Semester – III

16MBU303 INDUSTRIAL MICROBIOLOGY (4H – 4C)

Instruction Hours / week: L: 4 T: 0 P: 0 Marks: Internal: 40 External: 60 Total: 100

End Semester Exam: 3 Hours
SCOPE
To acquire the knowledge in the large scale production of industrial product, providing the trends to cater the needs of
industry
OBJECTIVES
 To encompasses the use of microorganisms in the manufacture of food or industrial products.

Unit I – Introduction to industrial microbiology

Brief history and developments in industrial microbiology. Sources of industrially important microbes and methods for
their isolation, primary and secondary strain improvement.

Unit II – Isolation of industrially important microbes and fermentation media

Preservation and maintenance of industrial strains. Crude and synthetic media - molasses, corn- steep liquor, sulphite
waste liquor, whey, yeast extract and protein hydrolysates.

Unit III – Types of fermentation processes, bio-reactors and measurement of fermentation parameters
Types of fermentation processes – Solid-state and liquid-state (stationary and submerged) fermentations; batch,
fed-batch and continuous fermentations. Components of a typical bio-reactor, Types of bioreactors – Laboratory,
pilot- scale and production fermenters, constantly stirred tank and air-lift fermenters. Measurement and control of
fermentation parameters - pH, temperature, dissolved oxygen, foaming and aeration.

Unit IV – Down-stream processing and Enzyme immobilization

Separation of cells – filtration and centrifugation. Cell disruption – physical, chemical and enzymatic methods. Product
separation – solvent extraction and precipitation. Lyophilization and spray drying. Methods of immobilization,
advantages and applications of immobilization, large scale applications of immobilized enzymes (glucose isomerase
and penicillin acylase).

Unit V – Microbial production of industrial products (micro-organisms involved, media, fermentation

conditions, downstream processing and uses)
Citric acid, Ethanol, Penicillin, Glutamic acid, Vitamin B12, Enzymes (amylase, protease, lipase) Wine, Beer.

SUGGESTED READINGS
1. Stanbury PF, Whitaker A and Hall SJ. (2006). Principles of Fermentation Technology. 2nd edition, Elsevier
Science Ltd.
2. Crueger W and Crueger A. (2000). Biotechnology: A textbook of Industrial Microbiology. 2nd edition. Panima
Publishing Co. New Delhi.
3. Okafor N. (2007). Modern Industrial Microbiology and Biotechnology. 1st edition. Bios Scientific Publishers
Limited. USA
4. Glaze A.N. and Nikaido H. (1995). Microbial Biotechnology: Fundamentals of Applied Microbiology. 1st
edition. W.H. Freeman and Company
5. Casida LE. (1991). Industrial Microbiology. 1st edition. Wiley Eastern Limited.
6. Patel A.H. (1996). Industrial Microbiology. 1st edition, Macmillan India Limited.
7. Waites M.J., Morgan N.L., Rockey J.S. and Higton G. (2001). Industrial Microbiology: An Introduction. 1st
edition. Wiley – Blackwell.
 DEPARTMENT OF MICROBIOLOGY
KARPAGAM ACADEMY OF HIGHER EDUCATION
(Deemed to be University Established Under Section 3 of UGC Act, 1956)
Eachanari Post, COIMBATORE - 641 021, INDIA

[Link]., Microbiology (Batch 2016-2019)

Industrial Microbiology (Semester-III) (16MBU303)
LECTURE PLAN
UNIT1

Duration Topic Reference

01 History and development in industrial microbiology. R1:3-14
02 History and development in industrial microbiology
03 Sources of industrially important microbes
04 Isolation of industrially important microbes
05 Primary screening of industrially important microbes R2:9-50
06 Primary screening of industrially important microbes
07 Secondary screening of industrially important microbes
08 Strain improvement R3:71-73
09 Unit revision and possible questions.
Total hours: 9 HRS

R1: Patel A. H., 2003. Industrial Microbiology. Macmillan India ltd, NewDelhi.
R2: Wulf Cruegar & A. Cruegar, 2000. A Textbook of Industrial Microbiology, Panima
Publishing Corp, Newdelhi.
R3: Kalaichelvan and Arulpandi, 2009. Bioprocess technology,MJP Publishers.

Dr [Link]
Assistant Professor
Department of Microbiology
 2017

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
Unit I
History and development of Industrial Microbiology

Industrial Microbiology

Use of microbes to obtain a product or service of economic value constitutes

industrial microbiology. Any process mediated by or involving microorganisms in which a
product of economic value is obtained is called fermentation. The terms industrial
microbiology and fermentation are virtually synonymous in their scope, objectives and
activities. The microbial product may be microbial cells (living or dead), microbial biomass,
and components of microbial cells, intracellular or extracellular enzymes or chemicals
produced by the microbes utilizing the medium constituents or the provided substrate.

Microbes have been employed for product generation, e.g., wines, bread, etc., since
thousands of years, but these activities were purely art. The science of industrial
microbiology is only about 150 years old. The first observations of microorganisms by
Leeuwenhoek were published in [Link] experiments of Spallanzani in 1799 and of
Schwan in 1837 not only disproved the idea of spontaneous generation of microorganisms,
but also provided a means of sterilization of liquids (by heat) and air (by heat), respectively.
Schwan's findings also suggested that alcoholic fermentation was due to a fungus or mold,
i.e., yeast, and inoculation resulted in quicker fermentation. But microbiology is widely
considered to have begun in 1857 when Pasteur reported his studies on lactic acid
fermentation, including the microscopic features of the microorganisms and a suitable
medium for the process; the scientific basis of industrial microbiology began with this paper.

In 1860, Pasteur reported the first synthetic medium for microorganisms, and used it
to study alcohol fermentation. In 1861, Pasteur showed that growth and physiology of yeast

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
(and hence the accumulation of fermentation product, alcohol) differs depending on the
presence or absence of °2' This phenomenon is known as Pasteur effect and is applicable to
other microorganisms as [Link] 1878, Lister described the dilution technique for obtaining
the first pure microbial culture of lactic acid bacterium. A simpler and more effective
technique for obtaining pure cultures from isolated separate colonies developed on solidified
medium was described by Robert Koch in 1881; this technique is widely followed even
today.

In 1876, Cohn showed that bacterial spores have a high level of heat resistance and
developed the technique of 'intermittent sterilization' for their inactivation. In 1897, Buchner
demonstrated alcohol fermentation by cell-free yeast juice; he suggested that a proteinaceous
enzyme was responsible for fermentation. Wildiers demonstrated in 1901 that yeast required
growth factors (vitamins) for growth, especially at low inoculum levels; vitamins are used in
fermentation even today. In 1929, Alexander Fleming accidentally discovered penicillin
produced by Penicillium growing as contaminant in a Petri plate of [Link]
developed the technique for assay of antibacterial activity of penicillin using bacteria and
showed its low toxicity to man and animals. This was followed by an intensive search for
antibiotics during the Second World War leading to the discovery of streptomycin,
chloramphenicol, tetracyclines, [Link] developments have resulted in the use of
metabolically blocked mutants of microorganisms, which accumulate large amounts of
metabolic intermediates.

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017

Industrially important strains- fermentation products

Bacteria Yeasts and filamentous fungi
Traditional products
Bread, beer, wine and spirits Mainly Saccharomyces cerevisiae
Cheeses, other dairy products Lactic acid bacteria
Ripening of blue and Penicillium species
Camembert-type cheeses
Fermented meats and vegetables Mostly lactic acid bacteria
Mushrooms Agaricus bisporus, Lentinula edodes
Soy sauce Aspergillus oryzae

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
Zygosaccharomyces rouxii
Sufu (soya bean curd) Mucor species
Vinegar Acetobacter species
Agricultural products
Gibberellins Fusarium moniliforme
Fungicides Coniothyrium minitans
Insecticides Bacillus thuringiensis
Silage Lactic acid bacteria
Amino acids
l-Glutamine Corynebacterium glutamicum
l-Lysine Brevibacterium lactofermentum
l-Tryptophan Klebsiella aerogenes
Enzymes
a-amylase Bacillus subtilis
b-amylase Aspergillus niger
amyloglucosidase Aspergillus niger
glucose isomerase Streptomyces olivaceus
invertase Kluyveromyces species
lactase (b-galactosidase) Kluyveromyces lactis
Cellulases Trichoderma viride
Lipases Candida cylindraceae
Pectinases Aspergillus wentii
Proteases
subtilisin (alkaline) Bacillus licheniformis
neutral Aspergillus oryzae
microbial rennet (acid) Rhizomucor miehei
Fuels and chemical feedstocks
Acetone Clostridium species
Butanol Clostridium acetobutylicum
Ethanol Zymomonas mobilis Saccharomyces cerevisiae
Glycerol Zygosaccharomyces rouxii
Methane Methanogenic archaeans
Nucleotides
5¢-Inosine monophosphate Bacillus subtilis
5¢-Guanosine monophosphate Brevibacterium ammoniagenes
Organic acids
Acetic Acetobacter xylinum
Citric Aspergillus niger
Yarrowia lipolytica
Fumaric Rhizopus species
Gluconic Acetobacter suboxydans
Itaconic Aspergillus itaconicus
Kojic Aspergillus flavus
Lactic Lactobacillus delbrueckii
Pharmaceuticals and related compounds &Hormones
Human growth hormone Recombinant Escherichia coli Recombinant Saccharomyces
cerevisiae
Insulin Recombinant Escherichia coli Recombinant Saccharomyces cerevisiae
Immunosuppressants
Cyclosporin Trichoderma polysporum

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
Interferon Recombinant Escherichia coli Recombinant Saccharomyces cerevisiae
Steroids Arthrobacter species Rhizopus species
Vaccines Bacillus anthracis
Clostridium tetani
Recombinant Escherichia coli
Salmonella typhi
Vitamins
B12 (cyanocobalamin) Pseudomonas denitrificans
b-Carotene (provitamin A) Blakeslea trispora
Ascorbic acid (vitamin C) Acetobacter suboxydans
Riboflavin Recombinant Bacillus subtilis Ashbya gossypii
Polymers
Alginates Azotobacter vinelandii
Cellulose Acetobacter xylinum
Dextran Leuconostoc mesenteroides
Gellan Sphingomonas paucimobilis
Polyhydroxybutyrate Ralstonia eutropha
Pullulan Aureobasidium pullulans
Scleroglucan Sclerotium rolfsii
Xanthan Xanthomonas campestris
Single cell protein Methylococcus capsulatus Candida utilis
Methylophilus methylotrophus Fusarium venenatum
Kluyveromyces marxianus
Paecilomyces variotii
Saccharomyces cerevisiae

Isolation and Screening of Microorganisms - The success of an industrial fermentation

process chiefly depends on the microorganism strain used. An ideal producer or economically
important strain should have the following characteristics.

1. It should be pure, and free from phage.

2. It should be genetically stable, but amenable to genetic modification.
3. It should produce both vegetative cells and spores; species producing only mycelium are
rarely used.
4. It should grow vigorously after inoculation in seed stage vessels.
5. Should produce a single valuable product, and no toxic by-products.
6. Product should be produced in a short time, e.g., 3 days.
7. It should be amenable to long term conservation.
8. The risk of contamination should be minimal under the optimum performance conditions.

Isolation of Microorganisms - The first step in developing a producer strain is the isolation
of concerned microorganisms from their natural habitats. Alternatively, microorganisms can
be obtained as pure cultures from organization, which maintain culture collections, e.g.,
American Type Culture Collection (ATCC). Rockville, Maryland, U.S.A., Commonwealth
Mycological Institute (CMI), Kew, Surrey, England, Fermentation Research Institute
(FERM), Tokyo, Japan, U.S.S.R. Research Institute for Antibiotics (RIA), Moscow,
U.S.S.R., etc.

The microorganisms of industrial importance are, generally, bacteria, actinomycetes,

fungi and algae. These organisms occur virtually everywhere, e.g., in air, water, soil, surfaces

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
of plants and animals, and plant and animals tissues. But most common sources of industrial
microorganisms are soils, and lake and river mud. Often the ecological habitat from which a
desired microorganism is more likely to be isolated will depend on the characteristics of the
product desired from it, and of process development. For example, if the objective is to
isolate a source of enzymes, which can withstand high temperatures, the obvious place to
look will be hot water springs.

A variety of complex isolation procedures have been developed, but no single method
can reveal all the microorganisms present in a sample. Many different microorganisms can be
isolated by using specialized enrichment techniques, e.g., soil treatment (UV irradiation, air
drying or heating at 70-120°C, filtration or continuous percolation, washings from root
systems, treatment with detergents or alcohols, preinoculation with toxic agents), selective
inhibitors (antimetabolites, antibiotics, etc.), nutritional (specific C and N sources), variations
in pH, temperature, aeration, etc.
The enrichment techniques are designed for selective multiplication of only some of the
microorganisms present in a sample. These approaches however take a long time (20-40
days), and require considerable labour and money. The main isolation methods used routinely
for isolation from soil samples are: sponging (soil directly), dilution, gradient plate, aerosol
dilution, flotation, and differential centrifugation. Often these methods are used in
conjunction with an enrichment technique. Microoorganisms are used extensively to
provide a vast range of products and services. They have proved to be particularly useful
because of the ease of their mass cultivation, speed of growth, use of cheap substrates (which
in many cases are wastes) and the diversity of potential products.

The most important factor for the success of any fermentation industry is of a
production strain. It is highly desirable to use a production Strain with the following four
characteristics:
(i) It should be a high-yielding strain.
(ii) It should have all adoptable characteristics.
(iii) It should not produce undesirable substances.
(iv) It should e easily cultivated on a large-scale.
Screening techniques
Both detection and isolation of high-yielding species from the natural, such as soil,
containing a huge microbial populationis called screening., There are many screening
techniques. Usually screening programmes include primary screening and secondary
screening.
Primary Screening
This consists of some elementary tests required to detect and to isolate new microbial species
exhibiting the desired property. With antibiotic producers,:primary screening programmes
serve to remove worthless micro-organisms on the basis of relatively simple, fundamental
criteria. The important selection critcria are the activity of antibiotics in vitro, and possibly in
vivo, against a small number of the most important test organisms/ Primary screening is also
needed in the case of other useful microbial species (e.g. micro-organisms capable of
producing organic acids, amines, [Link].). The evaluation of the primary screening in
industrial research programmes may be made by citing some specific examples of
screening procedures as under:
The crowded plate technique
The crowded plate technique is the simplest screening technique employed in detecting and
isolating antibiotic producing of the soil or other source material for the antibiotic
producing micro-organisms, followed b spreading of dilutions on the nutrient agar plate agar

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
plates having 300 to 400 or more colonies per plate are considered since they are helpful in
locating the colonies producing antibiotic of a colony to exhibit antibiotic activity is indicated
by the presence of a zone of growth inhibition surrounding the colony such a colony is
subcultured to a similar medium, and purified' by streaking, before making stock culture.
Thus necessary to carry on further testing to confirm the antibiotic activity associated with a
microrganism, since the zone of inhibition surrounding the colony may, sometimes,.because
to other cause.
The crowded plate techniqu ,has a limited application, since it merely provides information
regarding the inhibitory activity of colony against the unwanted microbes that may be resent
by chance on the,[Link] the technique has been improved upon by introducing the
use of a 'test-organism this modified agar plates which give well-isolated colonies after
incubation are flooded with a suspension of the test organism. Then the plates are subjected
to further incubation to allow the growth of the test organism. The formation of inhibitory
zones around certain colonies indicates their antibiotic activi~(!!te diameter of the zones of
inhibition are measured in millimeters, in a rough approximation of the relative amounts of
antibiotic(s) produced by various colonies of the antibiotic producers must be isolated and
purified.
Auxanography
This technique is rarely applied for detecting micro-organisms able to produce growth
factors (e.g. [Link] and vitamins) extracellularly. The two major steps of the
technique are as under: -
(A) Preparation of First Plate
A filter paper strip (1'5 X 12 em) is put across the bottom of a petri dish in such a way that
the ends pass over [Link] of the dish _
2.A filter, paper disc of petri dish size is placed over paper strip on the bottom of the dish.
[Link] nutrient agar (45°C) is poured on the paper disc in the dish and allowed to solidify.
[Link] source material such as soil, is subjected to dilution such that aliquots on plating
will produce well isolated colonies.
[Link] of aliquots dilluted soil sample is done.
(B) Preparation of Second Plate
I. A minimal medium lacking the growth factor under consideration is seeded with the test
organism.
[Link] seeded medium, is poured on the surface of a fresh petri dish.
[Link] plate is allowed to set.
The agar in the first plate, as prepared in step (A), is carefully and aseptically lifted out with
the help of tweezers and a spatula and, placed, without inverting, on the surface of the second
plate as prepared in major steps (B).
Growth factor(s) produced by colonies present on the surface of the first layer of agar can
diffuse into the layer of agar containing the test organism. The stimulated growth, of the test
organism around the colonies is an indication that they produce growth factor(s)
extracellularly.
Productive colonies are sub-cultured and are further tested. '
Enrich Culture technique
This technique was designed by a soil microbiologist, Beijerinck, to isolate the desired
microrganisms from a heterogeneous microbial population present in soil. Either medium or
incubation conditions are adjusted so as to favour the growth of the desired micro-organisms.
On the other hand, unwanted micro-organisms are eliminated, or develop poorly since they
do not find suitable growth conditions in the newly created environment. Today, this
technique has become a valuable tool in many screening programmes meant for isolating
industrially important strains.

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
Generally, it consists of the following steps:
[Link] broth containing an unusual substrate (e.g. Cellulose powder) is inoculated with
the microbial'source material (e.g. soil) and incubated.
2.A small'portion of inoculum from step (1) is plated onto a solid medium having the same
position. Well isolated colonies appear after incubation. '
[Link] from plate of step (2) are subcultured on fresh media and they are also
subjected to further testing.
An example of screening of enzyme, producing micro-organisms may be cited.
Miicro-organisms excreting alkaline proteases may be detected from the soil as under:
(i) Selected to serial dilution.
(ii) All soil dilutions are heated at 5O°Cfor 10 minutes. This treatment kills vegetative cells
,
(iii) The plating of heat treated sam les is done by spreading the samples (usually 0·1 ml.)
from dilutions on the surface of nutrient agar containing casein at pH 10-12.
(iv) The colonies surrounded by the clear zone are sub-cultured.
Use of an indicator dye
The pH indicating dyes may be employed in some screening methods for detecting
microorganisms capable of producing organic acids or amines, since a pH indicating dye
under .colour changes according to its pH. Such dyes (e.g. neutral red, bromophenol blue) are
added to the poor nutrient agar media. The change in the colour of a dye in the vicinity of the
colony suggests the capability of colonial cells to produce anionic acid(s or amine(s),
depending upon the nature of reaction. Such colonies are subcultured to make stock cultures.
Bdwever, further testing is needed, since inorganic acids or bases are also potential metabolic
products of microbial growth. In other words, these methods are not fool proof.
This differential technique may also be employed in finding out whether micro-organisms are
capable of certain microbial transformations or not. E.g. Eoestrogen formation by
bioconversion.
Secondary Screening
Secondary screening is strictly essential in any systematic screening programme intended to
isolate industrially useful micro-organisms, since primary screening merely allows the
detection and isolation of micro:Organisms that possess potentially interesting industrial
application Moreover, primary screening does not provide much information needed in
setting up a new fermentation process. Secondary screening helps in detecting really useful
micro-organisms in fermentation processes: This can be realized by a careful understanding
of the concept. The following points associated with secondary screening' very useful
insorting out micro-organisms that have real commercial value from many isolates obtained
during primary screening. At the same time, micro-organisms that have poor applicability in
a fermentation process are discarded. It is advisable to discard poor cultures.
Provides information whether [Link] produced by a microorganism is a new
one or
It is maybe-accomplished thin layer, other Chromatographic techniques.
The compound under consideration is compared with previously known
compounds.
It gives an idea about the economic position of the fermentation process
involving the use of a newly discovered culture.
Thus, one may have a comparative study of this process with processes that are
already known, so far as the economic status picture is concerned.

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
It helps in providing information regarding the product yield potentials of
different isolates
Thus, this is useful in selecting efficient cultures for the fermentation processes.
It determines the optimum conditions for growth or accumulation of a product
associated with a particular culture.
It provides information pertaining to the effect of different components of a
medium. This is valuable in design in the medium that may be attractive so far as
economic consideration is concerned.
It detects gross genetic instability in microbial cultures. This type of information
is very important, since micro-organisms tending to undergo mutation or
alteration in some way may
lose their capability for maximum accumulation of the fermentation products.
It gives information about the number of products produced in a single
fermentation additional major or minor products are of distinct value, since their
recovery and sale as by-products
can markedly improve the economic status of the prime fermentation.
Information about the solubility of the product in various organic solvents is
made available. This knowledge is useful in the recovery and the subsequent
purification of the product.
Chemical, physical and biological properties of a product are also determined
during secondary screening. Moreover, it reveals whether a product produced in
the culture broth occurs in more than one chemical form.
It reveals whether the culture is homoferrnentative or hetero fermentative.
Determination of the structure of the product is done.
With certain types of products (e.g. antibiotics) determinations of the toxicity for
.animal, plant br man are made if they are to be therapeutic purposes.
It reveals whether micro-organisms are capable of a chemical change or of even
destroying 'their own fermentation products. For instance, micro-organisms that
produce the adaptive enzyme, decarboxylase, CRn remove carbon dioxide from
the amino acid, leaving behind an organic amine.
Thus, secondary screening gives answers to many questions that arise during
the final sorting out of industrially useful micro-organisms. This is accomplished by
performing experiments on agar plates, in asks or small bioreactors containing liquid media,
or by a combination of these approaches. 'A specific example of antibiotic producing
Streptomyces species may be taken for an understanding of the sequence of events during a
screening programme. Those streptomycetes able to produce antibiotics are detected and
isolated in a primary screening programme. These streptomycetes exhibiting antimicrobial
activity are Subjected to an initial secondary' screening where their inhibition spectra are
determined. A simple, 'giant-colony', technique is used to do this. Each of the streptomycal
isolates is streaked in a narrow band across the centres of the nutritious agar plates. Then,
these plates are incubated until growth of a streptomycete occurs. Now, the test organisms are
streaked from the edges of the plates upto but not touching the streptomycete growth. Again,
the plates are incubated. At the end of incubation, growth inhibitory zones for each test
organism are measured in millimeters. Thus, the 'microbial inhibition spectrum helps in
discarding poor cultures. Ultimately, streptomycete isolates that have exhibited interesting
microbial inhibition spectra need further testing. With streptomycetes suspected to produce
antibiotics with poor solubility in water, the initial secondary screening is done/in some
different way which is out of the scope of this book.

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
Further screening is carried out employing liquid media in flasks'l ince such studies
give more information than that which can be obtained on agar media. At the same time, it is
advisable to use accurate assay techniques (e.g. paper disc-agar diffusion assay) to exactly
determine the amounts of antibiotic present in samples of culture fluids. Thus'/each of the
streptomycete isolates is studied by using several different liquid media in Erlenmeyer flasks
provided with baffles. These streptomycete cultures are inoculated into sterilized liquid
media. Then, such seeded flasks are incubated at a constant temperature. Usually, such
cultures are incubated at near room temperature. Moreover, such flasks are aerated by
keeping them on a mechanical shaker, since the growth of streptomycetes and production of
antibiotics occur better in aerated flasks than in stationary ones. Samples are withdrawn at
regular intervals under aseptic conditions and are tested in a quality control laborator .
Important tests to be carried out include:
(i) checking for contamination,
(ii) checking of pH,
(iii) estimation of critical nutrients,
(iv) assaying of the antibiotic, and .
(v) other determinations, if necessary.
The result of the above tests points out which medium is the best for antibiotic
formation, and at
which stage the antibiotic yields are greatest during the growth of the culture on the various
media. After performing all necessary routine tests in the screening of an actually useful
streptomycete for a fermentation process, other additional determinations are made. They are:
[Link] of fermentation media through the exploitation of which the highest antibiotic
yields may be obtained.
[Link] of whether the antibiotic is new.
[Link] of the number of antibiotics accumulated in the culture broth is made.
[Link] of different bioparameters On the growth of streptomycete culture, fermentation
process and accumulation of antibiotic.
[Link] picture of antibiotic in various organic solvents. Also, it is to be determined
whether antibiotic is adsorbed by adsorbent materials (e.g. ion-exchange resins or activated
carbon).This knowledge' is essential in the recovery and purification of an antibiotic from the
fermented broth.
[Link] tests are conducted on mice or other laboratory animals. An antibiotic is also
tested for the adverse effects if any, on man, animal or plant.
[Link] streptomycete culture is characterized and is classified upto species.
[Link] studies are made on a selected individual streptomycete culture. For example,
mutation and other genetic studies for strain improvement are carried out.
In [Link] are designed and conducted in such a way that production streptomycete
strains may be obtained with least expense Similar screening and analytical techniques could
be mployed for the isolation of microbial isolates important in the production of other
industrial chemical substances.

Strain Improvement - After an organism producing a valuable product is identified, it

becomes necessary to increase the product yield from fermentation to minimise production
costs. Product yields can be increased by (i) developing a suitable medium for fermentation,
(ii) refining the fermentation process and
(iii) improving the productivity of the strain.
Generally, major improvements arise from the last approach; therefore, all
fermentation enterprises place a considerable emphasis on this activity. The techniques and

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
approaches used to genetically modify strains, to increase the production of the desired
product are called strain improvement or strain development.

Strain improvement is based on the following three approaches:

(i) mutant selection,

(ii) recombination, and
(iii) recombinant DNA technology.

Mutant Selection - Large scale mutant selection programmes begin when favourable reports
of clinical trials are obtained. In the early stages, selection of spontaneous mutants may be
helpful, but induced mutations are the most common sources of improvements.
Many mutations bring about marked changes in a biochemical character of practical interest;
these are called major mutations. Some major mutations can be useful in strain improvement.
For example, a mutant strain (S-604) of Streptomyces aureofaciens produces 6-demethyl
tetracycline in place of tetracycline; this demethylated form of tetracycline is the major
commercial form of [Link] contrast, most improvements in biochemical production
have been due to the stepwise accumulation of so called minor genes. These genes lead to
small increases (or decreases) in the antibiotic or other biochemical production, and selection
may be expected to result in a 10-15% increase in yield. The selected strains are usually
subjected to successive cycles of mutagenesis and selection; after several cycles, a large
increase is yield is likely to be obtained. Mutants of Penicillium chrysogenum were selected
for increased penicillin production; each cycle of selection was preceded by Mutagen
(chemical) treatment and resulted in only small changes in penicillin yield.

Selective Isolation of Mutants - A majority of desirable mutants, especially the 'minor gene'
mutants, showing increased production are isolated by screening a large number of clones
surviving the mutagen treatment; this is called secondary screening. But this approach
requires a large amount of work. Therefore, efforts have increasingly focused on developing
techniques for the isolation of particular classes of mutants, which are likely to be
overproducers.

1. Isolation of auxotrophic mutants is the basis for commercial amino acid production in
Japan from the bacterium Corynebacterium glutamicus. For example, phe- mutants of C
glutamicus accumulate tyrosine.

2. Many analogue-resistant mutants have feed-back insensitive enzymes of the biosynthetic

pathway the analogue of whose product was used for selection of such cells. Such mutants
tend to overproduce the end product of the concerned pathway.

3. Sometimes revertants from nonproducing mutants of a strain are high producers, e.g., one
such reversion mutant of Streptomyces viridifaciens showed over 6-fold increase in
chlortetracycline production over the original strain from which the nonproducing mutant
was obtained.

4. Reversion mutants of appropriate auxotrophs may often be high producers.

5. In some cases, selection for resistance to the antibiotic produced by the organism itself
may lead to increased yields.

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
6. Sometimes, mutants with altered cell membrane permeability show high production of
some metabolites.

7. Mutants have been selected to produce altered metabolites, especially in case of

aminogycoside antibiotics. For example, Pseudomonas aureofaciens produces the
antibiotic pyrrolnitrin; a mutant of this fungus yields 4'- fluoropyrrolnitrin. Mutant
selection has been the most successful approach for strain improvement, but major advances
are being made in the exploitation of other strategies, i.e., recombination and recombinant
DNA technology.

Different Approaches in Utilization of Mutation and Genetic Recombination for Strain

Improvement -

Approach Chief feature Example/Remark

The main approach to strain
A. Mutant Selection :
improvement; produces new
Types
alleles of existing genes
Used in, the initial stages of strain
Occur without any
1. Spontaneous Mutations improvement; also for
treatment with a mutagen
maintenance of improved strains
Induced by chemical Mutagenesis followed by
2. Induced Mutations (mainly) or physical selection; several cycles
mutagens employed
Production of 6-demethyl
Affect the pattern of
3. Major Mutations tetracycline in place of
metabolite production
tetracycline by S. aureofaciens
Small gains in each cycle of
Affect the rate metabolite
4. Minor Mutations selection; substantial
production
improvement after several cycles
B. Mutant Selection
Strategies
Enhanced production of an amino
Defective biosynthesis of a
1. Auxotrophic mutants acid, e.g., phe mutants
biochemical
accumulate tyrosine
Overproduction of metabolites,
2. Analogue resistant Feedback insensitive
e.g., amino acids by C.
mutants enzymes
glutamicus
Some mutants are high producers,
3. Revertants of
e.g., chlortetracycline by S.
nonproducing mutants
viridifaciens
Some are high produces, e.g.
4. Revertants of
chlortetracycline by S.
auxotrophic mutants
viridifaciens
5. Resistance to the Increased production, e.g.,
antibiotic produced by chlortetracycline by S.
the organism itself aureofaciens

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
Produces new combinations of
C. Recombination
existing alleles
Conjugation; fusion of Some bacteria and
1. Sexual reproduction
gametes Actinomycetes; fungi and yeast
Nuclear fusion followed,
2. Heterokaryosis by mitotic recombination Fungi
and mitotic reduction
Protoplasts produced by
Bacteria, Actinomycetes, fungi;
3. Protoplast fusion lytic enzymes fusion by
quite; successful
PEG, recombinant recovery

Mutation

Each time a microbial cell divides there is a small probability of an inheritable change
occurring. A strain exhibiting such a changed characteristic is termed a mutant and the
process giving rise to it, a mutation. The probability of a mutation occurring may be
increased by exposing the culture to a mutagenic agent such as UV light, ionizing radiation,
and various chemicals, for example nitrosoguanidine, nitrous acid and caffeine. Such an
exposure usually involves subjecting the population to a mutagen dose which results in the
death of the vast majority of the cells. The survivors of the mutagen exposure may then
contain some mutants, the vast majority of which will produce lower levels of the desired
product. However, a very small proportion of the survivors may be improved producers.
Thus, it is the task of the industrial geneticist to separate the desirable mutants (the superior
producers) from the very many inferior types. This approach is easier for strains producing
primary metabolites than it is for those producing secondary metabolites, as may be seen
from the following examples.

The synthesis of a primary microbial metabolite (such as an amino acid) is controlled such
that it is only produced at a level required by the organism. The control mechanisms involved
are the inhibition of enzyme activity and the repression of enzyme synthesis by the end
product when it is present in the cell at a sufficient concentration. Thus, these mechanisms
are referred to as feedback control. It is obvious that a good ‘commercial’ mutant should
lack the control systems so that ‘overproduction’ of the end product will result. The
isolation of mutants of Corynebacterium glutamicum capable of producing lysine will be
used to illustrate the approaches which have been adopted to remove the control systems. The
control of lysine synthesis in C. glutamicum which it may be seen that the first enzyme in the
pathway, aspartokinase, is inhibited only when both lysine and threonine are synthesized
above a threshold level. This type of control is referred to as concerted feedback control. A
mutant which could not catalyse the conversion of aspartic semialdehyde into homoserine
would be capable of growth only in a homoserine-supplemented medium and the organism
would be described as a homoserine auxotroph. If such an organism were grown in the
presence of very low concentrations of homoserine the endogenous level of threonine would
not reach the inhibitory level for aspartokinase control and, thus, aspartate would be
converted into lysine which would accumulate in the medium. Thus, a knowledge of the
control of the biosynthetic pathway allows a ‘blueprint’ of the desirable mutant to be
constructed and makes easier the task of designing the procedure to isolate the desired type
from the other survivors of a mutation treatment.

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
The isolation of bacterial auxotrophs may be achieved using the penicillin enrichment
technique developed by Davis. Under normal culture conditions an auxotroph is at a
disadvantage compared with the parental (wild-type) cells. However, penicillin only kills
growing cells and, therefore, if the survivors of a mutation treatment were cultured in a
medium containing penicillin and lacking the growth requirement of the desired mutant only
those cells unable to grow would survive, i.e. the desired auxotrophs. If the cells were
removed from the penicillin broth, washed, and resuspended in a medium containing the
requirement of the desired auxotroph then the resulting culture should be rich in the required
type. Nakayama et used this technique to isolate a homoserine auxotroph of C. glutamicum
which produced 44 g 1- lysine.

An alternative approach to the isolation of mutants which do not produce controlling end
products (i.e. auxotrophs) is to isolate mutants which do not recognize the presence of
controlling compounds. Such mutants may be isolated from the survivors of a mutation
treatment by exploiting their capacity to grow in the presence of certain compounds which
are inhibitory to the parental types. An analogue is a compound which is similar in
structure to another compound and analogues of primary metabolites are frequently
inhibitory to microbial cells. The toxicity of the analogue may be due to any of a
number of possible mechanisms; for example, the analogue may be incorporated into a
macromolecule in place of the natural product, resulting in the production of a defective
compound, or the analogue may act as a competitive inhibitor of an enzyme for which the
natural product is a substrate. Also, the analogue may mimic the control characteristics of the
natural product and inhibit product formation despite the fact that the natural product
concentration is inadequate to support growth. A mutant which is capable of growing in the
presence of an analogue inhibitory to the parent may owe its resistance to any of a number of
mechanisms.

However, if the toxicity were due to the analogue mimicking the control characteristics of the
normal end product, then the resistance may be due to the control system being unable to
recognize the analogue as a control factor. Such analogue-resistant mutants may also not
recognize the natural product and may, therefore, overproduce it. Thus, there is a reasonable
probability that mutants resistant to the inhibitory effects of an analogue may overproduce the
compound to which the analogue is analogous. Sano and S h i im~a~de~ us e of this
approach in attempting to isolate lysine-producing mutants of Brevibacterium Jiavum. The
control of lysine formation in [Link] is the same. Sano and Shiio demonstrated that the
lysine analogue S-(2-aminoethyl)cysteine (AEC) only inhibited growth completely in the
presence of threonine, which suggests that AEC combined with threonine in the concerted
inhibition of aspartokinase and deprived the organism of lysine and methionine. Mutants
were isolated by plating the survivors of a mutation treatment onto agar plates containing
both AEC and threonine. A relatively high proportion of the resulting colonies were lysine
overproducers, the best of which produce more than 30 g 1-’. Thus, a knowledge of the
control systems may assist in the design of procedures for the isolation of mutants
overproducing primary metabolites. The design of procedures for the isolation of mutants
overproducing secondary metabolites has been more difficult owing to the fact that far less
information was available on the control of production and, also, that the end products of
secondary metabolism are not required for growth. Thus, many current industrial strains have
been selected using direct, empirical, screens of the survivors of a mutation treatment for
productivity rather than cultural systems which give an advantage to potential superior
producers.

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
Small volumes of liquid media in tubes or microtitre plates have been used coupled with the
use of robots to automate the process. However, as more information has accumulated on the
biosynthesis and control of secondary metabolites, directed selection approaches have also
been used, thus reducing the empirical nature of the screens. Mutants capable of producing
increased levels of secondary metabolite precursors have been isolated by techniques similar
to those used for the improvement of primary metabolite producers. For example, analogue-
resistant mutants have been isolated giving improved yields of pyrrolnitrin, candicidin and
~ephamycin.R~e lief of carbon repression has been achieved on of mutants resistant to 2-
deoxyglucose, a glucose analogue. \

Recombination

Recombination as any process which helps to generate new combinations of genes that were
originally present in different individuals. Compared with the use of mutation techniques for
the improvement of industrial strains the use of recombination was fairly limited in the early
years of improvement programmes. However, techniques are now widely available which
allow the use of recombination as a system of strain improvement. In vivo recombination may
be achieved in the asexual fungi (e.g. Penicillium chrysogenum, used for the commercial
production of penicillin) using the parasexual cycle. The technique of protoplast fusion has
increased greatly the prospects of combining together characteristics found in different
production strains. Protoplasts are cells devoid of their cell walls and may be prepared by
subjecting cells to the action of wall-degrading enzymes in isotonic solutions. Cell fusion,
followed by nuclear fusion, may occur between protoplasts of strains which would not
otherwise fuse and the resulting fused protoplast may regenerate a cell wall and grow as a
normal cell.

Protoplast fusion

Protoplast fusion has been achieved using filamentous fungi, yeasts, Streptomycetes and
bacteria. For example, rate of glucose consumption (and therefore lysine production) of a
high lysine producing strain of B. jlavum by fusing it with another B. jlavum strain which was
a non-lysine producer but consumed glucose at a high rate. Among the fusants one strain
exhibited high lysine production with rapid glucose utilization. Chang et al. used protoplast
fusion to combine the desirable properties of two strains of P. chrysogenum producing
penicillin V into one producer strain. Lein3 has described the use of protoplast fusion for the
improvement of penicillin production in the procedures used by Panlabs Inc. and DeWitt et
al. reviewed the technique for the improvement of actinomycete processes.

T'he manipulated organism was capable of more efficient ammonia metabolism which
resulted in a 5% improvement in carbon conversion. However, the strain was not used on the
large-scale plant due to problems of scale-up. In vitro DNA technology has been used to
amplify the number of copies in a critical pathway gene (or operon) in a process organism.
Although gene amplification is not an example of recombination it is best considered in the
context of DNA manipulative techniques. Threonine production by E. coli has been improved
by incorporating the entire threonine operon of a threonine analogue-resistant mutant into a
plasmid which was then introduced back into the bacterium. The plasmid copy number in the
cell was approximately 20 and the activity of the threonine operon enzymes was increased 40
to 50 times. The organism produced 30 g 1-' threonine compared with the 2-3 g 1-' ofthe non-
manipulated strain sim~ilar techniques in constructing an E. coli strain capable of
synthesizing 65 g 1- threonine.

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
The application of the techniques of genetic manipulation to the improvement of C.
glutamicum was hindered by the availability of a suitable vector. However, vectors have
been constructed and considerable progress has been made in the improvement of amino acid
fermentation. Threonine, histidine and phenylalanine production have been improved using
gene amplification techniques. In these examples the cloned genes were mutant forms which
were resistant to feedback control and had been obtained using the conventional
mutagenesislscreening [Link], the in vitro DNA techniques have built upon the
achievements of conventional strain improvement.

Phenylalanine has become a very important fermentation product because it is a precursor in

the manufacture of the sweetener, aspartame. Backman et ~ [Link]~ed ~the r ationale used
in the construction of an E. coli strain capable of synthesizing commercial levels of
phenylalanine. Escherichia coli was chosen as the producer because of its rapid growth, the
availability of DNA manipulative techniques, and the extensive genetic database. Several of
the phenylalanine genes are subject to control by the repressor protein of the tyr R gene. In
vitro techniques were used to generate tyr R mutations and introduce them into the
production strain. The promoter of the phe A gene, was replaced to remove repression and
attenuation control. As an alternative to the traditional technique of generating a tyrosine
auxotroph an excision vector carrying the tyr A gene was incorporated into the chromosome.
The vector is excised from the chromosome at a slightly increased temperature. Thus,
auxotrophy may be induced during the fermentation by careful temperature manipulation,
thus allowing tyrosine limitation to be imposed after the growth phase and at the beginning of
the production phase. However, the final step in the genetic manipulation of the organism
was the traditional step of isolating an analogue resistant mutant to relieve the feedback
inhibition of DAHP synthase by phenylalanine.

