PRODUCTION OF COMMERCIAL PRODUCTS

INTRODUCTION
Biotechnology is a science that has been explored in recent decades to provide the industrial
chemistry industry with products and solutions, and microbes are one of these solutions. As
well as being easily and readily available and cheap to produce, they are sustainable and
any remaining “spent” biomass can be utilized in other industries.

Possibly the most useful application of biotechnology is the utilization of microorganisms in

the production of alcohols (for example, ethanol and methanol) and acetone. These chemicals
have widespread uses. The fermentation of sugars by microorganisms has been used to
produce alcohol for thousands of years. These chemicals have many uses within modern
industry including biofuel production.

Fig1: Industrial production set up

The use of microorganisms in industrial processes is known as industrial microbiology. The

field has been revolutionized along with other applications of microbiology by the advent of
genetic engineering. Scientists can manipulate microorganisms such as bacteria and fungi at
the genetic level to produce new tailor-made strains that can produce a multitude of chemical
products.

PRODUCTION OF PRIMARY METABOLITES

PRIMARY METABOLITES
Primary Metabolites play an important role in the organism's growth, development, and
reproduction. The major metabolite is frequently referred to as a central metabolite because it
is typically a key component in maintaining normal physiological activities.
Primary metabolites are created as a result of energy metabolism during the growth phase and
are thought to be necessary for optimal growth. Alcohols like ethanol, lactic acid, and some
amino acids are examples of primary metabolites.

Fig2: types of metabolites

Alcohol is one of the most prevalent main metabolites used for large-scale synthesis in the
field of industrial microbiology. Alcohol is specifically employed in fermentation processes
that result in goods such as beer and wine. Primary metabolites like amino acids, L-glutamate
and L-lysine, which are often used as supplements, are also obtained by the mass production
of Corynebacteria glutamicum, a specific bacterial species.

Citric acid is another example of a primary metabolite often employed in industrial

microbiology. Aspergillus niger produces citric acid, which is one of the most extensively
used components in food production. It's also widely employed in the pharmaceutical and
cosmetic industries.

• Involved in growth, development and reproduction. Hence, essential for survival

and existence of the organism and reproduction.
• Formed at the same time as new cells.
• Production curve follows the growth curve.
• Formed in trophophase during exponential growth as normal end products of
primary metabolism.
• Also called central metabolites as these maintain normal physiological processes.
• Cells maintain optimum concentration of all macromolecules (proteins, DNA, RNA
etc.).
• Produced in adequate amount to sustain cell growth for example vitamins, amino
acids, nucleosides etc.
• Overproduction can be genetically manipulated. Auxotrophic (auxo, “increase,” and
trophos, ‘‘food’’) mutants having a block in steps of a biosynthetic pathway for the
formation of primary metabolite.
• Growth rate slows down due to limited supply of any other nutrient. Metabolism does
not stop but product formation stops.
• Industrially important for example ethanol, acetone, lactic acid, CO2.
• Common food supplements, L-glutamate and L-lysine, are produced and purified via
the mass production Corynebacterium glutamicum.
• Citric acid, commonly used in pharmaceutical and cosmetic industries is produced
by Aspergillus niger.

Fig3: Over production of primary metabolite.

To maximize production manipulation of feedback inhibition pathways is
performed. Another approach is to use auxotrophic mutant with defective metabolite
production.

PRODUCTION OF ORGANIC ACIDS

CITRIC ACID:
Citric acid was originally produced from lemons by an Italian cartel, the discovery of its
accumulation by Aspergillus niger (then named citromyces) in the early 1920s led to a rapid
development of a fermentation process which, 15 years later, accounted for more than 95% of
the world’s production of citric acid.

Today, citric acid is produced in bulk amounts with an estimated worldwide production of
400,000 tonnes per year, most of which is produced by fermentation with the fungus
Aspergillus niger.
Commercial Production:
Two different types of fermentation processes are basically used for commercial production
of citric acid, e.g., the surface fermentations and the submerged fermentations. In addition,
some citric acid is also produced by solid state fermentations, particularly in less developed
rural areas such as some East Asian countries. Citric acid production by yeast is exclusively
done by submerged cultivation.

Surface fermentation is the older and more labour intensive version of citric acid
fermentation, yet it is still in use, even by some major producers of citric acid. The main
reasons for this are the lower power requirements and the higher reproducibility of the
process due to its lower susceptibility to interference by trace metal ions and variations in the
dissolved O2 tension.

