Unit

Microbial technology products and

applications
3.1 Microbial production of
Organic acids (Lactic acid and Citric
acid)
3.2 Microbial production of Amino acids (Glutamic acid and Aspartic
acid)
3.3 Fermentation by microbes for food additives: dairy
products (Bread
and SCP), beverages (Beer and Wine) and antibiotics (Penicillin
and Streptomycin)

3.4 Therapeutic drugs: Monoclonal antibodies and insulin

3.5 Biofuel: Alcohol and Methane

3.6 Biomining (Extract of Copper, Aluminum, Uranium, and

Bioremediation)

3.1 Microbial production of Organic acids

Organic acids are chemical compounds widely distributed in nature as

normal constituents of plants or animal tissues. Organic acids represent a
TIsing chemical segment in which several bio based compounds such as funmaric,
 116
B.Sc. Microbial Technology- III(Sem-VI)
propionic, and itaconic acids are synthesized. Organic acids constitute a
Key group among the building block chemicals that can be produced by
microbial processes. Organic acids have been used for many years in the
food, chemical, agriculture, and phamaceutical industries. The chemical industries
use organic acids as basic compounds for a wide variety of polymer and
solvent production processes. Organic acids differ on the basis of the
nvolvement of carbon, hydrogen, and oxygen elements. Major types of organic
acid produced by microbial activity are citric acid, succinic acid, lactic acid,
1taconic acid, lactobionic acid, gluconic acid, fumaric acid, propionic acid,
and acetic acid.

3.1.1 Citric Acid

Citric acid was first discovered as a constituent of lemon. Today, we

know citric acid as an intermediate of ubiquitous Krebs cycle (citric acid
cycle), and therefore, it is present in every living organism. In the early days,
citric acid was isolated from lemons (that contain 7-9% citric acid), and
today about 99% of the world's citric acid comes from microbial fermentation.

Applications of Citric Acid

1. Citric acid, due to its pleasant taste and palatability, is used as a flavoring
agent in foods and beverages e.g., jams, jellies, candies, desserts, frozen
fruits, soft drinks, wine. Besides brightening the colour, citric acid acts
as an antioxidant and preserves the flavors of foods.

2. It is used in the chemical industry as an antifoam agent, and for the

treatment of textiles. In metal industry, pure metals are complexed
with citrate and produced as metal citrates.

3. In pharmaceutical industry, as trisodium citrate, it is used as a blood

preservative. Citric acid is also used for preservation of ointments and
cosmetic preparations. As iron citrate, it serve as a good source of
iron.

4. Citric acid can be utilized as an agent for stabilization of fats, oils or

ascorbic acid. It forms a complex with metal ions (iron, copper) and
 Unit- I11 Bloenergetien of Blomolecules 117

prevents metal catalysed reactions. Citric acid is also used as a stabilizer

of emulsions in the preparation of cheese.
5. In detergent/cleaning industry, citric acid has slowly replaced
polyphosphates.
Microbial Strains for Citric Acid Production

Many microorganisms can produce citric acid. The fungusAspergilius

niger is most commonly used for industrial production of citric acid. The
other organisms (although less important) include A. clavatus, A. wentii,

Penicillium luteum, Candida catenula, C. guilliermondii and

Corynebacterium sp.
For improved industrial production of citric acid, mutant strains oA.

strains that can tolerate high sugar

niger have been developed. The
of undesirable byproducts
concentration and low pH with reduced synthesis
are industrially important.
(oxalic acid, isocitric acid and gluconic acid)
Acid
Microbial Biosynthesis of Citric
metabolism)
Citric acid isprimary metabolic product (of primary
a
the predominant
acid (Krebs) cycle. Glucose is
formed in the tricarboxylic for citric
The biosynthetic pathway
carbon source for citric acid production.
to two
wherein glucose is converted
acid production involves glycolysis
and oxaloacetate
Pyruvate in turn forms acetyl CoA
molecules of pyruvate.
the biosynthesis
citrate. The major steps in
which condense to finally give
3.1.
ofcitricacid are depicted in Fig.
production
of citric acid
Enzymatic regulation
a tenfold increase in
the
of citric acid, there is
synthesis
During the activities of other enzymes
while the
citrate synthase
activity of the enzyme
that degrade citric acid are
reduced.
isocitrate dehydrogenase)
theory that reduction in
(aconitase, the
the
does not support
However, recent evidence contributes to
acid (i.e. degradation of citriceacid)
operation of tricarboxylic
accumulation of citric
acid.
 118 B.Sc. Microbial Technology -
Ill
(Sem-VD
Inereased citric acid is more likely due to enhanced biosynthesis rather
than inhibited degradation. Further, there are anaplerotic reactions that replenish
the TCA cycle intermediates to keep the cycle continuously in operation.
Pyruvate carboxylase that converts pyruvate to oxaloacetate is also a key
enzyme in citric acid production.
Yield of citric acid

