1. Fermentation Medium
(Substances used as raw materials for formulation of
fermentation media)
Dr. Deepak Bhusare
Department of Microbiology
DB, ACS College, Bhokar.
2. Medium improvement
• Medium designed for the initial production of antibiotic usually does not
have to be developed very skillfully since the potential for antibiotic
production is quite low with wild-type strains.
• Media for ultra-high antibiotic-producing strains, which have been
developed through repeated genetic manipulations, must be formulated
with utmost care.
• In the past, strain improvement and media development were the
responsibilities of different research groups. Today we know that each
higher- producing clone, after mutation and screening, requires a
medium optimized for its performance.
3. The importance of medium improvement
• Only small to moderate increases in the level of production can
result in an actual reduction in production cost so that it can
economically be sold in competition with others.
• There is little published literature on the complex substrates that
have been developed for the production of various products. Most
fermentation processes, which include the production of
fermentation media, are closely guarded trade secrets.
4. Different technical objectives of media formulation
• Inoculum (starter culture) propagation steps / pilot- scale
fermentations / main production fermentation.
• Biomass or primary metabolites production / secondary
metabolite production
5. Considerations in seed culture media formulation
• The seed stages are designed to give rapid and reproducible growth
without nutrient depletion, autolysis, or an adverse change in pH.
• There is less concern with the cost of ingredients in the seed stages
since the volume is usually only 5% of the fermentation volume and
excellent uniformity of medium ingredients is highly desirable.
• Some specially prepared dairy products have been used quite
extensively in primary and secondary seeds.
6. Constituents of medium
• Water
• Carbon source / Nitrogen source / Sources of phosphorous and
sulfur / Minor and trace elements / Vitamins such as biotin and
riboflavin
• Oxygen: even some anaerobic fermentations require initial
aeration, e.g. beer fermentations
• Buffers or controlled by acid and alkali additions
• Antifoam agents
• Precursor, inducer or inhibitor compounds
7. Considerations in media design
• Nutritional Requirements
• Environmental Requirements
• Techno-economic Factors
8. Nutritional requirements
• Nutritional requirements include elemental, specific nutrient, and
energy requirements
• Elemental requirements : the stoichiometry for growth and
product formation
C-source + N-source + O2 + minerals + specific
nutrients → cell mass + product + CO2 + H2O + heat
• Specific nutrient requirements:
Auxotroph: To use a complex medium or to identify the specific
nutrient
9.
10.
11. Environmental requirements
• Effect of growth temperature on cell yield / below optimal
temperature for growth.
• Combined effect of temperature and pH on growth and
production is not always the same.
• Environmental effect of substrate
12. Techno-economic factors that affect the
choice of individual raw materials:
• Cost: transport and storage, e.g. temperature control
• Availability: consistent quality and year round availability
• Ease of handling: solid or liquid forms
• Sterilization: thermal damage and inhibitory by product
• Operational characteristics: formulation, mixing, complexing and
viscosity characteristics that may influence agitation, aeration, foaming
and recovery
• Supply: the concentration of target product attained, its rate of
formation and yield per gram of substrate utilized
• Purification: levels and range of impurities, potential for generating
undesired products
• Pollution control
• Health and safety implications
14. Molasses
• Byproduct of cane or beet sugar production / residues remaining after
most of the sucrose has been crystallized from the plant extract
• Dark colored viscous syrup containing 50-60% (w/v) carbohydrate,
primarily sucrose, with 2% (w/v) nitrogenous substances, along with
some vitamins and minerals.
• Overall composition varies depending upon the plant source, the
location of the crop, the climatic conditions under which it was grown,
and the factory where it was processed
• The carbohydrate concentration may be reduced during storage by
contaminating microorganisms
• Hydrol molasses, containing primarily glucose, is a byproduct of
maize starch processing
15.
16. Malt extract
• Concentrated aqueous extracts of malted barley to form syrups /
particularly useful for the cultivation of filamentous fungi, yeasts and
actinomycetes
• App. 90% carbohydrate (w/w) and some vitamins and app. 5%
nitrogenous substances, proteins, peptides and amino acids / carbohydrate
comprising 20% hexoses (glucose and small amounts of fructose), 55%
disaccharides (maltose and traces of sucrose), 10% maltotriose, and
additionally contain 15-20% branched and unbranched dextrins, which
may or may not be metabolized, depending upon the microorganisms
• Careful sterilization to prevent over-heating reaction products (brown
condensation products resulting from the reaction of amino groups and
carbonyl groups) when heated at low pH / color change, loss of
fermentable materials, some toxic products
17.
