Download UNIT-II - E

1
UNIT-II
MICROBIAL SYNTHESIS OF COMMERCIAL PRODUCTS:
AMINO ACIDS
Explain about the production of amino acids by microbial fermentation process.
Introduction:
 Several amino acids are produced in commercial quantities via direct fermentation processes using
over producing microbial strains, or by microbial Biotransformation.
 They are mostly employed as food or animal feed supplements and flavour compounds.
 However, several amino acids also have uses in pharmaceuticals and cosmetics, and in the chemical
industry for the manufacture of polymers.
L-Glutamic acid:
 Of all the amino acid production processes, that of L glutamic acid is probably the most important
in terms of quantity.
 Its main use is as the flavour enhancer, monosodium L-glutamate (MSG), which can heighten and
intensify the flavour of foods without adding significant flavour of its own Glutamic acidproducing microorganisms include species of the clorelated genera Arthrobacter, Brevibacterium,
Corynebacterium, Microbacterium and Micrococcus.
 These are Gram-positive, biotin requiring, non-motile bacteria, which have intense glutamate
dehydrogenase activity.
 Species of Brevibacterium and Corynebacterium are used for most industrial fermentations.
 The wild-type Corynebacterium glutamicum, for example, exhibits feedback inhibition when
cellular glutamic acid concentrations rise to 5% on a dry weight basis
 However, the production strains developed using mutagenesis and selection programme are both
regulatory and auxotrophic mutants.
 These strains have been developed with a high steady-state cytoplasmic amino acid concentration.
They accumulate about 30% L-glutamic acid and yield 1 mol of glutamate per 1.4 mol o/f glucose,
and importantly are phage resistant.
 More recently, recombinant DNA technology has been used to increase the activity of specific
biosynthetic enzymes by transformation with multicopy plasmids bearing the structural genes for
these enzymes. The overall strategy for achieving overproduction of the amino acid involves:
 Increasing the activity of anabolic enzymes;
 Manipulation of regulation to remove feedback control mechanisms;
 Blocking pathways that lead to unwanted by products;
 Blocking pathways that result in degradation of the target product; and
 Limiting the ability to process the immediate precursor of L-glutanflc acid, namely oxoglutaric acid,
to the next intermediate of the tricarboxylic acid (TCA) cycle, succinyl coenzyme A (CoA), i.e use of
mutants lacking oxoglutaric acid dehydrogenase. During the growth phase these mutants produce
essential intermediates from isocitrate via the glyoxylate
 In addition, as these bacteria do not normally secrete glutamate, a range of treatments is employed
to render the cells more permeable and aid release of the amino acid into the medium.
 These treatments include: biotin limitation, restriction of phospholipid biosynthesis by adding C
saturated fatty acids during the growth phase, and inclusion of surfactants (e.g. Tween 40) and
penicillin in the production media.
WAYS OF GLUTAMIC ACID PRODUCTION:
 By the hydrolysis of wheat gluten, soybean cake or other proteinaceous materials
 By the cleavage of pyrrolidone carboxylic acid found in molasses.
 By a one – stage fermentation process involving one organism.
 By a two – stage fermentation process where α – ketoglutaric acid is produced by one microbe and
α – ketoglutaric acid, in turn, is converted to L – glutamic acid by another microbe.
2
Industrial production of l-glutamic acid:
 Industrial-scale fermenters are normally stainless steel stirred tank reactors of up to 450 m. These
are batch processes, operated aerobically at 30—37°C, the specific temperature depending on the
microorganisms used.
 Apart from carbon and nitrogen sources, the fermentation medium normally contains inorganic
salts, providing magnesium, manganese, phosphate and potassium, and limiting levels of biotin.
 Corynebacteria are nutritionally fastidious and may also require other vitamins, amino acids,
purines and pyrimidines
 The preferred carbon sources are carbohydrates, preferably glucose or sucrose.
 Cane or beet molasses can be used, but the medium requires further modification, as their biotin
levels tend to be too high.
 This can be overcome by the addition of saturated fatty acids, penicillin or surfactants, which
promote excretion. The nitrogen source (ammonium salts, urea or ammonia) is fed slowly to
prevent inhibition of L-glutamate production
 . Medium pH is maintained at 7-8 by the addition of alkali, otherwise the pH progressively falls as
the L-glutamate is excreted into the medium.
