UNIT-I
EXPLAIN ABOUT THE SCREENING PROCESS OF INDUSTRIALLY IMPORTANT
MICROORGANISMS ?
Introduction:
The wide variety of new or unusual compounds may be produced by various microbial
isolates due to variations in biochemical capabilities of microorganisms. There are five distinct approaches:
1) Screening for the production of new metabolites with new isolates and /or new test methods. This is
the only way to obtain completely new classes of substances.
2) Chemical modification.
3) Biotransformation.
4) Interspecific protoplast fusion and
5) Gene cloning.
Types of screening:
The goal of screening is always to detect and identify new substances of commercial interest
and to separate them in the quickest possible way from the numerous easily detected substances that are of
no commercial interest. Generally screening is by two stages, known as:
 Primary Screening
 Secondary Screening.
Explain in detail about primary screening.
Primary screening:

Definition: Screening may be defined as the use of highly selective producers to allow the
detection and isolation of only microorganisms of interest from among a large microbial
population.

Thus to be effective, screening must in one or a few steps, allow the discarding of many valueless
microorganisms, while at he same time allowing the easy detection of the small percentage of
useful microorganisms that are present in the population.

Screening of a new isolate with commercial interest can be basically starts with a method known
as Crowded Plate Technique.
Crowded plate technique:
Natural microbial source (soil)

Serially diluted

Aliquots spread, sprayed or applied (spread or pour plate technique)

Surfaces of agar plates

Colonies appear

The colonies are selected for screening the following:
1) Organic acid producer
2) Antibiotic producer
3) Miscellaneous metabolites
4) Enzyme producers
Using pH indicators, zone formation, around colonies, indicator microorganisms, chromogenic
substrates, can do the selection.
Screening of organic acid producing microorganism:
1
 Microbes producing organic acids (or amines) from various carbon substrates often can be detected
by the incorporation of a pH indicting dye, such as neutral red or bromothymol blue, into a poorly
buffered nutrient agar medium.
 The production of these compounds is indicated by a change in the color of the indicating dye in the
vicinity of the colony to a color representing an acidic or alkaline reaction.
 The usefulness of this procedure is increased if media of greater buffer capacity are utilized so that
only these microorganisms that produce considerable quantities of the acid or amine can induce
changes in the color of the dye.
Alternative procedure:
 The organic acid production can be detected by the incorporation of Calcium Carbonate (CaCo3) in
the medium.
 A cleared zone of dissolved CaCo3 around the colony indicates organic acid production.
 Thus, cultures yielding positive reactions require further testing to be sure that an organic acid or
base actually has been produced.
 The above screening approaches do not tell us just which organic acid or amine has been produced.
Therefore, further testing by some procedure such as Paper Chromatography is required.
Isolation of antibiotic producing strains:
The search for microorganisms capable of producing significant antibiotics can be done by
simple screening technique.
The simplest screening technique for antibiotic producers is the same crowded plate technique.
This technique is used when we are interested only in finding microorganisms that produce an
antibiotic.
Soil or other sources of microorganisms

