Nutritional Media Components

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Nutritional Media components essential for

growth of microorganisms and Product


formation

By,
Dr. Latika Shendre
Asst. Professor, DPU
COURSE DESCRIPTION:
Sr. No. Topic Description

 1.
Introduction to Biochemical Historical background of
Engineering and Bioprocess Biochemical engineering,
Technology Introduction of industrially
important biotechnologically
products

 2.
Isolation of microbes and Strain Isolation and preservation of
improvement industrially important microbes and
introduction of strain improvement
3. Design of fermentation media and Nutritional media components
inoculum development essential for growth of
microorganisms and product
formation, Media optimization
using conventional and statistical
designs, Inoculum development
for bacterial, fungal and yeast
strains
LEARNING OBJECTIVES
After this lecture, you will learn about:
 Microbial nutrition and role of different nutrients in the growth of
microbes.
 Growth medium and its various types.
 Microbial growth and Phases of growth curve.
 Effects of Different factors like temperature, pH etc. on the growth of
Microbes.
 Methods used for quantification of growth.
MICROBIAL NUTRITION
• The purpose of microbial nutrition is to obtain energy and construct
new cellular components for growth.
Nutrient Requirement:
 The major elements: these are also known as Macro Elements or
Macronutrients because these elements are required in high
amounts by the microbes. These includes C, H, O, N, S, and P.
 The minor elements: these are also known as micro elements or
micronutrients as these are required in low amounts by the microbes.
These includes Ca, K, Mg, Fe.
• The trace elements: these elements are required in very low
amounts.
These are not essential elements for the growth of the microbes but
these are involved in biological functions.
E.g. zinc (Zn2+) is present at the active site of several enzymes.

These includes Mn, Zn, Co, Mo, Ni, Cu


The sources of nutrients to create a medium should meet
the following criteria:
1. It will produce max. yield of product or biomass per gram of substrate.

2. It will permit the maximum rate of product formation.

3. There will be minimum yield of undesired products.

4. It will be of a consistent quality and be readily available.

5. It will cause minimal problems during media making and sterilization.

6. It will cause minimal problems in the production process particularly aeration and
agitation, extraction, purification and waste treatment.
1. The Basic Nutrient Requirements of
Industrial Media
• All microbiological media, whether for industrial or for laboratory
purposes must satisfy the needs of the organism in terms of carbon,
nitrogen, minerals, growth factors, and water.
• In addition they must not contain materials which are inhibitory to
growth.
NUTRIENT REQUIREMENT
1. Carbon (C), Hydrogen (H) and Oxygen (O):
 Each and every organism requires carbon, hydrogen, oxygen and
electrons for their growth and development.
 These elements are main constituent of cellular material.
 The requirement of C, H, and O can be satisfied together as most of
the carbon sources like glucose are often attached to hydrogen and
oxygen.
2. Nitrogen (N):
 Required for the synthesis of amino acids, purines, pyrimidines,
enzyme cofactors and other substances.

3. Phosphorus (P):
 Required for the synthesis of nucleic acids, phospholipids,
nucleotides, cofactors, some proteins and other cellular components.

4. Sulphur (S):
 Required for the synthesis of cysteine, methionine, biotin, thiamine
and some carbohydrates.
5. Potassium salts (K):
 It is cellular inorganic cation and cofactors for certain enzymes.

6. Magnesium (Mg):
 It is cellular inorganic cation and cofactors for certain enzymes.

7. Calcium (Ca):

 It is cellular inorganic cation and cofactors for certain enzymes.

