1.

Fermentation

Fermentation is the process involving the biochemical activity of organisms, during their
growth, development, reproduction, even senescence and death. Fermentation technology is the use
of organisms to produce food, pharmaceuticals and alcoholic beverages on a large scaleindustrial
basis.

The basic principle involved in the industrial fermentation technology is that organisms are
grown under suitable conditions, by providing raw materials meeting all the necessary requirements
such as carbon, nitrogen, salts, trace elements and vitamins.

The end products formed as a result of their metabolism during their life span are released
into the media, which are extracted for use by human being and that have a high commercial value.
The major products of fermentation technology produced economically on a large-scale industrial
basis are wine, beer, cider, vinegar, ethanol, cheese, hormones, antibiotics, complete proteins,
enzymes and other useful products.

Fermentation Methodology:

Fermentation process is carried out in a container called the fermentor or bioreactor. The
design and nature of the fermentor varies depending upon the type of fermentation carried out.
Invariably all the fermentors have facilities to measure some of the fermentation parameters like
temperature, pressure, pH, elapsed fermentation time, liquid level, mass etc.

The different types of fermentors are:

1. External recycle airlift fermentor—for producing bacterial biomass, with methanol as substrate.
2. Internal recycle airlift fermentor—for producing yeast with oil as substrate.
3. Tubular tower fermentor—Used for making beer, wine, vinegar etc.
4. Nathan fermentor—used in brewing industry.
5. Stirred fermentor—used for making antibiotics.
Types of Fermentation Processes:

1. Batch Fermentation: A batch fermentation is a closed culture system, because initial and limited
amount of sterilized nutrient medium is introduced into the fermenter. The medium is inoculated
with a suitable microorganism and incubated for a definite period for fermentation to proceed
under optimal physiological conditions. Oxygen in the form of air, an antifoam agent and acid or
base, to control the pH, are being added during the course of fermentation process.

During the course of incubation, the cells of the microorganism undergo multiplication and pass
through different phases of growth and metabolism due to which there will be change in the
composition of culture medium, the biomass and metabolites. The fermentation is run for a
definite period or until the nutrients are exhausted. The culture broth is harvested and the product
is separated. Batch fermentation may be used to produce biomass, primary metabolites and
secondary metabolites under cultural conditions supporting the fastest growth rate and maximum
growth would be used for biomass production. The exponential phase of growth should be
prolonged to get optimum yield of primary metabolite, while it should be reduced to get optimum
yield of secondary metabolites. The used medium along with cells of microorganism and the
product is drawn out from the fermenter. When the desired product is formed in optimum
quantities, the product is separated from the microorganism and purified later on.

2. Continuous Fermentation: It is a closed system of fermentation, run for indefinite period. In this
method, fresh nutrient medium is added continuously or intermittently to the fermenter and
equivalent amount of used medium with microorganisms is withdrawn continuously or
intermittently for the recovery of cells or fermentation products
As a result, volume of the medium and concentration of nutrients at optimum level are being
maintained. This has been operated in an automatic manner. The continuous fermenter has its
maximum use that take long time to reach high productivity, reduces down time and lowers the
operating costs. In continuous mode, starting medium and inoculum are added to the fermenter.
After the culture is grown the fermenter is fed with nutrients and broth is withdrawn at the same
rate maintaining a constant volume of broth in the fermenter. In continuous mode with cell cycle,
the cell mass is returned to the fermenter using micro filtrations with bacteria or screens with
fungal mycelium.

A continuous fermentation is generally carried out in the following ways:

1. Single Stage Fermentation: In this process, a single fermenter is inoculated and the nutrient
medium and culture are kept in continuous operation by balancing the input and output of
nutrient medium and harvested culture, respectively.