 Accumulation of L-glutamic acid does not become apparent until midway through the
fermentation, which normally lasts for 35—40 h and achieves L-glutamic acid levels in the broth of
80 g/L.
FLOW DIAGRAM:
Product recovery:
 Product recovery involves separation of the cells from the culture medium.
 The L-glutamic acid is then crystallized from the spent medium by lowering the pH to its isoelectric
point of pH3.2 using hydrochloric acid.
 Crystals of L-glutamic acid are then filtered off and washed.
 MSG is prepared by adding a solution of sodium hydroxide to the crystalline L-glutamic acid
followed by recrystallization.
ORGANIC ACID PRODUCTION
ACETIC ACID PRODUCTION
1. Acetic acid, CH3COOH, also known as ethanolic acid, is an organic acid that gives vinegar its sour
taste and pungent smell.
2. It is a weak acid, in that it is only a partially dissociated acid in an aqueous solution. Pure, waterfree acetic acid (glacial acetic acid) is a colourless liquid that absorbs water from the environment
(hygroscopy), and freezes at 16.5 °C (62 °F) to a colourless crystalline solid.
3. The pure acid and its concentrated solutions are quite corrosive.
4. Acetic acid is one of the simplest carboxylic acids. It is an important chemical reagent and industrial
chemical, used in the production of polyethylene terephthalate mainly used in soft drink bottles;
cellulose acetate, mainly for photographic film; and polyvinyl acetate for wood glue, as well as
synthetic fibres and fabrics.
5. In households, diluted acetic acid is often used in descaling agents. In the food industry, acetic acid
is used under the food additive code E260 as an acidity regulator and as a condiment.
6. The global demand of acetic acid is around 6.5 million tonnes per year (Mt/a), of which
approximately 1.5 Mt/a is met by recycling; the remainder is manufactured from petrochemical
feedstocks or from biological sources.
3
7. Dilute acetic acid produced by natural fermentation is called vinegar.
8. Glacial acetic acid is a trivial name for water-free acetic acid. Similar to the German name Eisessig
(ice-vinegar), the name comes from the ice-like crystals that form slightly below room temperature at
16.7 °C (62 °F).
9. The most common abbreviation for acetic acid is HOAc where Ac stands for the acetyl group
CH3−C(=O)−. In the context of acid-base reactions, the abbreviation HAc is often used where Ac
instead stands for the acetate anion (CH3COO−, abbreviated AcO−), although this use is often
regarded as misleading as HAc is also used for acetyladehyde.
10. In either case, the Ac is not to be confused with the abbreviation for the chemical element actinium.
Acetic acid has the molecular formula C2H4O2 (empirical formula CH2O).
11. To emphasize the role of the active hydrogen in forming the salt sodium acetate, some people write
the molecular formula as HC2H3O2.
12. To better reflect its structure, acetic acid is often written as CH3-CO2-H, CH3COOH, or CH3CO2H.
The ion resulting from loss of H+ from acetic acid is the acetate anion. The name acetate can also refer
to a salt containing this anion, or an ester of acetic acid.
Chemical properties
1. The hydrogen (H) atom in the carboxyl group (−COOH) in carboxylic acids such as acetic acid can
be given off as an H+ ion (proton), giving them their acidic character.
2. Acetic acid is a weak, effectively monoprotic acid in aqueous solution, with a pKa value of 4.75. Its
conjugate base is acetate (CH3COO−).
3. A 1.0 M solution (about the concentration of domestic vinegar) has a pH of 2.4, indicating that
merely 0.4% of the acetic acid molecules are dissociated.
Cyclic dimer of acetic acid; dashed lines represent hydrogen bonds
4. The crystal structure of acetic acid shows that the molecules pair up into dimers connected by
hydrogen bonds.
5. The dimers can also be detected in the vapour at 120 °C. They also occur in the liquid phase in
dilute solutions in non-hydrogen-bonding solvents, and a certain extent in pure acetic acid, but are
disrupted by hydrogen-bonding solvents.
6. The dissociation enthalpy of the dimer is estimated at 65.0–66.0 kJ/mol, and the dissociation
entropy at 154–157 J mol−1 K−1.This dimerization behaviour is shared by other lower carboxylic
acids.
7. Liquid acetic acid is a hydrophilic (polar) protic solvent, similar to ethanol and water. With a
moderate relative static permittivity (dielectric constant) of 6.2, it can dissolve not only polar
compounds such as inorganic salts and sugars, but also non-polar compounds such as oils and
elements such as sulfur and iodine.