Diluted

Dilutions were inoculated

Nutrient agar plates

After incubation

Colonies were observed

Colonies producing antibiotics, clear area around the colonies

Free of growth of other colonies

Such a colony is sub-cultured
And purified by streaking

Then ready for testing
“Microbial inhibition spectrum”.
Note:
 The crowded plate procedure does not necessarily select an antibiotic producing microorganism,
because the inhibition area around the colony sometimes can be attributed to other causes.
 Notable among these are a marked change in the pH value of the medium resulting from the
metabolism of the colony, or a rapid utilization of critical nutrients in the immediate vicinity of the
colony.
2
 Thus, further testing again is required to prove that the inhibitory activity associated with a
microorganism can really be attributed to the presence of antibiotic.
Using indicator organisms in isolation procedure. (Explain)
Introduction:
Finding a microorganism producing antibiotic activity against specific microorganisms and
not against the unknown microorganisms that were by chance on the plate in the vicinity of an antibiotic
producing microorganism.
Antibiotic screening:
Antibiotic screening is improved, therefore, by the incorporation into the procedure of a “test
organism”, that is, “an organism used as an indicator for the presence of specific antibiotic activity”.
 30 to 300 colonies / plate was obtained by serial dilution of the sample, which may be a soil or any
other microbial source.
 The plates were incubated until the colonies are a few mms in diameter, and so that antibiotic
production will have occurred for those organisms having this potential.
 Then a suspension of the test organism is sprayed or applied in some manner to the surface of the
agar medium and the plates were further incubated to allow the growth of the test organism.
 Antibiotic activity is indicated by zones of inhibited growth of he organism around antibiotic
producing colonies.
 Zone of inhibition can measured to detect the relative amounts of antibiotic produced by various
colonies, approximately.
 Antibiotic producing colonies again must be isolated and purified before further testing.
Screening of microorganisms for the production of different metabolites:
Microorganisms can be screened for extra-cellular product synthesis such as vitamins, amino
acids or other metabolites.
Steps:
The microbial source
Diluted
Plated to provide well-isolated colonies
The test organism used (may be auxotroph)
Increased growth of test organism around the specific colony present in the plate will be due
to the presence of specific metabolite.
Test organism:
 The choice of the particular test organism to be used is crucial.
 “It must possess a definite growth requirement for the particular metabolite”- auxotroph.
 Only if that metabolite is produced a zone of growth, or at least increased growth, of the test
organism adjacent to the colonies that have produced the metabolite is observed.
Isolation of enzyme producers:
Aim: In order to find microorganisms capable of utilizing a specific carbon or nitrogen for the growth and
biosynthesis.
3
To accomplish this objective,
 The plating medium is made up so us to contain the particular nutrient as its only sole source,
respectively, of carbon or nitrogen.
For e.g., starch for amylase producers.
 Dilutions of soil are plated and the utilization of that particular nutrient indicates the production
of enzymes.
 And this method is very much useful to find out the organisms having the ability to degrade
(utilize as a nutrient for their growth and activity) some critical pollutants.
 This is a method is useful to find out some significant enzyme producers.
 However, each organism must be tested further, since its growth under these conditions possibly
could be attributed to nutrient sources other than these incorporated in the medium. So low
dilutions of soil is recommendable for this kind of screening methods.
Other method:
 A modification of the latter technique is employed when volatile substrates such as
hydrocarbons, low molecular weight alcohols, and similar carbon sources are being
considered.
 In this case no need of incorporation of these substances into the medium.
Steps:
The specific substrate is placed in the lid of the petri plate
Inversion of agar containing the plate over the lid
Enough vapors from the volatile substrate rise to the surface of the agar
Within this closed atmosphere the substance is incorporated into the medium
Provide the specific nutrient for the microorganism
Growth of microorganism indicates its ability of utilization.
 These are but a few examples of the “Primary Screening” techniques.
EXPLAIN IN DETAIL ABOUT SECONDARY SCREENING ?
Need for secondary screening:
 Secondary screening is usually done to further testing of the capabilities of and gain information
about the organisms isolated by “Primary Screening Methods”.
 During primary screening procedure the microorganisms from the environment source are
isolated. There may be few or many microorganism of interest.
 Only a very small number of these organisms will have any real commercial value
 Secondary screening allows the further sorting out of those microbes that have real value for
industrial processes, and discarding of those lacking this potential.
Medium for secondary screening:
Secondary screening is performed on agar plates, in flasks or small fomenters containing
liquid media, or as a combination of these approaches.
The use of agar plates, although not as sensitive as liquid culture, is of advantage for initial
secondary screening, because more information obtained with the expenditure of a similar amount of effort.
Agar plates:
 Relatively little space
4
 Which is not enough for secondary screening
 It will provide only a limited indication of product yields potentials among various isolates.
Liquid culture:
 Provides much better picture of the nutritional, physical and production responses of an organism to
actual fermentation.
Qualitative and quantitative approaches:
Secondary screening can be qualitative or quantitative in its approach.
Qualitative approach:
 Tell us the spectrum or range of microbes, which is sensitive to a newly discovered antibiotic,
Quantitative approach:
 Tell us the yields of antibiotic which can be expected when the microorganism is grown in various
kinds of media.
Classification of microorganisms:
Based on pathogenicity:
The microorganism has to be callsifide base on pathogenicy as
1. Pathogenic
2. Non pathogenic
Pathogens :
Organismsms which has the abiliy to cause infection.The organisms may posses any pathogenicity for
plants, animals or humans, which would need to be considered in the handling of the organisms.
Non pathogens:
The do not cause infection. Usually non pathogenic organisms are used in fermenbtation industriaes.
Based on growth charactreistic:
Here the microorganisms are callsifed as
 Aeribic
 Anaerobic
 Facultative anaeraobic
 Types of media required for growth
 Bioparameters.
Note:
However, classification of microbes is an expenditure of time and effort, so that we may wish not to classify
them into broad groups.
Factors influenzing fermentation and product :
Media and incubation time:
 During secondary screening the organisms are grown for various periods of time and on
various media in liquid culture, so that quantitative (yield potential) assays may be
performed.
Bioparameters:
 It should be revealing whether there are pH, aeration or other critical requirements associated with
particular microorganisms, both for the growth of the organism and for the formation of chemical
products.
Screening for genetic stability:
 The screening also detects gross “genetic instability” in microbial cultures.
 Geneticaly insttable cultures are discarded.
Chemical nature of the product:
 It should determine whether the product has a simple, complex or even a macromolecular structure.
 Analysis of chemical nature are helpful in designing assay procedures for particular substance.
 Physical properties:
The physical properties, such as UV- light absorption or fluorescence or chemical properties, that can be
employed to detect the compound more easily.
Toxicity:
5
 The fermentation product must be tested for toxicity, weather toxic to plants, animals or human.
 It should be reveled that some of the fermentationproducts rea used as thepeutic agent for disease
treatment.
 Howere indiactionn of toxicity duerin screening process, allows to discard poor cultures.
Adaptive enzymes:
 It should reveal whether microbes are able to chemically alter or even destroy their own
fermentation products.
 The microorganisms may, because of a high-level accumulation of product in the culture broth,
produce “adaptive enzymes” that destroy the usefulness of the product.
 Thus, a microbe might produce a “racemase” enzyme that will change the L-configuration of an
amino acid product to a mixture of the D- and L-isomers.
 L amino acid ---> L or D amino acid.
4) EXPALIN ABOUT PRESERVATION TECHINQUE IN INDUSTRIALLY IMPORTANT
IMPORTANT MICRORGANISMS ?
Introduction:
The isolation of a suitable organism for a commercial process may be long and very
expensive
procedure and it is therefore essential that it retain the desirable characteristics that led to its
selection. This is
done by preservation technique.
Need for preservation:
The culture of industrial importance are stored to
 To eleminate genetic changes
 Protect against contamination
 To retain viability.
Subculture:
An organism may be kept viable by repeated subculture into fresh medium.
Disvantages:
Repated subculture may cause mutation that causes degenerationof strains.
Preservation:
The, preservation techniques has been developed to maintain cultures in a state of
“Suspended
animation” by storing either in two ways
1. Reduced temperature
2. Dehydrated form.
Storage at reduced temperatures:
Storage on agar slopes:
 Cultures grown on agar slopes may be stored in a refrigerator (5 C)
 sub-cultured at approximately 6-month intervals.
 The time of sub-culture may be extended to one year if the slopes are covered with sterile medicinal
grade mineral oil.
Storage under liquid nitrogen:
 The metabolic activities of microbes may be reduced considerably by storage at the very low
temperatures (150 C to 196 C), which may be achieved using a liquid nitrogen refrigerator.
 Fungi, bacteriophage, viruses, algae, yeasts, animal and plant cells and tissue cultures have all been
successfully preserved.
 Steps:
Culture
grown maximum stationary phase
6
re-suspending the cells in a cryoprotective agent (such as co. glycerol)
freezing the suspension
sealed ampoules
storage.
 Liquid nitrogen is the method of choice for the preservation of valuable stock cultures and the only
method for the long-term storage of cell that do not survive freeze – drying.
Demerits and Merits:
 The equipment is expensive
 However, the method has the major disadvantage that liquid nitrogen evaporates and must be
replenished regularly.
 If this is not done, then the consequences are the loss of collection.
Merit:
It does not require tedious labour process.
Storage in Dehydrated Form:
Dried Cultures:
 Dried soil cultures have been used widely for culture preservation, particularly for sporulating
mycelial organisms.
Steps:
Moist, sterile soil
inoculated with a culture
incubated for several days for some growth
then allowed to dry at room temperature (approximately 2 weeks).
The dry soil may be stored in a dry atmosphere are preferably, in a refrigerator.
 The technique has been used extensively for the storage of fungi and actinomycetes
 This method was extended by using the substrate other than soil.
 Silica gel and porcelain beads are suggested alternatives
Lyophilization:
Definition:
Lyophilization or freeze drying, involves the freezing of a culture followed by its drying under
vacuum which results in the sublimation of the cell water content.
The technique involves, growing the culture to the maximum stationary phase and resuspending the
cells in a protective medium such as milk, serum or sodium glutamate.
Steps:
Perpare culture
Stationary phase
7
Resuspend the cells in protective medium
Placed in vaccum, till sublimation
Transferred in ampoules & sealed
Stored in refigierator
Cell may remain viable for 10 yrs
Merits:
 Lyphilization is the major preservation method in culture collection centers
 once dried, the cultures need no further attention and the storage equipment (a refrigerator) is cheap
and reliable.
 Also, the freeze-dried ampoules may be dispatched as such still in a state of “Suspended animation”
where as liquid nitrogen stored cultures begin to deteriorate.
Demerits:
 Freeze-dried cultures are tedious to open the revitalize
 several sub-cultures may be needed before the cells regain their typical characteristics.
 Overall, the techniques appear to be second only to liquid nitrogen storage.
Quality Check:
Whichever technique is used for the preservation of an industrial culture it is essential to be certain
of the quality of the stocks. Each batch of newly preserved cultures should be routinely checked to ensure
their quality.
Microoragnism to be preserved
single colony of the culture to be preserved (agar plate)
culturing in a shake flask
growth of the organism observed
storage ampoules.
3% of the ampoules are reconstituted
the cultures assessed for purity, viability and productivity.
Culture viable
Retained
culture non viable
entire batch culture are destroyed
8
Used for industrial purpose
5) EXPLAIN IN DETAIL ABOUT THE NEED FOR STRAIN IMPROVEMENT
PROCEDURES ADAPTED IN INDISTRIES/ MENTION ABOUT THE OBJECTIVES OF
STRAIN
IMPROVEMENT :
(1) Strain improvement- are population obtained from single colony or pure isolate by the
screening process.
(2) Improvement exploitation of characters to produce more qualitative and quantitative
product of commercial value.
Objectives of strain improvement:
 The strain improvement techniques have following objectives.
 Higher yield producing strain-to obtain more quantity.
Shorter fermentation time.
(1) Early product time.
(2) Save energy
(3) Save time
To reduce undesirable pigment
(1) May affect product quality
(2) May reduce product commercial value
(3) Increases the cost of down stream process
Reduce oxygen demand
(1) To save energy
(2) To minimize foaming problem
Decreased foaming
(1) Excess of aeration and agitation leads to foaming
(2) Control of proper oxygen demand can also reduce foaming
Use of inexpensive substrates
(1) Use of naturally available products(agawaste, industrial waste etc)
To product single metabolite instead of mixture of compounds
Easy product recovery
(1) Extra cellularproducts are preferred than intracellular products.
(2) Reduce down stream cost.
Reduced contamination
(1) Use of stable strains, to reduce internal contamination.
Product with reduced toxicity.
Product with improved activity.
Eg: antibiotics and enzymes
EXPLAIN ABOUT THE DIFFERENT WAY BY WHICH MICROBIAL STRAINS CAN BE
IMPROVED ?
By following ways microbial strain improved can be performed
(1) Mutation
(2) Recombination
(3) Gene technology
(4) Regulation
(1) Mutation:
(2) Mutation may be defined as the sudden change in the genotype of an organisms.
(3) Strain improvements in industries are basically done by mutation techniques.
Example:
9
(4) Both physical and chemical agents can induce mutation.
(5) Penicillin is one of the best-known antibiotic produced by Penicillium chrysogenium and
P.notatum.
(6) In industries by using mutation techniques Penicillium strain has been improved to produce
quality of product. The steps are as follows:
Penicillium chrysogenum(wild type)
Penicillium chrysogenum(NRRL 1951-B25)
200units/ ml
uv irradiation
Penicillium chrysogenum
Penicillium chrysogenum( X-162)
(Stanford strain 25099)
500units/ ml
300units/ ml
Treating conidia with uv light
Penicillium chrysogenum Q-176
[ Wisconsin]
761units/ ml
Used as commercial strains
Advantages:
Improves strain can be obtained for the industrial production of various products.
Disadvantages:
 Mutation sometimes may cause serious problems with the person involved, due to improper
handling of the mutagens.
 At times silent mutation may not express its mutagenic effect in the strains.
 Due to non-sense mutations the incomplete polypeptide synthesis may occur.
 Structure of nature of the product may be altered.
 Growth and metabolism of the organism may be altered so that it may not be favorable condition
in the production.
 Quantity of mutagens and mutable ability should consider.
(2)Recombination:
(I)Recombination in bacteria:
(1) Transformation
(2) Transduction
(3) Conjugation
(II) In each case only a fragment of the donor cell is transformed into recipient cells, which thus become a
partial diploid(merozygote). After homologus pairing, recombination occurs, but not every DNA transfer
results auto-matically in recombination.
Transformation:
 Up take of forieng DNA from the surrounding environment.
 Recipient cells become competent
 Competence- ability to uptake DNA
 Take short pleces of DNA
10