2. Recycle Fermentation: In this method, a portion of the medium is withdrawn and added to the
culture vessel. Thus, the culture is recycled to the fermentation vessel. This method is
generally adopted in the hydrocarbon fermentation process. The recycling of cells provides
a higher population of cells in the fermenter which results in greater productivity of the desired
product.
 3. Multiple Stage Fermentation: In this process, two or more fermenters are employed
simultaneously and the fermentation is operated in a sequence. Different phases of
fermentation process like growth phase and synthetic phase are carried out in different
fermenters. Generally, growth phase is allowed in the first fermenter, synthetic phase in the
second and subsequent fermenters.

3. Fed Batch Fermentation: It is a modification to the batch fermentation. In this process substrate
is added periodically in instalments as the fermentation progresses, due to which the substratum
is always at an optimal concentration. This is essential as some secondary metabolites are
subjected to catabolite repression by high concentration of either glucose, or other carbohydrate
or nitrogen compounds present in the medium. For this reason, the critical elements of the nutrient
medium are added in low amount in the beginning of the fermentation and these substrates
continue to be added in small doses during the production phase. This method is generally
employed for the production of substances such as penicillin. Yoshida (1973) introduced this term
for the first time for feeding the substrates to the medium as the nutrients are exhausted, so as to
maintain the nutrients at an optimum level.

II. Basic concepts of Upstream and Downstream processing in Bioprocess

The current topics of debate on the Basic concepts of Upstream and Downstream processing in
Bioprocess:

1. Up-Stream Process: This process comprises of obtaining a desired microorganism, and its
improvement so as to enhance the productivity and yield. It also includes the maintenance of
strain purity, preparation of inocula and further efforts to improve the economic efficiency of
the process. There are two distinct processes involved in USP but both the processes run
simultaneously. The medium is prepared for the culture (inoculum) preparation as well as for
the production of desired product. The vessel of the fermenter is filled with production media and
inoculated with suitable microbial culture for the industrial product formations as shown in Fig.
20.4.
2. Down-Stream Process: This process includes the selection of suitable fermentation media,
optimization of important fermentation condition to give rise maximum yield. The main objective
of this process is to get reproducibility of result as far as possible besides safe recovery of the
target product. Further processing is carried out depending upon whether the product is
intracellular or present inside the cell or membrane bound or release outside the cell or
extracellular in nature. This leads to recovery of the product in pure form. The presence of
undesirable products (by products), impurities in media, antifoam agents, etc. affect the DSP
steps. Therefore, each step is required thorough check up in both USP as well as in DSP. For
example, a cheap carbon and energy source may increase DSP cost. At commercial level use of
existing equipment’s, ancillaries, etc. are more advantageous than that of creating new facilities
because the later step requires more investment which may lead to escalation of cost of the
product and processes. The DSP consists of series of distinct unity or processes which are
connected to each other for getting the final product as shown in Fig 20.5.

For the precautionary measures it is necessary to keep this unit process minimum. This will not
only save the cost of the plant but will also ensure not to loose the product during individual steps
(Fig. 20.5). Now-a-days, most preferred DSP is to link with the fermentation. This increases
productivity and reduce time and cost. There are two types of major processes for product
formation. The product formation is coupled with growth i.e. primary metabolites and another
where product is formed after growth as in case of antibiotics. In case of primary metabolites,
higher productivity is achieved by using integrated system of fermentation with DSP. This kind
of system maintains a high cell density through cell retention of recycling. If product is inhibitory,
various methods have been employed to partition fermenter as in case of formation of glucose by
breakdown of cellulose. This allows the rapid in-situ removal of product by extraction,
adsorption or stripping. The process can be ex-situ where the product is
removed outside the fermenter and the processed medium is returned to the fermentation. Such
processes have been successfully used for removing alcohols, solvents, proteins, etc. as shown
in Fig. 20.5. Following are the major steps for the product recovery.

1. Cell Harvesting: Since, microbial cells are in liquid medium or broth, solid-liquid separation
method is influenced by the size and morphology of the microorganisms (single cells,
aggregates or mycelia). Further, the process also depends upon the location of product
whether it is intracellular or extracellular it has been secreted into the medium. Besides,
other factors such as specific gravity, viscosity, and rheology of the medium also influence
cell harvesting method.