8. Iron (Fe):

 It is the component of cytochromes and certain non-heme iron proteins and cofactors for
enzymatic reactions.
Carbon: Provides Nitrogen: Needed for the Minerals:
both energy and synthesis of amino acids,
Required for the
carbon units for lipids, enzyme cofactors and
other substances. activity in cell.
biosynthesis

Water: Water is a raw Growth factors: Require


material of vital organic compounds that
importance in industrial cannot be synthesized by
microbiology. the organisms
Carbon or energy
• C or energy requirements are usually met from carbohydrates
(glucose, starch or cellulose & …., etc) .
• Energy sources may include hydrocarbons, alcohols, or even organic
acids.
• In formulation industrial medium, the carbon content must be
adequate for the production of cells.
Nitrogen
• is found in proteins including enzymes as well as in nucleic acids
hence it is a key element in the cell.
• Most cells would use ammonia or other nitrogen salts.
• Any nitrogen compound which the organism cannot synthesize must
be added.
Minerals
• form component portions of some enzymes in the cell and must be
present in the medium.
• The major mineral elements needed include P, S, Mg and Fe.
• Trace elements required include manganese, boron, zinc, copper and
molybdenum.
Growth factors

• include vitamins, amino acids and nucleotides and must be added to


the medium if the organism cannot manufacture them.
GROWTH MEDIA
 Since all microbe requires carbon source, energy source, nitrogen
source, water, oxygen, and micronutrients for growth.
 A growth medium or culture medium is a solid, liquid or semi-solid
substance which provides nutrients for the growth of microorganisms.
 Microbes can use the nutrients of culture media as their food is
necessary for cultivating them in vitro.
 Liquid culture medium is called nutrient broth.
• Liquid culture medium can be solidified by adding solidifying agent
agar known as Agar Media.
• Agar contains mainly D-galactose, D- glucuronic acid and 3,6 anhydro
L-galactose.
• Agar derived from red sea weed e.g., Gelidium
Classification of Growth Media
1. Based on chemical composition, media can be classified
into three categories as follows:

A. Natural medium

B. Semi-synthetic medium

C. Synthetic medium
A. Natural medium:
culture media which consist only of naturally occurring biological
fluids e.g. Milk, urine, diluted blood, vegetable juices, meat extracts,
blood etc is known as Natural media.
B. Semi-synthetic medium:
culture media whose chemical components are partially known
and partially unknown are termed as Semi-synthetic culture media. e.g.
Potato dextrose agar (PDA), nutrient agar etc.
C. Synthetic medium:
culture media whose chemical composition is completely known
e.g. Mineral glucose medium, Richard’s solution etc. These media
are very useful in studying the physiology of microbes.
2. Based on application or function, media can be classified
as follows:
A. Selective media:
 This media is used for growth of only selected microorganism.
 It provide nutrients that only enhance the growth of particular microbe.
 For example, if a microorganism is resistant to a certain antibiotic, such as
ampicillin or tetracycline, then that antibiotic can be added to the medium.
 Only those microbes will grow in this media that were resistant to incorporated
antibiotic while microbes that are not resistant will die due to the antibiotic
added to the medium.
 Example of selective media include MacConkey agar media which is used for
culture of Gram-negative bacteria.
B. Differential media or Indicator media:
• Differential distinguish one microorganism type from another,
growing on the same medium.
• This type of media uses the biochemical characteristics of a
microorganism growing in the presence of specific nutrients or
indicators (such as neutral red, phenol red, eosin y, or methylene
blue) added to the medium to visibly indicate the defining
characteristics of a microorganism. This type of media is used for the
detection and identification of microorganisms.
• Example of differential media include Blood agar media which
contains bovine heart blood that becomes transparent in the
presence of β- hemolytic organisms such as Streptococcus pyogenes
and Staphylococcus aureus.
C. Minimal media:
 A minimal medium is one which supplies only the minimal nutritional
requirements for the growth of a microorganism. It just has enough
ingredients to support growth hence called as “minimal medium”.