Fig4: citric acid

Their fermentation is usually carried out in aluminium trays (2 m x 2.5 m), filled with
nutrient medium to a depth of between 50 and 200 cm. Molasses solution containing
inorganic nitrogenous salts is used as fermentation medium.
The pH is maintained at about 2. The fermentation medium is first freed of trace elements
(e.g., Mn++, Zn++, Fe++, phosphates) through ion exchange resin because the trace elements
exert inhibition of citric acid formation above a critical level.

Following this, the requisite concentrations of metal ions are added as salts in the medium.
Spores are now distributed over the surface of the trays, and sterile air (serving both as an
oxygen supply as well as a cooling aid) is passed over them. The mycelium develops as a
coherent felt, becoming progressively more convoluted. The fermentation is completed
within a period of 7 to 12 days at 30°C.

The culture fluid is taken out from the bottom of the trays, leaving the mycelial mat of A.
niger more or less undisturbed. Citric acid is recovered from the fluid by precipitation. Fresh
medium is added into the trays for the next batch of fermentation.
 Fig5: surface fermentation used for commercial production of citric acid

The submerged fermentation process is desirable because of its higher efficacy due to higher
susceptibility to automatisation. Yet the severe influence of trace metal ions and other
impurities present in the carbohydrate raw materials and its disturbance by variations in
O2 supply make it more difficult to manage particularly because the quality of the
carbohydrate source is variable.
There are two types of fermentors in use:
 Stirred tanks and

 ii. Aerated tower fermentors.

Both types are constructed of high-grade stainless steel and contain facilities for cooling.
Sparging with O2 occurs from the base.

Fig6: submerged fermentation process used for commercial production of citric acid
One of the most prominent features of submerged fermentation is the mycelial development
which shows a characteristic pattern: the germinating spores form stubby, forked and bulbous
hyphae, which aggregate to small (0.2-0.5 mm) pellets having a firm, smooth surface, and
sediment quickly when harvested.

This striking morphology has been shown to be critical for attaining high yields by
submerged fermentation and is dependent on an appropriate nutrient composition. It is
therefore a convenient indicator for the progress of fermentation, e.g., by procedures
involving microscopy. A final yield of 0.8-0.9 kg kg-1 is obtained after 7 to 10 days.

Uses:
Due to its pleasant taste, low toxicity and excellent palatability, citric acid is widely used in
industry for the preparation of food and sugar confectionery (21% of total production) and
beverages (45%). Other major applications are in the pharmaceutical and detergent/cleaning
industry (8 and 19%, respectively).

Fig7: citric acid esters

It is also able to complex heavy metal ions, such as iron and copper, and therefore is applied
in the stabilisation of oils and fats or ascorbic acid against metal ion-catalysed oxidation. In
addition, citric acid esters of a wide range of alcohols are known and can be employed as
non-toxic plasticisers. Finally, some of its salts have commercial importance, e.g., trisodium
citrate as a blood preservative which prevents blood clotting by complexing calcium, or as a
stabiliser of emulsions in the manufacture of cheese.

INDUSTRIAL PRODUCTION OF AMINO ACIDS

About 20 amino acids are synthesized in the cell of the most microorganisms. They are
utilized in the synthesis of proteins and other essential substances required by the cell.
Fermentative production of amino acids has started by the discovery of glutamic acid
producing bacterium, Corynebacterium glutamicum (Micrococcus glutamicum), by Kinoshita
et al. (1957) Since then much research work has been carried out on fermentative production
of amino acids. Large number of microorganisms were isolated from nature which are
capable of producing amino acids by fermentation in commercially feasible
quantities, especially from the auxotrophic bacteria.

Fig8: layout of fermentation plant for production of amino acids

Of the various amino acids, L-glutamic acid and L-lysine are in great demand for commercial
production. Auxotrophs are used in the fermentation production of these two amino acids.
The major portion of the amino acid accumulation occurs after log phase of growth of the
microorganism. The concentration of specific growth factors is very crucial for the maximum
yield of the product because concentration above or below the optimum substantially, reduces
the yield.

Microorganisms, generally, do not produce amino acids in surplus or more than required
quantities. They accomplish this by regulating cellular metabolism. However,
overproduction of the amino acids by microorganisms can be achieved by controlling the
complex regulatory systems. This is done by raising auxotrophs by mutation.

