth of cells on a large scale is called industrial
fermentation. Industrial fermentation is normally performed in a bioreactor, which
controls aeration, pH and temperature. Microorganisms utilise an organic source
and produce primary metabolites such as ethanol,

Table 8 – Industrial Products Produced by Biological Processes

which are formed during the cells’ exponential growth phase. In some
bioprocesses, yeast or fungi are used to produce advanced valuable products.
Those products are considered as secondary metabolites, such as penicillin,
which is produced during the stationary phase. Yeasts are grown for wine- and
bread-making. There are other microbes, such as Rhizobium, Bradyrhizobium and

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Bacillus thuringiensis, which are able to grow and utilise carbohydrates and
organic sources originating from agricultural wastes. Vaccines, antibiotics and
steroids are also products of microbial growth.

PROCESS FERMENTATION

The term ‘fermentation’ was obtained from the Latin verb ‘fervere’, which
describes the action of yeast or malt on sugar or fruit extracts and grain. The
‘boiling’ is due to the production of carbon dioxide bubbles from the aqueous
phase under the anaerobic catabolism of carbohydrates in the fermentation
media. The art of fermentation is defined as the chemical transformation of
organic compounds with the aid of enzymes. The ability of yeast to make alcohol
was known to the Babylonians and Sumerians before 6000 BC. The Egyptians
discovered the generation of carbon dioxide by brewer’s yeast in the preparation
of bread. The degradation of carbohydrates by microorganisms is followed by
glycolytic or Embden–Myerhof–Parnas pathways. Therefore, the overall
biochemical reaction mechanisms to extract energy and form products under
anaerobic conditions are called fermentation processes. In the process of
ethanol production, carbohydrates are reduced to pyruvate with the aid of
nicotinamide adenine dinucleotide (NADH); ethanol is the end product. Other
fermentation processes include the cultivation of acetic acid bacteria for the
production of vinegar. Lactic acid bacteria preserve milk; the products are
yoghurt and cheese. Various bacteria and mold are involved in the production
of cheese. Louis Pasteur, who is known as the father of the fermentation process,
in early nineteenth century defined fermentation as life without air. He proved
that existing microbial life came from preexisting life. There was a strong belief that
fermentation was strictly a biochemical reaction. Pasteur disproved the chemical
hypothesis. In 1876, he had been called by distillers of Lille in France to investigate
why the content of their fermentation product turned sour.3 Pasteur found under
his microscope the microbial contamination of yeast broth. He discovered
organic acid formation such as lactic acid before ethanol fermentation. His
greatest contribution was to establish different types of fermentation by specific
microorganisms, enabling work on pure cultures to obtain pure product. In other
words, fermentation is known as a process with the existence of strictly anaerobic
life: that is, life in the absence of oxygen. The process is summarised in the
following steps:

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• Action of yeast on extracts of fruit juice or, malted grain. The biochemical
reactions are related to generation of energy by catabolism of organic
compounds.
• Biomass or mass of living matter, living cells in a liquid solution with essential
nutrients at suitable temperature and pH leads to cell growth. As a result,
the content of biomass increases with time.

In World War I, Germany was desperate to manufacture explosives, and

glycerol was needed for this. They had identified glycerol in alcohol fermentation.
Neuberg discovered that the addition of sodium bisulphate in the fermentation
broth favored glycerol production with the utilization of ethanol. Germany quickly
developed industrial-scale fermentation, with production capacity of about 35
tons per day.3 In Great Britain, acetone was in great demand; it was obtained by
anaerobic fermentation of acetone–butanol using Clostridium acetobutylicum.
In large-scale fermentation production, contamination was major problem.
Microorganisms are capable of a wide range of metabolic reactions, using
various sources of nutrients. That makes fermentation processes suitable for
industrial applications with inexpensive nutrients.

Molasses, corn syrup, waste products from crystallisation of sugar industries

and the wet milling of corn are valuable broth for production of antibiotics and
fine chemicals. We will discuss many industrial fermentation processes in the
coming chapters. It is best to focus first on the fundamental concepts of
biochemical engineering rather than the applications. There are various industries
using biological processes to produce new products, such as antibiotics,
chemicals, alcohols, lipid, fatty acids and proteins.

Deep understanding of bioprocessing may require actual knowledge of

biology and microbiology in the applications of the above processes. It is very
interesting to demonstrate bench-scale experiments and make use of large-scale
advanced technology. However, application of the bioprocess in large-scale
control of microorganisms in 100,000 liters of media may not be quite so simple to
manage. Therefore, trained engineers are essential and highly in demand; this
can be achieved by knowledge enhancement in the sheathe bioprocesses. To
achieve such objectives, we may need to explain the whole process to the skilled
labor and trained staff to implement bioprocess knowhow in biotechnology.

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APPLICATION OF FERMENTATION PROCESSES

Man has been using the fermentative abilities of microorganisms in various

forms for many centuries. Yeasts were first used to make bread; later, use
expanded to the fermentation of dairy products to make cheese and yoghurt.
Nowadays more than 200 types of fermented food product are available in the
market. There are several biological processes actively used in the industry, with
high-quality products such as various antibiotics, organic acids, glutamic acid,
citric acid, acetic acid, butyric and propionic acids.

Synthesis of proteins and amino acids, lipids and fatty acids, simple sugar
and polysaccharides such as xanthan gum, glycerol, many more fine chemicals
and alcohols are produced by bioprocesses with suitable industrial applications.
The knowledge of bioprocessing is an integration of biochemistry, microbiology
and engineering science applied in industrial technology. Application of viable
microorganisms and cultured tissue cells in an industrial process to produce
specific products is known as bioprocessing. Thus fermentation products and the
ability to cultivate large amounts of organisms are the focus of bioprocessing, and
such achievements may be obtained by using vessels known as fermenters or
bioreactors.

