UNIT 3 FOOD FERMENTATION TECHNOLOGY

Structure
3.0 Objectives
3.1 Introduction
3.2 Fermentation methodology
3.2.1 Primary metabolites
3.2.2 Secodory metabolites
3.3 Industrial bioprocesses, fermentation processes, and its operations
3.3.1 Batch fermentation
3.3.2 Fed-batch fermentation
3.3.3 Continuous fermentation
3.4 Basic designs of bioreactors and their types
3.4.1 Key concepts of bioreactor design
3.4.2 Structural components of the bioreactors
3.4.3 Types of bioreactors
3.5 Starter cultures
3.5.1 Single strain starter culture
3.5.2 Mixed pure culture
3.5.3 Mixed natural culture
3.6. Strain improvement
3.6.1 Mutation
3.6.2 Protoplast fusion
3.6.3 Recombination
3.6.4 Recombinant DNA Technology
3.7 Let Us Sum Up
3.8 Key Words
3.9 Answers to Check Your Progress Exercises
3.10 References/Suggested Readings

3.0 OBJECTIVES
After studying this Unit, you will be able to:
 Explain the basic technical concepts of fermentation technology;
 Describe designing and constructing different types of bioreactors;
 Differentiate various types of fermentation processes and operations involved in the
production of various types of value-added products;
  Elaborate starter cultures along with their significance; and
 Narrate various techniques for microbial strain improvement for fermentation.

3.1 INTRODUCTION
In the 4th Unit on “Beneficial role of microorganisms” of the course MVPI 001, you have
studied about the basics of food fermentation, their types and some common fermented foods.
So, you know that fermentation is a metabolic process in which natural sugars are into
alcohol or acid and/or carbon dioxide and hydrogen gas depending on the type of organisms,
substrate, and other process conditions. For example, yeast converts glucose into ethanol and
carbon dioxide without oxygen (i.e. anaerobic process), whereas lactic acid bacteria convert
milk sugar-lactose into lactic acid. The term “ferment” is derived from the Latin word
“fervere” which means “to boil”. The ancient people knew it and applied this process to make
different products-beverages, etc.
Fermenting food is an art, and various fermented foods are available globally. Every country
or region has its own type of fermented foods based on its local population's staple diet and
availability of raw materials. Nowadays, fermented foods have become a part of the
countries' cultural traditions. For example, certain popular milk-based fermented foods, such
as dahi, lassi, makkhan, etc. are prepared and consumed in India and considered part of
Indian mythology. Fermented foods are prepared from various raw materials like milk,
vegetables, fruits, cereals, legumes, meat, and fish. The production process of fermented food
preparation evolved from early civilisation to now and will be continued as well. Their
processes were standardised for commercial production based on the available scientific
evidence and expertise acquired with time. These are produced by bioconversion of raw
materials from different sources through microbial activities. The resultant product varies in
texture, flavour, stability, and nutritional value compared to the original raw materials. Most
of the fermentation processes are carried out by Lacto-fermentation, where natural flora
utilises sugar and starch of raw materials to produce lactic acid along with the production of
valuable enzymes, vitamins (B-vitamins), Omega-3 fatty acids, etc. The lactic acid serves as
a natural preservative in the product, thus extending the shelf-life in addition to beneficial
effects. Here, it is very important to note that the quality of fermented foods is dependent on
the below-mentioned factors (Fig.3.1):

Fig.3.1: Factors affecting the quality of fermented food

The demand and market potential for fermented foods is always rising because of their
claimed nutritional values and health benefits. Traditionally, the fermented process is initiated
by their natural microflora or present surrounding the food and as a result of spontaneous
fermentation, food acquires certain additional organoleptic and nutritional properties.
However, researchers have attempted to isolate and characterise the microorganisms involved
in the fermentation process over time. It was observed that different microbial groups, such as
bacteria, yeast, and molds were actively involved in the conversion process. Among the most
important bacterial groups that bring fermentation is “lactic acid bacteria (LAB)”, which are
most widely accepted and generally regarded as safe (GRAS) for human consumption.
The use of microorganisms in large-scale industrial production of food, pharmaceuticals, and
alcoholic beverages is known as fermentation technology. This unit will help you to learn in
detail about the fermentation methodology, industrial bioprocesses, bioreactors used for food
fermentation, starter cultures and microbial strain improvement. The fundamental concept
behind industrial fermentation technology is that organisms can grow in ideal environments
and utilise raw materials that meet all of the essential “carbon, nitrogen, salts, trace elements,
and vitamins”. The end products produced by their metabolism during their life cycle are
released into the fermentation medium, where they are recovered for human use and have a
high economic value. Beer, wine, cider, ethanol, vinegar, cheese, hormones, antibiotics,
complete proteins, enzymes, etc., are among the industrial products of fermentation
technology.