8. It readily mixes with other polar and non-polar solvents such as water, chloroform, and hexane.
With higher alkanes (starting with octane) acetic acid is not completely miscible anymore, and its
4
miscibility continues to decline with longer n-alkanes. This dissolving property and miscibility of
acetic acid makes it a widely used industrial chemical.
Chemical reactions
1. Acetic acid is corrosive to metals including iron, magnesium, and zinc, forming hydrogen gas and
metal salts called acetates.
2. Aluminium, when exposed to oxygen, forms a thin layer of aluminium oxide on its surface, which
is relatively resistant to the acid, allowing aluminium tanks to transport acetic acid.
3. Metal acetates can also be prepared from acetic acid and an appropriate base, as in the popular
"baking soda + vinegar" reaction. With the notable exception of chromium(II) acetate, almost all
acetates are soluble in water.
Mg(s) + 2 CH3COOH(aq) → (CH3COO)2Mg(aq) + H2(g)
NaHCO3(s) + CH3COOH(aq) → CH3COONa(aq) + CO2(g) + H2O(l)
4. Acetic acid undergoes the typical chemical reactions of a carboxylic acid, such as producing water
and a metal ethanoate when reacting with alkalis, producing a metal ethanoate when reacted with a
metal, and producing a metal ethanoate, water, and carbon dioxide when reacting with carbonates
and hydrogencarbonates.
5. Most notable of all its reactions is the formation of ethanol by reduction, and formation of
derivatives such as acetyl chloride via nucleophilic acyl substitution.
6. Other substitution derivatives include acetic anhydride; this anhydride is produced by loss of water
from two molecules of acetic acid. Esters of acetic acid can likewise be formed via Fischer
esterification, and amides can also be formed.
7. When heated above 440 °C, acetic acid decomposes to produce carbon dioxide and methane, or to
produce ethenone and water.
8. Acetic acid can be detected by its characteristic smell. A colour reaction for salts of acetic acid is
iron(III) chloride solution, which results in a deeply red colour that disappears after acidification.
9. Acetates when heated with arsenic trioxide form cacodyl oxide, which can be detected by its
malodorous vapours.
Biochemistry
1. The acetyl group, derived from acetic acid, is fundamental to the biochemistry of all forms of life.
When bound to coenzyme A, it is central to the metabolism of carbohydrates and fats. However, the
concentration of free acetic acid in cells is kept at a low level to avoid disrupting the control of the
pH of the cell contents.
2. Unlike longer-chain carboxylic acids (the fatty acids), acetic acid does not occur in natural
triglycerides. However, the artificial triglyceride triacetin (glycerin triacetate) is a common food
additive, and is found in cosmetics and topical medicines.
3. Acetic acid is produced and excreted by acetic acid bacteria, notable ones being the Acetobacter
genus and Clostridium acetobutylicum. These bacteria are found universally in foodstuffs, water, and
soil, and acetic acid is produced naturally as fruits and other foods spoil.
5
4. Acetic acid is also a component of the vaginal lubrication of humans and other primates, where it
appears to serve as a mild antibacterial agent.
Production
Purification and concentration plant for acetic acid in 1884
1. Acetic acid is produced both synthetically and by bacterial fermentation. Today, the biological route
accounts for only about 10% of world production, but it remains important for vinegar production,
as many nations' food purity laws stipulate that vinegar used in foods must be of biological origin.
2. About 75% of acetic acid made for use in the chemical industry is made by methanol carbonylation,
explained below.
Methanol carbonylation
Most virgin acetic acid is produced by methanol carbonylation. In this process, methanol and carbon
monoxide react to produce acetic acid according to the chemical equation:
CH3OH + CO → CH3COOH
The process involves iodomethane as an intermediate, and occurs in three steps. A catalyst, usually a metal
complex, is needed for the carbonylation (step 2).
1. CH3OH + HI → CH3I + H2O
2. CH3I + CO → CH3COI
3. CH3COI + H2O → CH3COOH + HI
Acetaldehyde oxidation
Acetic acid was produced by oxidation of acetaldehyde. This remains the second-most-important
manufacturing method, although it is uncompetitive with methanol carbonylation.
The acetaldehyde may be produced via oxidation of butane or light naphtha, or by hydration of ethylene.
