KWAME NKRUMAH UNIVERSITY OF SCIENCE & TECHNOLOGY, KUMASI

DEPARTMENT OF BIOCHEMISTRY AND BIOTECHNOLOGY: M. Phil. BTC 561

FERMENTATION AND ENZYME TECHNOLOGY 2020/2021 (Handout 2)

FERMENTATION TECHNOLOGY
The original definition of fermentation is ‘the anaerobic conversion of sugar to carbon dioxide and
alcohol by yeast.’ This original definition has been expanded over time to ‘the conversion of
organic materials into relatively simple substances by micro-organisms; essentially efficient,
flexible bio factories.’ During their growth and lifespan micro-organisms build a wide range of
different molecules types required for viability and multiplication; adaptation to changing
environment; stressful conditions and defence against hostile, competitive microbial threats.

The present definition of fermentation applies to both aerobic and anaerobic metabolic
activities of microorganisms in which specific chemical changes are brought about in an
organic substrate. From an industrial microbiology standpoint, the meaning includes almost any
process mediated by, or involving microorganisms in which a product of economic value
accrues.

The industrial usage of microorganisms often requires that they be grown in large vessels
containing considerable quantities of nutritive media. These vessels are called fermentors.
Fermentation usually is carried out in either of three ways, by the batch method, by the continuous
method or by the semi-batch method. In the laboratory on a small-scale fermentation is carried
out in batches.

The biotechnology industry originated in the 1970s, based largely on a new recombinant DNA
technique whose details were published in 1973 by Stanley Cohen of Stanford University and
Herbert Boyer of the University of California, San Francisco. Recombinant DNA is a method of
making proteins, such as human insulin and other therapies, in cultured cells under controlled
manufacturing conditions. Boyer went on to co-found Genentech.

Industrial fermentation is based on microbial metabolism. Microbes produce different kinds of

substances that they used for growth and maintenance of their cells. These substances can be
useful for humans. The goal of industrial fermentation technology is to enhance the microbial
production of useful substances.

In biology, fermentation is a process of harvesting energy of organic molecules in oxygen-free

conditions. Sugars are a prime example of what can be fermented, although, there are many other
organic molecules that can be used. Different fermentations are known and are categorised by
the substrate metabolised or the type of the product. In industry, any large microbiological process
is called fermentation. Thus, the term fermentation has a different meaning than in biology. Most
industrial fermentations require oxygen.

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 Figure: The stages of a biotechnological process.
It must not be forgotten that a crucial part of the input is research and development. Often research
and development (R&D) can take many years before a process becomes feasible or cost effective.

Industrial Fermentation Organisms

Different organisms, such as bacteria, fungi, and plant and animal cells, are used in industrial
fermentation processes. An industrial fermentation organism must produce the product of
interest in high yield, grow rapidly on inexpensive culture media available in bulk quantities, be
open to genetic manipulation, and be nonpathogenic (does not cause any diseases).

Microorganisms that are typically used within the fermentation industry include:
• Prokaryotes such as bacteria (e.g. Escherichia coli, Staphylococcus aureus) and
Streptomycetes (e.g. Streptomyces spp, Actinomyces spp);
• Eukaryotes such as filamentous fungi (e.g., Nigrospora spp, Aspergillus spp,) and,
• Yeast (e.g. Saccharomyces cereviciae, Pichia pastoris).

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Figure: Pharmaceutical technician working at one of a row of fermentation units, or bioreactors.

A strain of an organism is a type that is genetically distinct from other representatives of

the species to which the organism belongs, but which is not different enough to be called
a new species. Members of a strain are much more genetically similar to each other than to
members of other strains. The word “strain” is normally used for microorganisms to describe a
particular organism that has been isolated or engineered to have some property, like growing well
or making a lot of a product. Isolating and improving strains of microorganisms is a major
part of the process of making them suitable for an economic biotechnological process.

In microbial technology, microorganisms hold the key to the success or failure of a

biotechnological process. It is, therefore, important to select the most suitable microorganism(s)
to carry out the desired industrial process.

The first stage in setting up a microbial-based industrial process is to obtain suitable

microorganisms and screen their potential for industrial application. From a microbiologist's point
of view, this is probably the most critical step in the overall biotechnological process. Isolation and
screening require one to obtain either pure or mixed cultures, followed by their assessment to
determine which can carry out the desired reaction or produce the desired product most efficiently.
Assessment requires the cultivation of microorganisms under laboratory conditions where the
microorganisms are subjected to some screening procedure. It is desirable to design the isolation
procedures such that the desirable properties are screened for and recognised at early stages of
the isolation.

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There are a number of useful (ideal) attributes in an industrial microorganism, as listed
below:
1. The microorganism must be stable genetically. The organism should have a
reasonable genetic and hence physiological stability. An organism which mutates easily
is an expensive risk. It could produce undesired products if a mutation occurred
unobserved. The result could be reduced yield of the expected material, production of an
entirely different product or indeed a toxic material. None of these situations is a help
towards achieving the goal of the industry, which is the maximisation of profits through the
production of goods with predictable properties to which the consumer is accustomed.

2. The strain should be pure, free from other microorganisms and phages. The
organism should be reasonably resistant to predators such as Bdellovibrio spp or
bacteriophages. (Bdellovibrio is a genus of Gram-negative, obligate aerobic bacteria
that parasitise other Gram-negative bacteria by entering into their periplasmic space and
feeding on the biopolymers, e.g. proteins and nucleic acids, of their hosts). It should
therefore be part of the fundamental research of an industrial establishment using a
phage-susceptible organism to attempt to produce phage-resistant but high yielding
strains of the organism.

3. The strain should grow vigorously and rapidly after inoculation into the seed tank
or other fermentation vessels. One should avoid strains that might react with the
equipment. A slow growing organism no matter how efficient it is, in terms of the production
of the target material, could be a liability. In the first place the slow rate of growth exposes
it, in comparison to other equally effective producers which are faster growers, to a greater
risk of contamination. Second, the rate of the turnover of the production of the desired
material is lower in a slower growing organism and hence capital and personnel are tied
up for longer periods, with consequent lower profits.

4. The strain should exhibit the desired characteristic(s) or produce the desired
product(s) within a reasonable period of time (e.g. 3 days), preferably free of other
toxic by-products and cell lysis. Its end products should not include toxic and other
undesirable materials, especially if these end products are for internal consumption.

5. The strain should protect itself against contamination, if possible. Self-protection

can take the form of lowering the pH, ability to be cultured at high temperatures or
production of antimicrobial agents (e.g. antibiotics or killer factors). Note that growth
at a high temperature also reduces the cooling requirements significantly. Thus a
thermophilic efficient producer would be preferred to a mesophilic one.

6. The organism should lend itself to a suitable method of product harvest at the end
of the fermentation. If for example a yeast and a bacterium were equally suitable for
manufacturing a certain product, it would be better to use the yeast if the most appropriate
recovery method was centrifugation. This is because while the bacterial diameter is
approximately 1 µm, yeasts are approximately 5 µm. Assuming their densities are the
same, yeasts would sediment 25 times more rapidly than bacteria. The faster
sedimentation would result in less expenditure in terms of power, personnel supervision
etc which could translate to higher profit.

7. The strain should be amenable to genetic manipulation to enable the establishment

of strains with more acceptable properties.

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 8. The strain should be readily maintained for long periods of time.
9. The strain should (ideally) exhibit simple growth requirements. This characteristic
would depend on the feedstock available. Ideally, one would prefer a strain that can grow
on the available feedstock with the least nutrient requirements. The strain should
preferably not require growth factors (i.e., pre-formed vitamins, nucleotides, and acids)
outside those which may be present in the industrial medium in which it is grown. It is
obvious that extraneous additional growth factors may increase the cost of the
fermentation and hence that of the finished product.

10. Where practicable the organism should not be too highly demanding of oxygen as
aeration (through greater power demand for agitation of the fermentor impellers, forced
air injection etc) contributes about 20% of the cost of the finished product.

Not all microorganisms on initial isolation will possess all of the above characteristics.

Screening is the use of highly selective procedures to allow the detection and isolation of
only those microorganisms or metabolites of interest, from among a large population.
Screening and selection represent the first stage of setting the industrial microbiological process
in motion. The ideal industrial screen should have the following attributes:
1. Rapid.
2. Sensitive.
3. Inexpensive.
4. An industrial screen should be suitable for high throughput - this is being
addressed by automation.
5. Specific, i.e., low false-positives or false-negatives.
6. Predictive.
7. Simple.

No single screen will satisfy all the above conditions. One normally has to decide which
characteristics are important and tolerate less then perfection in the other characteristics. In
reality, every screen will miss some useful activities (false-negatives) and some of the selected
strains will turn out to be useless (false-positives).
Hopefully, most of the selected isolates should be active. One of the most important factors in
screening (which cannot be changed by further development or automation) is the participation
of an experienced investigator. The intuition, instinct and curiosity of a microbiologist, biochemist
or pharmacologist are absolutely vital for a successful screening program.

One of the most critical aspects in designing an effective screen is to establish a suitable testing
system and to decide the criteria for selection or rejection. Prior to undertaking an
isolation/screening project, it is best to do a thorough library search. The purpose is to see if
similar types of experiments/projects have been done before. Perhaps one can learn from these
previous attempts. Some of the things that are of interest include the sampling and screening
strategies, where microorganisms were isolated from, safety features associated with the work,
yield expected, etc
.
It is always a good idea to get a type strain as a control against which one can measure the
productivity of the isolated strains.
Type strains of microorganisms may be obtained from companies, universities and national or
international culture collections. Some of the advantages and disadvantages of these 3
sources are listed below:

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Companies
1. A large variety of strains are potentially available.
2. "Best strains" are not likely available since companies tend to keep their best
strains for their own use.
3. Strains have probably been tested industrially.
4. Probably not much basic research has been done, except for the "best" production
strains. However, these "best strains" are not likely to be provided. Therefore, the
strain obtained is likely poorly characterised.
5. One will most likely have to sign away some rights to get the strain(s).
6. Strains are usually provided free of charge.

Universities
1. A limited variety of strains are available in each laboratory, but collectively the
whole university system provides a large pool of strains.
2. "Best strains" are probably available and obtainable.
3. Strains have probably not been tested industrially.
4. Strains are better characterised and the results published.
5. Probably one will have to sign away some licensing and patenting rights to get the
strain(s), depending on the funding source of the researchers, and their inclination.
6. Strains are usually provided free of charge.

National or International Culture collections

1. A large variety of strains are available.
2. "Best strains" are available and obtainable.
3. Strains have probably not been tested industrially.
4. Strains are better characterised and the results published.
5. There is less concern about licensing and patent rights.
6. Normally there is a charge for the use of the strains.

Culture Collections (Culture Banks)

Microbial culture banks are the repositories of collection of various microorganisms. Microbial
culture banks are often referred to as “culture collections” or “culture banks". Culture collections
are the key repositories of biodiversity. These can provide abundant resources for plant
pathologists, microbiologists, molecular biologists, and other scientific professionals. However,
many such professionals (including graduate students, postdoctoral fellows, and technical staff)
lack adequate guidance on locating, obtaining, and processing such materials.

Since the early days of microbiology, astronomical numbers of microorganisms were isolated from
a wide variety of natural sources and used for the scientific research and for the industrial
fermentation. However, large numbers of microorganisms have been lost in the past, and they
are no longer available. Microbiologists often lose microbial cultures that they studied because of
the change of their interests and difficulties in keeping the cultures. This is due to the absence of
reliable culture collections in which microorganisms are maintained properly and supplied
promptly on demand.

Through the study of microbial cultures maintained in the culture collection, potential properties
of microorganisms have been developed, and the future perspective of microbiology will be

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presumed. Effective research needs adequate and reliable sources of properly preserved
cultures. In the near future, uncountable numbers of microbial strains will be isolated through the
study of microbial diversity, and the attributes of a large number of strains will be improved.
Therefore, reliable, and well-organised culture collections are needed as the depository and for
the promotion of research and application of the strains. In fact, culture collections play a role of
the depository of the type strains in bacteriology, and the study of bacterial systematics will not
be completed without culture collections consequently.

Major tasks of culture collections are the collection and maintenance of important and useful
microbial cultures, supply of the cultures on demand, and preparation of their informative
documents. Social needs for culture collections are increasing year by year, and effective and
smooth management is required for better services of culture collections.

Thus, improvement of culture collections is critical and crucial for the further development of
microbiology, microbial industry, and biotechnology. In addition, good operation and management
of culture collections are in great part due to the activity of personnel in the culture collections.
Examples of National or International Culture Collections
• ATCC (American Type Culture Collection Centre, Maryland,
• U.S.A.)
• NCIB (National Collection of Industrial Bacteria, Britain)
• DSM (Deutsche Sammlung von Mikroorganismen and Zelkulturen, Germany)
• NCTC (National Collection of Type Culture, London)
• MTCC (Microbial Type Culture Collection, Osaka Japan)
• MTCC (Microbial Type Culture Collection and Gene Bank Institute of Microbial
Technology, Chandigarh)
• ICIM (Indian Culture of Industrial Microorganisms, National Chemical
Laboratory, Pune)

In the United States there are several important microbial collections that are members of the
United States Federation of Culture Collections. These include:
• American Type Culture Collection (ATCC),
• United States Department of Agriculture ARS Collection.

The American Type Culture Collection (ATCC) is a private, not-for-profit biological resource
center whose mission focuses on the acquisition, authentication, production, preservation,
development and distribution of standard reference microorganisms, cell lines and other
materials for research in the life sciences. ATCC’s microorganism collection includes a collection
of more than 18,000 strains of bacteria from 900 genera, as well as 2,000 different types of animal
viruses and 1,000 plant viruses. In addition, ATCC maintains collections of
protozoans, yeasts and fungi with over 49,000 yeast and fungi strains from 1,500 genera and
2,000 strains of protists.

Both the Japanese and German national culture collections are fully supported by their respective
federal governments. Similarly, the Netherlands Royal Academy funds 85% of the cost of the
Netherlands Culture Collections (NCC) which arguably contains the world’s premier fungal
collection. Furthermore, active basic research programs are supported in many of the other
national culture collections.

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When new microorganisms are described and named, they should be deposited in culture
collections, thereby expanding accessibility. Indeed, national and international patent laws require
patent strains to be deposited in permanent collections.

The 16S or 18S rRNA of all microorganisms deposited in culture collections should be sequenced,
including not only those newly accessioned but also those already resident in collections. Serious
consideration should also be given to establishing a DNA collection for all microorganisms,
specifically bacteria, to expedite DNA/DNA reassociation analyses.
In addition to preserving type strains of microbial species, consortia and perhaps even entire
natural microbial community samples should be preserved, especially those of endangered
habitats. This can be accomplished by conservation under liquid nitrogen.

Culture collections represent a genetic resource for scientists and industry. Among microbial
strains deposited in culture collections are species that serve as “gold standard” for comparison
with newly discovered organisms.

However, there are some problems associated with culture collections. These include:
1. Strain degeneration (progressive loss of productivity) may occur on long term
storage.
2. A large part (e.g. in companies) may be unavailable.
3. Costs may be high if one were to obtain many strains.
4. Limitations on use of the strains placed by Materials Transfer Agreements
(MTA).

STRAINS FROM THE ENVIRONMENT

Cultivation of microorganisms greatly enhances the ability of the microbiologist to determine the
full complement of genetic and physiological characteristics of newly isolated species. Strains
available in pure or mixed culture can be examined to determine what novel features they have,
including those that might be of special interest to society.

Cultivation is the isolation and multiplication of organisms after removal from the
environment. It is difficult to recover, in viable, actively growing and reproducing cultures, most
organisms from typical soil and aquatic habitats. It may be a result of natural dormancy which
would be expected in certain microhabitats, such as soils during periods of low water activity. In
some cases, conditions for growth of the microorganisms may not have been met. In other cases,
the microorganisms may be injured and, therefore, difficult to recover.

Schut et al. (1993) and Bianchi and Giuliani (1996) were successful in using extinction dilution
procedures with water from the environment to grow many of the most numerous heterotrophic
marine bacteria. In this process, at the time of collection, the original sample from the habitat is
serially diluted several orders of magnitude using autoclaved water from the environment until the
highest dilution receives few, if any, organisms. In this manner, the most abundant species (from
the highest dilution tubes) can be obtained from the habitat, provided they grow in the medium.
Following prolonged incubation in sterile natural water samples, many of the most numerous
organisms from the environment were grown on ordinary laboratory media. This approach should
be attempted for other habitats as well.

‘Dilution to extinction’ or low-density partitioning of cells or CFUs in tubes or microwells with

low-nutrient media exploits the fact that culturable species diversity observed in microbial

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isolations apparently increases as inoculum density decreases. Partitioning of individual
cells in tubes or wells reduces interspecific interactions and raises the sensitivity of detection of
cells with low growth rates.

In situ hybridisation or fluorescent antibody techniques also should be used, if possible, to

confirm that the isolated strain indeed comprises a significant part of the original natural
community.

Many newer procedures are also available for isolation of fungi which show great potential. It
should be recognised that methods will need to be developed to cultivate the many dominant
microorganisms that can be found in natural communities and are not yet successfully cultured.
Therefore, research on new methodologies is not only strongly encouraged, but viewed as critical
to microbial discovery and harvest. When new microbial strains are cultivated, they need to be
characterised in a polyphasic manner, including determination of their phenotypic features
(morphology, physiology, biochemistry, and genetics) as well as their 16S/18S rRNA and other
sequences used to assess their phylogenetic position. Thus, the terms “polyphasic taxonomy”
and “polyphasic systematics” refer to the holistic characterisation of a species; when the terms
taxonomy and systematics are used, this connotation is implied.

Pure cultures are suitable for elucidation of physiologic, metabolic, and morphologic traits and
evaluation of the commercial potential of an organism. Illustrative features useful in
characterising the phenotype of bacteria are shown in the Table below.

Table: Typical Features Used for the Description of Bacteria

Morphology
• Cell shape
• Cell size (diameter, length)
• Motility
• Flagellation
• Type of cell division
• Cell differentiation
• Internal or external structures (endospores, gas vesicles, etc.)
• Gram stain
• Ultrastructure

Chemical composition and molecular analysis

• Colour of cell suspension
• Pigments
• Reserve materials
• DNA base composition
• 16S rRNA sequence
• DNA/DNA reassociation for species determination
• Whole cell fatty acid composition

Physiology
• Growth medium
• Temperature range and optimum
• pH range and optimum

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 • Phototrophic or lithotrophic growth
• Vitamin requirements
• List of carbon sources used for growth
• List of nitrogen sources used for growth
• Relation to oxygen
• Modes of energy generation
• Electron donors: either organic or inorganic
• Electron acceptors: oxygen, nitrate, sulfate, carbon dioxide, iron oxides, etc
• Extracellular enzymes:
Amylase
Lipase
Gelatinase
Cellulase
Xylanase
• Other enzymes:
Catalase
Oxidase
• Glucose fermentation
• Denitrification

ISOLATION OF PURE CULTURES

In natural habitats microorganisms usually grow in complex, mixed populations containing several
species. This presents a problem for the microbiologist because a single type of microorganism
cannot be studied adequately in a mixed culture. One needs a pure culture; a population of cells
arising from a single cell, to characterise an individual species.
Pure cultures are so important that the development of pure culture techniques by the German
bacteriologist Robert Koch transformed microbiology. Within about 20 years most pathogens
responsible for the major human bacterial diseases had been isolated. There are several ways to
prepare pure cultures.

