The composition of fermentation media is dependent on a number of factors
characteristic of the particular fermentation.
The major ingredients of a typical fermentation media are:
■
Water
■
Carbon source
■
Nitrogen source
■
Sources of P, S, minor and trace elements
■
Vitamins – Biotic, riboflavin etc
■
Buffers
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Antifoam
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Precursor
■
Inducer
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Inhibitor
Water
Water
is a major component of all fermentation media except solid-substrate
fermentation. It also provides trace mineral elements. Water is also important
for ancillary equipment and cleaning. Supply of large quantities of clean
water, of consistent composition, is therefore essential. The quality of water
in terms of pH, dissolved salts and effluent contamination is important. The mineral content is important in brewing
(mashing step) and has influenced the location of breweries and types of beer
produced. Before use, suspended solids,
colloids and microorganisms should be removed. If the water is ‘hard’, it should
be treated to remove salts such as calcium carbonate. Iron and chlorine should
also be removed. For some fermentations, especially, plant and animal cell
culture, the water must be highly purified. Since, water is becoming
increasingly expensive, its recycle/reusage wherever possible should be
encouraged. This minimizes water costs and reduces the volume requiring
waste-water treatment.
Carbon
Sources
A carbon source is
required for all biosynthesis leading to reproduction, product formation and
cell maintenance. In most fermentations
it also serves as the energy source.
Carbon requirements may
be determined from the biomass yield coefficient (Y), an index of the
efficiency of conversion of a substrate into cellular material.
Ycarbon(g/g)
= Biomass produced (g)
carbon substrate utilized (g)
For commercial
fermentations, the determination of yield coefficients for all nutrients is
usually essential. As most carbon
substrates also serve as energy sources, the organism’s efficiency of adenosine
triphosphate (ATP) generation and its utilization are key factors. Carbohydrates are traditional carbon and
energy sources for microbial fermentations, although other sources may be used,
such as alcohols, alkanes and organic acids.
Animal fats and plant oils may also be incorporated into some media,
often as supplements to the main carbon source.
Molasses
Pure glucose and sucrose are rarely used for industrial-scale fermentations,
primarily due to cost. Molasses, a
by-product of cane and beet sugar production, is a cheaper and more usual
source of sucrose. Molasses are
concentrated syrups or mother liquors recovered at any one of several steps in
the sugar refining process with different names depending on the step from
which it is recovered. Blackstrap
molasses from sugar cane is the cheapest and most used sugar source for industrial
fermentation. This is the residue
remaining after most of the sucrose has been crystallized from the plant
extract. It is a dark-coloured viscous
syrup containing 50-60% (w/v) carbohydrates, primarily sucrose, with 2%
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.
Refinery
blackstrap molasses is a similar product obtained from the recrystallization
refining of crude sucrose. High test or
invert molasses is produced after whole cane juice is partially inverted/partially
hydrolyzed to monosaccharides to prevent sugar crystallization. It contains
approximately 70-75% sugar and is preferable to blackstrap molasses as it has
lower levels of non-fermentable solids. Beet molasses are produced from
beetroot, in a similar process as for sugarcane. However, it may be limiting in biotin for
yeast growth and a small amount of cane molasses may need to be added in these
fermentations. Hydrol molasses, a by-product of maize starch processing
primarily contains glucose (60%) and a relatively high salt concentration.
Malt
Extract Aqueous extracts of malted barley can be concentrated
to form syrups that are useful carbon sources for the cultivation of
filamentous fungi, yeast and actinomycetes. The composition of malt extracts
varies to some extent, but they usually contain approximately 90% carbohydrate,
on a dry weight basis. This contains 20%
hexoses (glucose and small amounts of fructose), 55% disaccharides (mainly
maltose and traces of sucrose), along with 10% maltotriose, a
trisaccharide. These products contain a
range of branched and unbranched dextrins (15-20%), which may or may not be
metabolized, depending upon the microorganism.
Malt extracts also contain some vitamins and approximately 5%
nitrogenous substances, proteins, peptides and amino acids.
Sterilization
of media containing malt extract must be carefully controlled to prevent
overheating which produce Maillard reaction products. These are brown
condensation products due to the reaction of amino groups of amines, amino
acids and proteins with the carboxyl groups of reducing sugars, ketones and
aldehydes. This occurs when reducing sugars and amino acids are heated at low
pH. Such reactions cause loss of fermentable materials, colour change and some
reaction products may inhibit microbial growth.
