Code and Title of the Paper: F06FB Food Biotechnology

Code and Title of the Module: F06FB16 Fermentation Technology – definition, steps, bioprocess
Name of the Content Writer: Dr. D. Kavitha

Component – I

Role Name Affiliation

Principal Investigator Dr. S. Kowsalya Avinashilingam Institute for Home
Science and Higher Education for
Women, Coimbatore.
Co-Principal Dr. Sheela Ramachandran Avinashilingam Institute for Home
Investigators [Link] Subapriya Science and Higher Education for
Dr.G. Bagyalakshmi Women, Coimbatore.
[Link]
Paper Coordinator [Link] Avinashilingam Institute for Home
Science and Higher Education for
Women, Coimbatore.
Content Writer Dr.D. Kavitha Avinashilingam Institute for Home
Science and Higher Education for
Women, Coimbatore.
Content Reviewer Dr. K. Bhaskarachary National Institute of Nutrition
Jamai Osmania, Hyderabad – 500007
Language Editor Dr.D. Kavitha Avinashilingam Institute for Home
Science and Higher Education for
Women, Coimbatore.

Component-I (B) Description of Module

Items Description of Module

Subject Name Food and Nutrition
Paper Name Food Biotechnology
Module Name Fermentation technology – definition, steps, bioprocess
Module ID F06FB16
Pre-requisites Basic knowledge about culturing of microbes
Objectives · The learner will understand the steps in the bioprocess for the
production of products using integrated biochemical processes.
· The learner will be able to convert the promises of molecular
biology and genetic engineering into new processes to make
commercial products in economical feasible way
Keywords Cellular catalysts, fermentors, preservation, improvement, growth
phases, biomass
Code and Title of the Paper: F06FB Food Biotechnology
Code and Title of the Module: F06FB16 Fermentation Technology – definition, steps, bioprocess
Name of the Content Writer: Dr. D. Kavitha

Quadrant - I

6.16 Introduction

This module describes the fundamentals of fermentation technology, a multi-disciplinary area of

Biotechnology which involves the skills in science and engineering. Fermentation technology is
ubiquitous - they range from the production of food in a kitchen to the synthesis of sophisticated,
extremely high-value, cutting-edge therapeutics. The principle of bioprocess technology involve
the use of cells as micro-factories to manufacture the product of interest. The cellular products
can be directly utilized or, alternatively, the cells can be engineered to produce products of
interest. However, the production levels can be improved by employing several strategies for
formation or processing of the product.

6.16.1 Objectives

· The learner will understand the steps in the bioprocess for the production of
products using integrated biochemical processes.
· The learner will be able to convert the promises of molecular biology and genetic
engineering into new processes to make commercial products in economical
feasible way

6.16.2 Definition

Fermentation Technology is defined as any process that produces a product by the mass culture
of a microorganism. Also, its biochemical meaning is an energy–generation process in which
organic compounds act as both electron donors and terminal electron acceptors.

6.16.3 History of Fermentation technology

The cultivation of microorganisms to produce useful products or to undertake useful activities is

however not a recent invention. Fermented foods such as yoghurt, cheese, soya sauce, pickled
cabbage have been intentionally produced by man for centuries. Beer recovered from the
pyramids of the ancient Egyptians reveal that the Egyptians were able to produce the fermented
brew using almost pure cultures of yeasts. In Renaissance Europe, nitrates required for gun
powder manufacture were produced by allowing soil bacteria to nitrify ammonia in horse urine,
as it was flowed down channels dug into hills. The Aztecs of Mexico cultured Spirulina (a
singled cell alga) for both waste treatment purposes and as a source of protein. In the 20th
Code and Title of the Paper: F06FB Food Biotechnology
Code and Title of the Module: F06FB16 Fermentation Technology – definition, steps, bioprocess
Name of the Content Writer: Dr. D. Kavitha

century, biotechnologists have tried again to produce single celled protein to solve the world’s
protein shortage.