1. Spread Plate and Streak Plate

If a mixture of cells is spread out on an agar surface so that every cell grows into a completely
separate colony, a macroscopically visible growth or cluster of microorganisms called a colony
is observed. Each colony represents a pure culture.

The spread plate is easy, direct way of achieving this result. A small volume of dilute microbial
mixture containing about 100 to 200 cells or less is transferred to the center of an agar plate and
spread evenly over the surface with a sterile bent-glass rod. The dispersed cells develop into
isolated colonies. Because the number of colonies should equal the number of viable organisms
in the sample, spread plates can be used to count the microbial population.

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 Streak Plating

In both the spread-plate and streak-plate techniques, successful isolation depends on spatial
separation of single cells.

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2. Pour Plate

Pour plates are extensively used with bacteria and fungi, as they also can yield isolated colonies.
The original sample is diluted several times to reduce the microbial population sufficiently to obtain
separate colonies when plating. Then small volumes of several diluted samples are mixed with
liquid agar that has been cooled to about 45ºC, and mixtures are poured immediately into sterile
culture dishes. Most bacteria and fungi are not killed by brief exposure to the warm agar. After
the agar has hardened each cell is fixed in place and forms an individual colony. The total number
of colonies equals the number of viable organisms in the diluted sample. Colonies growing on the
surface also can be used to inoculate fresh medium and prepare pure cultures.

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The preceding techniques require the use of special culture dishes named petri dishes or plates
after their inventor Julius Richard Petri, a member of Robert Kock’s laboratory. They consist of
two round halves, the top half overlapping the bottom.

Figure: Plate counts and serial dilutions.

(a) In serial dilutions, the original inoculum is diluted in a series of dilution tubes. In our
example, each succeeding dilution tube will have only one-tenth the number of microbial
cells as the preceding tube. Then samples of the dilution are used to inoculate Petri plates,
on which colonies grow and can be counted. This count is then used to estimate the
number of bacteria in the original sample. The surface colonies are circular; subsurface
colonies would be lenticular or lens shaped.
(b) Methods for preparation of plates for plate counts.

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Figure: Example of a dilution series of E. coli plated at three dilutions. Dilutions
decrease from left to right. Here, plate A is the one which should be counted

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Industrial Fermentation
Industrial fermentation is the intentional use of fermentation by microorganisms such
as bacteria and fungi as well as eukaryotic cells like CHO cells and insect cells, to make products
useful to humans. Fermented products have applications as food as well as in general industry.
Some commodity chemicals, such as acetic acid, citric acid, and ethanol are made by
fermentation. The rate of fermentation depends on the concentration of microorganisms, cells,
cellular components, and enzymes as well as temperature, pH and for aerobic
fermentation oxygen. Product recovery frequently involves the concentration of the dilute solution.

Nearly all commercially produced enzymes, such as lipase, invertase and rennet, are made by
fermentation with genetically modified microbes. In some cases, production of biomass itself is
the objective, as in the case of baker's yeast and lactic acid bacteria starter cultures for cheese-
making. In general, fermentations can be divided into four types:
• Production of biomass (viable cellular material)
• Production of extracellular metabolites (chemical compounds)
• Production of intracellular components (enzymes and other proteins)
• Transformation of substrate (in which the transformed substrate is itself the
product)

These types are not necessarily disjoint from each other, but provide a framework for
understanding the differences in approach. The organisms used may be bacteria, yeasts, molds,
algae, animal cells, or plant cells. Special considerations are required for the specific organisms
used in the fermentation, such as the dissolved oxygen level, nutrient levels, and temperature.

In most industrial fermentations, the organisms or eukaryotic cells are submerged in a liquid
medium; in others, such as the fermentation of cocoa beans, coffee cherries, and miso,
fermentation takes place on the moist surface of the medium. There are also industrial
considerations related to the fermentation process. For instance, to avoid biological process
contamination, the fermentation medium, air, and equipment are sterilized.
Foam control can be achieved by either mechanical foam destruction or chemical anti-foaming
agents. Several other factors must be measured and controlled such as pressure, temperature,
agitator shaft power, and viscosity.

An important element for industrial fermentations is scale up. This is the conversion of a laboratory
procedure to an industrial process. It is well established in the field of industrial that what works
well at the laboratory scale may work poorly or not at all when first attempted at large scale. It is
generally not possible to take fermentation conditions that have worked in the laboratory and
blindly apply them to industrial-scale equipment. Although many parameters have been tested for
use as scale up criteria, there is no general formula because of the variation in fermentation
processes. The most important methods are the maintenance of constant power consumption per
unit of broth and the maintenance of constant volumetric transfer rate.

Natural biosynthesis of endogenous molecules involves specific multi-step complex routes, some
of which can be manipulated for the biosynthesis of foreign molecules. Micro-organisms may be
genetically modified (recombinant technology) or metabolically engineered by substantial
alteration of their endogenous routes.
The key elements of fermentation development are:
• Strain selection and optimisation;
• Media and process development,
• Scale-up to maximise productivity.

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Downstream processing utilises various technologies for extracting, concentrating and purifying
the product from a dilute fermentation broth. Fermentation derived product diversity, the recovery
and selective purification of the specific desired product out of the whole molecular repertoire,
makes fermentation technology a multi-disciplinary methodology encompassing microbiology,
organic chemistry, biochemistry and molecular biology.

When fermenting volumes larger than 10 litres, necessary biosafety measures are taken,
especially when Risk Group 2 (RG2) pathogens are used. These include Biosafety Level 2 Large
Scale (BSL2-LS) containment facility design and special operational procedures. As these
products can be toxic and hazardous, their recovery and purification require adequate
chemical/biochemical facilities and equipment including isolators.

Under cGMP fermentation procedures, quality is built into the entire process ensuring that
regulatory agencies requirements are met in terms of safety, product identity, quality and purity.
Deposited in temperature-controlled bio-storage, strains handled under strict aseptic procedures
will be identified and characterised for homogeneity (absence of foreign growth).

Special Features of Microbial Metabolites

The most important, inherent characteristics of the bioactive microbial metabolites are their
microbial origin, their interaction with the environment and their unique chemical structures. In
general, natural products including the microbial metabolites may be practically utilised in three
different ways:
1. Applying the natural/fermentation product directly in the medicine, agriculture, or
in any other fields.
2. Using as starting material for subsequent chemical or microbiological
modification (derivatization).
3. They can be used as lead compounds for chemical synthesis of new analogues or
as templates in the rational drug design (RDD) studies

Production of Extracellular Metabolites

Metabolites can be divided into two groups: those produced during the growth phase of the
organism, called primary metabolites and those produced during the stationary phase,
called secondary metabolites.

Some examples of primary metabolites are:

• Ethanol,
• Citric acid,
• Glutamic acid,
• Lysine,
• Vitamins and,
• Polysaccharides.

Some examples of secondary metabolites are:

• Penicillin,
• Cyclosporin A,
• Gibberellin, and,
• Lovastatin.

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Primary Metabolites
Primary metabolites are compounds made during the ordinary metabolism of the organism during
the growth phase. A common example is ethanol or lactic acid, produced during glycolysis. Citric
acid is produced by some strains of Aspergillus niger as part of the citric acid cycle to acidify their
environment and prevent competitors from taking over. Glutamate is produced by
some Micrococcus species, and some Corynebacterium species produce lysine, threonine,
tryptophan and other amino acids. All of these compounds are produced during the normal
"business" of the cell and released into the environment. There is therefore no need to rupture
the cells for product recovery.

Secondary Metabolites
Secondary metabolites are compounds made in the stationary phase; penicillin, for instance,
prevents the growth of bacteria which could compete with Penicillium molds for resources. Some
bacteria, such as Lactobacillus species, are able to produce bacteriocins which prevent the
growth of bacterial competitors as well. These compounds are of obvious value to humans
wishing to prevent the growth of bacteria, either as antibiotics or as antiseptics (such
as gramicidin S). Fungicides, such as griseofulvin are also produced as secondary
metabolites. Typically secondary metabolites are not produced in the presence of glucose or other
carbon sources which would encourage growth, and like primary metabolites are released into
the surrounding medium without rupture of the cell membrane.

Secondary metabolism is performed by many filamentous fungi, but is rather less widespread
amongst bacteria. Microorganisms that produce secondary metabolites exhibit two phases during
batch culture, the trophophase and idiophase (Figure).

Figure: Growth of a microorganism in a batch culture.

*Trophophase and idiophase

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Trophophase is the growth phase of a culture and idiophase is the following period when the
secondary metabolites are formed. Production frequently begins when active growth has ceased,
often as the growth-limiting substrate concentration approaches the Ks value for the
culture. The success of any further biosynthesis in the idiophase is dependent on the preceding
trophophase. Secondary metabolism during the idiophase utilises primary metabolites to produce
species-specific and chemically diverse end-products that are not essential for growth of the
microorganism.

Their production involves metabolic pathways which are not used during the growth phase, at
least not during exponential growth (Table).

Table: Some Metabolic Pathways Involved in the Production of Secondary

Metabolites by Fungi

These pathways are often not as well characterised as those for primary metabolism. Although
secondary metabolism can be easily demonstrated in batch culture, it can be studied in
continuous systems, as it occurs at low dilution (growth) rates. Here, secondary metabolism can
be demonstrated to operate at the same time as, and not after, primary metabolism. Much energy
can be expended in the production of secondary metabolites. However, many of these products
appear to have no recognisable metabolic function for the producing organism, yet they are not
merely end-products of metabolism, such as fermentation end-products. Those that perform
specific roles for their producer organism include:
• Sideramines (ferrichromes and ferrioxamines) and,
• Ionophore antibiotics (macrotetralides).

Both groups are involved in the uptake of cations.

Some compounds may inhibit competing organisms and others such as gramicidins are
associated with the promotion of spore formation. Fortuitously, many secondary metabolites
exhibit properties that have proved very useful and have become major industrial microbial
products (Table).

The synthesis of secondary metabolites is usually tightly regulated by the cell. Some regulatory
mechanisms are common to both primary and secondary metabolism, including the cell’s
adenylate EC and carbon catabolite repression. Carbon sources that support high growth rates
tend to be repressive; for example, glucose suppresses the production of several antibiotics
including penicillin and chloramphenicol.

18
In the cells there is correlation between primary and secondary metabolism. Secondary
metabolism succeeds primary metabolism (Fig.3). Alternatively, secondary metabolites are
produced from intermediates and end products of primary metabolites. A particular secondary
metabolite is produced by only a few selective microbes e.g. an antibiotic penicillin is produced
only by Penicillium spp.

Table: Some Examples of Industrially Important Secondary Metabolites and

Their Producer Microorganisms

19
Figure: Biosynthetic Pathways for the Production of Primary and Secondary
Metabolites

20
Microbial Biomass
In a few instances the cells i.e. biomass of microbes, has industrial application as listed in Table.
The prime example is the production of single cell proteins (SCP) which are in fact whole cells of
Spirullina (an algae), Saccharomyces (a yeast) and Lactobacillus (a bacterium). SCP is
essentially rich in amino acids which are either absent in vegetarian food or present in low
amounts e.g. lysine, threonine, methionine, leucine, isoleucine etc.

Table: Microbial Biomass Production

Recombinant Products
With the advent of gene cloning techniques, many industrially important genes from plants,
animals and microbes have been cloned in a few selected microbes like E.coli and
Saccharomyces and Pichia. The basic idea behind all these clonings is to produce large amount
of proteins and scientists have been highly successful in achieving this goal. Table 4 lists some
of the recombinant products.

Table 4: Recombinants of Industrial Importance

21
Important Microbial Products
Table: Microbial Enzymes of Industrial Importance

22
23
Table 6: Applications of Microbes in Healthcare Products

In addition to above mentioned microbial products, following Tables (5-8) will expose you to yet
more application of microbes or their derivatives in the commercial sector. Microbial enzymes are
biocatalysts which are primarily protein in nature though certain RNA molecules have also been
shown to possess catalytic activity. Enzymatic processes are fast replacing chemical processes
because such technology is eco-friendly, stereo specific and generate less undesirable waste
products. Table 5 lists the microbial enzymes of industrial importance.

24
Table 7: Applications of Microbes in Food and Beverage Fermentations

Table 8: Applications of Microbes in Food Additives and Supplements

25
Safe Microbes
For any microbe to be used at commercial level, it is imperative that it should not be pathogenic
and the final purified product should be free from toxic contaminants. Prior clearance from
regulatory authorities have to be sought for their use at industrial level. However, a few microbes
have been categorised as GRAS (generally regarded as safe). No permission from authorities is
needed for their use at industrial level, as long as they are grown under stipulated conditions.
Organisms listed under GRAS category mentioned in Table 9.

Table 9: Microbes Classified as GRAS

26
 DETERMINING GROWTH IN BACTERIAL POPULATIONS
The techniques used to measure growth obscure the fact that all bacterial cultures are grossly
heterogeneous, and the techniques really monitor average values, which describe the growth of
a population rather than of individual cells. Although all members of a particular population may
be genetically identical, the individual members vary with respect to doubling time, age,
composition, metabolic characteristics, and size.

The magnitudes of these variations are influenced by the environment and can therefore be
minimised by careful design and development of the system for cultivation. Most of the systems
to be described are designed to minimise heterogeneity within the population of a pure culture.
In such systems, the growth behaviour of a bacterial population can be predicted by the simple
relationship:

dx = μX Eqn. (1)
dt

where, dx is the increase in amount of cell mass (biomass)

dt is the time interval
x is the amount of biomass, and
μ is the specific growth rate, representing the rate of growth per unit
amount of biomass and having dimensions of reciprocal of time (1/t).

If μ is constant, integration of eqn. (1) shows that x will increase exponentially with time as follows:

In x = μt Eqn. (2)
xo

where, ln is natural logarithm (to the base e = 2.303), and

xo is the amount of biomass when t = 0

It follows by arrangement of eqn. (2), that the final biomass concentration x is:

x = xo e μt Eqn. (3)

Bacterial growth that follows this relationship is called exponential or logarithmic growth.

The relationship between the biomass doubling time (td) and the specific growth rate is found by
letting x = 2xo at t = td in eqn. (2) and solving the equation as follows:

td = In 2
μ
or,

td = 0.693 Eqn. (4)

μ
Eqn. (1) to (4) are used to predict the growth of bacteria in simple systems in which the factors
influencing growth are constant but do not allow prediction of deviation from constant-growth
(steady-state) conditions.

27
The techniques for continuous cultivation very nearly establish steady-state conditions of
theoretically infinite duration, whereas the techniques for batch cultivation allow significant
changes in the environment during the time course of cultivation.
In batch culture eqn. (1) to eqn. (4) will apply without adjustment to the value of μ only during the
portion of the growth cycle in which the changes in growth environment have no influence on
population growth (i.e., during the exponential growth phase).

The time taken for one complete cell cycle, the doubling time varies with species and with growth
conditions. For minimum doubling time, optimum growth conditions are necessary.
In E. coli the minimum doubling time is about 20 minutes, and in some species of Mycobacterium,
for example, it is many hours. It has been estimated that Mycobacterium leprae (the causative
agent of leprosy) has a doubling time of about two weeks in infected tissues.
Since following cell division, each daughter cell can itself grow and divide; one cell can quickly
give rise to a large population of cells if conditions are favourable. Given suitable conditions, such
populations may develop either on solid surfaces or within the body of a liquid. In bacteriology,
any solid or liquid prepared for bacterial growth is called a medium.

BATCH CULTURE
A batch culture system is one in which nothing (with the frequent exception of the gas
phase) is added to or removed from an environment after medium of appropriate
composition is inoculated with living cells. This is called a closed system.

It follows that a batch system can support cell multiplication for only a limited time and with
progressive changes in the original medium and environment. Suppose that a few bacterial cells
are introduced into a suitable liquid medium which is then held at optimum growth temperature
for that species. At regular intervals a small volume of the medium can be withdrawn and a count
made of the cells it contains. In this manner one can follow the development of a population, i.e.,
the increase in cell numbers with time. By plotting the number of cells against time, one obtains
a growth curve which, for a given species growing under given conditions, has a characteristic
shape.

By growing bacteria in, or on a medium, one produces a culture. Thus a culture is a liquid or solid
medium containing bacteria which have grown (or are still growing) in or on that medium.

The process of maintaining a particular temperature, and/or other desired conditions, for bacterial
growth is called incubation. The initial process of adding the cells to the medium is called
inoculation.

Table: Increase in Cell Numbers with Time for Escherichia coli Growing Under Optimal
Conditions in the Logarithmic Phase
Time (minutes) 10 20 40 60 80 100…..200…..300

Number of Generations 0 1 2 3 4 5…….10…….15

(i.e., Rounds of cell division)
Number of Cells 1 2 4 8 16 32…..1,024…32,768

Number of Cells as a Power of 2 20 21 22 23 24 25……210…….215

28
In the log phase of growth, a plot of cell numbers versus time gives a sharply rising curve on a
simple arithmetical scale (Figure, Lecture Notes).
Clearly, a simple arithmetical scale would not be adequate for large numbers of cells. There is a
better way of plotting growth in the log phase. The Table (bottom row) shows that cell numbers
can be expressed as powers of 2, for example, the 8 at 60 minutes can be written as 2 3 cells (in
which 3 is the index).

Each of the indices in the Table is, of course, the logarithm (to base 2, i.e., log2) of the
corresponding number of cells. Now, instead of plotting cell numbers directly, one can plot the
log2 of each number, the result is a straight-line graph [Fig.2(b)]
In such a graph each unit on the log2 scale represents a doubling in cell numbers; the doubling
time (time, in minutes, needed for a doubling in cell numbers). The doubling time can therefore
be read off directly from the time-scale of the graph. The doubling time is also called the
generation time.

Usually it is more convenient to use log10 rather than log2 when constructing a growth curve. The
log10 and log2 of any number can be interconverted by using the formula:

29
 log10 N = 0.301 log2 N

The log phase of the graph will still be a straight line. Only the slope of the graph will change.

The fact that constant exponential growth can occur for even a limited time in batch culture shows
that the growth rate can be virtually unaffected by changes in substrate concentrations over wide
ranges. Under these conditions the culture is said to be in balanced growth, whose rate can be
described by a single numerical value μ.
Eventually, the culture will deviate from constant exponential growth and can no longer be
described only by the value of μ even though it is possible to calculate this value for the case of
growth limitation by single nutrient substrate.