2. Broth Conditioning Technique: This technique allows separation of cells from large volume
of liquid medium. In this case, cells are allowed to form floccules or precipitate by using
chemical, physical and biological treatments. Coagulating materials such as simple
electrolytes, acids, bases, salts, multivalent ions etc. are added to form small floes from
dispersed colloids or suspended materials. Certain floes precipitation methods are also used
at the end of many traditional beer and wine fermentation processes for the precipitation of
yeast cells.

3. Sedimentation: This process is widely used for yeast separation during alcohol production and
in waste-water treatment. The rate of particle sedimentation is a function of both sizeand
density. If, larger the size, greater its density, and the faster rate of sedimentation. Therefore,
for quick separation, the difference in density between the particle and the medium must be
large with low viscosity.

4. Centrifugation: This process allows small particles or cells to separate from liquid. It is also
suitable for some liquid-liquid separations. Similar to sedimentation, its effectiveness also
depends upon cell size, density difference between the cells and the medium, and itsviscosity.
Higher speed of centrifugation requires for the separation of smaller microorganisms, such as
bacteria. In case of yeast cells present in beer, relatively, low centrifugation effectively
recovers residual yeast cells.

5. Filtration: There are certain filters such as clothes, glass wool or cellulose that retain the solids
and pass on the liquid. The solids accumulate above the filter. Such technique is
 useful in case of fungal mycelia separation. There are two types of filters used in industrial
process: plate and frame filters or filter press, and rotatory vacuum filter, which help in
harvesting the fungal mycelia during antibiotic manufacture, bakers yeast production and in
dewatering of sewage sludge.

6. Disruption of Microbial Cells: When the product is intracellular, it is necessary to disrupt the
cells/cell membrane so as to release the product. Cell disruption can be achieved by
mechanical and non-mechanical methods. Some of the methods used are ultra-sonication
and application of lysozyme. The ‘French-press’ is often used in laboratories, while Manton
and Gaulin homogenizer (high pressure homogenizer) is employed for pilot scale cell
disruption. These are used for disruption of bacterial, yeast cells and fungal mycelia. The non-
mechanical cell disruption is carried out by cell permeabilization. This can be accompanied
by autolysis, osmotic shock, ruptures by ice crystal or heat shock treatment. Some organic
solvents such as acetone, butanol, chloroform and methanol have been used to release enzymes
from microorganisms. Simple treatment with sodium dodecyl sulfate (SDS) or Triton X-100
is also found effective. Lysozyme is useful for Gram-positive organisms butaddition of EDTA
improves the effectiveness of lysozyme of Gram-negative bacteria. Some antibiotics namely
penicillin and cycloserine may also be used to lyse the actively growing bacterial cells.
Chitosan is effective for yeast cells.

1. Primary Metabolites

Primary metabolism, also referred to as trophophase, is characterized by balanced growth

of microorganisms. It occurs when all the nutrients needed by the organisms are provided in the
medium. Primary metabolism is essential for the very existence and reproduction of cells. In the
trophophase, the cells possess optimal concentrations of almost all the macromolecules (proteins,
DNA, RNA etc.).

It is during the period of trophophase, an exponential growth of microorganisms occurs.

Several metabolic products, collectively referred to as primary metabolites, are produced in
trophophase (i.e., during the period of growth).

The primary metabolites are divided into two groups:

1. Primary essential metabolites: These are the compounds produced in adequate quantizes to sustain
cell growth e.g. vitamins, amino acids, nucleosides. The native microorganisms usually do not
overproduce essential primary metabolites, since it is a wasteful exercise. However, for industrial
overproduction, the regulatory mechanisms are suitably manipulated.

2. Primary metabolic end products: These are the normal and traditional end products of fermentation
 process of primary metabolism. The end products may or may not have any significant function
to perform in the microorganisms, although they have many other industrial applications e.g.
ethanol, acetone, lactic acid. Carbon dioxide is a metabolic end product of Saccharomyces
cerevisiae. This CO2 is essential for leavening of dough in baking industry.