The application of in vitro recombinant DNA technology to the improvement of secondary

metabolite formation is not as developed as it is in the primary metabolite field. However,
considerable advances have been made in the genetic manipulation of the streptomycetes and
the filamentous fungi and a number of different strategies have been devised for cloning
secondary metabolite genes. The first such genes which were cloned were those coding for
resistance of the producer organism to its own antibiotic synthesizing pathways have now
been cloned and the genes for antibiotic biosynthesis have been shown to be clustered
together on the chromosome in both prokaryotes and the application of recombinant DNA
technology to the development of improved strains for the production of tylosin,
pristinamycin and daptomycin and the discovery of several global and pathway specific
secondary metabolism regulatory genes has opened the way to a new means of increasing
yields.

The yield of products will be much less when naturally available microorganisms are used for
fermentation in optimum growth medium. Providing optimum growth conditions increases
the yield only marginally. To increase the productivity of the microorganisms it is necessary
to modify their genetic structure since it is genomes that determine the productivity of
organisms. The culture medium and nutritional requirements also change slightly when the
genetic structure of the microorganism is changed and hence they are also modified
according to the new requirements to ensure maximum product yield. Genetic change of the
microorganism can be done by inducing mutations in the microorganisms, recombinant
technology, and selecting natural variants.

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 DEPARTMENT OF MICROBIOLOGY
KARPAGAM ACADEMY OF HIGHER EDUCATION
(Deemed to be University Established Under Section 3 of UGC Act, 1956)
Eachanari Post, COIMBATORE - 641 021, INDIA

[Link]., Microbiology (Batch 2016-2019)

Industrial Microbiology (Semester-III) (16MBU303)
LECTURE PLAN

UNIT II

Duration Topic Reference

01 Preservation and maintenance of industrial strains. R1:244-293
02 Preservation and maintenance of industrial strains.
03 Crude and synthetic media R2:315-317
04 Molasses
05 Corn- steep liquor
06 Sulphite waste liquor
07 Yeast extract R3:43- 53
08 Protein hydrolysates, Whey
09 Unit revision and possible questions.

Total hours: 9

R1: Peppler and Pearlman, 1979. Microbial Biotechnology. Academic Press.

R2:Kalaichelvan and Arulpandi, 2009. Bioprocess technology,MJP Publishers.
Umesh Kumar, 2014. Industrial Microbiology, MJP Publishers.
R3: Patel A. H., 2003. Industrial Microbiology. Macmillan India ltd, NewDelhi.

Dr [Link]
Assistant Professor
Department of Microbiology
 2017

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
Unit -II
Preservation of Microorganisms
There are different methods for microbial preservation. Suitable methods are selected
based on:
type of microorganism,
effect of the preservation method on the viability of the microorganism,
frequency at which the cultures are withdrawn,
size of the microbial population to be preserved,
availability of resources, and
cost of the preservation method.
All practising microbiologists have felt the need to preserve the viability of micro-organisms
with
which they work. In addition, all the cultural characteristics of a culture, as they were at the
time of
preservation, must be conserved. The nature of work being done will determine whether the
preservation requirement is only very short-term (e.g. a few days) or for an unlimited time
period (e.g. many years). Long-term preservation is required if a culture is to be deposited in
one of the service culture-collections with a view to preserving something of scientific value
"for perpetuity".
Many methods of preservation for micro-organisms have been developed. Here, it is to be
noted that
there exist different types of micro-organisms (bacteria viruses, algae, protozoa, yeasts and
moulds).
Therefore, there are two criteria for selecting a method of preservation for a given culture.
They are:
(i) The period of preservation desired, and
(ii) The nature of a culture to be preserved.
With the increasing importance of micro-organisms to industry (e.g. in biochemical and
antibiotic
production, bio-assay, as spoilage microbes, and the like), human, animal pathologists,
geneticists, taxonomists and teachers have felt the need for culture collections. There are
several large public service collections. These serve as repositories for cultures and as sources
of their distribution.
The best known of these are the Central Bureau voor Schimmelcultures (C.B.S.), founded
in 1906, the American Type Culture Collection (A.T.C.C.), founded in 1925 and the
collection of the Common wealth Mycological Institute (C.M.I.), founded in 1947. Several
other countries are developing their own national collections, and there are large collections
belonging to industrial concerns as well as specialized government departments.
However, any biologist dealing with living material must at least temporarily maintain his
own
cultures during the course of his studies and preserve them until they are ready for depositing
them
in one of these major collections. This depositing of important strains is most desirable as in
the past,
many organisms which have been the subject of intensive investigation, have been discarded
at the
end of the work or on the death of the biologist. Thus, much valuable material has been lost.
There are three basic aims in maintaining and preserving the micro-organisms. They are:
(i) to keep cultures alive

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
(ii) uncontaminated, and
(iii) as healthy as possible, both physically and physiologically, preserving their
originalproperties
until they are deposited in any major collections (i.e. unchanged in their properties). For very
long-term preservation, involving stocks of the strains (as opposed to single specimen of each
strain) and where withdrawals from stocks are regularly made, a fourth aim is to have
adequate stocks and appropriate systems for replenishing stocks when necessary.
iv) the concern of service culture collections, of course, but the
other three are the concern of any maintenance and preservation programme.
The running of the collection and the methods of maintenance used are designed to
minimize the following hazards to which cultures are exposed:
(i) By repated culturing, selection can occur, either of a mutant strain or of a purely
vegetative non-sporulating form. The transfer should, therefore, be done as far as possible by
an expert
with an eye for the wild strain. However, the fewer transfers made, the less is the risk.
(ii) Some strains, sometimes, tend to become attenuated under the artificial conditions of
culture. Others deteriorate to wet slimy disintegrated mycelium or spores. Simmons (1963)
suggests that this may be due to virus infections and there is considerable evidence to support
his theory.
(iii) The maintenance processes to which the micro-organism (e.g. the fungus) is subjected
are selective, and only adaptive strains survive. These may have somewhat 'a typical
characteristic.
(iv) Cultures are subject to contamination, infection with mites and adverse conditions of
temperature, light, humidity, etc., are responsible for their contamination. The latter may arise
through breakdown of apparatus, or by incomplete understanding of the organism.
(v) Adequate documentation of the strains must be made. In a culture collection of long
standing the strains may well survive several generations of microbiologists, so to assist in
maintaining them in their original condition a clear description of the cultural characteristics
supported by dried cultures should be provided at the time of depositing them.
Serial Subculture
This is the simplest and most common method of maintaining microbial cultures. Microbes
are
grown on slants and are transferred to fresh media before they exhaust all the nutrients or dry
out.
An exception to this is aerobic Streptomyces spp. where drying-up of the medium has been
found
successful, provided the initial growth showed the production of aerial hyphae. The drying-up
of the
medium appeared to encourage good sporulation and the preserved specimen became simply
a dried
out strand of agar coated with spores which remained viable for a few years at room
temperature. For
some microbial cultures, no other methods have been found satisfactory, but for the majority
of species other methods are available.
There are several factors to be borne in mind while choosing a suitable medium. Solid
media should be chosen in preference to liquid media, as growth of a contaminant can be
more readily observed. However, bacteriophages are often successfully maintained as
suspensions in liquid [Link], anaerobes, especially Clostridium spp., are frequently
maintained in a liquid medium ([Link]'s cooked meat). Some technicians prefer stab
cultures for maintenance. But there do not appear to be any published data to show these to

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
be any better than slope cultures. Obviously, if the micro-organism is oxygen sensitive, a stab
culture would be suggested as an extra safeguard while handlingon the bench. While a rich
medium may give the best initial growth for heterotrophs, it may also run the risk of
accumulating toxic end-products of metabolism. Therefore, the best medium for growth may
not necessarily be the best for maintenance and preservation of micro-organisms.
Substratum, other factors affect the growth of cultures for storage. They are: light
intensity, temperature, humidity, standard growth conditions, method of transfer, culture
vessel and storage.
The time period appropriate for subculture may range from a week to even a few years
Under normal conditions cultures have to be re-grown at fairly frequent intervals (e.g. every
four, six or eight months). With a large collection, this requires much labour. Moreover, there
is a risk of occurring hazards as discussed previously, every time a culture is handled. To cut
down the frequency of handling of the cultures, it is, therefore desirable to prolong the
intervals between subculturing. There are various means to accomplish this (e.g. cold storage
and mineral oil storage).

Preservation by overlaying cultures with oil

This method of preservation is a modification of serial subculture technique. It was first
extensively used by Buell and Weston (1947). Of 2000 fungus strains maintained under oil
for 10 years at the C.M.I., only forty-five were lost.
This method is cheap and easy, since it does not require special skills or apparatus such as a
centri
fuge, dessiccator, or vacuum pump. The steps involved in this method are:
(i) First of all, inoculation of the agar slant contained in a screw-cap tube with a given culture
is practised.
(ii) Inoculated agar slant is subjected to incubation until good growth appears.
(iii) Using sterial technique, a healthy agar slant culture (from above step) is covered with
sterile mineral oil to a depth of about I cm. above the top of the agar slant. If a short slant of
agar is used, less oil is required.
(iv) Finally, oiled culture from step (iii) can be stored at room temperature. But better
viability is obtained when stored at lower temperatures.
The oil used should be of good quality. British pharmacopoeia medicinal paraffin
oil of specific gravity 0·865 to 0·890 is quite satisfactory. Sterilization 0f oil at the C.M.1.
is done in McCartney bottles for 15 minutes at 15Ib/in2• However, Fennel (1960) insists that
the oil be autoclaved at lSlb/in2• for 2 hours and then dried in the oven at 170°C. for 1 to 2
hours. Simmons (1963) also stresses the need for high quality oil, initial sterility and dryness.
The covering of the culture with oil prevents drying out. The oil allows slow diffusion of
gases so growth continues at a reduced rate. This may induce change due to adaption to
growth in oil. Some fungus isolates appear stable and survivals of over 20 years have been
obtained at the C.M.1. Others change rapidly, producing a typical culture in a few months
(e.g. Fusarium species). If the McCartney bottles
are used the rubber liners should be removed from the metal caps as the oil tends to dissolve
the
rubber and this can be toxic to the cultures.
This method has the following advantages:
(i) Practically all bacterial species or strains tested live longer under oil than in the control
tubes
without oil. Some bacterial species have been preserved satisfactorily for 15 to 20 years.
(ii) Transplants may be prepared when desired without affecting the preservation of the stock
cultures.

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
(iii) The method is especially advantageous when working with unstable variants where
occasional
transfers to fresh media or growth in mass cultures results in changes in the developmental
stages of the strains.
iv)This method also appears to be an ideal method of storage for a busy laboratory with
limited funds
and a relatively small collection. Many workers have reported their experiences with the oiled
fungus
culture maintenance method (Sherf, 1943; Norris, 1944; Wemham, 1946; Buell and Weston,
1947;
and others).
Lyophilization or freeze-drying
Lyophilization is the most satisfactory method of long-term preservation of micro-organisms.
It
is universally used for the preservation of bacteria, viruses, fungi, sera, toxins, enzymes and
other
biological materials. While, it offers a convenient technique for preserving a large number of
cultures,
it is by no means the perfect method for storing yeasts with completely unchanged
characteristics. The process of lyophilization was first applied to microfungi on a large scale
by Raper and Alexander. They were successful in processing the cultures at the N.R.R.L.
(Northern Regional Research
Laboratory) at Peoria and presented their work in 1945. Lyophilization is perhaps the most
popular form of suspended metabolism. It consists of drying cultures or a spore suspension
from the frozen state under reduced pressure.
This can be accomplished in several ways.
There are various kinds of equipment available to do this. Major steps involved in this
technique
are:
(i) A cell or spore suspension is prepared in a suitable protective medium (at the
Commonwealth Mycological Institute 10 % skimmed milk and 5 % inositol in distilled water
is found suitable).
(ii) Using a sterile technique, the suspension from (i) is distributed in small quantities into
glass ampoules.
(iii) The ampoules are connected with a high vacuum system usually incorporating a
desiccant
(e.g. phosphorous pentoxide, silica gel or a freezing trap), and immersed into a freezing
mixture of dry
ice and alcohol (-78°C.).
(iv) The vacuum pump is turned on and the ampoules are evacuated till drying is complete,
after which
they may be sealed off.
The details of the methods used vary from one laboratory to another. Relatively simple
apparatus can be constructed for processing a few ampoules, though quite complex machines
are available for larger scale work. At the C.M.1. a centrifugal two-stage freeze dryer is used.
In this the cooling is by evaporation and no freezing mixture is required. After drying, the
small vials can be filled with sterile dry nitrogen instead of sealing under vacuum. Dewald
(1966) stressed the importance of the elimination of air and moisture from lyophilized
cultures prior to sealing of the ampoules. Similar results were reported by Nei et al. (1966).

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
The methods of revival vary from one laboratory to the other. In case of fungi, dry pellets
may be transferred to a suitable liquid and allowed to dissolve before it is streaked out on
agar. At the C.M.I.,a volume of sterile water equal to the original volume of the spore
suspension is placed in the ampoule at room temperature. Then, the ampoule is left for about
20 to 30 minutes for the water to be absorbed slowly before streaking out. This delay seems
to produce more satisfactory cultures. The degree of viability is assessed visually or by spore
counts.
Factors affecting the viability of freeze-dried cultures include:
(i) Chemical composition of the protective (suspending) medium;
(ii) Addition of certain compounds to the culture suspension before freeze-drying to give
protection to the
culture against the toxicity exerted by moisture and oxygen when stored in unsealed
ampoules;
(iii) Sealing the ampoules after freeze-drying to stop access of oxygen and moisture;
(iv) Insufficient elimination of oxygen and moisture on the survival rate of freeze-dried
culture;
(v) Storage temperature of freeze-dried and sealed cultures.
This method possesses the following advantages:
(i) As the ampoules are sealed there is no risk of contamination or infection with
mites.
(ii) The prepared ampoules are easily stored, they are not readily broken and most
species
(iii) There is less opportunity for cultures to undergo changes in characteristics (i.e.
they remain unchanged during storage period).
(iv) Owing to the small size of glass ampoules, hundreds of lyophilized cultures can be stored
in a small storage space. In addition to this, the ampoules' small size makes them ideal for
postage. Its remain viable for many years (more than 20 years in case of many bacterial
species).
(v) Lyophilization cuts down the number of transfers.
Checking of viability may be done at long intervals. This may be done when cultures are
required or by routine sampling. Usually, many replicates are made so that the material from
a constant source can be supplied over a considerable period. It makes the 'Seed Stock'
system possible, which is used at the American Type Culture Collection (Clark and
Loegering, 1967) for conserving living reference microbes over long periods. Clark and
Loegering describe this method as follows:
In this system the ampoules of freeze-dried or frozen culture material from original
stocks
are set aside as seed stock, and stored under optimum temperature conditions. This material is
never distributed. Periodically an ampoule of seed stock is opened, and the culture grown
from it re-frozen or re-Iyophilized in sufficient quantity for distribution, It is also used to
prepare new seed stock if the viability of original material declines or the original seed stock
becomes nearly depleted. In this way original material can be conserved over a long period'.

Storage at very low temperatures or Nitrogen Storage

This method is also called cryogenic storage. It is like lyophilization, a satisfactory method
for the long-term preservation of micro-organisms. It has also been successful with many
specimens which can be preserved by lyophilization. The maintenance of microbes is done by
suspended metabolism. According to Meryman (1956), life is regarded as 'at a standstill' at -
130°C. and below, so at the temperature of liquid nitrogen (--196°C.), provided the cultures
survive the treatment, the period of preservation should be indefinite. Thus, long-term

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
preservation (storage) without any change in the cultural characteristics is now attainable.
Hwang protects her material with 10 % glycerol and cools it slowly at the rate of about 1Cper
minute to a temperature of -3SOC. Thereafter, further cooling of the temperature from -
165°C. to -196°C. was uncontrolled and faster. The procedure and apparatus are fully
described (Hwang, 1966). She is able to preserve mycelial forms, Sapro/egniaceae,
Pythiaceae, Entomophthoraceae and other fungi which would not survive the vigorous
process of lyophilization as well as the more resistant moulds.
Major steps involved in the performance of this method are:
i) The culture is suspended as a cell or a spore suspension, as finely broken-up particles of
mycelium,
or as a piece of fungus mycelium in a suitable suspending medium (10 % glycerol is used at
the
Commonwealth Mycological Institute). As dimethyl sulfoxide is less pleasant to handle, it
would
appear most satisfactory to use glycerol as a suspending medium as a routine practice and
dimethyl
sulfoxide should-only be used when revivals are poor.
(ii) The suspension as prepared in step (i) is distributed into ampoules (These must be
resistant to
cold-shock). At the A.T.C.C. 1.2 ml. Wheaton 'Goldband' prescored borosilicate glass
ampoules have
been found satisfactory. The manufacturers of ampoules warn us that they must not be
overfilled (less
than 0·5 ml, being recommended).
(iii) Ampoules filled with a culture suspension are frozen and are hermetically sealed. Hwang
(1966) recommends precooling to T'C. before freezing begins. Freezing can be attained by
plunging the ampoules straight into the liquid nitrogen by suspending them over the liquid
nitrogen for a short period and then lowering them into the liquid nitrogen, or by controlled
cooling. There are many interacting factors concerning the choice of the method of freezing
and it will depend largely on the micro-organisms to be frozen, the degree of revival required
and the apparatus available.
(iv) The frozen ampoules prepared as in (iii) are usually clipped on metal (aluminium) canes,
one above the other and six to each cane. The canes, in turn, are packed in metal boxes or
canisters (aluminium), which hold about 20 canes. These are perforated to allow the free
running of the liquid nitrogen. The cultures are revived by removing from the container,
rapidly thawing and culturing them in the usual way.
Precautions: Cracked or faulty ampoules are dangerous. The reason is that the liquid
nitrogen
may penetrate and fill the ampoules. At the time of thawing, such ampoules (filled with the
liquid
nitrogen) may explode due to the sudden expansion of the nitrogen into gas. The storage
nitrogen may also become infected. If the thawing is done in water and if an explosion
occurs, the water will tend to hold the fragments of glass. But it is recommended that gloves
and eye or face shield be worn when thawing out ampoules. The nitrogen itself should be
handled carefully because of the risk of cold bums. Great care should be taken while handling
any metal parts in direct contact with the liquid nitrogen. There is a small risk of asphyxiation
while handling it in a confined space, since the gas is odourless and colour less. Hence, -the
presence of nitrogen in the atmosphere may not be noticed. Therefore, the nitrogen containers
should be housed in a well-ventilated room.
The method has the following advantages:

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
(i) It is an effective method of preservation.
(ii) No subculturing is required.
(iii) The cultural characteristics remain unchanged. This could be of particular importance
while
storing highly specialized strains employed in industrial processes.
(iv) The ampoules are not open to contamination or infection by mites, since they are sealed.
(v) The living material of a type which would not normally grow in a culture and would not
be preserved in a culture collection can be retained in a viable state.
The method has also some disadvantages. They are:
(i) The method is expensive, since a costly apparatus is required.
(ii) A reliable supply of nitrogen is needed.
(iii) There is a possibility of a minor accident, since the method involves the use of explosive
gas (the
liquid nitrogen).
A recent short review of current research has been made by CaIcott (1978).

Other Methods or Storage for Fungi

Every laboratory cannot afford lyophilization or liquid nitrogen storage. Many technicians
lack
the time for the periodic transfer to fresh media and they would find oil storage messy.
Indeed, in
these days, when the safety of the personnel is the first consideration, the spatter made by oily
needles when they are sterilized is undesirable. Therefore, various other methods of
preservations nave been developed.
Soil cultures: This method is particularly applied for the preservation of sporing microbes
(e.g.
Bacillus, Penicillium, Aspergillus and Streptomyces).
Steps involved in this method are listed. as under:
(i) A spore suspension is first prepared which may involve the use of a special medium.
(ii) A mixture of soil (20 %), sand (78 %) and calcium carbonate (2 %) is prepared and
distributed into
tubes (a few grams per tube). They are sterilized for 8 to 15 hours at 130°[Link] then cooled.
(iii) A spore suspension is added to the sterilized loam as prepared in step (ii) and allowed to
grow for about 10 days.
(iv) The inoculated tubes as in step (iii) are kept in a desiccator under vacuum. The reason
behind this is to evaporate the excess water. Then the tubes are sealed.
(v) The culture tubes are stored in a refrigerator at about 5 to 8°C. temperature.
Desiccation
This involves removal of water from the culture. Desiccation is used to preserve
actinomycetes (a form of fungi-like bacteria) for very long period of time. The
microorganisms can be preserved by desiccating on sand, silica gel, or paper strips.

Culture Media - Inoculum preparation media are quite different from production media.
These media are designed for rapid microbial growth, and little or no product accumulation
will normally occur. Many production processes depend on inducible enzymes. In all such
cases, the appropriate inducers must be included either in all the stages or at least in the final
stages of inoculum development. This will ensure the presence of the concerned inducible
enzymes at high levels for the production to start immediately after inoculation. The

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
inoculum used for production tanks must be contamination free. But the risk of contamination
is always present during inoculum development. Therefore, every effort must be made to
detect as well as prevent contamination.

Bioreactor Media - The medium composition is as critical to product yields as high

producing strains of microorganisms. The medium not only provides the nutrients needed for
microbial growth but also for the metabolite [Link] organisms vary greatly in their
nutrient requirements from autotrophs, which produce all the biochemicals required from
simple inorganic nutrients deriving their energy from oxidation of some inorganic component
of the medium to the difficult organisms like lactic acid bacteria, which require many organic
compounds for their growth.

The various media may be grouped into two broad categories:

(i) synthetic and
(ii) complex.

A synthetic or chemically defined medium is desirable for various studies, but product
yields from such media are generally low. Foaming is not a problem with such media. The
complex media contain undefined constituents like soybean meal, molasses, corn steep
liquor, etc., and give much higher yields of metabolites. Carbon source can be simple, e.g.,
sugar, alcohol, etc., or complex carbohydrates, proteins, molasses, potatoes, sweet potatoes,
etc. In many processes, precursors need to be provided, e.g., phenylacetic acid for penicillin
G, inorganic cobalt for vit. B12. Buffers are also added to prevent drastic changes in pH, and
anti foam would often be needed when complex media are used. For much fermentation, e.g.,
antibiotic production, medium suited for rapid cell growth is unsuitable for product
formation. In such cases, specialized media for production have to be devised.

Media for Industrial fermentation process

Fermentation Medium
Most fermentations require liquid media, often referred to as broth, although some solid-
substrate fermentations are operated.

Media formulation

All microbes require water, sources of carbon, mineral elements and probably vitamin and
oxygen if aerobic on a large scale, manufacturers normally use sources of cheap nutrient to
make a medium which will need the following criteria.
(i) It must produce the maximum yield of products or biomass per gram of substrate used.
(ii) It must permit the maximum rate of product formation.
(iii) The yield of undesirable products must be minimal.
(iv) It must be cheap and of consistent quality.
(v) It must cause minimum problems in the production process particularly
other aspects as aeration, purification extraction and waste management.

Medium improvement to what degree

1. Medium designed for the initial production of antibiotic usually does not have to be
developed very skillfully since the potential for antibiotic production is quite low with
wild-type strains.
2. Media for ultra-high antibiotic-producing strains, which have been developed through
repeated genetic manipulations, must be formulated with utmost care.

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
3. In the past, strain improvement and media development were the responsibilities of
different research groups. Today we know that each higher-producing clone, after
mutation and screening, requires a medium optimized for its performance.

The importance of medium improvement

1. Only small to moderate increases in the level of production can result in an actual
reduction in production cost so that it can economically be sold in competition with
others.
2. There is little published literature on the complex substrates that have been developed for
the production of various products. Most fermentation processes, which include the
production of fermentation media, are closely guarded trade secrets.

Different technical objectives of media formulation

1. Inoculum (starter culture) propagation steps / pilot-scale fermentations / main
Production fermentation
2. Biomass or primary metabolites production / secondary metabolite production

Considerations in seed culture media formulation

1. The seed stages are designed to give rapid and reproducible growth without nutrient
depletion, autolysis, or an adverse change in pH.
2. There is less concern with the cost of ingredients in the seed stages since the volume is
usually only 5% of the fermentation volume and excellent uniformity of medium
ingredients is highly desirable.
3. Some specially prepared dairy products have been used quite extensively in primary and
secondary seeds.

Constituents of medium
1. Water
2. Carbon source / Nitrogen source / Sources of phosphorous and sulfur / Minor and trace
elements / Vitamins such as biotin and riboflavin
3. Oxygen: even some anaerobic fermentations require initial aeration, e.g. beer
fermentations
4. Buffers or controlled by acid and alkali additions
5. Antifoam agents
6. Precursor, inducer or inhibitor compounds

Considerations in media design

Nutritional Requirements, Environmental Requirements and Techno-economic Factors.

Nutritional requirements
1. Nutritional requirements include elemental, specific nutrient, and energy requirements
2. Elemental requirements : the stoichiometry for growth and product formation
C-source + N-source + O2 + minerals + specific nutrients → cell mass + product +
CO2 + H2O + heat
3. Specific nutrient requirements:
Auxotroph: To use a complex medium or to identify the specific nutrient

Elemental requirements
1. Main elemental formula of microbial cells C4H7O2N (dry weight basis 48% C, 7% H,
32% O, 14% N), e.g. Baker’s yeast C3.72H6.11O1.95N0.61S0.017P0.035K0.056

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
2. Average: C 45-55%, N 6-14%, K 0.5-2%, P 1-3%, Mg 0.1-1%, S 0.02-1%, minor
minerals (mg /100g cell) Cu 0.1-1, Fe 1-10, Zn ~1, Mn 0-5 (e.g. 10g/L of cell mass
containing 0.4% magnesium will require at least 0.04 g/L of Mg or 0.2 g/L of MgSO4 or
0.4 g/L of MgSO4‧7H2O)
3. Chemical composition of fermentation product
4. Typical concentration of fermentation products in the broth (dry wt / vol, %): lactic acid
(13), citric acid (12), glutamic acid (10), ethanol (8), baker’s yeast (5), benzyl penicillin
(3), riboflavin (1), vitamin B12 (0.002)
Energy requirements
1. dS/dt = μX/Yx/s + mX + qpX/Yp/s
2. Mass synthesis + Maintenance + Product synthesis
3. Growth yield: YS = 0.4~1.0 g cell / g substrate ≈ 1.3 g cell / g C of substrate; YATP ≈
10.5 g cell / mole (可由X mole ATP / g substrate YS)
4. Maintenance: m = 0.01 ~ 0.04 g / g / h; m ≈ 4 m mole /g cell / h (可由X mole ATP / g
substrate m); short-term fermentation，long-term fermentation
5. Product yield: direct stoichiometry and theoretical yield

Environmental requirements
1. Effect of growth temperature on cell yield / below optimal temperature for growth.
2. Effect of water activity (Aw = Ps/Pw) on growth rate, vapor pressure of water in solution
(Ps) or in pure water(Pw).
3. Combined effect of temperature and pH on growth / opt pH for growth and production is
not always the same
4. Environmental effect of substrate.

Techno-economic factors that affect the choice of individual raw materials

1. Cost: transport and storage, e.g. temperature control
2. Availability: consistent quality and year round availability
3. Ease of handling: solid or liquid forms
4. Sterilization: thermal damage and inhibitory byproduct
5. Operational characteristics: formulation, mixing, complexing and viscosity characteristics
that may influence agitation, aeration, foaming and recovery
6. Supply: the concentration of target product attained, its rate of formation and yield per
gram of substrate utilized
7. Purification: levels and range of impurities, potential for generating undesired products
8. Pollution control
9. Health and safety implications

Major Carbon sources

Molasses
1. Byproduct of cane or beet sugar production / residues remaining after most of the sucrose
has been crystallized from the plant extract
2. Dark colored viscous syrup containing 50-60% (w/v) carbohydrate, primarily sucrose,
with 2% (w/v) nitrogenous substances, along with some vitamins and minerals.
3. Overall composition varies depending upon the plant source, the location of the crop, the
climatic conditions under which it was grown, and the factory where it was processed
4. The carbohydrate concentration may be reduced during storage by contaminating
microorganisms
5. Hydrol molasses, containing primarily glucose, is a byproduct of maize starch processing

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
Malt extract
1. Concentrated aqueous extracts of malted barley to form syrups / particularly useful for
the cultivation of filamentous fungi, yeasts and actinomycetes.
2. App. 90% carbohydrate (w/w) and some vitamins and app. 5% nitrogenous substances,
proteins, peptides and amino acids / carbohydrate comprising 20% hexoses (glucose and
small amounts of fructose), 55% disaccharides (maltose and traces of sucrose), 10%
maltotriose, and additionally contain 15-20% branched and unbranched dextrins, which
may or may not be metabolized, depending upon the microorganisms
3. Careful sterilization to prevent over-heating /Maillard reaction products (brown
condensation products resulting from the reaction of amino groups and carbonyl groups)
when heated at low pH / color change, loss of fermentable materials, some toxic products
Starch and dextrins
1. Can be directly metabolized by amylase-producing microorganisms, particularly
filamentous fungi. Maize starch is most widely used.
2. To allow use in a wide range of fermentations, the starch is usually converted into sugar
syrup, containing mostly glucose. It is first gelatinized and then hydrolyzed by dilute
acids or amylolytic enzymes, often microbial glucoamylases that operate at elevated
temperatures.
Sulfite waste liquor
1. Sugar containing wastes derived from the paper pulping industry are primarily used for
the cultivation of yeasts
2. Waste liquors from coniferous trees contain 2-3% (w/v) sugar, 80% hexoses (glucose,
mannose and galactose) and 20% pentoses (mostly xylose and arabinose) / Liquors
derived from deciduous trees contain mainly pentoses
3. Usually the liquor requires processing before use as it contains sulfur dioxide / The low
pH is adjusted with calcium hydroxide or calcium carbonate, and these liquors are
supplemented with sources of nitrogen and phosphorus
Cellulose
1. Predominantly as lignocellulose (composed of cellulose, hemicellulose and lignin)
2. Available from agricultural, forestry, industrial and domestic wastes
3. Relatively few microorganisms can utilize it directly / The cellulose component is in
part crystalline, encrusted with lignin, and provides little surface area for enzyme
attack
4. At present, mainly used in solid-substrate fermentations (e.g. mushrooms)
5. Potentially a very valuable renewable source of fermentable sugars once hydrolyzed,
particularly in the bioconversion to ethanol for fuel use
Whey
1. An aqueous byproduct of the dairy industry / Annual worldwide production is over 80
million tons, containing over 1 million tons of lactose and 0.2 million tons of milk
proteins.
2. Expensive to store and transport / Lactose concentrates are often prepared for later
fermentation by evaporation of the whey, following removal of milk proteins for use
as food supplements.
3. Lactose is less useful than sucrose / e.g. S. cerevisiae does not ferment lactose
4. Formerly used extensively in penicillin fermentation / Still employed for producing
ethanol, single cell protein, lactic acid, xanthan gum, vitamin B12 and gibberellic
acid.
Alkanes and alcohols
1. n-Alkanes (C10-C20): readily metabolized by certain microorganisms / industrial use
is dependent upon the prevailing price of petroleum.

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
2. Methane: utilized by a few microorganism, but its conversion product methanol is
often preferred for industrial fermentations.
3. High purity methanol is readily obtained / completely miscible with water / has a high
per cent carbon content and is relatively cheap / only limited organisms will
metabolize methanol / only low conc., 0.1-1% (v/v) are tolerated by microorganisms /
oxygen demand and heat of fermentation are high, but this is even more problematic
when growing on alkanes.
4. Ethanol is less toxic than methanol / used as a sole or cosubstrate / too expensive for
general use as a carbon source / its biotransformation to acetic acid remains a major
fermentation process.
Fats and oils
1. Hard animal fats (composed mainly of glycerides of palmitic and stearic acids) are
rarely used in fermentation.
2. Plant oils (primarily from cotton seed, linseed, maize, olive, palm, rape seed and soy)
and occasionally fish oil, may be used as the primary or supplementary carbon
source, especially in antibiotic production / Plant oils are mostly composed of oleic
and linoleic acids, but linseed and soy oil also have a substantial amount of linolenic
acid.
3. Oils contain more energy per unit weight than carbohydrates / Oils can be particularly
useful in fed-batch operations than carbohydrates (aqueous solutions less than 50%,
w/v; occupy a greater volume).
Major Nitrogen sources
Corn steep liquor
1. Byproduct of starch extraction from maize / first use in fermentations for penicillin
production in the 1940s
2. Exact composition varies depending on the quality of maize and the processing
conditions / Concentrated extracts generally contain about 4% (w/v) nitrogen,
including a wide range of amino acids, along with vitamins and minerals / Any
residual sugars are usually converted to lactic acid (9-20%, w/v) by contaminating
bacteria
3. Can sometimes be replaced by liquor derived from potato starch production
Yeast extract - 1
1. Produced from waste baker’s and brewer’s yeast, or other strains of S. cerevisiae / Or
Kluyveromyces marxianus (formerly K. fragilis) grown on whey and Candida utilis
cultivated using ethanol, or wastes from wood and paper processing
2. Extracts used in the formulation of fermentation media are normally salt-free
concentrates of soluble components of hydrolyzed yeast cells / Extracts with sodium
chloride concentrations greater than 0.05% (w/v) cannot be used in fermentation
processes due to potential corrosion problems
3. Yeast cell hydrolysis is often achieved by autolysis, which can be initiated by
temperature or osmotic shock, causing cells to die but without inactivating their
endogenous enzymes.
Yeast extract - 2
1. Temperature and pH are controlled throughout an optimal and standardized autolysis
process / Temperature control is particularly important to prevent loss of vitamins
2. Autolysis (50-55oC for several hours before the temperature is raised to 75oC to
inactivate enzymes), plasmolysis or mechanical disruption of cells / filtration or
centrifugation to remove cell wall materials and other debris / rapid concentration
3. Extracts are available as liquids containing 50-65% solids, viscous pastes or dry
powders

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
4. They contain amino acids (35-40%, w/v), peptides (30-45%, w/v), water-soluble
vitamins and some glucose derived from the yeast storage carbohydrates (trehalose
and glycogen)
Peptones
1. Peptones are usually too expensive for large-scale industrial fermentations
2. Prepared by acid or enzyme hydrolysis of high protein materials: meat, casein,
gelatin, keratin, peanuts, soy meal, cotton seed, etc.
3. Amino acids compositions vary depending upon the original protein source / Gelatin-
derived peptones are rich in proline and hydroxyproline, but almost devoid of
sulfur-containing amino acids / Keratin peptone is rich in both proline and cystine, but
lacks lysine
4. Plant peptones invariably contain relatively large quantities of carbohydrates
Soya bean meal
1. Residuals after extraction of soy oil
2. Composed of 50% protein, 7% non-protein nitrogenous compounds, 30%
carbohydrates and 1% oil
3. Often used in antibiotic fermentation because the components are only slowly
metabolized, thereby eliminating the possibility of repression of product formation
Water
1. Use for media, cleaning, cooling ?
2. A reliable source of large quantities of clean water, of consistent composition, is
essential
3. Before use, removal of suspended solids, colloids and microorganisms is usually
required
4. “Hard” water is treated to remove salts such as calcium carbonate
5. Iron and chlorine may also require removal
6. Water is becoming increasingly expensive / recycle / reuse wherever possible /
minimizes water costs and reduces the volume requiring waste-water treatment
Antifoams -1
1. Foaming is largely due to media proteins that become attached to the air-broth
interface where they denature to form a stable foam
2. If foaming is minimized, then throughputs can be increased
3. Three approaches to controlling foam production: modification of medium
composition, use of mechanical foam breakers and addition of chemical antifoams
4. Chemical antifoams are surface-active agents which reduce the surface tension that
binds the foam together
Antifoams -2
1. Ideal antifoam: 1. readily and rapidly dispersed with rapid action; 2. high activity at
low concentration; 3. prolonged action; 4. non-toxic to fermentation microorganisms,
humans or animals; 5. low cost; 6. thermostable; 7. compatibility with other media
components and the process , i.e. having no effect on oxygen transfer rates or
downstream processing operations (e.g. some may adversely affect membrane
filtration)
2. Natural antifoams include plant oils (e.g. from soy, sunflower and rapeseed),
deodorized fish oil, mineral oils.
3. Synthetic antifoams are mostly silicon oils, poly alcohols and alkylated glycols
Special compounds -1
1. Precursors: phenylacetic acid or phenylacetamide as side-chain precursors in
penicillin production / D-threonine in L-isoleucine production by Serratia marsescens
/ anthranillic acid for L-tryptophan production by yeast Hansenula anomala

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
2. Inducers and elicitors: Inducers are often necessary for genetically modified
microorganisms (GMMs) / Production of secondary metabolites, such as flavonoids
and terpenoids, in plant cell culture can be triggered by adding elicitors, which may be
isolated from various microorganism, particularly plant pathogens.
Special compounds -2
1. Inhibitors: 1. Used to redirect metabolism towards the target product and reduce
formation of other metabolic intermediates (e. g. sodium bisulfite in production of
glycerol by S. cerevisiae) 2. Antibiotics for some GMMS containing plasmids bearing
an antibiotic resistance gene
Cell permeability modifiers: e.g. penicillins and surfactants added to amino acid
fermentations, including processes for producing L-glutamic acid by members of the genera
Corynebacterium and Brevibacterium.

Antifoams

Chemicals controlling foams have been classified into antifoams or defoamers.

Defoamers are once they are formed. Most of the media used in culturing organisms contain
protein which is susceptible to foam formation due to the fine bubble which easily induce
foam. The problem of antifoam is widespread in fermentation process and can be
counteracted in a number of ways.

One possibility is to ensure that there:

(1) Is a sufficient space available in the fermenter for the foam produced. However this
reduces the effective volume of the fermenter as well as the additional changes of
contamination. Foaming can also be hindered/counteracted using chemical & mechanical
measures.

(a) Chemical antifoam agents such as animal vegetable oil reduce the surface tension of the
broth and at the same time they reduce the solubility of oxygen which in turn affects the
aeration requirement. It may also make down stream processing more difficult.

(b) Mechanical defoamers can be employed instead of chemical agents. Mechanical elements
mounted on the agitation shaft are that it will affect the speed of rotation for effective
defoaming.

To rectify this disadvantage, some defoamers are separately driven. It has a disadvantage in
that it increases the danger of contamination because of the additional shaft installation
through the fermenter.

Properties of Antifoams

(1) It must be non-toxic to microorganism and higher animals.

(2) It must have no effect on the taste & odour.

(3) It must not serve as a source of contamination.

(4) It must not be metabolized (i.e. organisms)

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
Preparation of sterilized media
Liquid media which are sterilized in their final containers should be cooled down to
room temperature as rapidly as possible. Screw caps should then be tightened. Containers of
agar media which have been sterilized should be placed in a 50°C water bath and the medium
dispensed as soon as it reaches this temperature, or within a maximum of 3 hours in the bath.
The medium should be mixed thoroughly, without bubble formation and aseptically
dispensed into sterile containers. Do not expose dishes of agar media to sunlight; it causes
excessive condensation on the lids and may cause the formation of inhibitory substances by
photo-oxidation.
Heat-labile supplements should be added to the medium after it has cooled to 50°C.
Allow the sterile supplement to come to room temperature before adding it to the agar
medium. Very cold liquids may cause agar to gel or form transparent flakes which can easily
be seen e.g. in blood enriched agar. Mix all supplements into the medium gently and
thoroughly, then distribute into the final containers as quickly as possible.
Blood used for the preparation of blood agar should be as fresh as possible and should
have been stored at 2-8°C (blood must not be frozen). Warm the blood in a 35°C incubator
before addition to sterile molten agar base, which has been cooled to 40-45°C. Adequate
mixing in a large head-space vessel is essential to ensure aeration of the blood. Poorly
oxygenated blood plates are purplish in colour whereas properly aerated blood agar is cherry-
red. Defibrinated blood is recommended for use rather than blood containing an
anticoagulant.