Microbial products can be classified in three major categories:

1. Growth-associated products are produced simultaneously to microbial growth.

qp = 1 dP = Yp/xμ
x dt
(i)
The production of a constitutive enzyme is an example of a growth-associated product.

2. Nongrowth-associated product formation takes place during the stationary phase when
the growth rate is zero. The specific rate of product formation is constant.

qP = β = constant
(ii)
Many secondary metabolites, such as antibiotics (for example, penicillin), are nongrowth-
associated products.

3. Mixed-growth-associated product formation takes place during the slow growth and
stationary phases. In this case, the specific rate of product formation is given by the
following equation:
qP = αμ + β
(iii)
Lactic acid fermentation, xanthan gum, and some secondary metabolites from cell culture
are examples of mixed-growth-associated products. Equation (iii) is a Luedeking-Piret
equation. If α = 0, the product is only growth associated, and if β = 0 the product would
be only growth associated and consequently α would then be equal to Yp/x

Most industrial bioreactors are operated in batch mode due to the relative simplicity of this
process. The whole batch operation consists of several steps, including medium formulation,
filling the bioreactor, sterilization in place (SIP systems), inoculation, cultivation, product
harvesting, and bioreactor cleaning in place (CIP systems). For efficient performance of batch
operation, it is important to minimise all nonproductive steps (all steps listed above except
cultivation), achieve a high rate of product synthesis, optimise productivity, and maximise the yield
of the end product. The performance of any particular batch operation is thus influenced by the
type of end product-an extension of exponential growth is advantageous for the efficient
production of biomass (baker’s yeasts, feed biomass) or primary metabolites (ethanol, acetic,

30
citric, or lactic acids), whereas in the case of secondary metabolite production, the exponential
phase is shortened (by the limitation of one nutrient, usually the source of nitrogen) and the
stationary phase is prolonged to achieve the maximum yield of the product.

Submerged batch cultivation can be used for the production of alcoholic beverages (beer, wine,
and distilled spirits such as whisky, brandy, rum, and others), organic acids used in the food
industry either as acidifiers or as preservatives (citric, acetic (vinegar), and lactic acids), and
amino acids used as flavor enhancers (e.g., monosodium glutamate) or sweeteners (e.g.,
aspartate).

For distilled spirits, the fermentation of wort during Scotch whisky production is taken as an
example. Washbacks, simple cylindrical fermentation vessels (volume 250 to 500 m3) for the
production of distilled spirits are made either from wood or from stainless steel. Although wood
washbacks are difficult to clean and sanitize, they are still used, especially in malt whisky
distilleries. Wort to be fermented is pumped to the washback, cooled to 20°C, and inoculated with
either fresh or dried yeast cells (Campbell, 2003).

The global production of citric acid reached 1.8 × 10 6 tonnes in 2010 (F.O. Licht data); 90% of
this was produced by microbial (Aspergillus niger) synthesis from sugar-containing or starch-
containing materials (sugar beet, sugarcane molasses, and corn) and about 60% of this amount
was consumed in the food industry. Although citric acid can be produced at an industrial scale
using surface liquid cultivation, solid-state cultivation, or submerged liquid cultivation, nowadays,
the latter predominates.

Submerged cultivation is carried out in stirred bioreactors (capacity 150 to 200 m3) or bubble
columns (capacity up to 1000 m3), usually operating aerobically for 4 to 10 days until the citric
acid concentration reaches 10 to 15% w/v (Moresi and Parente, 2000; Soccol et al., 2006).

The Bacterial Growth Curve

For microorganisms, growth is the most essential response to their physiochemical environment.
Growth is a result of both replication and changes in cell size. Microorganisms can grow under a
variety of physical, chemical, and nutritional conditions. In a suitable nutrient medium, organisms
extract nutrients from the medium and convert them into biological compounds. Part of these
nutrients is used for energy production and part is used for biosynthesis and product formation.
As a result of nutrient utilisation, microbial mass increases with time and can be described simply
as:
substrates + cells → extracellular products + more cells

∑S + x ──> ∑P + nX
Microbial growth is a good example of an autocatalytic reaction. The rate of growth is directly
related to cell concentration, and cellular reproduction is the normal outcome of this reaction.
The rate of microbial growth is characterised by the specific growth rate, which is defined as:
μ = 1 dx
x dt
Where x is cell mass concentration (g/l), t is time (h), and μ is specific growth rate (h-1).

31
Figure: Growth curve for an exponentially increasing population, plotted logarithmically
(dashed line) and arithmetically (solid line).

In the laboratory, under favourable conditions, a growing bacterial population doubles at regular
intervals. Growth is by geometric progression: 1, 2, 4, 8, etc. or 20, 21, 22, 23.........2n (where n =
the number of generations). This is called exponential growth. In reality, exponential growth is
only part of the bacterial life cycle, and not representative of the normal pattern of growth of
bacteria in nature. In normal practice an organism will seldom have totally ideal conditions for
unlimited growth; rather, growth will be dependent on a limiting factor, e.g. an essential nutrient.
As conditions of this factor drops, so also will the growth potential of the organism decrease.

In biotechnological processes there are three main ways of growing microorganisms in the
bioreactor, namely batch, semi-continuous or continuous. Within the bioreactor reactions can
occur with static or agitated cultures, in the presence or absence of oxygen, and in liquid or low
moisture conditions, e.g. on solid substrates. The microorganisms can be free or can be attached
to surfaces by immobilisation or by natural adherence.

In batch culture the microorganisms are inoculated into fixed volume of medium and as growth
takes place nutrients are consumed and products of growth (biomass, metabolites) accumulate.
The nutrient environment within the bioreactor is continuously changing and thus, in turn,
enforcing changes to cell metabolism. Eventually, cell multiplication ceases because of
exhaustion or limitation of nutrient(s) and accumulation of toxic excreted waste products.

32
 Figure 5: The Typical Bacterial Growth Curve
When bacteria are grown in a closed system (also called a batch culture), like a test tube, the
population of cells almost always exhibits these growth dynamics: cells initially adjust to the new
medium (lag phase) until they can start dividing regularly by the process of binary fission
(exponential phase). When their growth becomes limited, the cells stop dividing (stationary phase),
until eventually they show loss of viability (death phase). Note the parameters of the x and y axes.
Growth is expressed as change in the number viable cells vs. time. Generation times are calculated
during the exponential phase of growth. Time measurements are in hours for bacteria with short
generation times.

When a fresh medium is inoculated with a given number of cells, and the population growth is
monitored over a period of time, plotting the data will yield a typical bacterial growth curve
(Figure 3 below).

Four characteristic phases of the growth cycle are recognised.

1. Lag Phase. Immediately after inoculation of the cells into fresh medium, the population
remains temporarily unchanged. This occurs when the cells are introduced to their new growth
medium. Microorganisms reorganise their molecular constituents when they are transferred to a
new medium. Depending on the composition of nutrients, new enzymes are synthesised, the
syntheses of some other enzymes are repressed, and the internal machinery of cells is adapted
to the new environmental conditions. If their new environment is identical to their old medium,
then the lag phase can disappear: however, even the mechanical shock of moving some cells
around can cause a lag phase.
Although there is no apparent cell division occurring, the cells may be growing in volume or mass,
synthesising enzymes, proteins, RNA, etc., and increasing in metabolic activity.

The length of the lag phase is apparently dependent on a wide variety of factors including:
• Size of the inoculum;
• Time necessary to recover from physical damage or shock in the transfer;
• Time required for synthesis of essential coenzymes or division factors;
• Time required for synthesis of new (inducible) enzymes that are necessary to
metabolise the substrates present in the medium.

To minimise the duration of the lag phase, cells should be adapted to the growth medium and
conditions before inoculation, and the inoculum size should be large (5% to 10% by volume). The
nutrient medium may need to be optimised and certain growth factors may need to be included

33
to minimise the lag phase. Many commercial fermentation plants rely on batch culture; to obtain
high productivity from a fixed plant size the lag phase must be as short as possible.

Multiple lag phases may be observed when the medium contains more than one carbon source.
This phenomenon is known as diauxic growth and is caused by a shift in metabolic pathways in
the middle of a growth cycle. After one carbon source is exhausted, the cells adapt their metabolic
activities to utilise the second carbon source. The first carbon source is more readily utilisable
than the second, and the presence of more readily available carbon source represses the
synthesis of the enzymes required for the metabolism of the second substrate.

2. Exponential (log) Phase. The exponential growth phase is also known as the
logarithmic growth phase. During the exponential (log) phase, microorganisms are growing and
dividing at the maximal rate possible given their genetic potential, the nature of the medium, and
the environmental conditions. Their rate of growth is constant during the exponential phase; that
is, they are completing the cell cycle and doubling in number at regular intervals. The population
is most uniform in terms of chemical and physiological properties during this phase; therefore
exponential phase cultures are usually used in biochemical and physiological studies.

Exponential (logarithmic) growth is balanced growth. That is, all cellular constituents are
manufactured at constant rates relative to each other. If nutrient levels or other environmental
conditions change unbalanced growth results. During unbalanced growth, the rates of synthesis
of cell components vary relative to one another until a new balanced state is reached. Unbalanced
growth is readily observed in two types of experiments:
Shift-up, where a culture is transferred from a nutritionally poor medium to a richer one;
and
Shift-down, where a culture is transferred from a rich medium to a poor one.

In a shift-up experiment, there is a lag while the cells first construct new ribosomes to enhance
their capacity for protein synthesis. In a shift-down experiment, there is a lag in growth because
cells need time to make the enzymes required for the biosynthesis of unavailable nutrients. Once
the cells are able to grow again, balanced growth is resumed and the culture enters the
exponential phase. These shift-up and shift-down experiments demonstrate that microbial growth
is under precise, coordinated control and responds quickly to changes in environmental
conditions.

When microbial growth is limited by the low concentration of a required nutrient, the final
net growth or yield of cells increases with the initial amount of the limiting nutrient present
(Figure (a) below). The rate of growth also increases with nutrient concentration (Figure (b) below)
but in a hyperbolic manner much like that seen with many enzymes. The shape of the curve
seems to reflect the rate of nutrient uptake by microbial transport proteins. At sufficiently high
nutrient levels, the transport systems are saturated, and the growth rate does not rise further with
increasing nutrient concentration.

34
 Figure: Nutrient Concentration and Growth.
(a) The effect of changes in limiting nutrient concentration on total microbial yield. At sufficiently
high concentrations, total growth will plateau. (b) The effect on growth rate.

In this phase, the cells have adjusted to their new environment. After this adaptation period,
cells can multiply rapidly, and cell mass and cell number density increase exponentially with time.

Cell growth during the logarithmic (or exponential) phase is at a constant rate which is shown by:

Eqn. 1
where;
C0 = original cell concentration, Cb = cell concentration after time t, (biomass produced), μ (h-1) =
specific growth rate and t (h) = time of fermentation.
Graphically, the natural logarithm (ln) of cell concentration versus time produces a straight line,
the slope of which is the specific growth rate. The highest growth rate (μmax) occurs in the
logarithmic phase.

The productivity of a culture is the amount of biomass produced in unit time (usually per hour)
and is found using:

Eqn. 2
where;
Pb (gl-1h-1) = productivitiy, Cmax = maximum cell concentration during the fermentation, C0 = initial
cell concentration, t1 (h) = duration of growth at the maximum specific growth rate, t2 (h) = duration
of the fermentation when cells are not growing at the maximum specific growth rate and including
the time spent in culture preparation and harvesting.

35
Sample Problem 1.
An inoculum containing 3.0 x 104 cells ml-1 of Saccharomyces cerevisiae is grown on
glucose in a batch culture for 20 h. Cell concentrations are measured at 4h intervals and
the results are plotted in the Figure below. The total time taken for culture preparation and
harvest is 1.5 h. Calculate the maximum specific growth rate and the productivity of the
culture.

Solution to Sample Problem 1.

From equation (1) for the logarithmic phase,

From equation (2),

The exponential phase of growth is a pattern of balanced growth wherein all the cells are dividing
regularly by binary fission, and are growing by geometric progression. The cells divide at a
constant rate depending upon the composition of the growth medium and the conditions of
incubation. The rate of exponential growth of a bacterial culture is expressed as generation time,
also the doubling time of the bacterial population. Generation time (G) is defined as the time
(t) per generation (n = number of generations). Hence, G = t/n is the equation from which
calculations of generation time (below) derive.

36
3. Stationary Phase. The deceleration growth and stationary phases follows the
exponential phase. Exponential growth cannot be continued forever in a batch culture (e.g. a
closed system such as a test tube or flask). This stationary phase usually is attained by bacteria
at a population level of around 109 cells per ml. Other microorganisms normally do not reach such
high population densities. For instance, protist cultures often have maximum concentrations of
about 106 cells per ml. Final population size depends on nutrient availability and other factors, as
well as the type of microorganism being cultured. In the stationary phase, the total number of
viable microorganisms remains constant. This may result from a balance between cell division
and cell death, or the population may simply cease to divide but remain metabolically active.

Population growth is limited by one of three factors:

• Exhaustion of available nutrients: Aerobic organisms often are limited by O2 availability.
Oxygen is not very soluble and may be depleted so quickly that only the surface of a
culture will have an O2 concentration adequate for growth. The cells beneath the surface
will not be able to grow unless the culture is shaken or aerated in another way.

• Accumulation of inhibitory metabolites or end product: This factor seems to limit the
growth of many anaerobic cultures (cultures growing in the absence of O2). For example,
streptococci can produce so much lactic acid and other organic acids from sugar
fermentation that their medium becomes acidic and growth is inhibited.

• Exhaustion of space, in this case called a lack of "biological space": Finally, some
evidence exists that growth may cease when a critical population level is reached. Thus
entrance into the stationary phase may result from several factors operating in concert.

As we have seen, bacteria in a batch culture may enter stationary phase in response to starvation.
This probably occurs often in nature because many environments have low nutrient levels.
Prokaryotes have evolved a number of strategies to survive starvation. Some bacteria
respond with obvious morphological changes such as endospore formation, but many only
decrease somewhat in overall size. This is often accompanied by protoplast shrinkage and
nucleoid condensation. The more important changes during starvation are in gene expression
and physiology. Starving bacteria frequently produce a variety of starvation proteins, which
make the cell much more resistant to damage. Some increase peptidoglycan cross-linking
and cell wall strength. The Dps (DNA-binding protein from starved cells) protein protects DNA.

Proteins called chaperone proteins prevent protein denaturation and renature damaged
proteins. Because of these and many other mechanisms, starved cells become harder to kill
and more resistant to starvation, damaging temperature changes, oxidative and osmotic
damage, and toxic chemicals such as chlorine. These changes are so effective that some
bacteria can survive starvation for years. There is even evidence that Salmonella enterica serovar
typhimurium (S. typhimurium) and some other bacterial pathogens become more virulent when
starved. Clearly, these considerations are of great practical importance in medical and industrial
microbiology.
During the stationary phase, if viable cells are being counted, it cannot be determined whether
some cells are dying and an equal number of cells are dividing, or the population of cells has
simply stopped growing and dividing. The stationary phase, like the lag phase, is not necessarily
a period of quiescence. Bacteria that produce secondary metabolites, such as antibiotics, do so
during the stationary phase of the growth cycle (Secondary metabolites are defined as
metabolites produced after the active stage of growth). It is during the stationary phase that spore-

37
forming bacteria have to induce or unmask the activity of dozens of genes that may be involved
in sporulation process.

If different initial substrate concentrations are plotted against cell concentration in the stationary
phase, it is found that an increase in substrate concentration results in a proportional increase in
cell yield. This indicates substrate limitation of cell growth, which is described by:

Eqn. 3
where;
Cb = concentration of biomass, Y = yield factor, S0 (mgl-1) = original substrate concentration, Sr
(mgl-1) = residual substrate concentration.

For a typical bacterial culture, these changes occur over a very short period of time. The rapidly
changing environment results in unbalanced growth. In the exponential phase, the cellular
metabolic control system is set to achieve maximum rates of reproduction. In the deceleration
phase, the stresses induced by nutrient depletion or wastes accumulation cause a restructuring
of the cell to increase the prospects of cellular survival in a hostile environment. These observable
changes are the result of the molecular mechanisms of repression and induction.

The stationary phase starts at the end of deceleration phase, when the net growth rate is
zero (no cell division) or when the growth rate is equal to the death rate. Even though the
net growth rate is zero during the stationary phase, cells are still metabolically active and
produce secondary metabolites. Primary metabolites are growth-related products and
secondary metabolites are non-growth related. In fact, the production of certain metabolites is
enhanced during the stationary phase (for example, antibiotics, some hormones) due to
metabolite deregulation. During the course of the stationary phase, one or more of the following
phenomena may take place:
1. Total cell mass concentration may stay constant, but the number of viable cells may
decrease.
2. Cell lysis may occur and viable cell mass may drop. A second growth phase may
occur and cells may grow on lysis products of lysed cell (cryptic growth).
3. Cells may not be growing but may have active metabolism to produce secondary
metabolites. Cellular regulation changes when concentrations of certain
metabolites (carbon, nitrogen, phosphate) are low. Secondary metabolites are
produced as a result of metabolite deregulation.

During the stationary phase, the cell catabolises cellular reserves for new building blocks and for
energy-producing monomers. This is called endogenous metabolism. The cell must always
expend energy to maintain an energised membrane (i.e., proton-motive force) and transport of
nutrients and for essential metabolic functions such as motility and repair of damage to cellular
structures. This energy expenditure is called maintenance energy.

The reason for termination of growth may be either exhaustion of an essential nutrient or
accumulation of toxic products. If inhibitory product is produced and accumulates in the medium,
the growth rate will slow down depending on inhibitor production, and at a certain level of inhibitor
concentration, growth will stop. Ethanol production by yeast is an example of a fermentation in
which the product is inhibitory to growth. Dilution of toxified medium, addition of an
unmetabolisable chemical compound complexing with the toxin, or simultaneous removal of the
toxin would alleviate the adverse effects of the toxin and yield further growth.

38
4. Death Phase. If incubation continues after the population reaches stationary phase, a death
phase follows, in which the viable cell population declines. (Note, if counting by turbidimetric
measurements or microscopic counts, the death phase cannot be observed). During the death
phase, the number of viable cells decreases geometrically (exponentially), essentially the reverse
of growth during the log phase.

Figure: Loss of Viability

(a) It has long been assumed that as cells leave stationary phase due to starvation or toxic waste
accumulation, the exponential decline in culturability is due to cellular death. (b) The viable but
nonculturable (VBNC) hypothesis posits that when cells are starved, they become temporarily
nonculturable under laboratory conditions. When exposed to appropriate conditions, some cells
will regain the capacity to reproduce. (c) Some believe that a fraction of a microbial population dies
due to activation of programmed cell death genes. The nutrients that are released by dying cells
support the growth of other cells.

For many years, the decline in viable cells following the stationary phase was described simply
as the “death phase.” It was assumed that detrimental environmental changes such as nutrient
deprivation and the buildup of toxic wastes caused irreparable harm and loss of viability. That is,
even when bacterial cells were transferred to fresh medium, no cellular growth was observed.
Because loss of viability was often not accompanied by a loss in total cell number, it was assumed
that cells died but did not lyse.

