Spreading Knowledge is a great Legacy. MMG

1. Dr. Imran Sajid

2. Fermentation media
Nutritional requirements of microorganisms
• Carbon source -------- energy + carbon units for biosynthesis
• Nitrogen ------
• Phosphorus------
• Sulphur-------
• Vitamins--------
• Trace elements----------
• Etc------

3. The elemental formula of microbial cells
C4H7O2N
On the basis of dry weight is
48% C, 7% H, 32% O, 14% N
The elemental composition of Baker, s Yeast
C3.72 H6.11 O1.95 N0.61 S0.017 P0.035
Elemental Composition of the Microbial cells

4. • Most fermentations require--- liquid media (broth)
• Some times ------ solid substrate fermentations
Fermentation media must satisfy (two conditions)
1- Nutritional requirements of the microorganism
2- Technical objectives of the process (cell biomass + specific
metabolite)
Different stages of fermentation (media requirements)
1- Pre-culture or starter culture
2- Pilot-scale fermentation
3- Industrial scale (commercial fermentation)

5. Media formulation
Mixing of media components
• Water
• Carbon source
• Nitrogen
• Sulphur
• Vitamins
• Trace elements
• Buffers (Acid + alkali)
• Antifoaming agent
• Inducers or inhibitors (some cases)

6. Factors affecting media formulation
1- Cost and availability
2- Ease of handling and storage
3- Sterilization requirements (denaturation risk etc)
4- Formulation, mixing, viscosity characteristics
5- Yield of target compound
6- Level and range of impurities (byproducts etc)
7- Health and safety implications

7. The Carbon Sources
• Required for all biosynthesis
• Act as energy source
Determination of carbon requirement
Biomass yield coefficient (Y) = an index of the efficiency of
conversion of a substrate into cellular mass
Y carbon (g/g) =
Biomass produced (g)
Carbon substrate utilized (g)

8. The carbon sources
Yield coefficient (Y) of all the components can be determined
• By series of batch culture experiments
• Specific substrate is kept as growth limiting factor
• Culture conditions should be constant
• By plotting the total growth against substrate concentration
for each batch
Various organisms may exhibit different coefficients for same
substrate
Differences can also be seen with in one organism e.g.
Saccharomyces cerevisiae 0.56, 0.12 (g/g)

9. The carbon sources
• Molasses
• Malt Extract
• Starch and dextrins
• Sulphite waste liquor
• Cellulose
• Whey
• Alkanes and alkohols
• Fats and oils

10. The carbon sources
Molasses
• Byproduct of cane and beet sugar production
• Cheaper and most useful carbon source
• Residue remaining after the sugar crystallization
• Dark colored, viscous syrup
Composition
• Carbohydrates 50-60% (w/v) mainly sucrose
• Nitrogenous substances 2% (w/v)
• Vitamins and minerals

11. The carbon sources
Composition of molasses can vary depending on
• Plant source
• Location of the crop
• Climatic conditions
• Factory where it was processed
Carbohydrate concentration may vary during storage
Hydrol molasses
(a byproduct of maize starch) can also be used
Mainly contain glucose

12. Composition Sugar beet molasses Sugar Cane molasses
Dry matter %
Sucrose
Raffinose
Invert sugars
Missicillanous organic
materials
N
P2O5
CaO
MgO
K2O
SiO2
Al2O3
Fe2O3
Ash
Thiamine (ug/100g dry weight)
Riboflavin
Pyridoxin
Niacinamide
Pantothenic acid
Folic acid
Biotinn
78-85
48.5
1.0
1.0
20.7
0.2-2.8
0.02-0.07
0.15-0.7
0.01-0.1
2.2-4.5
0.1-0.5
0.005-0.06
0.001-0.02
4-8
130
41
540
5100
130
21
5.3
77-84
33.4
-
21.2
19.6
0.4-1.5
0.6-2.0
0.1-1.1
0.03-0.1
2.6-5.0
-
-
-
7-11
830
250
650
2100
2140
3.8
120
Composition of Molasses

13. The carbon sources
Malt extract
• Aqueous extract of malted barley
• Useful for cultivation of Fungi, Yeasts, Actinomycetes
Composition
1- Carbohydrates 90% (by dry Wt) this comprises
• 20% hexoses (glucose + small amount of fructose)
• 55% disaccharides (mainly maltose + some sucrose)
• 10% trisaccharide (maltotriose)
• 15-20% dextrins (branched or unbranched)
2- Nitrogenous substances 5%
3- Vitamins

14. Component % of dry weight
Maltose
Hexoses (glucose and fructose)
Sucrose
Dextrin
Other carbohydrates
Nitrogenous materials
Ash
Water content
52.2
19.1
1.8
15.0
3.8
4.6
1.5
2.0
Composition of Malt Extract