The enzymes responsible for the regulatory effect or repressor of an amino acid are made
inactive due to mutation which leads to the accumulation of the particular amino acid in
commercially feasible quantities which is being exploited commercially for fermentation
Apart from fermentative processes, some amino acids can be synthesized quite economically
by chemical processes. However, these chemical processes generally yield, D-isomers of
amino acids, which are biologically inactive and cannot be used as food flavouring
substances or food supplements. Only L-isomers are useful as food supplements or
flavouring
 substances and, therefore, most of the L-isomer of amino acids are produced by fermentation
process.
PENICILLIN
Penicillins are a class of β-lactam antibiotics of related structure with slightly different properties and
activities. They are produced by Penicillium molds. All penicillins have a common nucleus called 6-
aminopenicillanoic acid (6-APA). 6-APA is a fused β-lactamthiozolidine ring. The side chain
attached to this ring gives each penicillin its unique characteristic. Lactose is the universally
preferred carbon source. Glucose is also included in the medium but this is primarily for mycelial
growth. Abundant growth is very essential for the main fermentation. Glucose is a readily
assimilable carbon source and so the organism does not use it for the production of penicillin, which
is a secondary metabolite. The production of penicillin occurs in the idiophase. The biosynthesis is
the result of depletion of one or more nutrients. The mold, when forced to live on lactose (which is
not readily metabolizable), produces penicillin. Stated differently, the organism should experience
the condition of starvation for the synthesis of penicillin. Classical fermentations were most probably
of the batch type. Today, trade fermentations are carried out in fed-batch mode, in which lactose is
replaced by sucrose or even glucose. The feeding is controlled such that the organism suffers the
same fate as in the case of lactose medium. Cornsteep liquor (CSL) is universally used as nitrogen
source. Nitrogen is often supplied as NH3 because the cornsteep nitrogen may not be adequate. CSL
also contains growth factors, minerals, and above all, precursors of side chains. CSL is a
concentrated form of steepwater in which corn has been steeped for the manufacture of starch, gluten
and other corn products. CSL contains 50% solids. Although the composition is highly variable, the
concentrations of total nitrogen, lactic acid, amino nitrogen, reducing sugars and ash are around 7.4-
7.8%, 12-27%, 2.6-3.3%, 1.5-14%, and 18-20% respectively (on dry basis).
FERMENTATION
Following sterilization, the medium is transferred to the main fermenter, which may be of 40-
200MT capacity. The initial pH is kept at 6.5. Inoculum is built up the normal way and 10% vol/vol
is added to the main fermenter. The pure culture normally comes in the form of lyophilized spores.
The culture is initially grown in special sporulation agar. This is then transferred to shaker flask,
which contains a supplement of 2% sucrose. Incubation is done at 25°C for some days. The culture is
then transferred to propagator of considerable size. Aseptic and aerobic fermentation ensues here
also.
The main fermenter may be inoculated with either spores or mycelium. The methods of inoculum
build up, as also the method of inoculation, may vary here. The main fermentation is triphasic in
nature. During the active growth phase glucose and cornsteep components are rapidly utilized. The
pH remains constant. At the end of this phase, however, glucose and cornsteep components deplete.
Ammonia is liberated due to deamination reaction. Consequently, pH rises to 7-7.5. This pH is
optimum for the synthesis of penicillin. The antibiotic is very sensitive to pH changes. The latter can
be controlled by adding suitable amounts of H2SO4 or CaCO3. The late growth phase is followed by
idiophase, the phase in which penicillin synthesis occurs. Growth ceases in this phase because the
nutrients have depleted. The organism begins to utilize lactose. This condition forces the mold to
synthesize penicillin. Care must be taken not to allow the late idiophase to proceed too far, as this
leads to cell lyses and consequent degradation of penicillin. The duration of fermentation is typically
6 days. Since the organism is aerobic, aeration should be done at the rate of 0.5-1 vol/vol/min.
Turbine agitators are used to facilitate aeration because the rheology of the medium becomes very
complex as the mycelial growth progresses. Precursors are added constantly in regulated amounts.
The fermentation is normally carried out at 26°C.
During fermentation, periodic checks must be carried out for penicillin yield and contamination. The
penicillin yield for a typical fermentation is above 20g/L.
 Fig : Fermenter For Producing Penicillin

HARVESTING
Harvesting is done before the mycelia begin to lyse. The mycelia are filtered in rotary vacuum filter.
Washing is done repeatedly to elute the antibiotic. Filter aids such as hyflow may be used during the
filtration. The broth is cooled promptly to 0-4°C to minimize enzymatic and chemical degradation of
the antibiotic.