Example: bacillus subtilis, Bacillus punilis
Recombinant cell identified using genetic markers.
Eg: Antibiotic resistance, chromogenic substrates.
Conjugation:
 Transfer of DNA from one cell(donor) to the (recipient) through conjugation canal.
 Fertility factor play an important role.
 Conjugation involves the parti-cipation of ‘F’ plasmids.
 F plasmid containing cells are called as F+ cell.
 Cells without F plasmids are F-.
 Usually conjugation occurs between HFr strain and F- strains
Eg: E.coli, Pseudomonas sp are best examples.
Transduction:
 Transfer of DNA from one cell to another by means of phage partical.
 Transduction are of 3 types.
(a) Generalized
(b) Specialized
(c) Abortive transduction
 P1 and lambda phage are examples of generalized and specialized transduction play an important
role in recombination.
(3) Recombination in fungi:
Sexual Recombination:
Some fungi used industrially have a complete sexual cycle.
Example: Aspergillus. Claviceps and Saccharomyces
In these organisms, nuclear fusion( Karyogamy) results.
Fusion of hyphae has led to a mingling of nucleic in the heterokaryotic mycelium.
After diploid formation, recombination takes during the subsequent meiosis process.
A new genotype results either from the combination of parent chromosomes or through crossing over as
a result of segment exchange of paired homologus chromatids.
Parasexual recombination:
 The discovery of parasexual process in imperfect fungi has led to the development of suitable
breeding techniques.
 In parasexuality, the fusion of two hyphae of equal or different polarity result in a mycelium with
nuclei of both parent strains.
 This heterkaryon is normaly stable with the nuclei mingling but not interacting.
 In rare cases, nuclear fusion occurs and a diploid nucleus is formed.
 In such diploidnuclei, mitotic crossing over between chromosomes may occur resulting in genetic
recombination.
 To obtain a recombinant the formation of haploid cells or spores must occur.
 Spontaneous haploidization is relatively rare but can be induced with P-fluorophenyl alanine.
 Haploidy occurs not through meiosis but through random distribution of the chromosomes not be
progeny nuclei.
 Example: Penicillium chrysogenum, Cephalosporium acremonium.
 The most advantages is this process is more economical.
Protoplast fusion:
 Protoplast are cells from which the cell wall has been removed by enzyme treatment.
 Enzymes used are
(1) Bacteria- lysozyme
(2) Fungi- Chinase or cellulose
The protoplast must be stabilized against lysis by suspension in a medium containing an stabilizing
agent.
Protoplast are unstable under normal conditions. So kept in osmotic stabilizing agent.
Osmotic stabilizing agent: Sucrose solution.
11
Protoplast fusion can be used for the following:
Intra specific recombination of strain which lack sexual or parasexual system or whose recombination is
too low.
Ex: Mucor, bacillus, Lactobacillus, Aspergillus, Penicillium
Inter specific hybridization to obtain complementary new organisms capable of synthesis of modified
metabolites.
Eg: A.nidulans X A.rugulosus
P.citinum X P.cyancofulvum
Engineered genes in plasmids or virus DNA can also be to transform protoplast.
Electrofusion:
 When cells are placed in an alternative current fields, transient whole membrane merging and
cellfusion occurs.
 With this 2 or more protoplasts can be cause to fuse under microscopic control, or several cells can
be fused into the giant cells.
o Further protoplasts can be induced to fuse artificial phopholipid vesicles is called liposomes.
o The fusion rate is 0-90% than 60% rate obtained using PEG-fusogen.
Eg: Yeast and fungi protoplast can be fused by electrofusion.
Advantages:
Frequency of recombination high.
Enter genome can be transformed
Exanchange of genetic materials does not require the presence of fertility factors.
(3)Genetechnology:
The gene of interest is first selected.
The gene then isolated
The sequence of DNA is incorporated into a suitable vector.
The plasmid (vector) DNA is introduced by transformation into a host cell.
The recombinant clone is selected by suitable marker such as antibiotic resistance.
(4) Regulation:
Metabolism is generally so efficient that excess products are not formed.
Strains with less efficient regulation can be selected in a screening process.
It is well estabilished that strain development and the optimization of fermentation conditions lead to a
relaxation of regulation in the producing strains.
A broad understanding and biosynthesis, the enzymes involved in the processes and their regulation is
necessary for developing a rational approach to the alteration of the regulation of a fermentation
process.
Microbial metabolism is controlled by the regulation of broth
(1) Enzyme activity
(2) Enzyme synthesis.
Regulation of enzyme activity:
C enzymes activity can be controlled by
 Feed back inhibition
 Energy charge
 Breakdown of enzymes
 Modification of enzymes
Feed back inhibition:
In an unbranched biosynthetic pathway the end product inhibits the activity of the first enzyme of the
pathway, a process called feedback inhibition.
A conformation change and hence inactivation (allosteric site) occurs when an effector (end product) is
attached to a specified site of the enzymes (allosteric site).
The end product thus inhibits the activity of the enzyme non-competitively.
Regulation of enzyme synthesis:
(a) Induction
(b) Repression
12
(c) Attenuation
Induction:
1. Some enzyme is found irrespective of the culture medium, such enzymes are called constitutive.
2. Many catabolic enzymes are induced they are not formed until the substrate to be metabolized is
present in the medium.
3. The product of one enzyme can in turn induce the synthesis of another enzyme.
Repression:
Anabolic enzymes are generally present only when the end product is absent.
The excess end product suppresses enzyme synthesis, acting as a co-repressor.
Attenuation:
Ex: Trp operon in E.coli
Catabolic regulation:
Catabolic regulation is a general regulatory mechanism in which a key enzyme involved in a catabolic
pathway represented, inhibited or inactivated when a commonly used substrate is added.
9) EXPLAIN ABOUT MEDIA PREPARATION FOR INDUSTRIAL FERMENTATION/ WHAT IS
MEDIA FORMULATION EXPLAIN?
Introduction:
Media:
Media provides sources of nutrient for proper growth of microorganism and to carry out its metabolic
function properly.
Important criteria for media:
 It should produce maximum yeid of product
 It should produce maximum biomass per gram of substrate used.
 It should be of good consistency.
 Be readily available through out the year.
 There should be minimum yield of undesired products.
 It should posses minimal problems during media preparation and sterilization.
 Minimal problems in aeration, agitation and extraction and during waste treatment process.
Media formulation:
 Media formulation is an essential stage.
 The media should be design so as to suit the fermentor and fermentation process.
 Fermentation medium should be formulated with proper carbon source, nitrogen source, oxygen
and other requirement so as to yield biomass, products, Co2,H2O and heat.
Characteristics of ideal medium:
Chemical compositon:
Media should have suitable chemical composition (C,N, Minerals and growth factor).
Precurssors:
Media should be provided with precursors to get maximum yields.
Buffering capacity:
 pH of medium play a vital role.
 Buffering agents are added to controls pH variations.
Avoidance of foaming:
 Media with proper constituents.
 Addition of antifoaming agents.
Toxicity:
Media should not posses any toxic substance.
Consistency:
Liquid medium is better than solid or semi solid medium.
Contamination:
Certain conditions of production media are helpful in avoiding contamination.
13
Ex: acidic pH of the medium can avoid contamination of some organisms.
Components of media:
(1) Water
(2) Carbon sources
(3) Oils and fats
(4) Nitrogen sources
(5) Hydrocarbon and their derivatives
(6) Minerals
(7) Growth factors
(8) Buffers
(9) Precursors
(10)Inhibitors
(11) Inducers
(12) Non-nutritional media supplements.
(1) Water:
 It is important to consider pH, dissolved salts and efficient contamination of water.
 Minimal content is important in brewing industries.
 Hard waters containing high CaSo4 are better for English burton biter beers.
 Waters with high carbonate content are better for darker beer such as status.
 Now days the water may be treated by deionization or other techniques and salts are added or pH
adjusted to favor.
 Reverse or efficient use of water is of high priority.
(2) Carbon source:
Carbon source in medis are provided by carbohydrates such as
(a) Saccharide material
(b) Starchy material
(c) Cellulosic materials
(a) Saccharide material:
Saccharide materials includes
1. Molasses
2. Fruit juices
3. Cheese whey
4. Malt extract
Molases:
It is a byproduct of sugar production.
It is one of the cheapest sources of carbohydrate.
The composition of molasses varies depending on the raw material used for sugar production.
It is used in the production of ethanol, SCP, organic and amino acids and some microbial gums.
They are also useful in the production of antibiotics, enzymes, vaccines and the chemicals.
They contain impurities so these to be purified before usage.
The commonly used saccharide materials are sugarcane molasses and beef molasses.
S.No
1
2
3
Sugarcane molasses
Rich in biotin
Don’t have inositol
Organic content is less
Beef molasses
Less biotin
Containing inositol,Pyridxine
Organic content is more
Note:
These both have pathogenic acid thiamine,phosphorous and sulphur.
Fruite juice:
They have soluble sugars.
14
Desirable to have low nitroden content in grapes.
They contain glucose and fructose.
Grape juice is used in wine production.
Cheese whey:
 The straw-colored liquid production as a by-product of cheese making is called cheese whey.
 Used as feed for animals, lacticacid production, SCP production.
 It contains lactose, vitamins(riboflavin) nitrogenous substances and inorganic salts.
 lactose is primary source for the production of ethanol,Xanthangum and vitamin B12 ,2-3-butandiol,
lacticacid and gibberellic acid.
 Storage and transportation costs high. So it is often not economical as a substrate.
Malt extract:
It is an aqueous extract of malted barely, is an excellent substrate for many fungi, yeast and
Actinomycetes.
Nitrogenous substances are also present in malt extract includes proteins, peptides, aminoacids,
purines, pyrimidines and vitamins.
The aminoacid praline is present.
Culture media containing malt extract must be carefully sterilized.
When overheating occurs, the maillard reaction seen due to low pH and high proportion of reducing
sugars.
Starchy Materials:
 Starch is a good sourceof carbon.
 But it should be hydrolyzed before usage.
 Starch may also readily hydrolyzed by dilute acids and enzymes to give a variety of glucose.
 Hydrolysed Canava starch is used as a major carbon sources for glutamicacid production.
 Starch require pretreatment to bring about the conversion to fermentable sugars.
 There are 2 main sources of commercial starches:
Cereals: Ex., Potatose,Tapicoa (Moisture content is high)
Cellulosic Material:
 It is usually not possible to use cellulose directly as carbon sources.
 It must first be hydrolysed as that of starch chemically or enzymatically.