15. The carbon sources
Precautions for using malt extract
• Prevent overheating during sterilization
Maillard reaction
• Condensation products due to reaction between--- at low pH
• Amino groups of amines, amino acids and proteins with
carbonyl groups of reducing sugars ketones and aldehydes
• Cause a color change
• Results in loss of fermentable material
• Some reaction products may inhibit microbial growth

16. Maillard reaction

17. The carbon sources
Starch and dextrins
• Polysaccharides not readily utilized
• Can be metabolized by amylase producing microorganisms
(filamentous fungi)
• The extracellular enzymes hydrolyze the substrate to a
mixture of glucose, maltose, maltotriose, etc
• Maize starch is most widely used
• Can also be obtained from cereals, root crops etc
• To facilitate the utilization starch is converted into sugar syrup
containing mostly glucose

18. The carbon sources
Sulphite waste liquor
• Wastes derived from paper pulping industry
• Used for the cultivation of yeasts
Composition
Liquors from coniferous trees
• 2-3% (w/v) sugars (mixture of hexoses 80% and pentoses 20%)
• Hexoses includes: glucose, mannose and glactose
• Pentoses includes: xylose and arabinose
Liquors from deciduous trees contains mainly pentoses
• Require processing before use (contain SO2)
• pH adjusted with CaOH and supplemented (N- source)

19. The carbon sources
Cellulose
• Found as lignocelluloses in plant cell walls
Composition
• Three polymers: cellulose, hemicelluloses, and lignin
Availability/ utilization
• Agriculture, forestry, industrial and domestic wastes
• Only few of the organisms are able to use
• Mainly used in solid substrate fermentation (mushroom
production)
• May be a valuable material in ethanol production

20. The carbon sources
Whey
• Aqueous byproduct of dairy industry
• Annual production 80 million tons
• Containing 1 million tons of lactose + 0.2 million tons of milk
proteins
Utilization
• Milk proteins are removed to use as food supplements
• Lactose concentrates are prepared and is used for
• Penicillin fermentation, ethanol production, single cell
proteins, lactic acid, xanthan gum, vitamin B12 and gibbrellic
acid

21. The carbon sources
Alkanes and alcohols
• C10-C12 readily metabolized
• Mixture rather than single compounds are more useful
• Use depend on petroleum price
• Methane is rarely used but methanol is a good source of C
• Low concentration 0.1-1% (v/v) can be tolerated by
microorganisms
• 1970s – 1980s use of methanol in cell biomass production
• Ethanol less toxic but very expansive to use as raw material

22. The carbon sources
Fats and Oils
• Hard animal fats composed of glycerides is rarely used
• However plant oils may be good source of C e.g.
• Oils from- Cotton seed, corn , olive, palm, and soya etc
• Fish oil in antibiotic production
• Plant oils contains oleic and linoleic acids
• Oils contains more energy per gram of Wt than carbohydrates
• Can be used in fed batch fermentation
• Less space is required as compared to carbohydrates

23. The Nitrogen Sources
Most industrial organisms can use both
Inorganic and organic N source
Inorganic Nitrogen sources
• Ammonium salts
• Diammonium hydrogen phosphate
• Ammonia
Organic Nitrogen sources
• Amino acids, proteins, urea
Often supplied in crude form
Byproducts of other industries

24. The nitrogen sources
• Corn steep liquor
• Yeast extract
• Peptones
• Soy bean meal

25. The nitrogen sources
Corn Steep Liquor
• Byproduct of starch extraction from maize
• First utilization in penicillin production 1940s
Composition
• Nitrogen 4% (w/v) --- large range of amino acids
• Vitamins
• Minerals etc
• Can be replaced by similar liquors e.g.
• Derived from potato starch production

26. The nitrogen sources
Yeast extract
• Produced from waste backer’s or brewer yeast
• Other varieties of Saccharomyces cerevisiae
Alternative sources
• Kluyveromyces maraxianus (grown on whey)
• Candida utilis (grown on ethanol etc)
Yeast extract used are usually salt free, (0.05% Nacl con- cannot
be used)
Production: Cell hydrolysis is achieved by autolysis (by
temperature, or osmotic shock)

27. The nitrogen sources
• Autolysis is performed at 50-55° C (several hours)
• Temperature is raised to 75° C to inactivate the enzymes
• Finally the cells are disrupted by plasmolysis or mechanical
disruption
• Cell wall material or other debris are removed by filtration or
centrifugation
• Extracts are concentrated (50-65% solids)
• Viscous paste or dry powder
• composition: amino acids, peptides, water soluble vitamins
and some glucose