The cultivation of large amounts of organisms in vessels such as fermenters

and bioreactors with related fermentation products is the major focus of
bioprocess. A bioreactor is a vessel in which an organism is cultivated and grown
in a controlled manner to form the by-product. In some cases specialised
organisms are cultivated to produce very specific products such as antibiotics.
The laboratory scale of a bioreactor is in the range 2–100 litres, but in commercial
processes or in large-scale operation this may be up to 100 m3 . 4,5 Initially the
term ‘fermenter’ was used to describe these vessels, but in strict terms
fermentation is an anaerobic process whereas the major proportion of fermenter
uses aerobic conditions. The term ‘bioreactor’ has been introduced to describe
fermentation vessels for growing the microorganisms under aerobic or anaerobic
conditions.

Bioprocess plants are an essential part of food, fine chemical and

pharmaceutical industries. Use of microorganisms to transform biological
materials for production of fermented foods, cheese and chemicals has its
antiquity. Bioprocesses have been developed for an enormous range of

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commercial products, as listed in Table 8. Most of the products originate from

relatively cheap raw materials. Production of industrial alcohols and organic
solvents is mostly originated from cheap feed stocks. The more expensive and
special bioprocesses are in the production of antibiotics, monoclonal antibodies
and vaccines. Industrial enzymes and living cells such as baker’s yeast and
brewer’s yeast are also commercial products obtained from bioprocess plants.

BIOPROCESS PRODUCTS

Major bioprocess products are in the area of chemicals, pharmaceuticals,

energy, food and agriculture, as depicted in Table 9. The table shows the general
aspects, benefits and application of biological processes in these fields. Most
fermented products are formed into three types. The main categories are now
discussed.

1. Biomass

The aim is to produce biomass or a mass of cells such as microbes, yeast

and fungi. The commercial production of biomass has been seen in the
production of baker’s yeast, which is used in the baking industry. Production of
single cell protein (SCP) is used as biomass enriched in protein.6 An algae called
Spirulina has been used for animal food in some countries. SCP is used as a food
source from renewable sources such as whey, cellulose, starch, molasses and a
wide range of plant waste.

2. Cell Products

Products are produced by cells, with the aid of enzymes and metabolites
known as cell products. These products are categorised as either extracellular or
intracellular. Enzymes are one of the major cell products used in industry. Enzymes
are extracted from plants and animals. Microbial enzymes, on the other hand,
can be produced in large quantities by conventional techniques. Enzyme
productivity can be improved by mutation, selection and perhaps by genetic
manipulation. The use of enzymes in industry is very extensive in baking, cereal
making, coffee, candy, chocolate, corn syrup, dairy product, fruit juice and
beverages. The most common enzymes used in the food industries are amylase
in baking, protease and amylase in beef product, pectinase and hemicellulase
in coffee, catalase, lactase and protease in dairy products, and glucose oxidase
in fruit juice.

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Table 9 – Products and Services by Biological Processes

3. Modified Compounds (Biotransformation)

Almost all types of cell can be used to convert an added compound into
another compound, involving many forms of enzymatic reaction including
dehydration, oxidation, hydroxylation, amination, isomerisation, etc. These types
of conversion have advantages over chemical processes in that the reaction can
be very specific and produced at moderate temperatures. Examples of
transformations using enzymes include the production of steroids, conversion of
antibiotics and prostaglandins. Industrial transformation requires the production
of large quantities of enzyme, but the half-life of enzymes can be improved by
immobilisation and extraction simplified by the use of whole cells. In any

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bioprocess, the bioreactor is not an isolated unit, but is as part of an integrated

process with upstream and downstream components. The upstream consists of
storage tanks, growth and media preparation, followed by sterilisation. Also, seed
culture for inoculation is required upstream, with sterilised raw material, mainly
sugar and nutrients, required for the bioreactor to operate. The sterilisation of the
bioreactor can be done by steam at 15 pounds per square inch guage (psig),
121 °C or any disinfectant chemical reagent such as ethylene oxide. The
downstream processing involves extraction of the product and purification as
normal chemical units of operation. The solids are separated from the liquid, and
the solution and supernatant from separation unit may go further for purification
after the product has been concentrated.

PRODUCTION OF LACTIC ACID

Several carbohydrates such as corn and potato starch, molasses and whey
can be used to produce lactic acid. Starch must first be hydrolysed to glucose by
enzymatic hydrolysis; then fermentation is performed in the second stage. The
choice of carbohydrate material depends upon its availability, and pretreatment
is required before fermentation. We shall describe the bioprocess for the
production of lactic acid from whey. Large quantities of whey constitute a waste
product in the manufacture of dairy products such as cheese. From the
standpoint of environmental pollution it is considered a major problem, and
disposal of untreated wastes may create environmental disasters. It is desirable to
use whey to make some more useful product.

Whey can be converted from being a waste product to something more

desirable that can be used for the growth of certain bacteria, because it contains
lactose, nitrogenous substances, vitamins and salts. Organisms can utilise lactose
and grow on cheese wastes; the most suitable of them are Lactobacillus species
such as Lactobacillus bulgaricus, which is the most suitable species for whey. This
organism grows rapidly, is homofermentative and thus capable of converting
lactose to the single end-product of lactic acid. Stock cultures of the organism
are maintained in skimmed milk medium.