 The sugar syrup formed cellulose hydrolysis has been used for ethanol production, butanol,
acetone and isoproponal.
Sulfite waste material:
Digestion process:
 In the manufacture of paper pulp, wood is subjected to hydrolysis, this is called digestion
process.
 At the end of this process, the spend liquid is left behind it is referred to as sulfite waste liquor.
 It contains 10-12% solids of which sugars about 20%
 Sulfite waste liquor obtained from the paper-pulp industry cannot be used directly in the
fermentation.
 It is necessary to remove the free So2 or sulfurous acid present in the waste liquor, as these
compounds are toxic to molecules.
 There are mainly 2types of monosaccharides
(1) Hexoses D-glucose, D-galactose, D-mannose( Gymnosperms)
(2) Pentose D-xylose, L-arabinose (Angiosperms)
It is used in the ethylalcohol production.
S.cerevisiae requires only hexoses and pentoses.
Wood molasses:
It serves as carbon source.
It contains reducing sugars such as glucose and pentoses.
Rice straw:
Rice straw can serve as a good source of cellulose.
15
It is a major agricultural by product.
It is a poor quality animal feed in its natural state because of its balkiness, poor palatability, low
protein content and low digestibility.
They are used in the production of SCP, silage, mushroom cultivation etc.
Oils and fats:
 Oils were first used as anti foaming agents in antibiotic processes.
 Vegetable oils may also be used as carbon substances in some fermentation.
 By content of the fatty acids and based on their degree of unsaturation, they may be grouped into:
(1) Oleic:
 Non-drying type
 Olive and groundnut oil
(2) linoleic:
 Semi-drying type
 Sunflower, cottonseed oils
 They have higher content of double unsaturated fattyacids.
(3) Linolenic:
 Drying type
 Linseed, Soya bean oils
 They contain three double bonds.
Typical oil contains apporaximately 2-4 times the energy of glucose on a weight basis.
Oils also have antifoam properties which may make downstream processing simpler, but normally
they are not used solely for this purpose.
Glycerol trioleate is known to be used in some fermentation where substract purity is an important
consideration.
Methyl oleate has been used as a sole carbon substrate in cephalosprine production.
Hydrogen and their derivatives:
 Development work has been using n-alkanes for production of organic acids, aminoacids,
vitamins and Cofactors, nucleic acids, antibiotics, enzymes and proteins.
 Methane, methanol and n-alkanes have all been used as substances for biomass production.
 It was considered that hydrocarbons and their derivatives might have a potential role as feed
stocks in the microbial production of higher value products such as intermediates,
pharmaceuticals, fine chemicals and agriculture chemicals.
 These are not in use because of high costs.
Nitrogen sources:
1. They are produced from baker’s yeast through autolysis at 50-55’C or through plasmolysis in the
presence of high concentration of NACl.
2. It contains aminoacids and peptides, water soluble vitamins and carbohydrates.
3. The composition of yeast extract varies, because the substances used for yeast cultivation affect the
quality of the yeast extract.
 Trace elements may contribute to both primary
Micronutrients:
Or secondary metabolite production.
•Requirements for trace elements may include iron
(Fe2+and Fe3+), zinc (Zn2+), manganese (Mn2+),
 Manganese can influence enzyme production.
molybdenum (Mo2+), cobalt (Co2+), copper
Iron
(Cu2+), and
and zinc have been found to influence antibiotic
calcium (Ca2+).
production.
•The functions of each vary from serving in
 Primary metabolite production is usually not
coenzyme functions to catalyze many reactions,
very sensitive to trace element concentration,
vitamin synthesis, and cell wall transport. The
however, this is a different matter for secondary
requirements are generally in very low levels and
metabolite production.
can sometimes even be supplied from quantities
 K, Mg, Ca and Fe are normally required in
occurring in water or from leachates from
relatively large amounts and should normally
equipment.
always be included as salts in culture media
16
Oxygen source:
Oxygen is always provided in water.
•Some organisms require molecular oxygen as terminal
oxidizing agents to fulfill their energetic needs through
aerobic respiration.
•These organisms are obligatorily aerobic. For obligate
anaerobes molecular 02, is a toxic substance.
•Some organisms are facultative anaerobes and can
grow with or without molecular 02.
The combination of minerals is also important in
regulating the electrolytic and osmotic properties of
the cell interior.
•In most cases, the complex industrial carbon and/or
nitrogen sources supply sufficient minerals for proper
fermentation .
Each reaction that occurs within the cell has its own
optimum (range of) conditions.
•For instance, although a given medium may be suitable
for the initiation of growth, the subsequent development
of a bacterial strain may be severely limited by chemical
changes that are brought about by the growth and
metabolism of the microorganisms themselves.
•In the case of glucose containing media, organic acids
that may be produced as a result of fermentation may
become inhibitory to growth.
In contrast, the microbial decomposition or utilization
of anionic components of a medium tends to make the
medium more alkaline.
•To prevent excessive changes in the hydrogen ion
concentration, either buffers or insoluble carbonates are
often added to the medium.
•The phosphate buffers, which consist of mixtures of
mono-hydrogen and dihydrogen phosphates (e.g.,
K2HP04 and KH2P04), are the most useful ones.
EXPLAIN ABOUT THE INOCULUM DEVELOPMENT PROCESS IN INDUSTRIAL
FERMENTATION. (15 MARKS)
Inoculum:
The starter culture to carry out fermentation process is called as inoculum.
Characteristic of inoculum:
 It must be in active state.
 It must be available in sufficient quantity.
 It must be in a suitable morphological form.
 It must be free of contamination.
 It must retain its product forming capabilities
Factors influencing inoculum:
Medium:
There are two kinds of media employed in fermentation process
17
 Medium used for inoculum- the medium contains low carbon source and low amount of nutrients. The
medium is called as inoculation media or seed media. They have similar feature as that of media used for
the production (production media).
 Media used for production- called as production media. Used to obtain yield of product.
Media composition:
Major difference in pH, osmotic pressure, anion concentration may result in very sudden change in
up take rates, which in turn may affect viability.
Pure culture:
Pure culture with out contamination must be used.
Size of inoculum:
 Inoculum production is an important step in industrial fermentation process.
 A loop full of inoculum may not be sufficient for fermentation medium of 3000 gallons.
 A large time interval is required for the visible growth of the inoculum.
 So relatively large amount of inoculum is used to start the fermentation process.
 Large volume is required to shorten the fermentation time (growth phase) and to obtain product much
earlier.
 Inoculum development:
 The inoculum development has significant role in industrial production of specific product.
 The inoculum development includes many stages
Procedure:
 A subculture of production strain is inoculated in shake flask
 After incubation it is used as inoculum for larger volume of medium containing conical flask
 Like wise the procedure is carried out till sufficient quantity of inoculum is obtained.
 At each steep the culture is test for its purity (to detect contamination)
EXPLAIN ABOUT CRITERIA FOR INOCULUM SIZE. (6MARKS)
Introduction:
The physiological condition (growth stage) of the inoculum has major effect on the performance of
the fermentation and also the size of the inoculum should also be considered.
Size of the inoculum:
 Inoculum production is a critical stage in an industrial fermentation process.
 If a fermentation in a 3000 gallon tank receive only one loopful of inoculum, a prolonged period would
be required before visible growth would be evident and much longer period before production formation
could be detected. Thus, inoculum is prepared as a stepwise sequence employing increasing volumes of
media.
 A relatively large inoculum volume is used to minimize the length of the lag phase and to generate the
maximum biomass in the production fermenters in as short a time possible, thus increasing vessel
productivity.
 Thus starting from a stock culture, the inoculum must be built up in a number of stages to produce
sufficient biomass to inoculate the production stage fermenters.
 This may involve two or three stages in shake flasks and one to three stages in fermenters, depending
on the size of the ultimate vessel.
 Through out this procedure there is a risk of contamination and strain degeneration. So stringent quality
control of production strain is required.
 The greater the number of stages between the master culture and the production fermenters the greater
the risk of contamination and strain degeneration.
 Therefore a and strain degeneration.
 Another factor compromise must be reached regarding the size of the inoculum to be used and the risk
of contamination to be considered is the economics of the process. A seed fermenter 100% of the size of
the production fermenter represents a considerable financial investment and must be justified in terms of
productivity.
18
EXPLAIN IN DETAIL ABOUT GENERAL INOCULUM – DEVELOPMENT PROCEDURE.
(6MARKS)
 THe master culture is reconstituted and plated on to solid medium.
 Approximately 10 colonies of typical morphology of high producers are selected and inoculated on to
slopes as the sub master cultures.
 Each sub master culture being used for a new production run.
 At this stage, shake flasks may be inoculated to check the productivity of these cultures, the results of
such tests being known before the developing inoculum eventually reaches the production plant.
 A sub – master culture is used to inoculate a shake flask of volume 250 or 500Cm3 containing 50 or
100Cm3 medium, which in turn, is used as inoculum for a larger flask, or a laboratory fermenter, which
may then be used to inoculate a pilot – scale fermenter.
 Culture purity checks are carried out at each stage to detect the contamination as early as possible.
 Although the results of these tests may not be available before the culture has reached the production
plant, at least it can be detected at which stage in the procedure contamination has occurred.
 For a sporulating organism the process may be modified to facilitate the use of a spore suspension as
inoculum.
EXPLAIN ABOUT CRITERIA’S FOR THE TRANSFER OF INOCULUM. (15 MARKS)
The physiological condition of the inoculum when it is transferred to the next culture stage can have
a major effect on the performance of the fermentation.
The optimum time of transfer must be determined experimentally and then procedures established so that
inoculation with an ideal culture may be achieved routinely.
These procedures include the standardization of cultural conditions and monitoring the state of an
inoculum culture so that it is transferred at the optimum time, i.e., in the correct physiological state.
