BOTECHNOLOGY IS CHALLENGING SUBJECT TO TEACH AND UNDERSTAND ALSO .....THEIR INTERESTING PART IS TO LEARN ABOUT PRODUCTION OF CITRIC ACID , PENICILLIN, GLUTAMIC ACID , GRISIOFULVIN , VITAMIN B 12

1. BY
ASAWE TEJASWINI L
ASSISTANT PROFESSOR
SIDHHIS INSTITUTE OF PHARMACY
THANE

2.  Antibiotics, vitamins, enzymes and alcoholic products
is the main aim of the fermentation industry.
 Most pharmaceuticals are produced by the process of
fermentation using specific microbial strains .
 Antibiotics for therapy was discovered by scientists
Alexander Flaming in 1929 found that a world
Penicillin had an effect against Staphylococcus aureus.
 Penicillin was the first antibiotic commercially
produced from the Penicillin Species by the process of
fermentation.
 These antibiotics are mainly produced from fungi,
actinomycets and bacteria.
 This substances have wide application in medical
,veterinary and agriculture practices.
 These antibiotics produce by microbial fermentation
or chemical synthesis or combination of both.

3.  Production of Penicillin began in US in 1941 by
surface culture fermentation of Penicillin
notatum.
 Penicillin is produced from Penicillium
Chrysogenum by submerged culture techniques .
 Penicillin is effective against gram positive
bacteria and also some large viruses and
rickettsia.
 Natural penicillin penicillin V& G are effective
against several gram positive bacteria.
 They inhibit the bacterial cell wall synthesis and
destroy the cell.
 Penicillin is easily hydrolyzed by enzyme beta
lactamase.

4.  The selected strain of Penicillin Chrysogenum is
maintained in the form of a master culture and
preserve by mixing the spores in sterilized soils.
 For inoculum preparation spores from working solid
cultures are suspended in water .
 These spores are added in a flask containing nutrient
solution and incubated for four to six days at 25° C .
 The resulting force are used directly to inoculate
inoculum tank .
 The inoculum tanks are incubated for 48 hours with
agitation and aeration to grow more mycelium.
 The resulting inoculum is used for a production tank
or it is added to a second or even third stage
inoculum tank to produce more inoculum for large
scale fermentation .

5.  The exact composition of penicillin production media
used in the industry for production of penicillin is
unknown.
 A typical medium described by Jackson 1958 contains
fermentable carbohydrates such as corn steep liquor
solids 3.5%, lactose 3.5%, and glucose 1% ,
potential hydrogen phosphate 0.4%, calcium
carbonate 1% , edible oil 4% and Penicillin precursor .
 The PH of this medium after sterilization is 5 to 6.
 Inoculum Media are similar to production media
except the lactose and precursor are not added in the
Inoculum media.
 These medium compositions may be slightly changed
to increase the yields and meet economic changes.

6.  The media are placed in the fermentation vessel sterilize
and inoculated with a suspension of Penicillin
chrysogenum.
 The fermentation vessel is equipped with devices which
allow continuous addition of nutrients acid or bases to
maintain the PH 7 to 7.4 and cooling coils to maintain the
temperature 24 ° C .
 Maximum antibiotics are produced with four to five days
 The growth of mycelium (the mass of branched, tubular
filaments (hyphae) of fungi.)is occur in the 1st 20-30
hours.
 For fermentation the mycelium grows rapidly by utilizing
glucose , lactic acid and organic nitrogen compounds.

7.  fungal growth becomes very thick and heavy.
 Initially PH may decreases with glucose
utilization and production of acids ammonia is
liberated by Deamination(Removal of amino
group) of the amino acid of corn steep liquor
and ph becomes 7 to 7.5
 Glucose and lactic acid are utilized first them
lactose is available for continuation of
fermentation .
 Agar slant culture is used for culture bacterial
cells for identification

10.  Citric acid eats natural constituent and common metabolite of
plant and animals
 It is the most versatile and widely used organic acid in the field
of food and pharmaceuticals
 citric acid was isolated from lemons but today about 99 % of the
world’s citric acid produced from microbial fermentation .
 Citric acid fermentation was first observed by Wehmer in 1893
in the fungus species of Penicillium glaucum.
 Now Citric acid mainly produced by submerged fermentation
using aspargillus niger or Candida species from different
sources of carbohydrates .
 Citric acid is mainly used in food industry because of its high
solubility & pleasant taste .
 It is commonly used as a flavoring agent in food and beverages
example jam , jellies , candies, deserts , soft drinks etc .
 Citric acid also act as a antioxidant and preservatives .

11.  A large number of microorganisms are used
for production of citric acid including bacteria
example bacillus licheniformis,
corynebaterium sp.
 Molds like Aspargilius Niger , Penicillium
luteum, Penicillium citrinum , Candida
tropicals, Candida oleophila, Candida
Citroformans.
 Citric acid is primary metabolic product form
in tricarboxylic acid i.e kreb cycle

13.  Mutant species of Aspargillus Niger is commonly
used for production of Citric acid for commercial use
 Stock culture of species are maintained in the culture
tube in the form of lyophilized (freeze drying ) spores
or storage in refrigerator .
 Spores are produced in glass bottle on solid
substrates up to 12 to 14 days incubation at
25 to 27 °C
 A nutrient medium containing 15 % sugar from
molasses is used in seed fermentation to induce
mycelium formation in the form of pellets .
 The spores germinate at 30- 32 °C within 20 to 24
hours to form a pellets.
 These pellets are used as a inoculum for production
medium.