This view is currently under debate. There are two alternative hypotheses (Figure above). Some
microbiologists think starving cells that show an exponential decline in density have not
irreversibly lost their ability to reproduce. Rather, they suggest that microbes are temporarily
unable to grow, at least under the laboratory conditions used. This phenomenon, viable but

39
nonculturable (VBNC), is thought to be the result of a genetic response triggered in starving,
stationary phase cells.

Just as some bacteria form endospores as a survival mechanism, it is argued that others are able
to become dormant without changes in morphology, (Figure (b)). Once the appropriate conditions
are available (for instance, a change in temperature or passage through an animal), VBNC
microbes resume growth. VBNC microorganisms could pose a public health threat, as many
assays that test for food and drinking water safety are culture-based.

The second alternative to a simple death phase is programmed cell death, (Figure (b)). In
contrast to the VBNC hypothesis whereby cells are genetically programmed to survive,
programmed cell death predicts that a fraction of the microbial population is genetically
programmed to die after growth ceases. In this case, some cells die and the nutrients they leak
enable the eventual growth of those cells in the population that did not initiate cell death. The
dying cells are thus “altruistic”; they sacrifice themselves for the benefit of the larger population.

Long-term growth experiments reveal that an exponential decline in viability is sometimes

replaced by a gradual decline in the number of culturable cells. This decline can last months to
years (Figure below).

Figure: Prolonged Decline in Cell Numbers

Instead of a distinct death phase, successive waves of genetically distinct subpopulations of
microbes better able to use the released nutrients and accumulated toxins survive. Each successive
solid or dashed curve represents the growth of a new subpopulation.

During this time, the bacterial population continually evolves so that actively reproducing cells are
those best able to use the nutrients released by their dying brethren and best able to tolerate the
accumulated toxins. This dynamic process is marked by successive waves of genetically distinct
variants. Thus natural selection can be witnessed within a single culture vessel.

In the death phase the decrease of the number of cell occurs exponentially which happens when
the cell breaks open (lysed). The rate of death normally follows the first-order kinetics given by;

40
where Ns is the concentration of cells at the end of the stationary phase and at the beginning of
the death phase and k'd is the first order death rate constant. In both stationary and death phase,
it is important to recognise that there is a distribution of properties among the cells in a population.
A summary of the different phases of cell growth is given in Table below.

Table: Summary of the Growth Phases.

The length of the different phase varies enormously with different cell types. Thus, many common
bacteria have a stationary phase lasting only a day or two before death phase starts. In contrast,
mammalian nerve cells can last almost indefinitely in culture without dividing. A single mammalian
cell isolated from skin or muscle and put into culture medium could take a week to divide; a single
E. coli cell is unlikely to take more than 10 minutes to start growth.

A key idea in cell growth studies is doubling time. This is the time needed for the cell population
to double in number, and is equal to the time an ‘average’ member of that population takes to go
through one complete division cycle. The higher the doubling time, the lower the growth rate
of the culture and the longer it will take for an inoculum to reach stationary phase (‘growth
rate’ is sometimes defined as 1/doubling time). Doubling time depends on the growth
conditions and on the organism being grown; some bacteria (notably Clostridium perfringens) can
have a doubling time of 10 minutes in the right culture medium. Strictly speaking, the concept of
doubling only applies to organisms growing in log phase, i.e. growing exponentially.

A related concept is generation time, which is the time that a single organism takes to go through
one reproductive cycle. For organisms that grow by division, this is pretty much the same as
doubling time. However, it can be applied to organisms with more complicated life cycles, such
as flies and people, where you can get more than a doubling of the population every generation

41
if each parent has enough offspring. Thus growth cycle is not the same as the overall life and
senescence cycle of primary mammalian cells. Mammalian cells will stop dividing when they have
exhausted one of the critical components in their culture medium, or when their surroundings are
too crowded for their liking. However, if they are separated and put into new medium (a process
known as ‘splitting’ the cells), then healthy cells will start to grow again.

Senescence is what occurs when the cells have been split many times so that the total number
of divisions they have gone through is 40 to 60. Then, they slowly cease to be able to divide any
more, no matter how much new medium they are given.

Growth Rate and Generation Time

As mentioned, bacterial growth rates during the phase of exponential growth, under standard
nutritional conditions (culture medium, temperature, pH, etc.), define the bacterium's generation
time. Generation times for bacteria vary from about 12 minutes to 24 hours or more. The
generation time for E. coli in the laboratory is 15 to 20 minutes, but in the intestinal tract, the
coliform's generation time is estimated to be 12 to 24 hours. For most known bacteria that can be
cultured, generation times range from about 15 minutes to 1 hour. Symbionts such as Rhizobium
tend to have longer generation times. Many lithotrophs, such as the nitrifying bacteria, also have
long generation times. Some bacteria that are pathogens, such as Mycobacterium tuberculosis
and Treponema pallidum, have especially long generation times, and this is thought to be an
advantage in their virulence. Generation times for a few bacteria are shown in Table 2.

Table: Generation times for some common bacteria under optimal conditions of
growth.
Bacterium Medium Generation Time (minutes)
Escherichia coli Glucose-salts 17
Bacillus megaterium Sucrose-salts 25
Streptococcus lactis Milk 26
Streptococcus lactis Lactose broth 48
Staphylococcus aureus Heart infusion broth 27-30
Lactobacillus acidophilus Milk 66-87
Rhizobium japonicum Mannitol-salts-yeast extract 344-461
Mycobacterium tuberculosis Synthetic 792-932 (13-16 hours)
Treponema pallidum Rabbit testes 1980 (14 days)

Growth kinetics
Growth rate constant: number of generations that occur per unit time; (expressed as k or μ; h-1):
(1) k = (ln N2 - ln N1)/(t2 - t1)

where N2 and N1 = cells ml-1 at time t2 and t1 (in h)

(2) Convert (1) to log: k = (log N2 - log N1) (2.303)/(t2 - t1)

Generation time: time required for the cell population to double; (expressed as g; h):

42
(3) g = (ln 2)/k
Example I: A bacterial culture in exponential growth contained 104 cells/ml at time zero and
108 cells/ml at time 4 h. What is the growth rate constant (k)?
From
(1) k = (ln 108 - ln 104)/(4 - 0) = 2.3 h-1

What is the generation time?

From
(2) g = ln 2/k = 0.693/2.3 = 0.30 h = 18 min

Example II: E. coli grew exponentially from 5 x 10 5 cells/ml to 3.5 x 107 cells/ml in 5 h. Its
generation time was 40 min. Was there a lag phase?

From
(1) and (3) (t2 - t1) = (ln N2 - ln N1)/k = g (ln N2 - ln N1)/ln 2
= 40 [ln (3.5 x 107) - ln (5 x 105)]/0.693
= 245 min

Total growth period = 5 h

= 300 min

Lag = 300 - 245 = 55 min

Calculation of Generation Time

When growing exponentially by binary fission, the increase in a bacterial population is by
geometric progression. If we start with one cell, when it divides, there are 2 cells in the first
generation, 4 cells in the second generation, 8 cells in the third generation, and so on. The
generation time is the time interval required for the cells (or population) to divide.

G (generation time) = t(time, in minutes or hours)/n(number of generations)

G = t/n
G = generation time (time for the cells to divide)
t = time interval in hours or minutes
B = number of bacteria at the beginning of a time interval
b = number of bacteria at the end of the time interval
n = number of generations (number of times the cell population doubles during the time interval)
b = B x 2n (This equation is an expression of growth by binary fission)

and

G = t/n
Solve for n:
logb = logB + nlog2

n = logb - logB
log2

43
 n = logb - logB
0.301

n = 3.3 logb/B
but,

G = t/n

Solve for G

G = t ____________
3.3 log b/B

Sample Problem 2
What is the generation time of a bacterial population that increases from 10,000 cells to
10,000,000 cells in four hours of growth?

Solution to Sample Problem 2

First convert 4 hours to minutes = 240 minutes

G= t_________
3.3 log b/B
G= 240 minutes
3.3 log 107/104
G= 240 minutes
3.3 x 3

G = 24 minutes

44
 CONTINUOUS CULTURE
Continuous cultivation differs from batch cultivation in that a fresh supply of medium is added
continuously at the same rate that culture is withdrawn (an open system). If the culture is well
mixed, a sample that is representative of both the cell population and substrate concentration
within the fermentor can be withdrawn. The technique of continuous cultivation theoretically allows
continuous exponential growth of the culture in a system requiring constant addition of fresh
medium and constant withdrawal of culture so that the culture volume and cell concentration
remains constant with time.

In its broadest sense, continuous cultivation does not require a constant cell concentration, but
most literature accounts of quantitative study of continuous cultivation have dealt with such a
situation and the principles developed assume a constant cell concentration. The term “steady
state” is often applied to continuous cultivation and means literally that no change in status occurs
during the time span studied. In reality, this definition is too broad, since practical application of
the continuous-cultivation theory often results in changes in some parameters while others
remains constant.

A continuous culture is therefore defined at steady state by an invariant biomass concentration

with respect to the time span of observation. In contrast, a batch culture may have a steady-state
dissolved oxygen concentration maintained by constant replacement but is not a continuous
culture because the biomass concentration changes with time.

Continuous Culture of Bacteria

The cultures so far discussed for growth of bacterial populations are called batch cultures. Since
the nutrients are not renewed, exponential growth is limited to a few generations.
However, it is possible to grow microorganisms in a system with constant environmental
conditions maintained through continual provision of nutrients and removal of wastes. Such a
system is called a continuous culture system. These systems can maintain a microbial population
in exponential growth, growing at a known rate and at a constant biomass concentration for
extended periods.

Continuous culture systems make possible the study of microbial growth at very low
nutrient levels, concentrations close to those present in natural environments. These
systems are essential for research in many areas, including ecology. For example, interactions
between microbial species in environmental conditions resembling those in a freshwater lake or
pond can be modeled. Continuous culture systems also are used in food and industrial
microbiology.

Continuous culture represents an open system in which nutrients are aseptically and continuously
added to the bioreactor, and the culture broth (containing cells and metabolites) is removed at the
same time that is, the volume of the culture broth is constant due to a constant feed-in and feed-
out rate.

Frequently, continuous culture is used as a synonym for a chemostat, represented by a constant

specific growth rate of cells, which is equal to the dilution rate and is controlled by the availability
of the limiting nutrient, although other types of continuous operation such as turbidostat (a
constant concentration of biomass controlled by the dilution rate) or nutristat (a constant
parameter related to cell growth controlled by the dilution rate) can be employed.

45
 Figure: Continuous Fermentation System

The main advantages of continuous culture (chemostat) over the batch mode are:
(a) Possibility to set up optimum conditions for maximum and long-term
product synthesis,
(b) Ability to achieve stable product quality (the steady state is characterized
by a homogeneous cell culture represented by a constant concentration of
biomass and metabolites), and
(c) A distinct reduction in “unprofitable” periods of the bioreactor operation.

In spite of these advantages, there are also several problems that hamper the extensive
utilisation of continuous operation on a large scale.
These include:
(a) Increased risk of contamination due to the pumping of the medium in and out of
the bioreactor,
(b) The danger of genetic mutations in the production strain in a long-term operation,
and,
(c) Additional investments may be required for technical facilities.

In industrial usage, batch cultivation has been operated to optimise organism or biomass
production and then to allow the organism to perform specific biochemical transformations such
as end-product formation (e.g. amino acids, enzymes) or decomposition of substrates (sewage
treatment, bioremediation). Many important products such as antibiotics are optimally formed
during the stationary phase of the growth cycle in batch cultivation. However, there are means of
prolonging the life of a batch culture and thus increasing yield by various substrates feed methods,
namely:
1. Gradual addition of concentrated components of nutrients, e.g. carbohydrates, so
increasing the volume of the culture (fed batch). This is used for industrial
production of baker’s yeast.
2. Addition of medium to the culture (perfusion) and withdrawal of an equal volume of
used-free medium- used in animal cell cultivations.

46
 Figure: A Continuous Stirred Tank Reactor (CSTR) Configuration.

Bacterial cultures can be maintained in a state of exponential growth over long periods of time
using a system of continuous culture (Figure 6), designed to relieve the conditions that stop
exponential growth in batch cultures. The practice of continuous cultivation gives near-balanced
growth, with little fluctuation of nutrients, metabolites, cell numbers or biomass. This practice
depends on fresh medium entering a batch system at the exponential phase of growth with a
corresponding withdrawal of medium and cells. Continuous methods of cultivation will permit
organisms to grow under steady state (unchanging) conditions in which growth occurs at a
constant rate and in a constant environment.
Chemostat Setup

47
Figure: A continuous fermentation set-up that can easily be used in the
laboratory.
Feed is pumped into the reactor, increasing the volume within the reactor. At the same time, the
overflow weir allows the excess volume to flow into the product tank. This ensures that the volume
within the reactor remains constant

Two major types of continuous culture systems commonly are used: chemostats and
turbidostats.

Continuous culture in a chemostat can be used to maintain a bacterial population at a

constant density, a situation that is, in many ways, more similar to bacterial growth in natural
environments.

A chemostat culture system can be used for the following purposes:

• To vary the specific growth rate (or metabolic rate) of a culture without changing
the environmental condition, other than the concentration of growth-limiting
substrate. This application is particularly useful in the understanding of the relationship
between growth rate and product formation or a physiological function of the microbial
culture.

• To fix the specific growth rate while varying a culture condition, i.e. converse of
above. The application is useful in the study of the effect of the culture environment without
interfering the growth rate.

• To maintain substrate limited growth with a constant specific growth rate. This could
not be achieved in a batch culture, as the growth rate is determined by the concentration
of the substrate in the culture medium.

• To obtain time-independent balanced growth (in steady state), with phenotype fully
adjusted to environment, i.e. the culture is history independent, unlike cells in batch
culture which performance is often dictated by the physiological state of the inoculum and
culture conditions that the cells were exposed to prior to inoculation.

• To maintain maximum output rate of biomass or product. The rate of product

formation is ultimately determined by the growth rate of the culture.

Kinetics of Continuous Culture

Continuous cultivation at steady state is possible only when all factors contributing to
accumulation of biomass are exactly balanced by all factors contributing to the loss of biomass
from the system.
This is shown by the following general material balance on the bacteria cell

Cell added to - cells removed + Cells produced - Cells consumed = Cells accumulated
the system from the system through growth through death within the system

The balance is shown mathematically as follows:

Fx0 - Fx + μx - αx = dx (1)
v v dt

Where, F is medium flow rate to and from the fermentor (litres per hour)

48
 vis liquid volume within the fermentor (litres)
x0 and x are the cell masses (gram per liter) in the feed and fermentor, respectively
μ is the specific growth rate
α is the specific death rate (reciprocal hours), and
dx/dt is the rate of change in cell mass (gram per liter-hour).

At steady state, dx/dt = 0.

The volume of true continuous culture is fixed in theory and undergoes negligible variation in
practice. Therefore, the flow rates to and from the fermentor must be identical.
Finally, the specific death rate is almost always much lower than the specific growth rate, so the
death term (αx) may be ignored. (Exceptions for this rule may occur at very low growth rates, in
the presence of toxic substances, or in an extreme biophysical environment).

Since the feed to the fermentor is usually sterile so that x0 = 0, at steady state:

μ = F
v
or
μ = D (2)

That is, the specific rate of growth of the population within the fermentor is determined by the
dilution rate, D, where D = F/V

Many types of continuous cultures are possible. We shall limit our discussion to the two major
types of continuous culture.
1. Chemostat, and
2. Turbidostat.

Determination of Maximal Specific Growth Rate (Lecture Notes)

A chemostat is basically a culture vessel having an input aperture for the influx of sterile
nutrient medium from a reservoir and an overflow aperture for the efflux of exhausted
medium, living cells, and cellular debris. The device (and the term "chemostat") was invented
by Novick and Szilard.

In practice, a chemostat apparatus is complicated by various attachments for aeration of the

medium and for the prevention of contamination, and the overflow is often controlled by means of
a siphon. The rate of cell division within the chemostat can be varied by a factor of 10 by
appropriate adjustment of the rate of inflow, but an equilibrium will eventually be reached in which
the number of new cells created by division will be exactly balanced by the number of cells
siphoned off in the overflow.

This equilibrium cell density is determined almost exclusively by the concentration of limiting
nutrient in the reservoir. Thus, chemostats provide an environment in which cell division is
continuous but population size is held constant, and the two parameters can be independently
manipulated.

49
In a chemostat, the growth chamber is connected to a reservoir of sterile medium. Once growth
is initiated, fresh medium is continuously supplied from the reservoir. The volume of fluid in the
growth chamber is maintained at a constant level by some sort of overflow drain. Fresh medium
is allowed to enter into the growth chamber at a rate that limits the growth of the bacteria. The
bacteria grow (cells are formed) at the same rate that bacterial cells (and spent medium) are
removed by the overflow.

The rate of addition of the fresh medium determines the rate of growth because the fresh medium
always contains a limiting amount of an essential nutrient. Thus, the chemostat relieves the
insufficiency of nutrients, the accumulation of toxic substances, and the accumulation of excess
cells in the culture, which are the parameters that initiate the stationary phase of the growth cycle.
The bacterial culture can be grown and maintained at relatively constant conditions, depending
on the flow rate of the nutrients.

Both population size and generation time are related to the dilution rate, and population density
remains unchanged over a wide range of dilution rates (Figure below). The generation time
decreases (i.e., the rate of growth increases) as the dilution rate increases. The limiting nutrient
will be almost completely depleted under these balanced conditions. If the dilution rate rises too
high, microorganisms can actually be washed out of the culture vessel before reproducing
because the dilution rate is greater than the maximum growth rate. This occurs because fewer
microorganisms are present to consume the limiting nutrient.

Figure 6: Schematic Diagram of a Chemostat, a Device for the Continuous Culture of

Bacteria.
The chemostat relieves the environmental conditions that restrict growth by continuously
supplying nutrients to cells and removing waste substances and spent cells from the culture
medium.

50
At very low dilution rates, an increase in D causes a rise in both cell density and the growth rate.
This is because of the effect of nutrient concentration on the growth rate, sometimes called the
Monod relationship. When dilution rates are low, only a limited supply of nutrient is available and
the microbes can conserve only a limited amount of energy. Much of that energy must be used
for cell maintenance, not for growth and reproduction. As the dilution rate increases, the amount
of nutrients and the resulting cell density rise because energy is available for both maintenance
and reproduction. The growth rate increases when the total available energy exceeds the
maintenance energy.

Figure: Chemostat Dilution Rate and Microbial Growth: The effects of changing the
dilution rate in a chemostat.

Factors such as pH and the concentrations of nutrients and metabolic products that inevitably
change during batch cultivation can be held near constant in continuous cultivations. In industrial
practice continuously operated systems are of limited use and include only single cell protein and
ethanol productions and some forms of waste-water treatment processes.

