UNIT:4

MEDIA DESIGN
 What is a Nutrient?

a. "Something that nourishes or promotes growth and

repairs the natural wastage of organic life."

b. More specifically, nutrients are elements and specific

combinations of elements (chemicals) that specific
organisms require for their growth and repair.
Common Nutrient Requirement:
All micro-organisms require :

1. Water

2. Source of energy

3. Source of carbon

4. Source of nitrogen

5. Mineral elements

6. Possibly vitamins

7. Oxygen, if aerobic.
 Dry weight composition of microorganisms
Over 95% of the microbial cell dry weight composition is composed of the
following major elements:

1. Carbon
2. Oxygen
3. Hydrogen
4. Nitrogen
5. Sulfur
6. Phosphorus
7. Potassium
8. Calcium
9. Magnesium
10. Iron
11. Sodium
12. Chloride
Essential Nutrient: must be provided to the
organism
1. Macronutrients (Macro elements, Macronutrients):
• These are required by microorganisms in relatively large amounts.
• Play essential roles in cell structure and metabolism.
• C, O, H, N, S, and P are components of carbohydrates, lipids, proteins,
and nucleic acids.
• K, Ca, Mg, and Fe are cations and play a variety of roles.

2. Micronutrients(trace elements):
• Required by all organisms and the following are needed by most cells.
• Manganese (Mn 2+), Zinc (Zn2+), Cobalt (Co2+),Molybdenum
,(Mo2+),Nickel,Copper
Micronutrients
• Cells require such small amounts that they can obtain these trace
elements from contaminants in water, glassware, and regular media
components for growth.

• Micronutrients are normally part of enzymes or may be required for

enzyme function.

• Aid in catalysis of reactions.

• Maintenance of protein structure.

• Microorganism requires a balanced mixture of nutrients. If an essential

nutrient is in short supply, microbial growth will be limited regardless of
the concentration of other.
Criteria for an ideal medium
1. It must give maximum yield of product or biomass per gram of
substrate used.

2. It must produce maximum concentration of product or biomass .

3. It must give maximum rate of product formation.

4. It must give minimum yield of undesirable products.

5. It must be of consistent quality and be readily available through out

the year.

6. It must give minimum problems in media making and sterilization.

7. It must give minimum problems in other aspects of production

process.
Commonly used media constituents

• Carbon sources- molasses, cereal, grain, starch, glucose, sucrose,

lactose.
• Nitrogen source- ammonium salts, urea, nitrates, corn steep liquor
soya bean meal, slaughter house waste.
Effect of medium on fermenter design

• Medium selected for a particular fermentation process effects the

fermenter design to be used for the same.

• Examples:

1. Effect of metabolic rate on temperature control.

2. Viscosity of medium will effect the design of agitator blade.

Influences of medium used

Medium used affects the following aspects of a fermentation process:

1. Growth and product formation.

2. pH variation.
3. Foam formation.
4. Oxidation –reduction potential.
5. Morphological forms of the organism.
6. Recovery and effluent treatment.
Undefined complex natural raw materials
• Undefined complex natural raw materials are cheaper than pure substrates.

• Degree of impurities and variations in the composition is very high.

• Give unpredictable and variable results.

• Make recovery and effluent treatment much more difficult and costly.

• Pure substrates give more predictable and consistent results.

• Recovery and effluent treatment is much simpler and cheaper.

 Medium formulation

• Constituents of the medium must satisfy the elemental requirements for cell
biomass and metabolite production.

• It must supply adequate amount of energy for biosynthesis and cell

maintenance.

Carbon and energy source + Nitrogen source + Oxygen + other requirements

Biomass + Products + CO2 + H2O + heat
 Steps for Medium formulation:
Elemental - Express the
composition of equation in
the process quantitative
organism must terms
be known in - Calculate the
order to carry minimal
out these quantities of
Stoichiometric nutrients
calculations required

Consider the Substrate

equation based concentration
on required to
stoichiometry produce
for cell growth desired product
and product yield can be
formation. obtained.
• Few media components might be needed for product
formation but may not be required for biomass production.