Limitations in growth:

Due to insufficient/ limited supply of any nutrient (substrate or even O2), the growth rate of
microorganisms slows down. However, the metabolism does not stop. It continues as long as the cell
lives, but the formation of products differs.
 II. Secondary Metabolites

As the exponential growth of the microorganisms ceases (i.e. as the trophophase ends), they
enter idiophase. Idiophase is characterized by secondary metabolism wherein the formation of certain
metabolites, referred to as secondary metabolites (idiolites) occurs. These metabolites, although not
required by the microorganisms, are produced in abundance. The secondary metabolites however,
are industrially very important, and are the most exploited in biotechnology e.g., antibiotics, steroids,
alkaloids, gibberellins, toxins.

Characteristics of secondary metabolites:

1. Secondary metabolites are specifically produced by selected few microorganisms.

2. They are not essential for the growth and reproduction of organisms from which they areproduced.
3. Environmental factors influence the production of secondary metabolites.
4. Some microorganisms produce secondary metabolites as a group of compounds (usually
structurally related) instead of a single one e.g. about 35 anthracyclines are produced by a single
strain of Streptomyces.
5. The biosynthetic pathways for most secondary metabolites are not clearly established.
The regulation of the formation of secondary metabolites is more complex and differs from that
of primary metabolites.

Functions of secondary metabolites:

Secondary metabolites are not essential for growth and multiplication of cells. Their occurrence and
structures vary widely. Several hypotheses have been put forth to explain the role of secondary
metabolites, two of them are given below.

1. The secondary metabolites may perform certain (unknown) functions that are beneficial for the
cells to survive.
2. The secondary metabolites have absolutely no function. Their production alone is important for
the cell, whatever may be the product (which is considered to be useless).
After describing the various components of fermentation technology, we will next take a brief
look at the development process of microbial products using fermentation technology.
1. Isolation and Screening of Microorganisms
The success of an industrial fermentation process chiefly depends on the strain of the
microorganism used. An ideal producer or economically important strain should have the
following characteristics: It should be pure and free from phage. It should be genetically stable,
but amenable to genetic modification. It should produce both vegetative cells and spores; species
producing only mycelium are rarely used. It should grow vigorously after inoculation in seed
stage vessels. It should produce a single valuable product and no toxic by-products. Product
should be produced in a short time. It should be amenable to long-term conservation. The risk of
contamination should be minimal under optimum performance conditions.