Steady State
One of the most important features of chemostats is that microorganisms can be grown in a
physiological steady state. In steady state, all culture parameters remain constant (culture
volume, dissolved oxygen concentration, nutrient and product concentrations, pH, cell
density, etc.). Microorganisms grown in chemostats naturally strive to steady state: If a low
amount of cells are present in the bioreactor, the cells can grow at growth rates higher than the
dilution rate, as growth is not limited by the addition of the limiting nutrient.

The limiting nutrient is a nutrient essential for growth, present in the media at a limiting
concentration (all other nutrients are usually supplied in surplus). However, if the cell
concentration becomes too high, the amount of cells that are removed from the reactor cannot be
replenished by growth as the addition of the limiting nutrient is insufficient. This results in an
equilibrium situation (steady state), where the rate of cell growth is equal to the rate of cell
removal.

51
Because obtaining a steady state requires at least 5 volume changes, chemostats require
large nutrient and waste reservoirs.

Yield Factor
In order to obtain the amount of every element that took part in a growth process, the cellular
content of all elements should be known. These include carbon, nitrogen, oxygen and hydrogen
(C, N, O and H respectively). For the same strain that grows in different environment; composition
of the elemental basis of the cell mass is consistent. However, the formulation of the medium is
highly complicated due to:
• Some substrate elements are released in products and not assimilated into cell material.
• Consideration of the limiting rate and limiting stoichiometry.
• Specific nutrients may be limiting/specific products may be inhibitory; due to metabolic
properties of cell strain.

The two limiting factors above may not occur at the same time as the other. One type of nutrient
may limit the rate of cell growth, but it may also be cause by the depletion of a different compound
that stops the cell from growing. Such difficulties can only be observed by conducting an
experimental analysis which can distinguish between the rate limiting and stoichiometric limiting.

In order to obtain a balance amount of cell formed from the amount of substrate used, a ratio
between the cell and nutrient is used. It is normally termed the yield factor, YX/S
which is given by;

and has the unit of the cell and nutrient/substrate concentrations.

Substrate-Limited Growth: Monod Equation

If the concentration of one essential medium constituent is varied while the concentrations of all
other medium components are kept constant, the growth rate typically changes in a hyperbolic
fashion. A functional relationship between the specific growth rate, μ, and essential compound’s
concentration was proposed by Monod in 1942, and is of the same form as the standard rate
equation for enzyme-catalysed reactions with a single substrate (Henri in 1902 and Michaelis and
Menten in 1913).

This relationship of specific growth rate, μ, to substrate concentration often assumes the form of
saturation kinetics. Here we assume a single chemical species, S, is growth rate limiting (i.e., an
increase in S influences growth rate, while changes in other nutrient concentrations have no
effect. These kinetics are similar to the Langmuir-Hinshelwood (or Hougen-Watson) kinetics in
traditional chemical kinetics of Michaelis-Menten kinetics for enzyme reactions. When applied to
cellular systems, these kinetics can be described by the Monod Equation.

In his classic article Monod described the relationship between substrate concentration and
bacterial growth in simple systems at steady state as:

μ = μmaxS
(Ks + S)
Eqn. 5

52
 Where, μ = specific growth rate
μmax = maximum value of μ obtained when S >>Ks
Ks = saturation constant equivalent to a Michaelis-Menten constant, or
half-velocity constant and is equal to the conc. of the rate-limiting
substrate when the specific rate of growth is equal to one-half of the
maximum, i.e., Ks = S when μ = ½μmax .
S = instantaneous steady-state substrate concentration of a single
limiting nutrient.

The Monod equation is semi-empirical; it derives from the premise that a single enzyme
system with Michaelis-Menten kinetics is responsible for uptake of S, and the amount of that
enzyme or its catalytic activity is sufficiently low to be growth rate limiting.

Figure: Growth Curve Resulting from Equation of the Monod Model

Even when there are many substrates, one of these substrates is usually limiting, that is, the rate
of biomass production depend exclusively on the concentration of this substrate. At low
concentrations, [S] of this substrate, μ is proportional to [S] but for increasing values of [S], an
upper value of μmax for the specific growth rate is gradually reached.

The Monod equation describes substrate-limited growth only when growth is slow and population
density is low. Under these circumstances, environmental conditions can be related simply to S.
If the consumption of a carbon-energy substrate is rapid, then the release of toxic waste products
is more likely (due to energy-spilling reactions). At high population levels, the buildup of toxic
metabolic by-products become more important.

Monod’s equation can be linearised using the Lineweaver–Burke equation:

53
The Equation shows a plot of 1/μ vs. 1/S. The slope, y-intercept, and x-intercept are
(Ks/μmax), (1/μmax), and (-1/Ks), respectively. This plot allows the computation of Ks and μmax.

Deviation of the above relationship will be dealt with later. Several alternative models exist for
growth response to substrate concentration. Eqn. (5) may be used to describe growth response
to substrate limitation only under steady-state conditions with uncomplicated bacterial systems.

This particular form of the Monod equation is appealing; however, its simplicity urges several
warning upon the user.
• First, the value of Ks is often rather small. Thus, S, can be >> Ks rather easily and the term
S/(Ks + S) may be regarded simply as an adequate description of calculating the deviation
of μ from μmax as the concentration S becomes smaller.
• The relation also suggests that the specific growth rate is finite (μ ≠ 0) for any finite
concentration of the rate-limiting component. Generally, this implied behaviour is not well
tested for S << Ks.

Monod Relation suggests that the specific growth rate of a microbial culture is determined by the
highest possible growth rate of the culture (µm), the concentration of the growth-limiting substrate
(S) and the affinity for the substrate (Ks). The value of μm is determined by growth parameters
such as temperature, pH and medium osmolality.

Moreover, when
µ = 0.5 µm
s = Ks

54
Hence, Ks could be determined by measuring the value of µ at various values of S.

The Ks value for the same substrate (glucose) could be very different for two different
microorganisms. In this case, K. aerogenes has a higher affinity for glucose than S. cerevisiae.
The microorganism P. extorquens has higher affinity for methanol (lower Ks value) than formate.

Knowledge of Ks values is of practical important for:

1. Designing Culture Medium: In order to achieve the maximum specific growth rate,
concentration of all essential substrates needs to be higher than their respective Ks value.

2. Predicting Growth Kinetics: Knowledge of Ks would allow us to estimate the specific

growth rate of a microbial culture in respond to a certain concentration of the growth-
limiting substrate according to equation: µ = µm . s/(Ks + S). The theoretical maximum
specific growth rate could be obtained from the plot of 1/μ versus 1/S.

3. Interpreting Microbial Interactions in an Ecosystem: It is rare that a single species of

microorganism is found in a natural habitat. The co-existence of different microorganisms
competing for the same growth-limiting substrate could be understood from the following
example.

Figure; Growth curves of two bacteria, A and B, in a mixed culture competing for the
same rate-limiting substrate.
µ = specific growth rate; Ss = rate-limiting substrate concentration.

Assuming that two bacteria, A and B, are present in a small volume of water competing for the
same growth-limiting substrate. And μm of bacterium A is greater than that of bacterium B, while
KSA > KSB. As such, we would expect a cross over at Sc in the μ versus S plots of the two bacteria.
When some amount of the substrate, which exceeds the Sc value, is added into the ecosystem,
bacterium A (having a higher μm) would outgrow bacterium B in number, and the substrate
decreases in concentration. However, when the residual concentration of the substrate drops
below the Sc value, the specific growth rate of bacterium B would now be higher than that of

55
bacterium A. Thus, the two bacteria take turns to increase in number, and no one bacterium would
replace the other in the ecosystem.
The situation would be different if the concentration of the growth limiting substrate does not
decrease to below Sc or increase to above Sc. In this case, bacteria A and B would have same
population size.

When exponential growth ceases as a result of substrate limitation, conditions are no longer
steady state for cell mass accumulation or substrate concentration, and eqn. (1) dx/dt = μx, must
be used with eqns. (5) and (6) to adequately model the response of the culture to diminishing
substrate.
Simultaneous solution of eqns. (1), (5) and (6) yields a somewhat bulky model for batch growth,
which is best a rough estimation for very simple systems.

Growth Yield
It has been observed frequently that the amount of cell mass produced is proportional to the
amount of a limiting substrate consumed. Growth yield (YX/S, g-cells/g-substrate) is defined as
the amount of biomass produced (dX) through the consumption of unit quantity of a substrate
(dS),
i.e.
YX/S = dX/dS

It is an expression of the conversion efficiency of the substrate to biomass.

The maximum population in a batch culture can be estimated from experimental data relating the
increase in cell number or mass to the corresponding decrease in substrate concentration.
(X - X0) = Y Eqn. (6)
(S0 – S)
Where,
X and S are the cell and substrate concentrations at time t1.
X0 and S0 are the cell and substrate concentrations at an earlier time, t0,
Y is the overall yield coefficient.

Eqn. (6) accurately describes the relationship between cell concentration and substrate
concentration during exponential growth. If exponential growth continues unabated until the
stationary phase is reached and substrate is completely consumed during this exponential growth,
the maximum cell number or concentration will be given by eqn. (6).
This estimation assumes that the yield coefficient is constant throughout the growth cycle and
neglects substrate consumption during the lag and stationary phases.
In fact the yield coefficient cannot be assumed to be constant except when constant exponential
growth conditions exist at a single growth rate. It follows therefore that the prediction of a
maximum population from eqn. (6) will leads to an overestimation.

Monod Chemostat Model (Lecture Notes)

Knowledge of growth yield is of practical importance. In many industrial production processes, it

is desirable to maintain high growth yield to achieve efficient conversion of the substrates to
biomass and products. Biological waste treatment process represents the other extreme where it
is desirable to achieve the lowest possible sludge (biomass) formation, with the maximum
oxidation of carbon in the effluent to CO2.

56
Growth yield also determines the cost of process operation. In the example of Microbial Protein
(Single Cell Protein) production, the final product is biomass with a high protein content of about
50%. The yield value determines the biomass output rate as well as the cost of aeration and
cooling can be illustrated as below:

(i) The cost of aeration (O2 supply) in a fermentation process using methanol (a derivative
of a byproduct of petroleum refinery industry) is determined by the O2 demand of a
fermentation system. The mass balance of microbial growth could be expressed by
the following equation:

a(CH3OH) + (1.5a − 1.225c)O2 → c(CH2O0.55N0.25) + (a − c)CO2 + (2a − c)H2O

methanol biomass

For any value of growth yield on methanol (c/a) there is a corresponding O2 requirement (1.5a −
1.225c). Thus, a low growth yield of biomass (c) would result in a higher demand (1.5a − 1.225c)
of O2 (air supply) for the oxidation of unit quantity of methanol (a) into CO 2. In essence, as it
requires 2 moles of oxygen to produce 1 mole of carbon dioxide, while only 0.55 mole of oxygen
is necessary to produce 1 C-mole of biomass, there is a higher O2 demand if more carbon in
methanol is oxidised to CO2.
Hence this results in higher cost of production due to the increase in the cost of aeration.
Insufficient O2 supply would not allow the full oxidation of methanol, leading to even lower level
of biomass production.

Thus, the lower the growth yield on the energy substrate, the larger amount of the chemical energy
stored in the substrate would be released as heat through respiration process. In order to maintain
the optimal culture temperature in the fermentation system, energy supply is needed for the
circulation of cooling water through the system.
There are several biological factors and culture parameters that determine the growth yield of a
microbial culture.
(1) Type of Microorganism
(2) Substrates

Rate Laws
Many laws exist for the rate of new cell growth.

Monod Equation
The Monod equation is the most commonly used model for the growth rate response curve of
bacteria.

57
 (9)
Where,
rg = cell growth rate
Cc = cell cencentration

μ = specific growth rate

The specific cell growth rate, μ, can be expressed as

(10)
Where,
μmax = a maximum specific growth reaction rate
Ks = the Monod constant
Cs = substrate concentration

Tessier Equation
Two additional equations are commonly used to describe cell growth rate. They are the Tessier
and Moser Equations. These growth laws would be used when they are found to better fit
experimental data, specifically at the beginning or end of fermentation.

(11)

Moser Equation:

(12)

Where,

λ and k are empirical constants determined by measured data.

Contois Equation:
μ = μmax S (13)
BX + S

The first two examples render algebraic solution of the growth equations much more difficult than
the Monod form.
The equation of Contois contains an apparent Michaelis constant which is proportional to biomass
concentration X. The last term will therefore diminish the maximum growth rate as the population
density increases, eventually leading to μ = X-1.

58
Andrew’s Equation
The specific growth rate may be inhibited by medium constituents such as substrate or product.
An example due to Andrew (5), proposes that substrate inhibition be treated by the form:

μ = μmax S (14)
Ki + S + S2/Kp

Alcohol fermentation provides an example of product inhibition; the anaerobic glucose

fermentation by yeast has been treated by Aiba, Shoda and Nagatani (3,4), with specific-growth
function of the type:

μ = μmax S Kp (15)
Ki + S Kp + p

It is possible that two (or more) substrates may simultaneously be growth-limiting. While little data
is available, a Monod dependence on each limiting nutrient may be proposed, so that:

μ = μmax S1 S2 …. (16)
K 1 + S1 K 2 + S2

In the absence of convincing data for this form, we may regard it simply as a useful indicator that
growth depends on several limiting nutrients.

Death Rate
The death rate of cells, r d, takes into account natural death, k d, and death from toxic by-product,
kt, where Ct is the concentration of toxic by-product.

(17)
Death Phase: The death phase of bacteria cell growth is where a decrease in live cell
concentration occurs. This decline could be a result of a toxic by-product, harsh environments, or
depletion of nutrients.

Stoichiometry
In order to model the amount of substrate and product being consumed/produced in following
equations, yield coefficients are utilised.
Ysc and Ypc are the yield coefficients for substrate-to-cells and product-to-cells, respectively. Yield
cofficients have the units of g variable/g cells.

Equation (18) represents the depletion rate of substrate:

(18)
Equation (19) represents the rate of product formation:

59
 (19)

CONTROL FACTORS
The growth and survival of bacteria depend on the close monitoring and control of many
conditions within the chemostat such as the pH level, temperature, dissolved oxygen level, dilution
rate, and agitation speed.
As expected with CSTRs, the pumps delivering the fresh medium and removing the effluent are
controlled such that the fluid volume in the vessel remains constant.

pH level
Different cells favour different pH environments. The operators need to determine an optimal pH
and maintain the CSTR at it for efficient operation. Controlling the pH at a desired value during
the process is extremely important because there is a tendency towards a lower pH associated
with cell growth due to cell respiration (carbon dioxide is produced when cells respire and it forms
carbonic acid which in turn causes a lower pH). Under extreme pH conditions, cells cannot grow
properly, therefore appropriate action needs to be taken to restore the original pH (i.e. adding acid
or base).

GROWTH RATE VERSUS TEMPERATURE

Microbial growth is also affected by temperature. Growth can be described by:

Where µ is the specific growth rate and α is the specific death rate. Usually, conditions are such
that µ >> α so α is normally negligible. Both α and µ are temperature dependent, and thus, this
assumption may not always be true. Growth and death can be described by the Arrhenius
equation:

Where,
A and A' are constants, E and E' are the activation energies, R is the gas constant, and T is the
absolute temperature (K). Typical values of E and E' are 15-20 kcal/mol and 60 to 70 kcal/mole,
respectively.

The increased death rate and lower growth rate at higher temperatures are due mainly to the
thermal denaturation of proteins. This results in an increase in cell maintenance energy
requirement for repair mechanisms. At low temperatures, diffusional limitations (e.g., transport of
substrates into and within the cell) affect the regulatory mechanisms of the cell. Therefore, the
biomass yield falls at extremes in temperature (Scragg 1991).

60
Controlling the temperature is also crucial because cell growth can be significantly affected by
environmental conditions. Choosing the appropriate temperature can maximise the cell growth
rate as many of the enzymatic activates function the best at its optimal temperature due to the
protein nature of enzymes.

Changes in temperature markedly affect growth rate because they affect the cell’s growth-directed
chemical reactions. Over a limited range of temperature, the relationship between growth rate
and temperature is similar to that between chemical reaction rates and temperature.
This has been expressed in Arrhenius plots: in which the logarithm of growth rate is plotted
against the reciprocal of absolute temperature (1/K). However, over an extended temperature
range such plots are non-linear.

Ratkowsky et al., showed that for various species of bacteria, a linear relationship could be
demonstrated over the full biokinetic temperature range by plotting the square root of growth rate
against absolute temperature.

Dilution rate
One of the important features of the chemostat is that it allows the operator to control the cell
growth rate. The most common way is controlling the dilution rate, although other methods such
as controlling temperature, pH or oxygen transfer rate can be used. Dilution rate is simply defined
as the volumetric flow rate of nutrient supplied to the reactor divided by the volume of the culture
(unit: time-1). While using a chemostat, it is useful to keep in mind that the specific growth rate of
bacteria equals the dilution rate at steady state. At this steady state, the temperature, pH, flow
rate, and feed substrate concentration will all remain stable. Similarly, the number of cells in the
reactor, as well as the concentration of reactant and product in the effluent stream will remain
constant.
Negative consequences can occur if the dilution rate exceeds the specific growth rate. As can be
seen in Equation (20) below, when the dilution rate is greater than the specific growth rate (D >
μ), the dCC/dt term becomes negative.

(20)
This shows that the concentration of cells in the reactor will decrease and eventually become
zero. This is called wash-out, where cells can no longer maintain themselves in the reactor.
Equation (21) represents the dilution rate at which wash-out will occur.

(21)
In general, increasing the dilution rate will increase the growth of cells. However, the dilution rate
still needs to be controlled relative to the specific growth rate to prevent wash-out. The dilution
rate should be regulated so as to maximise the cell production rate. Figure 1 below shows how
the dilution rate affects cell production rate (DCC), cell concentration (CC), and substrate
concentration (CS).

Initially, the rate of cell production increases as dilution rate increases. When Dmaxprod is reached,
the rate of cell production is at a maximum. This is the point where cells will not grow any faster.

61
D = μ (dilution rate = specific growth rate) is also established at this point, where the steady-
state equilibrium is reached.
The concentration of cells (CC) starts to decrease once the dilution rate exceeds the Dmaxprod.

The cell concentration will continue to decrease until it reaches a point where all cells are washed
out. At this stage, there will be a steep increase in substrate concentration because fewer and
fewer cells are present to consume the substrate.

Figure 1: Cell concentration, cell production, and substrate concentration as a function

of dilution rate

Oxygen Transfer Rate

Since oxygen is an essential nutrient for all aerobic growth, maintaining an adequate supply of
oxygen during aerobic processes is crucial. Therefore, in order to maximise the cell growth,
optimisation of oxygen transfer between the air bubbles and the cells becomes extremely
important. The oxygen transfer rate (OTR) tells us how much oxygen is consumed per unit time
when given concentrations of cells are cultured in the bioreactor. This relationship is expressed
in Equation (22) below.