18. Starch and dextrins
• Can be directly metabolized by amylase-producing
microorganisms, particularly filamentous fungi.
• Maize starch is most widely used.
• To allow use in a wide range of fermentations, the starch is usually
converted into sugar syrup, containing mostly glucose. It is first
gelatinized and then hydrolyzed by dilute acids or amylolytic
enzymes, often microbial glucoamylases that operate at elevated
temperatures
19. Sulfite waste liquor
• Sugar containing wastes derived from the paper pulping industry
are primarily used for the cultivation of yeasts.
• Waste liquors from coniferous trees contain 2-3% (w/v) sugar, 80%
hexoses (glucose, mannose and galactose) and 20% pentoses
(mostly xylose and arabinose) / Liquors derived from deciduous
trees contain mainly pentoses.
• Usually the liquor requires processing before use as it contains
sulfur dioxide / The low pH is adjusted with calcium hydroxide or
calcium carbonate, and these liquors are supplemented with sources
of nitrogen and phosphorus.
20. Cellulose
• Predominantly as lignocellulose (composed of cellulose,
hemicellulose and lignin)
• Available from agricultural, forestry, industrial and domestic wastes
• Relatively few microorganisms can utilize it directly / The cellulose
component is in part crystalline, encrusted with lignin, and provides
little surface area for enzyme attack
• At present, mainly used in solid-substrate fermentations (e.g.
mushrooms)
• Potentially a very valuable renewable source of fermentable sugars
once hydrolyzed, particularly in the bioconversion to ethanol for
fuel use
21. Whey
• An aqueous byproduct of the dairy industry / Annual worldwide
production is over 80 million tons, containing over 1 million tons
of lactose and 0.2 million tons of milk proteins.
• Expensive to store and transport / Lactose concentrates are often
prepared for later fermentation by evaporation of the whey,
following removal of milk proteins for use as food supplements
• Lactose is less useful than sucrose / e.g. S. cerevisiae does not
ferment lactose.
• Formerly used extensively in penicillin fermentation / Still
employed for producing ethanol, single cell protein, lactic acid,
xanthan gum, vitamin B12 and gibberellic acid.
22. Alkanes and alcohols
• n-Alkanes (C10-C20): readily metabolized by certain microorganisms
/ industrial use is dependent upon the prevailing price of petroleum.
• Methane: utilized by a few microorganism, but its conversion product
methanol is often preferred for industrial fermentations.
• High purity methanol is readily obtained / completely miscible with
water / has a high per cent carbon content and is relatively cheap /
only limited organisms will metabolize methanol / only low conc.,
0.1-1% (v/v) are tolerated by microorganisms / oxygen demand and
heat of fermentation are high, but this is even more problematic when
growing on alkanes.
• Ethanol is less toxic than methanol / used as a sole or cosubstrate /
too expensive for general use as a carbon source/ its biotransformation
to acetic acid remains a major fermentation process
23. Fats and oils
• Hard animal fats (composed mainly of glycerides of palmitic and
stearic acids) are rarely used in fermentation
• Plant oils (primarily from cotton seed, linseed, maize, olive, palm,
and soy) and occasionally fish oil, may be used as the primary or
supplementary carbon source, especially in antibiotic production /
Plant oils are mostly composed of oleic and linoleic acids, but
linseed and soy oil also have a substantial amount of linolenic acid
• Oils contain more energy per unit weight than carbohydrates / Oils
can be particularly useful in fed-batch operations than
carbohydrates (aqueous solutions less than 50%, w/v; occupy a
greater volume)
25. Corn steep liquor
• Byproduct of starch extraction from maize / first use in
fermentations for penicillin production in the 1940s
• Exact composition varies depending on the quality of maize and
the processing conditions/Concentrated extracts generally
contain about 4% (w/v) nitrogen, including a wide range of
amino acids, along with vitamins and minerals / Any residual
sugars are usually converted to lactic acid (9-20%, w/v) by
contaminating bacteria
• Can sometimes be replaced by liquor derived from potato starch
production
26. Yeast extract - 1
• Produced from waste baker’s and brewer’s yeast, or other strains of
S. cerevisiae / Or Kluyveromyces marxianus (formerly K. fragilis)
grown on whey and Candida utilis cultivated using ethanol, or
wastes from wood and paper processing
• Extracts used in the formulation of fermentation media are
normally salt-free concentrates of soluble components of
hydrolyzed yeast cells / Extracts with sodium chloride
concentrations greater than 0.05% (w/v) cannot be used in
fermentation processes due to potential corrosion problems
• Yeast cell hydrolysis is often achieved by autolysis, which can be
initiated by temperature or osmotic shock, causing cells to die but
without inactivating their endogenous enzymes
27. Yeast extract - 2
• Temperature and pH are controlled throughout an optimal and
standardized autolysis process / Temperature control is particularly
important to prevent loss of vitamins
• Autolysis (50-55oC for several hours before the temperature is raised
to 75oC to inactivate enzymes), plasmolysis or mechanical disruption
of cells / filtration or centrifugation to remove cell wall materials and
other debris / rapid concentration
• Extracts are available as liquids containing 50-65% solids, viscous
pastes or dry powders
• They contain amino acids (35-40%, w/v), peptides (30-45%, w/v),
water-soluble vitamins and some glucose derived from the yeast
storage carbohydrates (trehalose and glycogen)
28.