The most widely used criterion for the transfer of vegetative inocula is biomass and such parameters
as;
 Packed cell volume.
 Dry weight
 Wet weight
 Turbidity
 Respiration
 Residual nutrient concentration and
 Morphological form has been used.
Criteria, which may be monitored on-line, are the most convenient parameters to use, as indicators of
inoculum quality and these would include,
 Dissolved oxygen
 pH (although pH would normally be controlled by seed fermentations) and
 Oxygen and carbon dioxide in effluent gas.
CO2 production rate (CPR):
 Parton and Willis (1990) advocated the use of the CO2 production rate (CPR) as a transfer criterion,
which requires analysis of the fermenter effluent air.
 This approach is suitable only when transfer is being made from a fermenter, but Parton and Willis
stressed the importance of adopting this strategy even for the inoculation of laboratory – scale
fermentations (for shake flask)
 In the transfer of inocula CPR has a significant effect on the biomass and product formation.
 For e.g., the effect of inoculum age on growth and productivity in a Streptomycetes fermentation. In
this type of fermentation mid-inoculum is selected as better choice and that time (mid-inoculum) is
suitable for the transfer of inocula.
 Thus, although the time of transfer had only a marginal influence on biomass in the production
fermentation, the effect on product formation was critical.
 It should be emphasized that the amount of biomass transferred was standardized for the three
fermentations and, thus, the differences in performance were due to the physiological stages of the
inocula.
19
Probes:
In recent years, probes have been developed for on-line assessment of biomass and these could be
invaluable estimating the time of inoculum transfer.
Biomass sensor:
 Boulton et al. (1989) reported the use of a biomass sensor (the Bug meter) to control the yeast-pitching
rate (inoculum level) in brewing.
 “The probe measures the di-electric permitivity of viable yeast cells and is un-affected by the presence
of dead cells, air bubbles or debris, making it ideal for the routine monitoring of yeast inoculum.
 Using the probe, these workers developed an automatic inoculum dispenser allowing a preset viable
yeast mass to be transferred from a yeast storage vessel to the brewery fermentation.
 Application of computers for inoculum transfer:
 A real-time expert computer system is used to predict the time of inoculum transfer for industrial scale
fermentation.
 The system involves the comparison of on-line fermentation data with detailed historical data of the
process. Some of the data are not available continuously (CPR) because the analyzer is not dedicated to
any one fermenter but analyzing process streams from a large number of vessels via a multiplexer system.
 Also, occasional false readings may be generated.
 Data from seed fermentations were analyzed by the expert system and the transfer time predicted.
Advantages:
 As a result of this approach, operators were able to plan their work more effectively, the need for
manual sampling was reduced and early warning of contamination was provided if the seed-culture
profile predicted from early readings was abnormal.
 Thus, this approach could be used to assess the cultural conditions giving rise to satisfactory inoculum,
but would be of less value in determining the time of transfer.
EXPLAIN IN DETAIL ABOUT INOCULUM PRODUCTION FOR BACTERIAL CULTURE.
Introduction:
Most of the fermentation process is operated using bacterial cultures. During the bacterial inoculum
development process step wise procedure are adapted. Lincoln proposed a model for inoculum production
and its storage.
Lincoln model of Inoculum Development for Bacterial Fermentations:
 He suggested procedure for the development of inoculum for bacterial fermentations.
 The procedure involved the use of one sub-master culture to develop a bulk inoculum, which was
subdivided, stored in a frozen state and used as inocula for several months.
 A single colony, derived from a sub-master culture, was inoculated into liquid medium and grown to
maximum log phase.
 This culture was then transferred into 19 times its volume of medium and incubated again to the
maximum log phase, at which point it was dispensed in 20 cm3 volumes, frozen and stored at below – 20
C
 At least 3% of the sample was tested purity and productivity in subsequent fermentation and provided
these were suitable, the remaining samples could be used as initial inocula for subsequent fermentations.
 To use one of the stored samples as inoculum it was thawed and used as a 5% inoculum for a seed
culture, which in turn was used as a 5% inoculum for the next stage in the program.
The Development of Inocula for Bacterial Processes:
 The main objective of inoculum development for traditional bacterial fermentations is to produce an
active inoculum, which will give as short a lag phase as possible in subsequent culture.
 A long lag phase is disadvantageous in that not only is time wasted but also medium is consumed in
maintaining a viable culture prior to growth.
 The length of the lag phase is affected by the size of the inoculum and its physiological condition.
 As already stated the inoculum size normally ranges between 3 and 10% of the culture volume.
 Lincoln (1960) stressed that bacterial inocula should be transferred in the lag phase of growth, when
the cells are still metabolically active.
20
 The age of the inoculum is particularly important in the growth of sporulating bacteria.
 Sporulation is induced at the end of the logarithmic phase and the use of an inoculum containing a high
percentage of spores would result in a long lag phase in subsequent fermentation.
Models for Bacterial Processes:
 Anustrup (1974) described a two-stage of inoculum development program for the production of
proteases by Bacillus subtilis.
 Inoculum for a seed fermenter was grown for 1-2 days on a solid or liquid medium and then transferred
to a seed vessel where the organism was allowed to grow for a further ten generations before transfer to
the production stage.
 Priest and Sharp (1989) cited the use of a 5% inoculum, still in the exponential phase, for the
commercial production of  amylase by Bacillus.
 Under Kofler (1976) emphasized that, in the production of bacterial enzymes, the lag phase in plant
fermenters could be almost completely eliminated by using inoculum medium of the same composition as
used in the production fermenter and employing large inocula of actively growing seed cultures.
 The inoculum development program for a pilot-scale process for the production of vitamins B12 from
Pseudomonas denitrificans.
Procedure:
Stock culture
(Lyophilized)
Maintenance culture (subculture)
Agar slope incubated 4 days at 28 C
Seed culture (first stage)
2dm3 flask containing 0.6-dm3 medium inoculated with culture
From one slop; incubated with shaking for 48h at 28 c
Seed culture (second stage)
40-80 dm3 fermenter containing 25- – 50-dm3 medium inoculated
With 1 –1.2% first stage seed culture. Incubated at 25 – 30 h at 32 c.
Production culture
500-dm3 fermenter with 300-dm3 medium inoculated with 5%
Second stage seed culture. Incubated at 32 c for 140 – 160 hrs.
Recombinant Bacteria as Inoculum:
 Strain stability is a major concern in inoculum development for fermentations employing recombinant
bacteria.
 Sabatie et al (1991) demonstrated that Plasmid stability and productivity in E.coli biotin fermentation
was greatly improved if stationary, rather than exponential, phase cells were used as inoculum.
 They postulated that the plasmid copy number might be higher in stationary cells than in exponential
ones, resulting in a lower plasmid less in the subsequent fermentation when a stationary culture is used as
inoculum.
21
 A stationary phase inoculum would result in a lag phase, but this disadvantage was more than
compensated for by the considerable improvement in plasmid retention and biotin production compared
with that obtained using an exponential inoculum.
EXPLAIN ABOUT THE DEVELOPMENT OF INOCULA FOR YEAST PROCESSES. (15 MARKS)
Even as the large industrial fermentations utilizing yeasts are the brewing of beer and the production
of biomass, recent processes have also been established for the production of recombinant products.
Brewing:
Crop:
 It is common practice in the British brewing industry to use the yeast from the precious fermentation to
inoculate a fresh batch are ‘crop’, referring to the harvested yeast from the previous fermentation, and
‘Pitch meaning to inoculate’.
 Generally yeast propagation systems are expensive to operate because of the sugar consumed by the
yeast during growth.
 It can then be appreciated that the reduced cost of using yeast from a previous fermentation is an
attractive proposition.
 The dangers inherent in this practice are the introduction of contaminants and the degeneration of the
strain, the most common degeneration being a ‘change in the degree of flocculance and attenuating
abilities of the yeast.
 In breweries employing top fermentations in open fermenters, these dangers are minimized by
collecting yeast to be used for future pitching from “Middle skimming”.
 During the fermentation the yeast cells flocculate and float to the surface, the first cells to do this being
the ‘most flocculent’ and the last cells the least flocculent.
Middle skimming:
 As the hand of yeast develops, the surface layer (the most flocculent and highly contaminated yeast is
removed and discarded and the underlying cells (the middle skimming) are harvested and used for
subsequent pitching.
 The pitching yeast may be treated to reduce the level of contaminating bacteria and remove protein and
dead yeast cells by such treatments as reducing the pH of the slurry to 2.5 to 3, washing with water,
washing with ammonium persulphate
 Treatment with antibiotics such as polymixin, penicillin and neomycin can also be employed.
 However, traditional open vessels are becoming increasingly rare and the bulk of beer is brewed using
cylindrical fermenters.
In these systems the yeast flocculates and collects in the cone at the bottom of the fermenter, where it is
subject to the,
1.
2.
3.
4.
5.
Stress of nutrient starvation.
High ethanol concentration
Low water activity
High carbon dioxide concentration and
High pressure
 Thus, the liability and physiological state of the yeast crop would not be ideal for an
inoculum.
 The viability of the crop may be assessed using a biomass probe, thus ensuring that At least
the correct amount of viable biomass is used to start the next fermentation.
 However, the physiological state of the biomass will not have been influenced by such
monitoring procedures.
 The situation is further complicated by the fact that the harvested yeast is stored before it is
used as inoculum. When the cells were suspended in beer and stored in the absence of
oxygen at reduced temperatures, metabolic activity of the cells were in reduced state.
 If oxygen is present during the storage period then the yeast cells consume their stored
glycogen, which renders them very much less active at the start of the fermentation.
 One of the key physiological features of yeast inoculum is the local of sterol in the cells.
22





