FERMENATION MEDIA

A hectic research is needed to develop the most suitable medium for an

Industrial fermentation process. All microorganisms requires water, Energy,
Nitrogen, Mineral elements and vitamins etc.,

The growth medium used for the cultivation of particular strain of

microorganism of subsequent production of microbial cells or Biochemical product
called “Production media or fermentation media.”

In industrial fermentation have to design suitable medium is very important

in all fermentation industry. This is a trial and error method. The composition of
fermentation media may be simple or complex and divided into two types.

1. Synthetic media
2. Nonsynthetic or Crude media
1. Synthetic media: contains all the components in definite proportions and
which requires for the growth of organisms. The synthetic media have
advantages and disadvantages.
Advantages:
1. The composition of the medium can be altered accordingly too the need.
2. Designed for either growth or product yield.
3. Foaming is not a problem, medium does not contain proteins.
4. The recovery and purification of the fermentation product easy.

Disadvantages:

1. The media is expensive

2. The yield is very low.
2. Nonsynthetic or Crude media: the crude media contains ill defined source
of nutrients and contain excess amount of nutrients and Growth factors. The
medium does not contain metals, inorganic salts and contain only organic
nutrients. The crude media contain high amount of Carbon and Nitrogen.
The crude medium is preferred for industrial fermentation because of the
following reason.
1. It gives high yield of fermentation product than synthetic media
2. Crude media is inexpensive.
3. The agricultural waste product can be used as crude media.
4. It should contain large quality of usable proteins and carbohydrates.
5. Crude media may be simple or complex crude media
 6. Adding or antifoam agents to Crude media during fermentation.

Crude Media components or Raw material:

Many different crude media or raw materials are used in industrial

fermentation process. The crude media raw material that are divided into several
categories mainly on the basis requirement of Carbon and Nitrogen Source. The
crude media enrich with C & N source, such kind of crude media can be used as
production media or fermentation media in fermentation industry..

Carbonaceous Material
1. Carbon Source (Carbohydrates):- Saccharine material, Starchy material,

Cellulosic material, Hydrocarbons and

Vegetable oils.

Saccharine material- Molasses -Black strap molasses, Refinery Molasses,

Beet Molasses, Hydrol & Fruit Juices.

Starchy material: Cereals, Underground Roots and tubers.

Cellulosic material: Sulfite waste liquor, Wood molasses & Rice straw.

2. Nitrogenous Material (Protein): Corn steep liquor, Soya bean Meal,

Pharmamedia & Distillers Solubles

1. Saccharine material: as the name suggest materials are sweet in taste,

simple, soluble form. The obtained from fruit juices or waste of sugar
industry called molasses.

Molasses: are the byproducts of Sugar cane and Beet sugar industry. It is
obtained during the sugar refining process. It is also called Black strap molasses
(BSM) and the chemical composition is variable from industry to industry. The
cane molasses contain 52 % of the sugars (30 % of Sucrose and 22% Invert
sugars.) and know as Black strap molasses. The BSM contain biotin.

The refinery molasses contain 70 to 75 % Invert sugars. The whole cane

juice is partially inverted to prevent sugar crystallization and the sugar is partially
hydrolyzed monosaccharide’s with heat and acid, then neutralized and
concentrated without removal of any sugars. It contains much amount of partially
hydrolyzed sugars of Glucose and Fructose.

Beet molasses: are produced from beet industry and it is limited in biotin

Hydrol: It is molasses obtained from the Corns starch after the preparation of
Dextrose. It contains 60% 0f sugars.

In India the Black strap molasses used to the large scale production of
Ethanol, Country liquor and other liquors like Rum, Brandy, Gin and Whisky.

Fruit Juices: contain soluble sugars. Example-Grape contain both glucose and
fructose and most of the fruit juices used in wine fermentation industries. The raw
juice of fruits is called “MUST” and obtained from various types of fruit, all the
fruit juices prominently used in the manufacture of wine.

Must is highly acidic liquid and it contains 17 % simple sugars, 0.3 ash, less
amount of nitrogen source.

Cheese whey: The waste is produced from Dairy industries during cheese
processing or making. It is a major waste product in all the cheese manufacturing
industry. It can be disposed into the environment without proper treatment.
Therefore it is desirable to use it of production of useful product. It is often more
profitable and sold as pig feed. It contains 0.82 – 0.95 % protein (Casein), 0.12 –
0.36 % Fat and 4.62- 0.36 % Lactose.

Mattic and Cook reported the cheese whey sample can be used in
fermentation industry for the production of Lactic acid and SSP, because the
presence of Carbohydrates (Lactose) and Nitrogenous material protein, Vitamins
and Inorganic salts.