29. Peptones
• Peptones are usually too expensive for large-scale industrial
fermentations
• Prepared by acid or enzyme hydrolysis of high protein materials:
meat, casein, gelatin, keratin, peanuts, soy meal, cotton seed, etc.
• Amino acids compositions vary depending upon the original
protein source / Gelatin-derived peptones are rich in proline and
hydroxyproline, but almost devoid of sulfur-containing amino
acids / Keratin peptone is rich in both proline and cystine, but lacks
lysine
• Plant peptones invariably contain relatively large quantities of
carbohydrates
30.
31. Soya bean meal
• Residuals after extraction of soy oil.
• Composed of 50% protein, 7% non-protein nitrogenous
compounds, 30% carbohydrates and 1% oil.
• Often used in antibiotic fermentation because the components
are only slowly metabolized, thereby eliminating the possibility
of repression of product formation.
32. Water
• Use for media, cleaning, cooling ?
• A reliable source of large quantities of clean water, of consistent
composition, is essential.
• Before use, removal of suspended solids, colloids and
microorganisms is usually required.
• “Hard” water is treated to remove salts such as calcium carbonate
• Iron and chlorine may also require removal.
• Water is becoming increasingly expensive / recycle / reuse
wherever possible / minimizes water costs and reduces the volume
requiring waste-water treatment.
33. Antifoams -1
• Foaming is largely due to media proteins that become attached to
the air-broth interface where they denature to form a stable foam
• If foaming is minimized, then throughputs can be increased
• Three approaches to controlling foam production: modification
of medium composition, use of mechanical foam breakers and
addition of chemical antifoams
• Chemical antifoams are surface-active agents which reduce the
surface tension that binds the foam together
34. Antifoams -2
• Ideal antifoam: 1. readily and rapidly dispersed with rapid action;
2. high activity at low concentration; 3. prolonged action; 4. non-
toxic to fermentation microorganisms, humans or animals; 5. low
cost; 6. thermostable; 7. compatibility with other media
components and the process , i.e. having no effect on oxygen
transfer rates or downstream processing operations (e.g. some may
adversely affect membrane filtration)
• Natural antifoams include plant oils (e.g. from soy, sunflower and
rapeseed), deodorized fish oil, mineral oils and tallow ( 獸脂 )
• Synthetic antifoams are mostly silicon oils, poly alcohols and
alkylated glycols
35. Special compounds -1
• Precursors: phenylacetic acid or phenylacetamide as side-chain
precursors in penicillin production / D- threonine in L-isoleucine
production by Serratia marsescens / anthranillic acid for L-
tryptophan production by yeast Hansenula anomala.
• Inducers and elicitors: Inducers are often necessary for genetically
modified microorganisms (GMMs) / Production of secondary
metabolites, such as flavonoids and terpenoids, in plant cell culture
can be triggered by adding elicitors, which may be isolated from
various microorganism, particularly plant pathogens.
36. Special compounds -2
• Inhibitors:
1. Used to redirect metabolism towards the target product and reduce
formation of other metabolic intermediates (e. g. sodium bisulfite in
production of glycerol by S. cerevisiae)
2. Antibiotics for some GMMS containing plasmids bearing an
antibiotic resistance gene.
• Cell permeability modifiers: e.g. penicillins and surfactants added to
amino acid fermentations, including processes for producing L-
glutamic acid by members of the genera Corynebacterium and
Brevibacterium.
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