3.2 FERMENTATION METHODOLOGY

The fermentation process takes place in a specialised vessel known as a “bioreactor or
fermenter”. The fermenter or bioreactor's design varies depending on the kind of fermentation
process used.
Regardless of the kind of fermentation, a well-established fermentation process may be
achieved through the below-listed steps:
i. i. Selection of microorganisms for fermentation
ii. ii. Medium formulation for cultivating microorganisms during inoculum formation
and in the production fermenter.
iii. iii. Sterilisation of the medium, fermenter, and other equipment associated with the
process.
iv. iv. Production process under controlled conditions
v. v. Product’s extraction and purification
vi. vi. Effluent disposal
During fermentation, several metabolites are produced by the microorganisms, which play a
significant role in the microorganism's metabolism to maintain life. Further, the metabolites
may be classified into below mentioned two categories.
3.2.1 Primary Metabolites
The primary metabolites are key components in maintaining the normal physiological
function of the organisms and are generally produced during the growth phase.
For example:
Some amino acids, vitamins, lactic acid, ethanol, etc., are recognised as primary metabolites.
3.2.2 Secondary Metabolites
The secondary metabolites do not play a role in microbial growth, development and
reproduction but are considered to play a role in ecological functions such as defense
mechanisms of the microorganisms. They are produced during the stationary phase of
microbial growth.
For examples:
Antibiotics, pigments, toxins, alkaloids, etc.
Check Your Progress Exercise 1
Note: a) Use the space below for your answers.
b) Compare your answers with those given at the end of the unit.
1) Fill in the blanks with appropriate words.
i. The specialised vessel where fermentation process takes place is known as………….
ii. The fermentation processe where natural flora utilises starch of raw materials to produce
lactic acid is known as……………….
2) Enlist various steps of a fermentation process.
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3) What are the factors that affect quality of fermented food?
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4) Differentiate between primary and secondary metabolites.
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3.3 INDUSTRIAL BIOPROCESSES, FERMENTATION

PROCESSES, AND ITS OPERATIONS
The fermentation process at a large scale is also termed as “industrial fermentation”, which
begins with the selected microorganism’s growth under controlled fermentation conditions. A
vast range of industrially important chemicals and compounds are being produced through
bioprocesses. Generally, bioprocess technology is divided into “upstream processes,
bioreactions, and downstream processes” (Fig. 3.2). The bioreactors are the key components
of bioprocesses technology, where biomass generation, metabolite biosynthesis, and
biotransformation occur. The numerous process factors that affect bioprocessing, the ideal
condition of each factor, and their interaction effect must be analysed to build a cost-effective
process. Further understanding and improving the separate functioning units, laboratory-scale
studies must be conducted.

Fig. 3.2: A schematic overview of the fermentation process.

Interestingly, the fermentation processes or bioprocessing consume less energy than

conventional procedures and often employ low-cost raw materials; in certain circumstances,
products cannot be made using chemical methods. The following products or metabolites are
examples of this technology:
 Enzymes
 Proteins
 Antibiotics
 Organic acids
 Pigments
 Biopolymers, etc.
As mentioned above, products/metabolites are an outcome of a closely monitored and
managed system accomplished by keeping track of and regulating critical factors during
fermentation and devising an effective control plan.
The primary types of industrial fermentation processes based on modes of operation are listed
below (Fig. 3.3):
 Fig. 3.3: The types of industrial fermentation processes based on modes of operation.

3.3.1 Batch fermentation

Batch fermentation is an example of a closed system in which materials (i.e. fermentation
medium, microbial culture, etc.) are fed into the bioreactor/fermenter and sanitised before the
start of the process. During the fermentation process, no addition and recovery of the end
product or byproduct is done; the end products are recovered only after the completion of the
fermentation process. As mentioned, the end/desired products, whether intracellular or
extracellular, are removed after the operation. The key disadvantage of the batch technique is
the significant downtime between batches, which necessitates bioreactor charge and
discharge, cleaning, sterilisation, and process restart. However, to obtain maximum product
recovery, the optimisation of pH and temperature should be considered.
Batch fermentation is the most widely used in the food industry to produce organic acids such
as lactic acids, citric acids, and acetic acids that are intended to be used as preservatives or
acidifiers. Moreover, this process is also used to produce alcoholic beverages, i.e. wine, beer,
and distilled spirits such as brandy, whisky, and rum, as well as sweeteners (such as
aspartate) and amino acids that are used as flavouring agents (e.g., monosodium glutamate).
Practically, the batch fermentation process has many disadvantages:
i. The microorganisms are continually exposed to a variety of fermentation variables
due to nutrient consumption and there is problem of waste deposition.
ii. Batch cultures need to be restarted after reaching an endpoint.
iii. The large bioreactors require a long time to empty, clean, and refill.
iv. Due to the significant downtime (spent washing, sterilising, and beginning another
batch of culture during non-production time) between two batches, batch culture has
low productivity. However, rarely, toxic metabolites might hinder cell growth and
product synthesis.