When butane or light naphtha is heated with air in the presence of various metal ions, including those of
manganese, cobalt, and chromium, peroxides form and then decompose to produce acetic acid according to
the chemical equation
2 C4H10 + 5 O2 → 4 CH3COOH + 2 H2O
The typical reaction is run at a combination of temperature and pressure designed to be as hot as possible
while still keeping the butane a liquid. Typical reaction conditions are 150 °C and 55 atm. Side-products
may also form, including butanone, ethyl acetate, formic acid, and propionic acid.
These side-products are also commercially valuable, and the reaction conditions may be altered to produce
more of them where needed. However, the separation of acetic acid from these by-products adds to the cost
of the process.
Under similar conditions and using similar catalysts as are used for butane oxidation, acetaldehyde can be
oxidized by the oxygen in air to produce acetic acid
2 CH3CHO + O2 → 2 CH3COOH
6
Using modern catalysts, this reaction can have an acetic acid yield greater than 95%. The major sideproducts are ethyl acetate, formic acid, and formaldehyde, all of which have lower boiling points than
acetic acid and are readily separated by distillation.
Ethylene oxidation
Acetaldehyde may be prepared from ethylene via the Wacker process, and then oxidized as above. In more
recent times, a cheaper, single-stage conversion of ethylene to acetic acid was commercialized by chemical
company Showa Denko, which opened an ethylene oxidation plant in Ōita, Japan, in 1997.
The process is catalysed by a palladium metal catalyst supported on a heteropoly acid such as tungstosilicic
acid. It is thought to be competitive with methanol carbonylation for smaller plants (100–250 kt/a),
depending on the local price of ethylene.
Oxidative fermentation
For most of human history, acetic acid, in the form of vinegar, has been made by acetic acid bacteria of the
genus Acetobacter. Given sufficient oxygen, these bacteria can produce vinegar from a variety of alcoholic
foodstuffs. Commonly used feeds include apple cider, wine, and fermented grain, malt, rice, or potato
mashes. The overall chemical reaction facilitated by these bacteria is:
C2H5OH + O2 → CH3COOH + H2O
A dilute alcohol solution inoculated with Acetobacter and kept in a warm, airy place will become vinegar
over the course of a few months. Industrial vinegar-making methods accelerate this process by improving
the supply of oxygen to the bacteria.
The first batches of vinegar produced by fermentation probably followed errors in the winemaking process.
If must is fermented at too high a temperature, acetobacter will overwhelm the yeast naturally occurring on
the grapes.
As the demand for vinegar for culinary, medical, and sanitary purposes increased, vintners quickly learned
to use other organic materials to produce vinegar in the hot summer months before the grapes were ripe
and ready for processing into wine. This method was slow, however, and not always successful, as the
vintners did not understand the process.
Anaerobic fermentation
Species of anaerobic bacteria, including members of the genus Clostridium, can convert sugars to acetic acid
directly, without using ethanol as an intermediate. The overall chemical reaction conducted by these
bacteria may be represented as:
C6H12O6 → 3 CH3COOH
It is interesting to note that, from the point of view of an industrial chemist, these acetogenic bacteria can
produce acetic acid from one-carbon compounds, including methanol, carbon monoxide, or a mixture of
carbon dioxide and hydrogen:
2 CO2 + 4 H2 → CH3COOH + 2 H2O
This ability of Clostridium to utilize sugars directly, or to produce acetic acid from less costly inputs, means
that these bacteria could potentially produce acetic acid more efficiently than ethanol-oxidizers like
Acetobacter. However, Clostridium bacteria are less acid-tolerant than Acetobacter.
7
Even the most acid-tolerant Clostridium strains can produce vinegar of only a few per cent acetic acid,
compared to Acetobacter strains that can produce vinegar of up to 20% acetic acid. At present, it remains
more cost-effective to produce vinegar using Acetobacter than to produce it using Clostridium and then
concentrate it. As a result, although acetogenic bacteria have been known since 1940, their industrial use
remains confined to a few niche applications.
Applications
1. 2.5-litre bottle of acetic acid in a laboratory. The bottle is made out of amber glass.
2. Acetic acid is a chemical reagent for the production of chemical compounds.
3. The largest single use of acetic acid is in the production of vinyl acetate monomer, closely followed
by acetic anhydride and ester production.
4. The volume of acetic acid used in vinegar is comparatively small.
5. Vinyl acetate monomer:
 The major use of acetic acid is for the production of vinyl acetate monomer (VAM). This
application consumes approximately 40% to 45% of the world's production of acetic acid.