Oxygen Transfer Rate (OTR) = QO2CC

(22)
Where CC is simply the concentration of cell in the reactor and QO2 is the microbial respiration
rate or specific oxygen uptake rate.
The chemostat is a very convenient tool to study the growth of specific cells because it allows the
operators to control the amount of oxygen supplied to the reactor. Therefore it is essential that
the oxygen level be maintained at an appropriate level because the cell growth can be seriously
limited if inadequate oxygen is supplied.

62
Agitation Speed
A stirrer, usually automated and powered with a motor, mixes the contents of the chemostat to
provide a homogeneous suspension. This enables individual cells in the culture to come into
contact with the growth-limiting nutrient and to achieve optimal distribution of oxygen when
aerobic cultures are present. Faster, more rigorous stirring expedites cell growth. Stirring may
also be required to break agglutinations of bacterial cells that may form.

Sample Problem 3.
Brewers’ yeast is grown continuously in a fermenter with an operating volume of 12 m 3.
The residence time is 20 h and the yeast has a doubling time of 3.2 h. A 2% inoculum,
which contains 5% yeast cells is mixed with the substrate. Calculate the mass of yeast
harvested from the fermenter per hour. (Assume that the density of the broth is 1,010 kgm-
3
.)

Solution to Sample Problem 3

63
Sample Problem 4.
Calculate the yield factor for the production of acetic acid using Acetobacter aceti bacteria.
The chemical equation for the conversion of acetic acid is given below;

Acetobacter aceti is added into a vigorously aerated medium containing 10 gl -1 ethanol.

After some time, ethanol and acetic acid concentrations become 2 gl -1 and 7.5 gl-1
respectively.
1. What is the overall/observed yield of acetic acid produced from the batch culture.
2. Determine the percentage of the observed yield in relation to the theoretical yield.

Solution to Sample Problem 4.

1. By using a basis of 1 litre, the overall yield over the entire culture period is given as;

2. The theoretical yield is based on the mass of ethanol actually used for synthesis of acetic acid,
thus, from the stoichiometric equation given above;

and the percentage of observed yield can be calculated by putting;

INDUSTRIAL APPLICATION
The chemostat may offer a significant increase in productivity over batch or fed-batch operation.
Increased productivity is the result of reduced fermentor downtime per unit of product
manufactured (6). Also, because the growth rate and cell concentration can be controlled
independently, critical demands of the fermentation for oxygen supply and heat removal can be
adjusted to match the equipment’s capabilities.

In addition, by controlling the dilution rate of the continuous fermentation, it may be possible to
maintain an optimal specific growth rate for product expression. The productivity gains which can
be achieved by continuous culture allow for smaller manufacturing plants and thus the reduction
of the capital costs for new facilities.

Fermentor Litres of Medium Required per Day at Dilution Rate

Working Volume 0.01 h-1 0.05 h-1 0.50 h-1 1.0 h-1
250 ml 0.06 0.3 3 6
1,000 ml 0.24 1.2 12 24
5 litres 1.2 6.0 60 120
10 litres 2.4 12.0 120 240

64
Advantages of Continuous Culture
• Productivity and growth rate can be optimised by changing the feed flow rate during
production;

• Longer periods of productivity with less down time. In theory, a continuous process can
be operated indefinitely; however, because long periods of operation can result in
mechanical failure, the process must be stopped occasionally to allow for system
maintenance;

• It can take advantage of cell immobilisation, which allows the maintenance of high cell
concentrations in the bioreactor at low substrate concentrations. In these systems, cells
are perfused via a membrane with a steady and continuous flow of fresh medium. The
cells are supplied with oxygen and nutrients, while waste and desired products are
continuously removed. The cells are retained by means of the membrane barrier and are
recycled into the bioreactor rather similar effects are achievable using acoustic perfusion
systems;

• The effects of environmental or physical factors are more easily analysed in a continuous
system, where any changes in the constant steady state are observed and can be
attributed solely to the change in those factors.

• Evolution in these cultures can be readily studied. Basically, continuous culture of a given
strain allows us to ‘direct the evolution’ of the strain. As the cells reproduce in an invariant
environment, the operator can select a particular evolutionary pressure, e.g. temperature,
nutrient limitation or pH, and study how the culture evolves in response to that selective
pressure in terms of physiological, metabolic or genetic change.

APPLICATIONS IN RESEARCH
• Chemostats in research are used for investigations in cell biology, as a source for large
volumes of uniform cells or protein.
• The chemostat is often used to gather steady state data about an organism in order to
generate a mathematical model relating to its metabolic processes.
• Chemostats are also used as microcosms in ecology and evolutionary biology. In the one
case, mutation/selection is a nuisance, in the other case; it is the desired process under
study.
• Chemostats can also be used to enrich for specific types of bacterial mutants in culture
such as auxotrophs or those that are resistant to antibiotics or bacteriophages for further
scientific study.
• Competition for single and multiple resources, the evolution of resource acquisition and
utilisation pathways, cross-feeding/symbiosis, antagonism, predation, and competition
among predators have all been studied in ecology and evolutionary biology using
chemostats.

Industry
Chemostats are frequently used in the industrial manufacturing of ethanol. In this case, several
chemostats are used in series, each maintained at decreasing sugar concentrations.

65
Concerns
• Foaming results in overflow with the volume of liquid not exactly constant.
• Some very fragile cells are ruptured during agitation and aeration.
• Cells may grow on the walls or adhere to other surfaces, which is easily overcome by
treating the glass walls of the vessel with a silane to render them hydrophobic.
• Mixing may not truly be uniform, upsetting the "static" property of the chemostat.
• Dripping the media into the chamber actually results in small pulses of nutrients and thus
oscillations in concentrations, again upsetting the "static" property of the chemostat.
• Bacteria travel upstream quite easily. They will reach the reservoir of sterile medium
quickly unless the liquid path is interrupted by an air break in which the medium falls in
drops through air.

Continuous efforts to remedy each defect lead to variations on the basic chemostat quite
regularly.
Examples in the literature are numerous.
• Antifoaming agents are used to suppress foaming.
• Agitation and aeration can be done gently.
• Many approaches have been taken to reduce wall growth.
• Various applications use paddles, bubbling, or other mechanisms for mixing.
• Dripping can be made less drastic with smaller droplets and larger vessel volumes.
• Many improvements target the threat of contamination.

Despite the producer’s proclamations, only a few food or feed production systems employing
microorganisms are operated in a genuine continuous mode, where continuous fermentation is
defined as a process running in one or more bioreactors at a stable dilution rate. One of the rare
examples of this is fodder yeast (Kluyveromyces fragilis) production using spent sulfite liquor as
the substrate, which is operated in the Czech Republic (http://www.biocel.cz/e_html/index.htm).
The main reason why this process can be performed continuously is the low sensitivity of the
production medium to possible contamination. At the same time, this process can be taken as a
rare example of industrial-scale production using a highly valued substrate, lignocellulosic
hydrolysate, which is obtained as a waste product from pulp production.

In other food applications such as in modern distilleries, a semi-continuous fermentation operated

in a series of fermentors is usually used instead of genuine continuous fermentation. The current
estimates are that only about 16% of ethanol in North America is produced in a continuous mode
due to problems with contamination (Ingledew and Lin, 2011). Nevertheless, both batch and semi-
continuous mode fermentations permit continuous distillation, which is the reason why the whole
distillery production is often considered as continuous.

Continuous fermentation systems based on immobilised cell technology have also been studied
in beer production. Although continuous beer fermentation has been tested as a promising
technology for several decades, the number of industrial applications is still limited. The reasons
include engineering problems (excess biomass and problems with CO2 removal, optimisation of
operating conditions, clogging, and channeling of the reactor) and unrealised cost benefits (carrier
price, complex, and unstable operation). The major obstacle hindering the extensive industrial
exploitation of this technology is the difficulty in achieving the correct balance of sensory
compounds in a short time typical for continuous systems. However, recent developments in
reactor design and in our understanding of immobilised cell physiology, together with applications
of novel carrier materials, could provide a new stimulus to research and potential applications of
this promising technology (Brányik et al., 2008).

66
DISADVANTAGES OF CONTINUOUS CULTURE
Continuous culture is not widely used in industrial processes. Three disadvantages must be
overcome before continuous culture becomes a viable alternative to the batch or fed-batch
process.
• Not all products are produced optimally in continuous processes, e.g. some fermented
foods and beverages require cellular products released from different phases of batch
culture growth for full flavour development. Nongrowth-associated products such as
antibiotics, monoclonal antibodies and toxins are also not produced well in continuous
culture. This is because antibiotics and toxins can exert a selective pressure in the
chemostat, and because there are risks of plasmid loss in genetically modified cultures
over number of generations.

• The continuous addition of feed streams over several weeks greatly increases the
probability of contamination. This risk can be minimised by careful operation and
equipment design.

• Equipment and instrumentation used to operate and control the process must be
extremely reliable for long periods of continuous operation. The overall failure rate can be
reduced by redundancy of critical equipment and by frequent monitoring of the process.

• The most difficult problem to correct is strain degeneration, a gradual and irreversible loss
of product expression (7, 8). Strain degeneration occurs when a spontaneous mutation
produces a new strain with a selective growth advantage but a reduced capacity for
product formation. In this way a population of low producers can become the dominant
species. For example, Clostridium acetobutylicum loses its ability to make acetone butanol
in continuous culture and instead makes acetate and butyrate.

• The US Food and Drug Administration (FDA) does not accept continuous culture in the
production of therapeutic products as a Current Good Manufacturing Practice (cGMP).
This is because they require the manufacturer to segregate such products into batches
for traceability purposes, precluding continuous culture as a means of production.

Most overproducing strains used for industrial production of biopolymers, amino acids, and
antibiotics are the result of mutation and selection programmes. These strains are generally
unstable and are highly likely to revert to poorer producers and better growers in continuous
culture.

Genetic instability is a major obstacle to the introduction of continuous fermentation technology

into the biochemical and biotechnology process industry.

67
OTHER CONTINUOUS-CULTURE TECHNIQUES:
• AUXOSTAT
An auxostat is a chemostat in which the dilution rate can vary. Usually a chemostat has a fixed
rate of dilution. This determines how fast the organism in the chemostat will grow. In an auxostat
the rate at which new material is added and old removed is determined by some feature of the
culture. For example, the amount of bacteria may be measured by measuring the cloudiness
(turbidity) of the culture, and the amount of material added adjusted to keep the turbidity constant.
Alternatively, if the bacteria reduce pH of the culture as they grow (as bacteria often do), pH may
be used to control the rate of dilution. The former is called a turbidostat, the latter a pH auxostat.

68
Figure: Typical layout of an auxostat using pH control coupled to the addition of
fresh medium

A simple feedback control based on nutrient concentration is used, and there is a wide variety of
feedback variables that can be used. The pH-auxostat is easily set up using commercially
available bioreactors, making it accessible to most researchers. The pH-auxostat couples the
addition of fresh medium to pH control. As the pH drifts from a desired set point, fresh medium is
added to bring the pH back to the desired set point.

The rate of medium addition is not directly determined by the pH set point, but rather by the
buffering capacity and the feed concentration of the limiting nutrient. For example, cultures of
filamentous fungi have been grown in pH-auxostats where it was shown that biomass production
could be controlled by the addition of alkali.

Other feedback variables that have been used to develop auxostat systems include dissolved
oxygen (DO), specific gravity, nutrients such as glucose and ammonium ion, inhibitory
substrates, carbon dioxide evolution rate (CER) and oxygen uptake rate (OUR).

Auxostats use feed rate to control a state variable, such as pH or dissolved oxygen, in continuous
culture. The microorganisms themselves establish their own dilution rate, and auxostats tend to
be much more stable than chemostats at higher dilution rates, especially at dilution rates
approaching the maximum specific growth rate of the microorganism. The high dilution rates
exert a selection pressure upon the microbial population, which leads to more rapidly growing
cultures. This method of continuous culture control is therefore ideal for such applications as high-
rate propagation and detoxification of waste materials at maximum rate concentrations.

Auxostats have the advantage that the maximum growth rate or yield can be obtained much more
easily than with a chemostat. If the dilution rate is not high enough in a chemostat, the culture will

69
grow at less than its maximum growth rate. If it is too high, the organisms will not be able to keep
up with the addition of new medium and so will be diluted out; and the end result will be an empty
chemostat.

An auxostat can be adjusted to keep up automatically with bacterial growth, and so maximise the
growth rate. At such high growth rates bacteria that can grow fast are selected over ones that
grow slowly. Thus, natural selection acts on the population, selecting fast-growing variants of the
bacteria. Depending on what the auxostat is to be used for, this can be a good or bad thing. In
practice, all large continuous industrial fermentation systems are auxostats rather than
chemostats, since they have many feedback controls, which allow the operator to adjust what
materials the fermentation receives as it proceeds.

• TURBIDOSTAT
The second type of continuous culture system, the turbidostat, has a photocell that measures the
absorbance or turbidity of the culture in the growth vessel. The flow rate of media through the
vessel is automatically regulated to maintain a predetermined turbidity or cell density. The
turbidostat differs from the chemostat in several ways. The dilution rate in a turbidostat varies
rather than remaining constant, and its culture medium lacks a limiting nutrient. The turbidostat
operates best at high dilution rates; the chemostat is most stable and effective at lower dilution
rates.

The turbidostat is a continuous-culture apparatus in which the cell concentration is not limited by
a nutrient. In cases where nutrient limitation is not desirable, such as degradation of toxic wastes,
the turbidostat can be a valuable tool. Cells are grown to the desired cell density as measured by
turbidity or light scattering. Medium with all nutrients in excess is then used to wash the culture
out of the vessel as quickly as it accumulates. This then maintains the culture at its maximal
growth rate. The medium inflow and outflow are balanced so that no increase in volume occurs.

Figure: Turbidostat Culture System.

The turbidity is constantly measured and used to control the exit pump, and a level controller is
used to maintain the liquid volume. The turbidostat suffers from the use of turbidity as the control
parameter. A biofilm usually develops over the optics with time, and special designs must be
employed to eliminate bubbles in the sample stream.
To date, turbidostats have found their greatest utility in the study of algae, where these problems
are not so likely to occur.

70
The turbidostat is commonly used for the selection of antibiotic resistant mutants and the
degradation of toxic wastes, where nutrient limitation is not desirable. It is also routinely used to
avoid the washout effects that are more common in chemostat systems, and to produce cells of
approximately uniform morphology and composition over prolonged periods. However, this
system has two major disadvantages: first, fouling of the optical surfaces of the probe used to
measure turbidity caused by unwanted cell growth, and second, gas bubbles trapped in the
circulating medium result in inaccurate optical density measurements and therefore the system is
often difficult to control.

The applications of turbidostat are as follows:

(i) To fix biomass concentration and allow the specific growth rate to adjust itself. This
application is particularly useful for slow growing microorganisms and those with complex
cell cycles. In the latter case, the μm of a culture may be very different at various stages
in the cell cycle, resulting in complete washout (total removal of cells from the culture
system through excessive dilution) of the culture in a fixed dilution rate chemostat.

(ii) To maintain a constant biomass concentration under unstable environment

conditions, such as for the treatment of wastewater containing variable concentration of
toxic compounds. Temporary suspension of specific growth rate by toxic compounds
would result in washout in a simple chemostat with fixed dilution rate.

(iii) To select for fast growing microbial strains, e.g. resistance to an inhibitor. The dilution
rate of a turbidostat is determined by the growth rate of the culture, thus evolution of a
resistant strain would cause an increase in the dilution rate and washout of the slow
growing cells (e.g. those could not tolerate the inhibitor) from the culture system.

Table: Other Continuous-culture Control Techniques.

Continuous-culture Techniques Control Parameter

Turbidostat Cell density (turbidity)

Nutristat Residual substrate concentration
pH-auxostat pH
CER-stat Carbon dioxide evolution rate
DO2-stat Dissolved oxygen
OUR-stat Oxygen uptake rate

Continuous culture systems are very useful because they provide a constant supply of cells in
exponential phase and growing at a known rate. They make possible the study of microbial
growth at very low nutrient levels, concentrations close to those present in natural environments.
These systems are essential for research in many areas; for example, in studies on interactions
between microbial species under environmental conditions resembling those in a freshwater lake
or pond. Continuous systems also are used in food and industrial microbiology.

71
Table: Comparison of the Chemostat and Turbidostat with Respect to Various
Parameters
OPERATING CHEMOSTAT TURBIDOSTAT
PARAMETER

Operation at or near Unstable Stable, very nearly steady

maximum specific growth state
rate
Operation at low specific Stable steady state Unstable, transient with
growth rate pulsatile response

Dilution rate equals Only at steady state At all times

specific growth rate

Cell concentration at Depends on substrate Depends on substrate

constant specific growth concentration in the feed concentration in the feed
rate

Dilution rate Predetermined Controlled as a function of

cell mass

Substrate concentration for Requires a single limiting All substrates may be

steady-state operation substrate present in excess

FED-BATCH CULTURE

Figure: A simple fed-batch fermentation set-up.

72
Feeding begins at a predetermined point during the process, often at the end of the exponential
growth phase. The feed consists of a super-concentrated version of the process medium, allowing
control of the growth rate and carbon flux through the system

In batch culture, the transition from exponential growth to stationary phase may occur for a variety
of reasons, including the depletion of an essential nutrient substrate or the buildup of a toxic
metabolite product.
When the transition results from nutrient depletion, growth will continue if fresh medium is added.
The medium addition rate and the culture volume must be increased exponentially to maintain a
constant rate of exponential growth. This technique for exponential growth maintenance is usually
called fed-batch culture (9, 10).

Two basic approaches to the fed-batch fermentation can be used: the constant volume fed-batch
culture or Fixed Volume Fed-Batch, and the Variable Volume Fed-Batch.

1. FIXED VOLUME FED-BATCH

In this type of fed-batch, the limiting substrate is fed without diluting the culture. The culture
volume can also be maintained practically constant by feeding the growth limiting substrate in
undiluted form, for example, as a very concentrated liquid or gas (e.g. oxygen).
Alternatively, the substrate can be added by dialysis or, in a photosynthetic culture, radiation can
be the growth limiting factor without affecting the culture volume.
A certain type of extended fed-batch, the cyclic fed-batch culture for fixed volume systems,
refers to a periodic withdrawal of a portion of the culture and use of the residual culture as the
starting point for a further fed-batch process. Basically, once the fermentation reaches a certain
stage, (for example, when aerobic conditions cannot be maintained anymore) the culture is
removed and the biomass is diluted to the original volume with sterile water or medium containing
the feed substrate. The dilution decreases the biomass concentration and result in an increase in
the specific growth rate. Subsequently, as feeding continues, the growth rate will decline gradually
as biomass increases and approaches the maximum sustainable in the vessel once more, at
which point the culture may be diluted again.