• Some nutrients added in excess- e.g. P,K

• Some added in limiting amount- e.g. Zn, Cu

• Organism specific requirements.

• Design of media influences the oxygen demand of culture

(More reduced carbon sources- higher oxygen demand).
 Water

• Water is the major component of all kind of fermentation

media as all the constituents are suspended in water.

• It is also required for various ancillary services like heating,

cooling, cleaning etc.

• So clean water of consistent quality is required.

• Reuse of water is normally a high priority.

• pH , mineral content and microbial load parameters to be

considered while choosing source of water.
Carbon &Energy sources
• Most industrial organisms are organotrophs, therefore the most
common source of energy is C-source.

• The carbon substrate has a dual role in biosynthesis and energy

generation.

1. The carbon substrate is required for product formation.

2. The carbon requirement for biomass production under aerobic
conditions may be estimated from cellular yield coefficient(Y).

Y= Quantity of cell dry mass produced / Quantity of carbon substrate

utilized
Types of C-sources

Carbon
sources

Hydrocarbons
Carbohydrates Oils and Fats and their
derivatives
 Types of C-sources
1. Carbohydrates
• Starch most common source of carbon- obtained from maize and other
sources like potato, cassava and cereals.

• Maize and other cereals are also used directly.

• Barley grains are also very common.

• Malt- contains variety of sugars – prepared by germinating and heating

barley grains.

• Sucrose from sugar cane and sugar beet.

• Molasses, lactose
• Corn steep liquor (by-product obtained after starch extraction from
maize) are also used.
2. Oils and fats

Vegetable oils like olive, soybean, linseed etc.

Advantages of oils/fats over carbohydrates:

1. Contain approximately 2.4 times more energy than glucose (per weight
basis).
2. More volume.
3. Antifoaming properties.
4. Allows the use of maximum working capacity of fermenter.
5. Easier downstream processing.
3. Hydrocarbons

Examples are n-alkanes(methane), alcohols (methanol, ethanol),petroleum

based products.
Advantages of Hydrocarbons over carbohydrates:

a) On per weight basis n-alkanes have twice the carbon and three times
the energy content of sugar.

b) Easier DSP.

Cost is the major hindrance in the use of hydrocarbons these days.

Factors affecting choice of C-sources
1. Rate of metabolism

2. Main end product of fermentation- Fast growth with high

concentration of fast metabolizing sugars
3. Purity

4. Method of media preparation

5. Government legislation

6. Local laws
 Types of N-sources
1. Organic sources: like amino acids, proteins, urea etc. Complex
sources like soybean meal, corn steep liquor, cotton seed meal, pea
nut meal etc.

2. Inorganic sources:

• Ammonia and ammonium salts (ACIDIC CONDITION)

• Ammonia also used for pH control.
• Nitrates (ALKALINE CONDITIONS)
Factors affecting choice of N- sources

1. Kind of enzyme system present.

2. End product required.

3. Predetermined aspects of the process.

4. Government legislation.

5. Local laws.
 Minerals
1. Macro elements- Ca, Mg- added distinctly as components

2. Microelements- Zn, Co - usually present as impurities in other elements.

Need to be added if synthetic media is being prepared.

3. Phosphorus - added in excess as helps in pH maintenance.

4. Phosphate -Concentration of much higher than that of other mineral

components.
• pH maintenance
• Sensitivity of processes to P concentration (secondary metabolites-lower tolerance
range). Examples: production of streptomycin, citric acid, polyenes.
• STREPTOMYCIN- Lower concentration, THIOPEPTIN/METHYLENOMYCIN- High.
• Calcium precipitates extra inorganic phosphate, thus improving YIELD.