Isolation of Microorganisms
The first step in developing a producer strain is the isolation of the relevant microorganisms from
their natural habitats. Alternatively, microorganisms can be obtained as pure cultures from
orga nizations that maintain culture collections, such as the American Type Culture Collection
(ATCC) in Rockville, Maryland, USA; the Commonwealth Mycological Institute (CMI) in Kew,
Surrey, England; the Fermentation Research Institute (FERM) in Tokyo, Japan; and the Research
Institute for Antibiotics, in Moscow, Russia. Microorganisms of industrial importance are usually
bacteria, actinomycetes, fungi, and algae. These organisms occur virtually everywhere, such as
in air, water, soil, surfaces of plants and animals, and in plant and animal tissues. Often, the
ecological habitat from which a desired microorganism is more likely to be isolated will depend
on the characteristics of the product desired from it and on the development process; for example,
if the objective is to isolate a source of enzymes that can withstand high temperatures, the obvious
place to look would be hot springs. The enrichment techniques are designed for selective
multiplication of only some of the microorganisms present in a sample. These approaches,
however, take a long time (about 20–40 days) and require considerable labor and money.
Screening of Microorganisms
After isolation of the microorganisms, the next step is their screening. A set of highly selective
procedures, which allows the detection and isolation of microorganisms producing the desired
metabolite, constitutes primary screening. However, this is possibly the most critical step, because
it eliminates the large bulk of unwanted useless isolates, which are either nonproducers or
producers of known compounds.
Rapid and effective screening techniques, which utilize either a property of the product or that of
its biosynthetic pathway for detection of desirable isolates, have been devised for a variety of
micro bial products. Some of the screening techniques are relatively simple, such as that for
extracellular enzymes and enzyme inhibitors. . For example, the search for new antibiotics now
focuses on rare actinomycetes, (i.e., actinomycetes other than those belonging to the genus
Streptomyces). Suitably designed specialized screening techniques may be used to detect
compounds having various pharmacological activities other than antibiotics.
Inoculum Development
The preparation of a population of microorganisms from a dormant stock culture to an active state
of growth that is suitable for inoculation in the final production stage is called inoculum
develop ment. As a first step in inoculum development, the inoculum is taken from a working
stock culture to initiate growth in a suitable liquid medium. Bacterial vegetative cells and spores
are suspended, usually in sterile tap water, which is then added to the broth. In case of non-
sporulating fungi and actinomycetes, the hyphae are fragmented and then transferred to the broth.
Inoculum develop ment is generally done using flask cultures. Flasks of 50 mL to 12 L may be
used and their number can be increased as needed. In some cases, small fermenters may be used.
Inoculum development is usually done in a stepwise sequence to increase the volume to the
desired level. At each step, the inoculum is used at 0.5%–5% of the medium volume, which allows
a 20–200-fold increase in inoculum volume at each step. Typically, the inoculum used for the
production stage is about 5% of the medium volume.
Culture Media
Inoculum preparation media are quite different from production media. These media are designed
for rapid microbial growth, and little or no product accumulation will normally occur. Many
production processes depend on inducible enzymes. In all such cases, the appropriate inducers
must be included either in all the stages or at least in the final stages of inoculum development.
This will ensure the presence of the concerned inducible enzymes at high levels for the production
to start immediately after inoculation.
Contamination
The inoculum used for production tanks must be free from contamination. However, as the risk
of contamination is always present during inoculum development, every effort must be made to
detect as well as prevent contamination.
Sterilization
Sterilization is the process of inactivating or removing all living organisms from a substance or
surface. In theory, it is regarded as absolute, in that all living cells must be inactivated or removed,
usually in a single step at the given time.
Strain Improvement
After an organism producing a valuable product is identified, it becomes necessary to increase
the product yield from fermentation to minimize production costs. Product yields can be increased
by developing a suitable medium for fermentation, refining the fermentation process, and by
improving the productivity of the strain. The techniques and approaches used to genetically
modify strains and to increase the production of the desired product are called strain improvement
or strain development. Strain improvement can be done by using mutant selection, recombination,
and rDNA technology.

Industrial Microbial Culture

The use of microbes to produce natural products and processes that benefit and improve human
healthcare has been a part of human history since early civilization. The production of secondary
metabolites (such as antibiotics) often occurs during the stationary growth phase (or idiophase)
of a bacterial culture. Secondary metabolites are nongrowth related and seem to play no obvious
role in cell maintenance. The production of secondary metabolites largely concerns the first
growth phase of a bacterial culture. This production is considered a two-stage process, with a
trophophase (growth phase) and an idiophase (production phase), which usually occurs when
culture growth has slowed or ceased. The major aim for mass production of secondary metabolites
is to maximize the biomass in a short trophophase, while optimizing the conditions for high,
sustained idiophase production.
The growth of a bacterial culture can be represented by a curve that consists of the fol lowing
four stages or phases: 1. Lag phase—growth and reproduction are just beginning 2. Log phase—
reproduction is occurring at an exponential rate 3. Stationary phase—the environmental
surroundings and food supply cannot support any more exponential growth 4. Death phase—
when all the nutrients have been exhausted and the population dies off A variety of parameters
are important to influence the production of desired secondary metabolites in bioreactor cultures,
which include substrate, slow or fast utilization, N2 requirements, amino acid supplements,
temperature, pH, oxygen concentration, and pressure.