Sterols are required for membrane synthesis but they are only produced in the presence of
oxygen.
But anaerobic conditions are required for ethanol production. This problem is resolved
traditionally by aerating the work before inoculation.
This oxygen allows sufficient sterol synthesis early in the fermentation to support growth of
the cells throughout the process (i.e., for anaerobic fermentation)
Generally the difficulties, such as strain degeneration and contamination may arise when the
yeasts are rarely used for more than five to ten consecutive fermentations, which signals the
importance of ‘the periodical production of a pure inoculum’.
This is done for developing sufficient biomass from a single colony to pitch a fermentation
at a level of approximately 2 gms of pressed yeast / liter.
Baker’s yeast:
 The commercial production of baker’s yeast involves the development of an inoculum through a
large number of aerobic stages.
 The development of inoculum for the production of baker’s yeast, a process involving 8 stages,
 The first 3 being aseptic while the remaining stages were carried out in open vessels.
 The yeast may be pumped from one stage to the next or the seed cultures may be centrifuged and
washed before transfer, which reduces the level of contamination.
7) EXPLAIN ABOUT THE DEVELOPMENT OF INOCULA FOR MYCELIA PROCESSES: (15
MARKS)

The preparation of inocula for fermentations employing mycelial (filamentous) organisms is more
involved than for unicellular bacterial and yeast processes.
 The majority of industrially important fungi and Streptomycetes are capable of asexual sporulation.
So it is common practice to use a spore suspension as seed during an inoculum development
program.
 A major advantage of a spore inoculum is that is contains for more ‘propagules’ than a vegetative
culture.
 Three basic techniques have been developed to produce a high concentration of spores for use as an
inoculum.
Sporulation on solidified media:
 Most fungi and Streptomycetes will sporulate on suitable agar media but a large surface area must be
an employed sufficient spore.
 Parker (1950) described the ‘roll- bottle” technique for the production of spores of
 Penicillium chrysogenum.
 Quantities of medium (300cm3) containing 3% agar were sterilized in 1dm3 cylindrical bottles, which
were then cooled to 45ºC and rotated on a roller mill so that the agar set as a cylindrical shell inside
the bottle.
 The bottles were inoculated with a spore suspension from a sub – master slope and incubated at 24ºC
for 6-7 days.
 Parker claimed that although the use of the ‘roll- bottle’ involved some sacrifice in ease of usual
examination, it provided a large surface area of cultivation of spores in a vessel of a convenient size
for handing in the laboratory.
 Hackenhall (1980) described the production of 1010 spores of P.chrysogenum in a ‘roux bottle’ and
107 and 108 cm-3 of spore suspensions are used for further applications.
 Use of roux bottle for the production a spore inoculum of Streptomycetes clamligerus for the
production of clamlanic acid.
 The spores produced from one bottle containing 200cm3 agar surface could be used to inoculate a
75dm3 seed fermenter, which, in turn, was used to inoculate a 1500dm3 fermenter.
Sporulation on solid media:
 Many filamentous organisms will sporulate profusely on the surface of cereal grains from which the
spores may be harvested.
23

