Over production of Metabolite

Production of Single Cell Protein

Protein malnutrition is usually far more severe than that of other nutrients deficiencies. However,
microorganisms could help meet the challenge posed by protein deficiency. The limitations of
conventional sources of proteins were recognized to include:
i. Possible crop failure due to unfavorable climatic conditions in the case of plants.
ii. The need to allow a time lapse for the replenishment of stock in the case of fish.
iii. The limited land available for farming in the case of plant production.

Production of Single cell protein (SCP) has a number of attractive features:

a. It was not subject to the vicissitudes of weather and can be produced every minute of the year.
b. Microorganisms have a much more rapid growth than plants or animals.

SCP is itself not entirely lacking in disadvantages. One of the most obvious is that many
developing countries, where protein malnutrition actually exists, lack the expertise and/or the
financial resources to develop the highly capital intensive fermentation industries involved. But
this short-coming can be bridged by the use of improvised fermentors and recovery methods which
do not require sophisticated equipment. Other criticisms of SCP are that microorganisms contain
high levels of RNA and that its consumption could lead to uric acid accumulation, kidney stone
formation and gout.
Properties of Organisms used for SCP
(a). Absence of pathogenicity and toxicity: It is obvious that the large-scale cultivation of
organisms which are pathogenic to animals or plants could pose a great threat to health and
therefore, should be avoided. The organisms should also not contain or produce toxic or
carcinogenic materials.
(b). Protein quality and content: The amount of protein in the organisms should not only be
high but should contain as much as possible of the amino acids required by man.
(c). Digestibility and organoleptic qualities: The organism should not only be digestible, but it
should possess acceptable taste and aroma.
(d). Growth rate: It must grow rapidly in a cheap, easily available medium.
(e). Adaptability to unusual environmental conditions.

The fermentation processes

The fermentation process requires a pure culture of the chosen organism that is in correct
physiological state, sterilization of the growth medium which is used for the organism, a
production fermenter which is the equipment used for drawing the culture medium in the steady
state, cell separation, collection of cell free supernatant, product purification and effluent
treatment.
Fermenters can vary in size from one or two litres capacity, to industrial models of several hundred
litres capacity. The most commonly used principle has been the chemostat: a perfectly mixed
suspension of biomass into which medium is fed at a constant rate and the culture is harvested at
the same rate so that the culture volume remains constant. Among the various designs which have
been put to effect, air-lift has enjoyed the greatest success as the configuration of choice for
continuous SCP production. The control of key process variables is a critical element of SCP
production, from oxygen transfer, substrate and product concentration, to the appearance of
minimal amounts of toxic compounds through undesired metabolic processes, which may
compromise the quality of the final product. The biomass from yeast fermentation processes is
harvested normally by continuous centrifugation. Filamentous fungi are harvested by filtration.
The biomass is then treated for RNA reduction and dried in steam drums of spray driers.
Removal of nucleic acids from SCP
(a) Growth and cell physiology method:
The RNA content of cell is dependent on growth rate: the higher the dilution rate (in continuous
cultures) the higher the RNA/ protein ratio. In other words the higher the growth rate the higher
the RNA content. The growth rate is therefore reduced as a means of reducing nucleic acid. It must
however be borne in mind that high growth is one of the requirements of reducing costs in SCP,
hence the method may have only limited usefulness.
(b) Extraction with chemicals: Dilute bases such as NaOH or KOH will hydrolyze RNA easily.
Hot 10% sodium chloride may also be used to extract RNA. The cells usually have to be disrupted
before using these methods. In some cases the protein may then be extracted, purified and
concentrated. Use of pancreatic juice: RNAase from bovine pancreatic juice, which is heat-stable,
has been used to hydrolyze yeast RNA at 80°C at which temperature the cells are more permeable.

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(c) Activation of endogenous RNA: The RNAase of the organism itself may be activated by heat-
shock or by chemicals. The RNA content of yeasts have been reduced in this way.
Industrial Alcohol
Ethyl alcohol, CH3CH2OH (synonyms: ethanol, methyl carbinol, grain alcohol, molasses alcohol,
grain neutral spirits, cologne spirit, wine spirit), is a colorless, neutral, mobile flammable liquid,
rarely found in nature.
Fermentable substrates
Following are the types of substrates used for alcohol production:
Sugary Materials: Examples of sugary materials are sugarcane and its by products/wastes
(molasses, bagasse) and sugar beet, tapioca, sweet potatoes, fruit juice, sweet sorghum, etc. Sugar
cane molasses is largely being used in many countries for alcohol production.
Starchy materials: Starchy materials used in ethanol production are tapioca, maize, wheat, barley,
oat, sorghum, rice and potatoes. But tapioca and corns are the two major substrates of the interest.
Lignocellulosic materials: The sources of cellulosic and lignocellulosic materials are the
agricultural wastes and wood. However, yield of ethanol from lignocellulose is low because of
lack of suitable technology and failure of conversion of pentoses into ethanol. On the basis of
technology available today about 409 liters of ethanol can be produced from one tonne of
lignocelluloses. Production of ethanol from lignocelluloses follows the following steps: (i)
hydrolysis, (ii) fermentation, and (iii) recovery.
Fermentation
Alcohol-resistant yeasts, strains of Saccharomyces cerevisiae are used, and nutrients such as
nitrogen and phosphate lacking in the broth are added.

a). Distillation
After fermentation the fermented liquor or beer contains alcohol as well as other volatile
compounds. The alcohol is obtained by several operations. First, steam is passed through the beer
which is said to be steam-stripped. The result is a dilute alcohol solution which still contains part
of the undesirable volatile compounds. Secondly, the dilute alcohol solution is passed into the
center of a multi-plate aldehyde column in which the following fractions are separated: esters and
aldehydes, fusel oil, water, and an ethanol solution containing about 25% ethanol. Thirdly, the

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dilute alcohol solution is passed into a rectifying column where a constant boiling mixture, an
azeotrope, distils off at 95.6% alcohol concentration.