 Minimal medium typically contains:


 A carbon source, which may be a sugar such as glucose
 Salts, these generally provide essential elements such as magnesium,
nitrogen, phosphorus, and sulphur to allow the bacteria to synthesize
proteins and nucleic acids.
 Water
MICROBIAL GROWTH
• Microbial growth is the result of both cell division and change in cell
size.
• It involves conversion of nutrients into biological compounds which
are used for energy production and also for biosynthesis and product
formation.
• In most bacteria, growth involves increase in cell mass and number of
ribosomes, duplication of the bacterial chromosome, synthesis of new
cell wall and plasma membrane, partitioning of the two
chromosomes, and cell division.
• This asexual process of reproduction is called binary fission.
FACTORS AFFECTING MICROBIAL GROWTH
1. Water
2. Oxygen
3. Carbon dioxide
4. Temperature
5. pH
6. Light
7. Osmotic pressure
1. WATER

1. Water is essential for the life of microorganisms. Most life processes


takes place in water base.

2. 80% of bacterial cell consists of water.

3. Spores are particularly resistant to desiccation and may survive in


the dry state for several decades.
2. OXYGEN

• Most life forms depend on oxygen for survival and growth.


• Microorganisms require oxygen to acts as terminal electron acceptor
in their respiratory chain.
• AEROBE:- Bacteria which require oxygen for growth.
• ANAEROBES:- Bacteria which do not require oxygen for growth.
• OBLIGATE AEROBE:- Bacteria which grow only in presence of oxygen.
e.g. Pseudomonas, Bacillus.
• FACULTATIVE ANAEROBES:- Bacteria which are aerobes but can grow
with lack of oxygen or in absence of oxygen e.g. Streptococci
• MICROAEROPHILIC:- Bacteria which grow with trace amount of
oxygen. E.g. Listeria monocytogenes

• OBLIGATE ANAEROBES:- Bacteria which strictly grow in the absence


of oxygen. They may die if oxygen is present. E.g. clostridia,
bacteroides.

• AEROTOLERANT ANAEROBES:- anaerobes which do not require


oxygen but tolerate the presence of oxygen.
3. CARBON DIOXIDE
 CO2 is provided by cellular metabolism and from environment.
 Autotrophic organisms are able to use carbon dioxide as source of
carbon.
 Heterotrophic bacteria require some amount of carbon dioxide from
exogenous sources.
 5-10% CO2 is supplied for them in culture.
 Capnophilic:- requiring excess amount of CO2 e.g. Brucella abortus
(10% CO2)
4. TEMPERATURE
• PSYCHROPHILES:- Bacteria which grow below 20°C, e.g. soil and water
saprophytes. Up to -7°C reported.
• MESOPHILES:- Bacteria which grow between 20-40°C. most pathogenic
bacteria are mesophiles. Wide range- e.g. Pseudomonas (5°C -43°C), narrow
range e.g. Gonococcus (30-39°C).
• THERMOPHILES:- Bacteria which grow at higher temperature i.e. 60-80 °C.
e.g. Bacillus stearothermophilus. Up to 250°C reported.
• THERMAL DEATH POINT:- The lowest temperature that kills a bacterium
under standard conditions in a given time.
• Under moist conditions most vegetative, mesophilic bacteria have a thermal death
point 50 to 65°C and most spores between 100 and 120 °C.
5. pH
• The pH requirement of bacteria is also variable. Most bacteria prefer
neutral pH (6.5-7.5).
• ACIDOPHILES:- Bacteria which grow in acidic environment i.e. below
pH 7.0, e.g. Lactobacilli grows in pH=3.
• NEUTROPHILES:- Bacteria which grow in neutral conditions i.e. at pH
7.0. Most bacteria have an average pH requirement of 7.2-7.6 which
matches with pH in human body environment.
• ALKALOPHILES:- Bacteria which grow in alkaline environment i.e.
above pH 7.0, e.g. Alkaligenes grows at pH= 10.5
6. LIGHT
 Most bacteria prefer darkness for growth.
 Cultures die if exposed to sunlight.
 However some bacteria require sunlight and are called phototropic.
 Exposure to light may influence pigment production.
 Photochromogenic mycobacteria form a pigment only on exposure to
light and not when incubated in the dark.
7. OSMOTIC PRESSURE
 There is wide range of osmotic tolerance found in bacteria.
 0.5% Sodium chloride (NaCl) is added in culture media to
provide suitable osmolarity.
 PLASMOLYSIS:- Sudden exposure to hypertonic solutions
may cause osmotic withdrawal of water and shrinkage of
protoplasm. This occurs more readily in gram negative than
in gram positive bacteria.
QUANTIFICATION OF GROWTH

