2.

0 PRODUCTION OF PRIMARY METABOLITES

2.1 PRODUCTION OF ORGANIC ACIDS

2.1.1 PRODUCTION OF CITRIC ACID

Citric acid is a 6-carbon containing tricarboxylic acid which was first

isolated from lemon juice. It is a natural component of many citrus fruits, and was
crystallized from lemon juice by Scheele in 1784. Approximately 70% of citric
acid produced is used in the food and beverage industry for various purposes, 12%
in pharmaceuticals and about 18% for other industrial uses. The estimated world
production of citric acid was reported as 350000 tons/year in 1986. It however was
recently reported that the world market requirement of citric acid is around 500000
tons/year.
The production of CA by fermentation include the nutritional composition
of the media, environmental conditions, deficiency of manganese and other metals,
pH, and dissolved oxygen tension. A. niger have also been studied.
At present time CA is produced commercially using mutant strains of A.
niger, and with a significant amount by Saccharomycopsis lipolytica , Pencillium
simplicissimum and A. foeitidus.

Other carbohydrates and wastes that have been considered, experimentally,

to produce CA by A. Niger includes inulin, date fruit syrup, sugar cane molasses,
soya whey and cheese whey.

Large amounts of whey are produced world wide as a by-product of cheese

and other dairy products manufacturing.

Whey in the Middle Eastern region is generally considered as a waste and

disposed in the sewage system leaving a small amount for drinking for domestic
animals.
 There are various fermentation methods used in the manufacture of citric
acid. These processes have been briefly dealt with in this chapter.

(a) Surface – culture process:

In this process, culture is inoculated across the surface of the
production medium. In addition, culture remains on the surface throughout the
fermentation, since this is a stationary fermentation. This is an old process and it is
still practiced. The details of the process are not easily available, since they are
treated as trade secrets.

Advantages of A.Niger:

There are so many organisms are available for the production of

citric acid. But only A.niger strains among these fungal strains are used for
following reasons,
1. They are efficient strains
2. They posses fairly uniform biochemical properties.
3. They produce a neglible or small amount of oxalic acid, provided
fermentation condition have been adjusted to favour the production of citric acid.
4. They can be easily cultivated.

Preparation of Inoculum:
The spores of A.niger strain required to inoculate shallow pants are
produced by growing the fungus from a stock culture on a suitable solid
sporulation medium at 250 C for 4-14 days.
In the composition formula of inoculum medium, the presence of trace
amounts of manganese salts, unless balanced against proper amounts of Zn or Fe
salts, may lower yields of citric acid in the actual fermentation receiving these
spores.
Suspension of spores is obtained by suspending the grown spores in a
suitable diluent, such as water containing wetting agent.
 The spores of inoculum are added to the production medium so as to keep
them floating on the surface, since this is a surface-culture process. This may
accomplished by a suitable special inoculating device.

Carbon Sources:
Sucrose, according to laboratory studies, is the best source of carbon among
various tested organic substances, particularly sugars, in producing high yields of
citric acid. It was also reported that sucrose concentration exceeding 15% should
not be used, since the excess amount of sugar (less than 3%) remained
unconverted to citric acid. When a part of sucrose was substituted by fructose or
glucose, it resulted in lower yields of citric acid than when controls containing
sucrose alone were used. Therefore, precaution is to be taken while using sucrose
to avoid partial hydrolysis. Beet molasses is extensively used as a carbon substrate
in the fungal production of citric acid on a commercial basis. Beet molasses
requires pretreatment, since it contains excessive amounts of trace metals.
Therefore, ferrocyanide or ferricyanide may be added to the production medium
before sterilization. The metals of the beet molasses form a complex with the
added chemical agent, thus eliminating themselves as the precipitate.
Alternatively, the clarified molasses may be passed through a cation-
exchange resin. Under laboratory conditions, the highest yields of citric acid are
secured, provided sucrose has been passed through an ion-exchange resin.