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 DEPARTMENT OF MICROBIOLOGY
KARPAGAM ACADEMY OF HIGHER EDUCATION
(Deemed to be University Established Under Section 3 of UGC Act, 1956)
Eachanari Post, COIMBATORE - 641 021, INDIA

[Link]., Microbiology (Batch 2016-2019)

Industrial Microbiology (Semester-III) (16MBU303)
LECTURE PLAN

UNIT III

Duration Topic Reference

01 Types of fermentation processes T1:43-57
02 Batch, fed-batch fermentations T1:56-69
03 Continuous fermentations T1:97-104
04 Solid-state and liquid-state fermentations
05 Components of a typical bio-reactor, T2:188-215
06 Types of bioreactors – Laboratory, pilot- scale and production R1:765-773
fermenters,
07 Constantly stirred tank and air-lift fermenters.
08 Measurement and control of fermentation parameters- pH, R2:223-225
temperature 375-395
09 Measurement and control of fermentation parameters-dissolved
oxygen, foaming and aeration.
10 Unit revision and possible questions
Total hours: 10

T1: Kalaichelvan and Arulpandi, 2009. Bioprocess technology,MJP Publishers.

T2:Umesh kumar, 2014. Industrial Microbiology, SBW Publishers.
R1: Doran, 2013. Bioprocess Enggineering Principles. Academic press,
R2: Peppler and Pearlman, 1979. Microbial Biotechnology. Academic Press.

Dr [Link]
Assistant Professor
Department of Microbiology
 2017

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
Unit-III

Fermentation

The term “fermentation” derives from the Latin work fevere meaning “to ferment.”
Fermentation is an ancient process dating back thousands of years. It was the means by which
bread, wine, beer, and cheese were made. Egyptians found that uncooked dough left standing
became lighter and softer. They and the Romans discovered that yeast produced lighter and
leavened bread. Around 4000 BC wine was made from grape juice through a fermentation
process. Beer making by the ancients came about by the soaking of barley in water, probably
a serendipitous by-product of bread making.

The Chinese used micro-organisms in the production of yogurt, cheese, wine, vinegar, and
different types of sauces. Cheese was made by storing milk in animal skins or bladders made
from animal stomachs. The bacteria and enzymes present in these containers would cause
separation of casein (milk protein) to form curd. Fermented rice, vegetables, and fruits were
extensively used by Ecuadorians.

Not until the work of Louis Pasteur in the late 19th century was it understood how the process,
which to that point was based on experience and tacit knowledge, actually worked. Antony
van Leeuwenhoek, a Dutch biologist in 1680 was the first to see micro-organisms in samples
of fermenting beer through a microscope. Pasteur, a French chemist, discovered that yeasts
convert sugars to alcohol and carbon dioxide during fermentation.

Fermentation is the process by which alcoholic beverages or acidic diary products (cheeses,
yogurt) are manufactured. It is a way for a cell to obtain energy without using oxygen. During
the process, complex organic substances are broken down into simpler ones. The cell
(microbial or animal) obtains energy through glycolysis– the splitting of a sugar molecule to
extract its electrons. The by-product of this process is excreted from the cell in the form of
substances such as alcohol, lactic acid, and acetone. With advances in the science of
microbiology and technologies like biotechnology, micro-organisms are exploited to produce
a wide variety of products using fermentation. These include:

• Dairy products– Cheese, yogurt

• Beverages– Beer, wine

• Single Cell Proteins (SCP) – SCP are a cell monoculture of bacteria, fungi, and algae. Since
the cells contain large amounts of protein, SCP is used as food or food supplement for
humans and cattle. It is regarded as a cheap source of dietary protein and is produced from
methanol and by-products of cheese production and paper making.

• Antibiotics– Antibiotics are one of the most important compounds produced by

fermentation. Alexander Fleming in 1929 was the first to discover “penicillin”, an antibiotic.
Large numbers of antibiotics are being produced now by fermentation using various bacteria
and fungi.

Chemicals– Citric and acetic acid, amino acids, enzymes, vitamins

• Fuels– Ethanol, methanol, methane

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21
 2017
Procedure of Fermentation:

(a) Depending upon the type of product required, a particular bioreactor is selected.

(b) A suitable substrate in liquid media is added at a specific temperature, pH and then
diluted.

(c) The organism (microbe, animal/plant cell, sub-cellular organelle or enzyme) is added to it.

(d) Then it is incubated at a specific temperature for the specified time.

(e) The incubation may either be aerobic or anaerobic.

i. Aerobic conditions are created by bubbling oxygen through the medium.

ii. Anaerobic conditions are created by using closed vessels, wherein oxygen cannot diffuse
into the media and the oxygen present just above is replaced by carbon dioxide released.

(f) After the specified time interval, the products are removed, as some of the products are
toxic to the growing cell or at least inhibitory to their growth. The organisms are re-
circulated. The process of removal of the products is called downstream processing.

End-products Obtained by Fermentation

Types of end products of fermentation include:

• Microbial cells (e.g. bacteria, yeast, fungal spores)

• Microbial enzymes (e.g. milk clotting enzymes or rennets, recombinant fungal and bacterial
rennets for cheese manufacture)

• Microbial metabolites (e.g. alcohols– ethanol, butanol, 2, 3-butanediol, isopropanol;

chemicals– lactate, propionate, proteins, vitamins, antibiotics; and fuels– methane)

• Recombinant products (e.g. hormones).

Submerged fermentation

Since microbial enzymes are mostly low volume, medium cost products, the
production methods using submerged liquid systems have generally relied on bioreactors
similar in design and function to those used in antibiotic production processes. Sterilization
and process control are easier. A typical enzyme- producing bioreactor is constructed from
stainless steel and has a capacity of 10-50 m3. Cultivation involves the suspension growth in
liquid medium. The reactor includes system for mixing by mechanical internal agitator.
External pumps, bubble columns, and air lift loop systems. The media prepared, after
adjusting to required pH value, is pumped to previously steam sterilized Fermenter vessel
through HTST sterilization units. Thermo labile additives are added separately to the sterile
medium. Inoculums build up before transfer to the production stage vessels. Sterile
compressed air is introduced into the fermentation broth through a sparger system of a ring

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
type or a single or multiple or inlet type. Agitation is by multiple, flat bladed disc turbines
mounted on a vertical shaft. Other types include axial flow propellers or counter current
stirrers. Temperature of the Fermenter during the process is maintained by using internal
cooling coils or external heat exchange surface. In situ probes are instruments are used to
monitor and control various physical parameters during inoculum development and
production stages. Analyses applied to culture samples include the measurement of pH value,
dissolved oxygen, CO2, redox potential, etc.

Batch Culture Fermentation

This type of fermentation is also called a closed culture system because nutrients and other
components are added in specific amounts at the start of the process and are not replenished
once the fermentation has started. At the end of the process the product is recovered; then, the
fermenter is cleaned, sterilized, and used for another batch process. In the initial stages
microorganisms grow at a rapid rate in the presence of excess nutrients but as they multiply
in large numbers they use up the nutrients. They also produce toxic metabolites which retard
further growth of microorganisms during the later stages of the fermentation process.

Fed-batch Culture

In this process the nutrients and substrates are added at the start of the process and at regular
intervals after the start. This is called controlled feeding. Inoculum is added to the
fermentation vessel when microorganisms are in exponential growth phase. Fed-batch culture
is controlled by feed-back control and control without feed-back.

1. Feed-back control– The fermentation process is controlled by monitoring process

parameters like dissolved oxygen content, carbon dioxide to oxygen ratio, pH, concentration
of substrate, and concentration of the product.

2. Control without feed-back– The substrates and nutrients are added at regular intervals.

Fed-batch culture requires special equipment such as a reservoir which holds the nutrients,
pH modifiers so that they can be added to the fermenter at regular intervals, and pumps to
deliver culture medium aseptically to the fermenter.

Continuous Fermentation

This method prolongs the exponential growth phase of microbial growth as nutrients are
continually supplied and metabolites and other wastes are continually removed thus
promoting continual growth of the microorganisms. Continuous culture fermentation is
advantageous because of its high productivity. Two control methods are used in continuous
culture fermentation, namely, chemostat and turbidostat.

Chemostat

This medium contains excess of all but one of the nutrients which determine the rate of
growth of the microorganism. At steady state of the chemostat the rate of input of medium
into the fermenter is equal to the rate of output out of the fermenter.

Turbidostat
Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21
 2017
This medium contains excess of all nutrients so the microbial growth is at its maximum
specific growth rate. The system consist of a photoelectric cell which is a turbidity sensor that
detects changes in turbidity of the contents in the fermenter and then controls the amount of
medium fed to the fermenter.

In other techniques, a fermenter variable, eg. turbidity or pH, will be monitored using an
appropriate detector and the liquid flow rate will be automatically adjusted so as to maintain
the variable at a constant level. Examples of these types of continuous fermenters are the pH-
stat, turbidostat and nutristat. Apart from the pH-stat, these reactors are however rarely used
as the necessary measurement-control systems are generally unreliable over long periods of
time.

A turbidostat is a continuous culturing method where the turbidity of the culture is held
constant by manipulating the rate at which medium is fed. If the turbidity tends to increase,
the feed rate is increased to dilute the turbidity back to its setpoint. When the turbidity tends
to fall, the feed rate is lowered so that growth can restore the turbidity to its set point.

The most widespread large scale application of continuous culture reactors is in wastewater
treatment. Activated sludge plants, trickle bed filters, anaerobic digester and ponds all operate
in an continuous manner. Cell immobilization is also often employed to improve the
efficiency of the [Link] cultures are well established in the wastewater industry
for several reasons:

Unlike pure cultre microbial and animal cell systems, contamination is not a
consideration, as the wastewater feed will always contain microorganisms.

Continuous reactors have long been used in waste treatment and their use is not
considered a risk.

Finally using batch cultures is simply not economically feasible. Wastewater flows
are often measured in mega litres per hour and batch reactors simply could not cope
with the load.

Types of fermenters

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
The main function of a fermenter is to provide a controlled environment for the growth of
microorganisms or animal cells, to obtain a desired product. Few of the bioreactor types are
discussed below:

Stirred tank fermenter

Microbial fermentations received prominence during 1940's namely for the production of life
saving antibiotics. Stirred tank reactor is the choice for many (more than 70%) though it is
not the best. Stirred tank reactor’s have the following functions: homogenization, suspension
of solids, dispersion of gas-liquid mixtures, aeration of liquid and heat exchange. The Stirred
tank reactor is provided with a baffle and a rotating stirrer is attached either at the top or at
the bottom of the bioreactor. The typical decision variables are: type, size, location and the
number of impellers; sparger size and location. These determine the hydrodynamic pattern in
the reactor, which in turn influence mixing times, mass and heat transfer coefficients, shear
rates etc. The conventional fermentation is carried out in a batch mode. Since stirred tank
reactors are commonly used for batch processes with slight modifications, these reactors are
simple in design and easier to operate. Many of the industrial bioprocesses even today are
being carried out in batch reactors though significant developments have taken place in the
recent years in reactor design, the industry, still prefers stirred tanks because in case of
contamination or any other substandard product formation the loss is minimal.

Fig: Stirred tank fermenter

The batch stirred tanks generally suffer due to their low volumetric productivity. The
downtimes are quite large and unsteady state fermentation imposes stress to the microbial
cultures due to nutritional limitations. The fed batch mode adopted in the recent years
eliminates this limitation. The Stirred tank reactor’s offer excellent mixing and reasonably
good mass transfer rates. The cost of operation is lower and the reactors can be used with a
variety of microbial species. Since stirred tank reactor is commonly used in chemical industry
the mixing concepts are well developed. Stirred tank reactor with immobilized cells is not
favored generally due to attrition problems; however by separating the zone of mixing from
the zone of cell culturing one can successfully.

Air-lift fermenter

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
Airlift bioreactors (ALB) are generally classified as pneumatic reactors without any
mechanical stirring arrangements for mixing and use the expansion of compressed gas to
bring about the mixing.

[Link] turbulence caused by the fluid flow ensures adequate mixing of the liquid.
2. The draft tube is provided in the central section of the reactor.
3. The introduction of the fluid (air/liquid) causes upward motion and results in circulatory
flow in the entire
reactor.
[Link] large fermenters doesn’t require internal cooling coils as a jacket can normally
provide sufficient heat transfer, due to the rapid movement of fluid within the vessel.
[Link] air/liquid velocities will be low and hence the energy consumption is also low.
6. ALBs can be used for both free and immobilized cells.
7. There are very few reports on ALBs for metabolite production.
[Link] advantages of Airlift reactors are the elimination of attrition effects generally
encountered in mechanical agitated reactors. It is ideally suited for aerobic cultures since
oxygen mass transfer coefficient are quite high in comparison to stirred tank reactors.
These fermenters do not have mechanical agitation systems (motor, shaft, impeller
blades) but contents are agitated by injecting air from the bottom. Sterile atmospheric air is
used if microorganisms are aerobic and “inert gas” is used if microorganisms are anaerobic.
This is a gentle method of mixing the contents and is most suitable for fermentation of animal
and plant cell cultures since the mechanical agitation produces high shearing stress that may
damage the cells. Air-lift fermenters are most widely used for large-scale production of
monoclonal antibodies.

a. Inner loop air lift fermentor [Link] loop air lift fermentor

FERMENTER DESIGN AND OPERATION

A fermenter is a vessel which does not permit contamination but provides conditions
necessary for the maximum production of the desired product.

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
In designing and
constructing a fermenter, a number
of factors must be considered.
(1) The vessel must be capable of
being operated aseptically for a
number of days and should be
reliable in long term operations.
(2) Adequate aeration and agitation
should be provided to meet the
metabolic requirements of
microorganisms.
(3) Power consumption should be
as low as possible.
(4) It must have a system of
temperature control.
(5) It must have a system of pH
control.
(6) Sampling facilities should be
provided.
(7) Evaporation losses from the
fermenter should not be excessive.
(8) The vessel should be designed to require the minimal use of labour in operation cleaning,
harvesting and maintenance.
(9) The vessel should be suitable for a range of processes.
(10) It should have smooth internal surface.
(11) The cheapest material which enables satisfactory result to be achieved should be used.
(12) The vessel should be of similar geometry to both smaller and larger vessels in the pilot
plant to facilitate scale-up.
(13) There should be adequate service provision for industrial parts.

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017

In a fermenter with strict aseptic requirement, it is important to select materials that

can withstand repeated steam sterilization. On a small scale, glass stainless steel can be used.
Glass gives smooth surfaces; it is not toxic and corrosion-free. It is usually easy to examine
the process while it is going on. Temperature can be achieved by placing a fermenter in a
thermostatically controlled bath or by use of internal cooling coil through which cold water is
circulated to achieve the correct temperature. The sparger is the aeration system. The primary
purpose of aeration is to provide microbes in submerged cultures with sufficient O2 for
metabolic requirements while agitation will ensure a uniform suspension of microbial cell is
achieved in a homogeneous nutrient medium. Agitation may be avoided if aeration provides
enough agitation for example in processes where broth of low viscosity and low focal solids
are used. In order to achieve and maintain aseptic condition during fermentation, the
following operations should be performed:
_ Sterilization of the fermenter
_ Sterilization of the air supply
_ Addition of inoculums, nutrients and other supplements must be done aseptically.
_ There must be a sampling point
_ Foam control
_ Correct monitoring and control of various parameters such as pH, dissolved O2,
temperature e.t.c.

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017

BODY CONSTRUCTION
Construction materials differ with small scale, pilot and large scale. In small scale for
vessel construction glass or stainless steel may be used. For pilot and large scale process,
stainless steel (>4% chromium), mild steel (coated with glass or epoxy material), wood,
plastic or concrete may be used as vessel construction material. Any vessel used should not
have any corners and smooth surface is essential. The construction material must be non toxic
and corrosion proof.
Glass vessel (borosilicate glass)
Type I – glass vessel round or flat bottom with top plate. It can be sterilized by autoclaving
and the largest diameter is 60cm.
Type II – glass vessel flat bottom with top and bottom stainless steel plate. This type is used
in in situ sterilization process and the largest diameter 30cm.
Stainless steel
Stainless steel is used as vessel construction material with the following modifications,
1. >4% chromium (atleast 10-13%) may be added
2. film of thin hydrous oxide - non-porous, continuous, self healing, corrosion resistance
3. inclusion of nickel - improves engineering
4. presence of molybdenum - resistance to halogen salts, brine, sea water
5. tungsten, silicone - improve resistance
Thickness of vessel should be increased with scale. Side plates have lower thickness than top
and bottom plates. Top and bottom plate are hemispherical to withstand pressures.
SEALING
Sealing between top plate and vessel is an important criteria to maintain airtight
condition, aseptic and containment. Sealing have to be done between three types of surfaces
viz. between glass-glass, glass- metal and metal-metal. There are three types of sealing. They
are gasket, lipseal and ‘O’ ring. This sealing ensures tight joint in spite of expansion of vessel
material during fermentation. The materials used for sealing may be fabric-nitryl or butyl
rubbers. The seals should be changed after finite time. There are two way of sealing in O ring
type simple sealing and double sealing with steam between two seals.
BAFFLES
Baffles are metal strips that prevent vortex formation around the walls of the vessel.
These metal strips attached radially to the wall for every 1/10th of vessel diameter. Usually
baffles are present but when the vessel diameter is over 3dm3 around 6-8 baffles are used.
There should be enough gap between wall and baffle so that scouring action around vessel is
facilitated. This movement minimizes microbial growth on baffles and fermentation walls. If
needed coolin coils may be attached to baffles.
Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21
 2017
AERATION SYSTEM (SPARGER)
Sparger is a device for introducing air into fermenter. Aeration provides sufficient
oxygen for organism in the fermenter. Fine bubble aerators must be used. Large bubbles will
have less surface area than smaller bubbles which will facilitate oxygen transfer to a greater
extent. Agitation is not required when aeration provides enough agitation which is the case
Air lift fermenter. But this is possible with only for medium with low viscosity and low total
solids. For aeration to provide agitation the vessel height/diameter ratio (aspect ration) should
be 5:1.
Air supply to sparger should be supplied through filter. There are three types of sparger viz.
porous sparger, orifice sparger and nozzle sparger.
1. Porous sparger: made of sintered glass, ceramics or metal. It is used only in lab scale-non
agitated vessel. The size of the bubble formed is 10-100 times larger than pore size. There is
a pressure drop across the sparger and the holes tend to be blocked by growth which is the
limitation of porous sparger.
2. Orifice sparger: used in small stirred fermenter. It is a perforated pipe kept below the
impeller in the form of crosses or rings. The size should be ~ ¾ of impeller diameter. Air
holes drilled on the under surfaces of the tubes and the holes should be atleast 6mm diameter.
This type of sparger is used mostly with agitation. It is also used with out agitation in some
cases like yeast manufacture, effluent treatment and production of SCP.
3. Nozzle sparger: Mostly used in large scale. It is single open/partially closed pipe
positioned centrally below the impeller. When air is passed through this pipe there is lower
pressure loss and does not get blocked.
4. Combined sparger agitator: This is air supply via hallow agitator shaft. The air is emitted
through holes in the disc or blades of agitator.
EXIT GAS COOLER
Similar to liebig condenser, condenses the moisture from the exhaust gas in the fermenter.
This removes as much moisture as possible from the gas leaving the fermenter and prevent
excess fluid loss.
AGITATION
Agitation provides uniform suspension of cells in homogenous nutrient medium. This
agitation provides bulk fluid and gas phase mixing, air dispersion, facilitates oxygen transfer
and heat transfer and uniform environment through out the vessel. There are four classes,
namely Disc turbine, Vaned disc, Open turbine of variable pitch and Marine impeller. Disc
turbine prevents flooding by air bubbles. Flooding occurs when the air bubble is not properly
dispersed the air pocket is formed one area. Flooded only at 120min/hour of air discharge
when disc turbine is used.
When open turbine and
propeller are used the medium
is flooded at 21min per hour of
air discharge.
Rushton disc turbine with 1/3
of fermentor diameter has
been optimum for some
fermentation process. Now
recent designs of agitator have been introduced. Scaba is a new
design of agitator that can handle high flow rate before flooding and has Radial flow. But this
is not ideal for top to bottom mixing. Prochem maxflow agitator has low power conception
with high hydrodynamic thrust. This design has increased downward pumping capacity of
blades. In this design agitator/ vessel diameter ratio is 0.4. Appoximately 66% less power
requirement even when viscous and oxygen transfer efficiency improved. Intermig agitator

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
has two units. Unlike the earlier design agitator/ vessel diameter ratio is 0.6-0.7. For this
agitator larger air sparger is used and top to bottom mixing not efficient. New turbine designs
with dual impeller have been introduced. One for gas disperser and other for aiding
circulation with multirod mixing.
STIRRER GLANDS AND BEARINGS
The entry point of stirrer into fermenter may be from top to bottom or sides. Mostly used
from bottom so that that leaves more space for entry ports on top. There are four types of
stirrer glands and bearings.
1) Stuffing box
a. sealed by several layers of packing rings of asbestos or cotton yarn-pressed against the
shaft by a gland follower
b. At high speeds- packing wears – pressure should be applied to ensure tightness
c. Difficult to sterilize- satisfactory heat penetration
d. Sufficient for GILSP containment
2) Mechanical seal
a. 2 parts; i) stationary in the bearing housing, ii) other rotates on the shaft.
b. Two parts pressed together by springs or expanding bellows
c. Steam condensate use to lubricate and cool seals
d. safe for containment
e. double mechanical seal for level 2
f. at level 2 and 3, the condensate is piped to a kill tank
g. Disinfectants flushed through the seal
h. steam condensate outlet monitoring indicates any seal failure
3) Magnetic drives (some animal cell cultures)
a. shaft does not pierce the vessel
b. two magnets- one driving, held in bearing in housing on outside of head plate and one
driven, placed on one end of impeller shaft held in bearing in suitable housing
c. ceramic magnets –magnetic power cross 16mm gap
d. 300 – 2000 rpm rotation possible
4) Simple bush seals
Disadvantage of double seals are more difficult to assemble, difficult to detect failure of seal
from normal and dead spaces and seals leading to contamination. Hence simple bush seal is
preferred in some cases.
Check valves
Valves used to prevent accidental reversal flow of liquid or gas due to break down. There
are three types – swing check, lift check, combined stop and check.
Pressure control valves
These types of valves are used for two purposes.
a) Pressure reduction
b) Pressure retaining
Safety valve
There are types of safety valve by which the increase in pressure is released. They are,
a) A spindle lifted from its seating against the pressure – releases pressure
b) Bursting / rupturing of discs to release pressure. In case of releasing the gas, the escaping
gas must be treated before release.

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 DEPARTMENT OF MICROBIOLOGY
KARPAGAM ACADEMY OF HIGHER EDUCATION
(Deemed to be University Established Under Section 3 of UGC Act, 1956)
Eachanari Post, COIMBATORE - 641 021, INDIA

[Link]., Microbiology (Batch 2016-2019)

Industrial Microbiology (Semester-III) (16MBU303)
LECTURE PLAN

UNIT IV

Duration Topic Reference

01 Separation of cells – filtration and centrifugation. R1: 111-123
02 Cell disruption – physical, chemical and enzymatic methods. R2: 64-86
03 Product separation – solvent extraction and precipitation. R3: 452-46
04 Lyophilization and spray drying. 438-563

05 Introduction to immobilization
06 Methods of immobilization R4:45-461
07 Advantages and applications of immobilization
08 Large scale applications of immobilized enzymes-glucose
isomerase
09 Large scale applications of immobilized enzymes-penicillin
acylase
10 Unit revision and possible questions
Total hours: 10

R1: Wulf Cruegar & A. Cruegar, 2000. A Textbook of Industrial Microbiology, Panima
Publishing Corp, Newdelhi.
R2: Patel A. H., 2003. Industrial Microbiology. Macmillan India ltd, NewDelhi.
R3: Kalaichelvan and Arulpandi, 2009. Bioprocess technology,MJP Publishers.
R4: [Link]. Kalyani Publishers, 1998. NewDelhi.

Dr [Link]
Assistant Professor
Department of Microbiology
 2017

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
Unit-IV

Down stream process

Downstream processing is an essential part of bioprocess technology in that the desired product
needs to be isolated, purified and for different end uses. A variety of microorganisms including
genetically engineered species are used for the production of desired products. The products formed
may be secreted into the broth or may be retained within the cell introducing complexity in the
recovery of the product. In view of the complexity, downstream processing involves various
techniques and methodologies. Bioproducts differ greatly in their nature hence different separation
principles and mechanisms depending on molecular mass, charge distribution, hydrophobicity,
distribution coefficient, structure and immunogenic structure and specific affinity towards other
biomolecules becomes necessary for their isolation and purification. The choice of the separation
methodology depends to a large extent on the nature of the product, its quantity and the extent of
purity required.

The various processes used for the actual recovery of useful products from a fermentation or any
other industrial process is called downstream processing. The cost of downstream processing (DSP)
is often more than 50% of the manufacturing cost, and there is product loss at each step of DSP. In
addition, the product is either present in the cells, in the medium or both. In either case, the
concentration of product is usually rather low, and it is generally mixed with other molecules from
which it has to be separated. Therefore, the DSP should be efficient, involve as few steps as possible
(to avoid product loss), and be costeffective.

Down stream processing involves the following primary steps:

Fermentation process

Foam separation

Primary separation – removal of insoluble products/cell (centrifugation, filtration and

sedimentation)

Cell disruption (mechanical, enzymatic and chemical)

Product isolation –solvent extraction, adsorption, aqueous two-phase system and

precipitation

Purification techniques

Chromatography (ion exchange, gel permeation and affinity)

Membrane separation (micro-filtration, ultra-filtration and reverse phase electrophoresis)

Product polishing (crystallization, drying and diafiltration).

Foam separation

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
Initial step in the down stream processing is the removal of foam without loss of cells and products.
Whole cells or proteins gets attached to the surface of the air bubbles rising through liquid forming
foam and when separated will be lost. To minimize the loss two ways of foam separation has been
adapted.

1. Excess foam collected in a separate outlet and the foam is mechanically broken. The cells and
protein from the foam is collected for further usage.

2. Materials are made surface active and collected termed as colligends and the Surfactants used for
that purpose are termed collectors. Addition of surfactants is found to improve the percentage of
removal of cells.

Separation of cells and insoluble products

Sedimentation

Following any industrial fermentation it is essential to sediment suspended particles which involves
coagulation and flocculation. Sedimentation process may be achieved naturally with selected strains
of brewing yeasts if chilled at the end of fermentation. Apart from temperature, other mechanisms
that induce flocculation are neutralization of opposing charges, reduction in surface hydrophilicity
and bridging with high molecular weight polymers. Addition of chemicals such as acids and bases
change the pH and hence the charge on the particles facilitating coagulation. Simple electrolytes
such as ferric chloride and alum screen the electrostatic repulsion between the colloidal particles
thereby permitting van der Waals’ force to operate and leads to coagulation. Sometimes the
polymers bridge between the particles leading to agglomeration of particles. The majority of
flocculating agents currently in use are poly electrolytes and polyamines. Other agents are alum,
calcium salts and ferric salts. After flocculation the particles are separated by filtration and
centrifugation.

Filtration

Filtration is defined as the separation of solid in a slurry consisting of the solid and fluid by passing
the slurry through a septum called filter medium. For filtration in some cases filter aids
(diatomaceous earth) are used to improve porosity and faster flow rate.

Types of filters

Batch filters

Plate and frame filters

In this plates and frames are arranged alternately assembled on a horizontal framework. Plates are
covered with filter clothes and held together by hand screw to prevent leakage between frames. The
slurry is fed through the continuous channel by the holes in the corners of the plates and frames.

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
The filtrate passes through the filter cloth or pad, runs down grooves in the filter plates and is then
discharged through outlet taps to a channel.

Fig: Plate and Frame filters

Pressure leaf filters

These filters incorporates a number of leaves, each consisting of a metal framework of grooved
plates which is covered with a fine wire mesh, or occasionally a filter cloth and often precoated with
a layer of cellulose fibers. The slurry is fed into the filter which operated under pressure or by
suction with a vacuum pump. There are three types based on the arrangement of filters. (a) Vertical
metalleaf filter – consists of number of vertical porous metal leaves mounted on a hollow shaft in a
cylindrical pressure level. The solids from the slurry gradually build up on the surfaces of the leaves
and the filtrate is removed from the plates via the horizontal hollow shaft (b) Horizontal metal-leaf
filter – consists of metal leaves mounted on a vertical hollow shaft within a pressure vessel. Filtration
is continued until the cake fills the space between the disc shaped leaves or when the operational
pressure has become excessive (c) stacked disc filter – this metafilter consists of a number of
precision made rings which are stacked on a fluted rod. The filtrate passes between the discs and is
removed through the grooves of the fluted rods, while solids are deposited on the filter coating.

Continuous filters

Rotary vacuum filtration

Drum covered with diatomaceous earth matter and allowed to rotate under vacuum with half
immersed in the slurry tank (Fig.). Small amount of coagulation agent added to broth and pumped
into the slurry tank. As drum rotates in the slurry tank under vacuum thin layer of coagulated
particles adhere to drum. The layer thickens to from cake. As the cake portion in the drum comes to
the upper region which is not immersed in the liquid it is washed with water and dewatered

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
immediately by blowing air over it. Then before the dried portion is again immersed into the liquid it
is cut off from drum by knife. The mechanism of cake discharge is achieved by three ways. (a) string
discharge – Long lengths of string 1.5cm apart are threaded over the drum and round two rollers.
The cake is lifted free from the upper part of the drum when the vacuum pressure is released and
carried to the small rollers where it falls free (b) scraper discharge – by using a knife or scraper
positioned accurately to slice off the cake (c) scraper discharge with precoating – to avoid blockage
of filter cloth in the drum by cells a scraper which is coated with a layer of filter-aid 2 to 10 cm thick.

Fig: Rotary vacuum filter

Micro or Ultra Filtration

Filtration of suspended particles can be achieved by either dead end filtration or cross flow filtration.

(a) Dead end filtration – Solution poured over the membrane and the filtrate is collected at the
bottom. On prolonged filtration pores become blocked which reduce the filtering capacity.

(b) Cross flow filtration – To prevent the blockage the solution is passed over the membrane. Cell
suspension enters laterally and flows over the membrane. The filtrate gets collected at the bottom
whereas the cells are pushed to the opposite end by the continuous flow of suspension which is sent
out via an outlet at the opposite end. The liquid is again passed through a tube which recycles back
to the flow. Since the cells do not block the pores the filtration process can be performed
continuously.

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
Fig. Cross flow filtration

Adsortion on filter aids

Filter aids, are inert incompressible discrete particles of high permeability. Solids such as wood pulp,
starch powder, cellulose, inactive carbon, when added as filter aid enhances their filterability. Filter
aids absorb small particles, which otherwise clog the filter pores. Filter aids also reduce the
compressibility of the accumulated biomass by adsorbing the colloidal particles.

Centrifugation

Centrifugation is a common method used to separate cells from cultured broth. It employs
centrifugal force to promote accelerated settling of particles in a solid-liquid mixture. Separation is
achieved by means of accelerated gravitational force by rapid rotation. Microorganisms and other
cells from the fermented slurry can be removed by using centrifuge when filtration is not a
satisfactory separation method. The particle size that can be separated range from 0.1μm to 100
μm. Separation is based on Stoke’s law, which states that the rate of Newtonian viscosity
characteristics is proportional to the square of the diameter of the particles which is expressed as,

Vg = d2g (ρP – ρL) / 18μ

Where, Vg – rate of sedimentation, d – particle diameter, g – gravitational force, ρP – liquid density,

ρL – particle density, μ - viscosity

Tubular centrifuges:

This is used of separate particle size of 0.1 - 200 μm. This is simple machine made of a tube rotating
between bearings at each end. The suspension enters at the bottom of the centrifuge and high
centrifugal forces act to separate the solids and liquids. The bulk of solids will adhere o the walls of
the bowl, while the liquids exit at the top of the centrifuge.

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017

Fig: (a) Tubular centrifuge and (b) Chamber bowl centrifuge

Chamber bowl centrifuge

This centrifuge has a number of tubular bowl arranged coaxially. The main bowl contains cylindrical
inserts that divide the volume of the bowl into a series of annular chambers, which operate in
sequence. The feed enters the centre of the bowl and passes through each chamber. The solids
settle on the outer walls of each chamber and the clarified liquid overflows from the largest
diameter chamber.

Disc centrifuge

This is a simplest design of disc stack separator is a closed bowl, containing the disc stack,
with an arrangement to collect residual solids at the outer part of the bowl, from where they have to
be removed manually after stopping rotation. The solids are discharged from the bowl through
nozzles, which are always open.

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017

Fig. Disc centrifuge

Decanter centrifuge

This used for continuous handling of slurry. This sedimentation centrifuge is designed to handle
significant solid concentration in feed suspension. It consists of horizontal cylindrical bowl rotating at
a high speed, with a helical extraction screw placed co-axially. The screw perfectly fits the internal
contour of the bowl, only allowing clearance between bowl and scroll. The differential speed
between the screw and scroll provides the conveying motion to collect and remove solids that
accumulate at the bowl wall.

Cell disruption

Chemical methods

Chemical treatment for disrupting cell include alkali, organic solvent, detergents and
chaotropic agents. Alkali acts on the cell wall and results in saponification of membrane lipids. Alkali
such as NaOH addition alters the pH and affect the integrity of the cell membrane. It is carried out at
pH range of 11 to 12 for about 20 to 30 min. Organic solvents results in disruption of cell wall. Cell
wall absorbs the solvent resulting in swelling and rupture of cell wall. At low concentration the cell
wall is not ruptured but the permeability is increased. Product stability should be considered when
choosing the solvent. Eg. Toluene is used for Agrobacterium radiobacter, Ethyl acetate used for yeast
and Dimethyl sulfoxide (DMSO) for plant cell wall. Other organic solvents used are benzene,
chlorobenzene, xylene, cumeme, octanol, etc.

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
Detergents permeabilize cells by solubilizing cell membranes. They are amphipathic capable
of interacting with both water and lipids and solubilize the cell wall. Eg. Anionic detergents such as
Sodium dodecyl sulphate (SDS), sodium sulphonate, Cationic detergents such as cetyltrimethyl
ammonium bromide (CTAB) and Non-ionic detergents such Triton X100. Chaotropic agents disrupt
the structure of water making it less hydrophilic and weaken the hydrophobic interactions and
increase permeability.

Biological methods

Enzymatic digestion is involved in two stages (i) cell wall disruption resulting in the release of cell
wall proteins leaving the protoplast intact and (ii) digestion of organelle membrane to release the
organelle proteins. Digestion may be achieved by hydrolyzing cell walls by specific enzymes such as
Lysozyme. Hydrolyzing cell wall by combination of enzymes (1,3 – glucanase, 1,6 – glucanase,
mannanase, chitinase etc) may be also used for digestion in cases like plants cell wall.

Physical methods

Mechanical methods

In Ultrasonic vibrators (sonicators) the ultrasound waves of frequencies greater than 20 kHz ruptures
the cell wall by a phenomenon known as cavitation. The passage of ultrasound waves creates
alternating areas of compression and rarefaction which change rapidly. The cavities formed in the
areas of rarefaction rapidly collapses as the area changes to one of compression. The bubbles
produced in the cavities collapse creating shock waves which disrupt cell walls.

In laboratory grinding with a ball mill or a waring blender may be used. Waring blender is
particularly effective with animal cells and tissues as well as with mycelial organisms. In industrial
scale, cell disruption is carried out using a bead mill or high pressure homogenizer. Vertical or
horizontal bead mill consists of a grinding cylinder with a central shaft fitted with a number of
impellers and driven by motor. The cell suspension is pumped into the cylinder and cell disruption
occurs due to shear forces produced between velocity gradients because of the rotary motion of
cells and beads. In addition, collision between beads and cells and grinding of cells between rolling
beads also contribute to the disruptive forces. High pressure homogenization consists of a high
pressure positive displacement pump couple to an adjustable discharge valve with a restricted
orifice. The cell suspension is pumped through the homogenizing valve at 200 – 1000 atmospheric
pressure depending on microbes and cell concentration. Cell disruption occurs due to stress due to
impingement, normal stress during passage through narrow channel and shear stress due to
pressure drop.

Non mechanical methods

Osmosic shock provided by adding cells into twice the volume of pure water. The cells swell
due to osmotic flow of water ultimately bursting. Freezing thawing cycles cause loss of membrane
integrity and cell wall is ruptured. In Thermolyis the heat inactivates the organism by disrupting the
cell walls without affecting the products. The effect of heat shock depends on pH, ionic strength,

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
presence of chelating or sequestering agents such as EDTA. Combination of mechanical and non
mechanical gives best results

Separation of soluble products

Liquid – liquid extraction

It is a classical method for recovery as well as concentration of various products. Solvent extraction
has several advantages such as selectivity of extraction directly from broth or reaction medium,
reduction in product loss as the product is just transferred to a second phase and easy scale up.
Solvent extraction involves extraction of compound in a liquid phase to another liquid. The solute
originally present in aqueous phase gets partitioned in both the phases. The distribution between
the two immiscible liquids and solubility in two liquids decide the efficacy of extraction. The choice
of solvent selection was based on dielectric point. The dielectric constant is a measure of the degree
of molar polarization of a compound. An increase dielectric pole increases the polarity of the solute.

D= C/C0

Where, D – dielectric constant, C – electrostatic capacity of a condenser containing the substance

Vbetween the plates, C0 – electrostatic capacity of the same condenser when completely evacuated.
The final choice of solvent is influenced by partition coefficient (k).

k = concentration of solute in extract / concentration of solute in raffinate High value of k required

single stage extraction and low value k requires multistage extraction. In single stage batch
extraction, the aqueous feed is mixed with the organic solvent and after equilibration; the extract
phase containing the desired solute is separated out for further processing. In some cases, a single
stage extraction may not be enough and multi stage process is required wherein fresh volume of
solvent is contacted with the raffinate.

Fig: (a) Single stage and (b) double stage extraction process

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
Continuous extraction can be carried out by co-current or counter current methods. In Cocurrent
extraction there are n mixer vessels in line and the raffinate goes from vessel 1 to vessel n. Fresh
solvent is added to each stage and the extracting solvent pass through the cascade in the same
direction. At every stage the extract is recovered. In Counter current extraction the extracted
raffinate passes from vessel 1 to vessel n while the product-enriched solvent is flowing from vessel n
to vessel 1. This is the most efficient method of extraction.

Fig: Counter current extraction process

Extraction is achieved by three mechanisms viz., physical extraction, dissociative extraction and
selective extraction. Physical extraction involves preferential dissolution of the desired solute in a
chosen organic solvent. Dissociative extraction involves the modification of the physical property of
the solute to increase the solubility in organic phase. For example for extraction of organic acids pH
is adjusted below pK value enhancing dissociation and thus extraction. Selective extraction involves
modifying the solute solubility through ion pair or complex or adduct formation. For example long
chain aliphatic amines are used for citric acid extraction. Solvent recovery after extraction process
Solvent recovery after extraction process is essential one which is usually done by distillation. The
distillation is performed in three stages (i) evaporation of solvent into vapour phase, (ii) vapour-
liquid separation and (iii) condensation to collect solvent.

Aqueous two phase extraction

The basic principle involves differential partitioning of solute in two immiscible phases. Phase
separation occurs when hydrophilic polymers are added to an aqueous solution. At low
concentration of polymers, homogenous solution is formed but at discrete concentration rise, two
immiscible phases are formed using two aqueous phases with incompatible polymers such as PEG
and dextran. Eg. PEG water / dextran water and PEG water/K-phosphate water, PEG phosphates.
Homogenates are prepared with the two incompatible polymers after which mixer – phase
separation is done by keeping idle. The bottom phase and top phase separated. Then the soluble
and nonsoluble substances are separated by ultra filtration and product recovered. The retentate
may be recycled for further recovery.