2. VARIABLE VOLUME FED-BATCH

As the name implies, a variable volume fed-batch is one in which the volume changes with the
fermentation time due to the substrate feed. The way this volume changes is dependent on the
requirements, limitations, and objectives of the operator. The feed can be provided according to
one of the following options:
(i) The same medium used in the batch mode is added;
(ii) A solution of the limiting substrate at the same concentration as that in the initial medium
is added;
(iii) A very concentrated solution of the limiting substrate is added at a rate less than (i) and
(ii).

This type of fed-batch can still be further classified as repeated fed-batch process or cyclic fed-
batch culture, and single fed-batch process. The former means that once the fermentation
reached a certain stage after which is not effective anymore, a quantity of culture is removed from
the vessel and replaced by fresh nutrient medium. The decrease in volume results in an increase
in the specific growth rate, followed by a gradual decrease as the quasi-steady state is
established.

73
The latter type refers to a type of fed-batch in which supplementary growth medium is added
during the fermentation, but no culture is removed until the end of the batch. This system presents
a disadvantage over the fixed volume fed-batch and the repeated fed-batch process: much of the
fermentor volume is not utilised until the end of the batch and consequently, the duration of the
batch is limited by the fermentor volume.

Figure: Growth kinetics in a cyclic variable volume fed batch culture.

V = volume of the culture, x = biomass concentration, μ = specific growth rate, S =
rate-limiting substrate concentration.

True fed-batch operation requires that the volume of the liquid medium in the fermentor increase
during the fermentation. This requirement places an upper limit on the culture time based on feed
rate and leads to changing conditions of aeration and agitation effectiveness. Furthermore, it is
possible to back-contaminate the feed reservoir from the fermentor; for this reason it is desirable
to use a medium breaker.

The fed-batch mode will allow substantial improvements in cell mass or product productivity over
an ordinary batch operation. The technique of fed-batch culture may also be used to supply large
quantities of a potentially toxic substrate while maintaining a low concentration of the toxic
substrate in the medium.
When a liquid feed stream enters the bioreactor, the culture volume is altered, and this must be
taken into account in the equations used to describe the bioreactor.

74
Table: Advantages and Disadvantages of Various Feeding Strategies

Fed-batch culture represents a semi-open system in which one or more nutrients are aseptically
and gradually added to the bioreactor while the product is retained inside; that is, the volume of
the culture broth in the bioreactor increases within this time.

The main advantages of fed-batch over batch cultures are:

(a) Possibility to prolong product synthesis,
(b) Ability to achieve higher cell densities and thus increase the amount of the
product, which is usually proportional to the concentration of the biomass,
(c) Capacity to enhance yield or productivity by controlled sequential addition of
nutrients, and,
(d) The feature of prolonged productive cultivation over the “unprofitable periods”
when the bioreactor would normally be prepared for a new batch.

Fed-batch is advantageously used in processes:

(a) Where substrate inhibition or catabolic repression is expected; this problem can
be overcome by using a “safe” concentration of the substrate in batch mode
followed by feeding the remaining substrate within fed-batch operation,
(b) Where a Crabtree effect (repression of yeast respiratory enzymes by high
concentrations of glucose) is expected (de Deken, 1966); by gradual feeding of the
substrate, the production of ethanol by yeasts can be eliminated under aerobic
conditions,
(c) Where a high cell density is required; a high and constant specific growth rate can
be maintained by exponential feeding of the substrate,
(d) Where a high production rate should be achieved; cell metabolism can be
regulated by precise sequential feeding of nutrients, and (e) where a high
viscosity of culture broth is expected (e.g., production of dextran or xanthan); a
gradual dilution of the medium can overcome the problems of mixing and oxygen
transfer.

75
There are many methods of adding a substrate to the bioreactor (either as a concentrated solution
of a sole carbon and energy source or as a medium containing carbon plus other nutrients); the
proper choice of the nutrient feeding rate can enhance the culture performance considerably since
it influences cellular growth rate, cell physiology, and the rate of product formation.

The common feeding strategies are:

(a) Discontinuous feeding, achieved by regular or irregular pulses of substrates and
(b) Regular continuous feeding of nutrients designed according to a pre-calculated
profile (Figure) or based on the feedback control of online measured variables
associated with cell growth and metabolism, for example, dissolved oxygen
concentration, pH, CO2, evolution rate, and biomass concentration.

The typical food fed-batch fermentations are large-scale production of baker’s yeast, pure ethanol,
which is further utilized for alcoholic beverages produced by mixing ingredients such as liquors or
cordials, and submerged acetification for vinegar production. Baker’s yeast (Saccharomyces
cerevisiae), which is distributed as compressed, dried, or instant biomass, is valued for its dough-
leavening ability.

Figure: Relationship between pre-calculated profiles of substrate feeding and specific

growth rates of cell culture. (a) Constant feeding rate, (b) linearly increasing feeding rate,
and (c) exponential feeding rate.

76
Yeast production is the only technology in which the respiratory metabolism of S. cerevisiae,
leading to high biomass yield, is stressed. Nevertheless, due to the Crabtree effect, that is, the
formation of ethanol under aerobic conditions in the presence of excess substrate, the only
alternative for producing baker’s yeast is fed-batch cultivation.

The main bottleneck of large scale baker’s yeast production is the control of nutrient medium
inflow, which was traditionally based on empirical data. Currently, many feeding methods utilising
different approaches have been developed, for example, a logistic feeding profile (Borowiak et
al., 2012), evolutionary optimization of genetic feeding algorithms (Yüzgeç et al., 2009), fuzzy
control (Karakuzu et al., 2006), pulsed feeding optimization (Kasperski and Mis´kiewitz, 2008),
and others.

In the European Union (EU), the annual production of vinegar, comprising 10% (w/v) acetic acid,
is estimated to be about 5 × 106 hL (García-García et al., 2009). Human knowledge of vinegar
production dates back to ancient history and several distinct production methods have been used,
for example, surface oxidation (Orleans process, slow vinegar production), the quick vinegar
process using a trickle-bed bioreactor, or submerged acetification. The most modern is
submerged acetification, in which acetic acid (vinegar) is produced by Acetobacter-mediated
oxidation of ethanol, and takes place in special “acetator” bioreactors. The most common
acetator, a Frings Acetator®, differs from the usual bioreactors by using a special rotor/stator
turbine aerator. The aerator with a double function (aeration and mixing), consists of a rotor placed
under the bioreactor and connected to an air-suction pipe surrounded by a stator. The aerator is
self-respiring, that is, during rotation (speed about 1500 rpm), it sucks air and pumps liquid, which
causes the formation of an air-liquid mixture that is radially injected into the culture medium. The
foam is broken by a mechanical defoamer and because oxidation is an exothermal process,
cooling is necessary. The acetators are operated in repeat fed-batch mode and one cultivation
cycle in a single acetator can produce vinegar containing 15% (w/v) of acetic acid. In addition, a
dual-stage high-strength fermentation process, which allows the culture to generate up to 20.5%
(w/v) acetic acid, has been developed (Ebner et al., 1996; García-García et al., 2009).

77
Figure: Different feeding regimes in fed-batch processes, (a) Variable feeding regime; (b)
continuous feeding regime; (c) intermittent feeding regime; (d) incremental feeding
regime

78
Advantages of Fed-Batch System
• Because cells are not removed during fermentation, therefore this type of fermentor is well
suited for production of compounds produced during very LOW or zero growth.
• The feed does not need to contain all the nutrients needed to sustain growth. Feed may
contain only the nitrogen source or metabolic precursor.
• Contamination is highly unlikely for fed-batch system unless it has occurred during the
early stage of a fermentation.
• Can operate in a number of ways; batch to fed-batch to batch in an alternate manner. The
feed for substrate/nutrient can be manipulated in order to optimise the product formation.
For instance, during fermentation, the feed composition or the flow rate can be adjusted
to match the physiological state of cells.

Fed-batch fermentation is a production technique in between batch and continuous fermentation.

A proper feed rate, with the right component constitution is required during the process. Fed-batch
offers many advantages over batch and continuous cultures. From the concept of its
implementation it can be easily concluded that under controllable conditions and with the required
knowledge of the microorganism involved in the fermentation, the feed of the required
components for growth and/or other substrates required for the production of the product
can never be depleted and the nutritional environment can be maintained approximately
constant during the course of the batch.

The production of by-products that are generally related to the presence of high
concentrations of substrate can also be avoided by limiting its quantity to the amounts
that are required solely for the production of the biochemical. When high concentrations of
substrate are present, the cells get "overloaded", this is, the oxidative capacity of the cells is
exceeded, and due to the Crabtree effect, products other than the one of interest are produced,
reducing the efficacy of the carbon flux. Moreover, these by-products prove to even "contaminate"
the product of interest, such as ethanol production in baker's yeast production, and to impair the
cell growth reducing the fermentation time and its related productivity.

Sometimes, controlling the substrate is also important due to catabolic repression. Since
this method usually permits the extension of the operating time, high cell concentrations can be
achieved and thereby, improved productivity [mass of product/(volume x time)]. This aspect is
greatly favoured in the production of growth-associated products. Additionally, this method allows
the replacement of water loss by evaporation and decrease of the viscosity of the broth
such as in the production of dextran and xanthan gum, by addition of a water-based feed. As
previously mentioned, fed-batch might be the only option for fermentations dealing with toxic
or low solubility substrates.

When dealing with recombinant strains, fed-batch mode can guarantee the presence of an
antibiotic throughout the course of the fermentation, with the intent of keeping the presence of an
antibiotic-marked plasmid. Since the growth can be regulated by the feed, and knowing that in
many cases a high growth rate can decrease the expression of encoded products in recombinant
products, the possibility of having different feeds and feed modes makes fed-batch an extremely
flexible tool for control in these cases. Because the feed can also be multi-substrate, the
fermentation environment can still be provided with required protease inhibitors that might
degrade the product of interest, metabolites and precursors that increase the productivity of the
fermentation.

79
Finally, in a fed-batch fermentation, no special piece of equipment is required in addition to
that one required by a batch fermentation, even considering the operating procedures for
sterilisation and the preventing of contamination. A cyclic fed-batch culture has an additional
advantage: the productive phase of a process may be extended under controlled conditions. The
controlled periodic shifts in growth rate provide an opportunity to optimise product synthesis,
particularly if the product of interest is a secondary metabolite whose maximum production takes
place during the deceleration in growth.

[The Crabtree effect, named after the English biochemist Herbert Grace
Crabtree, describes the phenomenon whereby the yeast, Saccharomyces,
produces ethanol (alcohol) aerobically in the presence of high
external glucose concentrations rather than producing biomass via the tricarboxylic acid
cycle, the usual process occurring aerobically in most yeasts e.g. Kluyveromyces spp.
Increasing concentrations of glucose accelerates glycolysis (the breakdown of glucose)
which results in the production of appreciable amounts of ATP through substrate-
level phosphorylation. This reduces the need of oxidative phosphorylation done by the
TCA cycle via the electron transport chain and therefore decreases oxygen consumption.
The phenomenon is believed to have evolved as a competition mechanism (due to the
antiseptic nature of ethanol)].

Table: Advantages of Batch and Fed-Batch Culture Techniques in Industry

• Products may be required only in relatively small quantities at any given time
• Markets needs may be intermittent
• Shelf-life of certain products is short
• High product concentration is required in broth to optimise downstream processing
operations
• Some metabolic products are produced only during the stationary phase of the
growth cycle
• Instability of some production strains require their regular renewal
• Continuous processes can offer many technical difficulties
______________________________________________________________________

Application of Fed-Batch System

Since the system can maintain the growth/reaction at LOW nutrient/substrate concentrations,
therefore;
• Fed-batch can be used to get the product or cells when the nutrient/substrate is inhibitory
to the cells or affect the mass transfer rate. This can be maintained by controlling the
substrate feeding such as during the production of citric acid, amylase enzyme and
vinegar.
• Can result in higher biomass, cells yield. LOW substrate concentration is important
especially in the production of mammalian cells, baker's yeast and antibiotic.
• Production is dependent on a specific nutrient composition, carbon to nitrogen ratio.
• Oxygen uptake rate (OUR) must be restricted in order to maintain LOW substrate
concentrations.

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 SOLID SUBSTRATE FERMENTATION
The term “solid substrate fermentation” (SSF) or “solid substrate cultivation” (SSC) is used
for systems where microorganisms are cultured on the surface of a concentrated water-insoluble
substrate (usually containing polysaccharides as a carbon and energy source) with a low level of
free water. This technique was developed in the Eastern countries, where it has been used for
centuries for the production of traditional foods such as soy sauce, koji, miso, or sake, using
different substrates and microorganisms. In the Western countries, it has not been widely
exploited and its application is limited mainly to the production of industrial enzymes, certain food
products, or feed supplements.

Typical Features of Solid Substrate Fermentation

Solid substrate fermentation is characterized by very low water activity (the relative humidity of
the gaseous phase in equilibrium with the moist solid is significantly below 1) (Hölker and Lenz,
2005); thus, the main features of this system are substantially different compared with classical
submerged cultivation as shown in the Table.

Table: Main Differences between Solid-Substrate Fermentation and Submerged

Cultivation

There are several advantages of SSF over the conventional submerged technology such as:
(a) Use of a concentrated medium, resulting in a smaller reactor volume and lower
capital investment costs,
(b) Lower risk of contamination with yeasts and bacteria due to low moisture levels
and substrate complexity,
(c) Simplicity of the technology and low production of effluent water from the process,

81
 (d) Higher product yield and easier product recovery, and,
(e) Use of agricultural wastes as substrates for certain applications (e.g., feed
supplements and cellulolytic enzymes). (Ali et al., 2011; Barrios-Gonzáles, 2012)

Microorganisms and Substrates Used in SSF Processes

Filamentous fungi are preferable for SSF processes, mainly due to their abilities to:
(a) Grow on substrates with reduced water activity,
(b) Penetrate their hyphae into the solid substrate, and (c) produce exoenzymes (e.g.,
amylolytic and cellulolytic enzymes), which decompose the polysaccharides (the
main carbon source often present in solid substrates).

When using yeast, it is necessary to integrate a material pretreatment step (such as steam
explosion, acid or alkali treatment, followed by enzymatic digestion or a combination of these) into
the process (Jacob, 1991) or to use a mixed culture, wherein the complex cellulosic or starchy
material is first degraded by other organisms (usually molds) that are able to produce extracellular
enzymes and the released glucose is then consumed by yeasts (or less frequently by bacteria)
yielding the desired product.

The efficiency of SSF is highly influenced by the selection of the solid substrate.

Substrates suitable for SSF should ideally meet the following requirements:
(a) Have a porous solid matrix with a large surface area per unit volume (103 to 106
m2/cm3),
(b) Should sustain gentle compression and mixing,
(c) Should contain biodegradable
carbohydrates,
(d) Its matrix should absorb water in the proportion of 1 to several times its dry
weight,
(e) Should have relatively high water activity on the solid/gas interface to support
microbial, and
(f) Should absorb the additionally added nutrients such as nitrogen sources
(ammonia, urea, and peptides) and mineral salts.
(Orzua et al., 2009; Raimbault, 1998; Singhania et al., 2009).

On the basis of these characteristics, the ideal material consists of small granular or fibrous
particles that do not break or stick together. The commonly used solid substrates include wheat,
wheat bran, soybean, rice barley, oats, and other cereals (Chisti, 1999).

Limitations and Challenges of Solid Substrate Fermentation

Although SSF has many advantages over liquid cultivation, the main challenges are poor heat
and mass transfers within the substrate, and limited potential to monitor, online, key cultivation
parameters (temperature, pH, dissolved oxygen, nutrient concentrations, or water content), and
thus an inability to precisely control the microbial environment (Mitchel et al., 1999). The heat
generated by microbial metabolism is a major bottleneck for process scale-up; the released heat
can reach up to 3000 kcal from 1 kg of assimilated substrate, causing a radial gradient of 5°C/cm
at the center of the reactor (Bellon-Maurel et al., 2003). Low water content in the system and poor
heat conductivity of SSF substrates promote the heat gradient, which is accentuated by limited
agitation (the molds are very sensitive to shear stress) and is reduced mostly by evaporative
cooling, which can conversely exacerbate further water loss.

82
In addition, the pH gradient caused mainly by the production of organic acids and the utilisation
of proteins poses another challenge; pH monitoring and control is difficult because no existing pH
electrodes can operate in the absence of free water (Bellon-Maurel et al., 2003). Therefore, the
variations in pH should be prevented by increasing substrate buffering capacity (e.g., by the
addition of CaCO3) or by the addition of urea, which can counteract acidification. Oxygen
gradients can be reduced by aeration with moist air, which also plays a role in the desorption of
carbon dioxide and the regulation of temperature and moisture levels.

Industrial Applications of SSF

In Europe, SSF in the food and feed contexts is usually used for the production of mold and
ripening cheeses, production of fermented vegetables (e.g., sauerkraut or pickled cucumbers) or
silage (preserved cattle feed), and in Asia for the production of fermented products from soya,
rice, or corn.

An example of Oriental SSF is red yeast rice (red rice, ang-kak, anka, ankak, beni-koji, and other
names), which is a product obtained after SSF of rice with different Monascus species, in the
most usual case with Monascus purpureus. This product has been known in various Asian
countries (China, Japan, Thailand, Indonesia, Taiwan, and Philippines) for centuries and can be
used for food coloring, such as red koji (for the production of Kaoliang liqueur) or as a food
supplement with an anti-cholesterolemic effect. The cultivation conditions of Monascus SSC of
rice differ depending on the intended use. For food coloring, high pigment content (especially
oligoketide, red-colored compounds, monascorubramine and rubropunctamine, and their
complexes with the amino group-containing compounds) is required; for koji production, the
creation of hydrolytic enzymes is most important and in red yeast rice food supplement, a high
concentration of monacolin K (lovastatin) is a necessary prerequisite (Martinkova and Patakova,
2000; Patakova, 2005).

The main drawback of red yeast rice is its possible contamination with citrinin, which can be by
the selection of an appropriate producer strain or by its modification (Jia et al., 2010).

Traditionally, red yeast rice used for coloring of fish meals, soya products, vinegar, Beijing duck,
and more recently, also frankfurters or sausages, is produced by successive washing, soaking,
draining, and steaming of nonglutinous rice, which is then followed by inoculation with Monascus,
7-days incubation at 30°C, and product drying at 45°C or 60°C. Originally, the production took
place in wobbled bamboo trays covered with banana leaves in which rice kernels were manually
mixed or moistened if necessary (Lin et al., 2008). Nowadays, different types of trays, roller drums,
or fluid-bed bioreactors are being used (Chiu et al., 2006).