Theoretically, the yield of citric acid for the most commonly used substrate
Sucrose has been calculated. It is worked out that from 100 g sucrose, 112
gof anhydrous citric acid or 123 g of citric acid hydrate can be formed.
However, due to oxidation of sugar to CO, during tropophase, the yield of
citric acid is lower than the calculated.

Factors in the Regulation of Citric Acid Production

Strict maintenance of controlled nutrient conditions is very crucial for

maximal production of citric acid. The optimal conditions that have been
worked out for A. niger for the production of citric acid are briefly described
(Table 3.1).
Table 3.1 Optima Parameters/ Conditions for Citrie acid production

Condition / parameter Optimum

Sugar concentration 10 25%

Trace metal concentration

Manganase <10- M
Zinc < 10-7 M

Iron 104 M
pH 1.5 2.5
Dissolved 0, lension 150 mbr
Ammonium salts concentration 0.2%
Time 150 250 hours
Unit-Ill Bioenergeties of Biomolecules 119

Carbohydrate source
A.wide range of raw materials can be used for the supply or

carbohydrates. These include molasses (sugar cane or sugar beet), starch

(from potatoes), date syrup, cotton wastes, banana extract, sweet potato
pulp, and brewery waste and pineapple waste water.

A high yield of citric acid production occurs if the sugars that are

rapidly metabolised are used e.g. sucrose, glucose and maltose. At present,
cane molasses and beet molasses are commonly used. The variations in the
composition of molasses (seasonal and production level), have to be carefülly
considered for optimising citric acid production.

The concentration of carbohydrate significantly influences citric acid

production. Ideally, the sugar concentration should be 12-25%. At a

concentration less than 5% sucrose, citric acid formation is negligible, and
increases as the concentration is raised to 10% and then stabilizes (Fig.
24.2). It is believed that a high sugar concentration induces increased glucose
uptake and consequently enhanced citric acid production.

Trace metals

Certain trace elements (Fe, Cu, Zn, Mn, Mg, Co) are essential for
the growth of A. niger. Some of the trace metals particularly Mn2+, Fe3+
and Zn2 increase the yield of citric acid. The effect of manganese ions has
been investigated to some extent. These ions promote glycolysis and reduce
respiration; both these processes promote citric acid production.

As regards iron, it is a cofactor for the enzyme aconitase (of TCA

cycle). It is estimated that an Fe concentration of 0.05-0.5 ppm is ideal for

optimal citric acid production. At higher Fe concentration, the yield is lower

which can be reversed to some extent by adding copper.

 120 III (Sem-vn
B.Sc. Microbial Technology
-

pH
The pH of the medium influences the yield of citric acid, and it is

maximal when pH is below 2.5. At this pH, the production of oxalic acid
and guconic acid is suppressed. Further, at low pH, transport of citric acid

1S much higher. If the pH is above 4, gluconic acid accumulates at the expense

acid accumulates.
Or Citric acid. And when the pH goes beyond 6, oxalic
contamination is very
Another advantage with low pH is that the risk of
minimal, since many organisms cannot grow at this pH.

Dissolved O
increases when the
The yield of citric acid production substantially
aeration or
dissolved O, tension is higher. This can be achieved by strong
observed that sudden interruptions
by sparging with pure O,. It has been
drastic reduction
O, supply (as occurs during power breakdowns)
cause
in
of the organism.
in citric acid production without harming the growth

Nitrogen source

used in
salts, nitrates and urea are the nitrogen
sources
Ammonium
the three compounds are equally
the media for citric acid production. All
do not adversely affect the pH of the medium.
good sources, as long as they

If molasses are used for nutrient supply, addition of extra nitrogen source
is not required. However,
some workers have shown that exogenous addition
stimulates citric acid production.
of ammonium ions