Precipitation - by decreasing the solubility of the solutes the solute can be separated by
precipitation Solubility of the particle can be changed by,

1. Salting out – by increasing ionic strength by adding salts as ammonium sulphate, disodium
sulphate

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
2. Solubility reduction at low temperature – by adding organic solvents at low temperature

3. Solvent precipitation – adding salt, pH adjustment and low temperature

4. Isoelectric precipitation – by the changing the pH to isoelectric pH (no charge in proteins)

5. Use of electrolytes – ionic polymers (ionic polysaccharides), non ionic polymer (dextrans)

Adsorption - This refers to the binding of molecules or particles to a surface or adsorption of solutes
from liquid media onto solids. Adsorption occurs due to van der Waals’ force, strong ionic bonds.
There are different types of solid – liquid contactors to facilitate adsorption viz., packed bed, moving
bed, fluidized bed, and agitated vessel contactors. Packed beds are adsorbent bed packed and liquid
sent through it. In Moving bed, adsorbent solid is continuously supplied, after adsorbing get
removed from vessel. Fluidized bed is in which adsorbent solid is suspended in liquid. Packed and
moving beds are widely used.

Dialysis is a membrane separation used to remove low molecular weight solutes (organic acids,
inorganic ions). Dialysis is the movement of molecules by diffusion from high concentration to low
concentration through a semi-permeable membrane. Only those molecules that are small enough
pass through the membrane pores and reach equilibrium with the entire volume of solution in the
system. Once equilibrium is reached, there is no further net movement of the substance because
molecules keep moving through the pores in and out of the dialysis unit at the same rate. Factors
affecting dialysis: dialysis buffer volume, buffer composition, number of buffer changes, time,
temperature and particle size. Membranes containing convoluted pores, not the tube-like pores
often found in traditional dialysis tubing.

Fig: Dialysis process

Reverse osmosis is a process where the solvent molecules are forced by an applied pressure to flow
through a semi permeable membrane in the opposite direction. Reverse osmosis from low to high by
applying pressure uses membrane (pore size 0.0001 – 0.001 μm) permeable to water but not
dissolved salts of low molecular weight. This method is applicable to concentrate smaller molecules.

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017

Fig: Reverse osmosis process

Supercritical fluid extraction

Supercritical fluid extraction involves the dissolution power of super critical fluids ie. fluids above
their critical temperature and pressure. Critical temperature is defined as the temperature above
which a distinct liquid phase cannot exist regardless of pressure. The vapour pressure of the
substance at its critical temperature is called the critical pressure. Alternately, pressure and
temperature required to liquefy a gas are critical temperature and pressure. At temperature and
pressure above but close to the critical point a substance exists as a supercritical fluid. For example
Carbon di oxide, NO, SO2 are used in extraction of β-carotene, vanilla, vegetable oil, etc.

Fig: Supercritical fluid extraction process. CO2 is fluidized and used for extraction

Concentration

Some concentration of the product may occur during the extraction step. Further concentration may
be achieved by: (i) evaporation, (ii) membrane filtration, (Hi) ion exchange methods and (iv)
adsorption methods.

(a) Evaporation. It is generally used in cases of solvent extraction usingvarious devices, e.g.,
continuous flow evaporators, falling film evaporators, thin film evaporators, centrifugal thin film
evaporators and spray-dryers. Efficient arrangements must be made for recovery of the evaporated
solvent to reduce costs. For low grade products, often evaporation of the whole broth is undertaken
using a spray-drier.

(b) Membrane Filtration. It generally achieves both concentration and separation of the products
usually based on the size of molecules. The different processes of membrane filtration are:
microfiltration, ultrafiltration, reverse osmosis and electrodialysis. Micro- and ultrafiltration work as
sieves and separate molecules of different sizes, but reverse osmosis can separate molecules of
similar size. Microfiltration can be used for cell separation as well.

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
(c) Ion Exchange Resins. These are polymers having firmly attached ionizable groups (anions or
cations) which ionize under a suitable [Link] may be solid, e.g., dextran, cellulose,
polyamine, acrylate etc., or liquid, e.g.,a solvent carrying a functional group like phosphoric acid
mono- or diester etc. Solid ion exchangers may be used in two ways: (i) they may be packed in
columnsor (ii) they may be added to the extract and removed by [Link] ion exchangers
dissolve only in nonaqueous solvent carrier and the separation is similar to liquid—liquid extraction.
Some antibiotics are recovered directly from the whole broth using ion exchange resins. The product
is recovered from the ion exchangers by ion displacement; this also regenerates the ion exchanger.

(d) Adsorption Resins . These are porous polymers without ionization. Most compounds are
adsorbed to the resins in non-ionized state. The porosity of the resin determines the surface
available for adsorption. These resins may be apolar (e.g.,styrene-divinyl beneze), polar (e.g.,
sulfoxide, amide etc.), or semipolar (e.g., acrylic ester). The products are recovered from such resins
by solvent (organic) extraction,changed pH etc.

Purification

The final step in the recovery of a product is purification which aims at obtaining the product in
highly purified state. The earlier steps will have achieved variable degrees of purification which may
determine the degree of resolution necessary during the purification step. The degree of resolution
will mainly depend on the similarities to the metabolite of other molecules present in the
concentrate, and the degree of purity required in the final product. Purification is achieved by: (a)
crystallization and(b) chromatographic procedures.

(a) Crystallization. It is mainly used for purification of low molecular weight compounds like
antibiotics, e.g., penicillin G is usually extracted from fermentation broth in butyl acetate and
cystallized by the addition of potassium acetate in ethanolic solution. Crystallization is the final stage
in purification of products like citric acid,sodium glutamate etc.

(b) Chromatographic Methods. These are used for purification of low molecular weight compounds
from mixtures of similar molecules, e.g., homologous antibiotics, and of macromolecules, especially
enzymes, which are similar in properties. The materials used for hromatography are generally
coated on particulate carrierswhich are packed in columns through which the liquid containing the
product is pumped either upward or downward. The separated product is recovered in some sort of
fraction collector. On a large, scale organic solvents are used for [Link], the whole
system has to be installed in a flame-proof and explosion-proof room. The different
chromatographic procedures are: (a) adsorption, (Hi) gel filtration, (iv) hydrophobic, (v) affinity, (vi)
covalent and (vif) partition chromatography. Adsorption chromatography separates molecules due
to their differential affinities for the surface of a solid matrix, e.g., silica gel, alumina, hydroxyapatite
(all inorganic) or an organic polymer. In case of ion exchange chromatography, resins or
polysaccharides, e.g., cellulose, sepharose, having attached ionized functional groups are used for a
high resolution separation of macromolecules, e.g., proteins. Gel filtration uses molecular sieves,
composed of neutral cross-linked carriers (e.g., polymers like agarose, dextrans), of different pore
sizes. Molecules smaller than the pore size enter the carrier and are retained; they are later eluted

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
(in order of molecule size) and collected. Gel filtration is used in aqueous systems. Hydrophobic
carriers are used for purification of hydrophobia molecules, e.g., many enzymes and other proteins.
Affinity chromatography uses molecules, called effectors, to which the product has high and specific
affinity, e.g., using an antibody (effector) for the purification of the antigen to which it is specific. The
effector is immobilized on a water insoluble carrier which is packed in a column through which the
mixture is passed. The effector binds only to the molecules for which it is specific and retains it in the
column; it is later recovered by elution using a buffer solution of a specified pH. For example, human
leucocyte interferon is recovered in high yield and in high purity by affinity chromatography using
monoclonal antibody immobilized on a sepharose column. Group specific affinity chromatography
and covalent chromatography are based on chemical interactions between the carrier and the
product molecules.

Drying

Drying makes the products suitable for handling and storage. It should be accomplished with a
minimum rise in temperature due to heat sensitivity of most products. Addition of sugars or other
stabilizers improves the heat tolerance of some products like enzymes and pharmaceutical
preparations. The most common approaches to drying are as follows: (/) vacuum drying, (ii) spray
drying, and (iii) freeze drying. In spray drying, the solution or slurry to be dried is atomized by a
nozzle or a rotating disc. A current of hot (150-250°C) air is passed; the drying is so rapid that the
temperature of particles remains very low. Spray drying is used for enzymes, antibiotics and food
products. Vacuum drying uses both heat and vacuum for drying; it can be applied both in batch
mode (e.g., chamber dryers) or in continuous mode (e.g., rotating drum vacuum dryers). In freeze
drying, the liquor to be dried is first frozen and the water is sublimed from the frozen mass. A very
low pressure (partial vacuum) is maintained to promote sublimation of water. The energy needed
for sublimation is provided by heated plates and radiation on to the surface. The temperature of
solid is regulated by regulating the pressure in the drying chamber. This is the most gentle method of
drying, and is used for many pharmaceutical products, e.g., viruses, vaccines, plasma fractions,
enzymes etc., and in food industries.

Enzyme immobilization:

Enzymes are protein molecules and known as biocatalysts, which serve to accelerate the
biochemical reactions by several orders of magnitude. Enzymes are not permanently modified by
their participation in reactions and display great specificity. It is cost-effective to use them more than
once as they are not changed during the reactions. However, if the enzymes are in solution with the
reactants and / or products it is difficult to separate them. Therefore, if they can be attached to the
reactor in some way, they can be used again after the products have been removed. The term
"immobilized" means unable to move or stationary. Thus, the immobilized enzyme is an enzyme that
is physically attached to a solid support, over which a substrate is passed and converted to product.
The immobilized enzyme can provide increased resistance to changes in conditions such as pH or
temperature. This also allows enzymes to be held in place throughout the reaction. Thus, the
immobilized enzyme can be easily separated from the products and may be used again. Therefore
the immobilized enzyme is widely used in industry for enzyme catalyzed reactions. Nelson and

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
Griffin discovered in 1916 that the invertase when absorbed on a solid (charcoal or aluminum
hydroxide) exhibited the same activity when uniformly distributed throughout the solution at the
bottom of the reaction vessel. This discovery was the first of various enzyme immobilization
techniques currently available. However, the proper selection of the immobilization techniques to
obtain active and stable immobilized enzyme mostly depends on physical and chemical properties of
an enzyme and support. This needs a careful investigation of different parameters to evolve most
suitable techniques of immobilization for the particular enzyme.

Immobilized enzymes are very important for commercial uses as they possess many benefits
which include:

1. Economical: The immobilized enzyme can be easily removed from the reaction thus making it easy
to recycle, multiple or repetitive use.

2. Stability: Immobilized enzymes typically have greater thermal and operational stability than the
soluble form of the enzyme and product is not contaminated with the enzyme.

3. Convenience: The ability to stop/start the reaction rapidly by removing/adding the enzyme from
the reaction solution, long half life, predictable decay rates, elimination of reagent preparation, etc.

Methods of immobilization: It is essential to choose a method for immobilizing an enzyme to a

surface which will prevent loss of enzyme activity. There are mainly three methods available for
immobilizing enzymes :

1. Adsorption: the binding of enzymes by physical / ionic interaction to water insoluble carriers. 2.
Covalent Binding: the binding of enzymes by covalent coupling to water-insoluble carriers.

3. Entrapping: incorporating enzymes into the lattices of a semi-permeable gel or enclosing the
enzymes in a semi-permeable polymer membrane.

Methods available for immobilizing enzymes: (a) enzyme (E) noncovalently adsorbed to an insoluble
particle; (b) enzyme (E) covalently attached to an insoluble particle; (c) enzyme (E) entrapped within
an insoluble particle (d) enzyme (E) entrapped within a semi-permeable membrane.

Adsorption: Adsorption is a very simple method of an enzyme immobilization which has wide
applicability. The immobilized enzyme in a directly usable form can be obtained by simply mixing an
enzyme with a suitable adsorbent under appropriate conditions of pH, ionic strength, incubation

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
period and washing off unbound enzyme. It is possible to achieve high enzyme loading using
adsorption. The binding between adsorbent and the enzyme is mostly due to a combination of
hydrophobic effects and the formation of several salt links per enzyme molecule. The physical links
between the enzyme molecules and the support generally weakened during use by inappropriate
changes in pH or ionic strength. Therefore the choice of adsorbent is most important to avoid
enzyme leakage during use. Although there are different choice of adsorbent mentioned in the
literature such as ion-exchange matrices, porous carbon, clays, hydrous metal oxides, glasses and
polymeric aromatic resins etc. But ion-exchange matrices can be used repeatedly to achieve desired
economy by just washing off the used enzyme with concentrated salt solutions and reloading the
active enzyme.

Covalent Binding: In comparison to other methods, the covalent binding method is most effective
and widely studied. The ultimate aim of the covalent binding is to avoid leaching of an enzyme into
the surrounding solution. However, binding reaction has to be performed under conditions that
should protect the catalytic activity of the enzyme and active site of the enzyme must be unaffected
by the reagents used. In general, the functional groups of proteins suitable for covalent binding and
which do not cause loss of the catalytic activity of the enzyme include (a) the alpha amino groups of
chain and epsilon amino groups of lysine and arginine, (b) the alpha carboxyl group of chain end and
beta and gamma carboxyl groups of aspartic and glutamic acids, (c) the phenol ring of tyrosine, (d)
the thiol group of cysteine, (e) the hydroxyl groups of serine and threonine, (f) the imidazile group of
histidine, and (g) the indole group of tryptophan. In some cases, it is possible to increase the number
of reactive residues of an enzyme in order to increase the yield of immobilized enzyme and to
provide alternative reaction sites to those essential for catalytic activity of the enzyme. The covalent
binding method has become mostly applicable method of immobilization because of the wide
variety of binding reactions, capabilities of covalent binding of functional groups with insoluble
carriers and possibilities of activation to generate such groups. Figure 2 shows some of the most
commonly used methods for the covalent immobilization of enzymes. The activation of sepharose by
cyanogen bromide is a simple, mild and often successful method of wide applicability. Sepharose is a
commercially available beaded polymer which is highly hydrophilic and generally inert to
microbiological attack. The cyanogen bromide combines with hydroxyl groups of sepharose to give
the reactive cyclic imido-carbonate, which reacts with primary amino groups on the enzyme under
mildly basic conditions (Figure 2a). However, the high toxicity of cyanogen bromide has led to the
investigation of alternative methods involving chloroformates to produce similar intermediates
(Figure 2b). The bifunctional reagents such as carbodiimides are very useful as they allow the
coupling of amines to carboxylic acids (Figure 2c). The mostly used bifunctional reagent to link
enzymes to the insoluble carriers is glutaraldehyde (Figure 2d). Glutaraldehyde is particularly useful
for producing immobilized enzyme membranes which is generally used in the preparation of
biosensors. The enzymes can be coupled to even inert materials such as glass with the help of
trialkoxysilanes (Figure 2e). There are numerous other methods available for the covalent
attachment of enzymes. However, selection of proper methods to achieve highly stable and active
immobilized is a most critical factor which varies from enzyme to enzyme.

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
Commonly used methods for the covalent immobilization of enzymes: (a) Activation of sepharose by
cyanogen bromide. (b) Chloroformates may be used to produce similar intermediates to those
produced by cyanogen bromide but without its inherent toxicity. (c) Carbodiimides may be used to
attach amino groups on the enzyme to carboxylate groups on the support or carboxylate groups on
the enzyme to amino groups on the support. (d) Glutaraldehyde is used to link enzymes to supports.
(e) The use of trialkoxysilane to derivatise glass.

Entrapping: In general, entrapment is a useful method of enzyme immobilization using gels or fibers
where substrates and products are low molecular weight. A purely physical caging or covalent
binding of an enzyme can be achieved using entrapment method. For instance, the lysine residues
available on the surface of the enzyme can be derivatised by reaction with acryloyl chloride
(CH2=CH-CO-Cl) to give the acryloyl amides and then it may be copolymerised and cross-linked with
acrylamide (CH2=CH-CO-NH2) and bisacrylamide (H2N-CO-CH=CH-CH=CH-CO-NH2) to form a gel.
The enzyme can also be entrapped in cellulose acetate fibers by making up an emulsion of the
enzyme plus cellulose acetate in methylene chloride and extrusion of this emulsion through a
spinneret into a solution of an aqueous precipitant. Generally, calcium alginate has been widely used
for the entrapment of microbial, animal and plant cells. Another method of entrapment is the
confinement of enzymes using membranes however, semipermeable nature of the membrane plays
a critical role. The entrapment using membranes should ensure confinement of the enzyme and free
passage for the reaction products. The entrapment of an enzymes using membrane can be achieved
by simply placing the enzyme on one side of the semipermeable membrane whereas the reactant
and product on the other side. Hollow fibers are the best example of entrapment of an enzyme using
membranes. They are available commercially with large surface areas relative to their contained
volumes (> 20 m2 l-1) and permeable only to substances of molecular weight substantially less than
the enzymes. Hollow fibers are very easy to use for a wide variety of enzymes (including
regenerating coenzyme systems) and the cost effect may be minimized by avoiding the cost of
additional research and development associated with other immobilization methods. Another
method is the encapsulation of an enzyme within small membrane bound droplets or liposomes. For
instance, the enzyme is dissolved in an aqueous solution of 1, 6-diaminohexane and then dispersed
in a solution of hexanedioic acid in the immiscible solvent, chloroform. The resultant reaction forms
a thin polymeric shell around the aqueous droplets which traps the enzyme. In general, liposomes
are formed by the addition of phospholipid to enzyme solutions. They form concentric spheres of
lipid membranes surrounding the soluble enzyme. After washing of the non-confined enzyme, the
micro-capsules and liposomes can be transferred back to aqueous solution before use.

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017

Applications of immobilized enzymes

The first industrial use of an immobilized enzyme is amino acid acylase by Tanabe Seiyaku
Company, Japan, for the resolution of recemic mixtures of chemically synthesized amino acids.
Amino acid acylase catalyses the deacetylation of the L form of the N-acetyl amino acids leaving
unaltered the N-acetyl-d amino acid, that can be easily separated, racemized and recycled. Some of
the immobilized preparations used for this purpose include enzyme immobilized by ionic binding to
DEAE-sephadex and the enzyme entrapped as microdroplets of its aqueous solution into fibres of
cellulose triacetate by means of fibre wet spinning developed by Snam Progetti. Rohm GmbH have
immobilized this enzyme on macroporous beads made of flexiglass-like material. By far, the most
important application of immobilized enzymes in industry is for the conversion of glucose syrups to
high fructose syrups by the enzyme glucose isomerase95. Some of the commercial preparations have
been listed in Table 2. It is evident that most of the commercial preparations use either the
adsorption or the cross-linking technique. Application of glucose isomerase technology has gained
considerable importance, especially in nontropical countries that have abundant starch raw
material. Unlike these countries, in tropical countries like India, where sugarcane cultivation is
abundant, the high fructose syrups can be obtained by a simpler process of hydrolysis of sucrose
using invertase. Compared to sucrose, invert sugar has a higher humectancy, higher solubility and
osmotic pressure. Historically, invertase is perhaps the first reported enzyme in an immobilized
form. A large number of immobilized invertase systems have been patented. The possible use of
whole cells of yeast as a source of invertase was demonstrated by D’Souza and Nadkarni as early as
1978. A systematic study has
been carried out in our
laboratory for the preparation
of invert sugar using
immobilized invertase or the
whole cells of yeast. These
comprehensive studies carried
out on various aspects in our
laboratory of utilizing
immobilized whole-yeast have

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
resulted in an industrial process for the production of invert sugar.

L-aspartic acid is widely used in medicines and as a food additive. The enzyme aspartase
catalyses a one-step stereospecific addition of ammonia to the double bond of fumaric acid. The
enzymes have been immobilized using the whole cells of Escherichia coli. This is considered as the
first industrial application of an immobilized microbial cell. The initial process made use of
polyacrylamide entrapment which was later substituted with the carragenan treated with
glutaraldehyde and hexamethylenediamine. Kyowa Hakko Kogyo Co. uses Duolite A7, a
phenolformaldehyde resin, for adsorbing aspartase used in their continuous process. Other firms
include Mitsubishi Petrochemical Co. and Purification Engineering Inc. Some of the firms, specially in
Japan like Tanabe Seiyaku and Kyowa Hakko, have used the immobilized fumarase for the
production of malic acid (for pharmaceutical use). These processes make use of immobilized
nonviable cells of Brevibacterium ammoniagenes or B. flavus as a source of fumarase. Malic acid is
becoming of greater market interest as food acidulant in competition with citric acid. Studies from
our laboratory have shown the possibility of using immobilized mitochondria as a source of
fumarase.

One of the major applications of immobilized biocatalysts in dairy industry is in the

preparation of lactose-hydrolysed milk and whey, using b -galactosidase. A large population of
lactose intolerants can consume lactose-hydrolysed milk. This is of great significance in a country like
India where lactose intolerance is quite prevalent102. Lactose hydrolysis also enhances the sweetness
and solubility of the sugars, and can find future potentials in preparation of a variety of dairy
products. Lactose-hydrolysed whey may be used as a component of whey-based beverages,
leavening agents, feed stuffs, or may be fermented to produce ethanol and yeast, thus converting an
inexpensive byproduct into a highly nutritious, good quality food ingredient. The first company to
commercially hydrolyse lactose in milk by immobilized lactase was Centrale del Latte of Milan, Italy,
utilizing the Snamprogetti technology. The process makes use of a neutral lactase from yeast
entrapped in synthetic fibres. Specialist Dairy Ingredients, a joint venture between the Milk
Marketing Board of England and Wales and Corning, had set up an immobilized b -galctosidase plant
in North Wales for the production of lactose-hydrolysed whey. Unlike the milk, the acidic b -
galactosidase of fungal origin has been used for this purpose. Some of the commercial b -
galactosidase systems have been summarized in Table 3. An immobilized preparation obtained by
cross-linking b -galactosidase in hen egg white (lyophilized dry powder) has been used in our
laboratory for the hydrolysis of lactose. A major problem in the large-scale continuous processing of
milk using immobilized enzyme is the microbial contamination which has necessitated the
introduction of intermittent sanitation steps. A co-immobilizate obtained by binding of glucose
oxidase on the microbial cell wall using Con A has been used to minimize the bacterial
contamination during the continuous hydrolysis of lactose by the initiation of the natural lacto-
peroxidase system in milk. A novel technique for the removal of lactose by heterogeneous
fermentation of the milk using immobilized viable cells of K. fragilis has also been developed.

One of the major applications of immobilized enzymes in pharmaceutical industry is the

production of 6-aminopenicillanic acid (6-APA) by the deacylation of the side chain in either
penicillin G or V, using penicillin acylase (penicillin amidase). More than 50% of 6-APA produced
today is enzymatically using the immobilized route. One of the major reasons for its success is in

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
obtaining a purer product, thereby minimizing the purification costs. The first setting up of industrial
process for the production of 6-APA was in 1970s simultaneously by Squibb (USA), Astra (Sweden)
and Riga Biochemical Plant (USSR). Currently, most of the pharmaceutical giants make use of this
technology. A number of immobilized systems have been patented or commercially produced for
penicillin acylase which make use of a variety of techniques either using the isolated enzyme or the
whole cells. This is also one of the major applications of the immobilized enzyme technology in India.
Similar approach has also been used for the production of 7-aminodeacetoxy-cephalosporanic acid,
an intermediate in the production of semisynthetic cephalosporins.

Immobilized oxidoreductases are gaining considerable importance in biotechnology to carry

out synthetic transformations. Of particular significance in this regard are oxidoreductase-mediated
asymmetric synthesis of amino acids, steroids and other pharmaceuticals and a host of speciality
chemicals. They play a major role in clinical diagnosis and other analytical applications like the
biosensors. Future applications for oxidoreductases can be in areas as diverse as polymer synthesis,
pollution control, and oxygenation of hydrocarbons. Immobilized glucose oxidase can find
application in the production of gluconic acid, removal of oxygen from beverages, and in the
removal of glucose from eggs prior to dehydration in order to prevent Maillard reaction. Studies
carried out in this direction in our laboratory have shown that glucose can be removed from egg,
using glucose oxidase and catalase which are co-immobilized either on polycationic cotton cloth57 or
in hen egg white foam matrix. Alternatively, glucose can also be removed by rapid heterogeneous
fermentation of egg melange, using immobilized yeast. Immobilized D-amino acid oxidase has been
investigated for the production of keto acid analogues of the amino acids, which find application in
the management of chronic uremia. Keto acids can be obtained using either L- or D-amino acid
oxidases. The use of D-amino acid oxidase has the advantage of simultaneous separation of
natural L-isomer from DL-recemates along with the conversion of D-isomer to the corresponding
keto acid which can then be transamina-ted in the body to give the L-amino acid. Of the several
microorganisms screened, the triangular yeast T. variabilis was found to be the most potent source
of D-amino acid oxidase with the ability to deaminate most of the D-amino acids. The permeabilized
cells entrapped either in radiation polymerized acrylamide Ca-alginate or gelatin have shown
promise in the preparation of a -keto acids. Another interesting enzyme that can be used profitably
in immobilized form is catalase for the destruction of hydrogen peroxide employed in the cold
sterilization of milk. A few reports are available on its immobilization using yeast cells.

Lipase catalyses a series of different reactions. Although they were designed by nature to
cleave the ester bonds of triacylglycerols (hydrolysis), lipase are also able to catalyse the reverse
reaction under microaqueous conditions, viz. formation of ester bonds between alcohol and
carboxylic acid moieties. These two basic processes can be combined in a sequential fashion to give
rise to a set of reactions generally termed as interesterification. Immobilized lipases have been
investigated for both these processes. Lipases possess a variety of industrial potentials starting from
use in detergents; leather treatment controlled hydrolysis of milk fat for acceleration of cheese
ripening; hydrolysis, glycerolysis and alcoholysis of bulk fats and oils; production of optically pure
compounds, flavours, etc. Lipases are spontaneously soluble in aqueous phase but their natural
substrates (lipids) are not. Although use of proper organic solvents as an emulsifier helps in
overcoming the problem of intimate contact between the substrate and enzyme, the practical use of

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
lipases in such psuedohomogeneous reactions poses technological difficulties. Varieties of
approaches to solve these, using immobilized lipases, have recently been reviewed.

Significant research has also been carried out on the immobilization and use of
glucoamylase. This is an example of an immobilized enzyme that probably is not competitive with
the free enzyme and hence has not found large-scale industrial application. This is mainly because
soluble enzyme is cheap and has been used for over two decades in a very optimized process
without technical problems. Immobilization has also not found to significantly enhance the
thermostability of amylase. Immobilized renin or other proteases might allow for the continuous
coagulation of milk for cheese manufacture. One of the major limitations in the use of enzymes
which act on macromolecular substrates or particulate or colloidal substrates like starch or cellulose
pectin or proteins has been the low retention of their realistic activities with natural substrates due
to the steric hindrance. Efforts have been made to minimize these problems by attaching enzymes
through spacer arms. In this direction, application of tris (hydroxymethyl) phosphine as a coupling
agent may have future potentials for the immobilization of enzymes which act on macromolecular
substrates. Other problem, when particulate materials are used as the substrates for an enzyme, is
difficulty in the separation of the immobilized enzyme from the final mixture. Efforts have been
made in this direction to magnetize the bicatalyst either by directly binding the enzyme on magnetic
materials (magnetite or stainless steel powder) or by co-entrapping magnetic material so that they
can be recovered using an external magnet. Magnetized biocatalysts also help in the fabrication of
magnetofluidized bed reactor

Applica
tion of
immobi
lized
Glucos
e
isomeri
se

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017

Glucose isomerase (EC [Link]) catalyzes the isomerisation of glucose into fructose. This is a
thermostable enzyme having wide applications. Glucose isomerase is one of the popular enzymes of
future market. This happens to be in demand due to the increasing requirement of health care
products. High Fructose Corn Syrup (HFCS) is formed by converting corn starch into glucose and
further isomerising it to fructose. It is extremely sweet and clear syrup, refined by carbon and ion
exchange systems to assure the highest food standards in terms of colour, clarity, composition,
flavour and ash. Fructose, also known as fruit sugar, is the sweetest natural sugar and is found in
fruits, vegetables, and honey. HFCS has wide applications in pharmaceutical and food industries. It is
added in medicated syrups, beverages, baking, canning, and confectionary items as a sweetening
agent. Glucose isomerase (GI) is commercially available as Sweetzyme produced by Bacillus
coagulans of Novo Nordisc industries, Maxazyme produced by Actinoplanes missouriensis of Gist
Brocades N V, Takasweet F produced by Flavobacetrium arborescens of Miles laboratories,
Ketozyme produced by Actinoplanes missouriensis of Universal Oil Products, Optisweet 22 produced
by Streptomyces rubiginosus of Kalie Chemie [Jao, 1985]. Fructose has higher sweetening index than
glucose and sucrose. HFCS an equilibrium mixture of glucose and fructose (1:1) is 1.3 times sweeter
than sucrose and 1.7 times sweeter than [Link] is essential for efficient use of immobilised
glucose isomerase that the substrate solution is adequately purified so that it is free of insoluble
material and other impurities that might inactivate the enzyme by chemical (inhibitory) or physical
(pore-blocking) means. In effect, this means that glucose produced by acid hydrolysis cannot be
used, as its low quality necessitates extensive and costly purification. Insoluble material is removed
by filtration, sometimes after treatment with flocculants, and soluble materials are removed by ion
exchange resins and activated carbon beads. This done, there still remains the possibility of
inhibition due to oxidised by-products caused by molecular oxygen. This may be removed by vacuum
de-aeration of the substrate at the isomerisation temperature or by the addition of low
concentrations (< 50 ppm) of sulphite.

Immobilization of glucose isomerase (GI)

In order to increase the productivity of a process involving enzyme technology, immobilization is a

very useful method. Immobilization of the enzyme aids in increasing it’s stability and reusability. The
development of immobilized glucose isomerase (IGI) and it’s application for the production of HFCS
has been a subject of great interest. With the growing demand of HFCS the increased reusability of
the enzyme would be a boon for industrial processes. This will help in reducing the cost of HFCS
production. Immobilization techniques of GI and cells containing glucose isomerase shall increase

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
the efficiency and economics of the process. It may also prolong the half life of the enzyme.
Affectivity of the process can be ascertained as it’s substrate is a low molecular weight compound as
compared to those having high molecular weight substrates where the enzymes on or near the
surface of gels are only available for the reaction. Several techniques for immobilization of glucose
isomerase have been reported by researchers. Whole cell as well as enzyme immobilization is
investigated. Streptomyces phaeochromogenes cells have been immobilized with collagen in
membrane form, and it’s enzyme in polyacrylamide. Crosslinking with gluteraldehyde, entrapment in
gelatin, acrylamide [Strandberg and Smiley, 1971] and collagen membrane and chitosan beads,
Indion-R, cobalt hydroxide gels, silica xerogel were tried [Kovalenko et al., 2011; Sapunova et al.,
2003; Pawar and Deshmukh, 1994]. It has also been immobilized by covalently bonding the ruptured
cells on gelatin or casein [Gong et al., 1980]. In evaluating an immobilized-enzyme system, the
important factors to consider are the amount of enzyme loaded per unit of reactor column, the flow
properties of, and the stability of, the enzyme complex. Immobilization of whole cells containing GI
rather than GI itself has advantages especially in the industrial perspective. This process saves the
isolation and purification of the enzyme. The enzymatic activity increased with repeated use at the
early stages and then reached a constant value. This initial increase is one of the characteristics of
cells immobilized by radiation induced polymerization. The increase can be attributed to the swelling
effect of the polymer matrix by water, which increases the diffusivity of the substrate into cells
entrapped in the inner part of the matrix. It was ascertained that swelling of the polymer matrix by
water occurred at the initial stage of repeated use and then reached equilibrium after two or three
batch reactions. Thus, swelling of the matrix has the apparent effect of increasing enzymatic activity
of the whole cells. It is assumed that the diffusion of the substrate to the position of trapped cells is
promoted by the swelling of the polymer matrix. In addition, the mobility of trapped cells might
increase by the swelling, taking the more favored configuration for contact and reaction with
substrate in the matrix.

Immobilisation of an enzyme for industrial application requires optimisation of various

factors. A low cost support, immobilized enzyme product with high activity per weight of support
material, adequate operational stability and efficient use of the product are important
considerations. The pH and temperature optima of the immobilized enzyme may vary a little bit
from the soluble enzyme. In some cases it also influences the Michaelis constant (Km) for the
isomerisation reactions.

The substrate of immobilized GI, glucose can be produced from a cheap and disposable non
sweet source starch or cellulose. As we are agricultural dominated country, huge amount of
cellulosic agro-residues are generated every year. Chitosan is an ideal support material for enzyme
immobilization because of its hydrophilicity, biocompatibility, biodegradability, and anti-bacterial
property. The macromolecule is derived chemically by deacetylation of natural polymer chitin.
Furthermore, chitosan exhibits a considerable protein binding capacity and the immobilized enzyme
remains considerably active. Benzyl DEAE-cellulose, triethylaminoethylcellulose, and DEAE-cellulose
are also effective for immobilization of partially purified glucose isomerase. The properties of
purified soluble enzyme, immobilized enzyme (DEAE-celluloseglucose isomerase), and heat-treated
whole cells may vary. Anionic exchangers are effective in adsorbing glucose isomerase, whereas
cationic exchangers were not. Benzyl DEAE-cellulose and TEAE-cellulose retained more enzyme

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
activity and protein than other ion exchangers. However, these two supports are much more
expensive than DEAE-cellulose. DEAE-cellulose suspensions can adsorb high amounts of protein and
thus high enzyme titre, 105 mg of protein per g of support and 1,052 U/g of support respectively.

Applications of Glucose Isomerase

GI can be used for various purposes like HFCS production, ethanol production, and
conversion of biodegradable wastes of food and agro-
industries into utilizable products.

High Fructose Corn Syrup production: The major

application is High Fructose Corn Syrup production
which has already been dealt earlier.

Ethanol production: Glucose isomerase also has great

importance in production of ethanol from
hemicelluloses. GI has been used to produce xylulose
from xylose which can further be fermented to
ethanol by yeasts. The ever increasing demand of fuels
can be served to some extent by bioconversion of
renewable biomass to fermentable sugars. The
biomass consists of cellulose (40%), hemicellulose
(30%), and lignin (30%). The economic feasibility of
biomass utilization depends on the hydrolysis of
cellulose and hemicellulose to glucose and xylose
respectively and their subsequent fermentation to
ethanol by yeasts. Hemicelluloses currently represent
the largest polysaccharide fraction.

Cofermentation of Glucose and Xylose The

hydrolysed waste streams of agricultural residues
contain both hexose and pentose sugars.
Fermentation of xylose in presence of glucose is not
efficiently feasible. An organism which can ferment
both the sugars will be useful or co-immobilization of
Saccharomyces cerevisiae and the enzyme glucose isomerase can be used for the production of
ethanol.

Production of fructose syrup from whey permeate Besides making use of agro residues GI can also
be employed for converting dairy industry waste to useful syrup which is as sweet as sucrose.
Lactase-hydrolyzed whey permeate can be used to convert glucose present in it to fructose. Excess
of minerals should be removed from whey permeate before treatment with the enzyme. The
optimum enzyme activity can be achieved by adding 250 and 100 ppm of Mg2+ and Co2+ ions
respectively.

Immobilization of penicillin acylase

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
One of the major applications of immobilized enzymes in pharmaceutical industry is the
production of 6-aminopenicillanic acid (6-APA) by the deacylation of the side chain in either
penicillin G or V, using penicillin acylase (penicillin amidase). More than 50% of 6-APA produced
today is enzymatically using the immobilized route. One of the major reasons for its success is in
obtaining a purer product, thereby minimizing the purification costs. The first setting up of industrial
process for the production of 6-APA was in 1970s simultaneously by Squibb (USA), Astra (Sweden)
and Riga Biochemical Plant (USSR). Currently, most of the pharmaceutical giants make use of this
technology. A number of immobilized systems have been patented or commercially produced for
penicillin acylase which make use of a variety of techniques either using the isolated enzyme or the
whole cells. This is also one of the major applications of the immobilized enzyme technology in India.
Similar approach has also been used for the production of 7-aminodeacetoxy-cephalosporanic acid,
an intermediate in the production of semisynthetic cephalosporins.

Penicillin acylases (penicillin amidohydrolase; EC [Link]) catalyze the hydrolysis of

penicillins into 6-aminopenicillanic acid (6-APA) and an organic acid that depends on the type of
penicillin hydrolysed. Biotechnological applications of penicillin acylases (PA) have emerged as a
serious alternative to traditional chemical procedures for the manufacture of β-lactam antibiotics,
small peptides and pure isomers from racemic mixtures. However, penicillin acylases are involved
mainly in the industrial production of semi synthetic penicillins. They are classified in three sub-
groups, according to their preferential substrate: penicillin V acylase, penicillin G acylase and
ampicillin amidase. Penicillin G acylase (PGA), catalyzes the hydrolysis of penicillin G, is the most
important acylase because penicillin G is the cheapest raw material for the production of 6-APA, the
nucleus from which a range of semi-synthetic penicillins are made.

The standard industrial practice to produce semisynthetic β-lactam antibiotics employs a

chemical route, with protection/de-protection of reactive groups, low temperatures (-30 оC or less),
and organochloride solvents. Hence, their enzymatic synthesis has received great attention as a
possible “green chemistry” alternative. To fully exploit the technical and economical advantages of
highly cost penicillin acylase, it is recommended to use them in an immobilized form. This is because
free enzyme, as a biocatalyst, is lacking of long-term stability under process conditions and difficult
to recover and recycle from the reaction mixture, making the reuse of the enzyme impossible.
Hence, the idea of immobilizing the enzyme on a rigid solid support enabling easy separation and the
possibility of operation in a packed-bed or fluidized-bed reactor, has been of great industrial interest
for many years. Among the immobilization methods, covalent attachment is the more advantageous
than the other methods since diffusional restrictions to substrate or products are decreased
considerably. Moreover, the formation of the rigid enzyme-support linkage provides both kinetic and
thermodynamic stabilization of the three-dimensional structure of the active catalytic site and often
improves the enzyme thermal stability. The technology for PGA immobilization has been improved in
the last decades and PGA has been covalently immobilized to various supports such as
microparticulate and monolithic silica supports, poly (vinyl acetate–co-diviyl benzene) beads,
activated agarose, grafted nylon membranes etc. However, various problems associated with these
supports were also reported. One common problem is the lack of active sites on the polymer.
Consequently, most previous studies used glutaraldehyde as a non-specific cross-linking agent to fix
the enzyme on the polymeric matrix, but the results were often unsatisfactory-with low

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 2017
immobilization yield and low final enzyme activity. Therefore, development of new techniques for
PGA immobilization on inexpensive and industrially applicable carriers is of economical significance.
Epoxy-activated supports are almost-ideal ones to perform very easy immobilization of enzymes at
both laboratory and industrial scale. Epoxide groups are convenient for the covalent binding of
enzymes since they are able to directly react with amino, hydroxyl, or sulfhydryl groups of enzymes
depending on pH of the buffer used. Thus, it is not necessary to activate the carrier or enzyme to
achieve covalent immobilization. Moreover, the N-C, O-C or S-C bonds formed by the epoxide groups
are extremely stable, so that the epoxide-containing commercial polymers such as Eupergit ® or
Sepabeads can be successfully used for the immobilization of enzymes and proteins

Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21

 DEPARTMENT OF MICROBIOLOGY
KARPAGAM ACADEMY OF HIGHER EDUCATION
(Deemed to be University Established Under Section 3 of UGC Act, 1956)
Eachanari Post, COIMBATORE - 641 021, INDIA

[Link]., Microbiology (Batch 2016-2019)

Industrial Microbiology (Semester-III) (16MBU303)
LECTURE PLAN

UNIT V

Duration Topic Reference

01 Microbial production of Citric acid
02 Microbial production of Ethanol
03 Microbial production of Penicillin R1: 162-169
04 Microbial production of Glutamic acid R1: 112-119
05 Microbial production of Vitamin B12
06 Microbial production of Enzymes (amylase, protease, lipase)
07 Microbial production of Wine & Beer
08 Revision of all units and possible questions
09 Last five year old question paper discussion
10 Last five year old question paper discussion
11 Last five year old question paper discussion
Total hours: 11

R1: Patel A. H., 2003. Industrial Microbiology. Macmillan India ltd, NewDelhi.