3.3.2 Fed-Batch Fermentation

In this fermentation process, one or more nutrients are added into the bioreactor along with
the microbial culture in an incremental pattern. However, the end products are kept in the
bioreactor till the completion of the fermentation process. Because of this technique's ability
to regulate the medium's substrate concentration, it has several advantages. The simplest fed-
batch culture is one in which a growth-limiting substrate is delivered constantly. In industrial
bioprocesses, fed-batch processes are widely utilised to obtain a high cell-density culture.
This kind of fermentation is more efficient, delivers better results with controlled sequential
nutrient inputs and higher cell densities, and allows for longer product synthesis in fed-batch
cultures.
As the substrates are used as a limiting factor to restrict or control the fermentation, hence
need to be added in small volumes throughout the production process to maintain consistency
in producing the desired product. Although substrates could be added during the process, the
byproducts are removed only after fermentation. However, if required, the desired or end
product may be recovered during the fermentation. As a result, the fermentation broth volume
increases throughout the process until it reaches its full capacity.
This type of fermentation technique has the following benefits over batch culture:
 The continuous product synthesis, by increasing the number of cells and hence the
quantity of product produced, the process may be made more efficient. As it is
proportional to the biomass concentration, it enables bioreactors to be used for
production during non-profitable periods rather than being ready for the next batch,
and increasing productivity and yield with the sequential addition of nutrients in a
controlled manner.
 Interestingly, the fermentation substrate (medium) is not eliminated throughout the
fermentation process using a fed-batch technique, but the amount of limiting nutrients
provided restricts the pace of the reaction.
There are several advantages of fed-batch fermentation. However, the key advantages of fed-
batch fermentation are listed below:
 A high cell density can be achieved during fermentation
 Allows evaporation to compensate for the loss of water.
 The viscosity of the fermentation medium/broth may be reduced/increased or adjusted
if required at any time during the fermentation.
 The fermentation process has become economically viable as the initial step is similar
to batch fermentation, production medium/substrates are introduced from the start of
the fermentation, and after the completion of the process, the fermentation
medium/broth with microbial culture is either fully or partially collected. In case it is
partially recovered, the remaining medium may be utilised as the inoculum for the
next repeated run. Further, a substrate is needed to be added in increments to initiate
this fermentation.
The types of fed-batch fermentation are given below (Fig.3.4):

Fig. 3.4: Various types of fed-batch fermentation

 i. i. Fixed volume fed-batch culture: It entails delivering the limiting substrate into
the fermenter in such a concentrated manner that volume will not increase. The
growth-limiting substrate (nutrient) must be supplied aseptically in a concentrated
liquid or gas form to maintain a nearly constant culture volume. For example, a
constant-volume fed-batch system is practised for the cultivation of hyperthermophile
Archaebacteria under aerobic conditions.
ii. ii. Variable volume fed-batch culture: As the name suggests, the working volume
keeps changing as the fermentation proceeds during this kind of fed-batch
fermentation.
iii. iii. Single-fed-batch process: The feeding mixture is introduced throughout
fermentation, but the wasted broth is not eliminated. Due to this approach, a large
portion of the fermentation broth is not used until the completion of the process. As a
result, the reactor capacity might be a significant limiting factor in the fermentation's
duration.
iv. iv. Cyclic or repeated fed-batch culture: As the fermentation reaches the stationary
phase and the cells' growth does not alter significantly (either due to depletion of
nutrient/substrate, or end-product inhibition), a part of the fermenter's discarded broth
is removed, followed by the addition of fresh fermentation medium. Subsequently, the
substrate concentration in the fermenter rises due to the removal of the end product
and the addition of fresh medium, resulting in a higher specific growth rate. Further,
once the substrate is used or depleted, the specific microbial growth rate drops and a
quasi-steady state is reached. For example, through a phosphorus-feeding approach,
the synthesis of penicillin G is optimised.

Various industrially important products or metabolites are produced by fed-batch

fermentation:
 Amino acids- Lysine
 Antibiotics-Penicillin
 Enzymes- amylases
 Solvents- Butanol
 Microbial cell mass- Saccharomyces
3.3.3 Continuous Fermentation
The continuous fermentation process is an example of an open system setup that involves
continually feeding nutrients to the fermenter while continuously withdrawing the microbial
cells, desired/end products, other metabolites, and toxic/waste materials; thus, the volume of
the fermentation medium/culture broth stays constant due to a steady feed-in and feed-out
pace as the depleted nutrients are replenished with fresh nutrients replaced and toxic
metabolites are removed from the culture as and when required.
During continuous fermentation, one or more feed ports are used to add nutrients to the broth
constantly, and the effluent contains microbial cells, products, and residuals continuously.
Finally, a steady state is achieved by maintaining the same volumetric flow rate for the feed
and effluent ports. Moreover, the microbial culture volume is maintained constant, and all
nutrient concentrations are kept at steady-state values. Therefore, the exponential growth
phase is extended throughout the fermentation process, and simultaneously the generated
byproducts are eliminated.
The monitoring of microbial growth activities or formation of the desired product in
continuous fermentation is performed by the below-listed processes:
A. Turbidostat method
In this method, the microbial cell growth is controlled and stabilised by manipulating the
flow rate of adding a fresh medium. For example, the fresh medium is constantly supplied,
and microbial cell density is controlled by a specified turbidity value determined by the cell
population. Suppose turbidity of the medium tends to increase, the feed (addition of fresh
medium) is increased to dilute back to a desired point, i.e. set point, or if turbidity of the
medium tends to decrease, in that case, the feed rate of the fresh medium is lowered so that
microbial growth can be restored turbidity to the desired point (set point).
B. Chemostat method
During the chemostat, nutrients are continuously delivered at a constant flow rate, and the
density of the microbial cells is adjusted as per the nutritional requirement for growth given.
The concentration of substrates, i.e. carbon, nitrogen, and phosphorus, are used to regulate
the growth rate of a chemostat.
The following fermentation conditions need to be maintained while performing continuous
fermentation either using chemostat or turbidostat methods:
 The steady state of microbial growth is to be maintained by keeping constant cell
density throughout the fermentation process.
 The continuous replacement of fermentation medium and microbial cells is needed.
Continuous fermentation has numerous industrial applications, such as the production of
Single-cell protein, organic solvents, starter cultures, and other products. It is also used in
synthesising secondary metabolites, including production antibiotics from a Penicillium or a
Streptomyces species.
Check Your Progress Exercise 2
Note: a) Use the space below for your answers.
b) Compare your answers with those given at the end of the unit.