The father of modern fermentation technology was however Louis Pasteur. This 19th Century
scientist discovered or perhaps re-discovered that the conversion of sugar to alcohol (in beer and
wine production) and the conversion of sugar to lactic acid (as occurs in cheese production) were
both microbiological conversions and both needed a microbial "seed" to start them. In the
process, Pasteur disproved the theory of spontaneous generation, discovered that some microbes
can grow in the absence of oxygen, and discovered a new way of sterilizing materials which was
subsequently called "pasteurization".

Following Pasteur, microbiology and fermentation technology grew hand in hand. Alexander
Fleming's discovery of penicillin eventually lead to the development of a huge antibiotic industry

The world wars and their aftermath of the 20th Century continued to spur on developments in
fermentation technology. Material shortages forced countries to look to biological sources of
chemical feedstocks. Scientists found ways of producing or enhancing the production of ethanol
and solvents such as acetone and butanol by fermentation. Antibiotic production was also scaled
up to meet growing needs derived from diseases and injuries..

The designs and technologies developed for improving antibiotic production found application in
many other products such as amino acids (e.g. lysine and glutamic acid) and food chemicals e.g.
gluconic acid and citric acid. The engineer and the scientist now had to work hand in hand to
achieve these improvements.

In more recent times, threats of diseases, environmental disasters, food shortages and juxtaposed
by growing demand in developed countries have created new opportunities for fermentation
technologists. Today, the fermentation industry is a multi-million dollar industry.

6.16.4 Basic principles of Fermentation

In developing a product, a fermentation technologist must consider all stages of the

manufacturing process. At the heart of the process is the fermenter. Large scale cell cultivation
is performed in specialized reaction vessels known as bioreactors or fermenters.

In the fermenter, cells convert raw materials (or substrates) to products and to more cells:
Code and Title of the Paper: F06FB Food Biotechnology
Code and Title of the Module: F06FB16 Fermentation Technology – definition, steps, bioprocess
Name of the Content Writer: Dr. D. Kavitha

The conversion of substrate to more cells is a major advantage of fermentation processes over
enzymatic and chemical processes. The production of more cells means that during a
fermentation, the concentration of catalyst (in this case the cells) can increase with time and the
process can actually speed up with time.

CELLULAR CATALYSTS

The major types of cells used in fermentation processes include:

Types of
Examples
cells

Chinese Hamster Ovary Cells

Animal
Hybridoma cells
cells
Insect cells

Penicillium spp.
Moulds
Aspergillus spp.

Saccharomyces spp.
Yeasts Saccharomycopsis spp.
Candida spp.

Escherichia coli, Bacillus spp.

Bacteria
Pseudomonas spp., Streptomyces spp.

6.16.5 Types of fermentation processes

Code and Title of the Paper: F06FB Food Biotechnology
Code and Title of the Module: F06FB16 Fermentation Technology – definition, steps, bioprocess
Name of the Content Writer: Dr. D. Kavitha

The commercially important fermentations are divided into five major groups

(i) The process that produce microbial cells

(ii) The process that produce microbial enzymes
(iii) The process that produce microbial metabolites
(iv) The process that produce recombinant products
(v) The transformation process

Apart from these types of processes, any established fermentation technique may be divided
into six basic component parts.

· Media formulation for culturing the process microorganism

· The sterilization of the medium, fermenters and accessories
· Active and pure culture production for inoculation in production vessel
· Growth of the organism under optimum conditions for product formation
· Extraction and purification of the product
· Effluent disposal produced by the process
Code and Title of the Paper: F06FB Food Biotechnology
Code and Title of the Module: F06FB16 Fermentation Technology – definition, steps, bioprocess
Name of the Content Writer: Dr. D. Kavitha

There are a number of steps involved in studying a fermentation process. The process
will initially be studied in laboratories using flasks and laboratory scale fermenters. If the process
looks commercially successful, the process will be "scaled-up" to a pilot scale process. During
this stage, the process will be looked at in terms of engineering factors such as "mass transfer"
and "heat transfer". Downstream processing will also be looked into. The pilot scale studies will
determine the commercial viability of the process and if successful the process will be scaled up
to industrial/commercial scale.

