Media for Industrial

fermentation

Reference: Principles of Fermentation

Technology- Stanburry
On a large scale one must normally use sources of nutrients to create a
medium which will meet as many as possible of the following criteria:
 Media Requirements and Media
formulations
• Large quantities of water is in need for
medium formulation.
• Media req. C, N, P & S.
• Minor req. as trace elements and vitamins
(biotin & riboflavin) may be in need.
• Media may include buffers or pH control (acid-
alkali additions).
• Antifoam agents, Inhibitors and Inducers may
be required.
• Historically – undefined complex natural materials used
– although they are cheaper but unpredictable
biomass/yield problems.
• Product recovery very difficult, residual components
my interfere with the recovery and contribute to the
BOD of the effluent.
• Although defined media is more expensive but it give
more predictable yield then undefined media.
• Control of pH and foam during growth in a fermenter
were indentified as two important parameters.
• Molasses would normally be used as the cheapest
carbohydrate to grow yeast biomass in a large scale
process. But this is not acceptable for Recombinant
protein production b/c of difficulties and cost for
purification.
 Medium Formulation
• Medium formulation is an essential stage in the design
of successful laboratory experiments, pilot – scale
development and manufacturing process.
• The constituent of the medium must satisfy the
elemental requirements for cell biomass & metabolite
production and there must be an adequate supply of
energy for biosynthesis.
C + N+ O2 +other req ------- biomass + product + CO2 +
H2O + Heat
• Thus, to calculate the minimal quantities of nutrients
which will be needed to produce a specific amount of
biomass, it should calculate substrate concentration
necessary to produce required product yields.
• Coonye 1981 – some nutrient are frequently added in
substantial excess of that required eg P, K, some trace eg.
Zn, Cu. Conc of P is deliberately raised in many media to
increase the buffering capacity. It depend on the
medium design.
• Some microorganism can't synthesize specific nutrients
eg AA, vitamins or nucleotides. Once a specific growth
factor has been identified it can be incorporated into a
medium.
• C has a dual role in Biosynthesis & energy generation. C
requirement for biomass production under aerobic
condition may be estimated the cellular yield Y which is
defined as
• Quantity of cell dry matter produced/quantity of C
substrate utilized.
 Energy Sources
• Energy comes either from oxidation of medium
components or from light.
• Most industrial microorganism are chemo-organotrophs,
therefore the commonest source of energy will be the
carbon (CHO), lipids & protein.
• Some micro-organisms can also use hydrocarbons or
methanol as carbon & energy sources.
Factor influencing the choice of carbon source:
Rate at which the C source is metabolized can often
influence the formation of biomass or production of
primary or secondary metabolites.
Fast growth due to high concentrations of rapidly
metabolized sugars is often associated with low
productivity of secondary metabolites.
• Main product of fermentation process depend on the choice
of C source.
• Eg. Ethanol, or SCP - 60-77% of the production cost depend
on cost of C source.
• it is the part of Company development program – to
determine the alternative C source to determine the yield.
• The purity of the C source may also affect the choice of
substance.
• The method of media preparation, particularly sterilization
may affect the suitability of CHO for individual fermentation
process.
• Its often best to sterilize sugar separately b/c they may react
with NH4 ions & AA to from black N containing compounds
which will inhibit the growth of many microorganism.
 Carbohydrates
• Its common practice to use CHO in microbial
fermentation e.g starch from maiz grain
• It may also obtained from, others cereals, potatoes &
cassava.
• Hydrolyzed cassava starch is used as a major C source
for glutamic acid production.
• Sucrose is obtained from sugar cane & sugar beet. It is
commonly used in fermentation media in a very impure
form as beet or cane molasses which are the residues
left after crystallization of sugar soln in sugar refining.
• Molasses is used in the production of high volume/low
value products such as ethanol , SCP, organic and AA
and some microbial gums.
• Molasses or sucrose also may be used for
production of higher value/low bulk products
such as antibiotics, specially enzymes, vaccines &
fine chemicals.
• However, molasses based fermentation will often
need a more expensive & complicated extraction
/ purification procedure to remove impurities &
effluents which make the procedure costly.
• Corn steep liquor is a by product after starch
extraction from maize. Although primarily used as
a N source, it does contain lactic acid, small
amounts of reducing sugars & complex
polysaccharides.
 Oils and Fats
• Oils were first used as carriers for antifoams in antibiotics
processes.
• Vegetables oils may also be used as C substrates, particularly for
their content of the fatty acid, oleic, linoleci and linolenic acid b/c
of cost are competitive with those of CHO.
• Bader 1984 – factors favouring the use of oils instead of CHO. A
typical oil contains approximately 2.4 times the energy of glucose
on a per weight basis.
• Oils also have a volume advantage as it would take 1.24 dm3 of soya
bean oil to add 10KCal energy to a fermenter whereas it would take
5dm3 of glucose or sucrose assuming that they are being added
50%w/w solutions.
• Oil also has antifoam properties
• Glycerol trioleat is known to be used in some fermentation
• Methly oleate used as the C substrate in cephalosporin production
Nitrogen sources:
• Most industrially used microorganism can utilize inorganic or
organic sources of nitrogen.
• Inorganic N may be supplied as NH3 gas, NH4 salts or nitrates.
NH3 has been used for pH control & as the major N source in a
defined medium for the commercial production of human
serum albumin by yeast.
• NH4 salts i.e (NH4)2SO4 will produce acid conditions & NH4NO3
normally cause an alkaline drift.
• When the NH4 ion has been exhausted, there is an alkaline
drift as the nitrate is used as n alternative N source.
• Organic N may be supplied as AA, protein or urea. Other
proteinaceous N compounds serving as sources of AA include
corn-steep liquor, soyas meal, peanut meal, cotton seed.
• In storage these products may be affected by moisture, temp,
& ageing.
 Factors influencing the choice of N source
• In Aspergilus nidulans NH3 also regulates the
production of alkaline & neutral proteases.
• In Antibiotic production by many microorganism
is influenced by the type and concentration of
the N in the culture medium.
• Antibiotic production only begins to increase in
the culture broth after most of the N consumed.
• Some complex N material cant be utilized by a
micro-organism & create problems in
downstream processing & effluent treatment.
This can be an important factor in the final choice
of substrate.
 Minerals
• All micro-organism requires certain mineral elements for growth &
metabolism.
• In many media, Mg, P, K, S, Ca & Cl are essential components, Co,
Cu, Zn, Fe, Mn, Mb & Zn are also essential
• P is used as a Buffer to minimize pH changes when external pH is
not being used.