Production Processes
for Citric Acid

which citric acid can be industrially produced

There are two processes by
and submerged process.
-
the surface process
Unit-II1 Bioenergetics of Biomolecules 121

Water Salts
Trace elements Water

Myc
Molasses
TSterili-
zation

Fermentation
chamber

Sulturic on
Oxalic Citric acid Charcoal
exchange
acid acid
remova precip
tation Evaporation
Crystalization

citric and process using the surface

Fig. 3.1 Block diagram of a

method

The surface process

This is characterized by growing the microorganisms as a layer or a

nutrient medium, which may be solid
film on a surface in contact with the
or liquid in nature. Thus, the surfäce process has supported-growth systems.

The submerged process

In this case, the organisms are immersed in or dispersed throughout

the nutrient medium. There are two types of submerged fermenters (bioreactors)
bioreactors.
Stirred bioreactors and airlift
 B.Sc. Microbial Technology - I l (Sem- VD
122

Surface Processes
Solid surface fermentation

Surface processes using solid substrates are particularly carried out in

less developed areas of some Asian countries. The solid substrates such as
wheat bran or pulp from sweet potato starch are used, as culture media.
The pH of the medium is adjusted to 4-5, and then sterilized. Now the

inoculum in the form of spores of A. niger is spread as layers (3-6 cm

thickness) and incubated at 28°C.

The growth of the organisms can be accelerated by the addition o -

amylase. Solid-state fermentation takes about 80to 100 hours for maximal
production of citric acid. At the end ofthe process, citric acid can be extracted

into hot water and isolated.

Liquid surface fermentation

Surface femmentation using liquid as nutrient medium is the oldest method

for citric acid production. It is still in use due to a simple technology, low
energy costs and higher reproducibility. Further, the interference of trace
metals and dissolved 0 tension are minimal. The labour costs are however,
higher since the manpower requirementsare more for cleaning the systems.
About 20% of the citric acid in the world is produced by surface processes.

The nutrient supply for surface fermentation normally comes from beet
molasses. The fermentation is usually carried out in aluminium trays filled
with sterile nutrient medium. The inoculum in the form of spores is sprayed
over the medium. A sterile air is passed for supplying Q as well as cooling.
The temperature is maintained around 30°C during fermentation.

As the spores germinate (that occurs within 24 hours of inoculation),

a layer of mycelium is formed over the medium. The pH of the nutrient
medium falls to less than 2, as the mycelium grows in size and forms a thick
layer on the surface of the nutrient solution. The fermentation is stopped
after 7-15 days.
 Unit-IlI Bioenergetics of Biomolecules 123

The mycelium and nutrient solution are separated. The mycelium 1s

mechanically pressed and thoroughly washed to obtain maximum amount or

citric acid. The nutrient solution is subjected to processing for the recovery
of citric acid. The final yield of citric acid is in the range of 0.7-0.9 of per

gram of sugar.

Submerged Processes
Around 80% of the world's supply of citric acid is produced by submerged
processes. This is the most preferred method due to its high efficiency and
easy automation. The disadvantages submerged fermentation are adverse
of
influence of trace metals and other impurities, variations in Q tension, and

advanced control technology that requires highly trained personnel.

Two types of bioreactors are in use- stirred tanks and aerated towers.
The vessels of the bioreactors are made up of high-quality stainless steel.
The sparging of air occurs from the base of the fermenter.

The success and yield of citric acid production mainly depend on the
structure of mycelium. The mycelium with forked and bulbous hyphae and
branches which aggregate into pellets is ideal for citric acid formation. On
the other hand, no citric acid production occurs if the mycelium is loose and
filamentous with limited branches. An adequate supply of O, (20-25% of
saturation value) is required for good production of citric acid. The ideal
aeration rate is in the range of 0.2-1 vvm (volume/ volume/ minute).