28. The nitrogen sources
Peptones
• Too expensive for large scale industrial fermentation
Production
• Acid or enzyme hydrolysis of high protein materials e.g.
• Meat, casein, gelatin, keratin, peanuts, soy meal, cotton
seeds, etc
• Amino acid composition vary according to source of protein
• Geletin derived: proline, hydroxyproline
• Keretin derived: proline, cystine, but lacks lysin
• Plant sources: reletively large quantity of carbohydrates

29. The nitrogen sources
Soya Bean Meal
• Residue obtained from the extraction of soya bean for oil
Composition
• Proteins 50%
• Non protein nitrogenous compounds 8%
• Carbohydrates 30%
• Oil 1%
Utilization
• Antibiotic fermentation

30. Other media components
Water
Should be clean and reliable (reverse osmosis)
Minerals
Sufficient quantities are present in water and
In raw materials as impurities (Co, Cu, Fe, Mn, Mo, Zn )
Some are added as specific salts (Ca, Mg, P, K, S, Cl etc)
Vitamins and growth factors
Most natural C and N sources contain vitamins
Bacteria usually can synthesize , required for fungi
Some are added as pure or less expensive plant extracts

31. Other media components
Antifoams
• The problem of foaming in the fermenters
Approaches to control foam production
• media modification
• mechanical foam breakers
• chemical antifoams
Properties of a good anti foam
• Disperse with rapid action, high activity, prolonged action,
non toxic, low cost, thermo stability, compatibility with media
components

32. Other media components
Natural antifoams
• Plant oils
• Deodorized fish oil
• Mineral oils
• Tallow
Synthetic anti foams
• Silicon oils
• Poly alcohols
• Alkylated glycols
Some may interfere with down stream processing

33. Other media components
Precursors
• Only specific fermentation processes
• Production of some secondary metabolites
• Added in controlled quantity
Examples
• Phenyl acetic acid- penicillin production
• D-threonine- L-isolucine production (Serratia marsences)
• Anthranilic acid- L-tryptophan production (yeast)

34. Other media components
Inducers
Some time product formation depends on inducers & structural
analogues etc
Examples
• Plant cell cultures (secondary metabolites)
• Flavonoids and terpenoids
• Inducers & elicitors can be isolated from microbes (plant
pathogens)
• Necessary in the fermentations by GMMs
• Cloned genes (switched on and off)

35. Other media components
Inhibitors
• Used to redirect metabolism towards the target compound
• Reduce formation of other intermediates
• Some intermediates can halt the process
Examples
• Sodium metabisulphite– glycerol production (S. cerevisiae)
• Incase of GMMs (plasmids contain antibiotic resistance gene)
• Addition of particular antibiotic can inhibit unwanted cells

36. Other media components
Cell permeability modifiers
• Increase cell permeability
• Effect on cell wall or cell membrane
• Promote the release of intracellular compounds in the
medium
Examples
• Penicillin and surfactants
• Used in amino acid fermentation
• As L-glutamic acid (corynebacterium and bravebacterium)

37. Other media components
Oxygen
• Quantity depends on the requirement of the organism
• Requirement may depends on carbon source
• Can be added as air (21% v/v)
• Occasionally in pure form (if requirement is high)
• In most cases filter sterlized prior to injection

38. Animal cell culture media
• Based on complex basal media (example)
Eagle’s cell culture medium
Components
• Glucose
• Mineral salts
• Vitamins
• Amino acids
For mammalian cells a serum is necessary
• Fetal calf serum, calf serum, new born calf serum, horse
serum etc

39. Animal cell culture media
Role of sera
• Provide essential growth factors (examples)
• Initiation and attachment factors
• Hormones
• Trace elements
• Protease inhibitors etc
Limitations of sera
• Highly complex composition makes the replacement with
lower cost ingredients difficult
• Sterilization is problematic (require filter sterlization)

40. Animal cell culture media
• Sera constitute (5-10% v/v) of the total medium
• Attempts have been made to reduce it
• In some cases (1-2% v/v)
• It should ultimately be eliminated (reasons)
• High cost
• Potential source of prions and viruses
• Some cell line have been developed (grow in serum free
media)

41. Plant cell culture media
• Chemically defined
Components
• An organic carbon source (cell grow heterotrophically) e.g.
sucrose, glucose, fructose, maltose and lactose
• A nitrogen source (nitrates supplemented with ammonium
salts & some time amino acids )
• Mineral salts
• Growth hormones (combination and concentration depends
on fermentation)
• Auxins- cytokinins promote cell division, two phase cultures

42. Culture maintenance media
• Used for storage and sub culturing (key industrial strains)
Designed to
• Retain good viability
• Minimize genetic variation
• Must reduce the production of toxic metabolites
• The naturally unstable strain are stored
• On media selective for specific character