Inorganic sources:
Apart from the carbon, hydrogen and oxygen supplied by the added
carbohydrate, the trace metals, namely nitrogen, potassium, phosphorus, sulphur
and magnesium are needed in the fermentation medium used for citric acid
production.
According to Currie (1917), the fermentation medium with the following
chemical composititon is the most favourable medium in the manufacture of citric
acid:
 COMPONENT gms/litre
Sucrose 125-150
NH4NO3 2.0-2.5
KH2PO4 0.75-1.0
MgSO4.7H2O 0.20-0.25
HCl to pH 3.4 to 3.5(5-4 ml of N/5HCl)

Dolger and Prescott (1934) reported the following medium to be most

statisfactory:
COMPONENT gms/litre
Sucrose 140
NH4NO3 2.23
KH2PO4 1.0
MgSO4.7H2O 0.23

Note:
Salts and sugars are dissolved and made up to 1 liter with distilled water.
The pH of the medium is adjusted in the range of 2.20 to 1.60 with N/Hcl. The
sterilization of the medium is done at 8to 10 lbs steam pressure per square inch for
30 minutes. It is highly essential to add NH 4NO3, KH2PO4 or K2HPO4, and
MgSO4.7H2O only in a minimum quantity, since the presence of these metallic
salts beyond their limits affects the fermentation adversely.
For example, more than 2.50 gms of NH 4NO3, 1.50 gms of
potassium monohydrogen phosphate, and 0.30 gms of MgSO 4.7H2O favour the
increased yield of oxalic acid and the citric acid yield decreases. It is highly
desirable that mycelial mats should be thin and the sporulation light or nearly
absent because high yields of citric acid may be secured.
pH:
According to curie, the pHof the medium should be adjusted to 3.4 to 3.5
with Hcl. also, low pH values (1.6 to2.20) have been found to be the most
favourable by Doolger and Prescott. These scholars also advocated the use of HCl,
which has been found superior to nitric, sulphuric, and acetic acids in producing
higher yields of citric acid.
Generally, calcium carbonate is not added to the production medium to
bring about the neutralization of citric acid produced because its presence favours
contamination. Moreover, its absence favours higher yields of citric acid and a
shorter fermentation time.
Low pH value in A. niger-citric acid fermentation is desirable for the
following reasons:
Sterilization of medium is more readily effected.
Formation of citric acid is favoured.
Formation of oxalic acid is suppressed.
The danger of contamination is minimized.
Lastly, stationary pans or trays must be protected from corrosion or they
should be made of stainless steel or aluminium because production media with low
pH values are used, and because of the sensitivity of the fermentation process to
excessive iron.
Temperature: the exact required temperature of incubation depends in
part on the fungal strain and the fermentation conditions. According to Doolger
and Prescott, a temperature range 0f 26 - 280C is considered to be satisfactory.

Aeration: it is necessary to supply air to the surface of the seeded medium.

From laboratory studies it has been found that, the rate of aeration, either higher or
lower than the optimum rate gives lower yields of citric acid. Therefore, it is
essential to determine the rate of the air supply required for each new apparatus
installed.
Time: with the shallow pan method (i.e. surface-culture process) the required
for the progress and termination of the fermentation process is in the range of 7to
10 days.
 Yields: It has been reported that yields of citric acid may be in the range 60
to 80 gms. (or higher) of anhydrous citric acid per 100 gms of incorporated sugar.
Wells, Moyer and May obtained a maximum yield of 90.7% of citric acid from
glucose on the sugar consumed.

Recovery of citric acid: the recovery operation for citric acid from the
harvested fermentation broth is difficult for following important reasons:
 The presence of unconverted sugars
 The presence of other acid fermentation
products(e.g.oxalic acid)
 The presence of trace salts as impurities.

Usually , recovery of citric acid is practiced as under:

 The fermentation liquor is drained off to separate the
mycelium. In addition, any intracellular citric acid
present in the mycelium is obtained by pressing the
mycellial mat.
 The recovered fermentation liquor, as in step 1 , is
treated with milk of lime forming the precipitate of
calcium citrate .
 The precipitate of calcium citrate obtained from hot
neutral aqueous solution, as in step 2 ,is filtered and
washed.
 Calcium citrate is treated with an equivalent of
sulphuric acid to liberate citric acid, leaving behind the
precipitate of calcium sulphate.
 The precipitated solution in step 4, is filtered and
washed.
  An impure solution of citric acid is subjected to
decolourisation by the activated carbon. Then, it is also
demineralised.
 Finally, the pure citric acid solution is evaporated and
is crysallised from the solution.
The alternative method is the counter current extraction method.
Organic solvents (100 parts of tri-n-butyl phosphate and 5 to 30 parts of n-butyl
acetate are employed for the purpose of extracting citric acid. Then citric acid is
extracted from the organic phase with lime water.