Mammalian Cell Culture

Cell culture is the process by which cells are grown under controlled conditions. In practice, the
term “cell culture” has come to refer to the culturing of cells derived from multicellular
eukaryotes, especially animal cells. Cells are grown and maintained at an appropriate temperature
and gas mixture (typically, 37°C, 5% CO2 for mammalian cells) in a cell incubator. Culture
conditions vary widely for each cell type, and variation of conditions for a particular cell type can
result in different phenotypes being expressed. Aside from temperature and gas mixture, the most
commonly varied factor in culture systems is the growth medium. Recipes for growth media can
vary in pH, glucose concentration, growth factors, and the presence of other nutrients. The growth
factors used to supplement media are often derived from animal blood, such as calf serum. Cells
can be grown in suspension or as adherent cultures. Some cells naturally live in suspension
without being attached to a surface, such as cells that exist in the bloodstream. There are also cell
lines that have been modified to be able to survive in suspension cultures so that they can be
grown to a higher density than adherent conditions would allow. Adherent cells require a surface,
such as tissue culture plastic or microcarrier, which may be coated with extracellular matrix
com ponents to increase adhesion properties and provide other signals needed for growth and
differentiation. Most cells derived from solid tissues are adherent. Another type of adherent
culture is the organotypic culture, which involves growing cells in a three-dimensional
environment as opposed to two-dimensional culture dishes. This three-dimensional culture
system is biochemically and physiologically more similar to in vivo tissue, but is technically
challenging to maintain because of many factors (e.g., diffusion).

Generation of Hybridomas
It is possible to fuse normal cells with an immortalized cell line. This method is used to produce
monoclonal antibodies. In brief, lymphocytes isolated from the spleen (or possibly from the
blood) of an immunized animal are combined with an immortal myeloma cell line (B cell lineage)
to pro duce a hybridoma that has the antibody specificity of the primary lymphocyte and the
immortality of the myeloma. Selective growth hypoxanthine-aminopterin-thymidine medium
(HAT medium) is used to select against unfused myeloma cells. Primary lymphocytes die quickly
in culture and only the fused cells survive. To start with, these are screened for production of the
required antibody, generally in pools, and then single cloning is done afterwards.

Industrial Plant Cell Culture

Plant secondary metabolites are important for plant interactions with their environments. These
metabolites give plants important properties such as antibiotic, antifungal, antiviral, UV
protection, and pest deterrence. These chemicals have also been used as medicines by humans for
thousands of years. Today, plants are responsible for the production of over 30,000 types of
chemicals, including pharmaceuticals, aromas, pigments, cosmetics, nutraceutics, and other fine
chemicals.

Bacterial/Yeast Culture Methods

For bacteria and yeast, small quantities of cells are usually grown on a solid support that contains
nutrients embedded in it, usually a gel such as agar, while large-scale cultures are grown with the
cells suspended in a nutrient broth.
Viral Culture Methods
The culture of viruses requires the culture of cells of mammalian, plant, fungal, or bacterial origin
as hosts for the growth and replication of the virus. Whole wild type viruses, recombinant viruses,
or viral products may be generated in cell types other than their natural hosts under the right
conditions. Depending on the species of the virus, infection and viral replication may result in
host cell lysis and formation of a viral plaque.

Downstream processing
The various processes used for the actual recovery of useful products from fermentation or any
other industrial process are called downstream processing (DSP).
Stages in DSP A widely recognized heuristic for categorizing DSP operations divides them into
four groups that are applied in order to bring a product from its natural state as a component of a
tissue, cell, or fermentation broth through progressive improvements in purity and concentration.
Removal of Insoluble
This first step involves the capture of the product as a solute in a particulate-free liquid, for
example the separation of cells, cell debris, or other particulate matter from fermentation broth
containing an antibiotic. Typical operations to achieve this are filtration, centrifugation,
sedimentation, 320 Biotechnology Fundamentals flocculation, electro precipitation, and gravity
settling. Additional operations such as grinding, homogenization, or leaching, which are required
to recover products from solid sources such as plant and animal tissues, are usually included in
this group.