Some fermentation processes however can involve the complex interactions of biological,
chemical and physical factors. To properly investigate a fermentation and to be able to predict
the effects that these factors play on fermentations, the process needs to be broken down into
meaningful units.

6.16.6 Various modes of operation

Fermentations can be carried out as batch, continuous and fed-batch processes.

[Link] BATCH CULTURE

Batch culture fermentation is a closed system where the required amount of nutrients and other
additives are added at the beginning of the process.
Code and Title of the Paper: F06FB Food Biotechnology
Code and Title of the Module: F06FB16 Fermentation Technology – definition, steps, bioprocess
Name of the Content Writer: Dr. D. Kavitha

Once the fermentation process has started there is no refill of nutrients and the product is
recovered only at the end of the process. At the end of the fermentation, the contents are
removed for down stream processing. The reactor is then cleaned, sterilized and refilled for the
next fermentation.

During the process, the inoculated microorganisms will pass through a number of phases.

· Lag phase : First there is a period during which growth does not occur referred to as the
lag phase. Lag phase is a time of adaptation.
· Log phase : Following the lag phase, the growth rate of the cells gradually increases. The
cells grow at a constant and maximum rate and the period is known as log phase or
exponential phase.
· Stationary phase : Eventually the growth rate has declined to zero and the cells entered
the so-called stationary phase.
· Decline phase: After a further period of time, the culture enters the death phase and the
viable cell number declines.

During the exponential phase, certain products are produced that are essential for the cell
growth namely aminoacids, nucleotides, proteins, lipids, carbohydrates etc. These
primary metabolites are produced during the log phase. Hence it is the task of the
industrial microbiologist to improve the productivity of these primary metabolites. The
secondary metabolites are synthesized during the stationary phase.
Code and Title of the Paper: F06FB Food Biotechnology
Code and Title of the Module: F06FB16 Fermentation Technology – definition, steps, bioprocess
Name of the Content Writer: Dr. D. Kavitha

Thus, batch fermentation can be used to produce biomass, primary metabolites and secondary
metabolites.

[Link] CONTINUOUS CULTURE

In continuous cultures, fresh media is continuously added into the bioreactor and at the same
time bioreactor fluid is continuously removed.

The cells thus continuously propagate on the fresh medium entering the reactor and at same time,
products, metabolic waste products and cells are removed in the effluent.

Continuous cultures have however been with us almost since life began on good old mother
earth. Our guts are in many ways a continuous culture. Food comes in, gets processed by
microorganisms and enzymes, some is absorbed, the rest is excreted.
Code and Title of the Paper: F06FB Food Biotechnology
Code and Title of the Module: F06FB16 Fermentation Technology – definition, steps, bioprocess
Name of the Content Writer: Dr. D. Kavitha

The important feature of this technique is that the microbial growth in a continuous culture takes
place under steady-state conditions; that is, growth occurs at a constant rate and in a constant
environment. Many factors as pH, concentrations of nutrients, metabolic products and oxygen,
which inevitably change during the growth cycle of a batch culture, are all maintained constant
in a continuous culture.

Today, continuous cultures find wide application in the waste treatment industry although to
date, limited application in fermentation industry. Continuous culture systems have many
advantages over batch reactors, but unfortunately a few critical disadvantages.

Comparison of batch and continuous cultures

Continuous cultures have several advantages over batch cultures:

· It is not necessary to shut the continuous fermenter down as frequently as a batch

fermenter. At the end of a batch fermentation, the reactor must be emptied, cleaned,
sterilize and re-filled. The time required for these operations is known as turnaround
time. Theoretically, a continuous fermenter could operate indefinitely without having to
be shut down. In practice, however this is not possible.