The 3–5% of inoculum is prepared and transferred to the main bioreactor,

and the culture is stored in pasteurized, skimmed milk at an incubation
temperature of 43 °C. During fermentation, pH is controlled by the addition of
slurry of lime to neutralize the product to prevent any product inhibition. The

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accumulation of lactic acid would retard the fermentation process because of

the formation of calcium lactate. After 2 days of complete incubation, the
material is boiled to coagulate the protein, and then filtered. The solid filter cake
is a useful, enriched protein product, which may be used as an animal feed
supplement. The filtrate containing calcium lactate is then concentrated by
removing water under vacuum, followed by purification of the final product. The
flow diagram for this process is shown in Figure 5.

Figure 5 – Production of Lactic Acid from Whey

PRODUCTION OF VINEGAR

The sugars in fruits such as grapes are fermented by yeasts to produce

wines. In winemaking, lactic acid bacteria convert malic acid into lactic acid in
malolactic fermentation in fruits with high acidity. Acetobacter and
Gluconobacter oxidise ethanol in wine to acetic acid (vinegar). The word ‘wine’
is derived from the French term ‘vinaigre’ meaning ‘sour wine’. It is prepared by

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allowing a wine to get sour under controlled conditions. The production of vinegar
involves two steps of biochemical changes: (1) Alcoholic fermentation in
fermentation of a carbohydrate. (2) Oxidation of the alcohol to acetic acid. There
are several kinds of vinegar. The differences between them are primarily
associated with the kind of material used in the alcoholic fermentation, e.g. fruit
juices, sugar and hydrolysed starchy materials. Based on US Department of
Agriculture (USDA) definitions, there are a few types of vinegar: vinegar, cider
vinegar, apple vinegar. The products are made by the alcoholic and subsequent
acetous fermentations of the apple juice. The acetic acid content is about 5%.
Yeast fermentation is used for the production of alcohol. The alcohol is adjusted
to 10–13%, then it is exposed to acetic acid bacteria (Acetobacter species),
whereby oxygen is required for the oxidation of alcohol to acetic acid. The
desired temperature for Acetobacter is 15–34 °C. The reaction is:

PRODUCTION OF AMINO ACIDS (LYSINE AND GLUTAMIC ACID) AND INSULIN

Many microorganisms can synthesize amino acids from inorganic nitrogen

compounds. The rate and amount of some amino acids may exceed the cells’
need for protein synthesis, where the excess amino acids are excreted into the
media. Some microorganisms can produce certain amino acids such as lysine,
glutamic acid and tryptophan.

1. Stepwise Amino Acid Production

One of the commercial methods for production of lysine consists of a two-stage

process using two species of bacteria. The carbon sources for production of
amino acids are corn, potato starch, molasses, and whey. If starch is used, it must
be hydrolysed to glucose to achieve higher yield. Escherichia coli is grown in a
medium consisting of glycerol, cornsteep liquor and di-ammonium phosphate
under aerobic conditions, with temperature and pH controlled.

• Step 1: Formation of diaminopimelic acid (DAP) by E. coli.

• Step 2: Decarboxylation of DAP by Enterobacter aerogenes.

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E. coli can easily grow on corn steep liquor with phosphate buffer for an
incubation period of 3 days. Lysine is an essential amino acid for the nutrition of
humans, which is used as a supplementary food with bread and other foodstuffs.
This amino acid is a biological product which is also used as a food additive and
cereal protein. Many species of microorganisms, especially bacteria and fungi,
are capable of producing large amounts of glutamic acid. Glutamic acid is
produced by microbial metabolites of Micrococcus, Arthrobacter, and
Brevibacterium by the Krebs cycle. Monosodium glutamate is known as a flavour-
enhancing amino acid in food industries.

The medium generally used consists of carbohydrate, peptone, inorganic

salts and biotin. The concentration of biotin has a significant influence on the yield
of glutamic acid. The -ketoglutaric acid is an intermediate in the Krebs cycle and
is the precursor of glutamic acid. The conversion of -ketoglutaric acid to glutamic
acid is accomplished in the presence of glutamic acid dehydrogenase,
ammonia and nicotinamide adenine dinucleotide dehydrogenase (NADH2). The
living cells assimilate nitrogen by incorporating it into ketoglutaric acid, then to
glutamic acid and glutamine. Therefore glutamic acid is formed by the reaction
between ammonia and -ketoglutaric acid in one of the tricarboxylic acid (TCA)
cycle or Krebs cycle intermediates.

2. Insulin

Insulin is one of the important pharmaceutical products produced commercially

by genetically engineered bacteria. Before this development, commercial insulin
was isolated from animal pancreatic tissue. Microbial insulin has been available
since 1982. The human insulin gene is introduced into a bacterium like E. coli. Two
of the major advantages of insulin production by microorganisms are that the
resultant insulin is chemically identical to human insulin, and it can be produced
in unlimited quantities.

ANTIBIOTICS, PRODUCTION OF PENICILLIN

The commercial production of penicillin and other antibiotics are the most
dramatic in industrial microbiology. The annual production of bulk penicillin is
about 33 thousand metric tonnes with annual sales market of more than US$400
million. The worldwide bulk sales of the four most important groups of antibiotics,
penicillins, cephalosporins, tetracyclines and erythromycin, are US$4.2 billion per
annum. The mold isolated by Alexander Fleming in early 1940s was Penicillium

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notatum, who noted that this species killed his culture of Staphylococcus aureus.
The production of penicillin is now done by a better penicillin-producing mould
species, Penicillium chrysogenum. Development of submerged culture
techniques enhanced the cultivation of the mould in large-scale operation by
using a sterile air supply.