1) Describe industrial fermentation processes based on modes of operation.

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2) What are the disadvantages of batch fermentation?
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3) Differentiate between turbidostat and chemostat methods of fermentation.
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4) Name industrially important products produced by fed-batch fermentation.
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3.4 BASIC DESIGNS OF BIOREACTORS AND THEIR

TYPES
A bioreactor or fermenter is a fermentation tank or vessel that may produce valuable
metabolites/food products through certain microbial cultures under control conditions. These
devices are used to produce microbial biomass, industrially important metabolites, enzymes,
antibiotics, etc. The fermenter is designed in such a way that it has a provision for sufficient
aeration, agitation, temperature, and pH control, as well as a drain or overflow vent for
removing the waste biomass of cultivated microorganisms and their products.
3.4.1 Key concepts of bioreactor design
In fermentation, the starter culture or microorganisms plays a significant role in the
biochemical conversion of substrate to a desired metabolite or end product. However, these
conversions can be achieved only under controlled conditions, i.e. pH, temperature, oxygen
concentration, etc., which are to be maintained throughout the production process. Hence,
such conditions must be created to yield the maximum productivity of microbial culture.
Therefore, the operation and scale of production are dependent on the design of the
fermenter. An excellent fermenter design will aid in increasing productivity and delivering
higher-quality products at cheaper costs in a short time.
An ideal bioreactor or fermenter should have the following:
i. In case of aerobic fermentation, an aeration port is required to ensure sufficient
oxygen supply during the fermentation.
ii. There should also be a provision of ports for the addition of acid, base, nutrients,
etc., and ports for removal of spent medium and or recovery of microbial cells as
well as sampling as per the need of fermentation.
iii. Provision of agitation for uniform mixing of microbial cells and fermentation
medium components during the process.
iv. Need for installation of heating devices.
 v. The controlling devices or sensors for the regulation of dissolved oxygen, pH,
temperature, aeration, nutrients, etc., should be in place.
vi. As the entire fermentation process is to be carried out under aseptic conditions,
hence there should be a provision for sterilisation and maintenance of sterility
facilities.
The fermenter's size varies greatly depending on the use. For example, some fermenters are
built for small-scale fermentation and others for large-scale (industrial applications), ranging
from the shaking flask (100-1000 ml) to the laboratory-size fermenter (1–50 L) to the pilot
level (10-350 L) to the plant scale (more than 350 L) for a large volume.

3.4.2 Structural components of the bioreactors

Basic components such as flow rates, aeration, temperature, pH, foam control, agitation rate,
etc., are needed in most bioreactors. However, additional parameters, sensors, and other
controlling devices may be installed depending on the production requirements.
Ideally, a fermenter has an agitator system, an oxygen supply system, a foam control system,
and a variety of other systems such as temperature and pH control, sampling ports, cleaning
and sterilisation systems, and lines for charging and emptying the reactor. The material used
in constructing a bioreactor/fermenter should not be corrosive, sustain high pressure and
provide efficient steam sterilisation.
An ideal bioreactor should be equipped with below listed basic components, accessories,
instrumentation, and sensors (Fig. 3.5):

Impeller

Fig.3.5 Schematic illustration of a typical bioreactor

Source: [By GYassineMrabetTalk- Own work, CC BY-SA 3.0,
https://commons.wikimedia.org/w/index.php?curid=8301774]
 i. Fermentation vessel: It is a large cylinder with top and bottom closures with
various tubes and valves. It is made up of either double-jacketed glass or stainless
steel. However, as per the need and suitability of various materials are also being
used in the construction of fermenters.
ii. Sparger (aeration system): It is one of the most important parts of a fermenter as
it is very important to make a provision of supply of sterile air to ensure optimal
aeration and oxygen availability to the culture throughout the fermentation. The
key function of spargers is to ensure efficient dispersal of filtered air to the
impeller.
iii. Heating and cooling device: The exterior of the vessel is covered with a double
jacket with two ports, in which cold water is circulated to reduce the temperature
to a set point if needed. In addition, thermostatically controlled baths or internal
coils are often utilised to generate heat as per the requirement.
iv. Baffles: Inside the vessel, the baffles are used to prevent vortex formation and
improve aeration and is made up of radially fastened metal strips on the wall. It
also prevents sedimentation on the side walls. The baffle's numbers and design
may vary; however, 4, 5 or 8 may generally be used. The size is about 1/10th of the
fermenter vessel diameter.
v. Impellers: These are used to maintain a consistent suspension of microbial cells
and uniform distribution of air in the fermentation medium/broth throughout the
operation. The blades of the impeller are connected to the motor. Furthermore,
impeller blades ensure the dissemination of air bubbles uniformly throughout the
fermentation medium by reducing the volume of air bubbles. Fig. 3.6 shows
different types of impellers used in fermenters depending on the process
requirements.