· Because continuous cultures are shut down with less frequency as compared to batch
reactors, there is less loss of productivity during lag phases
· Most downstream processing operations are most productive when operated in a
continuous manner. Using a continuous culture allows the fermentation to be in-tune with
other operations in the plant. Thus, overall plant productivity is easier to optimize.

The disadvantage of a continuous culture is

· Contamination of a continuous fermenter can have disastrous consequences. A

contaminant can cause the wash out of the resident organism and then completely take
over a fermenter.

[Link] FED BATCH CULTURE

Code and Title of the Paper: F06FB Food Biotechnology
Code and Title of the Module: F06FB16 Fermentation Technology – definition, steps, bioprocess
Name of the Content Writer: Dr. D. Kavitha

In a fed batch fermenter, the feed is continuously added until the maximum liquid fermenter
volume is reached. The fermenter may be then allowed to continue or be partially or completely
emptied depending on the process.

Although low nutrient and substrate concentrations can be maintained in continuous cultures, as
we have seen, continuous reactors do suffer from a number of significant problems.

In fed batch reactors, feed is added, but effluent (and cells) are not removed. Thus fed-batch
reactors can be used to maintain cells under low substrate or nutrient conditions without washout
occurring.

Fed batch reactors are thus primarily used for producing products under low nutrient or substrate
conditions and play an important role in industry.

Comparison of fed-batch and continuous bioreactor

· With both fed-batch and continuous fermenters, it is possible to set and maintain the
specific growth rate and substrate concentration at optimal levels.
· The major physical difference between a fed-batch and continuous fermenter is that the
effluent is not continuously removed and thus washout does not occur.

This provides a fed-batch reactor a number of advantages over continuous reactors:

· Because cells are not removed during the fermentation, fed-batch fermenters are well
suited for the production of compounds produced during very slow or zero growth.
· Unlike a continuous fermenter, the feed does not need to contain all the nutrients needed
to sustain growth. The feed may contain only a nitrogen source or a metabolic precursor.
Code and Title of the Paper: F06FB Food Biotechnology
Code and Title of the Module: F06FB16 Fermentation Technology – definition, steps, bioprocess
Name of the Content Writer: Dr. D. Kavitha

· Contamination will also not have the same dramatic effect on a fed-batch fermenter as a
contaminant will not be able to completely take over the fermenter (unless the
contamination occurred during the early stages of the fermentation).

Of the three systems,

· Fed batch bioreactors are most commonly used to produce biological products.
· Batch reactors are the second most commonly used.
· Although continuous reactors are rarely used for large scale production of biochemicals,
they are widely used in waste treatment processes. In addition, natural ecosystems, such
as the rumen and gut, soil and rivers, are more closely represented by a continuous
culture system

6.16.7 Screening of industrially important microorganisms

The tasks of both discovering the new microbial compounds and improving the synthesis
of known strains have become more and more [Link] microorganisms of industrial
importance are generally, bacteria, actinomycetes, fungi and algae. These organisms occur
virtually everywhere, e.g., in air, water and soil, on the surfaces of plants and animals, and in
plant and animals tissues. But most common sources of industrial microorganisms are soils, lake
and river mud.

There are three important stages in the screening strategies namely isolation, preservation and
improvement of the strains of commercial importance

(i) Isolation

Involves obtaining either pure or mixed cultures based on their production of industrially
relevant products. A variety of complex isolation procedures have been developed, but no single
method can reveal all the microorganisms present in a sample. Specialized enrichment
techniques are used to isolate many different microorganisms. Selective pressure can be used in
the isolation of organisms to favour the growth of the desired types or addition of inhibitors to
destroy the undesired strains.