The reaction is of ethylene and acetic acid with oxygen over a palladium catalyst.
2 H3C-COOH + 2 C2H4 + O2 → 2 H3C-CO-O-CH=CH2 + 2 H2O
 Vinyl acetate can be polymerized to polyvinyl acetate or to other polymers, which are
applied in paints and adhesives.
6. Ester production:
 The major esters of acetic acid are commonly used solvents for inks, paints and coatings. The
esters include ethyl acetate, n-butyl acetate, isobutyl acetate, and propyl acetate. They are
typically produced by catalysed reaction from acetic acid and the corresponding alcohol:
H3C-COOH + HO-R → H3C-CO-O-R + H2O, (R = a general alkyl group)
7. Acetic anhydride:
 The condensation product of two molecules of acetic acid is acetic anhydride. The
worldwide production of acetic anhydride is a major application, and uses approximately
25% to 30% of the global production of acetic acid. Acetic anhydride may be produced
directly by methanol carbonylation bypassing the acid, and Cativa production plants can be
adapted for anhydride production.

Acetic anhydride is a strong acetylation agent. As such, its major application is for cellulose
acetate, a synthetic textile also used for photographic film. Acetic anhydride is also a reagent
for the production of aspirin, heroin, and other compounds.
8. Vinegar:
In the form of vinegar, acetic acid solutions (typically 4% to 18% acetic acid, with the percentage
usually calculated by mass) are used directly as a condiment, and also in the pickling of vegetables
and other foods. Table vinegar tends to be more diluted (4% to 8% acetic acid), while commercial
food pickling, in general, employs more concentrated solutions.
9. Organic or inorganic salts are produced from acetic acid, including:




Sodium acetate, used in the textile industry and as a food preservative (E262).
Copper(II) acetate, used as a pigment and a fungicide.
Aluminium acetate and iron(II) acetate—used as mordants for dyes.
Palladium(II) acetate, used as a catalyst for organic coupling reactions such as the Heck reaction.
8

Silver acetate, used as a pesticide.
10. Substituted acetic acids produced include:



Monochloroacetic acid (MCA), dichloroacetic acid (considered a by-product), and trichloroacetic
acid. MCA is used in the manufacture of indigo dye.
Bromoacetic acid, which is esterified to produce the reagent ethyl bromoacetate.
Trifluoroacetic acid, which is a common reagent in organic synthesis.
---------------------------------------------------------------------------------------------------------------------------------CITRIC ACID PRODUCTION
1. Citric acid is a weak organic acid. It is a natural preservative and is also used to add an acidic, or
sour, taste to foods and soft drinks.
2. In biochemistry, it is important as an intermediate in the citric acid cycle and therefore occurs in the
metabolism of virtually all living things. It can also be used as an environmentally benign cleaning
agent.
3. Citric acid exists in greater than trace amounts in a variety of fruits and vegetables, most notably
citrus fruits. Lemons and limes have particularly high concentrations of the acid; it can constitute as
much as 8% of the dry weight of these fruits (about 47 g/L in the juices).
4. The concentrations of citric acid in citrus fruits range from 0.005 mol/L for oranges and grapefruits
to 0.30 mol/L in lemons and limes. Within species these values vary depending on the cultivar and
the circumstances in which the fruit was grown.
Properties
1. At room temperature, citric acid is a white crystalline powder. It can exist either in an anhydrous
(water-free) form or as a monohydrate.
2. The anhydrous form crystallizes from hot water, where as the monohydrate forms when citric acid
is crystallized from cold water.
3. The monohydrate can be converted to the anhydrous form by heating above 78 °C. Citric acid also
dissolves in absolute (anhydrous) ethanol (76 parts of citric acid per 100 parts of ethanol) at 15
degrees Celsius.
4. In chemical structure, citric acid shares the properties of other carboxylic acids. When heated above
175°C, it decomposes through the loss of carbon dioxide and water. Citric acid leaves a white
crystalline precipitate.
5. Citric acid is a slightly stronger acid than typical carboxylic acids because the anion can be stabilised
by intramolecular hydrogen-bonding from other protic groups on citric acid.
Measurement
1. Citric acid has been used as an additive to soft drinks, beer, and seltzer, and occurs naturally in
many juices. For soft drinks and orange juice the best measure of sweetness is the sugar/acid ratio.
2. Recently, the use of infrared sensors has allowed measurement of both Brix (sugar content) and
acidity by detecting sugars and citric acid through their characteristic molecular vibrations; this
gives an accurate assessment of a drink's sweetness.