▪ Streptomycin produced by Actinomycetes

▪ Molasses, corn steep liquor, waste product from sugar industry, and wet
milling corn are used for the production of penicillin
▪ Penicillium chrysogenum can produce 1000 times more penicillin than
Fleming’s original culture8

The major steps in the commercial production of penicillin are:

(1) Preparation of inoculum.

(2) Preparation and sterilisation of the medium.
(3) Inoculation of the medium in the fermenter.
(4) Forced aeration with sterile air during incubation.
(5) Removal of mould mycelium after fermentation.
(6) Extraction and purification of the penicillin.

PRODUCTION OF ENZYMES

Many molds synthesize and excrete large quantities of enzymes into the
surrounding medium. Enzymes are proteins; they are denatured by heat and
extracted or precipitated by chemical solvents like ethanol and by inorganic salts
like ammonium sulphate.11 Coenzymes are also proteins combined with low
molecular mass organics like vitamin B. It is industrially applicable and
economically feasible to produce, concentrate, extract and purify enzymes from
cultures of molds such as Aspergillus, Penicillium, Mucor and Rhizopus. Mold
enzymes such as amylase, invertase, protease, and pectinase are useful in the
processing or refining of a variety of materials.

Amylases hydrolyze starch to dextrin and sugars. They are used in preparing
sizes and adhesives, desizing textile, clarifying fruit juices, manufacturing
pharmaceuticals and other purposes. Invertase hydrolyses sucrose to form
glucose and fructose (invert sugar). It is widely used in candy making and the
production of non-crystallizable syrup from sucrose, which is partly hydrolyzed by
this enzyme. The proteolytic enzymes such as protease are used for bating in

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leather processing to obtain fine texture. Protease is also used in the manufacture
of liquid glue, degumming of silks and clarification of beer protein. It is used in
laundry detergents and as an adjunct with soaps. Pectinase is used in the
clarification of fruit juice and to hydrolyze pectins in the retting of flax for the
manufacture of linen. Apoenzyme is the protein portion of the enzyme, which is
inactive. The reaction between low molecular mass coenzymes and apoenzyme
gives active holoenzyme.

Figure 6 – Commercial Production of Baker’s Yeast

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Figure 7 - One Complete Set of Fermenters with All Accessory Controlling Units

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PRODUCTION OF BAKER’S YEAST

The use of yeast as a leavening agent in baking dates back to the early
histories of the Egyptians, Greeks and Romans. In those days, leavened bread was
made by mixing some leftover dough from the previous batch of bread with fresh
dough. In modern baking, pure cultures of selected strains of Saccharomyces
cerevisiae are mixed with the bread dough to bring about desired changes in the
texture and flavor of the bread. Characteristics of S. cerevisiae strains are selected
for commercial production of baker’s yeast.

It can ferment sugar in the dough vigorously and rapidly. The selected
strains must be stable and produce carbon dioxide, which results from the
fermentation process for leavening or rising the dough. The quality of the bread
depends on the selected strain of yeast, the incubation period and the choice of
raw materials. Sugars in the bread dough are fermented by yeast to ethanol and
CO2; whereby the CO2 causes the bread to rise. In the manufacture of baker’s
yeast, the stock strain is inoculated into a medium that containing molasses and
corn steep liquor. The pH of the medium is adjusted to be slightly acidic at pH 4–
5. The acidic pH may retard the bacterial growth. The inoculated medium is
aerated during the incubation period.

At the end, the cells are harvested by centrifuging out the fermentation
broth, and they are recovered by filter press. A small amount of vegetable oil is
added to act as plasticizer, and then the cell mass is molded into blocks. The
process is shown in Figure 6. A full set of bioreactors with pH and temperature
controllers are shown in Figure 7. The complete set of a 25 liter fermenter with all
the accessory controlling units creates a good opportunity to control suitable
production of biochemical products with variation of process parameters.
Pumping fresh nutrients and operating in batch, fed batch and continuous mode
are easy and suitable for producing fine chemicals, amino acids, and even
antibiotics.

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II. BASIC BIOCHEMISTRY

Biochemistry is the study of life cyclic processes in terms of chemicals, It is
not the study of chemistry of living organisms. It obtains information from the basic
microorganisms viz., bacteria, viruses, algae and fungi. It draws information on
energitics from basic thermodynamics, It couples them to show a path how life
cycle proceeds with the mutual cooperation of various activities of the living
beings. Energy is required for various metabolic activities in the living organisms.
This energy is released by breaking of some of the high energy strong molecules,
usually phosphate containing molecules. Oxidation of NADH (nicotinamide
adenine dinucleotide, reduced form) in the mitochondria is one of the main
reactions. Some Of the chemical/biochemical reactions in the living organisms
are facilitated by another type of compounds known as enzymes. The facilitation
of the reaction is known as catalysis, and hence enzymes are known as
biocatalysts or biological catalysts.
The cells themselves contain some of the enzymes. The cell regulates the
type of enzyme and its number by which it regulates both the type of biochemical
reaction and its rate. Genetic engineering deals with the information flow of the
living organisms. Biochemistry, coupled with genetic engineering principles, plays
a vital role in devising techniques for manipulating most of the biochemical
transformations. Thus, of late, the theoretical knowledge of biochemistry is being
diverted to applied aspects in regulating the cell metabolism.
Living organisms contain various biomolecules which are the building
blocks of the cell and also help in storing and releasing energy for
biotransformations, and at the same time carrying forward genetic information_
They include carbohydrates, lipids, proteins, nucleic acids, vitamins, etc.
Biomolecules Living organisms contain many biomolecules which are essentially
composed of carbon and nitrogen. The bacteria which are the smallest cells
contain a large number of (5000) different kinds of compounds including 3000
kinds of proteins, 1000 kinds Of nucleic acids. These biomoleeules have high
molecular weights and are complex in structure. They form the basic building
blocks for the cells and serve various purposes. For example, the amino acids
serve not only as building blocks of protein molecules but also as precursors of
hormones, alkaloids, porphyrins, pigments etc. Similarly, nucleotides serve not only
as building blocks of nucleic acids but also as co-enzymes and as energy carrying
molecules.