The submerged fermenters have the problem of foam formation which

may occupy about 1 /3rd

of the bioreactor. Antifoam agents (e.g. lard oil)
and mechanical antifoam devices are used to prevent foaming. Nutrient
concentration is very important in the industrial production of citric acid. A
diagrammatic representation of sucrose, citric acid and biomass concentration
with respect to cultivation time is shown in Fig. 24.4. It is estimated that
under optimal conditions, in about 250-280 hours, 100- 110 g/l of citric
acid is obtained from 140 g/l of sucrose with a biomass (dry weight) of 8-

12 g/l
 124 B.Sc. Microbial Technology -III (Sem-V)

Production of Citric Acid from Alkanes:

Both yeasts and bacteria can be used for citric acid production from
n-alkanes (C-C2 hydrocarbons).The citric acid yield is better from
hydrocarbons compared to sugars i.e. 145% of citric acid from paraffin.
The most commonly used organism is Candida lipolytica The fermentation
can be carried out in batch, semi-continuous or continuous modes. The pH
should be kept above 5. The major limitations of citric acid production from
alkanes are very low solubility of alkanes and increased production of unwanted

isocitric acid.

Recovery of Citric Acid

The steps for the recovery of citric acid either from surface process
or submerged process are comparable The recovery starts with the filtration

ofthe culture broth and washing of mycelium (which may contain about
10% of citric acid produced). Oxalic acid is an unwanted byproduct and
it can be removed by precipitation by adding lime at pH < 3.

The culture broth is then subjected to pH 7.2 and temperature 70-

90°C for precipitating citric acid. For further purification, citric acid is dissolved
in sulfuric acid (calcium sulfate precipitate separates). The final steps for
citric acid recovery are treatment with activated charcoal, cation and anion-
exchangers and crystallization.

Citric acid monohydrate formed below 36°C is the main commercial

product. Above 40°C, citric acid crystallizes in an anhydrous form. The
degree of purity of citric acid produced depends on the purpose for which
it is required. For instance, pure forms of citric acid are needed for use in
food preparations, while for industrial use it can be crude form.

3.1.2 Lactic Acid

Lactic acid occurs in two isomeric forms i.e. L (+) and D (-) isomers,
and as a racemic mixture (DL-lactic acid). The isolation of lactic acid from
milk was done in 1798. It was the first organic acid produced by microorganismns
Unit-IlI Bioenergetics ofBiomolecules 125

Lime
Sugar/
Molasses Filtration
Ncatirako
Sulfuric acid w
Fermentation AcidficationL Concentration

Filtration
Crystalization Citric acid

Fig. 3.2 Flow chart for production of citric acid

Applications of Lactic Acid
There are different grades of lactic acid mainly based on the percentage
of lactic acid.

Microorganisms for Production of Lactic Acid

Lactic acid producing bacteria are broadly categorized into two types.

Hetero-fermentative bacteria:- produce other byproducts, besides lactic

acid, and therefore are not useful for industrial production of lactic acid.
These bacteria are employed in food or feed preservation.

Homo-fermentative bacteria:- specialised for exclusive production of lactic

acid and therefore are suitable for industrial purpose.

Lactobacillus sp are used for lactic acid production. However, there

are variations in the substrates utilised as indicated below.

Biosynthesis of lactic acid

he synthesis of lactic acid occurs through glucose oxidation by

glycolysis to produce pyruvate which on reduction gives lactic acid. The
reducing equivalents (NADH"+H) produced during the oxidation of
gyceraldelyde 3-phosphate are utilised by the enzyme lactate dehydrogenase
to form lactate. Most of the lactic acid producing microorganisms normally
produce only one isomer of lactic acid L{+) or DE). However, some bacteria
which usually occur as infection can form racemic mixture.
 126 B.Sc. Microbial Technology - I1I (Sem-V)

Production Process for Lactic Acid

The fermentation medium contains 12-15% of glucose, nitrogen and

phosphate containing salts and micronutrients. The process is carried out at
pH 5.5-6.5 and temperature 45-50°C for about 75 hours Fig. 3.2. Generally,
the strains operating at higher temperature (45-60°C) are preferred, since
it reduces the need for medium sterilization.
Fermentation broth

Heated to dissolve calcium loctate

Addition H,SO
(removes calcium sulfate)

Filter and concentrate

Addition of hexacyanoferrant
(to remove heavy metals)

Purificatiom
ion exchange)

Concentration

LACTIC ACID

Fig. 3.3 Flow chart for recovery of lactic acid from fermentation
broth

As the lactic acid is produced, it has to be removed since it is toxic

to the organisms. This can achieved either by a continuous culture technique
or by removal of lactic acid by electro dialysis. Theoretically, every molecule
of glucose forms two molecules of lactic acid. About 90% of theoretical

yield is possible in fermentation industry. L(+) Lactic acid is

predominantly produced.