SUBMERGED-CULTURE PROCEES USING Aspergillus niger

In this process the fungus is grown dispersed through a liquid production
medium. Usually, fermentation is carried out in the fermentation vessel consisting
of a sterilizable tank of the capacity of thousands of gallons. The fermentation tank
is equipped with a mechanical agitator and a sparger.
The origins of the submerged-culture method using a small flask to
stimulate deep culture conditions date back to the work of Amelung. Perquin
established that phosphate limitation plays a significant role in the production of
citric acid by the submerged-culture process using A. niger. Martin and Waters
established the relationship of the effect of A. niger morphology with citric acid
production from the beet molasses. Subsequently, Clark found that, specific
quantities of ferrocyanide lead to form of an optimum mycelial form.
In practice the mould spores are produced under controlled aseptic
conditions. When harvested, they are used in specific quantities to seed the
inoculum tank containing a medium designed to develop cellular mass and to
control morphology rather than to produce citric acid. Then it is transferred to the
production tank under aseptic conditions. In contrast to the composition of the
inoculum medium, production medium contains constituents so as to favour citric
acid production rather than growth. High aeration rates (i.e. 0.5 to 1.5 volumes per
minute) producing much foam are necessary. Samples are regularly withdrawn to
determine the citric acid and sugar content revealing the progress of the
fermentation process. In addition to this, pH, dissolved oxygen and solids content
are determined. The initial sugar concentrations and the citric acid yield is
determined , compared favourably with those of the surface-culture process. This
method also utilizes pretreated molasses medium like the surface-culture process.
The selection of raw materials for this submerged-culture process suffers from
the same constraints as the surface-culture process, except that it allows a wider
choice of raw materials.
A. niger in the submerged-culture process employing a production medium
consists of pure sucrose or a solution of cation-depleted molasses. Subsequent
patents cover production by A. niger on a substrate produced from starch treated
with amylase and amyloglucosidase. Very recently, glucose solutions derived
directly from corn have been employed. It has been reported that yields of citric
acid from sugar substrates may exceed 90%.
SUBMERGED-CULTURE PROCESS USING A YEAST:

The belief that industrial production of citric acid could be possible only by
A. niger , disproved in about 1969 or 1970. a challenging patent was issued in
1970 by demonstrating the production of citric acid by the species of yeast(e.g.
Candida guilliermondii) grown submerged in a medium containing either glucose
or black-strap molasses with an equivalent amount of sugar. With this
fermentation period was shorter than that of the submerged-culture process
involving A. niger. A subsequent improvement patent quotes citric acid
concentrations of 110 gms/ltr.
Using certain strains of Candida lipolytica, the possibility of using
hydrocarbons as raw materials in citric acid fermentation has been shown. A
patent was issued in 1970 for the bioconversion of c9 toc20 normal paraffins to
CA by C. lipolytica. Thus CA weight yields exceeding 100 % were claimed. In
1972, other patentee were issued describing a method to select high-yielding
mutant strains of C. lipolytica. By using a chemical agent, monofluroacetate, in
media, Japanese workers have succeeded in isolating mutants with only one 100th
of the cis-aconitase activity exhibited by the parent strain . these strains are able to
accumulate 112 gms. Of CA per liter after 3 days incubation at 30 C. on the basis
of the substrate consumed, the yield is 145%. In 1974, Pfizer patented continuous
process for C. lipolytica. CA fermentation. The process utilizes single vessel.
Continuous feeding of paraffin and continuous withdrawal of fermented broth can
be practiced.