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A. LIPIDS
Lipids are a class of compounds which are fatty/oily in nature and present
in cells and tissues. They have the following distinct characteristic features.

➢ They are insoluble in water

➢ They are soluble in non-polar solvents like n-hexane, alcohol, chloroform,
benzene, etc. and hence can be extracted from the organisms using these
solvents
➢ They release a lot of energy on break-down and hence are considered as
the energy-storage media
➢ They contain a large proportion of C-H bonds
➢ Upon saponification, they release fatty acids and glycerol, and hence
glycerol is considered as a binding agent for fatty acids
➢ They are synthesized by the cells from sugars

Some lipid compounds, such as vitamins and hormones have intense biological
activity. Lipids also include a heterogeneous group of structural components. In
Table 1, they are grouped based on their chemical composition. Some lipids are
combined with other classes of compounds, and they are known as:
➢ Lipoproteins
➢ Proteolipids
➢ Phosphatidopeptides
➢ Lipoaminoacids
➢ Lipopolysaccharides

Table 1 – Various Types of Lipids

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As biomolecules, they are a constituent of cell walls and form a protective

coating to the cell and encourage some species-specific reactions. They are also
energy carriers and release the energy as and when the cell requires it.

Fatty Acids

The fatty acids are generally available in the esterified form in cells and
tissues and are rarely available in free form. They are named on the basis of the
hydrocarbon from which they are derived. These hydrocarbons may be saturated
or unsaturated. Accordingly, the fatty acids are also classified as saturated and
unsaturated fatty acids. In saturated fatty acids, the carbon atoms hold as many
hydrogen atoms as possible in addition to the carboxylic grouping, whereas in the
unsaturated form, the carbon atoms hold double and triple bonds also. Fatty
acids differ from each other primarily in chain length and in the number and
position of their unsaturated bonds. They are generally represented by a
shorthand notation based on the length of the carbon chain and the number,
position and configuration of the double bonds. For example, palmitic acid is
symbolized as 16:0 and oleic acid as 18:1.
Table 2 – Some Naturally Occurring Fatty Acids

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Table 2 lists some naturally occurring fatty acids. The palmitic acid contains
16 carbon atoms without having any unsaturated double bonds, whereas oleic
acid contains eighteen carbon atoms and contains one unsaturated double
bond between 9th and 10th carbon atoms. Fatty acid esters of glycerol are called
acyl-glycerols or glycerides. Depending upon the number of hydroxyl groups of
the glycerol is esterified, it is known as monoacyl-, diacyl- or triacyl-glycerol. They
are shown as follows.

Lipids Containing Glycerol

In the above RI, R2 and R3 may be the same fatty acid group or different
fatty acid groups. If they are all same, then the fat is known as simple
triacylglycerol. If R's are stearic acid, the lipid is known as tristearoyl- glycerol or
simply known as tristearin.

Phospholipids
Phospholipids consist of a glycerol connected to a phosphate group. They
consist of two fatty acids linked to glycerol molecules. They are generally crystal
white solids. They turn brown when exposed to air due to polymerization of
unsaturated fatty acids. They are characterized also by the presence of nitrogen
group. They generally possess a negative charge on the phosphate group. Most
of the phospholipids are derivatives of L-a-phosphoric acid

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L-a-phosphatidic acid on hydrolysis yields one mole each of glycerol,

phosphoric acid and two moles of fatty acid. In phosphatides, the phosphoric
acid is bound in ester linkage to one of the following nitrogenous compounds

Lipids Not Containing Glycerols

Sphingolipids Plant and animal cells contain sphingolipids as important
components. The sphingolipids are present in the brain and nerve tissues in large
quantities. They are lipids containing sphingosine in place of glycerol as their
backbone. Sphingomyelins are the sphingolipids in higher animals which contain
phosphoryl ethanolamine or phosphoryl choline. Sphingomyelins have the
following generalized structural formula:

Terpenes
Terpenes contain multiples of the five-carbon hydrocarbon isoprene (2-
methyl-l,3 butadiene), and they are classified based on the number of isoprene
units (Table 3).
Terpenes are known for their characteristic odor and flavor and are mostly
present in the essential oils of plants.

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Table 3 – Classification of Terpenes

Steroids
Steroids are a group of compounds derived from the saturated tetracyclic
hydrocarbon perhydrocyclopentanophenanthrene. Many steroids have different
functions and distinct metabolic activity. Mostly they are derived from natural
sources. All steroids originate from the linear triterpenesqualene which cyclizes
readily. Lanosterol is the precursor of cholesterol, which is the most abundant
steroid in animal tissues. Lanosterol and cholesterol are steroid alcohols, which
occur either as free alcohols or as long-chain fatty acid esters of the hydroxyl
group at carbon 3.

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Lipoproteins
Lipoproteins are a class of compounds formed by the association of certain
lipids with proteins. Hence they exhibit properties characteristic of both lipids and
proteins. They are classified into the following two major groups:
➢ transport lipoproteins
➢ membrane systems
The transport lipoproteins are so named because they carry water-insoluble lipids
to various organs of the body through blood, They are also known as plasma
lipoproteins and are further classified into the following three groups depending
upon the type of lipid
➢ very low density lipoproteins (VLDL)
➢ low density lipoproteins (LDL)
➢ high density lipoproteins (HDL)
Membrane systems are the lipoproteins present in the membrane of the cell
structure. They contain about 40% lipid and 60% protein. They are mainly meant
for protecting the cell from external environment. They also facilitate the transport
of certain enzymes and other transport systems.