Dr [Link]
Assistant Professor
Department of Microbiology
 2017

1
Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21
 2017
Unit V

PENICILLIN PRODUCTION:

Penicillin was the first naturally occurring antibiotic discovered. It is obtained in a number of forms from
Penicillium moulds. Penicillin is not a single compound but a group of closely related compounds, all with
the same basic ring-like structure (a β-lactam) derived from two amino acids (valine and cysteine) via a
tripeptide intermediate. The third amino acid of this tripeptide is replaced by an acyl group (R) and the nature
of this acyl group produces specific properties on different types of penicillin.

There are two different types of penicillin.

Biosynthetic penicillin is natural penicillin that is harvested from the mould itself through fermentation.
Semi-synthetic penicillin includes semi synthetic derivatives of penicillin - like Ampicillin, Penicillin V,
Carbenicillin, Oxacillin, Methicillin, etc. These compounds consist of the basic Penicillin structure, but have
been purposefully modified chemically by removing the acyl group to leave 6-aminopenicillanic acid and
then adding acyl groups that produce new properties. These modern semi-synthetic penicillins have various
specific properties such as resistance to stomach acids so that they can be taken orally, a degree of resistance
to penicillinase (or β-lactamase) (a penicillin-destroying enzyme produced by some bacteria) and an
extended range of activity against some Gram-negative bacteria. Penicillin G is the most widely used form
and the same one we get in a hypodermic form.

PENICILLIN G
Penicillin G is not stable in the presence of acid (acid-labile). Since our stomach has a lot of hydrochloric
acid in it (pH2.0), if we were to ingest penicillin G, the compound would be destroyed in our stomach before
it could be absorbed into the bloodstream, and would therefore not be any good to us as a treatment for
infection somewhere in our body. It is for this reason that penicillin G must be taken by intramuscular
injection - to get the compound in our bloodstream, which is not acidic at all. Many of the semi-synthetic
penicillins can be taken orally.
Penicillium chrysogenum that produce antibiotics, enzymes or other secondary metabolites frequently
require precursors like purine/pyrimidine bases or organic acids to produce said metabolites. Primary
metabolism is the metabolism of energy production for the cell and for its own biosynthesis. Typically, in
aerobic organisms (Penicillium chrysogenum) it involves the conversion of sugars such as glucose to pyruvic
acid2 and the production of energy via the TCA cycle. Secondary metabolism regards the production of
metabolites that are not used in energy production for example penicillin from Penicillium chrysogenum. In
this case the metabolite is being utilized as a defence mechanism against other microorganisms in the
2
Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21
 2017
environment. In essence Penicillium chrysogenum can kill off the competition to allow itself to propagate
efficiently. It should be noted that these secondary metabolites are only produced in times of stress when
resources are low and the organism must produce these compounds to kill off its competitors to allow it to
survive.

MEDIA FORMULATION:
Lactose: 1%
Calcium Carbonate: 1%
Cornsteep Liquor: 8.5%
Glucose: 1%
Phenyl acetic acid: 0.5g
Sodium hydrogen phosphate: 0.4%
Antifoaming Agent: Vegetable oil

FERMENTATION

To begin the fermentation process, a number of these spores will be introduced into a small (normally 250-
500ml) conical flask where it will be incubated for several days. At this stage, explosive growth is the most
desired parameter and as such the medium in the flask will contain high amounts of easily utilisable carbon
and nitrogen sources, such as starch and corn-steep liquor. At this stage, the spores will begin to revive and
form vegetative cells. Temperature is normally maintained at 23-280C and pH at ~6.5, although there may be
some changes made to facilitate optimum growth. The flask will often have baffles in it and be on a shaking
apparatus to improve oxygen diffusion in the flask.
Once the overall conditions for growth have been established and there is a viable vegetative culture active
inside the flask, it will be transferred to a 1 or 2 litre bench-top reactor. This reactor will be fitted with a
number of instruments to allow the culture to be better observed than it was in the shake flask. Typical
parameters observed include pH, temperature, and stirrer speed and dissolved oxygen concentration. This
allows tweaking of the process to occur and difficulties to be examined. For example, there may not be
enough oxygen getting to the culture and hence it will be oxygen starved. At this point, the cells should be
showing filamentous morphology, as this is preferred for penicillin production. As before, cell growth is
priority at this stage. At this stage, growth will continue as before, however, there are often sudden changes
or loss in performance. This can be due to changes in the morphology of the culture (Penicillium
chrysogenum is a filamentous fungi and hence pseudoplastic) that may or may not be correctable.
At this stage the medium being added to the reactor will change. Carbon and nitrogen will be added sparingly
alongside precursor molecules for penicillin fed-batch style. Another note is that the presence of penicillin in
the reactor is itself inhibitory to the production of penicillin. Therefore, we must have an efficient method for
the removal of this product and to maintain constant volume in the reactor. Other systems, such as cooling
water supply, must also be considered. If all goes well we should have penicillin ready for downstream
processing. From here it can be refined and packaged for marketing and distribution to a global market.
PRODUCTION OF 6-AMINO PENICILLANIC ACID
The penicillins G and H are mostly used as the starting material for the production of several
synthetic penicillins containing the basic nucleus namely 6-amino penicillanic acid (6- APA). Immobilized
penicillin amidases enzymes have been developed for specific hydrolysis of penicillin G and V. penicillin
salt of either G or V can be used for hydrolysis by immobilized enzyme system. The pH during hydrolysis is
kept around 7-8, and the product 6-APA can be removed by bringing down pH to 4 by precipitation in the
3
Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21
 2017
presence of water immiscible solvent. In general, the enzymatic hydrolysis is more efficient for penicillin V
than for G. However, penicillin G is more versatile compound, as it required for ring expansions.

PRODUCTION OF VITAMINS
Definition:
Vitamins are organic substances that are essential in small amounts for the health, growth, reproduction, and
maintenance of one or more animal species, which must be included in the diet since they either cannot be
synthesized at all or in sufficient quantities in the body. Each vitamin performs specific function; hence, one
cannot replace or, act for, another. In general, the human body cannot synthesize them, at least in
large enough amounts to meet its needs. However vitamin D is exception; when a person is exposed to
ultraviolet rays, the vitamin is synthesized from its precursor, which is found in the skin.
Vitamins: General Aspects
Nomenclature and Classification:
There is no universal agreement on the nomenclature of the vitamins. But the modern tendency is to use the
chemical name, particularly in describing! Members of the B complex. Today vitamins are generally classed
as (1) fat-soluble vs water-soluble vitamins, (2) vitamin B complex and (3) vitamin like substances.
Fat-Soluble vs Water-Soluble Vitamins: Several phenomena of vitamin nutrition are related to solubility.
Vitamins are soluble in either fat or water. Consequently, it is important that both! nutritionists and
consumers be well informed about solubility differences in vitamins and make use of such differences in
programs and practices. Based on solubility, vitamins may be grouped as follows;

The Fat-Soluble Vitamins The Water-Soluble Vitamins

Vitamin A Choline
Folacin (folic acid)
Vitamin E Niacin (nicotinic acid; nicotinamide)
Pantothenic acid (vitamin B-3)
Vitamin D Thiamin (vitamin B-1)
Riboflavin (B-2)

Vitamin K Vitamin B-6 (pyridoxine; pyridoxal;

Qyridoxamine)
Vitamin C (ascorbic acid; dehydroascorbic
acid)
Vitamin B-12 (cobalamins)

It is necessary that vitamin C is the only member of the water soluble group that is not a member of the B
family.
Chemical composition: The fat-soluble vitamins contain only carbon, hydrogen, and oxygen, whereas the
water-soluble B vitamins contain these three elements plus nitrogen.
Occurrence: -Vitamins originate primarily in plant tissues which includes vegetable products (Cabbage,
carrot, orange, lemon, corn or maize oil, and corn or maize bran etc.) and microorganisms (brewers yeast”
bakers yeast etc.). Vitamins are also obtained from animal products like fish ox or calf liver oil, eggs etc.
Vitamins C and D are present in animal tissues only. If an animal consumes foods containing them or harbors
microorganisms that synthesize them. Fat-soluble vitamins can occur in plant tissue in the form of a pro-
vitamin (or precursor of a vitamin), which can be converted into a vitamin in the animal body. But no pro-
4
Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21
 2017
vitamins are known for any water soluble vitamin. Also, the B vitamins are universally distributed in all
living tissues, whereas the fat-soluble vitamins are completely absent from some.

Storage: The fat-soluble vitamins are stored in appreciable quantities in the body, whereas the water-soluble
vitamins are not. Any of the fat-soluble vitamins can be stored wherever fat is deposited; and the greater the
intake, the greater the storage.

Excretion: The fat-soluble vitamins are excreted exclusively in the faces via the bile. The water-soluble
vitamins ~e excreted primarily in the urine, although limited amounts may be present in the faeces. This
difference in pathway of excretion reflects the difference in solubility.
Vitamin B Complex
With the exception of vitamin C, all of the water-soluble vitamins 2an,be grouped together under the vitamin
B complex, of which there are the following nine: biotin, choline, folacin (folic acid), niacin. (nicotinic acid;
nicotinamide), pantothenic acid (vitamin B3), riboflavin (vitamin B-2), thiamin (vitamin B-1)” vitamin B6
(pyridoxine; pyridoxal; pyridoxamine), vitamin B12 (cobalamins) Vitamin B12 belongs to a group of
compounds known as cobalamins. Chemically, vitamin B12is composed of a central cobalt atom coordinated
by a porphyrin-like group called corrin. The axial coordination sites are occupied by a base,
dimethylbenzimidazole,. and a cyano group. Other cobalamins are also known. The compounds containing
bases different from dimethylbenzimidazole are known as B12analogs.
Vitamin B12 (Cobamide) Production
Vitamin B12, like so many other members of the B complex, is not a single substance; rather, it consists of
several closely related compounds with similar activity. The term cobalamins is applied to this group of
substances because, all of them contain cobalt. VitaminB-12, which is the most active member, is
cyanocobalamin, name after the cyanide ion in the~molecule. Other chemically related compounds known to
have vitamin B12activity include hydroxocobalamin, nitritocobalamin, and thiocyanate cobalamin.
Chemistry: Vitamin B12is the largest and the most complex of vitamin molecules. The main part of the
molecule consists of a porphyrin ring containing cobalt as the central element. A cyanide (CN) group may be
attached to the cobalt, in which case the compound is called cyanocobalamin (or vitamin BI2); the
commercially available form of the vitamin, little of which occurs naturally. A hydroxy group can replace the
cyanide group attachment to the cobalt (-OH), giving hydroxocobalamin, the common naturally occurring
form of the vitamin; or it can be replaced by a nitrite group (-NO2), giving nitritocobalamin, a form found in
certain bacteria. Vitamin BI2 is, the only vitamin produced by fermentation. For the commercial production
of riboflavin, chemical or semi
synthetic procedures are feasible. Various types of microorganisms like :
 Pseudomonas (i.e. P.,fmitrificans) and
 Propionibacterium (P. shermanii and P. freudenreichii) are
used for commercial production of vitamin BI2, These were preferred to Streptomycin and other species
because of their rapid growth and high productivity of selected mutants.
Vitamin BI2 production includes a laboratory-scale fermentation process from Pseudomonas denitrificans
and pilot plant-scale fermentation process from Propionibacerium shermanii. Vitamin B12 from
Pseudomonas denitrificans:
In this one stage process vitamin B12 is produced during the entire fermentation. Cobalt and 5,6-
dimethylbenzimidazole must be added as supplements. It has also been observed that addition of the
compound betaine results in increased yield, sugar beet molasses is used as low cost betaine source. Mutant

5
Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21
 2017
strains have been developed from the original wild type of P. denitrificans for commercial production of
vitamin B12
Medium:
Medium for laboratory-scale fermentation process for vitamin B12 from Pseudomonas denitrificans includes
(in g/L)
beet molasses (60.0),
brewers yeast (1.0),
N2 amine (1.0),
(NH4hHPO4 (2.0),
MgSO4.7H2O (1.0), MnSO4.7H} O (0.2),
ZnSO4.7H2O (0.02), NazMoO4.2H2O (510-3),
Agar (25.0),
and
pH (to 7.4).
The inoculated medium is incubated at 28°Cfor 4 days.
Seed culture medium includes
the same composition as mentioned above but it is devoid of agar. It is incubated for 3
days at 28°C on rotary shaker.
Carbohydrate source: Sugar beet molasses containing 5.)0% betaine (trimethyl-glycine). It stimulates the
production of vitamin B12 by promoting synthesis of Ala-synthetase. It helps in production of 8-
aminotvulinic acid (the first intermediate of the pathway of cobalamin biosynthesis). The Cobalamin
biosynthesis by Pseudomonas denitrificans require external supply of 5,6- dimethylebenzimidozole(5,6-
DBf) and cobalt salt.
Fermentation: Aerated condition with agitation bioreactor is used for growth of. Pseudomonas and
biosynthesis of vitamin B12
Flow chart for production of vitamin B12 from Pseudomonas denitrificans:
Pseudomonas denitrificans

Inoculum cultivation


Preculture


Production culture

Vitamin B12 from Propionibacterium Shermanii’ These and other mutantstrains are used in a two stage
process with added cobalt. In a preliminary anaerobic phas (2-4days),5’- deoxyadenosylcobinamide is
mainlyproduced ,in a second aerobic phase(3-4 days) the biosynthesis of 5,6- dimethylbenzimidiazole takes
place, so that 5’-deoxyadenosylcobalamin can be produced. Only traces of other cobamides are synthesized
in this process. As an alternative to the two stage batch process in a fermenter
,both stages can also be operated continuously in two tanks operated in cascade fashion. During the recovery
process ,the cobalamins which are almost completely bound to the cell are brought into solution by heat
treatment (10-30min at 80-120degree C,pH 6.5-8.5).They are then converted chemically into the more stable
cyanocobalamin. The raw product 80% purity is used as a feed additive.
6
Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21
 2017
Seed Culture Medium: This is different types and prepared according to stages.
1. First stage medium: It is similar to maintenance medium
but is devoid of agar. It is incubated for 2 days at 30°C without agitation.
2. Second stage medium: It includes
corn steep 20g/L,
glucose 90 g/l
and pH maintained at 6.5.
Stainless steel bioreactor is used. The medium is incubated at 30°C for 24 hrs without aeration, pH is
adjusted to 6.5.
Main culture medium: The production media includes
(in g/l)
corn steep liquor (40.0),
glucose (100.0), COCI2.6H2O (0.02),
and pH (to 7.0).
It is incubated at 30DC. The first 80 hrs is allowed to proceed without aeration, with slight nitrogen with
agitation and slight aeration (0.1 v/v/m). pH is adjusted to 7.0. Propionic organisms are grown on
carbohydrate-based media in an ul1aerated conditions. These organisms produce cobaltocorrinoids. Cobalt
supplement is necessary for cobalamin production. It also depends on the internal formation or external
supply of 5,6-dimethylbenzimidazole (5,6-DBI). The mutant strains of p. [Link] their own 5,6-
DBI. It increases the yield up to 65 mg/L on pilot-scale. Aeration promotes formation of 5,6-DBI, but it
decreases vitamin BI2 biosynthetic pathway at one of the stages. Therefore, at first stage (80 hrs)
fermentation is conducted in absence of oxygen (anaerobic condition), but with little agitation until all the
sugar in the media is utilized for the growth and the formation of cobinamide. This kind of operation will
have no repressive effect. The subsequent stage (next 88 hrs) is followed with agitation and slight-aeration.
Aeration at this stage induces biosynthesis of 5,6-DBI and cobinamide is converted in to cobalamin.
Isolation and Purification
The extraction process depends on the release of unstable cobalamin from the cells lysed and subsequent
treatment of this with cyanide to convert into more stable cyanocobalamin. Cells are separated from the
culture broth. Cells are lysed by heat treatment at 80 to l20°C for 1O-3O minutes at pH 6.5-8.5. The cells on
lysis release various cobalamin. These are then solubilized with potassium cyanide in the presence of sodium
nitrite. The obtained cobalamin gets converted into cyanocobalamin. The purification of the product is done
using adsorption method for substances like amberlite IRC SO, Dowex Ix2, alumina, silanized silica gel, and
Amberlite XAD2. It is then followed by elution with water-alcohol or water-phenol mixtures.

Enzymes are biological catalysts which are an indispensible component of biological reactions. The
use of chemical catalysts has been followed for a very long time. Chemical catalysis though widely used was
very cumbersome. The disadvantages that this method poses include need for high temperature and pressure
for catalysis and the moderate specificity. These limitations were overcome by the use of enzymes. Enzymes
work at milder conditions when compared to that required by chemical catalysts for operation. Also enzymes
are highly specific and catalyze reactions faster than chemical catalysts [1]. Enzymes are now being used in
various sectors of industry. They are used in detergents, paper industry, textile industry, food industry and
many others industrial applications. Enzymes have been in use since ancient times [2] and they have been
used in saccharification of starch, production of beverages like beer, treatment of digestive disorders and
production of cheese from milk [3]. Among the many enzymes that are widely used α-Amylase has been in

7
Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21
 2017
increasing demand due to its crucial role of starch hydrolysis and the applications of this hydrolytic action.
The following sections elaborate on the types of amylases and their roles in enzymatic reactions.
Production of α-Amylase

Sources

α-Amylase can be isolated from plants, animals or microorganisms. The enzyme has been isolated
from barley and rice plants [7]. It has been found that cassava mash waste water is a source of α-Amylase
which is active in wide range of pH and temperature [8]. In the recent past, there has been extensive research
on microbial production of α-Amylase. There are 2 major reasons for the increasing interest in microbial
sources:

1) The growth of microorganisms is rapid and this will in turn speed up the production of enzyme.
Microorganisms are easy to handle when compared to animals and plants. They require lesser space and
serve as more cost effective sources.

2) Microorganisms can be easily manipulated using genetic engineering or other means. They can be
subjected to strain improvement, mutations and other such changes by which the production of α-Amylase
can be optimized. Also, the microorganisms can be tailored to cater to the needs of growing industries and to
obtain enzymes with desired characteristics like thermostability for example. Thermostable α-Amylases are
desired as they minimize contamination risk and reduce reaction time, thus saving considerable amount of
energy. Also when hydrolysis is carried out at higher temperatures, the polymerization of D-glucose to iso-
maltose is minimized [9].

α-Amylase is produced by several bacteria, fungi and genetically modified species of microbes. The
most widely used source among the bacterial species is
the Bacillus spp. B. amyloliquefaciens and B. licheniformis are widely used for commercial production of the
enzyme. Other species which have been explored for production of the enzyme
include [Link] and B. subtilis to name a few. α-Amylases produced
from Bacillus licheniformis, Bacillusstearothermophilus, and Bacillus amyloliquefaciens show promising
potential in a number of industrial applications in processes such as food, fermentation, textiles and paper
industries [9, 10]. Bacillus subtilis, Bacillusstearothermophilus, Bacillus licheniformis and Bacillus amylolique
faciens are known to be good producers of thermostable α-Amylase. Thermostability is an important
characteristic as enzymatic liquefaction and saccharification of starch are performed at high temperatures
(100–110°C). Thermostable amylolytic enzymes are being investigated to improve industrial processes of
starch degradation and are useful for the production of valuable products like glucose, crystalline dextrose,
dextrose syrup, maltose and maltodextrins. Use of enzyme produced by thermophiles has the added
advantage of reduced risk of contamination by mesophiles. Enzymes produced by some halophilic
microorganisms are stable at high salinities and therefore could be used in many harsh industrial processes
where the concentrated salt solutions are used [4]. The halophilic nature the enzyme prevents inhibition of its
activity under these conditions which would otherwise occur if a normal enzyme is used. In addition, most
8
Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21
 2017
halobacterial enzymes are considerably tolerant to high temperatures and remain stable at room temperature
over long periods [4]. Halophilic amylases from halophilic bacteria such
[11]
as Chromohalobactersp., Halobacillus sp., Haloarcula hispanica, Halomonas meridiana ,
and Bacillus dipsosauri have been characterized.
Fungal sources of α-Amylase are confined to terrestrial isolates, mostly to Aspergillus species and to
only few species of Penicillium, P. brunneum being one of them [12]. Penicillium fellutanum has been used in
the recent past to produce α-Amylase by submerged fermentation [12]. Penicillium expansum MT-1 has been
used to produce the enzyme by solid state fermentation. Here, Loquat (Eriobotrya japonica Lindley) kernels
were used as the substrate for growth of the fungi [13]. Penicillium chrysogenum was used as the microbial
source for producing amylase by solid state fermentation using various substrates such as, corncob leaf, rye
straw, wheat straw and wheat bran [14]. The fungal source used predominantly for commercial production of
α-Amylase are the strains of Aspergillus spp. Aspergillus oryzae, A. niger and A. awamori are most
commonly used species for commercial production among several others [9]. Aspergillus fumigatus has been
employed for production of the enzyme by submerged fermentation technique [15].

Genetically modified organisms are also being used for production of α-Amylase. There are various
methods by which microorganisms can be manipulated at a genetic level in order to improve and optimize
the production of this enzyme. The microbes can be mutated by chemical agents. Nitrous acids or ethyl
methane sulphonate (EMS) have been used for genetic manipulation of bacterial
strains [16]. Bacillus amyloliquefaciens UNG-16 was subjected to mutation by both the chemical (EMS) and
radiation method. The mutant strain exhibited an activity of 102.78±2.22 U/ml/min which was 1.4 times
greater than the parent strain [17]. α-Amylase production in [Link] Marburg was improvised by treatment
with N-methyl-N'-nitro-N-nitrosoguanidine. One of the mutants, YN9, showed a threefold increase in
production of the enzyme when compared to the parent strain [18]. The microbial sources and methods used to
produce α-Amylase are listed in Table 1.

Production Methods

There are mainly two methods which are used for production of α-Amylase on a commercial scale.
These are: 1) Submerged fermentation and 2) Solid State fermentation. The latter is a fairly new method
while the former is a traditional method of enzyme production from microbes which has been in use for a
longer period of time.

Submerged fermentation (SmF) employs free flowing liquid substrates, such as molasses and broths. The
products yielded in fermentation are secreted into the fermentation broth. The substrates are utilized quite
rapidly; hence the substrates need to be constantly replenished. This fermentation technique is suitable for
microorganisms such as bacteria that require high moisture content for their growth. SmF is primarily used
for the extraction of secondary metabolites that need to be used in liquid form [19]. This method has several
advantages. SmF allows the utilization of genetically modified organisms to a greater extent than SSF. The
sterilization of the medium and purification process of the end products can be done easily. Also the control

9
Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21
 2017
of process parameters like temperature, pH, aeration, oxygen transfer and moisture can be done
conveniently [20].

Solid state fermentation is a method used for microbes which require less moisture content for their growth.

The solid substrates commonly used in this method are, bran, bagasse, and paper pulp. The main advantage is
that nutrient-rich waste materials can be easily recycled and used as substrates in this method. Unlike SmF,
in this fermentation technique, the substrates are utilized very slowly and steadily. Hence the same substrate
can be used for a longer duration, thereby eliminating the need to constantly supply substrate to the
process [20]. Other advantages that SSF offers over SmF are simpler equipments, higher volumetric
productivity, higher concentration of products and lesser effluent generation [19]. For several such reasons
SSF is considered as a promising method for commercial production of enzymes.

Purification of α-Amylase

Enzymes used for industrial applications are usually crude preparations and require less downstream
processing. Whereas the enzymes used clinical and pharmaceutical industry need to be highly purified. Also
when used for study of structure function relationships and biochemical properties the enzymes have to be in
purified form [2]. Purification methods commonly employed are precipitation, chromatography and liquid-
liquid extraction depending on the properties of the enzyme desired. A combination of the above methods is
used in a series of steps to achieve high purity. The number of steps involved in purification will depend on
the extent of purity that is desired [30, 59, 60, 61]. The crude extracellular enzyme sample can be obtained from
the fermented mass by filtration and centrifugation. In the case of intracellular enzymes, raw corn starch may
be added followed by filtration and subsequent steps. The crude amylase enzyme can be precipitated and
concentrated using ammonium sulphate precipitation or organic solvents. The precipitated sample can be
subjected to dialysis against water or a buffer for further concentration [68]. This can be followed by any of
the chromatographic techniques like ion exchange, gel filtration and affinity chromatography for further
separation and purification of the enzyme. In a method of purification of the enzyme produced
by Aspergillus falvus var. columnaris, the enzyme was precipitated was followed by dialysis and then
column chromatography [68]. In a study of Amylase production by Preussia minim, the sample was
precipitated using trichloroacetic acid (TCA)/acetone and applied to a Sephadex G-200 gel filtration column.
The resulting fractions were pooled and further applied to a DEAE-Sepharose ion exchange column [57]. In a
study of purification and characterization of an extracellular α-Amylase from Clostridium perfringens Type
A, crude enzyme concentrate was prepared by precipitation with polyethylene glycol. This concentrated
sample was then separated by DEAE Sephacel chromatography resulting in three distinct amylolytic peaks.
Fractions from a single peak were then collected and chromatographed on the same column, followed by
separation of obtained active fractions on a Sephacryl S-100 HR column [69]. Purification of α-Amylase
produced by a mutant Bacillus subtilis strain was done in a series of steps employing precipitation with 80%
ammonium sulfate, TSK Toyopeal column chromatography, ultrafiltration, dialysis and SP Sepharose
column chromatography [70]. In case of thermotolerant amylases, the cell extract obtained after centrifugation

10
Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21
 2017
which is free of cell debris can be subjected to high temperatures, in order to denature thermolabile proteins.
For purification of α-Amylase produced by Thermotoga maritima MSB8, this step was followed by anion
exchange chromatography. The purity was then analysed by using SDS-PAGE [71]. The purity can also be
analysed by size exclusion chromatography wherein the molecular weight of the purified protein can be
determined.

Industrial Applications of α-Amylase

α-Amylase is gaining increased attention due to its starch hydrolyzing properties and the activities that can be
carried out owing to this property. There are many potential and widely used applications of this enzyme on
the industrial front. Enzymes have replaced the previously used chemical methods of hydrolysis in various
industrial sectors to make the process environment friendly and make processes easier.

MICROBIAL PRODUCTION OF CITRIC ACID

Citric acid (C6H8O7, 2 - hydroxy - 1,2,3 - propane tricarboxylic acid), a natural constituent and
common metabolite of plants and animals, is the most versatile and widely used organic acid in the field of
food (60%) and pharmaceuticals (10%). It has got several other applications in various other fields.
Currently, the global production of citric acid is estimated to be around 736000 tones/year, and the entire
production is carried out by fermentation. In Brazil, almost the entire demand of citric acid is met through
imports. There is constant increase (3.5-4%) each year in its consumption, showing the need of finding new
alternatives for its manufacture.
Historical developments
Citric acid was first isolated by Karls Scheels in 1874, in England, from the lemon juice imported
from Italy. Italian manufacturers had monopoly for its production for almost 100 years, and it was sold at
high cost. This led extensive attempts all over the world to find alternatives way for its production, which
included chemical and microbial techniques. In 1923, Wehmer observed the presence of citric acid as a by-
product of calcium oxalate produced by a culture of Penicillium glaucum. Other investigations showed the
isolation of two varieties of fungi belonging to genus Citromyces (namely Penicillium). However, industrial
trials did not succeed due to contamination problems and long duration of fermentation. The industrial
process was first open by Currie, in 1917, who found that Aspergillus niger had the capacity to accumulate
significant amounts of citric acid in sugar based medium. He also showed that high concentrations of sugar
favoured its production, which occurred under limitation of growth. In the thirties, some units were
implanted in England, in Soviet Union, and in Germany for the commercial production. However, the
biochemical basis was only cleared in the fifties with the discovery of the glycolytic pathway and the
tricarboxylic acid cycle (TCA). Consequently, an improved process employing submerged fermentation was
developed in United States. Although methods were well developed to synthesis citric acid using chemical
means also, better successes were achieved using microbial fermentations, and over the period of time, this
technique has become the method of ultimate choice for its commercial production, mainly due to economic
advantage of biological production over chemical synthesis. Much attention has been paid on research to
improve the microbial strains, and to maintain their production capacity.
Applications of citric acid
Citric acid is mainly used in food industry because of its pleasant acid taste an its high solubility in water. It
is worldwide accepted as "GRAS" (generally recognized as safe), approved by the Joint FAO/WHO Expert
11
Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21
 2017
Committee on Food Additives. The pharmaceutical and cosmetic industries retain 10% of its utilization and
the remainder is used for various other purposes.

MICRO-ORGANISMS USED FOR CITRIC ACIC PRODUCTION

A large number of micro-organisms including bacteria, fungi and yeasts have been employed to produce
citric acid. Most of them, however, are not able to produce commercially acceptable yields. This fact could
be explained by the fact that citric acid is a metabolite of energy metabolism and its accumulation rises in
appreciable amounts only under conditions of drastic imbalances. Kubicek and Rohr (1986) reviewed the
strains reported to produce citric acid. Among these, only A. niger and certain yeasts such
as Saccharomycopsis sp. are employed for commercial production. However, the fungus A. niger has
remained the organism of choice for commercial production. The main advantages of using this micro-
organism are: (a) its ease of handling, (b) its ability to ferment a variety of cheap raw materials, and (c) high
yields.
Strains selection and improvement
The two principal methods of selecting populations, namely, "the single-spore technique" and the "passage
method" have been used for selecting citric acid producing micro-organisms The single-spore technique has
the disadvantage that mineral acid or organic acids (gluconic acid, oxalic acid) simulate the presence of citric
acid. Rohr et al. (1979) improved this method by incorporating a specific stain for citric acid (para-di-
methylamino benzaldehyde), instead of using the indicator.
The most employed technique to improve citric acid producing strains has been by inducing mutations in
parental strains using mutagens. Among physical mutagens, g-radiation (Bonatelli and Azevedo, 1983 ;
Gunde-Cimerman, 1986 ; Islam et al., 1986) and UV-radiation (Pelechova et al., 1990) have often used. To
obtain hyper-producer strains, frequently UV treatment could be combined with some chemical mutagens,
e.g. aziridine, N-nitroso-N-methylurea or ethyl methane-sulfonate (Musilkova et al., 1983). By using a
suitable selection technique on model medium with non-specific carbon sources, a strain yielding high
amounts of citric acid from unusual substrates can be obtained from the mutants produced.
PRODUCTION TECHNIQUES AND RAW MATERIALS

12
Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21
 2017
Although citric acid is mostly produced from starch or sucrose based media using liquid fermentation, a
variety of raw materials such as molasses, several starchy materials and hydrocarbons have also been
employed. Rohr et al. (1983) classified raw materials used for citric acid production in to two groups: (i) with
a low ash content from which the cations could be removed by standard procedures (e.g. cane or beet sugar,
dextrose syrups and crystallized dextrose); (ii) raw materials with a high ash content and high amounts of
other non sugar substances (e.g. cane and beet molasses, crude unfiltered starch hydro-lysates).
Several attempts have been made to produce citric acid using molasses, which is preferred due its low cost
and high sugar content (40-55%). The composition of molasses depends on various factors, e.g. the kind of
beet and cane, methods of cultivation of crops and fertilizers and pesticides applied during cultivation,
conditions of storage and handling (e.g. transport, temperature variations), production procedures, etc. Both,
cane and beet molasses are suitable for citric acid production. However, beet molasses is preferred due to its
lower content of trace metals. Generally, cane molasses contains calcium, magnesium, manganese, iron and
zinc, which have a retarding effect on the synthesis of citric acid. Consequently, some pre-treatment is
required for the removal/reduction of trace metals. Despite that, cane molasses posses difficulties in
achieving good fermentation yields.
Various other agro-industrial residues such as apple pomace, cassava bagasse, coffee husk, wheat straw,
pineapple waste, sugar beet cosset, kiwi fruit peel, etc. have been investigated with solid state fermentation
techniques for their potential to be used as substrates for citric acid production (Pandey and Soccol, 1998,
Pandey et al. 1999, Vandenberghe et al., 1999a,b, c). In fact, these residues are very well adapted to solid-
state cultures due to their cellulosic and starchy nature. However, despite the fact that these solid residues
provide rich nutrients to the micro-organisms, and are good substrates for growth and activity of micro-
organisms, much remains to be done for developing commercially feasible process utilizing these residues
(Pandey 1992, 1994, Pandey and Soccol 1998).
Liquid fermentation
Submerged fermentation: The submerged fermentation (SmF) process is the commonly employed
technique for citric acid production. It is estimated that about 80% of world production is obtained by SmF.
Several advantages such as higher yields and productivity and lower labour costs are the main reasons for
this. Two types of fermenters, conventional stirred fermenters and tower fermenters are employed, although
the latter is preferred due to the advantages it offers on price, size and operation (Rohr et al., 1983).
Preferentially, fermenters are made of high-grade steel and require provision of aeration system, which can
maintain a high dissolved oxygen level. Fermenters for citric acid production do not have to be built as
pressure vessels since sterilization is performed by simply steaming without applying pressure. Cooling can
be done by an external water film over the entire outside wall of the fermenter.
In SmF, different kinds of media are employed such as sugar and starch based media (Table 3). Molasses and
other raw materials demand pre-treatment, addition of nutrients and sterilization. Inoculation is performed
either by adding a suspension of spores, or of pre-cultivated mycelia. When spores are used, a surfactant is
added in order to disperse them in the medium. For pre-cultivated mycelia, an inoculum size of 10% of fresh
medium is generally required. Normally, submerged fermentation is concluded in 5 to 10 days depending on
the process conditions. It can be carried out in batch, continuous or fed batch systems, although the batch
mode more frequently used.
Surface fermentation: The first individual process for citric acid production was the liquid surface culture
(LSC), which was introduced in 1919 by Société des Produits Organiques in Belgium, and in 1923 by Chas
Pfizer & Co. in US. After that, other methods of fermentation, such as submerged fermentation were
developed. Although this technique is more sophisticated, surface method required less effort in operation
and installation and energy cost (Grewal and Kalra, 1995).
13
Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21
 2017
In the classical process for citric acid manu-facture, the culture solution is held in shallow trays (capacity of
50-100 L) and the fungus develops as a mycelial mat on the surface of the medium. The trays are made of
high purity aluminium or special grade steel and are mounted one over another in stable racks. The
fermentation chambers are provided with an effective air circulation in order to control temperature and
humidity. Fermentation chambers are always in aseptic conditions, which might be conserved principally
during the first two days when spores germinate. Frequent contamination are mainly caused by Penicilia,
other Aspergilli, yeast and lactic bacteria (Rohr et al, 1983; Morgant, 1988). Refined or crude sucrose, cane
syrup or beet molasses are generally used as sources of carbon. When applied, molasses is diluted to 15-20%
and is treated with hexacyanoferrate (HFC).
Solid-state fermentation
Solid-state fermentation (SSF) has been termed as an alternative method to produce citric acid from agro-
industrial residues (Pandey 1991, 1992, 1994, Soccol 1994, Pandey and Soccol 1998). Citric acid production
by SSF (the Koji process) was first developed in Japan and is as the simplest method for its production. SSF
can be carried out using several raw materials. Generally, the substrate is moistened to about 70% moisture
depending on the substrate absorption capacity. The initial pH is normally adjusted to 4.5-6.0 and the
temperature of incubation can vary from 28 to 30°C. The most commonly organism is A. niger. However
there also have been reports with yeasts (Maddox and Kingston, 1983; Tisnadjaja et al., 1996). One of the
important advantages of SSF process is that the presence of trace elements may not affect citric acid
production so harmfully as it does in SmF. Consequently, substrate pre-treatment is not required.
Different types of fermenters such as conical flasks, glass incubators and trays, etc. have been used for citric
acid fermentation in SSF. Vandenberghe et al. (1999a,b) used Erlen-meyer flasks and glass columns for the
production of citric acid from gelatinized cassava bagasse. Higher yields were obtained in flasks without any
aeration, and very little sporulation was observed. The same yields were found in column reactors only with
variable aeration. This showed great perspective to use SSF process for citric acid production in simple tray
type fermenters.
Aeration: Aeration has been shown to have a determinant effect on citric acid fermentation (Rohr et al.,
1983; Dawson et al., 1986). Increased aeration rates led to enhanced yields and reduced fermentation time
(Grewal and Kalra, 1995). The influence of dissolved oxygen concentration on citric acid formation has been
examined. It is important to maintain the oxygen concentration above 25% saturation and interruptions in
oxygen supply may be quite harmful (Kubicek et al., 1980). The high demand of oxygen is fulfilled by
constructing appropriate aeration devices, which is also dependent on the viscosity of the fermentation broth.
This is an additional reason why small compact pellets are the preferred mycelial forms of A. niger during
fermentation (Kubicek and Rohr, 1986). When the organism turns into filamentous developments, e.g. due to
metal contamination, the dissolved oxygen tension rapidly falls to less than 50% of its previous value, even if
the dry weight has not increased by more than 5%. Aeration is performed during the whole fermentation with
the same intensity through the medium at a rate of 0.5 to 1.5 vvm. However, because of economic reasons,
it's usually preferred to start with a low aeration rate (0.1 to 0.4 vvm). High aeration rates lead to high
amounts of foam, especially during the growth phase. Therefore, the addition of antifoaming agents and the
construction of mechanical “defoamers” are required to tackle this problem.
PRODUCT RECOVERY The recovery of citric acid from liquid fermentation is generally accomplished by
three basic procedures, precipitation, extraction, and adsorption and absorption (mainly using ion exchange
resins). Citric acid extraction has been described by the Food and Drug Administration (1975) of the United
States and by Colin (1960,1962). Citric acid extracted by this method has been recommended suitable for use
in food and drugs. Precipitation is the classical method and it is performed by the addition of calcium oxide
hydrate (milk of lime) to form the slightly soluble tri-calcium citrate tetrahydrate. The precipitated tri-
14
Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21
 2017
calcium citrate is removed by filtration and washed several times with water. It is then treated with sulphuric
acid forming calcium sulphate, which is filtered off. Mother liquor containing citric acid is treated with active
carbon and passed through cation and anion exchangers. Several anion-exchange resins are commercially
available. Finally, the liquor is concentrated in vacuum crystallizers at 20-25°C, forming citric acid
monohydrate. Crystalization at temperatures higher to this is used to prepare anhydrous citric acid.
Glutamic Acid:
The history of the first amino acid production dates back to 1908 when Dr. K. Ikeda, a chemist in
Japan, isolated glutamic acid from kelp, a marine alga, after acid hydrolysis and fractionation. He also
discovered that glutamic acid, after neutralization with castic soda, developed an entirely new, delicious
taste. This was the birth of the use of monosodium glutamate (MSG) as a flavour-enhancing compound. The
breakthrough in the production of MSG was the isolation of a specific soil-inhabiting gram-positive
bacterium, Corynebacterium glutamicum, by Dr. S. Ukada and Dr. S. Kinoshita in 1957. The successful
commercialization of monosodium glutamate (MSG) with this bacterium provided a big boost for amino acid
production and later with other bacteria like E. coli as well.
Commercial Production of Glutamic Acid:
Glutamic acid commercial production (Fig. 40.6) by microbial fermentation provides 90% of world’s
total demand, and remaining 10% is met through chemical methods. For the actual fermentation the
microbial strains are grown in fermentors as large as 500 m3. The raw materials used include carbohydrate
(glucose, molasses, sucrose, etc.), peptone, inorganic salts and biotin. Biotin concentration in the
fermentation medium has a significant influence on the yield of glutamic acid. Fermentation completes
within 2-4 days and, at the end of the fermentation, the broth contains glutamic acid in the form of its
ammonium salt. In a typical downstream process, the bacterial cells are separated and the broth is passed
through a basic anion exchange resin. Glutamic acid anions get bound to the resin and ammonia is released.
This ammonia can be recovered via distillation and reused in the fermentation.
Elution is performed with NaOH to directly form monosodium glutamate (MSG) in the solution and
to regenerate the basic anion exchanger. From the elute, MSG may be crystallized directly followed by
further conditioning steps like decolourization and serving to yield a food-grade quality of MSG.