• For unicellular organisms such as bacteria, growth can be measured in


terms of two different parameters:

• 1. Changes in cell mass

• 2. Changes in cell numbers


1. Measurement of Cell Biomass
I. Direct physical measurement of dry weight, wet weight of cells after
centrifugation.
II. Direct chemical measurement of some chemical component of the
cells such as total nitrogen, total protein, or total DNA contents.
III. Indirect measurement of chemical activity such as rate of O2
production or consumption, CO2 production or consumption, etc.
IV. Turbidity measurements employ a variety of instruments to
determine the amount of light scattered by a suspension of cells. As
bacteria multiply in media, it becomes turbid. The turbidity or optimal
density of a suspension of cells is directly related to cell mass or cell
number.
2. Measurement of Cell Numbers
1. Direct Microscopic counts (DMC):
Specific volume of a bacterial suspension (0.01 ml) is placed on a
microscope slide with a special grid. Stain is added to visualize
bacteria. Cells are counted. Dead cells can not be distinguished
from living ones.
2. Electronic counting chambers:
this is done to measure size distribution of cells. For cells size of
the bacteria, the suspending medium must be very clean.
3. Plate counts:
• This value plating out (spread plate or pouring plate) a sample of a
culture on a nutrient agar surface.
• The sample or cell suspension can be diluted in a nontoxic diluent
(e.g. water or saline) before plating.
• If plated on a suitable medium, each viable unit grows and forms a
colony.
• Each colony that can be counted is called a Colony forming Unit (cfu)
and the number of cfu’s is related to the viable number of bacteria in
the sample.
MICROBIAL GROWTH CURVE
Phases of Growth curve:

1. Lag Phase
2. Log or Exponential phase
3. Stationary Phase or Plateau phase
4. Death phase
PHASES OF GROWTH CURVE
1. Lag Phase:
 There is NO increase in cell number in this phase.
 In this phase, cells adapt to a new environment.
 There is NO change in number, but an increase in mass in this phase.
 Length of the lag phase depends on characteristics of microbial
species also in part by the media conditions.
2. Log Phase or Exponential Phase

 Growth rate is higher in Log or Exponential phase.


 This phase involves increase in cell mass and cell number with time
exponentially.
 It is the period of balanced growth, in which all the components of a
cell grow at the same rate.
 Composition of biomass remains constant in this phase.
 The exponential phase is followed by deceleration phase, which is the
period of unbalanced growth.
Growth rate
 This phase is also known as Plateau phase.
 In this phase, net growth rate is zero because numbers of cells divide
is equal to numbers of cells died.

Growth rate = Death rate

 Although the net growth rate is zero during the stationary phase, but
cells are metabolically active and produce secondary metabolite.
4. Death Phase:
 After the period of the stationary phase, the bacterial population
decreases due to the death of cells.
 The death phase starts due to the death of cells.
 The phase starts due to exhaustion of nutrients, accumulation of
toxic products and autolytic enzymes.
 There is a decline in the variable count and not in the total count.
Fermentation medium:

• The growth medium is required for cultivation of production strain.


• Not a single medium is ideal or best for all the fermentation process.
While designing/formulating fermentation medium following points
are considered:-

• Chemical composition: Medium must have a suitable chemical


composition i.e., it must contain source of carbon, nitrogen, growth
factors and minerals.