2.1.2 PRODUCTION OF LACTIC ACID

Lactic acid-LA is commercially produced by the synthetic method as well

as by the fermentation process. It is manufactured in Japan and US by the
synthetic method. On the other hand, European manufacturers produce LA by
fermentation process. Half of the estimated world capacity for LA production
(28,000-30,000 metric tons per annum) is by European manufacturers. LA is
largely used in pharmaceutical industries (e.g. iron lactate) and is some plastic
industries.
Applications of lactic acid in different Application
industries Industry
Food Preservative, acidulant, buffering
agent, pickling agent and dough
conditioner
Meat Prolongs the shelf life of poultry and
fish during packaging
Textile Finishing, antimony lactate as a
mordant during dyeing
Metal Electroplating bath, plasticizer and
corrosion inhibitor.
Leather Acidulant
Pharmaceutical Ointments, lotions, anti acne
solutions, humectants, and parenteral
solutions, calcium lactate (anti caries
 agent) and biodegradable polymers
for sutures in medicine
Medical Orthopedic implants, controlled drug
release
Chemical Ethyl/butyl lactate are used as green
solvents

THE MICRO-ORGANISM
There are numerous microbial species capable of producing large
quantities of Lactic Acid.
These includes
 Lactobacillus bulgaricus
 L. delbrueckii
 L. leichmanni
 L. casei
 L. pentoses
 Streptococcus lactis
 Rhizopus oryzae
The selection of micro-organism largely depends on the carbon sources being used
in the fermentation process. For example, L. bulgaricus is used as a source of
carbohydrate.

CARBON SOURCES:
Organism Carbon source
Lactobacillus delbrueckii Glucose
Lactobacillus leichmannnii Glucose
Lactobacillus bulgaricus Lactose
Lactobacillus helviticus Lactose and galactose
Lactobacillus amylophyllus Starch
Lactobacillus amylovirus Starch
Lactobacillus lactis Glucose, sucrose and
galactose
Lactobacillus pentosus pentoses of sulfite waste
 liquor
Pre-treatment of starchy materials by enzymes or by acids is
necessary to bring hydrolysis e.g. sulfuric acid. Jerusalem artichokes is a potential
raw material for LA production.

NITROGEN SOURCES:
Ammonium salts have been used giving satisfactory results .
.e.g. ammonium hydrogen phosphate.

GROWTH FACTORS & MINERAL RESOURCES:

Lactobacilli require complex nutrients particularly vitamin B-
complexes. These may be supplied by enriching the medium with crude vegetable
sources( e.g. malt sprouts). Minerals, based on the requirement of a selected
organism are added to the production medium. Calcium carbonate in higher
concentration (e.g.10%) is used as a buffer.
[

pH
pH of the broth should be in the range of 5.5-6.5 is considered satisfactory.
Neutralization of the accumulated acid is carried out by the continuous addition of
slurry of calcium hydroxide. If it is not so, it results in high acidity. L. bacilli
cannot tolerate high acidity. Hence fermentation is not completed. Low pH values
are advantageous, since most contamination problems are eliminated & it allows
sterilization at low temperatures.

Temperature
It depends on the microorganism involved.For example, Lactobacillus
bulgaricus & L. delbrueckii needs a temperature range of 45 to 50 C . L. casei,
L. pentoses & Streptococcus lactis needs a temperature of 30 C.

Aeration and agitation

The supply of sterile air is not necessary in the fermentation process
using lactobacilli, since this is an aerobic fermentation. Moreover, complete
removal of air is not necessary because these bacteria are facultative aerobes. The
broth is usually gently stirred to keep CaCO3 in suspension.

TIME
The duration of plant fermentation is commonly 5-10 days. It is possible to
complete fermentation in 72 hrs, using 12 to 13% glucose in the medium, provided
the pH is controlled at 6.3-6.5 by the continuous neutralization of the acid
produced.
YIELD
Commercial fermentation yields are in the range of 93-95% of the weight of
the glucose supplied.