Starchy material-there are two main sources of commercial starch used in the
fermentation industry. 1. Cereals 2. Root & Tubers.

Cereals:- Wheat, Rice, Maize, Barely etc., (Moisture content is too low)
Root & Tubers: Corn, sweet potato, potatoes, tapioca etc., (Moisture content is
high).

The polysaccharide are utilized for the fermentation medium as a source of

carbon for the growth of microorganism, starch require pretreatment to bring about
the conversion to simple or eatable sugars.

The pretreatment process accomplished by enzymatic or chemical treatment.

The conversion of starchy material or polysaccharide to sugars the process is
called “Saccharification” by enzymes (Amylases) or chemicals ( Dilute acids or
combination of both).

3). Cellulosic Material: it is a Homopolysaccharide, made up of repeating units of

Glucose. Before using as a raw material in fermentation process the cellulose
require Saccharification.

The cellulose breakdown and it gives 2-molecule sugar “Cellobiose”

(Disaccharide) and gives 1,000 to 10,000 units of cellobiose.

Cellulose abundantly occurred in Agricultural Waste- Rice straw, Wood is

called wood molasses, Sulfite waste liquor. Etc.

a) Sulfite waste liquor (SWL):

It arises from Paper & Pulp manufacturing industries.
The wood is subjected by hydrolysis with the help of Calcium Bisulfite
under heat and Pressure.
At the end of hydrolysis left the spent liquid and it is called Sulfite waste
liquor.
This SWL contain 10-12 % sugars and make upto 20%. It is a dilute sugar
solution and having 2 % sugar content. There are two types monosaccharide’s.
Hexoses- D series of Glucose, Galactose, Mannose
Pentoses- D xylose, Arabinose.
 SWL cannot be used directly in the fermentation industry. It is necessary to
remove SO2 or Sulfurous acid present in the waste liquor. This is removed
by Steam stripping or Precipitation with lime.
After the treatment with lime, the spent liquor may be used as dilute
fermentation medium in the fermentation industry.
It is mainly used for the production of ethyl alcohol by S.cerevisiae (utilize
Hexoses) and Torula utilis (utilizes pentoses)
The sulfite waste liquor composition varies depending upon the varity of
wood being used manufacture of paper and pulp.
b) Wood Molasses: it is similar to sulfite waste liquor, produced by hydrolysis
of wood. (Aspen, Douglas-fir, white pine, Redwood , yellow pine, red oak
dust, Sugar maple, Conifers woods, Broad tree leafs.) and wood waste that
is called saw dust.
The cellulosic wood contain 65 to 85 & fermentable sugars.
Several methods of wood hydrolysis were developed during the second
world war. The wood immersed in 0.5 % concentration of H 2SO4 at 150 to
165OC and obtain a syrup called wood molasses, contain 4-5% reducing
sugars (mixtures of hexoses and Pentose’s).
The wood molasses can be prepared by various woods.
Conifers woods- 85 % Hexoses and 15% pentoses
Broad leaf trees- 65% hexoses 35 % pentoses.
Ethyl alcohol, 2-3 butylene glycol, butanol, lactic acid, acetic acid, and other
products by fermentation.
c) Rice Straw: is an agricultural waste.
It is a good source of Cellulose.
Rice straw is major agricultural byproduct in Asian regions.
It can be directly used in fermentation process in industries without
Saccharification.
Because several microorganisms are capable of using cellulose medium for
their growth by producing extracellular enzymes.
 It is used for the production of SCP, Silage and Mushroom cultivation.

4). Hydrocarbons and Vegetable oils:

Hydrocarbons used as fermentation substrate, usually mixture of hydrocarbons.

They are relatively crude hydrocarbons are cheap

The purified hydrocarbon com[pounds are more expensive.

Example: Gasolin, n-parafinns are used to produce SCP, oroduction of Biomass of

yeast under aerobic condition.

Vegetable Oils : oils obtained by deoiling of vegetable seeds is called vegetable

oils.

On the basis of unsaturation the oils grouped into three types.