Mushroom cultivation, which is well accepted by the public, can be considered as a product of a
special single-cell protein (SCP) system, which utilises various types of agricultural and forestry
wastes and is relatively simple to perform. The special types of SSF are used for the production
of edible mushrooms of Agaricus, Pleurotus, Lentinula, Flammulina, and other genera. These
comprise the following steps: maintenance of mushroom culture, seed or spawn production,
substrate pretreatment, growing, and cropping. The primary rot fungi such as oyster mushroom
(Pleurotus spp.), shiitake (Lentinula edodes), winter mushroom (Flammulina velutipes), or paddy
straw mushroom (Volvariella spp.) degrade the moistened lignocellulosic material such as straw,
corn stover, saw dust, wood logs, or stumps as substrates (Rai, 2003). The button mushroom
(Agaricus bisporus), unlike other species, is a secondary rot fungus that requires compost
preparation prior to culture (Rai, 2003). To get high mushroom yields, and because many
mushroom species need changes in the environmental conditions to form fruiting bodies, the

83
original methods of outdoor or extensive cultivation have been replaced by intensive mushroom
farming that requires the construction of specialised facilities that allow the precise control of
environmental factors (temperature, humidity, light, and atmospheric gases). After thermal
pretreatment, the substrate is placed into different immobile (shelves) or mobile (plastic bags or
containers) systems. Substrate handling, spawn spreading (inoculation), substrate mixing, and
moistening, along with cropping, can be mechanized, for example, special combined harvesters
are used for button mushrooms (Chakravarty, 2011; Sánchez, 2004).

Among a wide range of SSF applications, the processes yielding protein-enriched agro-industrial
materials that can be used as animal feed play an important role (Ugwuanyi et al., 2009). In the
literature, there are reports on the production of protein-enriched animal feeds by SSF using
starchy materials as substrates. Besides their characteristics described above, all organisms
intended for animal consumption must comply with certain nutritional requirements such as amino
acid composition and digestibility, and the absence of toxins, antibiotics, and mainly mycotoxins
(Ghorai et al., 2009). SSF has also been described as a protein enhancement factor for cereal
grain and potato residues (Gélinas and Barrete, 2007). The protein content of wheat bran was
increased fourfold by SSF of Aspergillus terreus (Sabry, 1993).

INTENSIFICATION OF FERMENTATION PROCESSES

There are several strategies available for the intensification of bioprocesses. Some of them focus
on engineering aspects, whereas others exploit the tools of physiological modulation (selection
or adaptation of microorganisms), mutagenesis, or genetic manipulation to improve the production
strains. Examples of areas where a significant improvement of the fermentation processes can
be achieved by engineering approaches are improved mass and heat transfer, reduction of power
consumption, high-density cell cultures, and low-shear mixing (Chisti and Moo-Young, 1996). The
performance of the bioprocess is both individually and synergistically influenced by all
components of the production unit and related know-how (strain, bioreactor, media composition,
feeding strategy, etc.). In addition, the biological elements of the process (microorganisms, animal
and plant cells, and enzymes) are subject to many processing constraints (fragility, temperature,
pH range, and hygienic design of the equipment). These facts place important practical limitations
on the bioreactor and bioreaction engineering.

In the last few decades, there has been a significant progress in the area of process control and
instrumentation for bioreactors. This has an economic importance because the optimum operation
of a fermentation process is associated with improved productivity (high product concentration,
high production rate) and savings in product separation. The ability to operate a process at high
productivity requires a sound understanding of the biological requirements, process kinetics
(limitation, inhibition), and transport phenomena (Erickson, 2011). The following sections provide
the principles and examples of some bioprocess intensification methods.

Immobilised Cell Technology

The increased productivity of bioprocesses can be achieved through controlled contact of
substrates with a high concentration of the active biocatalyst, enzyme, or microbial cells. These
high-cell-density cultures can be created by feeding strategies, cell retention/recycle, or cell
immobilisation (Bumbak et al., 2011; Schiraldi et al., 2003; Verbelen et al., 2006). Among the
strategies to create high cell-density cultures, cell immobilisation is the most widely studied and
applied in the food and beverage industries (Karel et al., 1985; Kosseva, 2011). Table 4.2 lists
some references of research papers on food applications of immobilised cells

84
Table: Examples of Immobilised Cell Applications in Food and Beverage
Production

The maximum immobilised biomass concentration achieved in continuous beer fermentation was
up to 10 times greater than the free cell concentration at the end of the conventional batch
fermentation (Nakanishi et al., 1993). However, the immobilisation of microorganisms provokes
different physiological responses when compared to low-cell-density cultures of free cells (Junter
et al., 2002) and therefore, their application has to be carefully considered in terms of product
quality. For example, the application of immobilised microorganisms in fermentation processes
induces modifications in cell physiology due to mass transfer limitations, concentration gradients
created by an immobilisation matrix, and by aging of the immobilised biomass.

Immobilised Cell Physiology

An important factor influencing the growth and metabolic activity of immobilised cells is the
microenvironment of the solid immobilisation matrix, represented by parameters such as water
activity, pH, oxygen, substrate and product concentration gradients, and mechanical stress. The
interplay between the appropriate production strains and immobilisation methods is very important
in immobilised cell reactors and their suitable combination can improve both system performance
and product quality. The importance of careful matching of the chosen yeast strain with the
immobilisation method and the suitable reactor arrangement was demonstrated in beer
production (Mota et al., 2011).

Although there are a variety of methods for investigating the metabolic state of immobilised cells
(monitoring of cellular activity, microscopic, noninvasive, and destructive methods), acquiring the
reliable data is still the limiting factor for process optimization (Kosseva, 2011; Pilkington et al.,
1998). In addition, the data concerning the physiological conditions of immobilised microbial cells
are rather complex, due to different matrices and variable system configurations, and therefore
their interpretation is difficult (Junter and Jouenne, 2004).

85
Immobilisation has been reported to activate some metabolic functions (substrate uptake, product
formation, enzyme expression, and activity) of microbial cells (Lohmeier-Vogel et al., 1996; Norton
and D’Amore, 1994 ; Van Iersel et al., 2000).

According to some authors, the enhanced metabolic activity can also be attributed to surface-
sensing responses in immobilised microbial cells (Prakasham et al., 1999) but the reasons are
still a matter of controversy. Overall, conclusions should be very carefully drawn from the results,
since sampling and sample treatment may also influence the measurements of immobilised cell
physiology.
It has been shown that immobilised cells exhibit increased levels of deoxyribonucleic acid (DNA),
structural carbohydrates (Doran and Bailey, 1986), glycogen (Galazzo and Bailey, 1990), and
fatty acids (Hilge-Rotmann and Rehm, 1991), as well as modifications of cell proteome, cell wall,
and cell membrane composition (Jirku˚, 1995; Parascandola et al., 1997).

Not surprisingly, alterations in plasma membrane composition have a profound impact on several
enzymes, sensor proteins, transporters, and membrane fluidity. Many reports also underline
increased stress resistance of immobilised cells (Junter and Jouenne, 2004; Reimann et al.,
2011).

The increased resistance to inhibit substances (ethanol, pollutants, antimicrobial agents, etc.) can
be ascribed to changes in the composition and organization at the level of the cell wall and plasma
membrane (Jirku˚, 1999) and/or to the protective effect of the immobilisation support (Norton et
al., 1995).

Mass Transfer in Immobilised Cell Systems

The diffusional resistance to substrate transport from the bulk solution to the biocatalyst and the
hindered diffusion of products in the opposite direction may represent the most significant mass
transfer limitations arising from the use of immobilised cell technology. These mass transfer
limitations constitute the most evident hypothesis to explain the often-observed decrease in
immobilised cell growth rate and specific productivities as compared to free cell cultures (Abdel-
Naby et al., 2000; Taipa et al., 1993).

The typical immobilisation materials exhibiting internal mass transfer limitations are polymeric
matrices (Willaert and Baron, 1996). In these materials used for cell entrapment, the internal mass
transfer limitations of cells by nutrients can be further influenced by the position of the cells, bead
size, and structure of the polymer. Mass transfer limitations are crucial in immobilised cell systems
when oxygen supply to cells and the removal of carbon dioxide are required.

Oxygen transfer from the gas phase to the immobilised biocatalyst has long been recognised as
the major rate-limiting step in aerobic immobilised cell processes. The most common option to
improve mass transfer in these systems is to reduce the bead diameter (Groboillot et al., 1994).

Unlike polymeric matrices, the preformed porous (sintered glass) and nonporous (DEAE
cellulose, wood chips, and spent grains) carriers do not have the additional gel-diffusion barrier.
However, depending on the porosity of the carrier and on the amount of biomass adsorbed in the
pores, internal mass transfer limitations may also occur (Norton and D’Amore, 1994). In the case
of nonporous carriers, internal

86
Engineering Aspects of Process Intensification
Fermentation processes can be divided into two main categories based on the characteristic state
of matter of the medium: solid substrate fermentation and submerged fermentation.
Among the latter, the most common bioreactor configurations used in food and beverage
applications are stationary particle bioreactors, such as packed-bed/fibrous-bed, trickle-bed
reactors, and mixed (particle) bioreactors, such as fluidized bed, gas lift, bubble column, and
stirred tank (Kosseva, 2011; Raspor and Goranovic, 2008; Willaert and Nedovic, 2006).

The stationary particle bioreactors are either operated with immobilised cells (enzymes) or a
mixture of free and immobilised cells (enzymes). Mixed bioreactors may contain solely free or
immobilised cells as well as their mixture. There are also bioreactor configurations that do not fit
into the two previous categories. For example, rotating biological contactors (RBCs), also
classified among moving surface reactors, where a biofilm grows on rotating disks partially or
completely immersed in a liquid medium. The use of RBCs in food applications is rare, and is
limited to the production of citric acid (Wang, 2000).

Table: Factors Influencing the Selection of Bioreactor Type

The selection of a suitable reactor design from numerous available types and configurations
(Zhong, 2011) is a complex task and depends on various factors (Table). The importance of
individual factors may change depending on the process requirements and the product
characteristics. However, there is a need to have a fundamental understanding of the kinetics and
transport limitations when a bioreactor is selected or when a new bioreactor is designed and
constructed. Two-phase bioreactors are generally limited to anaerobic processes or to processes
where gas–liquid mass transfer plays a marginal role. Conversely, in three-phase bioreactors,
efficient mass transfer usually requires an intimate mixing of all three phases.
Three-phase bioreactor design is an area in which significant process intensification can be
achieved through the enhancement of gas–liquid mass transfer (Chisti and Moo-Young, 1996;
Suresh et al., 2009).

87
Gas-Liquid Mass Transfer Considerations
In aerobic bioprocesses, oxygen is the key substrate due to its low solubility in aqueous media.
Consequently, a continuous supply of oxygen into aerobic bioreactors is often needed. Therefore,
the oxygen transfer rate (OTR) should be predicted prior to the choice/design and scale-up of a
bioreactor. Many studies have been conducted to estimate the efficiency of oxygen transfer in
different bioreactors and these have been reviewed in various works (Clarke and Correia, 2008;
Garcia-Ochoa and Gomez, 2009; Kantarci et al., 2005; Suresh et al., 2009).

Another area where gas-liquid mass transfer rate is crucially important (CO2 supply and O2
removal) is the construction and operation of photobioreactors used for the cultivation of
photoautotrophic microorganisms (microalgae) with nutritional potential (Carvalho et al., 2006).
The dissolved oxygen concentration in aerobic cultures depends on the rate of oxygen transfer
from the gas phase (usually air bubbles) to the liquid, on the rate at which oxygen is transported
into the cells, and on rate of oxygen uptake by the microorganism. The transport of oxygen from
air bubbles to the site of oxygen consumption can be described in a number of steps, among
which oxygen diffusion through the liquid film surrounding the bubble shows the greatest
resistance.

The gas-liquid mass transfer rate is usually modeled according to the two-film theory and is
characterised by the volumetric (gas-liquid) mass (oxygen) transfer coefficient (kLa), while the
driving force of the process is the difference between the concentration of oxygen at the interface
(C*) and that in the bulk liquid (CL). In the case of large microbial pellets, immobilised cells, or
fungal hyphae, the resistance in the liquid film surrounding the solid can also be significant (Blanch
and Clark, 1997).

Oxygen transfer in aerobic bioprocesses is strongly influenced by the hydrodynamic conditions in

the bioreactors. These conditions are known to be affected by the operational conditions (stirrer
speed, superficial gas velocity, liquid circulation velocity, etc.), physicochemical properties of the
culture (viscosity, density, and surface tension), bioreactor geometry, and also by the presence
of oxygen-consuming cells (Garcia-Ochoa and Gomez, 2009).

Stirred Tank Bioreactors (STBRs) are widely used in a large variety of bioprocesses taking
advantage of free cell (enzyme) suspensions. An industrial-scale STBR usually consists of a
stainless-steel vessel, motor-driven impeller, and gas sparger positioned below the impeller.

Aerated STBRs generally have high mass and heat transfer coefficients, good homogenization,
and the capability of handling a wide range of superficial gas velocities. Mass transfer and mixing
in STBRs are most significantly affected by stirrer speed, type and number of stirrers, and the gas
flow rate (Garcia-Ochoa et al., 2011).

In bioreactors with a height-to-diameter ratio (H/T) above two, standard single-impeller systems
were often found to have unsuitable operating parameters. Oxygen transfer in these geometries
can be improved by using multiple-impeller configurations (approximately one impeller per each
H/T = 1) that exhibit efficient gas distribution, increased gas holdup, superior liquid-flow
characteristics, and lower power consumption per impeller as compared to single-impeller
systems (Gogate et al., 2000).

Pneumatic bioreactors consist of a cylindrical vessel, into the bottom (usually) of which air (gas)
is introduced to ensure aeration, mixing, and liquid circulation, without any moving mechanical
parts. In pneumatically agitated reactors such as bubble columns (random liquid circulation) and

88
airlift reactors (streamlined liquid circulation), the homogeneous shear environment compared to
the local shear extremes in STBRs has enabled the successful cultivation of shear-sensitive cells
such as mammalian and plant cells or mycelial fungi (Guieysse et al., 2011). In contrast, the lack
of mechanical agitation can cause poor mixing in a highly viscous medium and serious foaming
under high aeration. Airlift and stirred tank reactors exhibit comparable mass transfer capacities;
however, airlift reactors can be superior to STBRs in terms of operating costs because of lower
power consumption (Chisti, 1998). A further increase in the overall volumetric gas-liquid oxygen
transfer coefficient (kLa) in bubble column and airlift reactors was achieved by the installation of
static mixers (Thakur et al., 2003) into the draft tubes and riser sections, respectively (Chisti et
al., 1990; Goto and Gaspillo, 1992).

The improvement of OTR achieved by static mixers is a result of air bubble breakup increasing
the specific gas-liquid interfacial area (a). Industrial applications include the cultivation of a
filamentous mold (Gavrilescu and Roman, 1995) and ethanol production in an airlift reactor
(Vicente et al., 1999).

However, in the second example, increased ethanol productivity was also achieved as a
consequence of size reduction of yeast flocs, and thus improved liquid-solid mass transfer,
provoked by the new riser design.

The predictions of OTR determined by a dynamic method in sterile culture medium in the absence
of biomass, often underestimates the kLa value for the real bioprocess (Djelal et al., 2006). In
fermentation processes, an enhancement of OTR was found to be due to oxygen consumed by
the microorganisms, leading to a lower dissolved oxygen concentration in the bulk liquid (CL).
Simultaneously, mass transfer enhancement was also attributed to the presence of a dispersed
phase (microorganisms) adsorbed onto the gas-liquid interface, influencing the oxygen adsorption
rate and gas-liquid interfacial area. The extent of this enhancement was expressed as the
biological enhancement factor (E). According to some studies, the E value of kLa can be up to
1.3 times that of the mass transfer coefficient determined for the system without microbial cells
(Garcia-Ochoa and Gomez, 2005).

Table: Characteristics of Cultivation Methods

TYPE OF CULTURE OPERATIONAL APPLICATION
CHARACTERISTICS

Solid Simple, cheap, selection of Maintenance of strains,

colonies from single cell
possible; process control
limited
Film Various types of bioreactor;
trickling filter, rotating disc,
packed bed, sponge
reactor, rotating tube
Batch: Submerged “Spontaneous” reaction,
homogeneous various types of reactor:
distribution of cells continuous stirred tank
reactor, air lift, loop deep
shaft, etc.; agitation by
stirrers, air, liquid process
control for physical
genetic studies; production
of enzymes; composting
Waste-water treatment,
monolayer culture (animal
cells); bacterial leaching;
vinegar production
Standard type of cultivation;
antibiotics, solvents, acids,
etc.

89
 parameters possible; less
for chemical and biological
parameters

Fed-batch Simple method for control of Production of baker’s yeast

regulatory effects, e.g.
glucose repression

Continuous one-stage Proper control of reaction; Few cases of application in

homogeneous excellent for kinetic and industrial scale; production
regulatory studies; higher of single cell protein; waste-
costs for experiment; water treatment
problem of aseptic
operation; the need for
highly trained operators

Variations
Fermentation setups closely related to the chemostats are the turbidostat, the auxostat and the
retentostat. In retentostats culture liquid is also removed from the bioreactor, but a filter retains
the biomass. In this case, the biomass concentration increases until the nutrient requirement for
biomass maintenance has become equal to the amount of limiting nutrient that can be consumed.

It is highly difficult to control the delivery of nutrient and the removal of cell so that an equal amount
of medium is maintained in the vessel. This can be tackled by changing the configuration of the
bioreactor into a semi-continuous or fed-batch type bioreactor.

Studying the growth of bacterial populations in batch or continuous cultures does not permit any
conclusions about the growth behaviour of individual cells, because the

Future Perspectives
The development of fermentation processes for the food and beverage industries aims at
improving the productivity and product quality by means of process design, strain
selection/construction, and process monitoring. In all these areas, there have emerged some very
innovative ideas that could lead to economically attractive solutions. With regard to (online)
process monitoring, significant progress is required, particularly in the area of advanced
instrumentation and sensor development, for solid substrate fermentations. The innovative
techniques described so far include different sensor technologies, respirometry, x-rays, image
analysis, infrared spectrometry, magnetic resonance imaging, and so on. However, for some of
them, the main drawback is high cost, which makes these techniques unsuitable for large-scale
applications (Bellon-Maurel et al., 2003).

One of the significant challenges in the bioreactor design is the improvement of large-scale
photobioreactors and phycocultures (seaweed farms) for the production of micro- and macroalgae
and algae-derived food products (Carvalho et al., 2006; Luening and Pang, 2003).

Another prospective strategy to increase the metabolic productivity in bioprocesses is the use of
suitably controlled ultrasonication. The beneficial effects of ultrasound can be exploited at the
level of biocatalysts (cells and enzymes) and their function (e.g., cross-membrane ion fluxes,
stimulated sterol synthesis, altered cell morphology, and increased enzyme activity) and

90
sonobioreactor performance (mass transfer enhancement) (Chisti, 2003; Kwiatkowska et al.,
2011).

The potential for genetic engineering in the field of food fermentation is indisputable and has been
reviewed (Leisegang et al., 2006). However, the nutritional status of fermented foods can also be
improved by the rational choice of food-fermenting microbes based on the understanding of their
interaction with diet and human gastrointestinal microbiota. In this respect, fermented foods can
be regarded as an extension of the food digestion and fermentation processes and can be steered
toward beneficial health attributes (Vlieg et al., 2011).

Lecturer: Zakpaa Domakyaara Hilary (Ph.D.)

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