• Buffering reagent: Optimum pH is crucial for the successful yield of


fermentation product. Any change in pH have significant effect on
yield, therefore it is necessary to incorporate buffering reagent in
medium e.g. CaCO3.
• Anti Foaming Reagent: Foaming is a serious problem and lead
contamination of fermentation medium. These reagents are added at
the time of medium preparation or incorporated after sterilization.

• Toxicity: An ideal production medium should not have any toxic


effect on culture or product formation.
• Consistency: Medium should be consistent in uniform diffusion of
oxygen throughout medium, that’s why most fermentation process
make use of liquid medium. Medium should not be viscous as it
affect oxygen diffusion and its absorption.

• Contamination: Certain conditions of the production medium are


helpful to check contamination. E.g., In citric acid production from
Aspergillus niger, low pH keep the contamination of medium under
check.
• Recovery: Components of the medium should be such that it should
not effect the extraction and purification of products.
• Availability of raw materials: The raw materials required for the
designing of the production should be easily available in large
quantities at reasonable cost. That’s why non synthetic or crude
medium have wide application in large scale processes whereas
synthetic medium are limited only for research purpose as these
are expensive and production yield is low.

• Precursor (a substance from which another, usually more active or


mature substance is formed): It should be ideal to have precursor
molecule in the medium for better yield of the Product e.g., Phenyl
acetic acid in medium for penicillin fermentation.
Medium formulation

• Medium formulation is an essential stage in design of

successful lab experiments, Pilot-scale development and

manufacturing processes.

• The constituents of medium must satisfy the elemental

requirements for cell biomass and metabolite production &

supply of energy for biosynthesis and cell maintenance.


• The first step consider is an equation based on the stoichiometry (of growth

and product formation. From this it is possible to calculate the minimal

quantities of nutrients needed to produce specific amount of biomass.

• Some nutrients are frequently added in substantial excess of that required, e.g.

P, K; however, others are often near limiting values, e.g. Zn, Cu.

• The concentration of P is deliberately raised in many media to increase the

buffering capacity.
Different constituents of medium:

• Water:
• It is the major component of the most fermentation media.
• Clean water with consistent composition is required for medium
preparation.
• Mineral content of the water is important in brewing during mashing
process.
• Hard water containing high CaSO4 concn are better for English Burton
bitters beers
• Similarly, high content of carbonate in water is better for darker beers.
• Nowadays, the water may be treated by deionization or other techniques
and salts added, or the pH adjusted, to favour different beers.
• Energy sources
• Light for autotrophs,
• Most industrial MO are chemo-organotrophs & common source of energy is carbon
source from carbohydrates, proteins , lipids.
• Factors influencing the choice of carbon source
• Rate at which carbon source is metabolized directly influence the formation of biomass
& formation of primary and secondary metabolite.
• The main product of fermentation process will determine the choice of carbon source.
• The purity of carbon source may affect the choice of substrate.
• The method of media preparation mainly sterilization may affect the suitability of
carbohydrates for individual fermentation process.
• Regional availability of carbon source (Pfizer use 10 different carbon source for
antibiotic production depending on the geographical location of production site.)
• The choice of substrate is often controlled by Government legislation and local laws –
in EEC beet sugar is encouraged rather cane sugar.
Commonly used carbon sources:
• Carbohydrates are the most commonly used as carbon source.
• Most widely available carbohydrate source is starch obtained from maize grain.
Other sources of starch are cereals, potatoes and cassava.
• Partially germinated barley grains when heated gives a material known as malt, it
contains variety of sugars besides starch.

• Sucrose is obtained from sugar cane and sugar beet. In fermentation media it is
commonly used in impure form known as molasses, which is the residues left after
the crystallization of sugar.
• Molasses can be used in the production of both:-
• Low value/ bulk products e.g., ethanol, SCP, organic and amino acids and some
microbial gums as well as high value/low bulk products e.g., antibiotics, enzymes,
vaccines and fine chemicals.
• In India cane molasses are mainly used in alcohol industry for the production of
sprit, country liquor or other liquor brands like rum, brandy, whisky.