1. Oleic oils (Nondrying) Ex: Groundnut oil

2. Linoleic oils (Semi drying) Ex: Sunflower or cotton seed oil.
3. Linolenic oils: (Drying type) Ex: Soybean oil.

NITROGENEOUS MATERIAL

1. Corn Steep liquor (CSL): it is a steep water extract obtained during the
manufacture of Starch, Glutin and other corn products from the corm by
steeping process.
the steep water subjected to concentration to approximately 50% of solids
and this concentrated liquor is called Corn steep Liquor .
CSL contain 7.4 – 7.8% Nitrogen.
It is used for the production of Antibiotic Pencillin
Nowadays used for the production of food stuffs.
2. Soya bean Meal: The waste obtained after deoiling of soya bean seeds.
It contains 8 % of Nitrogen
It is differ from corn steep liquor.
 It is a complete medium.
It is used as a ingredient media for the commercial production of antibiotic
Streptomycin.
3. Pharmamedia:
It is prepared form the Embryo of Cotton seed.
It is clean, yellow, finely ground powder.
It contain 56% protein, 24% Carbohydrates, 5% oil and 5% ash.
The ash inturns contain Ca, Fe, Cl, PO4, SO4.
It is used as a ingredient production media for the production of Tetracyclin.
4. Distillers Solubles: the Maize or Grains allowed for production alcohol, the
fermented broth allowed for alcohol distillation the remaining leaving
residues.
The solids are separated or eliminated from the residue and leaving a
effluent, subjected to concentration until reaches 35%. This syrup is called
“Distillers Solubles”
It can be used as fermentation media in the fermentation industry.
ANTIFOAM AGENTS

In most microbial fermentation process, foaming is a problem. Foaming may

be due to many reasons (Vigorous agitation, Aeration, Proteins of Crude media
(CSL, Pharmamedia, Penutmeal, Soybean meal, Yeast extract.) and
Microorganisms autolysis release protein.

These proteins may denature at air broth form foam. The foaming can cause
removal of cells from the medium and decreasing the efficiency of fermentation
process, because of shredding organisms in the foam. Further stabilization of foam
enhanced or increased by release of protein by autolysis of organisms. If foam not
controlled, numerous changes occur and physical and chemical changes created.
The foam is controlled by foam suppressing agents called Antifoam agents.
 “Antifoam agents are surface agents, reducing the surface tension in the
foam and destabilizing protein films”

Ideal antifoam agents should have following properties.

1. It should easily disperse and fast action on an existing foam.
2. It should be active at low concentration.
3. It should be prevent new foam formation.
4. It should not be metabolized by microorganisms.
5. Should be nontoxic to microorganisms, humans and animals.
6. Should not create any problem during extraction and purification
of product.
7. Should be low price.
8. Should have no effect on oxygen transfer.
9. Should be heat stable.

Examples: Vegetable oils – Sunflower oil, Castor oil, Soybean oil. Groundnut
oil etc., and Inorganic Silicone, Esters, Alcohol (Stearyl and decanol) And Fatty
acid derivatives, Glycerol.

These antifoam agents are generally added when the foaming occur during the
fermentation process at a low level in to fermentation media.

CHELATORS
Many media cannot be prepared or autoclaved without formation of visible
precipitate of insoluble metal phosphate. When the media contain metal Ions (Fe,
Ca, Mn and Zn) forms a insoluble white precipitate in the medium and further not
available for the growth of microorganisms.

Formation of insoluble metal precipitated can be prevented by incorporating

low concentration of Chelating agents. (EDTA, Citric acid, polyphosphate).
 These chelating agent are complex with metal ion and further prevent the
formation of precipitate during the autoclaving or preparation of medium.

These chelating does not cause inhibition of growth of microorganisms

during fermentation or culturing.

During large scale production these chelating agents are not required
because ingredients of medium such as Yeast extract, Protease peptones complex
with metal ion.

METABOLIC REGULATORS
Some components of fermentation medium help to regulate the production of
desired product rather than support for the growth of microorganisms. Such
additives include Precursors, Inhibitors and Inducers. These regulators are
incorporated in fermentation media to enhance the progress of fermentation
processes.

1. PRECURSORS: are substance added prior to fermentation process into

fermentation media, which incorporated without any major change into the
molecule of fermentation product. These precursors increase the yield or
Quality of the product and very important in all fermentation industries.

Example:

Precursors Product Organism

Phenylacetic acid + CSL Penicillin G P. chrysogenum
Corn steep liquor 6 types of Penicillin P. chrysogenum
Chloride Chlorotetracyclin Streptomyces aurefacieum.
Chloride Griscofulvin P. griseofulum
Propionate Riboflavin Lactobacillus bulgaricus
Anthranillic acid Tryptophan Hansenula onamala
Propiobacterium,
Cobalt Vitamin b12 Streptomyces spp.
Aminobutyric acid Cyclsporin A Tolypocladium inflantum

2. INDUCERS: inducers are external substance and most of Industrial enzymes

are Inducible. Theses induced enzymes are called adaptive enzymes and are
synthesized only in the presence of specific substrate which is called inducer.
These inducers are incorporated prior to fermentation process or during
fermentation process to fermentation media.
Example:

Enzyme Inducer Organisms

Amylase Starch or Dextrin Aspergillis spp
Bacillus spp.
Pullulanase Maltose Aerobacteraerogenosa
Mannosidase Yeastmanns Streptomyces griseus.
E.coli, Bacillus spp.,
Streptomyces spp
Penicillinase Phenylaceticact
Streptococcus spp
Aspergillus spp
Mucor spp.
Cellulase Cellulose Trichofdrma viridae
Pectinases Pectin Aspergillus spp

3. INHIBITORS: Are the chemical substance inhibit the synthesis of intermediate

metabolites. Inhibitors are added to the fermentation media during the fermentation
process to enhance the production of desired product and suppress the formation of
undesired product or unwanted products. These inhibitors further metabolism of
desired product and enhance the accumulation or permeability of desired product.