Fig. 3.6: Schematic illustration of different types of impellers.

vi. Valves: Various valves are used in the fermenter to control the flow of liquid in
the vessel.
vii. Sensors: Numerous probes/sensors/electrodes are used in the vessel to monitor
temperature, foam level, pH, dissolved oxygen level, etc.
3.4.3 Types of Bioreactors
As mentioned above, the fermenter design can be varied depending on the production
requirements. However, the below-described types of fermenters are most commonly used by
industries to produce various types of products (Fig. 3.7):
 Fig. 3.7.: Most commonly used types of bioreactors in industries

i. Continuous stirred tank fermenter: It comprises a cylindrical container with a main

shaft supporting one or more agitators and is powered by an electric motor
(impellers). When used with impellers (agitators), the sparger enhances gas
distribution throughout the vessel. During fermentation, the temperature control is
easily controlled, construction is affordable, and the operation is simple, resulting in
low labour expenses and easy cleaning. It is one the most common types of bioreactor
used in industry.
ii. Airlift fermenter: It is also called a “tower bioreactor” and is similar to a bubble
column bioreactor but differs because it contains an internal tube, which improves
heat circulation and oxygen transfer, thus, balances shear forces in the bioreactor. The
airlift bioreactors are most suited for the cultivation of aerobic cultures. This type of
bioreactor is ideal for single-cell protein (SCP) production using methanol as a carbon
substrate. Mechanically, this bioreactor has several advantages, including its ease of
use because it has no moving parts or agitators, quick sterilisation, low energy
consumption, and resultant low operational cost.
iii. Fluidised bed bioreactor (FBB): Nowadays, this type of bioreactor has gained much
attention over the other types of bioreactors. Here, microbial cells or enzymes are
“immobilised” on small particles suspended in the fermentation medium. As the small
particles provide a large surface area for the cells to stick to and allow a high rate of
oxygen transfer and nutrient supply to the microbial cells. The immobilised cells or
enzymes serve as the biocatalyst.
iv. Packed bed/fixed-bed bioreactor: In this type of bioreactor, the immobilised cells
are more densely packed in the column and fed with substrates or nutrients from the
top or bottom. Such bioreactors are simple to construct along with operation but have
the issue of blockages and poor oxygen transfer. Still, these bioreactors are the most
successful because they can be used where substrate inhibition is a concern. Such a
bioreactor has a high catalyst conversion rate, is simple to operate, has low
construction and operational costs, has more reactant-to-catalyst contact, and can be
operated at high temperatures and pressures.

Check Your Progress Exercise 3

Note: a) Use the space below for your answers.
b) Compare your answers with those given at the end of the unit.
1) Briefly describe structural requirements of an ideal fermenter.
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2) What is the key function of a sparger in a bioreactor?
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3) Why are different sizes of fermenters designed? Explain.
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4) List different types of biorecators.
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3.5 STARTER CULTURES

The production of value-added food products needs either a single or groups of microbial
cultures, which could efficiently convert into the desired products or perform value addition
to the food under certain fermentation conditions such as availability of nutrients, pH,
temperature, oxygen, etc. The microorganism that has the potential to perform the above-
mentioned biochemical conversion is termed a starter culture. Such culture initiates the
fermentation process. For example, lactic acid bacteria are used as a starter culture in
converting milk to curd under defined conditions. During the fermentation, the lactic acid
bacteria convert the milk sugar lactose to lactic acid, and the final product is termed curd.
Simply, we can say that these starter cultures (microorganisms) are introduced directly into
food components to achieve desired value addition in the food or food products, and have
been traditionally used to produce certain food, generally termed fermented food products.
Comparatively, the developed product has enhanced preservation, nutritional content, sensory
qualities, and economic values over non-fermented food.
For the large-scale production of the food products, the starter culture should have
demonstrated the following characteristics:
i. Culture must be in pure culture form.
ii. Culture must grow and reproduce faster.
iii. Culture must be genetically stable, but the scope of genetic manipulation toward
better performance.
iv. Culture must produce a constant product in a short time.
 v. Culture must not produce undesirable byproducts or toxic substances.
vi. Culture must have certain protective mechanisms, including organic acid or
bacteriocin, that could be effective against other undesired contaminating
microorganisms.
Following microorganisms are most widely used as a starter culture in food industries:
i. Bacteria: Lactobacillus, Streptococcus, Bifidobacterium, Lactococcus, Leuconostoc,
Pediococcus, Bacillus, etc.
ii. Yeast: Saccharomyces (S. cerevisiae and S. carlsbergensis), Candida, Torulopsis, etc.
iii. Mold: Aspergillus, Rhizopus, Penicillium, etc