Lipids Combined with Other Compounds

Lipids combined with other classes of compounds, in addition to
lipoproteins, are as follows:
➢ Proteolipids: Proteolipids contain proteins associated with lipids in which
lipid content is more.
➢ Phosphatidopeptides: Lipid category ofphosphatides associated with
peptides are phosphatidopeptides.
➢ Lipoamino acids: Lipids associated with amino acids are lipoamino acids.
➢ Lipopolysaccharides: Lipids found in nature combined with
polysaccharides are lipopolysaccharides.

B. PROTEINS
The word protein is derived from a Greek word meaning 'of the first rank'.
They are the beginners and builders of biochemical reaction. Thus, they occupy
a central position in the construction and functioning of living matter. All enzymes
are proteins and they catalyze biochemical reactions as biocatalysts. Collagen is
an elongated protein which is responsible for the tensile strength of skin and
bones. In addition, proteins perform various other functions as well; viz., they help

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in transport of oxygen into blood, and transmission of nerve impulses, etc. Proteins
constitute a major part of the animal tissues. Proteins are a large molecule
consisting of as many as twenty amino acids. Their molecular weights range from
5000 to few millions. The amino acids are linked through the peptide linkages.

Amino Acids
Amino acids are the building blocks of proteins. There are twenty amino
acids present in all living organisms, which is a clear manifestation of the biological
unity in all living organisms. These twenty amino acids have different structural
configurations and are represented by three lettered symbols (Table 4). They are
also represented by single alphabets which help in writing the amino acid
sequence in the proteins. Some proteins contain a large combination of several
amino acids. The amino acids have the structure:

These are known as a-amino acids in which the amino group and the
carboxyl group are attached to the same carbon atom. Different amino acids
possess different groups in place of R. Amino acids are also classified as acidic,
basic, and neutral amino acids. The above classification is based on the number
of carboxyl groups and number of amino groups present. If they are same, it is
known as neutral amino acid. Structures of some amino acids are shown in Figs 1
to 4.

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Table 4 – Amino Acid Symbols, Chemical Group, and Charge on the Molecule

Figure 1 – Structure of some neutral amino acids

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Figure 2 – Structures of Some Amino Acids

Figure 3 – Structures of Some Basic Amino Acids

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Figure 4 – Structures of More Amino Acids

Absorption Spectra
Spectrophotometric method is a rapid method for estimating the protein
content in a solution. Tyrosine and other two amino acids namely tryptophan and
phenylalanine absorb light in the ultraviolet region. Hence, the measurement of
light absorption at 280 nm in a spectrophotometer is used for the estimation of
proteins in a solution, since most proteins contain tyrosine.

Analysis of Amino Acid Mixtures

Chromatographic separation methods are useful for the separation,
identification and quantitative analysis of amino acid mixtures. A few
chromatographic separation methods are described as follows:
(i) Partition Chromatography The relative distribution of the solute between
two immiscible phases is taken as an advantage for separation of the mixture of
amino acids. It is like counter-current extraction principle. When a solute is brought
into contact with a two-solvent System (in which the two solvents are immiscible)
the solute gets distributed between the two solvents depending upon the
partition coefficient of the solute. The immiscible solvent systems could be phenol-
water or n-butanol-water, etc. Subsequently, the solute is separated from the
solvents.
(ii) Amino Acid Analyzer This technique of amino acid analysis was first
pioneered at the Rockefeller Institute, New York in which the entire procedure of
cation exchange chromatography is automated so that elution, collection of

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fractions, analysis of fractions and recording data are all performed

automatically.

Other chromatographic methods include:

➢ column chromatography
➢ paper chromatography
➢ TLC
➢ Ion-exchange chromatography

Purification of Proteins
The characteristic properties of proteins which are used to separate
mixtures of proteins are:
➢ molecular size,
➢ solubility, electric charge, differences in absorption characteristics
➢ biological affinity for other molecules.
The following are some methods employed to separate proteins:

(i) Dialysis and Ultra-filtration Proteins can be separated from low molecular
weight solutes by dialysis. This method employs a semipermeable membrane on
which the bigger protein molecules are retained. Small solute molecules and
water pass through the membrane. Thus, separation takes place. In ultra-filtration,
pressure or centrifugal force is used to filter the aqueous medium and small solute
molecules through a semipermeable membrane. Cellophane and other
synthetic materials are employed as membranes (also see Section 17.7).
(ii) Gel Filtration Separation is carried out based on the relative size of the
proteins. Gel filtration or molecular-sieve chromatography consists of a column
packed with a polysaccharide called Sephadex. The hydrated sephadex gel will
have molecular size of 0.1 mm in diameter. Thus, it holds smaller protein molecules.
The bigger protein molecules pass through the column and get separated out.
(iii) Ion-exchange Chromatography In this method, the separation is based
on the charge of the proteins. The ion-exchange column is packed with
negatively charged carboxylate ions. They retain the positively charged proteins
at pH below 7. The negatively charged proteins will pass through the column and
get separated. Subsequently, the adsorbed proteins are separated from the
column by pouring a solution of different concentrations of sodium chloride

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(iv) Affinity Chromatography The method is based on the ligand-ligand

interaction. The chromatographic column consists of a polysaccharide molecule
(like agarose). The protein mixture is passed through the column. Those proteins,
which have the property of binding non-covalently to ligand molecules of
agarose, will get attached to the agarose particles. Other proteins, which do not
have the affinity for such ligand interaction, will pass through the column and get
separated. This method is extensively used for separation of enzymes.
(v) Electrophoresis The isoelectric point of a protein is that value of pH at
which the net electric charge on the protein molecule is zero (i.e. the positive
charge equals the negative charge). Hence, at isoelectric point, the mobility of
the protein under an electric field will be zero. This property of a protein is used to
separate a protein mixture by the electrophoresis method. The protein mixture is
subjected to electric field in the retarding gel support. Proteins with different
isoelectric points will have different mobilities. Thus, the separation takes place in
the electrophoresis method. This method can also be effectively used as an
analytical tool for proteins.