RECOVERY
A number of methods can be applied for the separation of lactate salt from
fermented medium which involve extraction by solvents or separation by ion-
exchange, adsorption, vacuum distillation and membrane filtration. The medium
after completion of fermentation consists of either pure lactic acid or its salt or the
mixture of the two. Earlier method used addition of excess of calcium carbonate to
the medium at the end of the fermentation and pH adjusted to 10, heated and
filtered.
In this procedure all the lactic acid is converted in to calcium lactate, the
bacteria are killed and protein in the medium gets coagulated. A number of other
methods have been developed to recover and purify lactic acid. In one such
method, the filtrate is concentrated to crystallize calcium lactate following which
sulphuric acid is added to precipitate calcium as calcium sulphate and removed by
filtration.

Lactic acid is again recrystallized as calcium lactate and passed through

activated carbon to remove coloured impurities. Lactic acid is also extracted with
isopropyl ether directly from the heated and filtered fermentation broth, by
counter-current continuous extraction method, and lactic acid is further recovered
from the solvent by counter-current washing with water.
A preferred process for the lactic acid recovery from the mixture
containing free lactic acid and the dissolved lactate salt involves first lowering
down of the pH of fermented broth to 3.0-4.2 and then hydrophilic membrane and
the volatile amine weak base (VAWB) are used to separate lactic acid from the
fermented broth. The lactic acid is finally regenerated from salts of weak amine
base by selectively vaporizing the volatile amine base.

2.1.3 PRODUCTION OF ACETIC ACID:

The term “vinegar” has originated from the French word vinaigre (sour
wine). Formation of vinegar involves souring of wine under specified conditions.
Thus there are 2 stages in the formation of vinegar using sugar-containing media:
=>Alcoholic fermentation
=>Acetic acid fermentation
Alcoholic fermentation:
During this stage sugar- containing materials (e.g. fruit juices, honey or
hydrolysed starchy materials) are fermented to ethyl alcohol. This bioconversion is
brought about by the agency of yeast (i.e. a selected strain of saccharomyces
cerevisiae). According to Gay-Lussac eqn:
C6H12O6 ------- 2CO2 + 2C2H5OH
The product made by the alcoholic and subsequent acetous fermentations of the
juice of apples. It contains, in 100 cc (20 C), not less than 4 gms of acetic acid.
Yeast fermentation is used for production of the alcohol. The alcohol
concentration is adjusted to between 10 to 13 % and then exposed to the action of
acetic acid bacteria.
Many types of equipment have been designed for industrial production of
vinegar. All depend upon providing a suitable environment for the bacterial
oxidation of alcohol to acetic acid. The essential features of one of the industrial
processes for vinegar production, the Fringes method. A mix is prepared which
consists of an adjusted solution of alcohol acidified with acetic acid bacteria.
Acetic acid bacteria, species of the genus Acetobacter, are inoculated onto the
beechhood shavings. As the alcohol solution passes over the shavings, the
acetobacters oxidize some of the alcohol to acetic acid. This is applied in a trough
at the top of the chambers and allowed to trickle down over the shavings. As the
alcohol passes over the shavings, the acetobacter oxidize some of the alcohol to
acetic acid.
2CH3CH2OH + 2CO2 → 2CH3COOH + 2H2O
The mix is collected at the bottom of the unit and may be
recirculated over the shavings, resulting in oxidation of alcohol until vinegar of the
desired strength is produced. An abundant supply of air must be available
throughout the chamber. It is also necessary to keep the temperature between 15 0
and 340 C.
The Frings vinegar generator is equipped with various accessories which
permit permit control of these factors. Deviation in temperature below or above
this range not only has an adverse effect on the acetobacters but permits growth of
the other microorganisms with different metabolic characteristics.