Based on the origin and type of strain, the starter culture may be grouped into the followings:
3.5.1 Single-strain starter culture
It is the starter culture which uses only one type of starter strain to produce fermented food
products. The product formation is achieved under aseptic conditions. For example,
fermented milk is produced by Lactococcus lactis subsp. lactis.
3.5.2 Mixed pure culture
Certain product formation is achieved by mixing two or more pure forms of known
microorganisms in different combinations. Here, the pure mixed cultures can be a mixture of
either bacteria or bacteria with a combination of yeast and mold in a defined ratio. It is very
important to use a balance to achieve the maximum performance of both cultures. For
example, in the production of yoghurt, the desired ratio of cocci, i.e. Streptococcus
thermophilus to the rod Lactobacillus delbreuckii subsp. bulgaricus is 5:1 in cell number;
however, 1:1 ratio by weight.
3.5.3 Mixed natural culture
In several parts of the world, particularly in developing countries, the flora of various
traditional fermented foods are mixed with natural cultures. The exact composition of
microflora used in preparing these foods is yet to be studied. However, the microbial culture
dynamics vary with the traditional food preparation processing.
To check purity and activity of starter cultures, certain quality control tests such as
microbiological, chemical and organoleptic tests need to be performed.
The purity of starter cultures is confirmed using microscopic examination (i.e., Grams
staining) and chemical tests (i.e., catalase). Moreover, the activity of the starters can be
examined by performing titratable acidity and dye reduction tests.

You will learn in detail about the microorganisms used as starter cultures to prepare various
dairy and non-dairy fermented foods in unit-5.

3.6 STRAIN IMPROVEMENT

Microorganisms are potent sources of various metabolites and value-added products.
However, naturally, these substances are produced less because they produce only for their
benefit. Therefore, the microorganisms tend not to overproduce their valuable metabolites. If
we go by their natural production strategies, in that case, the cost of the metabolites increases
manifolds while adopting the fermentation technology. To overcome the cost of production
of the metabolites through advanced fermentation, great scientific and technological efforts
need to be made to extend the potential of these microorganisms to have an increased ability
to produce valuable products or metabolites of public interest at a faster rate using the same
substrate under controlled conditions. This can be achieved by subjecting selected
microorganisms to a “strain improvement program” by altering the microbial genetics using
the techniques mentioned below to obtain the desired goal of metabolite production.
The traditional approach to strain improvement is to use mutagens (chemical mutagens or UV
light) to generate mutations and then screen the mutants for products. The alternative way is
protoplast fusion, which involves fusing two strains with two distinct, desirable qualities to
create a hybrid having both, such as greater growth from one strain and improved product
production from another. Molecular methods and recombinant DNA technologies are at the
core of today’s modern approach to strain enhancement. It may be accomplished by inducing
overproduction of a gene product using molecular methods and insertion of additional genes
into a known excellent strain to enhance it further.
In general, mutation, protoplast fusion, recombination, and recombinant DNA technology can
be applied to obtain the goal of fermentation technology (Fig. 3.8). Detailed information and
procedure and suitable examples are also below for better understanding.

Fig. 3.8: Strain Improvement techniques

3.6.1 Mutation
Mutants can be generated by modifying the target organism's genome (genetic material,
usually DNA) through physical, chemical, and biological methods. A process through which
mutants are obtained is called mutagenesis. It is performed by a substance termed mutagen. It
is a chemical or physical agent which has the potential to irreversibly alter the genetic
material in exposed organisms. Though not all mutations are caused by mutagens, a few
mutations occur naturally due to spontaneous hydrolysis, defects in DNA during replication,
repair, and recombination. Such type of mutation is termed as “spontaneous mutation”.
Several mutagens, which may be of physical, chemical, or biological origin, are thought to
work in different ways. Most of them may act directly on genetic material, causing direct
damage to the DNA and most frequently resulting in errors in replication, while some may
act on the replication process and chromosomal partition.
Following are examples of mutagens:
 Physical: Ionising radiations, UV rays, etc.
  Chemical: Sodium azide, benzene, ethidium bromide, ethylnitrosourea, metals, etc.
 Biological: Virus, transposon, etc.
Changing mutagens regularly throughout a long-term strain enhancement program is
recommended to take full advantage of these varied mechanisms of action. A small number
of these cells may be the source of a vast number of bioactive components of human
interest. As most of the mutations cause significant changes in the biochemical
characteristics of an organism, hence are considered valuable for strain enhancement
programs. For example, the wild strain of Streptomyces griseus produced tiny amounts of
streptomycin, but the mutated strain produced significantly more streptomycin. As
mentioned above, mutations are often infrequent, occurring once per 10 7 to 1011 cells,
necessitating the use of a very sensitive detection technique. The finest example of direct
mutant detection is an approach for detecting auxotrophic mutants that involves replica
plating. Auxotrophic mutants have a deficiency in one of their biosynthetic pathways,
requiring a particular biochemical to grow and develop normally. At a practical level, once
the biosynthetic pathways for the relevant product and the precursors and regulatory
mechanisms are understood, then only mutant selection procedures can be perfectly applied.
3.6.2 Protoplast fusion
A protoplast is a cell that has been stripped of its cell wall. To perform protoplast fusion,
bacteria, actinomycetes, and fungal protoplasts are extracted by exposing these cells to wall-
degrading enzymes in isotonic solutions. This method can be used to combine beneficial
qualities from two lineages or parental cultures. The protoplasts require an osmoticum for
stability, and PEG (polyethene glycol) treatment causes fusion. For example, a strain of
Streptomyces griseus that produces indolizomycin (a novel indolizine antibiotic), was
developed through protoplast fusion between non-producing strains of Streptomyces griseus
and Streptomyces tenjimariensis.
3.6.3 Recombination
It has been used to create a variety of industrially important strains. In recombination, a new
gene arrangement is formed through DNA exchange, elimination, or insertion. Because of the
intricacy of the method and the lack of genetic literature for industrial strains, the use and
practical application of this technique for strain improvement are fairly limited in the context
of improving industrial strains. However, In certain bacteria and actinomycetes, sexual
reproduction through conjugation results in the generation of partial diploids, which cross
over to generate recombinant genotypes. Nuclear fusion and gene segregation may occur
outside the sexual organs of fungi such as Aspergillus nidulans, Penicillium chrysogenum,
and others during the parasexual cycle.
3.6.4 Recombinant DNA Technology
You already have studied this technology in the previous unit. It has been used to achieve
goals such as producing recombinant proteins by transferring commercially valuable genes
into target microorganisms (i.e. bacteria and altering the metabolic pathway of an organism)
to acquire new, modified, or larger quantities of metabolites.
Check Your Progress Exercise 4
Note: a) Use the space below for your answers.
b) Compare your answers with those given at the end of the unit.
1) What is mutagenesis?
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2) What type of characteristics should be demonstrated by the starter culture to be used in the
large-scale production of the food products?
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3) What is the process through which the microbial strain can increase metabolite production?
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4) Name the most widely used starter cultures in food industries.
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5) Differentiate between single strain starter culture and mixed pure starter culture.
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6) List the mutagens that can be used for strain improvement of starter culture.
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3.7 LET US SUM UP