C. CARBOHYDRATES
The carbohydrates or saccharides are a large group of compounds which are
poly-hydroxyaldehydes or poly-hydroxyketones and their derivatives. They
contain carbon, hydrogen and oxygen, with the ratio of hydrogen and oxygen
being 2: l. They have a general chemical formula: and may be classified as:
➢ monosaccharides
➢ oligosaccharides (di- and tri- saccharides)
➢ poly-saccharides
➢ high molecular weight poly-saccharides.

Monosaccharides
Formaldehyde (HCHO) and hydroxy-acetaldehyde (OH-CH2-CHO)
conform to the empirical formula of carbohydrates, (CH2O)n, n being equal to 1
and 2 respectively. The smallest three-carbon sugar molecules are
glyceraldehyde and dihydroxyacetone, and they are termed as trioses.

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Various other monosaccharides are shown in Table 5, which are named

based on the number of carbon atoms. All the simple monosaccharides are white
crystalline solids, sweet in taste, and are freely soluble in water, but are insoluble
in non-polar solvents. The carbon skeleton of the common monosaccharides is
unbranched, and each carbon atom except one, contains a hydroxyl group. The
carbon atom has a carbonyl oxygen which is combined in an aldehyde or ketose
linkage. If the carbonyl group is at the end of the chain, the monosaccharide is
an aldehyde derivative, and such monosaccharides are called aldoses. If it is at
any other position, the monosaccharide is a ketone derivative, and is called as
ketoses. Among monosaccharides, the hexoses are abundant. Aldopentoses are
important components of nucleic acids and various polysaccharides.

Table 5 – Various Monosaccharides

Figure 5 – Structure of D and L Monosaccharides

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Epimers are defined as the sugars which have the same chemical formula
and structure except around one specific carbon atom. Thus D-glucose and L-
glucose are epimers with respect to carbon atom 2 (Fig. 5). The families of D-
ketoses and D-aldoses are shown in Figure 6 and Figure 7.

Figure 6 – Family of D-ketoses

Figure 7 – Family of D-aldoses

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Pyranoses are also six carbon sugars but with a ring structure. Thus a-ID-
glucose in the ring form is known as a-D-glucopyranose. The structural formulae
of a-D-glucopyranose and a-D-glucopyranose are shown as follows

Pyranoses are also represented by two other conformations, viz. boat-

form and chair-form (Fig. 8). The conformational forms are represented to show
the sugar structures in planar form. Of the two forms, the chair form is more rigid
and stable.

Figure 8 – Pyranose Rings (in boat and chair forms)

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Disaccharides
Disaccharides are anhydrides of two monosaccharides, which include
sucrose, maltose, lactose and cellobiose (Fig. 9). Sucrose occurs in sugarcane or
sugar beets, and it is a disaccharide of glucose and fructose (Fig. 9(a)). Sucrose
yields an equimolar mixture of glucose and fructose on hydrolysis. The hydrolysis
of sucrose is called the inversion of sucrose because during the reactions, the sign
of rotation changes from dextro to levo. It is an enzymatic reaction by invertases.
The equimolar mixture of glucose and fructose is called 'invert sugar'. It is sweeter
than sucrose in taste. Hence hydrolysis of sucrose is carried out in confectionary
industry. Fructose is present naturally in honey.

Figure 9 – Structures of Sucrose, Maltose, and Lactose

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Oligosaccharides
Oligosaccharides are anhydrides of several monosaccharide residues.
Compounds containing ten or less monosaccharide units are called
oligosaccharides, while those containing more than ten are called
polysaccharides.

Polysaccharides
Polysaccharides are also known as glycans. They are anhydrides of one
or more monosaccharides in which many units are combined. Many
polysaccharides are composed of only one repeating monosaccharide, and
even where a large number exists, they appear to be arranged in a systematic
fashion. Homopolysaccharides possess one repeating unit, while
heteropolysaccharides contain more than one monosaccharide unit. D-Glucose
is the most prevalent monosaccharide unit though polysaccharides of D-
mannose, D-fructose, D- and L-galactose, D-xylose and D-arabinose are
common. D-glucosamine, D-galactosamine, D-glucuronic acid, N-
acetylmuramic acid and N-acetylneuraminic acid are the monosaccharide
derivatives which are commonly found in natural polysaccharides.

Starches: Starches are nutritional reservoirs in plants corresponding to α(1->4)

linkages. The repeating disaccharide unit in starch is maltose (Fig. 10). Native
starches are a mixture of two compounds, viz. amylose and amylopectin.
Amylose is an unbranched chain. Amylopectin is a branched chain
polysaccharide (Fig. 11). Amylose consists of long unbranched chains in which D-
glucose units are bound in α(1->4)linkages. The molecular weight of amylose
varies from a few thousands to whereas that of amylopectin is of the order of 109.
It contains about 24—30 glucose residues. The backbone glycosidic linkage is α(1-
>4), but the branch points are α(1->6) linkages.