2.2.0 AMINO ACID PRODUCTION

Amino acids are extensively used in food industry, as feed additives, in
medicine and cosmetics and as starting materials for the synthesis of myriads of
compounds in chemical industry. In the last 20 years, the demand for amino acid
has been growing with a rate of 5-7 % annually. The amino acids - L-lysine, L-
methionine, L-threonine, and L-tryptophan used as feed additives have the largest
share (56%) of the total amino acid market (US $4.5 billion) estimated in 2004.
The three amino acid – L-glutamic acid (monosodium glutamate, MSG) as flavor
enhancer, and L-aspartic acid and L-phenylalanine for synthesizing peptide
sweetener L-aspartyl - L-phenylalanyl methyl ester (Aspartame) are widely used
in food industries. Total worldwide consumption of amino acids is estimated to be
more than 2 million tons per year. Monosodium glutamate, L-lysine
hydrochloride, L-methionine and L-threonine are the major ones.
The production of amino acids may involve any of these
processes:
 Microbial fermentation,
 Extraction from animal or plant protein hydrolysates,
 Chemical synthesis, and
 Enzymatic transformation.
Fermentation processes mainly employ stains of
Corynebacterium glutamicum and Escherichia coli to produce L-glutamic acid
(monosodium glutamate, MSG), L-aspartic acid, L-phenylalanine, L-lysine, L-
methionine, L-threonine, and L-tryptophan, from sugar sources such as molasses,
sucrose, or glucose. These amino acids serve as building blocks for active
ingredients that are applied as pharmaceuticals, cosmetics, and agricultural
products (Leuchtenberger et al., 2005).
In the future the demand of amino acids in food, feed,
pharmaceuticals, cosmetics, agriculture, will lead to further exploration of the
potential of microorganisms, plants and enzymes to develop more efficient
processes for amino acid production.

2.2.1 Industrial production of L-glutamic acid

Glutamic acid bacteria convert 50-60% of the added carbon source to L-

glutamic acid under the optimal culture conditions. Stirred tank reactor up to 450
m3 is used for industrial production of glutamic acid.
The fermentation is carried out aerobically at 30-37°C, depending on the
microorganisms used. Glucose, fructose, sugar cane and sugar beet molasses, and
starch hydrolysates are some carbon sources used in production of L-glutamic
acid. Penicillin or fatty acid derivatives (e.g. Tween 60) are added in the sugar
cane or sugar beet molasses based medium upsetting the cell wall synthesis of
these bacteria as these carbon sources contain high biotin (0.02-0.12 mg/Kg)
content favoring the formation of cell membrane with high lipid content. Acetate,
methanol, ethanol, acetaldehyde, or n-alkane have also been employed as carbon
source in the production of of L-glutamic acid by bacteria, but still cane sugar
molasses or starch hydrolysates are the main carbon sources. Ammonium salts or
ammonia are generally used as nitrogen source. In case of glutamic acid bacteria
having high urease activity, urea can also be used as nitrogen source in the
medium.
The optimal biotin concentration in the production medium
depends on the carbon source used, i.e., 5μg/L biotin for media with glucose and
0.2 – 1.0 μg/L in case of media containing acetate. L-Cysteine as an additional
growth factor is required by some strains and media based on n-alkane require
thiamine supplementation. Oxygen supply is necessary for glutamic acid
production and under oxygen deficiency, excretion of lactate and succinate occurs,
whereas excess oxygen results in ammonium ion deficiency, ceasing the growth
and production of α-ketoglutarate, thus lowering the L-glutamic acid yield in both
cases. Medium pH during fermentation is maintained at 7-8 by the addition of
alkali/ammonia. L-glutamic acid starts accumulating from the mid way of the
fermentation process which normally lasts for 30-35 h and finally L-glutamic acid
level reaches to 100g/L in the fermentation broth in case of Brevibacterium
divaricatum (NRRL-B-231) (Miescher, 1975). In acidic pH with excess ammonia,
glutamine is produced instead of L-glutamic acid. L-Glutamic acid is recovered
from the fermentation broth by separating the cells from the culture medium and
its crystallization is done by lowering the pH to 3.2 (isoelectric point) of the cell
free broth using HCl. Crystals are then filtered, washed and monosodium
glutamate (MSG) is prepared by adding sodium hydroxide to the crystalline L-
glutamic acid followed by recrystallization.
2.2.2 PRODUCTION OF PHENYL ALANINE
L-Phenylalanine production has been much improved using a
regulatory mutant after the review of oishi (1972). A prototrophic mutant resistant
to p-fluorophenylalanine produced 5.5gm of L-phenylalanine per liter and trace
amounts of L-tyrosine in a medium containing 10% reducing sugars as invert (as
cane molasses). A tyrosine auxotrophic mutant, resistant to PFP and PAP,
produced 9.5gm of L-Phenylalanine per liter in the molasses containing medium.