Recently, food scientists widely appreciated and adopted the potential of fermentation
technology in food production and value addition to existing products. Fermentation
processes employ selected microorganisms to obtain desired product under controlled
conditions. These microorganisms are known as starter culture and these can be single strain
starter culture, mixed pure culture and mixed natural culture depending on the requirement of
the fermented food. Various selective strain improvement approaches are being used to
improve microbial cultures' productivity, i.e., mutation, protoplast fusion, recombination, and
recombinant DNA technology.
The fermentation process is carried out in a specially designed vessel (bioreactor) depending
upon the product requirements. In this unit you learnt about the basic design and type sof
bioreactors and different typs of fermentation processes used at industrial level. Various
industrial bioprocesses (fermentations) are batch, Fed-batch and continuous fermentation.
The industrially important products (metabolties of fermentation) like enzymes, proteins,
antibiotics, organic acids, pigments, biopolymers, etc.are developed from fermentation.
The advancement in fermentation technology opens new avenues and opportunities for young
scientists to find a sustainable solution for combating various concerns related to nutritional
deficiencies among the population.

3.8 KEYWORDS
Mutagen : It is a chemical or physical agent which has the potential
to irreversibly alter the genetic material in exposed
organisms.

Mutagenesis : A process through which mutants are obtained.

Spontaneous mutation : A few mutations occur naturally due to spontaneous

hydrolysis, defects in DNA during replication, repair, and
recombination.

Mixed pure culture : A mixture of two or more pure forms of known

microorganisms is used in different combinations as a
starter culture.

Bioreactor : A device in which fermentation is carried out.

Industrial Fermentation : Large-scale production of metabolites begins with the

selected microorganism’s growth under controlled
fermentation conditions in a bioreactor of 350 L capacity
or more.

3.9 ANSWERS TO CHECK YOUR PROGRESS EXERCISES

Check Your Progress Exercise 1
1) i.Bioreactor or fermenter.
ii.Lacto-fermentation
2) The steps of fermentation process: Selection of microorganisms for fermentation; Medium
formulation for cultivating microorganisms; during inoculum formation and in the production
fermenter; Sterilisation of the medium, fermenter, and other equipment associated with the
process; Production process under controlled conditions; Product’s extraction and
purification; and Effluent disposal.
3) The factors that affect quality of fermented food are: type sof raw materials,
microorganisms involve din fermentation, incubation temperature an dfermentation duration,
heat treatment and other processing parameters, packaging and storage.
4)

Primary metabolites Secondary metabolites

These are key components in They do not play a role in microbial growth,
maintaining the normal physiological development and reproduction but are considered
function of the organisms to play a role in ecological functions such as
defense mechanisms of the microorganisms

These are generally produced during the These are produced during the stationary phase of
growth phase. microbial growth.

Example : amino acids, vitamins, lactic Example : Antibiotics, pigments, toxins, alkaloids
acid, ethanol, etc.

Check Your Progress Exercise 2

1) The primary types of industrial fermentation processes based on modes of operation

are Batch, fed-batch, and continuous process.
2) The disadvantages of batch fermentation process are:
i. Microorganisms are continually exposed to a variety of fermentation variables due to
nutrient consumption and there is problem of waste deposition.
ii. Batch cultures need to be restarted after reaching an endpoint.
iii. The large bioreactors require a long time to empty, clean, and refill.
iv. Due to the significant downtime between two batches, batch culture has low
productivity.
3)
Turbidostat method Chemostat method
The microbial cell growth is controlled and Nutrients are continuously delivered at a
stabilised by manipulating the flow rate of constant flow rate, and the density of the
the addition of a fresh medium. microbial cells is adjusted as per the
nutritional requirement for growth given.
Microbial cell density is controlled by a The concentration of substrates (carbon,
specified turbidity value determined by the nitrogen, and phosphorus) are used to
cell population regulate the growth rate of a chemostat.
4) Various industrially important products produced by fed-batch fermentation are: amino
acids- Lysine, antibiotics-Penicillin, enzymes- amylases, solvents- Butanol, microbial cell
mass- Saccharomyces .