• Difference in cane molasses and beet molasses:-


• Cane molasses are rich in biotin whereas as beet molasses has less biotin (limiting),
therefore while using beet molasses for yeast culture in fermentation, small amount
of cane strap molasses or biotin supplying material should be incorporated. Because
yeasts require biotin for their growth.

• Cane molasses are deficient in organic nitrogen content as compared to beet


molasses because beet molasses contains betaine.

• Cane molasses contains 21.2 % Invert sugar, 33.4 % sucrose and lacks raffinose,
whereas beet molasses contains 1% invert sugar, 1% raffinose and 48.5%sucrose.
• Oils and fats:
• Oils were first used as carriers for antifoams during antibiotic production.
• Vegetable oils such as olive, maize, cotton seed, linseed, soya bean are used as carbon
source because of their content of fatty acids, oleic, linoleic, linolenic acids.

• Why oils are preferred over carbohydrates as energy source?

• Oil contains much more energy approximately 2.4 times energy from glucose per
weight basis and thus oil also have volume advantage i.e., more energy requirement,
oils require less volume in fermenter as compared to glucose or sucrose.

• During fermentation if substrate purity is of upmost concern Glycerol trioleate is
used.
• Methyl oleate is used as the sole carbon substrate source in cephalosporin production.
• Hydrocarbon and their derivatives:
• Several n- alkanes have been used as carbon source for the production of :-
• Organic acids
• Vitamins and cofactors
• Nucleic acids
• Antibiotics
• Enzymes

• Advantages and disadvantages of using hydrocarbon and its derivative Advantage:


• Hydrocarbon or their derivatives has twice more carbon and thrice more energy as compared to
sugar of same weight.
• These have stable priced as compared to agriculture derived feedstocks whose cost is quite
fluctuating.
• Disadvantage:
• These are initially impure and need to be refined to obtain pure products.
• These are not economically attractive for the bulk production.
• Nitrogen sources:
• MO used in industry utilize both inorganic & organic sources of nitrogen.
• Inorganic sources of nitrogen:
• Ammonia gas
• Ammonium salts
• Ammonium nitrates
• Ammonium sulphates

• Ammonia is used for pH control and major source of nitrogen in defined medium for the
production of human serum albumin by Saccharomyces cerevisiae.

• Organic source of nitrogen:


• Amino acids
• Protein
• Urea
• Nitrogen sources:

• Other proteinaceous nitrogen compounds which serve as source of amino acids are:-
• Corn steep liquor
• Soya meal
• Cotton seed meal
• Distiller solubles
• Peanut meal

• Corn steep Liquor (CSL): It is the byproduct left after the extraction of starch from maize.
• It is primarily used as a nitrogen source, also contain lactic acid, traces of reducing sugars
and complex polysaccharides (both C & N source).
• It is originally used for the production of penicillin. But now it is used in many fungal
antibiotic production medium.
• Nitrogen sources

• Soya meal: It is material left after deoiling of soya bean seeds. It contains about 8% w/w
Nitrogen. It has complex nitrogenous source than CSL. It is used in production medium
for Streptomycin.

• Cotton seed meal: It is clean, yellow, finely ground powder prepared from the embryo of
Cotton seed. It contains 56% protein, 24% carbohydrate, 5% oil and 5% ash. Ash basically
contains Fe, Ca, P, Cl and SO4. It is used in production medium of tetracycline.

• Distiller solubles: Residues (6-8% w/v total solids) left after the distillation of alcohol
from fermented grain or maize are further screened (removal of suspended solid residues),
leaving the effluent. Thereafter, effluent is further concentrated until solid reaches 35% w/v.
• To yield evaporator syrup, it is drum dried to yield Distiller solubles ,which contain
proteins.
• Minerals:
• These are very crucial for growth and metabolism of organism.
• Essential minerals are Ca, Mg, K, Cl, P & S. These are required in large conc (major
ingredients) as compared to other essential minerals like cobalt, copper, iron, manganese,
molybdenum and zinc (due to their presence as impurities in major ingredients do not
required in large amount).