α-ketoglutaric acid serves as the precursor of glutamic acid and the conversion of the α- ketoglutaric acid to
glutamic acid occurs in presence of enzyme glutamic acid dehydrogenase. It has been found that if penicillin
is added in he medium, the glutamic acid production can be increased manifold.

15
Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21
 2017
Uses of Glutamic Acid:
As stated earlier, glutamic acid is widely used in the production
of monosodium glutamate (MSG) which is commonly known
as the ‘seasoning salt’. The world production of glutamic acid
is to the tune of 800,000 tonnes/year. Monosodium glutamate
is condiment and flavour-enhancing agent, it finds its greatest
use as a common ingredient in convenient food-stuffs.
Beer Production
Brewing is the production of beer by steeping a starch source
(commonly cerealgrains, the most popular of which is barley)[1] in water and fermenting the resulting sweet
liquid with yeast. It may be done in a brewery by a commercial brewer, at home by a homebrewer, or by a
variety of traditional methods such as communally by the indigenous peoples in Brazil when
making cauim.[2] Brewing has taken place since around the 6th millennium BC, and archaeological evidence
suggests that emerging civilizations including ancient Egypt[3] and Mesopotamia brewed beer.[4] Since the
nineteenth century the brewing industry has been part of most western economies.
The basic ingredients of beer are water and a fermentable starch source such as malted barley. Most beer is
fermented with a brewer's yeast and flavoured with hops.[5] Less widely used starch sources
include millet, sorghum and cassava.[6]Secondary sources (adjuncts), such as maize (corn), rice, or sugar,
may also be used, sometimes to reduce cost, or to add a feature, such as adding wheat to aid in retaining the
foamy head of the beer.[7] The proportion of each starch source in a beer recipe is collectively called the grain
bill.
Steps in the brewing process
include malting, milling, mashing, lautering, boiling, fermenting, conditioning, filtering, and packaging.
There are three main fermentation methods, warm, cool and spontaneous. Fermentation may take place in an
open or closed fermenting vessel; a secondary fermentation may also occur in the cask or bottle. There are
several additional brewing methods, such as barrel aging, double dropping, and Yorkshire Square.
The basic ingredients of beer are water; a starch source, such as malted barley, able to be fermented
(converted into alcohol); a brewer's yeast to produce the fermentation; and a flavouring, such as hops,[5] to
offset the sweetness of the malt.[21]A mixture of starch sources may be used, with a secondary saccharide,
such as maize (corn), rice, or sugar, often being termed an adjunct, especially when used as a lower-cost
substitute for malted barley.[7] Less widely used starch sources include millet, sorghum, and cassava root in
Africa, potato in Brazil, and agave in Mexico, among others.[6] The amount of each starch source in a beer
recipe is collectively called the grain bill.
Hops
Hops are the female flower clusters or seed cones of the hop vine Humulus lupulus,[28] which are used as a
flavouring and preservative agent in nearly all beer made today.[29] Hops contain several characteristics that
brewers desire in beer: they contribute a bitterness that balances the sweetness of the malt; they provide
floral, citrus, and herbal aromas and flavours; they have an antibiotic effect that favours the activity
of brewer's yeast over less desirable microorganisms; and they aid in "head retention", the length of time that
a foamy head will last.[36] The preservative in hops comes from the lupulin glands which contain soft resins
with alpha and beta acids.[37][38]Though much studied, the preservative nature of the soft resins is not yet fully
understood, though it has been observed that unless stored at a cool temperature, the preservative nature will
decrease.[39][40] Brewing is the sole major commercial use of hops.[41]

16
Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21
 2017
Yeast is the microorganism that is responsible for fermentation in beer. Yeast metabolises the sugars
extracted from grains, which produces alcohol and carbon dioxide, and thereby turns wort into beer. In
addition to fermenting the beer, yeast influences the character and flavour.[42] The dominant types of yeast
used to make beer are Saccharomyces cerevisiae, known as ale yeast, and Saccharomyces pastorianus,
known as lager yeast; Brettanomyces ferments lambics,[43] and Torulaspora delbrueckii ferments
Bavarian weissbier.[44] Before the role of yeast in fermentation was understood, fermentation involved wild
or airborne yeasts, and a few styles such as lambics still use this method today. Emil Christian Hansen, a
Danish biochemist employed by the Carlsberg Laboratory, developed pure yeast cultures which were
introduced into the Carlsberg brewery in 1883,[45] and pure yeast strains are now the main fermenting source
used worldwide.
Clarifying agent
Some brewers add one or more clarifying agents to beer, which typically precipitate (collect as a solid) out of
the beer along with protein solids and are found only in trace amounts in the finished product. This process
makes the beer appear bright and clean, rather than the cloudy appearance of ethnic and older styles of beer
such as wheat beers.[47]
Examples of clarifying agents include isinglass, obtained from swimbladders of fish; Irish moss, a seaweed;
kappa carrageenan, from the seaweed Kappaphycus cottonii; Polyclar (artificial); and gelatin.[48] If a beer is
marked "suitable for Vegans", it was generally clarified either with seaweed or with artificial
agents,[49] although the "Fast Cask" method invented by Marston's in 2009 may provide another method.
Brewing process
There are several steps in the brewing process, which may include malting, mashing,
lautering, boiling, fermenting, conditioning, filtering, and packaging.[51]
Malting is the process where barley grain is made ready for brewing.[52]Malting is broken down into three
steps in order to help to release the starches in the barley.[53] First, during steeping, the grain is added to a vat
with water and allowed to soak for approximately 40 hours.[54] During germination, the grain is spread out on
the floor of the germination room for around 5 days.[54] The final part of malting is kilning when the malt
goes through a very high temperature drying in a kiln; with gradual temperature increase over several
hours.[55]
When kilning is complete, the grains are now termed malt, and they will be milled or crushed to
break apart the kernels and expose the cotyledon, which contains the majority of the carbohydrates and
sugars; this makes it easier to extract the sugars during mashing.[56] Milling also separates the seed from
the husk. Care must be taken when milling to ensure that the starch reserves are sufficiently milled without
damaging the husk and providing coarse enough grits that a good filter bed can be formed during lautering.
Grains are typically dry-milled with roller mills or hammer mills. Hammer mills, which produce a very fine
mash, are often used when mash filters are going to be employed in the lautering process because the grain
does not have to form its own filter bed. In modern plants, the grain is often conditioned with water before it
is milled to make the husk more pliable, thus reducing breakage and improving lauter speed.
Mashing converts the starches released during the malting stage into sugars that can be fermented.
The milled grain is mixed with hot water in a large vessel known as a mash tun. In this vessel, the grain and
water are mixed together to create a cereal mash. During the mash, naturally occurring enzymes present in
the malt convert the starches (long chain carbohydrates) in the grain into smaller molecules or simple sugars
(mono-, di-, and tri-saccharides). This "conversion" is called saccharification. The result of the mashing
process is a sugar-rich liquid or "wort", which is then strained through the bottom of the mash tun in a
process known as lautering. Prior to lautering, the mash temperature may be raised to about 75–78 °C (167–

17
Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21
 2017
172 °F) (known as a mashout) to free up more starch and reduce mash viscosity. Additional water may be
sprinkled on the grains to extract additional sugars (a process known as sparging).[57]
The wort is moved into a large tank known as a "copper" or kettle where it is boiled with hops and
sometimes other ingredients such as herbs or sugars. This stage is where many chemical and technical
reactions take place, and where important decisions about the flavour, colour, and aroma of the beer are
made.[58] The boiling process serves to terminate enzymatic
processes, precipitate proteins, isomerize hop resins, and concentrate and sterilize the wort. Hops add
flavour, aroma and bitterness to the beer. At the end of the boil, the hopped wort settles to clarify in a vessel
called a "whirlpool", where the more solid particles in the wort are separated out.[59]
After the whirlpool, the wort is drawn away from the compacted hop trub, and rapidly cooled via
a heat exchanger to a temperature where yeast can be added. A variety of heat exchanger designs are used in
breweries, with the most common a plate-style. Water or glycol run in channels in the opposite direction of
the wort, causing a rapid drop in temperature. It is very important to quickly cool the wort to a level where
yeast can be added safely as yeast is unable to grow in very high temperatures, and will start to die in
temperatures above 60 °C (140 °F).[56][60] After the wort goes through the heat exchanger, the cooled wort
goes into a fermentation tank. A type of yeast is selected and added, or "pitched", to the fermentation
tank.[58] When the yeast is added to the wort, the fermenting process begins, where the sugars turn into
alcohol, carbon dioxide and other components. When the fermentation is complete the brewer may rack the
beer into a new tank, called a conditioning tank.[57] Conditioning of the beer is the process in which the beer
ages, the flavour becomes smoother, and flavours that are unwanted dissipate.[59] After conditioning for a
week to several months, the beer may be filtered and force carbonated for bottling,[61] or fined in the cask.[62]
Mashing
Mashing is the process of combining a mix of milled grain
(typically malted barley with supplementary grains such as corn, sorghum, rye or wheat), known as the
"grain bill", and water, known as "liquor", and heating this mixture in a vessel called a "mash tun". Mashing
is a form of steeping,[63] and defines the act of brewing, such as with making tea, sake, and soy
sauce.[64] Technically, wine, cider and mead are not brewed but rather vinified, as there is no steeping process
involving solids.[65] Mashing allows the enzymes in the malt to break down the starch in the grain into
sugars, typically maltose to create a malty liquid called wort.[66] There are two main methods –
infusionmashing, in which the grains are heated in one vessel; and decoction mashing, in which a proportion
of the grains are boiled and then returned to the mash, raising the temperature. [67] Mashing involves pauses at
certain temperatures (notably 45–62–73 °C or 113–144–163 °F), and takes place in a "mash tun" – an
insulated brewing vessel with a false bottom.[68][69][70] The end product of mashing is called a "mash".
Mashing usually takes 1 to 2 hours, and during this time the various temperature rests activate
different enzymes depending upon the type of malt being used, its modification level, and the intention of the
brewer. The activity of these enzymes convert the starches of the grains to dextrins and then to fermentable
sugars such as maltose. A mash rest from 49–55 °C (120–131 °F) activates various proteases, which break
down proteins that might otherwise cause the beer to be hazy. This rest is generally used only with
undermodified (i.e. undermalted) malts which are decreasingly popular in Germany and the Czech Republic,
or non-malted grains such as corn and rice, which are widely used in North American beers. A mash rest at
60 °C (140 °F) activates β-glucanase, which breaks down gummy β-glucans in the mash, making the sugars
flow out more freely later in the process. In the modern mashing process, commercial fungal based β-
glucanase may be added as a supplement. Finally, a mash rest temperature of 65–71 °C (149–160 °F) is used
to convert the starches in the malt to sugar, which is then usable by the yeast later in the brewing process.
Doing the latter rest at the lower end of the range favours β-amylaseenzymes, producing more low-order
18
Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21
 2017
sugars like maltotriose, maltose, and glucose which are more fermentable by the yeast. This in turn creates a
beer lower in body and higher in alcohol. A rest closer to the higher end of the range favours α-
amylaseenzymes, creating more higher-order sugars and dextrins which are less fermentable by the yeast, so
a fuller-bodied beer with less alcohol is the result. Duration and pH variances also affect the sugar
composition of the resulting wort.[71]
Lautering is the separation of the wort (the liquid containing the sugar extracted during mashing)
from the grains.[72] This is done either in a mash tun outfitted with a false bottom, in a lauter tun, or in a mash
filter. Most separation processes have two stages: first wort run-off, during which the extract is separated in
an undiluted state from the spent grains, and sparging, in which extract which remains with the grains is
rinsed off with hot water. The lauter tun is a tank with holes in the bottom small enough to hold back the
large bits of grist and hulls.[73] The bed of grist that settles on it is the actual filter. Some lauter tuns have
provision for rotating rakes or knives to cut into the bed of grist to maintain good flow. The knives can be
turned so they push the grain, a feature used to drive the spent grain out of the vessel.[74] The mash filter is a
plate-and-frame filter. The empty frames contain the mash, including the spent grains, and have a capacity of
around one hectoliter. The plates contain a support structure for the filter cloth. The plates, frames, and filter
cloths are arranged in a carrier frame like so: frame, cloth, plate, cloth, with plates at each end of the
structure. Newer mash filters have bladders that can press the liquid out of the grains between spargings. The
grain does not act like a filtration medium in a mash filter.[75]
Boiling
After mashing, the beer wort is boiled with hops (and other flavourings if used) in a large tank known
as a "copper" or brew kettle – though historically the mash vessel was used and is still in some small
breweries.[76] The boiling process is where chemical and technical reactions take
[58]
place, including sterilization of the wort to remove unwanted bacteria, releasing of hop flavours, bitterness
and aroma compounds through isomerization, stopping of enzymatic processes, precipitation of proteins, and
concentration of the wort.[77][78] Finally, the vapours produced during the boil volatilise off-flavours,
including dimethyl sulfide precursors.[78] The boil is conducted so that it is even and intense – a continuous
"rolling boil".[78] The boil on average lasts between 45 and 90 minutes, depending on its intensity, the hop
addition schedule, and volume of water the brewer expects to evaporate.[79] At the end of the boil, solid
particles in the hopped wort are separated out, usually in a vessel called a "whirlpool".[59]
Brew kettle or copper
Copper is the traditional material for the boiling vessel, because copper transfers heat quickly and
evenly, and because the bubbles produced during boiling, and which would act as an insulator against the
heat, do not cling to the surface of copper, so the wort is heated in a consistent manner.[80] The simplest boil
kettles are direct-fired, with a burner underneath. These can produce a vigorous and favourable boil, but are
also apt to scorch the wort where the flame touches the kettle, causing caramelisation and making cleanup
difficult. Most breweries use a steam-fired kettle, which uses steam jackets in the kettle to boil the
wort.[78] Breweries usually have a boiling unit either inside or outside of the kettle, usually a tall, thin
cylinder with vertical tubes, called a calandria, through which wort is pumped.[81]
Whirlpool
At the end of the boil, solid particles in the hopped wort are separated out, usually in a vessel called a
"whirlpool" or "settling tank".[59][82] The whirlpool was devised by Henry Ranulph Hudston while working
for the Molson Brewery in 1960 to utilise the so-called tea leaf paradox to force the denser solids known as
"trub" (coagulated proteins, vegetable matter from hops) into a cone in the centre of the whirlpool tank.
Hopback

19
Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21
 2017
A hopback is a traditional additional chamber that acts as a sieve or filter by using whole hops to clear debris
(or "trub") from the unfermented (or "green") wort,[87] as the whirlpool does, and also to increase hop aroma
in the finished beer.[88][89] It is a chamber between the brewing kettle and wort chiller. Hops are added to the
chamber, the hot wort from the kettle is run through it, and then immediately cooled in the wort chiller before
entering the fermentation chamber.
Fermenting
Fermentation takes place in fermentation vessels which come in various forms, from enormous
cylindroconical vessels, through open stone vessels, to wooden vats.[94][95][96] After the wort is cooled
and aerated – usually with sterile air – yeast is added to it, and it begins to ferment. It is during this stage that
sugars won from the malt are converted into alcohol and carbon dioxide, and the product can be called beer
for the first time.
Most breweries today use cylindroconical vessels, or CCVs, which have a conical bottom and a cylindrical
top. The cone's aperture is typically around 60°, an angle that will allow the yeast to flow towards the cone's
apex, but is not so steep as to take up too
much vertical space. CCVs can handle both
fermenting and conditioning in the same tank.
At the end of fermentation, the yeast and
other solids which have fallen to the cone's
apex can be simply flushed out of a port at
the apex. Open fermentation vessels are also
used, often for show in brewpubs, and in
Europe in wheat beer fermentation. These
vessels have no tops, which makes harvesting
top-fermenting yeasts very easy. The open
tops of the vessels make the risk of infection
greater, but with proper cleaning procedures
and careful protocol about who enters
fermentation chambers, the risk can be well
controlled. Fermentation tanks are typically
made of stainless steel. If they are simple
cylindrical tanks with beveled ends, they are
arranged vertically, as opposed to
conditioning tanks which are usually laid out
horizontally. Only a very few breweries still
use wooden vats for fermentation as wood is
difficult to keep clean and infection-free and
must be repitched more or less yearly
Conditioning
After an initial or primary fermentation, beer
is conditioned, matured or aged,[125] in one of several ways,[126] which can take from 2 to 4 weeks, several
months, or several years, depending on the brewer's intention for the beer. The beer is usually transferred into
a second container, so that it is no longer exposed to the dead yeast and other debris (also known as "trub")
that have settled to the bottom of the primary fermenter. This prevents the formation of unwanted flavours
and harmful compounds such as acetylaldehyde.[127]

20
Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21
 2017
Kräusening
Kräusening is a conditioning method in which
fermenting wort is added to the finished
beer.[128] The active yeast will restart
fermentation in the finished beer, and so
introduce fresh carbon dioxide; the
conditioning tank will be then sealed so that
the carbon dioxide is dissolved into the beer
producing a lively "condition" or level of
carbonation.[128] The kräusening method may
also be used to condition bottled beer.[128]
Lagering
Lagers are stored at near freezing
temperatures for 1–6 months while still on the
yeast.[129] The process of storing, or
conditioning, or maturing, or aging a beer at a
low temperature for a long period is called
"lagering", and while it is associated with
lagers, the process may also be done with ales,
with the same result – that of cleaning up various chemicals, acids and compounds.[130]
Secondary fermentation
During secondary fermentation, most of the remaining yeast will settle to the bottom of the second fermenter,
yielding a less hazy product.[131]
Bottle fermentation
Some beers undergo an additional fermentation in the bottle giving natural carbonation.[132] This may be a
second or third fermentation. They are bottled with a viable yeast population in suspension. If there is no
residual fermentable sugar left, sugar or wort or both may be added in a process known as priming. The

resulting fermentation generates CO2 that is trapped in the

bottle, remaining in solution and providing natural carbonation. Bottle-conditioned beers may be either filled
unfiltered direct from the fermentation or conditioning tank, or filtered and then reseeded with yeast.[133]
21
Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21
 2017
Cask conditioning
Cask ale or cask-conditioned beer is unfiltered and unpasteurised beer that is conditioned (including
secondary fermentation) and served from a cask, either pumped up from a cellar via a beer engine (hand
pump), or from a tap by gravity.[134]Sometimes a cask breather is used to keep the beer fresh by allowing
carbon dioxide to replace oxygen as the beer is drawn off the cask. [135] The term "real ale" as used by
the Campaign for Real Ale (CAMRA) refers to beer "served without the use of extraneous carbon dioxide",
which would disallow the use of a cask breather.[136][137]
Filtering
Filtering the beer stabilizes the flavour, and gives beer its polished shine and brilliance. Not all beer is
filtered. When tax determination is required by local laws, it is typically done at this stage in a calibrated
tank. There are several forms of filters, they may be in the form of sheets or "candles", or they may be a fine
powder such as diatomaceous earth, also called kieselguhr. The powder is added to the beer and recirculated
past screens to form a filtration bed.
Kieselguhr filters
Filters that use a powder medium are considerably more complicated to operate, but can filter much more
beer before regeneration. Common media include diatomaceous earth and perlite.

Wine production
Wine is an alcoholic beverage produced through the partial or total fermentation of grapes. Other fruits and
plants, such as berries, apples, cherries, dandelions, elder-berries, palm, and rice can also be fermented.

Grapes belong to the botanical family vitaceae, of which there are many species. The species that are most
widely used in wine production are Vitis labrusca and, especially, Vitis vinifera, which has long been the
most widely used wine grape throughout the world.

The theory that wine was discovered by accident is most likely correct because wine grapes contain all the
necessary ingredients for wine, including pulp, juice, and seeds that possess all the acids, sugars, tannins,
minerals, and vitamins that are found in wine. As a natural process, the frosty-looking skin of the grape,
called "bloom," catches the airborne yeast and enzymes that ferment the juice of the grape into wine.

The cultivation of wine grapes for the production of wine is called "viticulture." Harvested during the fall,
wine grapes may range in color from pale yellow to hearty green to ruby red.

Wine can be made in the home and in small-, medium- or large-sized wineries by using similar methods.
Wine is made in a variety of flavors, with varying degrees of sweetness or dryness as well as alcoholic
strength and quality. Generally, the strength, color, and flavor of the wine are controlled during the
fermentation process.

Wine is characterized by color: white, pink or rose, and red, and it can range in alcohol content from 10
percent to 14 percent. Wine types can be divided into four broad categories: table wines, sparkling wines,
fortified wines, and aromatic wines. Table wines include a range of red, white, and rose wines; sparkling

22
Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21
 2017
wines include champagne and other "bubbly" wines; aromatic wines contain fruits, plants, and flowers; and
fortified wines are table wines with brandy or other alcohol added.

The name of a wine almost invariably is derived from one of three sources: the name of the principal grape
from which it was made, the geographical area from which it comes, or—in the case of the traditionally
finest wines—from a particular vineyard or parcel of soil. The year in which a wine is made is only printed
on bottles that have aged for two or more years; those aged less are not considered worthy of a date. Wine
years are known as "vintages" or "vintage years." While certain wines are considered good or bad depending
on the year they were produced, this can vary by locality.

In general, red wines are supposed to age from seven to ten years before being sold. Because white and rose
wines are not enhanced by additional ageing, they are usually aged from only one to four years before being
sold. And, since the quality of wine can depend on proper ageing, older wines are generally more expensive
than younger ones. Other factors, however, can affect the quality of wine, and proper ageing does not always
ensure quality. Other factors affecting quality include the grapes themselves, when the grapes are picked,
proper care of the grapes, the fermentation process, as well as other aspects of wine production.

Most wineries bottle wine in different size bottles and have different product and graphic designs on their
labels. The most common bottle sizes are the half bottle, the imperial pint, the standard bottle, and the gallon
bottle or jug. Most red and rose wine bottles are colored to keep light from ageing the wine further after they
are on the market.
While viticulture has remained much the same for centuries, new technology has helped increase the output
and variety of wine.
Raw Materials

As mentioned above, the wine grape itself contains all the necessary ingredients for wine: pulp, juice, sugars,
acids, tannins, and minerals. However, some manufacturers add yeast to increase strength and cane or beet
sugar to increase alcoholic content. During fermentation, winemakers also usually add sulfur dioxide to
control the growth of wild yeasts.

The Manufacturing Process

The process of wine production has remained much the same throughout the ages, but new sophisticated
machinery and technology have helped streamline and increase the output of wine. Whether such advances
have enhanced the quality of wine is, however, a subject of debate. These advances include a variety of
mechanical harvesters, grape crushers, temperature-controlled tanks, and centrifuges.

The procedures involved in creating wine are often times dictated by the grape and the amount and type of
wine being produced. Recipes for certain types of wine require the winemaker (the vintner) to monitor and
regulate the amount of yeast, the fermentation process, and other steps of the process. While the
manufacturing process is highly automated in medium- to large-sized wineries, small wineries still use hand
operated presses and store wine in musty wine cellars.

23
Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21
 2017
A universal factor in the production
of fine wine is timing. This includes
picking grapes at the right time,
removing the must at the right time,
monitoring and regulating
fermentation, and storing the wine
long enough.

The wine-making process can be

divided into four distinct steps:
harvesting and crushing grapes;
fermenting must; ageing the wine;
and packaging.

Harvesting and crushing grapes

1 Vineyardists inspect sample clusters of wine grapes with a refractometer to determine if the grapes
are ready to be picked. The refractometer is a small, hand-held device (the size of a miniature
telescope) that allows the vineyardist to accurately check the amount of sugar in the grapes.

2 If the grapes are ready for picking, a mechanical harvester (usually a suction picker) gathers and
funnels the grapes into a field hopper, or mobile storage container. Some mechanical harvesters have
grape crushers mounted on the machinery, allowing vineyard workers to gather grapes and press them
at the same time. The result is that vineyards can deliver newly crushed grapes, called must, to
wineries, eliminating the need for crushing at the winery. This also prevents oxidization of the juice
through tears or splits in the grapes' skins.

Mechanical harvesters, or, in some cases, robots, are now used in most medium to large vineyards, thereby
eliminating the need for hand-picking. First used in California vineyards in 1968, mechanical harvesters have
significantly decreased the time it takes to gather grapes. The harvesters have also allowed grapes to be
gathered at night when they are cool, fresh, and ripe.

3 The field hoppers are transported to the winery where they are unloaded into a crusher-stemmer
machine. Some crusher-stemmer machines are hydraulic while others are driven by air pressure.

The grapes are crushed and the stems are removed, leaving liquid must that flows

Once at the winery, the grapes are crushed if necessary, and the must is fermented, settled, clarified, and
filtered. After filtering, the wine is aged in stainless steel tanks or wooden vats. White and rose wines may
age for a year to four years, or far less than a year. Red wines may age for seven to ten years. Most large

24
Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21
 2017
wineries age their wine in large temperature-controlled stainless steel tanks that are above ground, while
smaller wineries may still store their wine in wooden barrels in damp wine cellars.

Fermenting the must

4 For white wine, all the grape skins are separated from the "must" by filters or centrifuges before the
must undergoes fermentation. For red wine, the whole crushed grape, including the skin, goes into the
fermentation tank or vat. (The pigment in the grape skins give red wine its color. The amount of time
the skins are left in the tank or vat determines how dark or light the color will be. For rose, the skins
only stay in the tank or vat for a short time before they are filtered out.)

5 During the fermentation process, wild yeast are fed into the tank or vat to turn the sugar in the must
into alcohol. To add strength, varying degrees of yeast may be added. In addition, cane or beet sugar
may be added to increase the alcoholic content. Adding sugar is callchaptalization. Usually
chaptalization is done because the grapes have not received enough sun prior to harvesting. The
winemaker will use a handheld hydrometer to measure the sugar content in the tank or vat. The wine
must ferments in the tank or vat for approximately seven to fourteen days, depending on the type of
wine being produced.

Ageing the wine

6 After crushing and fermentation, wine needs to be stored, filtered, and properly aged. In some
instances, the wine must also be blended with other alcohol. Many wineries still store wine in damp,
subterranean wine cellars to keep the wine cool, but larger wineries now store wine above ground in
epoxylined and stainless steel tanks. The tanks are temperature-controlled by water that circulates
inside the lining of the tank shell. Other similar tanks are used instead of the old redwood and
concrete vats when wine is temporarily stored during the settling process.

After fermentation, certain wines (mainly red wine) will be crushed again and pumped into another
fermentation tank where the wine will ferment again for approximately three to seven days. This is done not
only to extend the wine's shelf life but also to ensure clarity and color stability.

The wine is then pumped into settling ("racking") tanks or vats. The wine will remain in the tank for one to
two months. Typically, racking is done at 50 to 60 degrees Fahrenheit (10 to 16 degrees Celsius) for red
wine, and 32 degrees Fahrenheit (0 degrees Celsius) for white wine.

7 After the initial settling (racking) process, certain wines are pumped into another settling tank or vat
where the wine remains for another two to three months. During settling the weighty unwanted debris
(remaining stem pieces, etc.) settle to the bottom of the tank and are eliminated when the wine is
pumped into another tank. The settling process creates smoother wine. Additional settling may be
necessary for certain wines.

25
Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21
 2017
8 After the settling process, the wine passes through a number of filters or centrifuges where the wine
is stored at low temperatures or where clarifying substances trickle through the wine.

9 After various filtering processes, the wine is aged in stainless steel tanks or wooden vats. White and
rose wines may age for a year to four years, or far less than a year. Red wines may age for seven to
ten years. Most large wineries age their wine in large temperature-controlled stainless steel tanks that
are above ground, while smaller wineries may still store their wine in wooden barrels in damp wine
cellars.

10 The wine is then filtered one last time to remove unwanted sediment.

The wine is now ready to be bottled, corked, sealed, crated, labeled, and shipped to distributors.

Packaging

11 Most medium- to large-sized wineries I now use automated bottling machines, and most
moderately priced and expensive wine bottles have corks made of a special oak. The corks are
covered with a peel-off aluminum foil or plastic seal. Cheaper wines have an aluminum screw-off
cap or plastic stopper. The corks and screw caps keep the air from spoiling the wine. Wine is usually
shipped in wooden crates, though cheaper wines may be packaged in cardboard.

Quality Control

All facets of wine production must be carefully controlled to create a quality wine. Such variables as the
speed with which harvested grapes are crushed; the temperature and timing during both fermentation and
ageing; the percent of sugar and acid in the harvested grapes; and the amount of sulfur dioxide added during
fermentation all have a tremendous impact on the quality of the finished wine.

26
Prepared by Dr S. Ramalakshmi, Asst Prof, Dept of Microbiology, KAHE, CBE-21
UNIT-I 16mbu303 INDUSTRAL MICROBIOLOGY
Industrial microbiology deals with areas of from
microbiology
costlyfromsubstrates
cheaper
involving
fromandunavailable
economic
disposable
from foreign
aspects,
substrates
substrates
fromcountries
where
cheapervaluable
and dis
p
In history of industrial microbiology, the period of pre-1800
ignorance 1800-1900
is post-1900post-2000pre-1800
The period of discovery in history of industrial microbiology
pre-1800 1800-1900 is from post-1900post-20001800-1900
The period of industrial development in history of pre-1800
industrial 1800-1900
microbiologypost-1900post-2000post-1900
is
st
Bread was 1 baked around ---------------B.C 1000 2000 3000 4000 4000
Wine is produced from malt molasses grapes sugarcanegrapes
The compound Microscope produced by Zaccharies resolution
Jensenfocusing
had
power no provision
light facility
forspecimen holding
focusing
Anton von Leewanhoek was able to obtain magnification150-300 upto 160-270-------------------diameters.
140-250 150-200 160-270
In middle of last century fermentation was consider biological
to be aphysical
------process. chemical electrical chemical
Fermentation was first described as chemical process Pasteur by Robert Koch Liebig Anton vanLiebig
Leewanhoek
In 1873,-------------------described that yeastsare involved
Pasteur inSchwann
fermentation Robertprocess
KochBerzelius
for alcohol
Schwann
production
In fermentation, yeast converts -------------to---------------and
carbohydrate, fatty
-----alcohol,
acids,sugar,
alcohol,
carbonic
alcohol,
carbonic
starch,
acidcarbonic
alcohol,
acid
sugar,
acid
carbonic
alcohol, acid
carbon
who isolated the microbes associated fementation. Schwann Bertholet Pasteur Koch Pasteur
The optimum temperature condition for fermentation 20-40process 30-50 was in40-60range from-----
20-30 30-50
Pasteur identified the organisms involved in the pyruvic
transformation
acid
lactic of acidcitric
sugar toacid -----------
stearic acid lactic acid
Lactic acid organism is a -------------- fungi bacteria virus protozoa bacteria
who made an important discovery that fermentation Schwanntakes Pasteur
place in absence
Koch of [Link] Pasteur
In 1861 pasteur did experiments on ---------and-----------fermentation
butyric acid acetone
and acetic
andlactic
butanol
acidacid acetic
and acetic
acidbutyric
acid
and citric
acidacid
and acet
)----------and----------requested Pasteur to study the
Dumasproblem
andDumas
Napoleon
of sowingandNapoleon111
Flemming
111
wine that Duman
thereatened
and and
Schwann
Dumas
Leewanhoek
French
and Napoleon
wine ind
The process of sterilization of wine introduced bypasteurization
Pasteur is ultrafiltration
called low temperature,high high temperature,low
pasteurization
holding timeholding t
which method is most satisfory method for long time mineral preservation
oillyophilisation
overlayofcryopreservation
microbes periodic transfer lyophilisation
The major antibiotics such as streptomycin and neomycin
Bacillus spStaphylococcus
etc were isolatedStreptococcus
spfrom------------
Streptomyces
sp Streptomyces
sp sp
During 1910-1920,-----------and---------were producedethanol by and
Industrial
lactic
glycerol
acid fermentation
acetone
and amylasesand
acetone
n-butanol
and
acetone
lactic acid
and n-butano
During 1920-1930,------------- was produced by industrial
lactic acidfermentation
acetic acidcitric acid glutanic acid citric acid
During 1930-1940, first vitamin to be produced by riboflavin
industrialvitamin
fermentation
B12vitamin is------
C vitamin D riboflavin
The acetone-butanol fermentation is also called as Watsmann
----------------process
WalksmanWebsmann WeizmannWeizmann
The important quality of production strain is should be unstable
a high yieldingbiochemical
producestrainunderisable
not
characteristics
easily should
cultivate
substances
be a high yield
The screening techniques involves ---------------and-------------
primary and secondary
secondary primary
and tertiaryand
secondary
quartenary
primary
and quartenary
and seconda
Primary screening technology involves the isolation desined
of newcolor
desired
microbial shape
desired
speciesquality
exhibiting
desired property
the
desired property
Crowded plate technique is an example of -----------screening
primary secondarytertiary quartenaryprimary
The primary screening technique which is employed crowdedfor a plate
detect
auxanography
technique
and enrichment
isolating antibiotic
useculture
of indicator
producing
technique
crowded dyeplate
strain
techni
is
Enrichment culture technology was designed by Pasteursoil microbiologist------------
Koch Ehrlich BeijerinckBeijerinck
which technique is largely employed to identify the crowded
growthplate
auxanography
factor technique
producing
enrichment strain
useculture
of
extracellularly
indicator
technique
auxanography
dye
Neutral red, bromothymol blue dyes are added tovitaminspartly buffered
growthnutrient
factors
organicagar acids
amines
media to detect
organic microorganism
acids
Example of enrichment substrate used is nutrient brothcellulose powder peptone minimal media cellulose powder
which screening helps in segregation microbe that primary
have real secondarytertiary
potential in fermentation quartenaryindustry.
secondary
The suitable protective medium used at the ---------------------is
Commonwealth American
10% Mycological
inositol
African
type culture
intype
Institute
dissolved
Indian
collection
culture
Mycological
water
Commonwealth
collectionInstitute Myco
The fermentation product produced by the identified old industrialnovelstraingold should becritical
-----------------
novel
The selected industrial strain is--------------by secondary
optimizedpriotized
screening. compareddeselectedoptimized
The process of lyophilization was first applied toRaper
microfungi
andThomas
Alexander
on layers andKoch
scale
Alexander
andby Alexander
---------and
Koch and Thomas
Raper
----------in
and Alexander
1942
The important criteria in handling the industrially stability
productive contamination
strain is oxidation
to prevent reduction contamination
Mutation is done by--------------and-------------methods
physical and chemical
chemical and
physical
political
and
chemical
botanical
and
physical
zoological
and chemica
Phosphorous pentoxide silica gel/freezing trap are crypreservation
examples desiccant
of------------------
preservants mineral oildesiccant
overlay
In multivalent regulatory mechanism of a branched single
biosynthetic
doublepathway,
triple -------------end
single, double,
products
single,
triple
double,
inhibit
triple
the e
The two categories of mutants are-----------------and---------------
autotropicphototropic
mutants and auxenic
mutants
mutants
mutants
auxotropic
and
resistant
mutants
andautotropic
mutants
to resistant
analogues
resistant
and
mutants
tomutan
analoto
a
The wild strain of Coryrebacterium glutamicus secretes both
lysine and -------and--------------
lysine
threonine
andthreonine
methionine threonine
and methionine
lysine
and pectin
and threonine
The mutant strain of Coryrebacterium glutamicus 50 60 70 80 60
The oil used in oil overlay method is British Pharmacopoeia
American Australian
Pharmacopoeia
MedicinalAfrican
Pharmacopoeia
Paraffin
Medicinal
Pharmacopoeia
British
oil. Paraffin
Medicinal
Pharmacopoe
Medicin
[Link]
The lysine biosynthesis, the end products lysine andaspartate
threonine
kinase
homoserine
inhibitserine
the
phosphatase
enzyme.
kinase
tryptophan aspartate
synthasekinase
An analogue of threonine is ----------------- α-amino,β-hydroxyvaleric
β-amino,β-hydroxyvaleric
ч-amino,β-hydroxyvaleric
acid)£-amino,acidα-amino,β-hydroxyva
acid
The analogue is --------------to sensitive mutant cells
ecofriendly
in the population.
toxic nutrient non-toxic toxic
An example of analogue resistant mutant is --------------capable
Brevibacterium
Brevibacterium
offlavum
excreating
Brevibacterium
lactum
threomine
Brevibacterium
aseptum
upto
Brevibacterium
12.6g/l.
glutans flavum
An example of revertant mutant is---------------forHydrophiles
the enzymeHydromonas
threonine Hydrogenomonas
deaminase.
Hydromonothrobis
Hydrogenomonas
.----------------mutants are important in fermentation
auxotrophic
industry
mutants
as produce
resistant
constitutive
hightoyields
mutants
analogue
of particular
sensitive
constitutive
to
enzymes
analogues
in a
Microbes are grown in especially designed vessels Fermentors
called-----------,
Batch cookers
containing
swap mediumspecial
conicalmedia
flasks
Fermentors
for its growth.
Secondary metabolites are produced during Lag Log Stationar death Log
_________. phase phase y phase phase phase