The next step after isolation of microorganisms is their screening. A set of highly selective
procedures, which allows the detection and isolation of microorganisms producing the desired
metabolite, constitutes primary screening. Ideally, primary screening should be rapid,
inexpensive, predictive, specific but effective for a broad range of compounds and applicable on
a large scale. Primary screening is time-consuming and labour intensive since a large number of
isolates have to be screened to identify a few potential ones.
Code and Title of the Paper: F06FB Food Biotechnology
Code and Title of the Module: F06FB16 Fermentation Technology – definition, steps, bioprocess
Name of the Content Writer: Dr. D. Kavitha

Rapid and effective screening techniques have been devised for a variety of microbial products,
which utilize either a property of the product or that of its biosynthetic pathway for detection of
desirable isolates. Some of the screening techniques are relatively simple, e.g., for extracellular
enzymes and enzyme inhibitors.

Suitably designed specialized screening techniques may be used to detect compounds having
various pharmacological activities (other than antibiotics).

A sample of the screening approaches used for detection of microbial isolates

producing useful products

Therefore, it is necessary to preserve the isolated strain without loosing its desirable
characteristics.

(ii) Preservation techniques

The isolated cultures can be stored by various methods

· Storage at reduced temperature

Ø If the cultures are subcultured at 6-month interval, it can be stored on agar slopes
in refrigerator at 4°C or in freezer at -20°C. The time of subculture may be
extended to 1 year if the cultures are covered with mineral oil
Code and Title of the Paper: F06FB Food Biotechnology
Code and Title of the Module: F06FB16 Fermentation Technology – definition, steps, bioprocess
Name of the Content Writer: Dr. D. Kavitha

Ø For the long term preservation of cells that do not survive freeze drying, liquid
nitrogen (-156°C to-196°C) is the suitable method for preservation of valuable
stock cultures. This technique involves growing a culture to the maximum
stationary phase and then resuspending the cells in a cryoprotective agent such as
10% glycerol
· Storage in a dehydrated form
Ø Dried soil cultures are commonly used especially for sporulating mycelia
organisms
Ø Lyophilization technique is used to store the culture in refrigerator for 10 years.
This is done by freezing the culture followed by its drying under vaccum leading
to the sublimation of water

The industrial cultures must be stored to eliminate genetic change, protect against contamination
and retain viability.

(iii) Improvement

Wild type microbes usually produce a very low compound titre. Therefore, In order to increase
the productivity of the wild strain, various improvement strategies are adopted. Traditional
methods for strain improvement are random mutagenesis and selection. The selected strains are
usually subjected to successive cycles of mutagenesis and selection; after several cycles, a large
increase is yield is likely to be [Link] a random approach is often a good start and
sufficient to generate a high producing strain.

Increased yields may be achieved by optimizing the culture medium and growth conditions, but
this approach will be limited by the organism’s maximum ability to synthesize the product. The
potential productivity of the organism is controlled by its genome, therefore, the genome must be
modified to increase the potential yield.

The techniques and approaches used to genetically modify strains, to increase the production of
the desired product are called strain improvement or strain development.

Genetic modification may be achieved by selecting natural variants, by selecting induced

mutants and by selecting recombinants. There is a small probability of a genetic change
occurring each time when a cell divides, hence a microbial culture will undergo a vast number
of such divisions, the culture will become more heterogeneous.. However, variants have been
isolated which are superior producers and this has led to the development of a natural product
from a newly isolated organism. Recombinant DNA technology has enabled the production of
heterologous products and has built on the achievements of directed selection to increase yields
of conventional products still further.
Code and Title of the Paper: F06FB Food Biotechnology
Code and Title of the Module: F06FB16 Fermentation Technology – definition, steps, bioprocess
Name of the Content Writer: Dr. D. Kavitha

6.16.8 Conclusion

This module has presented the overview of fermentation technology where the microorganisms
are capable of growing on a wide range of substrates and can produce a spectrum of
commercially valuable products. Bioprocess technolgy deals with various biotechnological
processes used in industries for large scale production of biological product for optimization of
yield in the end product and the quality of end product. Further the bioreactor design and
downstream processing will be discussed in the next modules