Example:
 Product Inhibitor Main affect Organism
Glycerol Sodiumbisulphite Actetaldehyde Saccarhromyces spp
production
repressed
Tetracycline Bromide Chlorotetracycline Streptomyces spp.
repressed
Glutamic acid Penicillin Cellwall Micrococcus
permeability glutamicus
Citric acid Alkalimetal Oxalic acid A.niger
phosphates pH 2.0 repressed
Rifamycin B Diethyl Barbutarate Other rifamycin Nocardia mediterranei
inhibited

BUFFERS: Any chemical substances that control pH value are called buffers. The
Control of pH extremely important during the fermentation to obtain Maximum
amount of desired product. An increase or decrease in pH during fermentation
may affect on product yield because of acidity or Alkalinity on the metabolism of
product.

During fermentation Decarboxylation of organic acids or deamination of

amines from the medium will raises or lower the pH. Deamination of proteins,
peptides, amino acids can raise the pH value in fermentation media during
fermentation.

Generally CaCO3 is incorporated in fermentation media to provide

neutralization of Acidic product or Phosphate buffers (Na 2HPO4, NaH2PO4,
K2HPO4, KH2PO4) are used for neutralization of acidic components. In addition the
NaOH or H2SO4 are used as buffering agents.

MEDIA COMPONENTS AND FORMULATION

 An ideal production media should have the following characteristic and
single media is not used, the media is variable form one fermentation media to
another fermentation media. The media may be simple or complex.

1. Chemical Composition: fermentation media have suitable chemical

composition, the synthetic or crude media should contain a sufficient source
of Carbon, Nitrogen, Growth factors and Mineral salts.
2. Precursors: supply or incorporation to the fermentation media to obtain
desired product.

Phenylacetic acid + CSL-Penicillin G

Corn steep liquor 6 types of Penicillin
Chloride- Griscofulvin, Chlorotetracyclin
Propionate -Riboflavin
Cobalt -Vitamin b12
3. Buffering Capacity: maintenance of optimum pH ranges is essential for
successful of fermentation process. During fermentation process release of
Acidic or Basic components and it traces accumulation. In order to control
the optimum pH of the fermentation medium buffers should have added to
the medium. Example: CaCO3, Amino acids are act as good buffers and
Phosphate buffers (Na2HPO4, NaH2PO4, K2HPO4, KH2PO4).
4. Avoidance of foaming: Foaming is serious problem in fermentation
industry. Hence Defomers or Antifoam agents are added to the fermentation
media. The antifoam agents are added before sterilization or after
sterilization. Example: Vegetable oils – Sunflower oil, Castor oil, Soybean
oil. Groundnut oil etc., and Inorganic Silicone, Esters, Alcohol (Stearyl and
decanol) And Fatty acid derivatives, Glycerol.
5. Toxicity: The fermentation media should be free from toxic components.
Example: Sulphite waste liquor.
6. Consistency: in aerobic fermentation it is necessary to supply of sterile air
into the fermentation medium by agitation in liquid medium it diffuse the
 air in to the medium. The media should be liquid it should not be viscous,
the proper air is difficult to diffuse in high viscous media.
7. Recovery: after the fermentation process the recovery or separation or
Extraction of the product is easy and cheap. During this process it should not
create any problem of DSP.
8. Availability of raw material: the raw material for the fermentation media
and for the production of desired product should be freely available in large
quantities at a reasonable price.
It should renewable and available throughout the year.
In Large scale industrial fermentation processes the use of Synthetic
media are limited application, because they are expensive and low yield of
desired product.
The raw material in fermentation industry always crude media and
most of the Agricultural waste abundantly occurred, renewable, Cheap rate
used in fermentation processes.
Many different types of raw materials are used in industrial
fermentation process. The crude media raw material that are divided into
several categories mainly on the basis requirement of Carbon and Nitrogen
Source.
The crude media enrich with C & N source, such kind of crude media
can be used as production media or fermentation media in fermentation
industry.
Example: Saccharine material, Starchy, Cellulosic, Vegetable oils ,
Hydrocarbons, cheese whey, Corn steep liquor, Sulphite waste liquor,
Peanut meal, soya bean meal,