Uses of ethanol
(i) Use as a chemical feed stock: In the chemical industry, ethanol is an intermediate in many
chemical processes because of its great reactivity as shown above. It is thus a very important
chemical feed stock.
(ii) Solvent use: Ethanol is widely used in industry as a solvent for dyes, oils, waxes,
explosives, cosmetics etc.
(iii) General utility: Alcohol is used as a disinfectant in hospitals, for cleaning and lighting
in the home, and in the laboratory second only to water as a solvent.
(iv) Fuel: Ethanol is mixed with petrol or gasoline up to 10% and known as gasohol and
used in automobiles.

Production of Organic acids

A large number of organic acids with actual or potential uses are produced by microorganisms.
Citric, itaconic, lactic, mallic, tartaric, gluconic, mevalonic, salicyclic, gibberelic, diamino-
pimelic, and propionic acids are some of the acids produced using microorganisms.

a. Production of Citric Acid

Citric acid is a tribasic acid. It crystallizes with large rhombic crystals containing one molecule of
water of crystallization, which is lost when it is heated to 130°C. At temperatures as high as 175°C
it is converted to itaconic acid, aconitic acid, and other compounds.

Biochemical basis of the production of citric acid

Citric acid is an intermediate in the citric acid cycle. The acid can therefore be caused to
accumulate by one of the following methods:
(a). By mutation – giving rise to mutant organisms which may only use part of a metabolic
pathway, or regulatory mutants; that is using a mutant lacking an enzyme of the cycle.
(b) By inhibiting the free-flow of the cycle through altering the environmental conditions, e.g.
temperature, pH, medium composition (especially the elimination of ions and cofactors

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 considered essential for particular enzymes). The following are some of such
environmental conditions which are applied to increase citric acid production:
(i) The concentrations of iron, manganese, magnesium, zinc, and phosphate must be limited.
(ii) The dehydrogenases, especially isocitrate dehydrogenase, are inhibited by anaerobiosis,
hence limited aeration is done on the fermentation so as to increase the yield of citric acid.
(iii) Low pH and especially the presence of citric acid itself inhibits the TCA and hence
encourages the production of more citric acid.
Citric acid can be caused to accumulate by using a mutant lacking an enzyme of the cycle
or by inhibiting the flow of the cycle.
Fermentation for citric acid production
In recent times yeasts, especially Candida spp. (including Candida quillermondi) have been used
to produce the acid from sugar.
(a) Surface fermentation: Surface fermentation using Aspergillus niger may be done on rice
bran as is the case in Japan, or in liquid solution in flat aluminium or stainless steel pans.
Special strains of A. niger which can produce citric acid despite the high content of trace
metals in rice bran are used. The citric acid is extracted from the bran by leaching and is
then precipitated from the resulting solution as calcium citrate.

(b) Submerged fermentation: As in all other processes where citric acid is made the
fermentation the fermentor is made of acid-resistant materials such as stainless steel. The
carbohydrate sources are molasses decationized by ion exchange, sucrose or glucose. The pH
is never allowed higher than 3.5. Copper is used at up to 500 ppm as an antagonist of the
enzyme aconitase which requires iron. 1-5% of methanol, isopranol or ethanol when added to
fermentations containing unpurified materials increases the yield; the yields are reduced in
media with purified materials. As high aeration is deleterious to citric acid production,
mechanical agitation is not necessary and air may be bubbled through. Antifoam is added. The
fungus occurs as a uniform dispersal of pellets in the medium. The fermentation lasts for five
to fourteen days.

Extraction
The broth is filtered until clear. Calcium citrate is precipitated by the addition of magnesium-free
Ca(OH)2. Since magnesium is more soluble than calcium, some acid may be lost in the solution as

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magnesium citrate if magnesium is added. Calcium citrate is filtered and the filter cake is treated
with sulfuric acid to precipitate the calcium. The dilute solution containing citric acid is purified
by treatment with activated carbon and passing through iron exchange beds. The purified dilute
acid is evaporated to yield crystals of citric acid.
b. Lactic Acid
Lactic acid is produced by many organisms: animals including man produce the acid in muscle
during work.
Fermentation for lactic acid
The organisms which produce adequate amounts and used in industry are the homofermentative
lactic acid bacteria, Lactobacillus spp., especially L. delbruckii. In recent times Rhizopus
oryzae has been used. Both organisms produce the L- form of the acid, but Rhizopus fermentation
has the advantage of being much shorter in duration; further, the isolation of the acid is much easier
when the fungus is used.
Lactic acid is very corrosive and the fermentor, is made of wood. Alternatively special stainless
steel (type 316) may be used. They are sterilized by steaming before the introduction of the broth
as contamination with thermophilic clostridia yielding butanol and butyric acid is common. Such
contamination drastically reduces the value of the product. During the step-wise preparation of the
inoculum, which forms about 5% of the total beer, calcium carbonate is added to the medium to
maintain the pH at around 5.5-6.5. The carbon source used in the broth has varied widely and has
included whey, sugars in potato and corn hydrolysates, sulfite liquour, and molasses. However,
because of the problems of recovery for high quality lactic acid, purified sugar and a minimum of
other nutrients are used.