Substrates such as barley, hard wheat bran, ground mirage and rice are all suitable for the sporulation
of a wide range of fungi.
 The sporulation of a given fungus is particularly affected by the amount of water added to the cereal
before sterilization and the relative humidity of the atmosphere, which should be as high as possible
during sporulation.
 Cooked rice can be used for the production of spores of Penicillium and Cephalosporium in
penicillin and cephalosporin respectively in inoculum development.
 The mass production of spores of several Aspergillus and Penicillium species on whole loaves of
white bread and the use of millet for the sporulation of Streptomycetes aurofaciens in the
development of inoculum for the chlortetracycline fermentation.
Solidified media suitable for the sporulation of some representative Streptomycetes:
Organism
Product
Medium
Composition
Malt extract
1.0%
Yeast extract
0.4%
S .aureofaciens
Tetracycline
Glucose
0.4%
S .ethythreus
Erythromycin
Beef extract
Yeast extract
Amino acids
Glucose
0.1%
0.1%
0.1%
0.1%
Sporulation in submerged culture:
 Many fungi will sporulate in submerged culture. This technique is more convenient than the use of
solid or solidified media because it is easier to operate aseptically and it may be applied on a large
scale.
 This technique was first adopted for the sporulation (inoculum development) in Penicillium notatum
by including 2.5% calcium chloride in a defined nitrate – sucrose medium.
 For e.g. production of inoculum using this mode for an industrial fermentation is the Griseofulvin
process.
 Rhodes et al described the conditions necessary for the submerged sporulation of the Griseofulvin –
producing fungus, Penicillium patulum, for prolific sporulation.
 The nitrogen level should be limited condition
 The good aeration to be maintained.
 Submerged sporulation was induced by inoculating 600Cm3 of above medium in a 2dm3 shake flask,
with spores from a well sporulate Czapekdox agar culture and incubating at 25ºC for 7 days.
 The results of suspension of spores were then used as a 10% inoculum for a vegetative seed stage in
a stirred fermenter.
 Most Actinomycetes do not sporulate in submerged culture, and thus, solid or solidified media tend to
be used for the production of spore inocula.
The use of the spore inoculum:
 The stage in an inoculum development program at which a large – scale spore inoculum is used
varies according to the process and it will be depend on the scale of the production fermentation.
 In the inoculum development program for the early penicillin fermentation described by Parker
(1950) the penultimate stage was inoculated with a spore suspension (from a roll – bottle) and this
stage may have produced either a vegetative or a submerged spore inoculum for the final
fermentation.
 When considering the production of gluconic acid by Aspergillus niger, the inoculating the final
fermentation directly with a spore suspension is better as compared with germinating the spores in a
seed tank to give a vegetative inoculum.
 Direct spore inoculation would avoid the cost of installation and operation of the seed tanks where as
the use of germinated spores would reduce the fermentation time of the final stage. However, labor
costs for the production of the vegetative inoculum could be almost as high as for the final
fermentation.
24