•in specific processes the conc of certain minerals may be very

critical.

•It also influence the production of bactricin, citric acid, ergot,

monomycin,novobiocin, oxytetracycline, polyene, ristomycin,
rifamycin, streptomycin, vacomycin & viomycin
 Chelators
• Many media can’t be prepared or autoclaved
without the formation of a visible ppt of insoluble
metal PO4 .
• The problem of insoluble metal PO4 may be
eliminated by incorporating low conc of chelating
agents i.e EDTA, citric acid,polyphosphate etc
• These chelating agents form complexes with the
metal ions in a medium. The metal ions then my
be gradually utilized by the micro-organism.
• Chelating agent does not cause inhibition of
growth.
 Growth Factors
• Vitamins, specific AA, fatty acids or sterols.
• Many of the natural C & N sources used in media
formulation contain all or some of the required
growth factors.
• If only one vitamins is required it may be move
economical to add a pure vitamin instead of large
bulk of cheaper multiple vitamins source. E.g Ca
pantothenate has been used in one medium
formulation for vinegar production. -
• In glutamic acid, limited conc of biotin must be
present in the medium, some requires thiamin.
 Buffers
• The control of pH may be extremely important if
optimal productivity is to be achieved.
• Many media are buffered at about pH 7.0 by the
incorporation of CaCO3, if pH decreases the CO3 is
decomposed.
• PO4 which are the part of many media also play an
important role in buffering. High PO4 conc are critical in
the production of many secondary metabolites.
• C & N sources will also a basis for pH control as
buffering capacity can be provided by the protein,
peptides & AA such as in corn steep liquor.
• The pH may also be controlled externally by addition of
NH3 or NaOH & H2SO4.
 The addition of precursors & metabolic regulators to media
• Some components of a fermentation medium help to
regulate the production of the product rather than support
the growth of the micro-organism. Such additive include,
precursors, inhibitors,
Precursors:
• Some chemicals when added to certain fermentation are
directly incorporated into the desired product.
• E.g Penicillin yields
• A range of diff side chain can be incorporated into the
penicillin moleucle, eg corn steep liquor increased the yield
of penicillin form 20 units to 100 units.
• Corn steep liquor contain phenylethlamine when
incorporated, it yield benzyl penicillin.
• Phenylacetic acid is widely used precursor in penicillin
production.
 Inhibitors
• When certain inhibitors are added to
fermentations more of specific product may
be produced.
• Inhibitors have also been used to affect cell
wall structure and increase the permeability
for release of metabolites.
• The best example is the use of penicillin &
surfactants in glutamic acid production.
 Inducers
• The majority of enzymes which are industrial interest
are inducible.
• Induced enzymes are synthesized only in response to
the presence in the environment of an inducer.
• Inducers are often substrate such as starch or dextrins
for amylase, maltose for pullulanase & pectin for
pectinase.
• Most inducers which are included in microbial enzyme
media are substrate or substrate analogues but
intermediates and products may sometime be used as
inducers.
• E.g maltodextrins will inudce amylase & fatty acids
induce lipase. (use depend upon the cost)
Fermentation Media
Thank you