• Concentration of Zn, Mn and Fe - critical for production of secondary

metabolites.
• Chlorine- Required for halophilic bacteria, but no role in growing fungi.
 Chelators
• Some insoluble metal phosphates (visible as white precipitate) are
generally formed during sterilization of the media.

• Examples- EDTA, citric acid, polyphosphates

• Mechanism of action- bind to the metal ions in the medium which are
then gradually utilized by the microorganisms.

• Chelating agent used should not inhibit the growth of microorganism.

• Complex ingredients like yeast extract also act as chelators.

 Growth factors
• GROWTH FACTORS- Special compounds that are required for
growth and product formation but cannot be synthesized by the
organism, hence supplied from outside.

• Examples-amino acids, vitamins, fatty acids etc.

• Most of the C & N sources used have all or some of the growth
factors.
 Buffers
• Productivity affected by pH.

• A compound may be specifically added to act as buffer or as a

nutrient source.

• Examples- calcium carbonate (decomposes if pH decreases),

phosphate, NaOH, ammonia, sulphuric acid.

• Balanced use of C& N sources helps in maintaining pH.

 Precursors
• COMPONENTS of fermentation medium that are directly
incorporated into the desired product.

• They are neither metabolized nor involved in any form of growth

requirement.

• They help to regulate the production of the product rather than

support the growth of microorganism.

• Example- phenyl acetic acid added as a precursor to be

incorporated as a side chain into the penicillin molecule.
Examples of precursors
 Inhibitors
• Inhibitors are the chemical compounds which when added into
fermentation medium enhance the production of a specific
metabolite.
• Examples of inhibitors
 Inducers
• Inducers are required for the production of inducible enzymes.

• Examples- substrates (starch, maltose) or substrate analogues

(should not be attacked by enzymes).
 Oxygen requirement
Oxygen affects- growth rate and metabolic productivity.
Factors that affect oxygen requirement include-

1. Fast metabolism: rapidly metabolizing sugars at high

concentration lead to high oxygen demand.

2. Rheology: the individual components of the medium may

affect the viscosity of the medium and hence the aeration and
agitation rate.

3. Antifoams: surface active agents, hence reduce the oxygen

transfer rates and increase oxygen requirement.
 Foaming
• Foaming may occur due to
1. a component present in the medium.
2. some factors produced by the micro-organism.

• Main cause- proteins in the medium.

• Proteins denature at the air-broth interface and forms a thin
film which doesn’t rupture easily.
 Effects of foaming
• Physical effects:

1. Reduction in the working volume of fermenter due to

oxygen exhausted gas bubbles circulating in the system
2. Change in bubble size
3. Lower mass and heat transfer rates
4. Invalid process data
5. Incorrect monitoring and control

• Biological effects:

1. Deposition of cells in the upper part of system

2. Problem in sterile operation as air filters become wet
3. Possibility of siphoning leading to loss of product.
 Patterns of foaming

1. Constant level through out the fermentation process.

2. Steady fall in the early part, thereafter constant.

3. Steady fall in the early part, then rises.

4. Low initial foaming which rises later on.

5. Complex foaming pattern.

 Methods for handling the foaming

1. To avoid foam formation by defined medium and a modification in the

physical parameters.

2. Mechanical foam breakers.

3. Antifoaming agents(antifoams).
 Antifoams

• Antifoams are surface active agents - reduce surface tension in

foams and destabilize protein films by :

1. forming a hydrophobic bridge between two surfaces.

2. displacement of the absorbed proteins.

3. rapid spreading on the surface of the film.

• Examples: alcohols, esters, silicones, sulphonates.

 Properties of an ideal antifoam
Should
1. disperse readily and have fast action on existing foam.
2. be active at low concentrations.
3. be long acting in preventing new foam.
4. not be metabolized by microorganisms.
5. be non-toxic to microorganisms/humans.
6. not cause any problem in extraction and purification of product.
7. not cause any handling hazard.
8. be cheap.
9. not affect oxygen transfer.
10. be heat sterilizable.