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sporulating fungi or actinonmycetes,
Com IrAgnei
ponent
FERMENTATION MEDIA the success of
The choice of a good medium
is very important for
nitrogen, carbon,
industria
require water and
sources of minera
fermentation. All micro-organisms also require
elements, vitamins amino acids etc. for growth. The microbial cells
gases,
different
bufiers, antifoamin
dissolved
growth factors, precursors of fermentation products, One of the major problems for
agents for the synthesis of specific fernentation
products.
production medium. This is only done by atria
fermentation industry is to design a suitable fermentation medium should meet the
characteristics of
d error method. The ideal
following criteria.
maximum yield of product or biomass in short duration.
roduction of the
Aminimum yield of undersized other products.
production medium.
Asimple, suitable chemical composition of
Free from any toxic effect on culture or product.
Low cost, good quality and easily available.
easily available at a low cost.
Kaw materials required for production medium are
extraction, purification, waste
Most suitable for production process such as agitation,
treatment etc.
and
Production medium must be suitable for adjustment pH, consistency
composition.
Inhibition or slowing of the growth of contaminating micro-organisms.
Allowing the proper growth and maintenance of the genetic stability of active micro
organisms.
Foaming must be minimum.
Media components must not interfere with the extraction and purification of desired
product.
Pharmaceu°ical BioBechnolooy 3.9 Fermentation Procass
The composition of a fermentation medium may be simple or complex depending on the
DArticular micro-organism, its nutritional needs and the product required. Asynthetie
medium contains known components and these media components are easily redesigned to
increaso possible yield of the product. Foaming is not a problem of fermentation which
contains synthetic media because these media do not contain any high molecular weight
peptides and proteins. The purification of fermentation products is also simple with the
synthetic media. The synthetic media may be exDensive because of the high cost of pure
ingredients and the yields obtained from thege media are low, Crude or non-sysnthetic
nedium mainly produce higher yield fermentation products. Crude medium mainly
contains soyabean meal, black strap molasses and cornsteep liquor as a source of carbon and
nitrogen.
Carbon sources:

The common carbon sources used in pharmaceutical fermentation processes are glucose,
fructose, sucrose, molasses, hydrolyzed starch, organic acids, bydrocarbons plant oils etc.
Beet and cane molasses are the concentrated syrups formed in the sugar-refining process as
by-products of sugar industry. Blackstrap molasses is the cheapest and most useful sugar
source for industrial fermentation. In addition to sucrose, blackstrap molasses also contain
small amounts of complex polysacharides and invert sugars. Glucose, sucrose and fructose
are more expensive as compared to molasses but it gives more clean medium. Complex
starch-containing substrates such as cornmeal are widely used in secondary metabolite
fermentation. Plant oils are a rich source of carbon than carbohydrates.
Cellulose material are complex corbohydrates made up of repeating units of f- glucose.
Sulfite waste liquor is the spent sulfite liquor obtained from the paper-pulping industry. It is
a dilute sugar solution having approximately 2% sugar content. Hexoses (D-glucose,
D-galactose, -mannose) and pentoses (D-xylose, L-arabinose) are mainly present in sulfite
waste liquor as monosaccharides.
Nitrogen sources :

The most common nitrogen sources are yeast extract, peptone, ammonia, distillers
solubles
solubles, soyabean meal, corn steep liquor, fish meal, cottonseed meal etc. Distillers
alcohol. Eluent is
are prepared from fermented grain or maize by distillation using
concentrated to reach the solid content 35% w/v (evaporator syrup). This syrup is then dried
with
to yield 'distillers solubles'. It is used as a production medium and it supplies nitrogen
by-product resulting from
many accessory food factors. Corn-steep liquor is the water extract
products. The
the steeping of corn during the production of starch, gluten and other corn
50% solids and this concentrate is
spent steep waters are concentrated to approximately
in fermentation media for the
called corn-steep liquor. It was first extensively used
acids, glucose,
production of penicillin. Corn-steep liquor mainly contains lactic acid, amino
 FermentationProcese

harmaceutical Biotechnology 3.10

deoiling
ofsoyabeanseeda
Balts and vitamina.
and it contains Soyabean meal is the componenta left
Buffers: approximatoly 8% wlv nitrogen. after
capacity
for their bufferingalkalin.
Buffers are
During microbial generally added in the production mediumConstituents scidieo r
growth in the fermentation, pH may be changed mayraisethe
by pH
products. media
and Decarboxylation
deamination of
of organic acids
among the mediumthe pH of
fermentation
neutralization
strongly basic organie lower
Calcium carbonate is generally added in emines nediato provideaminoacids posses
acidic fermentation products. fermentation peptidesand
good buffer capacity in the pH Media containing proteins,
range near neutrality.
Antifoaming agent ini the process
of
producefoam ie
Fermentation media containing proteins or peptides can levels offoam. If thisfoam
agitation and aeration) Proteolytic bacteria mainly produce high mainly esponsible
not controlled, it This condition is forced out
may rise in the head space of the tank. medium maybe
for causing contamination of the fermenter. In high levels of foam, control
the media to
from the tank as foam. Therefore, added in
antifoam agents are decrease the stability of the
foam. These antifoam agents can lower the and
surface tension used in fermentation i.e. crude
foam bubbles. Two types of antifoam agents are commonly organic materials are
crude
organic materials and inert antifoams. Antifoams made from soyabean oil, lavd ojl
sterilized separately and then added in the fermentation. Corn oil,
and other fatty acids are commonly used as antifoam agents. These anttoam
anol nutrients to cultures. On
gents nay be toxic or lower the pH of media or nrovide specificagents are not utilized by the
compounds are mainly used as inert antifoam agents. These
micro-organisms and they are also nontoxic.
Minerals :
of micro-organisms. Minerals
Most fermentation process require minerals for the growth
manganese and zinc are
Such as potassiumn, phosphorus, sulfur, copper, chloride, cobalt, iron,
frequently added in the media. These compounds are mainly present in complex nitrogen or
carbon sources.
Precurors :
Precurors are added prior to or simultaneously with the fermentation. These substances
are used to increase the yield or improve the quality of the product. Phenylacetic acid is
added the fermnentation medium of penicillin G and inorganic cobalt in the medium of
vitamin B1 as a precursor.
All fermentation media are generally sterilized by boiling or passing steam through the
medium or steam under pressure i. e. autoclaving. In asmall -scale laboratory fermenter, the
medium is directly placed in the fermenter and then the fermenter is sterilized by autoclave.
WHAT IS STERILIZATION:

Sterilization can be defined as any process that

effectively kills or eliminates transmissible agents
(such as fungi, bacteria, viruses and prions) from a
surface, equipment, foods, medications, or
biological culture medium.

METHODS OF STERILIZATIC
o The various methods of sterilization are:
1. Physical Method
a. Thermal (Heat) methods
b. Radiation method
c. Filtration method
2. Chemical Method
a. Gaseous method
 PHYSICAL METHODS:
1. HEAT STERILIZATION:
o Heat sterilization is the most widely used and
reliable method of sterilization, involving
destruction of enzymes and other essential cell
constituents.
This method of sterilization can be applied only to
the THERMO STABLE PRODUCTS, but it can be
used for MOISTURE-SENSITIVE MATERIALS.
i) Dry Heat (160-1800*C) Steilization for
thermo stable products
ii) moist heat (121-1340 'C) sterilization is
used for moisture- resistant materials.
o The efficiency with which heat is able to
inactivate microorganisms is dependent upon
i) the degree of heat, the exposure time and
i) the presence of water.
O The action of heat will be due to induction of
lethal chemical events mediated through the
action of water and oxygen.
o In the presence of water much lower
temperature time exposures are required to kill
microbe than in the absence of water.
 THERMAL (HEAT) METHOD:
Thermal methods includes:
i) Dry Heat Sterilization
Ex:1. Incineration
2. Red heat
3. Flaming
4. Hot air oven
i) Moist Heat Sterilization
1.Dry saturated steam - Autoclaving
2. Boiling water/ steam at
atmospheric
pressure
3. Hot water below boiling point
Dry Heat Sterilization
olt employs higher temperatures in the range of
160-180C and requires exposures time up to
2 hours,depending upon the temperature
employed.
o The benefit of dry heat includes good
penetrability and non-corrosive nature which
makes it applicable for sterilizing glass wares
and metal surgical instruments. It is also used
for sterilizing non-aqueous thermo stable
liquids and thermo stable powders.
o Dry heat destroys bacterial endotoxins (or
pyrogens) which are difficult to eliminate by
other means and this property makes it
applicable for sterilizing glass bottles which
are to be filled aseptically

Moist Heat Sterilization

o Moist heat sterilization involves the use of

steam in the range of 121-134'C. Steam under
pressure is used to generate high temperature
needed for sterilization. Saturated steam acts
as an effective sterilizing agent.
Autoclave
oAutoclaves use pressurized steam to destroy
microorganisms, and are the most dependable
systems available for the decontamination of
laboratory waste and the sterilization of
laboratory glassware, media, and reagents.
For efficient heat transfer, steam must flush
the air out of the autoclave chamber.
Generally the conditions employed are
Temperature upto121-134'C for 15-20 min under
15 Ibs pressure,based on type of metiral used.
 RadiationSterilization
o Many types of radiation are used for
sterilization like electromagnetic radiation (e.g.
gamma rays andUV light), particulate
radiation (e.g. accelerated electrons).The
major target for these radiation is microbial
DNA.
Radiation sterilization with high energy gamma
rays or accelerated electrons has proven to be
useful method for the industrial sterilization
of heat sensitive products.
Filtration Sterilization

o Filtration process does not destroy but

removes the microorganisms. It is used for
both the clarification and sterilization of liquids
and gases as it is capable of preventing the
passage of both viable and non viable
particles.
oThe major mechanisms of filtration are
sieving, adsorption and trapping within the
matrix of the filter material.
Ex:HEPAFILTERS
o There are two types of filters used in filtration
sterilization:
(a) Depth filters:
(b) Membrane filters: These are porous
membrane about 0.1 mm thick, made of
cellulose acetate, cellulose
nitrate, polycarbonate, and polyvinylidene
fluoride,or some other synthetic material.

CHEMICAL STERILIZATION
METHOD
GASEOUS METHOD
o The chemically reactive gases such as
formaldehyde, (methanol, H.CHO) and
ethylene oxide (CH2)20 possess biocidal
activity. Ethylene oxide is a colorless,
odorless, and flammable gas.
oThe mechanism of antimicrobial action of the
two gases is assumed to be through
alkylations of sulphydryl, amino, hydroxyl and
carboxylgroups on proteins and amino groups
of nucleic acids.