PURIFICATION
Penicillin can be purified by two methods, viz., (a) carbon process, and (b) solvent extraction. The
carbon process is an obsolete process. Solvent extraction is now used for purification/ concentration
of penicillin. This latter method is thus an industry standard.

Fig : Production Of Penicilline

SOLVENT EXTRACTION PROCESS
Penicillin is very sensitive to temperature and pH. At pH 2 and 20°C, the half-life is only 15 min. It
is therefore very important that extraction cycles be very fast. In practice, each extraction cycle takes
not more than 60-90 seconds. The temperature during the extraction is maintained at 0-3°C. The
most common solvents used for the extraction are amyl- or butyl acetate. Basically the process
entails extraction with organic solvent followed by back-extraction in alkaline aqueous phase.
To begin with, the pH of the penicillin broth is adjusted to 2.5-3.5 with H3PO4 or H2SO4 (dilute,
10% vol/vol). Emulsifiers may be added at the rate of 0.003-0.1% in the organic solvent. At this pH,
penicillin is preferentially soluble in the solvent. The extraction takes place in continuous,
countercurrent, multistage, centrifugal extractor (Podbielniak D-36). The ratio of broth to solvent is
kept 5-7:1. A single extraction will produce 5-7 fold concentration and the concomitant 5-10% loss
of penicillin.
This extraction in organic solvent is immediately followed by back-extraction in water. Water is
added to the penicillin-rich solvent in the ratio water:solvent = 0.1-0.2. The pH of the mixture is
adjusted to 5-7.5 by adding NaOH or KOH in the water. At this pH, penicillin shifts its selectivity
towards water. The resulting aqueous extract is rich in Na- or Pot salt of penicillin. In neutral pHs in
water, penicillin is ionized. In acid conditions this ionization is suppressed and the penicillin is more
soluble in solvents.
The extract is again dissolved in organic solvent by adjusting the pH to 2.5-3.5. This time the amount
of solvent is reduced. This is again followed by aqueous extraction. The cycle is repeated until the
required degree of concentration is achieved. The final extract will necessarily be an aqueous extract
of Na- or Pot salts of penicillin. The spent solvent is recovered for reuse.
The resulting aqueous solution is treated with 0.25-0.5% carbon to remove pigments and other
impurities. The solution is subjected to filtration to remove trace impurities, bacteria and pyrogens.
Crystallization is done by salting out using sodium acetate as the salt. The crystals are washed with
water and dried in anhydrous isopropanol, butanol, etc. The final drying is done in warm air,
vacuum, radiant heat, or large horizontal bed driers. The penicillin thus produced must conform to
the FDA standards. The strength and dosage of penicillin is expressed in terms of international units.
Each of these units is equal to 0.0006 g of the crystalline fraction of penicillin G. The yield from
modern strains can be as high as 20g/L. The loss figure (during handling, filtration, extraction,
crystallization, drying) is about 22%.

COMMERCIAL PRODUCTION OF ALCOHOLS

Ethyl alcohol has been produced on large scale for centuries. However, much study could not be
accomplished because of hazards on human consumption. In 1865 Alcohol Act was passed thereby
free sale of alcohol after its denaturing by adding methylated spirit was allowed.
Early production was primarily used for human consumption. But today, apart from being used for
human consumption, it is also used as universal solvent and as chemical raw material for the
production of other industrial products.
Along with gasoline it is also used as motor fuel. Because of the above utilities the demand for
alcohol increased enormously today, which lead to establishment of many distilleries throughout
the world. Ethyl alcohol is produced, besides yeasts, by large number of bacteria and fungi
Fermentative Production of Ethyl Alcohol:
On commercial scale ethyl alcohol production process consists of four steps.
They are:
(i) Inoculum production,
(ii) Production medium,
(iii) Fermentation process, and
(iv) Harvest and recovery

Fig9: Commercial alcohol production

CONCLUSION
The application of microorganisms to the production of useful industrial chemicals is still a
fairly recent scientific development that can solve many problems inherent to the industrial
chemical industry. With research such as that illustrated above, industrial microbiology is at
the cutting edge of research to provide sustainable solutions for the field of industrial
chemistry.

BY

Tharani.K
1036
2 nd Year
Bsc. Biotechonology