The document discusses the history and production process of penicillin, the first naturally occurring antibiotic, discovered by Alexander Fleming in 1928. It details the fermentation process using the high-yielding strain Penicillium chrysogenum, methods of maintaining and preparing inoculum, as well as the recovery and purification of penicillin. Emphasis is placed on the optimal environmental conditions and raw material requirements necessary for maximizing penicillin yield.

2. THE WONDER DRUG
PRESENTED BY:
Anamika- M.Sc. MB
Nayan Dhiman - M.Sc. MB
Jyoti Bayalkoti - M.Sc. MB
Date- 6/12/2021
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3. ANTIBIOTICS
• The term antibiotic has been defined by Selman Waksman as being an organic
compound produced by one microorganisms that inhibits the growth of or kills a
group of harmful microorganism.
• These enter and stick to important parts of bacterial cell and interfere with its
ability to survive and multiply.
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4. FIRST NATURALLY OCCURRING ANTIBIOTIC
Fleming was already well known upon his
return from a long holiday in September
1928,he noted that one petri dish contained
colonies of staphylococcus bacteria expect
for clear area contaminated with a fungus
that appeared to inhibit bacterial growth
The mold was found to be Penicillium
notatum.
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5. GENERAL STRUCTURE OF PENICILLIN
• Penicillin is not a single compound but a group of closely related compounds,all
with the same basic ring like structure (a beta lactam) derived from two amino
acid(valine and cysteine) via a tripeptide intermediate .
• The third amino acid of this tripeptide is replaced by an acyl group(R) and the
nature of this acyl group produces specific properties on different types of
penicillin
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6. PENICILLIN PRODUCTION
Aim: To produce sterile,controlled and homogenous environment in which the
fermentation can carry out in a manner that is safe and practical and which
optimizes the Penicillin production for ultimate use.
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7. .
INOCULUM PREPARATION
Inoculum : A small amount of material
containing bacteria,viruses or other
microorganisms that is used to start a culture.
Out of various species of the fungus
Penicillium ,mainly two species used in the
fermentation .These are Penicillium notatum
and Penicillium chrysogenum
WHY Penicillium chrysogenum?
Penicillium chrysogenum is high yielding
strain and therefore most widely used as
production strain.
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8. MAINTENANCE OF STRAIN
• The production strain should be carefully
maintained because appropriate maintenance and
production of reliable pure culture with desirable
quality is a key operation.
• Production strains are stored in dormant form by
any means-
a) A spore suspension may be mixed with a sterile,
finely divided inert support
b) Spore suspension can be lyophilized in
appropriate media
c) Spore suspension can be stored under liquid
nitrogen
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9. INOCULUM BUILD UP
• The purpose - to develop a pure inoculum in an adequate amount and in the fast
growing phase for the production stage fermenter.
• The starter culture- it is transferred into agar-plate to allow growth.
• When spores are placed on a vegetative media, the spores start to germinate and form
mycelia.
• After growth on agar plate, cultures were incubated for approx. 70h in a rotary shaker
at 200 rpm an 25 to improve oxygen diffusion.
• Two growth stages ( 2 days incubation) are allowed upon transfer into seed fermenter.
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10. • Initial stages of fermentation are designed for considerable microbial growth and the can
be distinguished from main Bioreactors by several factors ,these are :
1. Smaller in size than the main fermenters approx.2 litre capacity.
2. Made of stainless steel.
3. Equipped with agitators which allow continuous mixing of growth medium
4. Provided with pump to deliver sterilized ,filtered air .
Must contain all the nutrients including Growth factors .
After about 24-28 hrs, the content of the seed tanks is transferred is transferred to fermentation
tank.
Seed Fermenters
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11. RAW MATERIAL
• An abundant growth of mycelium
• Maximum accumulation of penicillin and
• Ease of extraction and purification of the antibiotic.
• Raw materials are primary requirement to design the
fermentation broth.
CARBON SOURCE NITROGEN SOURCE AMMONIUM SOURCE
● Lactose acts as a carbon
compound.
● Corn oil supplemented
with lactose.
● Corn steep liquor (CSL)
is used as important
sources as it results in
higher penicillin yields.
● Cottonseed /soybean
meal can be used.
● Continuous
addition of
ammonium sulfate
is required.
● Concentration
around 250-300
mg/L is required. 10

12. PRECURSORS
• Certain precursor of the penicillin side
chain needed to be added into the
fermentation medium.
• This constitute a major cost item-
Penicillin G, requires 0.47g Phenyl
acetate.
• The precursor must be added
repeatedly in small amounts during the
fermentation process. BUT WHY?
• PAA can be substituted by other ring
system. It is transported across the
plasma membrane by free diffusion into
mycelium.
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13. PRODUCTION
INSIDE THE BIOREACTOR
FIRST PHASE SECOND PHASE THIRD PHASE
● Growth of mycelium occurs,
yield of antibiotic is low.
● Lactic acid present in corn
steep liquor is utilized at the
maximum rate by
microorganisms
● Lactose is used slowly
● Ammonia is liberated into the
medium resulting into the rise
in pH.
● There was intense synthesis of
penicillin in this phase due to
rapid consumption of lactose
and ammonia, nitrogen.
● The mycelia mass increase.
● The pH remain unchanged.
● The concentration of
antibiotic decreases in
the medium.
● The autolysis of
mycelium starts
liberation of ammonia
and slight rise in pH.
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15. RECOVERY
a) Filtration:
• The penicillin rich filtrate is cooled to 2- 4 to avoid chemical or enzymatic degradation
of penicillin. It is usually achieved by using high capacity, rotary vacuum filters for
separation of mycelia. Mycelia is filtered and disposed.
a) Extraction:
• Solvent extraction is accomplished by at low pH such as- 2.5- 3 using amyl acetate as
solvent.
• Continuous, countercurrent, multistage centrifugal extractor are used for this purpose to
avoid degradation of penicillin during solvent extraction at low pH, temperature is kept
around 2-4 and filtration for 1-2 min.
• Two extractors used in series result in 99% penicillin recovery.
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16. 1. Adsorption:
• Carbon adsorption is used to remove impurities and pigments from penicillin-
rich solvent after extraction. Several activated carbon columns in series can be
used.
1. Crystallization:
• Sodium and penicillin concentrations, pH and temperature need to be
adjusted for this purpose.
• Excess amount of sodium are added to the penicillin- rich solvent before
crystallization in an agitated vessel.
• The crystals are separated by a rotary vacuum filter.
• The crystals are washed and pre-dried with anhydrous butyl alcohol to
remove some impurities.
• Large horizontal belt filters are used for collection and drying of the
crystals.
PURIFICATION
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18. RESULT

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