Wine is produced from ---------- malt molasses grapes sugarcanegrapes

The great break through came with observation of----------------under
plant animals microbes
simple microscope
God by
microbes
Anton Van Leewan
Anton van Leewanhoek observed ----------------in bacteria
his own scarpings,
fungi water.
protozoa viruses protozoa
---------------made an important discovery that fermentation
Schwann Pasteur
takes place
Kochin absence
Bertholet
of oxygen.
Pasteur
UNIT-II
ATCC is ------------- founded in 1925 American African
type culture
type
Auxenic
collection
culturetype
Australian
collection
cultureAmerican
type
collection
culture
typecollecti
cultur
The specific gravity of oil used in presence of cultures0.821-0.860
is )0.865-0.890 0.752-0.812 0.718-0.835 )0.865-0.890
.------------is the simplest and common method ofserial maintaining
subculture
lyophilisation
microbial
cryopreservation
[Link] serial subculture
The Drying-up of medium encourages good ---------of growth sporulation storage collection sporulation
.-------------and-----------are usually maintained in BacteriophagesBacteriophages
liquid medium and Viruses andand
Bacteriophages
Bacteriophages
and and
The mineral oil overlay method was firstused by Bwell 1945
and Weston
1947in -----------------
1949 1950 1947
Temperature of liquid nitrogen is -130˚C -150˚C -176˚C -196˚C -196˚C
In Pencillin fementation the precursor added is ------------
phenyl acetic
benzyl
acidalcohol
Benzene pyridine phenyl acetic acid
Buffering agents used in media formulation is mono and triiodosodium dihydropotassium
Tetraiodiosodium
phosphates
phosphates
monophosphates
phosphates
mono and dihydropo
Foaming during fermentation process creates oxidation reduction contamination productioncontamination
The antifoaming agent used in pencillin fermentation lard oil
is lard oil with decanol
ocetadecanol
mustard oil lard
with
oildecanol
with ocetadec
The citric acid fermentation Aspergillus niger culture low is grownhighat --------------pH
medium very values prevent
high low contamination
.------------media is mainly used in fermentation process.
synthetic semi-syntheticnon-syntheticdifferentialnon-synthetic
Which is the common raw material source used in food
fermentation
wasteagricultural
process
industrial
waste Biofuel
toxic waste
waste
agricultural waste
--------------------is rich in biotin, panthothenic acidcane
, thiamine,
molasses
beet
phorphorus
molasses
fruit and
molasses
sulphur.
cheese molasses
cane molasses
In Beet molasses ------------------is limiting compared biotin
to canepyridoxine
molassesthiamine pantothenic biotin
acid
----------------require biotin for growth in [Link] yeasts viruses phages yeasts
In India there is very large utilization of cane blackstrap
textile molasses
animal fodder
in -----------------industry
alcohol dyes alcohol
The ---------------contain 17% sugar, 1%acid and 0.3%ashrust just must bust must
In grapes the nitrogen content should be ----------------as
high it may mediumresultlow
in underisable
no fermentation.
low
-----------is 6.6-7.1% in cheddar whey total solidsprotein lactose fat total solids
Cheese whey is an important raw material in ---------production
lactic acidaspartic acidglutamic acidcitric acid lactic acid
.----------------and----------------are the main sourcesmolasses
of starchand
cheese
cereals
whey
cereals,
and tubers
roots
cereals
and and
tubers
cereals,
cornsteep
rootsliquor
and tub
Wheat, maize and rice are example of ----------------- molasses cereals roots tubers cereals
Cellulose are carbohydrates made of repeating units α-glucose β-glucose α-galactose
of -------- β-galactose β-glucose
In -------------industry the digestion process of wood
cheese,
withcheese
calcium
starch,whey
bisulfate
starch
paperliquor
under
pulp,wood,
sulfite
heatmolasses
and
waste
paper
pressure
liquor
pulp,tosulfite
give spe
wa
Sulfite waste liquor contains ----------sugars 1% 2% 3% 4% 2%
Wood molasses syrup contain ------of fermentable 60-80%
sugars 65-85% 70-90% 75-95% 65-85%
The untreated cellulosic wastes have been used in ethanol
productionsingle
of cell fuel
protein vitamins single cell protein
During manufacture of starch, gluten from corn---------is
sulfite waste
formed
cornliquor
steep
by steeping
wood
liquormolasses
of corn.
distillers
syrup
soluble
corn steep liquor
The clean, yellow, fine powder prepared from embryo corn-steep
of cotton
soya
liquor
bean
seed Pharmamedia
is
meal
called asdistiller’s
------------soluble
Pharmamedia
Pharmamedia is used as production media for ---------production.
penicillin streptomycin tetracycline griseofulvin tetracycline
Initially fermentation industries used corn steep mushroom
liquor for penicillin
-------production.
vitamin organic acid penicillin
Soya bean meal is used as production medium forpenicillin
--------------
streptomycin tetracycline griseofulvin streptomycin
Vegetable oils are used as animal feed antifoamsmushroompH production
adjustment
antifoams
media
which is the most preferred method compared tosteam other agentsUV light
for mass
ethidium
sterilization.
bromide
chlorine steam
Sugar containing medium can’t be sterilized by prolonged
reaction with
heating
charring
contaminants
because
caramelization
sugars
reaction
undergowith
caramelization
phosphates
------------are simplest method employed for sterilizing
continous
production
batch
fermentors
cookers
medium.
filtration radiation batch cookers
Lyophilization is the most satisfactory method oflonglongterm
termshort
preservation
termbothofamicroorganisms
andnone
b of thelong above
term
------------------ is perhaps the most popular form of
overlaying
suspended lyophilization
cultures
metabolism.
with
nitrogen
mineral
storage
none
oil of thelyophilization
above
Hwang in ----------- year recommends precooling 1966to 7 degree 1969
Celsius. 1974 1980 1966
Aim of ATCC is to Prevent contamination
To maintain Preserve
antibiotics
cultures
None of the Preserve
above cultures
Oil should be autoclaved at ___________ 15 lb/in2 for30 lb/in2
2 hr for45 lb/in2
2 hr for60 lb/in2
2 hr for 15 lb/in2
2 hr for 2 hr
Lyophilization Short termlong preservation
term killing
preservation
ofofmicroorganisms
Microorganisms
Noneof of
Microorganisms
thelong
above
term preservatio
Dessicant used in lyophilizer are Phosphorus silica gel Magnesium Copper silica gel
________ is used in storage at very low temp glycerol mineral oilParaffin wax All the aboveglycerol
The soil culture tubes are kept in refrigerator at above
5-80 C________
4-60temperature
C 2-40 C 6-80 C 5-80 C
De Becze and liebmann in -------- year used the first large
1941scale 1944fermentor1948for the production
1940 of
1944
yeast.
Fungus Mycelium should be suspended in Dimethyl Rose Saboura nutrient Dimethyl
________ medium to maintain its structure. sulphoxi Bengal ud’s agar sulphoxi
de medium dextrose de
agar
medium

The volume of inoculum used to cultivate 0.1-2% 0.5-5% 3-5% 06-Apr 0.5-5%
bacteria are
Carbon source used for production of Molasses Corn Barley None of Molasses
tctracycline is ________ steep the
liquor above
Phosphorous pentoxide silica gel/freezing trap are crypreservation
examples
desiccant
of------------------
preservants mineral oildesiccant
overlay
-------------- stressed the importance of the Meryma Dewald leogetrin Louis Dewald
elimination of air and moisture from n g Pasteur.
lyophilized cultures prior to sealing of
ampoules.
The main success of fermentation industry depends production
mainlyinoculums
on
temperature
-------------------
production
developmentproduction
strain production
medium strain
Secondary screening reveals whether the culturezygofermentative
is homofermentative
heterofermentative
perifermentative
and -------------.
afermentativeheterofermentative
In fermentation process, the---------of product in solubility
various organic
insolubility
solvents
emulsion
has toviscosity
be checked solubility
for purification pr
The important criteria in handling the industriallystability
productive
contamination
strain isoxidation
to preventreduction
----------contamination
The analogue is --------------to sensitive mutant cells ecofriendly
in the population.
toxic nutrient non-toxic toxic
-----------method is used specially for preserving sporing agar slant microbes
agar
culturesdeepsuch
soil ascultures
cultures cryopreservation
soil cultures
The special medium used in soil culture method is 20%,78%
soil, sand 18%,78%
andand2% calcium
10%,88%
and carbonate
4% 30%,68%
and 2% in ratio20%,78%
and of 2%
-------,--------and
and 2%
UNIT-III
Microbes are grown in especially designed vessels Fermentors
called-----------,
Batch cookers
containing
swap medium special
conical media
flasks
Fermentors
for its growth.
Fermentation tank should be provided with portscontamination for ----------------
[Link] inoculums
------------are used in side of fermentors to avoid vortex spargers formation
bearing glands rotameterbaffles baffles
)-------------tanks are used in production of all andstainless
lactic acidsteel
glass
fermentationcopper wooden wooden
The ------------in stainless steel fermentor gives resistance
chromiummolybdenum
to halogen nickelsalts, lodinetungsten
and sea molybdenum
water
-----are used to regulate the addition of medium,nutrients,syringe pumps defoamers.
peristalticfeed pumps pumps pressure pumps feed pumps
The fermentor vessel ----------------should be increased diameterwiththickness
scale. height design thickness
-------between top plate and vessel is very important baffles
to maintain
sealingairtightsparger/ aseptic
[Link]
---------device is used for giving air into fermentorsparger baffles shaft bearings box sparger
--------------type of bubbles facilitate high oxygen larger,
transfersmaller
than
smaller,
--------------------bubbles
larger
medium, large very small,smaller,
very mediumlarger
The ideal aspect ratio for a fermentor is 03:01 04:01 05:01 06:01 05:01
The number of baffles used in fermentor of diameter 2 TO3dm3
4 4isTO6 6 TO 8 8 TO 10 6 TO 8
)------------spargers are used widely large scale fermentation
porous orifice process nozzle combined nozzle sparger agitator
---------removes enough moisture from the gas leaving bafflesfermentor
heat exchange
andcooler
prevent excess
exit gas fluid
cooler
exit
[Link] cooler
In high quality bioreactor, all the processes in fermentation
agitator )aeration
are controlled
process by controller
cooler process controller
In-line, on-line and off-line are types of -------------- foamers agitator glands shaft sensors sensors
Example of In-line sensors are Ion-specific mass
sensors
spectrophotometer
antifoam probemedium additionantifoam probe
probe
------------sensors don’t form integral part of fermentor. in-line on-line off-line fermentoroff-line
)---------------is generated due to mixing by agitator energy
and microbes
heat action resistancecurrent
on substrates during heatfermentation pro
--------are semiconductors of Iron, Michel oxides exhibiting
mercury in-glass
electrical
large change
thermometers
resistance
thermistors
in resistance
electricalwithimpedance
thermistors
small change of tem
Stainless steel ---------------- sensors are used for temperature
Pt 100 Pl 100
measurements
Pb 100 inPs fermentation
100 Pt 100 system
Gas flow rate is measured by -------------- thermometers rotameterspistonmeters torsion dynamometer
rotameters
The liquid flow rate is measured by using thermometers thermal mass pistonmeters
flowmeter
torsion dynamometer
thermal mass flowme
Which gauge is used for measuring pressure under peristalticdiaphragm
aseptic conditiondiaguls bourbon tube diaphragm
-------is used for measuring the speed of agitator voltmeter torsion dynamometer ammeter taometer torsion dynamomete
Peristaltic pump is mainly used for addition of ---------and--------
medium and acid inoculums
and basebuffers and salts
antifoamers
and growth
acid and factors
base
Dissolved oxygen in fermentation process medium galvanic
measuredelectrode
pH by electrode
using thermometers
-----------------
thermistors galvanic electrode
If initially foam has started forming interior ---------------has
antifoamers mechanical
been used water
to
foamcontrol
breaker
controlling
foam mechanicalagitator/aeration
foam bre sp
Oxygen diffuses from tubing into medium is measured galvanic by electrode
paramagnetic platinum
gas analyzer
electrode
thermistors paramagnetic gas ana
pH denotes the presence of --------------in aqueoushydrogen solution ionhydroxyl ion carboxyl ions carbonyl ion hydrogen ion
The voltage difference between two electrodes istemperature used to determine
pH ------------of
moisture dissolved
unknownoxygenpHsolution
--------------,---------------and-------------- are three distinct
loggingareas
of data
process
ofanalysis
computer
data
process function.
control
logging of logging
data, data of data,
analysis
dataana
-----------system controls the addition of liquid from analog
reservoir
control
direct
to fermentator
control
direct digitalhumancontrol
control
direct digital control
At ------------ cultures may be aerated by means ofpilot the scalelaboratory-scale industrial semi-industrial
scale laboratory-scale
scale
In ----------- fermentations broth or culture is aerated Pilot-byand pilot
industrial-scale
scaleindustrial laboratory-scale
scale Pilot- and industrial-
In ----------- bioreactor are so designed that adequate bubble supply
columns
CSTRof oxygen PACKED
is obtained
BED
FLUIDISED
without bubble
BED
agitation
columns
In ----------- bioreactor are so designed that adequate AIR LIFT
supply CSTRof oxygen PACKED
is obtained
BED
FLUIDISED
without AIRBED
agitation
LIFT
Maximum -------------production may be achievedwaste by satisfying biomass
the organism's
toxic maximum specific biomass oxygen dema
______ is to provide microorganisms in submergedAeration culture withAgitation
sufficient
Impeller
oxygenBaffler
for metabolicAerationrequirements.
_________ ensures uniform suspension on microbial Aeration
cells. Agitation Sparger Baffler Agitation
---------device is used to introduce air in fermenter
Spargers Impellers Baffles Turbines Spargers
Aeration and agitation of a liquid medium may lead Acidto the formation
Alkali of
Foam Air Foam
In fermentation, it is very important to find the ---------------or-------------
gases or distribution
transport or productivity
energy
of product
concentration
orfrom
conversion
the
productivity
given
or yield
consumable
carbon
or conve
sour
---------and-------------developed methods to analyse
Humpthe and
biomass
Humphery
Honey and product
Hughes
and Cooney
and
concentration
Hyhes
Humphery
andHumphery
Cooney
during fermentatio
and Coon
The capacity of the batch fermentors _________ 10 – 12 12 – 15 20 -40 6–8 10 – 12
litre litre litre litre litre
Large fermentors range from ________ 2000 – 5000 – 10,000 none of 2000 –
5,000 10,000 gallons the 5,000
gallons gallons above. gallons
Clogging problems occur in _________ Bacteria Algae Mycebial Mycorhi Bacteria
za
Sparger size ranges from 1/64 – 1/32 – 1/48 – 1/24 – 1/64 –
1/32 1/18 1/32 1/12 1/32
inch inch inch inch inch
The first pilot fermenter was erected in India at 1920 1930 1940 1950 1950
Hindustan Antibiotic ltd, Pune in the ---------
year.
-------- type of bioreactor is used for vinegar packed photo pulsed bubble packed
production. tower bioreact column column tower
or
-------------- is used for sep and other algal packed photo pulsed bubble photo
protein production. tower bioreact column column bioreact
or or
--------------- is used as a enzyme bioreactor. packed photo pulsed bubble pulsed
tower bioreact column column column
or
--------------- are provided to maintain constant baffles cooling stirrer sparger cooling
temperature inside the bioreactor coils gland coils

The impeller should be ---------- of the vessel 1\1 none of 1\4 1\3 1\3
diameter. the
above
Range of fermentation tank used in enzyme 1500 1000 – gallons none of 1500
production. 30,000 30,000 c the 30,000
above
-------- fermenter is called as elongated non- Tower Airlift Cylindra Deep jet Tower
mechanically stirred fermenter conical

_______ fermentor is a gas tight baffled rise tube Tower Air lift Cylinder Deep jet Air lift
connected to a down comer tube. conical

Multiple air lift fermenter Is designed by Bakker Okabe Bacon Dawsa Bakker
etal etal etal etal
The inoculum level introduced into a 0.5-5% 5 – 25% 20 – 50% 5 – 25%
production tank is usually ______. 40%
-------- fermenter is called as elongated non-mechanically
Tower stirred Airlift fermenter
Cylindraconical
Deep jet Tower
_______ fermentor is a gas tight baffled rise tube connected
Tower to Aira down
lift comer
Cylinder tube.
conical
Deep jet Air lift
UNIT-IV
Ancillary equipments in fermentors means the -----------------
seed tank fermentation extramedium
connection
antifoamers extra connection
The sterilization temperature of the fermentation120°C equirement
– 15120°C
minis – 20115°C
min – 15min115°C – 20min120°C – 20 min
After sterilization, all parts of fermentor are keptpositive
sterile bynegative
maintaining no at ---------------pressure
zero positive
There should be no permanent direct connectionmedium below -----------and-------------parts
and
sterile
air andmixing
non-sterile
andprobes
air of and
thesterile
fementor
mediumand non-steril
system
High quality valves such as------------should be used plastic β-hydroxybutyrate
where joints are needed
silica gelconnecting
rubber torubberfermentor
Mycellium undergoes autolysis with raise in ___________
pH value temp Ionic concnone pH value
Example for non ionic detergents Tween 80 Tween 20 Tween 40 none of theTween above20
Silicon compound are example are of inert ------- Antibacterial
agent Antifoam Anti fungalAntiprotozal Antifoam
----------- is the main compound in corn steep liquor Lactic acidAmino acidTartaric acid Lactose Lactic acid
Impeller are used in the fermentor helps in Aeration Antifoaming Agitation AbsorptionAgitation
_______ or ______ is added to adjust pH if too [Link] Sodium hydroxide Both a or bSulphuric Both acid a or b
_________ chromatography separates according to Adsorption
the affinityAffinity
of the protein,
Ion exchange
for the
Column
surfaceAdsorption
of the solid matrix
-------- chromatography is a powerful and highly selective
Adsorption purification
Affinity Ion technique.
exchangeColumn Affinity
Microbial cells and other insoluble materials are Filtration
normally separated
Centrifugation
Filtration
from the harvested
Sedimentation
OR centrifugation
broth
Filtration
by_______
OR centrif
or ____
Salts such as ammonium and --------------are usedNH3SO4for the discover
Na2SO4of protein
CaCl2 Ca Na2SO4
Dextrans can be precipitated out of a broth by the Methanol
additionEthanol
of ______. Butanol Alcohol Methanol
----------- method is used for large scale enzyme purification
Liquid shear Solid shearUltrasonicationFreeze thawing
Liquid shear
Ultrasonication has frequency of -----------khz 200 2 20 2000 20
------------- damage the cell membrane and lead toOsmotic
the release
shock
Alkali
of intracellular
treatment
Detergent components
Enzyme Osmotic shock
_______ caused by a sudden change in salt concentration
Osmotic will
shock
Alkali
cause disruption
Proteaseof SDSa numberOsmotic
of cell types.
shock
_ is the separation process where the solvent molecules
Ultra filtration
areReverse
passed to osmosis
Liquid
flow through
membranes
pumping
Semipermiable
Reverse osmosis
membrane
______ of any product is often the last stage of a manufacturing
Filtration Centrifugation
[Link] Packing Drying
--------- drier is mostly used for drying of biological
Freeze
materials Spray Drum Tray Spray
_______ device are the most economical available Freeze
for handling Spray
large volumes.
Drum Tray Spray
------- drying is an important operation in the production
Freeze ofDrum biologicalSpray
and pharmaceuticals
Tray Freeze
_____ is an established and final purification of a diverse
Drying range Crystallization
of compounds.
Filtration HPLC Crystallization
______ are metal strips roughly one- tenth of the vessel
Spargerdiameter
Bafflerand attached
Magneticradically
devices
Impellersto the
Baffler
wall
A combined sparger and agitator may be used in Laboratary
---------fermenter
Tower Airlift Batch Laboratary
---------were first used as careeier for antifoam inOils
antibioticFatsprocessesCarbohydrate Acids Oils
Which is the by-product after starch extraction from Cornmaize.
steepBarley
liquor Molasses Soybean oil Corn steep liquor
Chemically defined amino acid media devoid of protein Acids areVitamin
used in production
Vaccines of Antibiotics
______ Vaccines
________does not appear to play a nutritional role in Chlorine
the metabolism
FluorideofCopper
fungi. Cadmium Chlorine
_________ is used to carry out microbiological processBatch onfermentor
batchContinuous
basis. Fed fermentor
batch Semi
fermentor
continuous
Batch fermentor
fermentor
Small lab fermentor is in the size range of 1-21+ 0.5-11+ 1-101+ 5-101+ 1-21+
Larger fermentor range from _________ gallous. 5000-10000 100-1000 1000-500010000-20000 5000-10000
pH control is achieved by _________ device. Anti titrator Aerator Baffler Impeller. Anti titrator
___________ consists of circular discs to which bladesImpellers
are fitted
Sparger
with bolts.
Baffler Aerator Impellers
Size of the holes in the sparger ranges from_______1/64-1/321/32- 1/64 1/32- 1/32 1/64 -1/64 1/64-1/32
Steady state condition can be achieved by operation Chemostatic
on _________
Turbidostatic
principles.
Both chemostat
Photostat
and Both
turbidostat
chemostat and t
CSTF is expanded as Continuous Continuous
stirred tank
Cell
solid
fermentor
suspended
tank
Continuous
fermentor
tank Continuous
fermentor
solid type stirred
fermentota
The temperature of _______ in the production tanks 80°F
is satisfactory
70°F during90°Ffermentation.
100°F 80°F
Industrial alcohol production can be carried out in 12500
very large
125fermentor
25000
upto _______
100000Gallous.
12500
_________ chromatography separates according Adsorpti Affinity Ion Column Adsorpti
to the affinity of the protein, for the surface of on exchang on
the solid matrix e
-------- chromatography is a powerful and Adsorpti Affinity Ion Column Affinity
highly selective purification technique. on exchang
e
Microbial cells and other insoluble materials Filtratio Centrifu filtration Sedimen filtration
are normally separated from the harvested n gation or tation or
broth by_______ or ______. centrifug centrifug
ation ation
-------------------is added along with stainless steelchromiumnickel
fermentor during construction
tungsten silicone
improvesnickel
engineering.
In antibiotic manufacturing processes, the 2-3 1-2 4-5 2-4 4-5
fermentation time ranges from________ weeks weeks weeks weeks weeks

Micro filtration refers to the separation of

suspended material such as bacteria by using a 0.02 to 0.02 to
membrane with spore sizes of________ 10μm 1-10A° 20-30μm 10-200A° 10μm

separati
on of one
solute
from
other
different constitue different different
rate of nts by rate of rate of
moveme being moveme moveme
nt of the captured nt of the nt of the
solute in on the solvent solute in
the adsorbe in the the
Chromatography is based on the________ column nt column solvent differnces
column
The purification and recovery of the production after
Upstream
fermentation
Downstream
process isSurface
called________
process
fermentation
None of these
Downstream process
Batch fermentation is also called________ Closed system
Open system Fed-batchNonesystemof these
Closed system
The capacity of laboratory fermentors is________ 12–15 liters 2000 gallons 500 liters 10000 gallons12–15 liters
Cell lysis becomes an important operation if the product
Extra cellular
is Heat
________
labileToxic Intracellular
Intracellular
Precipitation is done by________ PEG(polyethylene
Triazine glycol)
dyes
Ammonium PEG,
andtriazine,
sodium
PEG,ammonium
sulphate
triazine, ammon
and s
If the product formed is extracellular then the method
Reversewhich
osmosis
Ultra
is not
filtration
used
Chromatography
is________
Freeze thawing
Freeze thawing
Which process uses a porous medium for the separation
Precipitation
ofFiltration
the solid material
Centrifugation
from
Foamgasseparation
or Filtration
liquids________
A centrifuge is used to separate molecules on theSize
basis of their
Shape--------Density Size, shapeSize, andshape
density
and densi
Which is the preferred method for clarification ofPrecipitation
wine________
Chromatography
? Centrifugation
Foam separation
Centrifugation
Chilled________ and________ can be used in the precipitation
Ethanol, Ethane
ofAcetone,
proteinsKetone
Glycol,
mainlyGlycerol
dueEthanol,
to changes
Acetone
Ethanol,
in theAcetone
dielectric pr
Which separation technique is based on differential Filtration
partitioning
Precipitation
betweenCentrifugation
two phases
Chromatography
thatChromatography
is mobile and statio
Two phase aqueous extraction requires ---------- Low waterLow content
water and
High
content
lowwater
surface
and
High
content
high
water
tension.
surface
and
High
content
lowwater
tension.
surface
and
content
high
tensi
su
a
UNIT-V
The Aspergillus niger spores are developed by growing production stock
sporulation
medium
culture growth
onto
mediuma solid
medium
optimization
------------ sporulation
atmedium
25oC formedium
4 to 14
Alpha amylase are produced by ___________________AspergillusMucor Pencillium fusarium Aspergillus
Proteases are produced by __________________ B .subtilis [Link] theBabove .subtilis
Acid proteases are produced by the fungi AspergillusRhizopus
oryzae stoleniferous
fusarium sp Altemaria Aspergillus
sp. oryzae
__________ are usually employed for commercial prodution
Sacchcromyces ofCandida
ethyl cerevasiae
alcohol
albucans
Cryptococcus none of the Sacchcromyces
above cereva
The most efficient industrial strain used in productionAspergillus
of citric
Aspergillus
nigeracid isAspergillus
------------------
terreus Aspergillus
clavatus Aspergillus
oryzae niger
------------- is often the best carbon substrate for production
sugarcane molasses
of citric aci(d)
wood molasses corn steepbeet liquormolasses
The optimum pH for citric acid production is -------------
2.60 to 3.20 3.6 to 4.202.20 to 1.60 1.2 to 2.6 2.20 to 1.60
The optimum temperature for citric acid production 25oCaccording
to 27oC
26oCtotoDoelger
28oC
28oC and to 30oC
Prescott
27oC to 29oC is 26oC
---------------
to 28oC
The citric acid fermentation using Aspergillus niger sterilization
at low pHcitric
ofismedium
highly
acid oxalic
is favourable
produced
is not
acideffective
There
formation
atasvery
is high
low
ANS:
is rich
suppresse(d)
yields
(c)oxalic
of contamina
acid fo
The production vessel for citric acid must be made stainless
of -------------------
steel
iron as
aluminium
they arestainless
less prone steel
stainless
to and
conversion
aluminium
steel and
with
alu
The citric acid fermentation takes up to -----------5days to 8 days6 to 9 days7 to 10 days 8 to 11 days 7 to 10 days
-------------have various structures, composition and antibiotics
are of low
hydrocarbons
molecularproteins
weightlipids
compounds. antibiotics
Natural and semi-synthetic penicillins and cephalosporins
peptide antifungalmacrolidepencillin
are included in -------------group pencillin
of antibiotics.
In ------------ group of antibiotics, β-lactum ring ispeptide
foun(d) antifungalmacrolidepencillin pencillin
------------ employed in production media yields high pharmamedia
amounts corn-steep
of penicillin.
sulfite-waste
liquor hydrocarbons
liquor corn-steep liquor
For the commercial production of penicillin highPenicillium
yielding strains
Penicillium
notatum havePenicillium
flavum
been selected
Penicillium
geiseofulvicus
fromPenicillium
the
chrysogenum
ancestral
chrysogen
fungus
The examples of two important naturally occurring pencillin
pencillinGpenicillin
and
areV-------------
B
penicillin
and VandM penicillin
--------------
and V O pencillin
and V G and V
Staphylococcus aureus produces an enzyme called amylase
------- which
pectinase
cleaves protease
the β-lactum
penicillinase
ring penicillinase
of penicillin.
The precursor for penicillin G is ----------------- phenyl sulphate phenylacetic phenyl
acid phosphate
phenychloride phenylacetic acid
The ----------- medium contains glycerol, cane molasses,
production corn-steep
sporulation liquor
preservation
for production
mother culture of
sporulation
large number of sp
For removal of fungal mycelium --------------with rotoary
centrifugation
vacuum
filtration
filterheating
has beenprecipitation
employe(d) filtration
For extraction of penicillin, --------------- solvent extraction
counter currenttechnique
charcoal co-currentbi-currentcounter
is use(d) current
The organic solvents such as amylacetate, butyl acetatecounterare current
charcoal
used in ------------
co-currentbi-currentcounter
solvent extraction metho(d) current
The final industrial product is treated with ------------
alkalito eliminate
charcoal pyrogens.
glycerol acid charcoal
.------------- is a penicillin without an acyl group, prepared
natural penicillin
from
synthetic
6-amino penicillin
semi-synthetic
penicillanic
GN penicillin
aci(d)
penicillin
semi-synthetic penic
----------- of 6-amino penicillanic acid is carried outacylation
by usingchlorination
derivative carboxylation
of carboxylic
methylation
aci(d)acylation
Citric acid is used as ------------ is food and pharmaceutical
acidulant industries.
basifier precursoranti-foaming acidulant
agent
Several species such as Aspergillus clavatus, Penicillium
lactic acidsuccinic
luteum, Penicillium
acid
tartaric citrinium,
acid
citric acid Mucor
citricpiriformis
acid are us
The foremost important bioparameter in citric acid temperature
fermentation
pH is----------
ratio of surface
aeration area ratio
to volume
of surface area t
When the ratio of surface area to volume is -----------lower there ishigher
high yield medium
of citric equal
aci(d) lower
The recovered fermentation liquor is treated with NaOH
-------- forming
Sulphuric precipitate
Nacl
acid ofMilk
calcium of limecitrate.
Milk of lime
Another method for extraction and purification of counter
citric acid
current
co-currentbi-currentTri-current
is byextraction
using tri-n-butyl phosphate counter andcurrent
n-butylextra
acet
The problems faced during recovery of citric acidpresenceare -------- of
presence
unconverted of
presence
oxalic
sugars
acid
of
PRESENCE
trace saltsOF unconverted sug
The yield of citric acid by submerged fermentation are -----------------------
80 90 95 92 90
In 1970, ---------------- yeast was used for submerged Candida
culturebrumptil
Candida
processlipolytica
of
Candida
citric acid
hydrocarbofumarica
Candida
production albicans
Candidausinglipolytica
hydrocarbo
One gram of glucose yields ----------- gms of ethanol0.4111 in fermentation
0.5111process. 0.6111 0.7111 0.5111
The bioconversion of sugars to alcohols are mediated proteinsby -------------
lipids ofsaccharides
years. enzymes enzymes
The french word “vinaigre” means --------------- fresh grape fresh
juicewinesour wineethanol sour wine
o o o o
70-80oF 72-82 F 75-80 F 76-82 F 75-80 F
The temperature required for fermentation is -------------
There are ---------- stages in alcoholic fermentation. two three four five two
The primary stage of alcoholic fermentation is carried
2 to 5 out in3 to
open
7 fermentation
4 to 8 5vats
to 10for -----------
3 to 7 days.
The secondary stage of alcoholic fermentation proceeds
closed foropen several weeks
plasticin ------------
iron containers.
closed
---------- is generally added in alcoholic productionsulfur
medium.
dioxide
sulfur oxide ethylene gas nitrous oxidesulfur dioxide
--------- inhibits the growth and activity of unwanted
sulfur
bacteria
dioxide
sulfur
andoxide
yeasts.
ethylene gas nitrous oxidesulfur dioxide
The microorganismis namely Aspergillus niger, Rhizopus
⍺-amylaseGlucoamyase
niveus, Rhizopus
Protease delemar
Cellulaseare Protease
sources of -------------
Lipase is produced industrially using ----------------------.
AspergillusRhizopus
niger sp Candida spAspergillusAspergillus
niger, Rhizopus
niger,sp
Rhi
a
During completion of enzymatic fermentation, the rapid
spent
heating
liquid
precipitation
is subjected
condensation
to rapid
-----------------to
cooling
rapid reduce
coolingdeterior
The presence of -----------------inhibits the formation
glucose
of lactose sucrose galactose glucose
The enzymes are purified using solvents such as---------------------
acetone alcohols inorganic-salts(ammonium
acetone, salts
acetone,
andsulfate)
solvents
salts and sol
which enzyme is very important in bread making,⍺-amylases
beer makingpectinase
for flood
cellulase
industry.
proteases ⍺-amylases
The preservation of bacterial ⍺-amylase enzyme in liquid5formulation 10 is--------%
15 NaCl 20 20
The optimum pH for amylase industrial fermentation process 5 is-------------
6 7 8 7
Proteases enzymes are a complex mixture of -----------------
amylases & cellulase
&---------------------------
amylopectinases
&proteinases
cellulobiaseinvertase&
& peptidases
proteinases
phosphatase & peptid
which enzyme are unstable & loss of activity can⍺-amylaseGlucoamyase
occur during fermentationProteaseprocess.
Cellulase Protease
The enzyme protease is produced from bacteria such Bacillus
as ----------------
subtilis
Bacillus lichenifermis
Bacillus subtilis
Bacillus
& Bacillus
anthrax
Bacilluslichenifermis
subtilis & Bac
The optimum temperature & pH for protease enzyme 20-30°C,fermentation
730-40°C, 730-40°C,
is-----------------
840-50°C, 730-40°C, 7
Vitamin B12 is formed by linking of a cobinamidenucleotidenucleoside
linked to a ---------------
nucleode nucleic acid nucleotide
(DNA/RNA)
The nucleotide of cyanocobalamine has ---------------5,7-dimethyl
as its base.
5,8-dimethyl
benziminazole
5-6-dimethyl
benziminazole
5,4-dimethyl
benziminazole
5-6-dimethyl
benziminazolebenzim
Microorganisms namely Streptomyces griseus, Streptomyces
vitamin A vitaminolivaceus,
B12
vitamin
Bacillus
C vitamin
megaterium,
D vitamin
Pseudomonas
B12 de
The strain used for vitamin B12 production is -------Streptomyces Streptomyces
olivaceusStreptomyces
NRRL
olivaceus
A-1125
Streptomyces
NRRL
olivaceus
B-1125
Streptomyces
NRRL
olivaceus
C-1125
NRRL
olivaceu
D
The vitamin B12 fermentation takes about ----------- 2 todays
4 3 to 5 4 to 6 5 to 7 3 to 5
After removal of cobalamine from mycelium of Streptomyces
cyanide chlorine olivaceus,cobalt
-----------
citrate
is addedcyanide
with broth to form c
The temperature of ------------ is favourable during 60oF
fermentation
70oF of vitamin
80oF B12.90oF 80oF
The yield of cobalamine is in range of ------------- mg/l
0.5-1 at end 0.8-1
of fermentation
1 to 2 process.
1.2-2.5 1 to 2
from cheaper and disposable substrates

1800-1900
post-1900

sugar, alcohol, carbonic acid

lactic acid

butyric acid and acetic acid

Dumas and Napoleon 111
pasteurization
lyophilisation
Streptomyces sp
acetone and n-butanol
citric acid
riboflavin
Weizmann
should be a high yielding strain
primary and secondary
desired property

crowded plate technique

Beijerinck
auxanography
organic acids
cellulose powder
secondary
Commonwealth Mycological Institute

optimized
Raper and Alexander
contamination
physical and chemical
desiccant
single, double, triple
autotropic mutants and mutants resistant to analogues
lysine and threonine

British Pharmacopoeia Medicinal Paraffin oil.

aspartate kinase
-amino,β-hydroxyvaleric acid

Brevibacterium flavum
Hydrogenomonas
constitutive
Fermentors

American type culture collection

)0.865-0.890
serial subculture
sporulation
Bacteriophages and clostridium

phenyl acetic acid

mono and dihydropotassium phosphates
contamination
lard oil with ocetadecanol

non-synthetic
agricultural waste
cane molasses

total solids
lactic acid
cereals, roots and tubers
paper pulp, sulfite waste liquor

single cell protein

corn steep liquor
Pharmamedia
tetracycline

streptomycin
antifoams

caramelization
batch cookers
long term
lyophilization

Preserve cultures
15 lb/in2 for 2 hr
long term preservation of Microorganisms

5-80 C

desiccant

production strain
heterofermentative

contamination
soil cultures
20%,78% and 2%

Fermentors
inoculums

molybdenum
feed pumps

smaller, larger

exit gas cooler

process controller

antifoam probe

thermistors

rotameters
thermal mass flowmeter
diaphragm
torsion dynamometer
acid and base
galvanic electrode
mechanical foam breaker
paramagnetic gas analyzer
hydrogen ion

logging of data, data analysis and process control

direct digital control
laboratory-scale
Pilot- and industrial-scale
bubble columns
Spargers

productivity or conversion yield

Humphery and Cooney
Tower

extra connection
120°C – 20 min

sterile and non-sterile

Tween 20

Lactic acid
Agitation
Both a or b
Adsorption

Filtration OR centrifugation

Methanol
Liquid shear
20
Osmotic shock
Osmotic shock
Reverse osmosis

Freeze
Crystallization

Laboratary
Oils
Corn steep liquor
Vaccines
Chlorine
Batch fermentor

5000-10000
Anti titrator
Impellers
1/64-1/32
Both chemostat and turbidostat
Continuous stirred tank fermentor

Downstream process
Closed system
12–15 liters
Intracellular
PEG, triazine, ammonium and sodium sulphate
Freeze thawing
Filtration
Size, shape and density
Centrifugation
Ethanol, Acetone
Chromatography
High water content and high surface tension.

sporulation medium
Aspergillus
B .subtilis
Aspergillus oryzae
Sacchcromyces cerevasiae
Aspergillus niger
beet molasses
2.20 to 1.60
26oC to 28oC
ANS: (c)oxalic acid formation is suppresse(d)
stainless steel and aluminium
7 to 10 days
antibiotics

corn-steep liquor
Penicillium chrysogenum
pencillin G and V
penicillinase
phenylacetic acid
sporulation

counter current
counter current

semi-synthetic penicillin

citric acid
ratio of surface area to volume

Milk of lime
counter current extraction
E OF unconverted sugars, oxalic acid, trace salts

Candida lipolytica

sour wine
sulfur dioxide
sulfur dioxide

Aspergillus niger, Rhizopus sp and Candida

rapid cooling

acetone, salts and solvents

⍺-amylases

proteinases & peptidases

Bacillus subtilis & Bacillus lichenifermis

30-40°C, 7
nucleotide
5-6-dimethyl benziminazole
vitamin B12
Streptomyces olivaceus NRRL B-1125
 [16MBU303]
KARPAGAM ACADEMY OF HIGHER EDUCATION
KARPAGAM UNIVERSITY
(Established Under Section 3 of UGC Act, 1956)
Eachanari Post, Coimbatore, Tamil Nadu, India – 641 021
B. Sc., DEGREE FIRST INTERNAL EXAMINATION AUGUST 2017
Microbiology
INDUSTRIAL MICROBIOLOGY
TIME: 2 Hours Max Marks: 50 Marks
Date:
PART –A 20 x 1= 20 marks
1. In the lysine biosynthesis, the end products lysine and threonine inhibit the enzyme_______.
(a) Aspartate kinase (b) Homoserine phosphatase
(c) Serine kinase (d) Tryptophan synthase
2. In fermentation, yeast converts -------------to---------------and -----
(a)carbohydrate, alcohol, carbonic acid (b)fatty acids, alcohol, carbonic acid
(c)sugar, alcohol, carbonic acid (d)starch, alcohol, carbonic acid
3. Chemical stability of the product is identified by _______.
(a) Primary screening (b) Secondary screening
(c) Enrichment method (d) Tertiary method.
4. Growth factor producing organisms are identified by using
(a) Crowded plate technique (b) Enrichment technique
(c) Auxanography (d) Indicator dye method.
5. Crowded plate technique is used in the detection _________ producing organisms.
(a) Growth factor producers (b) Amino acid producers
(c) Antibiotic producers (d) Organic acid producers.
6. Indicator dye are used in the medium
(a) To detect temp change (b) To detect nutrient concentration
(c) To detect ph change (d) To detect aw availability.
7. The medium which doesn’t contain particular essential nutrient is called as _______.
(a)Enriched media (b) Minimal media
(c) Molden agar (d) Selective media
8. Enrichment technique was designed by _________.
(a)Beijernick (b) Pasteur
(c) Bacon (d) Hooke
9. The size of paper strip used in auxanography was ________.
(a)5 x12 cm (b) 2.5x12 cm
(c) 1.5x10 cm (d) 3x20 cm
10. --------- drier is mostly used for drying of biological materials
(a)Freeze (b) Spray
(c)Drum (d)Tray
11. Primary screening technique employed for detect and isolating antibiotic producing strain is
(a)Crowded plate technique (b)Auxanography
(c)Enrichment culture technique (d)Use of indicator dye
12. The chemical agent which causes mutation is __________.
 (a)Sodium sulfate (b) Ethyl methyl sulfonate
(c)CaCl2 (d) Ethyl sulfonate
13. In 1861 Pasteur did experiments on ---------and-----------fermentation
(a)butyric acid and acetic acid (b)acetone and butanol
(c)lactic acid and acetic acid (d)acetic acid and citric acid
14. Primary metabolites are produced during ________.
(a) Lag phase (b) Log phase
(c) Stationary phase (d) Death phase
15. Mutants can be obtained by treating the cells with ________.
(a) UV radiation (b) Nitrogen oxide
(c) Uric acid (d)Diamines
16. Different microbial species are fused by _________
(a) r-DNA technology (b)Protoplast fusion
(c)Mutation (d)Plasmid fusion
17. The clean, yellow, fine powder prepared from embryo of cotton seed is called as ------------
(a)corn-steep liquor (b)soya bean meal
(c)Pharmamedia (d)distiller’s solubles
18. In Pencillin fementation the precursor added is ------------
(a)phenyl acetic acid (b)benzyl alcohol (c)Benzene (d)pyridine
19. The specific gravity of oil used in presence of cultures is
(a)0.821-0.860 (b)0.865-0.890 (c)0.752-0.812 (d)0.718-0.835
20. Protoplast fusion is mainly used to improve _________ industrial microbes
(a)Algae (b)Fungi
(c)Yeast cells (d)Bacteria

PART –B 3 x 2= 6 marks
21. What are auxotrophs?
22. How will you store the fungal cultures?
23. Write two differences between crude and synthetic media?
PART –C 3 x 8= 24 marks
24. a. Detail on history and development of industrial microbiology.
(Or)
b. Outline the primary screening methods with figures.
25. a. Write notes on isolation of industrially important strain with examples.
(Or)
b. Explain the secondary screening methods.