Krebs cycle
1. Citric acid is one of a series of compounds involved in the physiological oxidation of fats, proteins,
and carbohydrates to carbon dioxide and water.
2. This series of chemical reactions is central to nearly all metabolic reactions, and is the source of twothirds of the food-derived energy in higher organisms.
9
3. Hans Adolf Krebs received the 1953 Nobel Prize in Physiology or Medicine for the discovery. The
series of reactions is known by various names, including the citric acid cycle, the Krebs cycle, and the
tricarboxylic acid cycle (or TCA cycle).
Uses
Food additive


As a food additive, citric acid is used as a flavoring and preservative in food and beverages,
especially soft drinks.
The buffering properties of citrates are used to control pH in household cleaners and
pharmaceuticals.
Water softening















Citric acid's ability to chelate metals makes it useful in soaps and laundry detergents. By
chelating the metals in hard water, it lets these cleaners produce foam and work better
without need for water softening.
Citric acid is the active ingredient in some bathroom and kitchen cleaning solutions. A
solution with a 6% concentration of citric acid will remove hard water stains from glass
without scrubbing. In industry it is used to dissolve rust from steel.
Citric acid is commonly used as a buffer to increase the solubility of brown heroin. Singleuse citric acid sachets have been used as an inducement to get heroin users to exchange their
dirty needles for clean needles in an attempt to decrease the spread of AIDS and hepatitis.
Other acidifiers used for brown heroin are ascorbic acid, acetic acid, and lactic acid; in their
absence, a drug user will often substitute lemon juice or vinegar.
Citric acid is one of the chemicals required for the synthesis of HMTD, a highly heat-,
friction-, and shock-sensitive explosive similar to acetone peroxide. For this reason,
purchases of large quantities of citric acid may rouse suspicion of potential terrorist activity.
Citric acid can be added to ice cream to keep fat globules separate, and can be added to
recipes in place of fresh lemon juice as well. Citric acid is used along with sodium
bicarbonate in a wide range of effervescent formulae, both for ingestion (e.g., powders and
tablets) and for personal care (e.g., bath salts, bath bombs, and cleaning of grease).
Citric acid is commonly employed in wine production as a substitute or improver where
fruits containing little or no natural acidity are used. It is mostly used for inexpensive wines
due to its low cost of production.
Citric acid can be used in shampoo to wash out wax and coloring from the hair. It is notably
used in the product "Sun-in" for bleaching, but is generally not recommended due to the
amount of damage it causes.
Citric acid is also used as a stop bath as part of the process for developing photographic
film. The developer is normally alkaline, so a mild acid will neutralize it, increasing the
effectiveness of the stop bath when compared to plain water.
Citric acid is used as one of the active ingredients in the production of anti-viral tissues.
Citric acid can be used in food coloring to balance the pH level of the normally basic dye.
Citric acid is used as an odorless alternative to white vinegar for home dyeing with acid
dyes.
Citric acid may be used as the main ripening agent in the first steps of making mozzarella
cheese.
Citric acid was the first successful eluant used for total ion-exchange separation of the
lanthanides, In the 1950s, it was replaced by the far more efficient EDTA.
Citric acid is used as a successful alternative to nitric acid in the process of stainless steel
passivation (ie "Citrisurf")
10


Citric acid can be used as a delay to prompt natural cement. It can delay the very rapid
setting time substantially.
Citric acid is one of several acids that is used by home brewers to modify brewing water for
making beer.
Safety
1. Contact with dry citric acid or with concentrated solutions can result in skin and eye irritation, so
protective clothing should be worn when handling these materials.
2. Excessive consumption is capable of eroding the tooth enamel.
3. Contact to the eyes can cause a burning sensation, and may cause blindness with prolonged
exposure in extremely high concentrations (as anything with low enough pH will).
4. Sometimes a high concentration of citric acid can damage hair and bleach it.
VITAMIN
Explain about vitamin B12 production by fermentation.
Introduction:
 Vitamins are low molecular weight compounds indispensable for vital activity of the organism.
 In 1926, G.R Minot & W.B.Murphy used liver extract to treat pernicious anaemia and was awarded
Nobel Prize in medicine in 1934.
 Later in 1948 E.L Rickie and L.Smith isolated crystalline form of Vitamin B12 from liver extract.