Check Your Progress Exercise 3

1) Structural requirements of an ideal fermenter:
  For aerobic fermentation, an aeration port is required to ensure sufficient oxygen
supply during the fermentation.
 Provision of ports for the addition of acid, base, nutrients, etc., and ports for removal
of spent medium, and or recovery of microbial cells as well as sampling.
 Provision of agitation for uniform mixing of microbial cells and fermentation medium
components during the process.
 Need for installation of heating devices.
 The controlling devices or sensors for the regulation of dissolved oxygen, pH,
temperature, aeration, nutrients, etc., should be in place.
 Provision for sterilisation and maintenance of sterility facilities.
2) The key function of spargers is to ensure efficient dispersal of filtered air to the impeller to
provide optimal aeration and oxygen availability to the culture in a fermenter throughout the
operation.
3) The fermenter's size varies greatly depending on the use. For example, some fermenters
are built for small-scale fermentation and others for large-scale (industrial applications),
ranging from the shaking flask (100-1000 ml) to the laboratory-size fermenter (1–50 L) to the
pilot level (10-350 L) to the plant scale (more than 350 L) for a large volume.
4) Types of bioreactors are: Continuous stirred tank fermenter, Airlift fermenter, Fluidised
bed bioreactor (FBB), Packed bed/fixed-bed bioreactor.
Check Your Progress Exercise 4
1) A process through which mutants are obtained is called mutagenesis. It is performed
by a substance termed mutagen. It is a chemical or physical agent which has the potential
to irreversibly alter the genetic material in exposed organisms.
2) The starter culture should have demonstrated the following characteristics for the
large-scale production of the food products: Culture must be in pure culture form;
Culture must grow and reproduce faster; Culture must be genetically stable but the
scope of genetic manipulation toward better performance; Culture must produce a
constant product in a short time; Culture must not produce undesirable byproducts or
toxic substances; Culture must have certain protective mechanisms (organic acid or
bacteriocin) that could be effective against other undesired contaminating
microorganisms.
3) This can be achieved by subjecting selected microorganisms to a “strain improvement
program” by altering the microbial genetics using various techniques to obtain the
desired goal of metabolite production. The traditional approach to strain improvement
is to use mutagens (chemical mutagens or UV light) to generate mutations and then
screen the mutants for products.
4)The most widely used starter cultures in food industries are:
 Bacteria: Lactobacillus, Streptococcus, Bifidobacterium, Lactococcus, Leuconostoc,
Pediococcus, Bacillus, etc.
 Yeast: Saccharomyces (S. cerevisiae and S. carlsbergensis), Candida, Torulopsis, etc.
 Mold: Aspergillus, Rhizopus, Penicillium, etc
5) Single strain starter culture uses only one type of starter strain to produce fermented food
products. For example, fermented milk is produced by Lactococcus lactis subsp. lactis.
Whereas, mixed pure culture comprises of two or more pure forms of known microorganisms
in different combinations. For example, in the production of yoghurt, the desired ratio of
cocci, i.e. Streptococcus thermophilus to the rod Lactobacillus delbreuckii subsp. bulgaricus
is 5:1 in cell number; however, 1:1 ratio by weight.
6) Mutagens that can be used for strain improvement of starter culture are:
 Physical: Ionising radiations, UV rays, etc.
 Chemical: Sodium azide, benzene, ethidium bromide, ethylnitrosourea, metals, etc.
 Biological: Virus, transposon, etc.

3.10 REFERENCES/SUGGESTED READINGS

 Casida L.E.J.R. (1968), Industrial Microbiology, New age international Ltd,
Publishers. New Delhi.
 Casida LE. (1991). Industrial Microbiology. 1st edition. Wiley Eastern Limited.
 Crueger W and Crueger A. (2000). Biotechnology: A textbook of Industrial
Microbiology. 2nd edition. Panima Publishing Co. New Delhi.
 Glaze A.N. and Nikaido H. (1995). Microbial Biotechnology: Fundamentals of
Applied Microbiology. 1st edition. W.H. Freeman and Company.
 Manay, S. and Sharaswamy, M. (1987). Food Facts and Priniciples. Wiley Eastern
Limited.
 Okafor N. (2007). Modern Industrial Microbiology and Biotechnology. 1st edition.
Bios Scientific Publishers Limited. USA.
 Patel A.H. (1996). Industrial Microbiology. 1st edition, Macmillan India Limited.
 Stanbury PF, Whitaker A and Hall SJ. (2017). Principles of Fermentation
Technology. 3nd edition, l Butterworth-Heinemann publications, Elsevier Science Ltd
 Waites M.J., Morgan N.L., Rockey J.S. and Higton G. (2001). Industrial
Microbiology: An Introduction. 1st edition, Wiley – Blackwell.