The medium used had the following composition:

Reducing sugars - 10%
(NH4)2SO4 - 2%
KH2PO4 - 0.05%
K2HPO4 - 0.05%
MgSO4.7H2O - 0.025%
NZ-amine - 0.25%
CaCO3 - 2%
pH - 7.2

L-Phenylalanine production in these mutants was inhibited by L-

Tyrosine and stimulated by L-Tryptophan.

A mutant of B.subtilis resistant to 5-FT produced 6.0gm of L-

Phenylalanine per liter in addition to 4.0gm of L-Tryptophan per liter. Maximum
L-Phenylalanine production by a mutant of Brevibacterium lactofermentum was
attained at an oxygen deficiency degree between 0.45 and 0.65. This oxygen
deficiency degree was attained at the redox potential of the medium between -
200mv and -275mv. A tyrosine auxotroph of a Corneybacterium species, an n-
parafin utilizing glutamic acid producer, has also been reported to produce L-
Phenylalanine.
2.2.3 PRODUCTION OF ASPARTIC ACID
Aspartase activity was exploited for the production of L-
Aspartic acid from Ammonium fumerate. Escherichia coli, E.freundii and
Pseudomonas fluorescence are good sources of aspartase. E.coli is a facultative
aerobe, so that cell yield is strongly affected by culture conditions such as the
degree of aeration. To avoid glucose repression of aspartase formation, the glucose
concentration in the medium should be low, though a small amount of glucose
sometimes gives good results causing improved growth of the bacterium.

PROCEDURE
A large concentration of ammonium fumerate, corresponding to
50gm of fumaric acid was suspended in 100ml water and subjected to the action of
aspartase. Crystals of ammonium fumerate gradually disappeared and were
converted to a solution of acid ammonium aspartate. Hydrochloric acid was added
to bring the pH value of the solution to 2.8, the isoelectric point of aspartic acid.
The solubility of aspartic acid being only 0.6% at this point, the greater part
immediately crystallizes out.

CHCOONH4 CHCOONH4 CHNH2COOH CHNH2COOH

ll ↔ ll ↔ ll → l + NH4Cl
CHCOONH4 CHCOONH4 CH2COONH4 HCl (pH 2.8) CH2COOH
Solid phase liquid phase solubility 60%
Solubility 20%

Later Alcaligenes and Pseudomonas ovalis were selected as

bacteria which produce L-aspartate from ammonium maleate. The activity of
A.faecalis was high when it was grown in an acidic medium due to the permeation
of maleate, an inducer of maleate cis-trans-isomerase. Malonic acid was found to
be a gratuitous inducer.
Continuous production of L-aspartic acid from ammonium
fumerate was attained by an employing an enzyme column packed with the
immobilized aspartase, a preparation of partially purified aspartase from E.coli
entrapped in a polyacrylamide gel lattice. Furthermore, E.coli was used in place of
the enzyme. When a solution of 1M ammonium fumerate (pH 8.5) containing
1mM Mg2+ was passed through the immobilized cell column at a flow rate of
space velocity 0.8 at 370C, the highest rate of reaction was attained. L-aspartic acid
was obtained in good yield from the column effluent. The half life of the column
was 120 days.

2.3.0 PRODUCTION OF ALCOHOL

2.3.1 BUTANOL PRODUCTION

Butanol fermentation from renewable carbohydrates used to be the largest

biotechnological process second only to yeast ethanol fermentation and the largest
process ever run under sterile conditions. With the rising prices for mineral oil, it
has now the economical and technological potential to replace petrochemistry for
the production of renewable resources.