Thus, lock wood claimed that the choice of inoculum for the production stage depends on the length
of the cycle of the fermentation process, plant size and the availability and cost of labor.
Inoculum development for vegetative fungi:
 Some fungi will not produce asexual spores and therefore, an inoculum of vegetative mycelium must
be used. E.g. Gibberella fujikuroi is a fungus and is used for the commercial production of
gibberellins.
 The major problem is using vegetative mycelium, as initial seed is the difficulty to obtaining a
uniform, standard inoculum.
 The procedure may be improved by fragmenting the mycelium in a homogenizer, prior to the use as
inoculum.
8) EXPLAIN ABOUT SOLID STATE AND SUBMERGED FERMENTATION. (6 MARKS)
Introduction:
Based on the point of fermentation reaction the fermentation can be classified in to four types. They
are
1. Submerged fermentation
2. Solid state fermentation
3. Surface fermentation
4. Deep-bed fermentation
Submerged fermentation:
 The nature of medium used in this process is liquid and rarely semi solid media.
 Microorganisms are cultured in defined medium and used as inoculum.
 Aeration is necessary for this process, which is provided using the devise called sparger.
 Here during fermentation the microorganisms are dispensed throughout the medium.
 Similar to other fermentation process, here also the product is recovered from the fermented
broth.
 The product recovery is done by
1. Extra cellular or
2. Intracellular process.
 The recovery and purification of the product from the fermented broth is called as down stream
process.
Solid State Fermentation:
 The processes, which take place in the absence or near absence of free water in substrate, are termed
as solid-state fermentation (SSF).
 In this process the wheat bran, rice bran, sawdust, wheat, paddy straw, fruit pulps, etc., are all used as
a substrate.
 SSF can be classified into two types. They are namely,
1. Solid-state fermentation:
The solid substrates only act as a supportive material (solid bed) for the growth of the organisms.
2. Solid -substrate fermentation:
The solid substances such as agricultural products (corn, wheat etc) are act as a nutrient substrate
(i.e. medium) for the growth of the organisms and for the complete biological processes.
FOR E.G.: ENZYME ORGANIC ACIDS ETC ARE PRODUCED THROUGH THE SSF.
Surface fermentation:
1. Surface fermentation is more suitable for obligate aerobes mostly fungi some bacteria.
2. Microbes generally fungi grow on liquid medium and form a mat (net like structure) on the
surface.
3. If extra cellular product is produced then the fermented broth is processed.
4. If the product is intracellular then the microbial mat or cells are processed and the product is
further purified.
25
Deep bed fermentation:
1. It is also called as bottom fermentation.
2. Here neither liquid nor semisolid medium is used.
3. After addition of the inoculum the microorganisms settle down at the bottom and carry out
fermentation.
4. Mostly these fermentation processes are carried out by anaerobes or facultative anaerobes.
5. Eg: production of acetone-butanol using Clostridium acetobutylicum.
6. Production of beverages like beer using Saccharomyces calbergensis (bottom yeast)
9) EXPLAIN IN DETAIL ABOUT SOLID-STATE FERMENTATION: (15 MARKS)
INTRODUCTION:
SOLID-STATE FERMENTATION IS CLASSIFIED IN TO 2 TYPES, NAMELY
1. SOLID SUPPORT FERMENTATION E.G.
IMMOBILIZATION OF CELLS
2. SOLID SUBSTRATE FERMENTATION
SOLID SUBSTRATE FERMENTATION:
 Solid-substrate fermentations have been used for producing various fermented foods.
 It involves the growth of microorganisms on solid, normally organic materials in the absence or near
absence of free water.
 The substrates used are often cereal grains, bran, legumes & lignocellulosic materials, such as straw,
wood chippings, etc.
 Traditional processes are largely food fermentations producing oriental tempeh & sufu, cheeses &
mushrooms along with compost & silage making. In addition, enzymes, organic acids & ethanol are
now produced by solid – substrate fermentations, particularly in areas where modern fermentations
equipment is unavailable.
 Solid-substrate fermentations lack the sophisticated control mechanisms that are usually associated
with submerged fermentations.
 Their use is often hampered by lack of knowledge of the intrinsic kinetics of microbial growth under
these operating conditions.
 Control of the environment within the bioreactors is also difficult to achieve, particularly the
simultaneous maintenance of optimal temperature & moisture.
 However, in some instances, solid-substrate fermentations are the most suitable methods for the
production of certain products.
 For example, most fungi do not form spores in submerged fermentations, but sporulation is often
accomplished in solid-substrate fermentations.
 This method is successfully employed in the production of Coniothyrium minitans spores for the
biocontrol of the fungal plant pathogen, Sclerotinia sclerotiorum.
 Solid-substrate fermentations are normally multi-step processes, involving:
 Pretreatment of a substrate that often requires mechanical, chemical or biological processing,
1. Hydrolysis of primarily polymeric substrates, eg. Polysaccharides & proteins:
2. Utilization of hydrolysis products and
3. Separation & purification of end products.
 The microorganisms associated with solid-substrate fermentations are those that tolerate relatively
low water activity down to Aw values of around 0.7.
 They may be employed in the form of:
1. Monocultures, as in mushroom production, eg. Agaricus bisporus;
2. Dual cultures, eg. Straw bioconversion using Chaetomium cellulilyticum & Candida
tropicalis;
3. Mixed cultures, as used in composting & the preparation of silage, where the microorganisms
may be indigenous or added mixed starter cultures (inoculants).
26