• Conc of Zn, Mn & Fe are most critical in secondary metabolite formation.

• The yield of product formation varies linearly with the logarithmic concentration of key
metal.

• Incorporation of chlorine during the formation of chlorine containing secondary


metabolite is very important. e.g. Chlorotetracyclin, griesofulvin, caldriomycin,
nornidulin, mollisin.
• Chelators:
• Chelators are an important constituents of media.
• a heterocyclic compound having a metal ion attached by coordinate
bonds to at least two nonmetal ions.

• When medium are autoclaved without the addition of chelators, it


result in the formation of visible white precipitate of insoluble metal
phosphate containing all the iron, calcium, manganese and zinc.

• Chelators such as ethylene diamine tetraacetic acid (EDTA), citric


acid and polyphosphate are added in small amount.
• These chelators form complex with metal ions and causes gradual
release of metal ions from the complex.

• It is important to check that a chelating agent does not cause inhibition


of growth the micro-organism which is being cultured.
• Growth factors:

• Theses are preformed factors added to the medium as some microorganism cannot
synthesize it.

• Vitamins are the most commonly required growth factor, besides vitamins

• Some amino acids, fatty acids or sterols are used as growth factors.
• Calcium panthothenate used in vinegar production,
• biotin in glutamic acid production.
• Some production strains may also require thiamine

• Many of the natural carbon and nitrogen sources used in media formulations contain
all or some of the required growth factors.
• Buffers:

• It is incorporated into medium to maintain required pH optimal for growth of organism.

• A compound may be added to the medium to serve specifically as a


• buffer, or may also be used as a nutrient source.

• Many media are buffered at about pH 7.0 by the incorporation of CaCO3.



• Presence of certain proteins, peptide, amino acids in corn steep liqour may serve as
buffering agent.

• pH may be controlled by the external addition of ammonia, NaOH, and H 2 SO4. (acid or
alkali)
Besides the factors that regulate the growth of the cells, there are the
factors that regulate the product formation are incorporated in the media
formulations.

Precursor:
These are the molecules when incorporated into the medium they are
directly converted into the desired product.

When corn steep liqour is used in the production of penicillin the yield of
penicillin is increased from 20 unit /cm3 to 100 unit /cm3 as compared to
grown in other media.

When molecule analyzed it was phenylethylamine.

Phenyl acetic acid is the most commonly used precursor for Penicillin
production.

DYPBBI Amol Salagare 64


Precursor Product Micro-organism

DYPBBI Amol Salagare 65


Inhibitors:
These are the molecules when incorporated during media formulation
it results in:-
• Increase formation of specific product
• Accumulation of metabolic intermediates which were normally
metabolized when inhibitors were not incorporated.

Glycerol production depends on the modification of ethanol fermentation


by removing Acetaldehyde.
Addition of sodium bisulphite to broth leads to the formation of
acetaldehyde bisulphite addition compound (Sodium hydroxy ethyl
sulphite).
Since, acetaldehyde is no longer available for re-oxidation of NADH2, its
place as hydrogen acceptor is taken by dihydroacetone phosphate,
produced during glycolysis.
The product of this reaction is glycerol-3-phosphate, which is converted
to glycerol.
DYPBBI Amol Salagare 66
Product Inhibitor Effect Micro-organism

DYPBBI Amol Salagare 67


Inducers:

• These are the molecules when incorporated during media


formulation it results in induced production.

• Induced enzymes are synthesized only in response to presence of


inducer in the environment.

• Inducers are often substrates such as starch or dextrin for amylases,


maltose for pullulanase, pectin for pectinases.

• Besides substrate, its analogous molecule which is not acted by the


enzyme can also be used as inducer.

DYPBBI Amol Salagare 68


Enzyme Inducer Micro-organism

DYPBBI Amol Salagare 69

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