Two -Step Process for Butanol Production

Two-Step
Butyric acid Butanol
Butanol

Immobilized Separation
Cell Unit
Reactor

Solvent
Starch / Glucose

Separating acidogenesis and solventogenesis into two reactors to optimize the

process and increase butanol yield - U.S. Patent 5,753,474
E
2
i

To our knowledge, the AB plants in Russia were the only fullscale

industrial plants which used hydrolyzates of lignocellosic waste for butanol
fermentation.
 These plants were further developed into the 1980s, and the process was
finally run in a continual mode different from plants in Western countries. A
biorefinery concept for the use of all by-products has been elaborated and was
partially put into practice. The experience gained in the Soviet Union forms a
promising basis for the development of modern largescale processes to replace a
considerable fraction of the current chemical production of fuel for our future
needs on a sustainable basis.
Butanol production by Clostridium beijerinckii NCIMB 8052 was
investigated using both batch and continuous cultures containing suspended or
immobilized cells.
In the batch reactor, the initial addition of acetate and butyrate into the
culture media was found not only to enhance solvent production but also to affect
the ratio of acetone/butanol, which might result from the metabolic changes in
solvent production.
Furthermore, the addition of butyrate to the medium prevented strain
degeneration during an extended subculturing, significantly induced butanol
production (11.2 g/L butanol versus 0.45 g/L butanol with and without 36 mM
butyrate, respectively), and shifted the acetone/butanol ratio to 1:3, which resulted
in a higher yield (0.45 g of butanol/g of glucose) when compared to other studies.
The beneficial effects of butyrate were also observed in continuous reactor
tests containing suspended cells as the solvent production was maintained over
more than 300 h of continuous operation.
During a continuous butanol production with immobilized cells, using
porous hydrophilic media and a dilution rate of 0.04 h−1, the overall butanol
productivity and yield were 0.40 g L−1 h−1 and 0.44 g of butanol/g of glucose,
respectively, which are approximately twice the values seen in a continuous
reactor with suspended cells.
The butanol production was maintained over 150 days without apparent
degeneration, even in the presence of high butanol concentrations (10−13 g/L).
 These results validate the effectiveness of producing butanol with an
immobilized cell system supplemented with butyrate.

2.3.2 ETHANOL PRODUCTION

94% denatured ethanol sold in a bottle for household useEthanol is
produced both as a petrochemical, through the hydration of ethylene, and
biologically, by fermenting sugars with yeast. Which process is more economical
is dependent upon the prevailing prices of petroleum and of grain feed stocks.
Ethylene hydration
Ethanol for use as an industrial feedstock or solvent (sometimes referred to
as synthetic ethanol) is often made from petrochemical feed stocks, primarily by
the acid-catalyzed hydration of ethylene, represented by the chemical equation

C2H4 (g) + H2O (g) → CH3CH2OH (l)

The catalyst is most commonly phosphoric acid, adsorbed onto a porous
support such as diatomaceous earth or charcoal. This catalyst was first used for
large-scale ethanol production by the Shell Oil Company in 1947. The reaction is
carried out with an excess of high pressure steam at 300°C. In the U.S., this
process was used on an industrial scale by Union Carbide Corporation and others;
but now only LyondellBasell uses it commercially.
In an older process, first practiced on the industrial scale in 1930 by
Union Carbide,but now almost entirely obsolete, ethylene was hydrated indirectly
by reacting it with concentrated sulfuric acid to produce ethyl sulfate, which was
then undergo hydrolysis to yield ethanol and regenerate the sulfuric acid.

C2H4 + H2SO4 → CH3CH2SO4H

CH3CH2SO4H + H2O → CH3CH2OH + H2SO4
Ethanol fermentation
Ethanol for use in alcoholic beverages, and the vast majority of ethanol for
use as fuel, is produced by fermentation. When certain species of yeast (e.g.,
Saccharomyces cerevisiae) metabolize sugar they produce ethanol and carbon
dioxide. The chemical equation below summarizes the conversion:

C6H12O6 → 2 CH3CH2OH + 2 CO2

The process of culturing yeast under conditions to produce alcohol is called

fermentation. Ethanol's toxicity to yeast limits the ethanol concentration obtainable
by brewing. The most ethanol-tolerant strains of yeast can survive up to
approximately 15% ethanol by volume.

In order to produce ethanol from starchy materials such as cereal grains, the
starch must first be converted into sugars. In brewing beer, this has traditionally
been accomplished by allowing the grain to germinate, or malt, which produces
the enzyme, amylase. When the malted grain is mashed, the amylase converts the
remaining starches into sugars. For fuel ethanol, the hydrolysis of starch into
glucose can be accomplished more rapidly by treatment with dilute sulfuric acid,
fungally produced amylase, or some combination of the two.