Product Isolation
Product isolation is the removal of those components whose properties vary markedly from that
of the desired product. For most products, water is the chief impurity and isolation steps are
designed to remove most of it, reducing the volume of material to be handled and concentrating
the product. Solvent extraction, adsorption, ultrafiltration, and precipitation are some of the unit
operations involved.
Product Purification
Product purification is done to separate those contaminants that resemble the product very closely
in physical and chemical properties. Steps in this stage are expensive to carry out and require
sensitive and sophisticated equipment. This stage contributes a significant fraction of the entire
DSP expenditure. Examples of operations include affinity, size exclusion, reversed phase
chromatography, crystallization, and fractional precipitation.
Product Polishing
Product polishing describes the final processing steps which end with packaging of the product
in a form that is stable, easily transportable, and convenient. Crystallization, desiccation,
lyophilization, and spray drying are typical unit operations.

Antibiotic production
Antibiotics are produced industrially by a process of fermentation, where the source
microorganism is grown in large containers (100,000 – 150,000 liters or more) containing a liquid
growth medium. Oxygen concentration, temperature, pH, and nutrient levels must be optimal and
are closely monitored and adjusted if necessary. As antibiotics are secondary metabolites, the
population size must be controlled very carefully to ensure that maximum yield is obtained before
the cells die. Once the process is complete, the antibiotic must be extracted and purified to a
crystalline product. This is simpler to achieve if the antibiotic is soluble in organic solvent.
Otherwise, it must first be removed by ion exchange, adsorption, or chemical precipitation.
Hormones production
Recombinant DNA technology was used to produce large-scale quantities of human insulin (a
hormone) in E. coli as early as 1978. Previously, it was only possible to treat diabetes with pig
insulin, which caused allergic reactions in humans because of differences in the gene product. In
recent times, human growth hormone (HGH) has been used to treat growth disorders in children.
The HGH gene was cloned from a cDNA library and inserted into E. coli cells by cloning it into
a bacterial vector. The bacteria were then grown and the hormone isolated, enabling large scale
commercial production.
Vaccine production
Vaccines are weakened or inactive forms of microorganisms to mount the initial immune response
through the use of antigens, which are produced through use the genes of microbes that are cloned
into vectors. Vaccines contain attenuated disease-causing organisms or toxins that initiate an
immune response in the body. The first step in vaccine production is to prepare the antigen.
Viruses are grown in the primary or cultured cells, bacteria and yeast are grown in the bioreactors.
With the advent of biotechnology, recombinant antigenic protein is produced in bacteria or yeast.
Then antigen is isolated and inactivated, the recombinant protein is processed and ultra filtrated.
Then vaccine is prepared by adding adjuvant, preservatives and stabilizers. Adjuvants enhance
the immune response and stabilizers increase the shelf life. The final step in vaccine production
is packaging. Every vaccine has to be licensed by FDA before being brought into use.
 Fig: Steps of vaccine production

1. Microbial Production of Gluconic Acid:

1. Gluconic acid can be produced by a wide variety of prokaryotic and eukaryotic
microorganisms.
2. Bacterial species of the genera— Gluconobacter, Acetobacter, Pseudomonas, Vibrio.
3. Fungal species of the genera—Aspergillus, Penicillium, Gliocladium.

2. Production Process for Lactic Acid:

Hetero-fermentative bacteria—produce other byproducts, besides lactic acid, andtherefore

are not useful for industrial production of lactic acid. These bacteria are employed in food
or feed preservation.

Homo-fermentative bacteria—specialised for exclusive production of lactic acid and

therefore are suitable for industrial purpose.
The fermentation medium contains 12-15% of glucose, nitrogen and phosphate containing
salts and micronutrients. The process is carried out at pH 5.5-6.5 and temperature 45-50°C
for about 75 hours. Generally, the strains operating at higher temperature (45-60°C) are
preferred, since it reduces the need for medium sterilization.As the lactic acid is produced,
it has to be removed since it is toxic to the organisms. This can achieved either by a
continuous culture technique or by removal of lactic acid by electro dialysis. Theoretically,
every molecule of glucose forms two molecules of lactic acid. About 90% of theoretical
yield is possible in fermentation industry. L(+) Lactic acid is predominantly produced.