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Figure 10 – Repeating Maltose Unit of Starch

Figure 11 – An α(1->6) Branch Point and α(1->4) Main Chain of Amylopectin

Cellulose: Cellulose is the compound which gives strength to plant tissues. On acid
hydrolysis of cellulose, glucose is formed in yields of 95—96%. The cellulose is
abundantly available in nature with the purest source being cotton containing at
least 90% pure cellulose. It is insoluble in water. Its molecular weight is in the range
of 50,000 and corresponding to 300—15000 glucose residues per molecule.
Glucose units are joined in /3(1,4) linkages (Fig. 12).

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Figure 12 – Repeating Cellobiose Units of Cellulose

On complete hydrolysis, cellulose yields D-glucose. Partial hydrolysis yields

cellobiose. Cellulase enzyme can hydrolyze cellulose to ID-glucose. But cellulase
enzyme is not secreted in the digestive tract of mammals except cows.

D. NUCLEIC ACIDS AND NUCLEOTIDES

Nucleic acids are the polymers of nucleotides, which are the units that
store genetic information in each cell. Thus, nucleic acids contain all the
hereditary information. Hence, they are also known as informational bio-
polymers. When the cells divide, a copy of the parent cell's nucleic acids is passed
on to the daughter cell.

Nucleotides
Nucleotides are the building blocks of nucleic acids and, they are also
energy carriers. They contain essentially the following three components:
➢ phosphoric acid
➢ ribose or deoxyribose (five-carbon sugars), and
➢ a nitrogenous base.
The nitrogenous base is derived either from purine or pyrimidine.

The above three components of the nucleotides are joined as shown here

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The five nitrogen bases found in DNA and RNA (shown in Fig. 13) are
adenine, guanine (purines) and cytosine, thymine and uracil (pyrimidines).
Ribonucleic acid (RNA) is a polymer of ribose containing nucleotides while
deoxyribonucleic acid (DNA) is a polymer of deoxyribose containing nucleotides.

Figure 13 – Five Nitrogen Bases Found in DNA and RNA

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DNA contains 2-deoxy 2-1)-ribose whereas RNA contains D-ribose sugars

which are in their furanose form in nucleotides. The base in the form of purine or
pyrimidine is joined to the pentose sugar by a β-N-glycosyl bond between carbon
I of pentose and nitrogen atom of the bases. This is known as nucleo- side. The
phosphate group is joined to the pentose of the nucleoside in the form of the ester
at the carbon 5 position. When phosphate group is added to the nucleoside, it is
called nucleotide. Thus, nucleotides are the 5 '-phosphates of the corresponding
nucleosides.
Ribonucleotides and deoxyribonucleotides occur in free form in cells, and
most of them are 5'-mono- phosphates. Some of them also occur as 5'-
diphosphates and 5'-triphosphates. Adenosine may occur in all the three forms as
adenosine 5'-monophosphate (AMP), adenosine 5'-diphosphate (ADP) and
adenosine 5'-triphosphate (ATP). ATP is a carrier of phosphate and
pyrophosphate and is involved in the transfer of chemical energy. ATP and ADP
are interconverted by dephosphorylation and rephosphorylation during
respiration. ATP—ADP system is responsible for transferring phosphate groups in
the cell. Nucleoside triphosphates (NTPs) function as energy-rich precursors of
mononucleotide units in the biosynthesis of DNA and RNA.

DNA
Deoxyribonucleic acid (DNA) consists of deoxyribonucleotides linked with
covalent bonding (Fig. 14). The backbone of DNA consists of alternating
phosphate diesters and pentose groups with the covalent bonding in which the
continuity is maintained by the phosphodiester bridges. The purines and
pyrimidines constitute the side chains. Different sources of DNA molecules differ in
terms of the ratio of the four major types of nucleotide monomers. Hence, their
molecular weights will also differ. J.D. Watson and F.H.C. Crick postulated the DNA
structure with two strands in complementary double helical arrangement in 1953
(Fig. 14).
DNA is present in prokaryotic cells, eukaryotic cells and bacteria in different
forms. In prokaryotic cells, it is present in single chromosome while in eukaryotic
cells, it is present in different chromosomes as different molecules. Hence its
molecular weight will also change considerably. In bacteria, it is generally present
in the nucleus. In some cases, it may be present in the cytoplasm in the form of
extra-chromosomal DNA. In eukaryotic cells also, in some cases it may be present

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in the mitochondria; but in such cases its molecular weight and base composition
are different from that of the DNA present in the nucleus of the cell.

Figure 14 – Structures of DNA and RNA

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RNA
Ribonucleic acid (RNA) was discovered in the year 1955 by Frankel-
Conrat and Williams and was assigned a single helical structure. In this respect, it
differs from the DNA, which has a double helical structure. Unlike DNA, it contains
ribose sugars, and the nitrogen group is provided by uracil. There are three major
types of RNAs, namely
➢ messenger RNA (mRNA)
➢ ribosomal RNA (rRNA)
➢ transfer RNA (tRNA)
Each type of RNA has a characteristic range of molecular weights in between
25,000—11 Each RNIA occurs in multiple molecular forms. RNA is present in the
cytoplasm in bacteria. Mitochondrial rRNAs and tRNAs are different from the
extramitochondrial forms.

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REFERENCES
Najafpour, G. D. (2007). Industrial Microbiology. Biochemical Engineering and
Biotechnology, 1–13. doi:10.1016/b978-044452845-2/50001-x

Rao, D. G. (2010), Introduction to Biotechnology, 2nd Edition, Tata McGraw Hill,

pp. 9-54, ISBN 0-0701-5138-5.

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