14.  Citric acid is produced by both surface as well as
submerged method
 In Surface fermentation method Microorganisms can grow
as a layer or a film on a surface of nutrient medium which
may be liquid or solid in nature.
 liquid surface fermentation is the oldest method for
production of citric acid the more sophisticated and simple
method requires less effort in operation and installation,
less energy cost and higher reproducibility .
 The inoculum in the form of spores is spread over the
medium and fungus develops as a mycelial mat on the
surface of the medium
 The trays are made of high purity of aluminum or special
grade steel .
 Sterilized nutrient solutions automatically flow over a
distribution system onto the trays .

15.  Microbial culture is inoculated in the incubation
chamber at 30 to 40 °C by blowing dry spore or
by spraying spore suspensions.
 The fermentation chambers are provided with
an effective air circulation to control the
temperature 30 °C and humidity .
 Within 24 hours after inoculation germinating
spores form a thin cover of mycelium on the
surface of medium.
 The mycelium is a part of a mushroom that is
very similar to the root system of any plant .
 The fermentation is stopped after 7 to 14 days

17.  Vitamin B12 is water soluble vitamin commonly known as
Cobalamin.
 It is an important dietary component for normal growth in human
being and animal Ricke E.L and Smith L in 1948 isolated small
amount of active material from liver and crystallized it and
determine Vitamin B12 which was active in the treatment of
pernicious anemia
 (a deficiency in the production of red blood cells through a lack of
vitamin B12).
 Vitamin B12 is one of the largest and most complex molecules
 The main part in the structure of B12 is porphyrin ring containing
cobalt as a central element.
 The cynide (CN)group, hydroxyl group(-OH) or nitride group (-
NO2) attached to the cobalt is called CynoCobalamine,
hydroxocobalamine and Nitritocobalamine respectively .
 Vitamin B12 is produced by bacteria and pseudomonas
denitrificans, bacillus subtilis, bacillus magaterium and
actinomycetes Nocardia species, streptomyces olivaceus ,
streptomyces griseus .

18. The main part in the
structure of B12 is porphyrin
ring containing cobalt as a
central element.
The cynide (CN)group,
hydroxyl group(-OH) or
nitride group (-NO2)
attached to the cobalt is
called CynoCobalamine,
hydroxocobalamine and
Nitritocobalamine
respectively .

19.  Pure culture of streptomyces olivaceus is
inoculated in inoculum medium contained in
Erlenmeyer flasks.
 Bennett's broth [ east extract 1% , Beef extract 1
%, Glucose 1 % and enzymatic hydrolysate of
casein 25% ] is employed for development of
Inoculum.
 Flash are kept on mechanical shaker during
incubation for the aeration
 These flask cultures are used to inoculate the
large amount of inoculums media the required
amount of inoculums ( 5% of volume of
production medium) is prepared by successive
transfers.

20.  The culture of streptomyces olivaceus is grown with aeration at
20 degrees Celsius in nutritionally reached crude medium
 distiller soluble 4% dextrous 1% and calcium carbonate 0.5% are
present in typical production medium used in production of
vitamin B12
 cobalt chloride( COCL2. 6H2O at approximately 2 -10 ppm is
added on this medium as a precursor
 The p H of the medium is adjusted to 7 to 7.5 before
sterilization and may or may not be controlled during
fermentation .
 In the 1st 24 hours the PH of fermentation is reduced due to
rapid sugar consumption and again ph increases after 48 to 96
hours due to lysis of mycelium.
 A correct rate of aeration and speed of agitation are required for
proper growth of microbial strains
 The optimum rate of aeration is about 0.5 volume air /volume
medium /minute
 Soya bean oil corn oil etc are used as a antifoaming agent to
suppress the foam formation

22.  Glutamic acid was first amino acid to be
produced by microorganisms cynebacterium
glutamicum
 Glutamic acid is widely used in the
production of monosodium glutamate which
is commonly known as a seasoning salt
 Monosodium glutamate is flavor enhancing
agent and it is commonly used in convenient
food stuffs
Glutamic acid

23.  L- glutamic acid can be synthesized from
wide species of bacteria actinomycets or
bacteria and fungi.
 In addition of corynebacterium glutamicum
the other different strains are used for the
production of acid such as E- coli, bacillus
subtilis, cephalosporium , bacillus
megatherium etc.

24.  A suitable strain of Corynebacterium
glutamicum from stock culture is used for
Inoculum development .
 This srain is inoculated in sterilized medium
and incubated at 35 °C for 16 hours.
 Sufficient inoculum 6% can be developed and
then added in a final production fermenter

25.  The production medium consists of carbon
sources nitrogen sources minerals and vitamins
one of production medium for glutamic acid
contains glucose k2HP of four KH2PO4 , KH2PO4
, MnSO4.7H2O, urea and biotin .
 Carbon sources Such as glucose , sucrose ,
fructose , maltose , sugarcane molasses are
commonly used in fermentation.
 Since Mueller says he had high biotin content
penicillin or fatty acid derivatives must be added
to the fermentation
 Glutamic acid is intracellular components hence
production and excretion is depend upon cell
permeability

26.  Permeability (the quality liquid or gas passed
through medium ) of acid producing bacteria can
be increased by growth under biotin limiting
condition , addition of penicillin and saturated
fatty acids and use of oleic acid and glycerol
auxotroph.(does not contains nutrient medium )
 The concentration of ammonia Is very important
for converting carbon sources of glutamic acid
 Biotin is an important growth factor an essential
coenzyme in fatty acid synthesis .
 Seed fermenter are used to regenerate or refresh
the microorganism during fermentation process.
 Anion exchangers are used for separation and
purification of acid from fermentation broth
(liquid medium contains nutrients)