26. a. What are the ideal characteristics of a fermentation media? Explain the components of
media?
(Or)
b. Describe the methods of preservation of microorganisms with its advantages and
disadvantages.
 KARPAGAM ACADEMY OF HIGHER EDUCATION
KARPAGAM UNIVERSITY
(Established Under Section 3 of UGC Act, 1956)
Eachanari Post, Coimbatore, Tamil Nadu, India – 641 021
B. Sc., DEGREE FIRST INTERNAL EXAMINATION AUGUST 2017
Microbiology
INDUSTRIAL MICROBIOLOGY

ANSWER KEY
PART A
1]
2] C
3]
4] C
5] C
6] C
7] B
8] A
9] C
10] B
11] A
12] B
13] A
14] B
15] A
16]
17] C
18] A
19] B
20] C

PART B
21] auxotrophs: a mutant organism - especially a bacterium or fungus- requires
additional nutrient which the normal strain does not.
22] storage of fungal culture : Periodic serial subcultures - Freezing - Freeze
drying, -. Sterile mineral oil overlay.
23] crude media : used in fermentation – rough composition – high yield- high
level of nutrients vitamins , growth factors etc.
Synthetic media : every component is chemically known - useful in controlling
the growth and yield of product in a lab environment.

PART C
24] (a) History of industrial microbiology

first observations of microorganisms - Leeuwenhoek in 1677- experiments

of Spallanzani in 1799 and of Schwan in 1837 - disproved the idea of spontaneous
generation of microorganisms: provided a means of sterilization of liquids and air.
Schwan's - alcoholic fermentation - due to a fungus or mold. But microbiology-
considered - begun in 1857 when Pasteur studies on lactic acid fermentation.

In 1860- Pasteur - first synthetic medium for microorganisms - used to

study alcohol fermentation. In 1861- Pasteur showed - growth and physiology of
yeast - hence the accumulation of fermentation product, alcohol - phenomenon -
Pasteur effect .1878- Lister described - dilution technique for obtaining the first
pure microbial culture of lactic acid bacterium. technique for obtaining pure
cultures - developed on solidified medium- by Robert Koch in 1881.

In 1876- Cohn showed - bacterial spore - high level of heat resistance -

develop technique of 'intermittent sterilization' for - inactivation. In 1897- Buchner
- alcohol fermentation - suggested that a proteinaceous enzyme - responsible for
fermentation. Wildiers in 1901 t- yeast required growth factors - In 1929
Alexander Fleming - penicillin produced by Penicillium growing as contaminant in
a Petri plate of [Link] - followed - search for antibiotics -
discovery of streptomycin, chloramphenicol, tetracyclines, etc.

(b) primary screening:

some elementary tests- to detect and isolate new microbial species -desired
property- antibiotic producers- serve to remove worthless micro-organisms- The
important selection critcria are the activity of antibiotics -other useful microbial
species -. micro-organisms capable of producing organic acids, amines,
[Link]-.:
The crowded plate technique- detecting and isolating -antibiotic producing -
soil or other source material- spreading of dilutions on the nutrient agar plate agar
plates- exhibit antibiotic activity is indicated by the presence of a zone of growth
inhibition surrounding the colony
Auxanography- applied for detecting micro-organisms able to produce growth
factors - [Link] and vitamins.
Enrich Culture technique- designed by, Beijerinck,- to isolate the desired
microrganisms from a heterogeneous microbial population present in soil. Either
medium or incubation conditions are adjusted so as to favour the growth of the
desired micro-organisms.
Use of an indicator dye- pH indicating -detecting microorganisms -producing
organic acids or amines, since a pH indicating dye under .colour changes according
to its pH. Such dyes (e.g. neutral red, bromophenol blue) are added to the poor
nutrient agar media. The change in the colour of a dye in the vicinity of the colony
suggests the capability of colonial cells to produce anionic acid(s or amine(s),
depending upon the nature of reaction.

25] (a) isolation of industrially important strains

Two methods- primary screening and secondary screeing
Primary screeining -The crowded plate technique- detecting and
isolating -antibiotic producing - soil or other source material- spreading of
dilutions on the nutrient agar plate agar plates- exhibit antibiotic activity is
indicated by the presence of a zone of growth inhibition surrounding the colony
Auxanography- applied for detecting micro-organisms able to produce growth
factors - [Link] and vitamins.
Enrich Culture technique- designed by, Beijerinck,- to isolate the desired
microrganisms from a heterogeneous microbial population present in soil. Either
medium or incubation conditions are adjusted so as to favour the growth of the
desired micro-organisms.
Use of an indicator dye- pH indicating -detecting microorganisms -producing
organic acids or amines, since a pH indicating dye under .colour changes according
to its pH. Such dyes (e.g. neutral red, bromophenol blue) are added to the poor
nutrient agar media. The change in the colour of a dye in the vicinity of the colony
suggests the capability of colonial cells to produce anionic acid(s or amine(s),
depending upon the nature of reaction
Secondary screening – following points-
Providesinformation whether [Link] produced by a microorganism
is a new one or
thin layer, other Chromatographic techniques.
compared with previously known compounds.
idea about the economic position of the fermentation proces.
It helps in providing information regarding the product yield potentials
of different isolates
It determines the optimum conditions for growth
valuable in design in the medium that may be attractive so far as
economic consideration is concerned.
It detects gross genetic instability
number of products produced in a single fermentation additional major
or minor productssolubility of the product in various organic solvents is
made available.
Chemical, physical and biological properties of a
homoferrnentative or hetero fermentative.
the structure of the product is done.
determinations of the toxicity [Link]

(b)secondary screening
 strictly essential in any systematic screening programme -to isolate
industrially useful micro-organisms, since primary screening merely
allows the detection and isolation of micro:Organisms that possess
potentially interesting industrial application Moreover, primary
screening does not provide much information needed in setting up a new
fermentation process. Secondary screening helps in detecting really
useful micro-organisms in fermentation processes:

Secondary screening – following points-

Providesinformation whether [Link] produced by a microorganism
is a new one or
thin layer, other Chromatographic techniques.
compared with previously known compounds.
idea about the economic position of the fermentation proces.
It helps in providing information regarding the product yield potentials
of different isolates
It determines the optimum conditions for growth
valuable in design in the medium that may be attractive so far as
economic consideration is concerned.
It detects gross genetic instability
number of products produced in a single fermentation additional major
or minor productssolubility of the product in various organic solvents is
made available.
Chemical, physical and biological properties of a
homoferrnentative or hetero fermentative.
the structure of the product is done.
determinations of the toxicity [Link]

26] (A) characteristics of fermentation media

All microbes require water, sources of carbon, mineral elements and probably
vitamin and oxygen if aerobic on a large scale- use sources of cheap nutrient to
make a medium which will need the following criteria.
(i) produce maximum yield of products or biomass per gram of substrate used.
(ii) the maximum rate of product formation.
(iii) The yield of undesirable products must be minimal.
(iv) cheap and of consistent quality.
(v) cause minimum problems in the production process particularly
Different technical objectives of media formulation- Inoculum (starter culture)
propagation steps / pilot-scale- Biomass or primary metabolites production /
secondary metabolite production
Constituents of medium
1. Water
2. Carbon source / Nitrogen source / Sources of phosphorous and sulfur / Minor
and trace elements / Vitamins such as biotin and riboflavin
3. Oxygen: even some anaerobic fermentations require initial aeration, e.g. beer
fermentations
4. Buffers or controlled by acid and alkali additions
5. Antifoam agents
6. Precursor, inducer or inhibitor compounds
Considerations in media design - Nutritional Requirements, Environmental
Requirements and Techno-economic Factors.

(b) methods of preservation

Different methods – on type of cultures – they are – Serial Subculture -
simplest and most common method of maintaining microbialcultures. Microbe are
grown on slants.
Preservation by overlaying cultures with oil - This method is cheap and easy,
since it does not require special skills or apparatus such centrifuge, dessiccator, or
vacuum pump. - The covering of the culture with oil prevents drying out - strains
tested live longer under oil than in the control tubes
without oil
Lyophilization or freeze-drying most satisfactory method of long-term
preservation of [Link] is universally used for the preservation of
bacteria, viruses, fungi, sera, toxins, enzymes and other biological materials.
Storage at very low temperatures or Nitrogen Storage - also called cryogenic
storage- like lyophilization, a satisfactory method for the long-term preservation of
micro-organisms. It has also been successful with many specimens which can be
preserved by lyophilization. The maintenance of microbes is done by suspended
metabolism. According to Meryman (1956), life is regarded as 'at a standstill' at -
130°C. and below, so at the temperature of liquid nitrogen (--196°C.)
Desiccation - involves removal of water from the culture. Desiccation is used to
preserve actinomycetes (a form of fungi-like bacteria) for very long period of
time. The microorganisms can be preserved by desiccating on sand, silica gel, or
paper strips.
 Reg. No.___________________
[16MBU303]
KARPAGAM ACADEMY OF HIGHER EDUCATION
KARPAGAM UNIVERSITY
(Established Under Section 3 of UGC Act, 1956)
Eachanari Post, Coimbatore, Tamil Nadu, India – 641 021
B. Sc., DEGREE SECOND INTERNAL EXAMINATION AUGUST 2017
Microbiology
INDUSTRIAL MICROBIOLOGY
TIME: 2 Hours Max Marks: 50 Marks
Date:
PART –A 20 x 1= 20 marks
1. ----------- culture where medium is fed to the culture resulting in an increases in volume
(a) Batch (b)Fed batch
(c)Continuous (d)Semi continuous
2. Steady state condition can be achieved by operation on _________ principles.
(a)chemostatic (b)turbidostatic
(c)chemostatic and turbidostatic (d)photostat
3. Sulfite waste liquor contains ----------sugars
(a)1% (b)2%
(c)3% (d)4%
4. In ----------- bioreactor are so designed that adequate supply of oxygen is obtained without
agitation.
(a)air lift (b)CSTR
(c)packed bed (d)fluidised bed
5. CSTF is expanded as
(a)Continuous Stirred Tank Fermentor (b) Continuous Solid Tank Fermentor
(c)Cell Suspended Tank Fermentor (d)Continuous Solid Type Fermentor
6. ___are metal strips roughly one- tenth of the vessel diameter and attached radically to the wall
(a)Sparger (b) Baffler
(c)Magnetic devices (d)Impellers
7. --------- is a culture system which contain a initial limited amount of nutrient.
(a)Fed batch (b)Batch
(c)Semi continuous (d)Continuous
8. Cellulose are carbohydrates made of repeating units of --------
(a)α-glucose (b)β-glucose
(c)α-galactose (d)β-galactose
9. The ideal aspect ratio for a fermentor is ------
(a)3:1 (b)4:1
(c)5:1 (d)6:1
10. The first pilot fermenter was erected in India at Hindustan Antibiotic ltd, Pune in the ----
year.
(a)1920 (b)1930
(c)1940 (d)1950
11. Large fermentors range from ________
(a)2000 – 5,000 gallons (b)5000 – 10,000 gallons
 (c)10,000 gallons (d)10,000-30,000 gallons.
12. ----------- is the main compound in corn steep liquor
(a)Lactic acid (b) Amino acid
(c)Tartaric acid (d)Lactose
13. Impeller are used in the fermentor helps in
(a)Aeration (b) Antifoaming
(c)Agitation (d)Absorption
14. ______ is added to adjust pH if too acidic.
(a)Ammonia (b) Sodium peroxide
(c)Sodium nitrate (d)Sulphuric acid
15. In -------a portion of the culture is harvested and replaced by an equal volume of medium.
(a)Fed batch culture (b) Batch culture
(c) Semi continuous culture (d) Continuous culture
16. --------spargers are used widely large scale fermentation process.
(a)Porous (b)orifice
(c)nozzle (d)combined sparger agitator
17. -------is generated due to mixing by agitator and microbes action on substrates during
fermentation process.
(a)energy (b)heat
(c)resistance (d)current
18. --------are semiconductors of iron, michel oxides exhibiting large change in resistance with
small change of temperature.
(a) mercury in-glass thermometers (b)electrical resistance
(c)thermistors (d)electrical impedance
19. Stainless steel ------ sensors are used for temperature measurements in fermentation system.
(a) Pt 100 (b) Pl 100
(c) Pb 100 (d) Ps 100
20. Dissolved oxygen in fermentation process medium measured by using -----------------
(a)galvanic electrode (b)pH electrode
(c)thermometers (d)thermistors
PART –B 3 x 2= 6 marks
21. What is solid state fermentation?
22. Name two anti-foaming agents used in fermentation medium?
23. What is mean by upstream and down stream processes?
PART –C 3 x 8= 24 marks
24. a. Elaborate on the basic design of a fermentor.
(Or)
b. Explain the fed-batch fermentation.
25. a. With clear figure explain the principle and application of air lift reactors.
(Or)
[Link] are the process parameters such as pH and temperature in fermentation are measured
and controlled?.
26. a. Elaborate on the various carbon sources for fermentation medium.
(Or)
b. Describe the various nitrogen sources for fermentation medium.
 DEPARTMENT OF MICROBIOLOGY
KARPAGAM ACADEMY OF HIGHER EDUCATION
(Deemed to be University Established Under Section 3 of UGC Act, 1956)
Eachanari Post, COIMBATORE - 641 021, INDIA

[Link]., Microbiology (Batch 2016-2019)

Industrial Microbiology (Semester-III) (16MBU303)

ANSWER KEY
PART A
1] A
2] C
3] B
4] A
5] A
6] B
7] B
8] B
9] C
10] D
11] B
12] A
13] C
14] A
15] D
16] C
17] B
18] C
19] B
20] A
PART B
21] Solid state fermentation - biomolecule manufacturing process- used in the
food, pharmaceutical, cosmetic etc- metabolites by microorganisms grown on
solid support - alternative to liquid or submerged fermentation
22]Antifoam agents in fermentation - Natural antifoams - soy, sunflower and
rapeseed oil deodorized fish oil, mineral oils - Synthetic antifoams - silicon oils,
poly alcohols and alkylated glycols.

23] Upstream process - first step - microbes/cells are grown - in bioreactors.

Upstream processing - inoculum development, media development, improvement
of inoculum by genetic engineering process.
Downstream process - three main sections - cell disruption, a purification
and a polishing.
PART C
24] A]Design of a fermentor
BODY CONSTRUCTION - small scale - vessel construction glass or stainless
steel – used -For pilot and large scale process,- stainless steel (>4% chromium),
mild steel (coated with glass or epoxy material), wood, plastic - construction
material - non toxic and corrosion proof.
Glass vessel -Type I – glass vessel round or flat bottom with top plate - Type II –
glass vessel flat bottom with top and bottom stainless steel plate
Stainless steel - as vessel construction material with the following modifications -
Thickness of vessel - increased with scale - Side plates - lower thickness than top
and bottom plates
SEALING - Sealing important - to maintain airtight condition, aseptic and
containment - three types of surfaces - between glass-glass, glass- metal and metal-
metal - three types of sealing- gasket, lipseal and ‘O’ ring- sealing ensures tight
joint - materials used for sealing - fabric-nitryl or butyl rubbers.
BAFFLES - are metal strips that prevent vortex formation - attached radially to
the wall for every 1/10th of vessel diameter - enough gap between wall and baffle
- scouring action around vessel is facilitated.
AERATION SYSTEM (SPARGER) - for introducing air into [Link]
sufficient oxygen for organism in the fermenter. Fine bubble aerators must be used.
Large bubbles will have less surface area than smaller bubbles which will facilitate
oxygen transfer to a greater extent - three types of spargerviz. porous sparger,
orifice sparger and nozzle sparger..
EXIT GAS COOLER - removes as much moisture as possible from the gas
leaving the fermenter and prevent excess fluid loss.
AGITATION - provides uniform suspension of cells in homogenous nutrient
medium - provides bulk fluid and gas phase mixing, air dispersion, facilitates
oxygen transfer and heat transfer and uniform environment through out the vessel -
four classes, namely Disc turbine, Vaned disc, Open turbine of variable pitch and
Marine impeller
STIRRER GLANDS AND BEARINGS - more space for entry ports on top.
There are four types of stirrer glands and bearings.1) Stuffing box2) Mechanical
seal3) Magnetic drives (some animal cell cultures)4) Simple bush seals
Check valves - prevent accidental reversal flow of liquid or gas due to break down
- three types – swing check, lift check, combined stop and check.

(b) fed batch fermentation - technique in biotechnological processes - one or

more nutrients (substrates) are fed (supplied) to the bioreactor - during cultivation -
in which the product(s) remain in the bioreactor until the end of the run – needs - a
base medium supports initial cell culture and a feed medium is added to prevent
nutrient depletion - type of semi-batch culture - nutrients are fed into the
bioreactor - advantage - control concentration of fed-substrate in the culture -
desired levels - superior to conventional batch

In this process the nutrients and substrates are added at the start of the process and
at regular intervals after the start - called controlled feeding - Inoculum is added
to the fermentation vessel - microorganisms are in exponential growth phase -
controlled by feed-back control and control without feed-back.
1. Feed-back control – process parameters like dissolved oxygen content, carbon
dioxide to oxygen ratio, pH, concentration of substrate, and concentration of the
product.

2. Control without feed-back– The substrates and nutrients are added at regular
intervals.

Fed-batch culture requires special equipment - reservoir which holds the nutrients,
pH modifiers so that they can be added to the fermenter at regular intervals, and
pumps to deliver culture medium aseptically to the fermenter.

25] Airlift fermentor - classified as pneumatic reactors - without any mechanical

stirring arrangements for mixing and use the expansion of compressed gas to bring
about the mixing - turbulence caused by the fluid flow ensures adequate mixing of
the liquid - The draft tube is provided in the central section of the reactor -
introduction of the fluid (air/liquid) causes upward motion - results in circulatory
flow in the
advantages - elimination of attrition effects generally encountered in mechanical
agitated reactors - suited for aerobic cultures since oxygen mass transfer
coefficient are quite high in comparison to stirred tank reactors - do not have
mechanical agitation systems (motor, shaft, impeller blades) - contents are agitated
by injecting air from the bottom - Sterile atmospheric air is used -
microorganisms are aerobic - “inert gas” is used if microorganisms are anaerobic -
gentle method of mixing the contents - most suitable for fermentation of animal
and plant cell cultures since the mechanical agitation produces high shearing -
stress that may damage the cells - widely used for large-scale production of
monoclonal antibodies.
26]A] Carbon sources
Molasses - Byproduct of cane or beet sugar production / residues remaining after
most of the sucrose has been crystallized from the plant extract -Dark colored -
containing 50-60% (w/v) carbohydrate, primarily sucrose - vitamins and minerals -
Hydrol molasses, containing primarily glucose, is a byproduct of maize starch
processing
 Malt extract - Concentrated aqueous extracts of malted barley - useful for the
cultivation of filamentous fungi, yeasts and actinomycetes - App. 90%
carbohydrate (w/w) - some vitamins - app. 5% nitrogenous substances, proteins,
peptides and amino acids - carbohydrate comprising 20% hexoses - 55%
disaccharides - 10% maltotriose, - 15-20% branched and unbranched dextrins-
when heated at low pH / color change - loss of fermentable materials - some toxic
products
Starch and dextrins - metabolized by amylase-producing microorganisms -
filamentous fungi- Maize starch is most widely used- use in a wide range of
fermentations - starch converted into sugar syrup, containing mostly glucose - first
gelatinized and then hydrolyzed by dilute acids or amylolytic enzymes
Sulfite waste liquor - Sugar containing wastes derived from the paper pulping
industry are - used for the cultivation of yeasts - Waste liquors from coniferous
trees contain 2-3% (w/v) sugar, 80% hexoses and 20% pentoses - are
supplemented with sources of nitrogen and phosphorus
Cellulose - Predominantly as lignocellulose (composed of cellulose, hemicellulose
and lignin) - Available - agricultural, forestry, industrial and domestic wastes - few
microorganisms can utilize directly - mainly used in solid-substrate fermentations
(e.g. mushrooms) - very valuable renewable source of fermentable sugars -
bioconversion to ethanol for fuel use
Whey - aqueous byproduct of the dairy - containing over 1 million tons of lactose
and 0.2 million tons of milk proteins - Expensive to store and - Formerly used
extensively in penicillin fermentation - employed for producing ethanol, single cell
protein, lactic acid, xanthan gum, vitamin B12 and gibberellic acid.
Alkanes and alcohols - Ethanol is less toxic than methanol / used as a sole or
cosubstrate / too expensive for general use as a carbon source / its
biotransformation to acetic acid remains a major fermentation process.
Fats and oils - Hard animal fats (composed mainly of glycerides of palmitic and
stearic acids) - Plant oils (primarily from cotton seed, linseed, maize, olive, palm,
rape seed and soy) - fish oil, may be used as the primary or supplementary
carbon source - Plant oils - of oleic and linoleic acids - Oils - more energy per
unit weight than carbohydrates - useful in fed-batch operations
(b)Nitrogen sources
Corn steep liquor - Byproduct of starch extraction from maize - first use in
fermentations for penicillin - Concentrated extracts - contain about 4% (w/v)
nitrogen- amino acids, along with vitamins and minerals- Can sometimes be
replaced by liquor derived from potato starch production
Yeast extract – 1 - Produced from waste baker’s and brewer’s yeast - cultivated
using ethanol, or wastes from wood and paper processing - used in the formulation
of fermentation media - salt-free concentrates of soluble components of
hydrolyzed yeast cells
Yeast extract – 2 - Extracts are available as liquids containing 50-65% solids -
viscous pastes or dry powders - contain amino acids (35-40%, w/v), peptides (30-
45%, w/v), water-soluble vitamins and some glucose derived from the yeast
storage carbohydrates
Peptones - too expensive for large-scale industrial fermentations - Prepared by
acid or enzyme hydrolysis of high protein materials - Amino acids compositions
vary depending upon the original protein source peptones are rich in proline and
hydroxyproline - devoid of sulfur-containing amino acids / Keratin peptone is rich
in both proline and cystine, but lacks lysine - Plant peptones invariably contain
relatively large quantities of carbohydrates
Soya bean meal - Residuals after extraction of soy oil - Composed of 50% protein,
7% non-protein nitrogenous compounds, 30% carbohydrates and 1% oil- used in
antibiotic fermentation .
Water - Use for media, cleaning, cooling - source of large quantities of clean
water, of consistent composition- Hard water is treated to remove salts such as
calcium carbonate .
 Reg. No.___________________
[16MBU303]
KARPAGAM ACADEMY OF HIGHER EDUCATION
KARPAGAM UNIVERSITY
(Established Under Section 3 of UGC Act, 1956)
Eachanari Post, Coimbatore, Tamil Nadu, India – 641 021
B. Sc., DEGREE THIRD INTERNAL EXAMINATION SEPTEMBER 2017
Microbiology
INDUSTRIAL MICROBIOLOGY
TIME: 2 Hours Max Marks: 50 Marks
Date:
PART –A 20 x 1= 20 marks
1. In ____ the solvent molecule are passed through a semi permeable membrane in opposite
direction.
(a) ultra filtration (b)reverse osmosis
(c)liquid membranes (d)pumping
2. ______ of any product is often the last stage of a manufacturing process.
(a)Filtration (b)Centrifugation
(c)Drying (d)Packing
3. -----------------require biotin for growth in production medium.
(a)Bacteria (b)yeasts
(c)viruses (d)phages
4. Salts such as ammonium and --------------are used for the discover of protein
(a)NH3SO4 (b)Na2SO4
(c)CaCl2 (d)Ca
5. _____ is an established and final purification of a diverse range of compounds.
(a)Drying (b)Crystallization
(c)Filtration (d)HPLC
6. Vitamin B12 production using Streptomyces olivaceus NRRLB ____strain.
(a)1125 (b) 1127
(c)1161 (d)1141
7. Alpha amylase are produced by ___________________
(a)Aspergillus (b) Mucor
(c)Pencillium (d)Fusarium
8. The most efficient industrial strain used in production of citric acid is ------------------
(a)Aspergillus niger (b) Aspergillus terreus
(c)Aspergillus clavatus (d)Aspergillus oryzae
9. An example of anionic surfactants is __________
(a)antiseptics (b) shampoo
(c) disinfectants (d) emulsifiers
10. The examples of two important naturally occurring pencillin are ------------- and --------------
(a)pencillin G and V (b)penicillin B and V
(c) penicillin M and V (d)penicillin O and V
11. --------- chromatography is a powerful and highly selective purification technique.
(a)Adsorption (b) Affinity
(c)Ion exchange (d)Column
12. ------------- is a penicillin without an acyl group, prepared from 6-amino penicillanic acid
(a) synthetic penicillin (b)GN penicillin
(c)semi-synthetic penicillin (d)natural penicillin.
13. The yield of cobalamine are usually in the range of _______mg/ litre.
 (a)2 – 3 mg (b) 1 – 2 mg
(c) 2 - 4 mg (d)2–5mg
14. The enzymes are purified using solvents such as---------------------
(a)acetone (b)alcohols
(c)inorganic-salts(ammonium sulfate) (d)acetone, salts and solvents
15. The enzyme protease is produced from bacteria such as ----------------
(a)Bacillus subtilis (b)Bacillus lichenifermis
(c)Bacillus subtilis & Bacillus lichenifermis (d)Bacillus anthrax
16. Penicillin antibiotic is produced from -------------------
(a)Penicillium Sp (b) Aspergillus Sp
(c)Fusarium Sp (d)Staphylococcus Sp
17. The optimum temperature & pH for protease enzyme fermentation is-----------------
(a)20-30°C, 7 (b) 30-40°C, 7
(c)30-40°C, 8 (d)40-50°C, 7
18. The preservation of bacterial ⍺-amylase enzyme in liquid formulation is--------% NaCl
(a)5 (b)10
(c)15 (d)20
19. The french word “vinaigre” means ---------------
(a)fresh grape juice (b) fresh wine
(c)sour wine (d)ethanol
20. Staphylococcus aureus produces an enzyme called ------- which cleaves the β-lactum ring of
penicillin.
(a)amylase (b) pectinase
(c)protease (d)penicillinase

PART –B 3 x 2= 6 marks
21. Name the applications of immobilized pencillin acylase?
22. Give the production medium for ethanol?
23. Give applications for lipase.
PART –C 3 x 8= 24 marks
24. a. Write notes on cell disruption methods.
(Or)
b. Give a note on lyophilization.
25. a. Detail on the microbial production of Beer.
(Or)
b. Brief on production of amylase enzyme.
26. a. Detail on the microbial production of citric acid.
(Or)
b. What is immobilization? Explain the methods of immobilization with figures.
 DEPARTMENT OF MICROBIOLOGY
KARPAGAM ACADEMY OF HIGHER EDUCATION
(Deemed to be University Established Under Section 3 of UGC Act, 1956)
Eachanari Post, COIMBATORE - 641 021, INDIA

[Link]., Microbiology (Batch 2016-2019)

Industrial Microbiology (Semester-III) (16MBU303)
ANSWER KEY
PART A
1] B
2] C
3] B
4] B
5] B
6] A
7] A
8] A
9]
10] A
11] B
12] C
13] B
14] D
15] C
16] A
17] B
18] D
19] C
20] D

PART B
21] Immobilised penicillin acylase :catalyze the hydrolysis of penicillins into 6-
aminopenicillanic acid - manufacture of β-lactam antibiotics, small peptides and
pure isomers from racemic mixtures.
22] Production media for ethanol : jiggery – KH2PO4 – yeast extract
23] Application of lipase : important - yogurt and cheese fermentation - cheap
and versatile catalysts to degrade lipids - applications such as baking, laundry
detergents and even as biocatalysts - convert vegetable oil into fuel

PART C
24] Cell disruption method

Chemical methods - include alkali, organic solvent, detergents and

chaotropic agents - Alkali saponification of membrane lipids - integrity of the cell
membrane - Organic solvents - disruption of cell wall - At low concentration the
cell wall is not ruptured but the permeability is increased. Product stability should
be considered when choosing the solvent. Eg. Toluene , Ethyl acetate used for
yeast and Dimethyl sulfoxide .

Detergents - solubilizing cell membranes - are amphipathic - solubilize the

cell wall - Sodium dodecyl sulphate (SDS), sodium sulphonate, Cationic detergents
such as cetyltrimethyl ammonium bromide (CTAB) and Non-ionic detergents such
Triton X100

Biological methods - Enzymatic digestion - cell wall disruption resulting in the

release of cell wall proteins leaving the protoplast intact - digestion of organelle
membrane to release the organelle proteins. Digestion - by hydrolyzing cell walls
by specific enzymes - Lysozyme. Hydrolyzing cell wall - combination of enzymes

Physical methods- Mechanical methods - Ultrasonic vibrators (sonicators) -

ultrasound waves frequencies - greater than 20 kHz ruptures the cell wall –
cavitation - ultrasound waves creates compression and rarefaction - rarefaction
rapidly collapses as the area changes to one of compression. The bubbles produced
in the cavities collapse creating shock waves which disrupt cell walls - In
laboratory grinding -Waring blender - animal cells and tissues as well as with
mycelial organisms. In industrial scale - bead mill or high pressure homogenizer

Non mechanical methods - Osmosic shock - The cells swell due to osmotic flow
of water ultimately bursting - Freezing thawing cycles cause loss of membrane
integrity - cell wall is ruptured- In Thermolyis the heat - disrupting the cell walls
without affecting the products. The effect of heat shock depends on pH, ionic
strength, presence of chelating or sequestering agents such as EDTA.

(b) Lyophilisation - or freeze drying - method of long-term preservation of

micro-organisms - preservation of bacteria, viruses, fungi, sera, toxins, enzymes
and other
biological materials - convenient technique for preserving a large number of
cultures,
perfect method for storing yeasts with completely unchanged characteristics - first
applied to microfungi on a large scale by Raper and Alexander - consists of drying
cultures or a spore suspension from the frozen state under reduced pressure.
Majorsteps - suitable protective (ii) Using a sterile technique, the suspension from
is distributed in small quantities into glass ampoules - The ampoules are connected
with a high vacuum system usually incorporating a desiccant - The vacuum pump
is turned on and the ampoules are evacuated till drying is complete, after which
they may be sealed off.
After drying, the small vials can be filled with sterile dry nitrogen instead of
sealing under vacuum
Advantages - no risk of contamination or infection with mites - stored, they are
not readily broken and most species - changes in characteristics (i.e. they remain
unchanged during storage period) - hundreds of lyophilized cultures can be stored
in a small storage space - Its remain viable for many years (more than 20 years in
case of many bacterial species - Lyophilization cuts down the number of transfers.

25]A] Beer production

Brewing - production of beer by steeping a starch - The basic
ingredients - water and a fermentable starch source such as malted barley -
fermented with a brewer's yeast and flavoured with hops - starch sources
include millet, sorghum and cassava - Secondary sources (adjuncts), such as maize
(corn), rice, or sugar – Steps in brewing process
include malting, milling, mashing, lautering, boiling, fermenting, conditioning, filt
ering, and packaging. three main fermentation
methods, warm, cool and spontaneous
The basic ingredients - water; a starch source, such as malted barley - a brewer's
yeast to produce the fermentation; and a flavouring, such as hops, A mixture of
starch source - adjunct
Hops - female flower clusters or seed cones of the hop vine - used as a flavouring
and preservative agent - contain several characteristics - contribute a bitterness
that balances the sweetness of the malt; they provide floral, citrus, and herbal
aromas and flavours; they have an antibiotic effect that favours the activity
of brewer's yeast over less desirable microorganismsYeast is
the microorganism that is responsible for fermentation in beer -
Yeast metabolises the sugars extracted from grains, which
produces alcohol and carbon dioxide, and thereby turns wort into beer
Clarifying agent - which typically precipitate (collect as a solid) out of the beer
along with protein solids and are found only in trace amounts in the finished
product - makes the beer appear bright and clean, rather than the cloudy
appearance of ethnic and older styles of beer such as wheat beers.[47]
Brewing process - There are several steps in the brewing process, which may
include malting, mashing, lautering, boiling, fermenting, conditioning, filtering,
and packaging.[51]
Malting is the process where barley grain is made ready for brewing. when the
malt goes through a very high temperature drying in a kiln; with gradual
temperature increase over several hours - When kilning is complete, the grains are
now termed malt, and they will be milled or crushed to break apart the kernels and
expose the cotyledon, which contains the majority of the carbohydrates and sugars;
this makes it easier to extract the sugars during mashing - Milling also separates
the seed from the husk.
Mashing converts the starches released during the malting stage into sugars
that can be fermented - milled grain is mixed with hot water in a large vessel
known as a mash tun - saccharification. The result of the mashing process is a
sugar-rich liquid - The boiling process serves to terminate enzymatic
processes, precipitate proteins, isomerize hop resins, and concentrate
and sterilize the wort
Mashing - Mashing is the process of combining a mix of milled grain - known as
the "grain bill - a form of steeping - Mashing usually takes 1 to 2 hours - Lautering
is the separation of the wort (the liquid containing the sugar extracted during
mashing) from the grains
Boiling - After mashing, the beer wort is boiled with hops -
including sterilization of the wort to remove unwanted bacteria, releasing of hop
flavours, bitterness
Whirlpool - At the end of the boil, solid particles in the hopped wort are separated
out, usually in a vessel called a "whirlpool" or "settling tank
Hopback - traditional additional chamber that acts as a sieve or filter by using
whole hops to clear debris (or "trub") from the unfermented (or "green") wort.

(b) amylase enzyme production

Sources - α-Amylase can be isolated from plants, animals or microorganisms -
major reasons for the increasing interest in microbial sources - The growth of
microorganisms is rapid and this will in turn speed up the production of enzyme -
Microorganisms can be easily manipulated using genetic engineering or other
means.

α-Amylase - produced by several bacteria, fungi and genetically modified

species of microbes. - the bacterial - B. amyloliquefaciens and B. licheniformis -
industrial applications in processes such as food, fermentation, textiles and paper
industries Fungal sources - to terrestrial isolates, mostly to Aspergillus species and
to only few species of Penicillium, P. brunneum Penicillium fellutanum
Production Methods - two methods which are used - 1) Submerged fermentation
and 2) Solid State fermentation - Submerged fermentation (SmF) - free flowing
liquid substrates, such as molasses and broths - primarily used for the extraction of
secondary metabolites - several advantages - allows the utilization of genetically
modified organisms - The solid substrates commonly used in this method are,
bran, bagasse, and paper pulp - advantage is that nutrient-rich waste materials can
be easily recycled and used as substrates in this method generation

Purification of α-Amylase - Purification methods commonly employed are

precipitation, chromatography and liquid-liquid extraction depending on the
properties of the enzyme desired. A combination of the above methods is used in a
series of steps to achieve high purity - The crude amylase enzyme can be
precipitated and concentrated using ammonium sulphate precipitation or organic
solvents - subjected to dialysis against water - chromatographic techniques like
ion exchange, gel filtration and affinity chromatography for further separation and
purification of the enzyme

26]A] Citric acid production

Citric acid (C6H8O7, 2 - hydroxy - 1,2,3 - propane tricarboxylic acid), a
natural constituent and common metabolite of plants and animals, is the most
versatile and widely used organic acid in the field of food (60%) and
pharmaceuticals (10%).
MICRO-ORGANISMS USED FOR CITRIC ACIC PRODUCTION - bacteria,
fungi and yeasts - only A. niger and certain yeasts such as Saccharomycopsis sp.
are employed for commercial production. However, the fungus A. niger has
remained the organism of choice for commercial production - The main advantages
of using this micro-organism are: (a) its ease of handling, (b) its ability to ferment
a variety of cheap raw materials, and (c) high yields.
Strains selection and improvement - The two principal methods of selecting
populations - "the single-spore technique" and the "passage method" - The most
employed technique to improve citric acid producing - mutations in parental
strains using mutagens. Among physical mutagens, g-radiation) and UV-radiation.
- chemical mutagens, e.g. aziridine, N-nitroso-N-methylurea or ethyl methane-
sulfonate
PRODUCTION TECHNIQUES AND RAW MATERIALS - raw materials used
for citric acid production in to two groups: (i) with a low ash content procedures
(e.g. cane or beet sugar, dextrose syrups and crystallized dextrose); (ii) raw
materials with a high ash content substances (e.g. cane and beet molasses, crude
unfiltered starch hydro-lysates).
Liquid fermentation - Submerged fermentation: The submerged fermentation
(SmF) process is the commonly employed technique for citric acid production -
advantages such as higher yields and productivity and lower labour costs are the
main reasons for this. In SmF, different kinds of media are employed such as sugar
and starch based media
Surface fermentation: In the classical process for citric acid manu-facture, the
culture solution is held in shallow trays (capacity of 50-100 L) and the fungus
develops as a mycelial mat on the surface of the medium. The trays are made of
high purity aluminium or special grade steel and are mounted one over another in
stable racks
Solid-state fermentation - termed as an alternative method to produce citric acid
from agro- - advantages of SSF process is that the presence of trace elements may
not affect citric acid production so harmfully as it does in SmF
PRODUCT RECOVERY The recovery of citric acid from liquid fermentation is
generally accomplished by three basic procedures, precipitation, extraction, and
adsorption – Precipitation - addition of calcium oxide hydrate (milk of lime) to
form the slightly soluble tri-calcium citrate tetrahydrate - removed by filtration -
treated with sulphuric acid forming calcium sulphate - Mother liquor containing
citric acid is treated with active carbon and passed through cation and anion
exchangers - the liquor is concentrated in vacuum crystallizers at 20-25°C, forming
citric acid monohydrate. Crystalization at temperatures higher to this is used to
prepare anhydrous citric acid.
(b) immobilisation
The term "immobilized" - unable to move or stationary - immobilized
enzyme - physically attached to a solid support, over which a substrate is passed
and converted to product - can provide increased resistance to changes in
conditions such as pH or temperature - allows enzymes to be held in place
throughout the reaction - can be easily separated from the products - used in
industry for enzyme catalyzed reactions
for commercial uses as they possess many benefits which include: 1.
Economical - easily removed from the reaction thus making it easy to recycle,
multiple or repetitive use - 2. Stability - greater thermal and operational stability
than the soluble form of the enzyme and product is not contaminated with the
enzyme - 3. Convenience - The ability to stop/start the reaction rapidly by
removing/adding the enzyme from the reaction solution, long half life, predictable
decay rates, elimination of reagent preparation, etc.
There are mainly three methods - Adsorption - 2. Covalent Binding- 3.
Entrapping
Adsorption - simple method of an enzyme - wide applicability - simply mixing
an enzyme with a suitable adsorbent under appropriate conditions of pH, ionic
strength, incubation period and washing off unbound enzyme - achieve high
enzyme loading using adsorption - ion-exchange matrices can be used repeatedly
to achieve desired economy
Covalent Binding - method is most effective and widely studied - aim - to avoid
leaching of an enzyme into the surrounding solution - Commonly used methods -
(a) Activation of sepharose by cyanogen bromide. (b) Chloroformates may be used
to produce similar intermediates to those produced by cyanogen bromide but
without its inherent toxicity. (c) Carbodiimides may be used to attach amino
groups on the enzyme to carboxylate groups on the support or carboxylate groups
on the enzyme to amino groups on the support. (d) Glutaraldehyde is used to link
enzymes to supports. (e) The use of trialkoxysilane to derivatise glass.
Entrapping - useful method of enzyme immobilization using gels or fibers where
substrates and products are low molecular weight
Application - Amino acid acylasecatalyses the deacetylation of the L form of
the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid, that can be
easily separated, racemized and recycled - L-aspartic acid - in medicines and as a
food additive - major applications - in dairy industry is in the preparation of
lactose-hydrolysed milk and whey, using b –galactosidase - major applications in
pharmaceutical industry is the production of 6-aminopenicillanic acid (6-APA) -
Immobilized oxidoreductases are gaining considerable importance - in clinical
diagnosis and other analytical applications like the biosensors.