Starch
and Dextrins These polysaccharides are not as readily
utilized as monosaccharides and disaccharides. It can be metabolized by
amylase-producing microorganisms, particularly filamentous fungi which
hydrolyze the substrate to a mixture of glucose, maltose or maltotriose. The
product composition is similar to that of malt extracts. Maize starch is most widely used, but may
also be obtained from other cereal or root crops. Starch is usually converted
into sugar syrup, containing mostly glucose before use in fermentations. It is first gelatinized and then hydrolyzed
by dilute acids or amylolytic enzymes, often microbial glucoamylases.
Sulphite
Waste Liquor Sulphite waste liquor is the product of
the paper pulping industry. It is obtained after wood for paper manufacture is
digested to cellulose pulp. It can be
used as a dilute fermentation medium for ethanol production by S. cerevisiae and the growth of Torula utilis for feed. Waste liquors from coniferous trees contain
2-3% (w/v) sugar, which is a mixture of hexoses (80%) and pentoses (20%). Hexoses include glucose, mannose and
galactose, whereas the pentose sugars are mainly xylose and arabinose. The liquors derived from deciduous trees
contain mainly pentoses. Sulphite Waste
Liquor requires processing before use as it contains sulphur dioxide or calcium
hydroxide or calcium carbonate which need to be stripped or removed by
precipitation with lime. Supplementation with sources of nitrogen and
phosphorous is also required.
Cellulose
Cellulose is mainly present as lignocellulose in plant cell walls, which has
cellulose, hemicellulose and lignin.
Lignocellulose is available from agricultural, forestry, industrial and
domestic wastes. Very few microorganisms
can utilize it directly, as it is difficult to hydrolyze. The cellulose component is encrusted with
lignin and provides little surface area for enzyme attack. It is mainly used in solid-substrate
fermentations to produce various mushrooms. Preliminary processing by
hydrolysis may be required in case of cellulose to expose fermentable sugars. It has potential as a valuable renewable
source of fermentable sugars particularly in the bioconversion to ethanol for
fuel use.
Whey
Whey is an aqueous by-product of the dairy industry. The annual worldwide production is over 80
million tonnes, containing over 1 million tonnes of lactose and 0.2 million
tonnes of milk protein. It is expensive
to store and transport. Milk proteins can
be removed from whey to be used as food supplements. Whey is then evaporated
and lactose concentrates are obtained which can be used for later fermentation.
Lactose is generally less useful as a fermentation foodstock than sucrose, as
it is metabolized by fewer organisms. S. cerevisiae, for example, does not
ferment lactose. It was used extensively
in penicillin fermentations and is still employed for producing ethanol, single
cell protein, lactic acid, xanthan gum, vitamin B12 and gibberellic acid.
Alkanes
and Alcohols n-Alkanes of chain length C10-20 are
readily metabolized by certain microorganisms.
Mixtures, rather than a specific compound, are usually most suitable for
microbial fermentations. However, their
industrial use is dependent upon the prevailing price of petroleum. Methane is utilized as a carbon source by a
few microorganisms, but its conversion product methanol is often preferred for
industrial fermentations as it presents fewer technical problems. High purity methanol is readily obtained and
it is completely miscible with water.
Methanol has a high percent carbon content and is relatively cheap,
although only a limited number of organisms will metabolize it. Also unlike many other carbon sources, only
low concentrations, 0.1-1% (v/v), are tolerated by microorganisms, higher
levels being toxic. During fermentations
on methanol, the oxygen demand and heat of the fermentations are high, but this
is even more problematic when growing on alkanes. Several companies used methanol in microbial
protein production in the 1970s and early 1980s, but these processes are
currently uneconomic.
Ethanol is less toxic than methanol and is
used as a sole or co-substrate by many organisms, but it is too expensive for
general use as a carbon source. However,
its biotransformation to acetic acid by acetic acid bacteria remains a major
fermentation process.
Fats
and Oils
Hard animal fats that are
mostly composed of glycerides of palmitic and stearic acids are rarely used in
fermentations. However, plant oils
(primarily from cotton seed, linseed, maize, olive, palm, rape seed and soya)
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 soya oil also have a substantial amount of linolenic acid. The oils contain more energy per unit weight
than carbohydrates. In addition, the
carbohydrates occupy a greater volume because they are usually prepared as
aqueous solutions of concentrations no greater than 50% (w/v). Consequently, oils can be particularly useful
in fed-batch operations as less spare capacity is needed to accommodate further
additions of the carbon source.
References
- Industrial Microbiology:
An Introduction. Michael J. Waites, Neil L. Morgan, John S
- Principles of Fermentation Technology- Peter Stanbury,
Allan Whitaker, Stephen Hall
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