Production by various modes:
 Activated sludge process (recover process expensive).
 Chemical process (tedious process).
 Microbial fermentation process (By-product during streptomycin production and during
production of chloramphenicol and Neomycin).
Function of Vitamin B12:
 It acts as coenzyme.
 Essential for formation of matured RBC involved in synthesis of nucleic acids.
 Stimulates the production of WBC and platelets.
 Helps in growth of microorganisms.
 Prevents hyperglycemias and pernicious anaemia.
Deficiency diseases:
 Deficiency of vit B12 leads to pernicious anaemia and hyperglycemia.
 In pig and rats it slows done the rate of growth, nervousness & immobility.
Production of vitamin B12:
Organism used: Streptomyces olivaceous NRRL B-1125.
Preparation of inoculum:
Streptomyces olivaceous NRRL B-1125
100-250 ml broth (Bennets broth)
Transferred to Erlenmeyer flask
11
Seed stage I
Seed stage II
Seed stage III
Final inoculums volume 5% of total volume
Production Medium:
Distiller soluble 4%
Dextrose
1%
CaCo3
0.5%
CoCl.6H2O
1.5 to 10 ppm
Bioparameters:
Temperature:
80 F
pH:
5
Aeration and agitation:
optimum
Time Duration:
5 days
Production using improved strains:
Bacillus megaterium (4mg/l).
Streptomyces olivaceous (3.3mg/l).
Production of vitamin B12: (Propioni bacterium)
The fermentation process can be divided in two phases:
 Phase I: Anaerobic (2-4 days).
 Phase II: Aerobic (3-4 days).
Recovery:
Vitamin B12
Bound to cell
Separated by heat treatment (80-120 c for 30 min)
Purified by pH alteration (6.5-8.5)
Further chemical treatment (80% purity)
Production of Vit B12 (Pseudomonas denitrificans MB 2436):
Preparation of inoculum:
 Lyophilized culture.
 Seed culture development.
Production media:
 Beet molasses.
 Yeast extract.
 MgSO4.
12
 ZnSO4.
 Dimethyl benimidazole.
 Cobalt salt.
Bioparameters:
Parameters
pH
Temperature
Duration
Specifications
7.4
30C
5 days
Recovery:
Cobalamin remain attached to the cell
PH 5, heat
Released
Filtration
Chromatography
Extraction
Drum Dry/ spray dry
Yield:
1-2 ml per Litre.
ANTIBIOTIC PRODUCTION
Antibiotics:
Industrial microbiology techniques are used to produce many antibiotics produced by microorganisms
mainly by actinomycetes belongs to the genus Streptomyces and also filamentous fungi. Synthesis of the
several of the most important antibiotics such as penicillin and streptomycin are discussed along with the
importance of media formulation and also environmental influence on the production of these antibiotics.
Penicillin:
Antibiotic penicillin is produced by Penicillium chrysogenum in large quantities. Production of penicillin
requires precise adjustment of the culture media composition to achieve maximum yield. Rapid cell
division can be achieved by adding high level of glucose as carbon source, but this will not yield high
antibiotics. But by using slowly hydrolyzed disaccharide lactose, in addition with limited nitrogen leads to
high production of antibiotic penicillin after growth has stopped. Same kind of yield can also be achieved
by using a slow and continuous feed of glucose as carbon source. To get a particular type of antibiotic
penicillin specific precursors are added to the culture media. For example antibiotic penicillin G, which has
got a benzyl side chain, can be produced in large quantity using precursor phenyl acetic acid. The pH of the
fermentation medium is maintained at around neutral by adding sterile alkali. This neutral pH provides
maximum stability to the newly synthesized antibiotic penicillin. Usually fermentation completes in six to
13
seven days, then the broth is separated from the fungal mycelium cells and antibiotic is downstream
processed using techniques such as absorption, precipitation and crystallization to get final pure form of
penicillin. This penicillin is then undergoes some chemical procedures to get a variety of semi synthetic
antibiotic
penicillin.
Streptomycin:
Streptomycin is produced by Streptomyces griseus, as secondary metabolite. Streptomycin production or
accumulation is influenced by the changes in environmental condition and also substrate availability. For
this fermentation process soybean-based medium along with glucose is used as carbon source. That is
nitrogen source is provided in this combined form or soybean meal form, this also acts as growth limiting
factor. After the initial growth, antibiotic production and also accumulation increases in the fermentation
medium.