Penicillin Fermentation
Introduction:
Fermentation is the process used for the large-scale production of an antibiotic. The first
discovered natural antibiotic was Penicillin.
Penicillin was obtained from multicellular fungi, “Penicillium molds”.
Penicillin is a group of compounds having common basic nucleus, 6-amino penicillinic acid (6-
APA). 6-APA contains ring like structure termed as a β-lactam ring.
Penicillin are of two different types,
i. Natural Penicillin
ii. Semi-synthetic Penicillin
Natural penicillin is directly harvested from the penicillium mold.
Semi-synthetic penicillin consists of the basic Penicillin nucleus (6-APA), but with new side
chain that change properties of natural penicillin. Semi synthetic penicillin includes Ampicillin,
Methicillin, Penicillin V, etc. They are produced by modifying natural penicillin by removing the
natural acyl group to leave 6-APA and then adding new acyl groups having different properties
like,
i. resistance to stomach acids so it can be taken orally
ii. a degree of resistance to penicillinase (penicillin degrading enzyme)
iii. an extended range of activity against some Gram-negative bacteria.
USES:
It is a broad spectrum antibiotic. It acts against gram positive and gram negative bacteria.
Used to treat the infections caused by streptococcus, meningococcal infections, syphilis,
anthrax, gas gangrene and rat bite fever etc.
History
In 1928, Sir Alexander Fleming made one of the most important contributions to the field of
antibiotics. He first observed the antibiotic properties & therapeutic value of penicillin.
It interferes with cell wall synthesis of actively growing sensitive organisms. It mainly inhibit the
cross linking steps of peptidoglycan synthesis in the cell wall.
The World War II had brought a demand for penicillin on a large scale for the treatment of burns
and wounds. In 1945 Fleming, Florey and Chain were awarded the Nobel Prize in Physiology
and Medicine.
Penicillin Fermentation
Microorganisms
Out of various species of the fungus Penicillium mainly two species are used in the fermentation
are P. notatum & P. chrysogenum.
Even from these two P. chrysogenum is high yielding strain and therefore most widely used as
production strain. The production strain is improved by mutation with the help of X-rays or any
other agents to give high yield.
Inoculum preparation
Here the chief purpose is to develop a pure inoculum in an adequate amount and in the fast
growing phase for the production stage fermenter.
1. A starter culture which is available in cold-stored form is transferred to an agar containing plate
to allow growth.
2. After getting growth on solid media, one or two growth stages should allowed in shaken flask
cultures to create a suspension, which can be transferred to seed tanks for further growth.
3. After about 24-28 hours, the content of the seed tanks is transferred to the primary fermentation
tank and then to the main fermentation tank, which contains same growth media and also
provides proper growth promoting environment.
Fermentation: Submerged fermentation
Fermentation Parameters
pH: near 6.5
Temperature: 26°C to 28°C
Aeration: a continuous stream of sterilized air is pumped into it - 0.5 – 1 Vol of air/min
Agitation: have baffles which allow constant agitation.
Fermentation time: 5 – 6 days
Raw Materials
Raw materials are primary requirement to design the fermentation broth for antibiotic production.
Generally, it contains a carbon source, nitrogen source, mineral source, precursors and antifoam
agents if necessary.
Carbon Source
Lactose acts as a very satisfactory carbon compound if it is used in a concentration of 6%.
Other carbohydrates like glucose & sucrose may be used but it has to provide with slow feeding
rate.
Complex as well as cheap sources like molasses or soy meal can also be used which are made up
of lactose and glucose sugars. These materials are desired as a food source for the organisms.
Nitrogen Source
Ammonium salts such as ammonium sulfate, ammonium acetate and ammonium lactate or
ammonia gas are used for this reason. Sometime corn steep liquor may be used.
Mineral Source
These elements include phosphorus, sulfur, magnesium, zinc, iron, and copper which generally
added in the form of water soluble salts.
Precursors
Various types of precursors are added into production medium to produce specific type of
penicillin. Eg: if phenyl acetic acid is provided then only penicillin-G will be produced but if
hydroxy phenyl acetic acid is provided then penicillin-X will be produced.
Phenoxy acetic acid is provided as precursor for penicillin-V production.
When corn steep liquor is provided as nitrogen source, it also provides phenyl acetic acid
derivatives; therefore it is widely used in the production of penicillin-G.
Anti-foam agents
Anti-foaming agents such as lard oil, octadecanol and silicones are used to prevent foaming
during fermentation.
Following three points should be kept in mind before choosing raw materials for manufacture of
penicillin,
1. An abundant growth of mycelium
2. Maximum accumulation of penicillin
3. Ease of extraction and purification of antibiotics.
Production medium
Components Percent (%)
Lactose 3.5 to 6
Calcium Carbonate 1.0
Cornsteep Liquor 3.5
Glucose 1.0
Phenyl acetic acid 0.5
Sodium hydrogen phosphate 0.4
Antifoaming Agent: Edible oil 0.25
In inoculum medium lactose is generally absent because it induces penicillin production and
retarding the growth of production strain.
Recovery
The recovery of penicillin is carried out in three successive stages:
1. Removal of mycelium
2. Counter current solvent extraction of penicillin
3. Treatment of crude extracts
The resulting crystalline penicillin salts, sodium or potassium penicillinate are then washed and
dried.
Sometimes the crude extract of penicillin is passed out from charcoal treatment to eliminate
pyrogens; even sterilization can also be done.
Sterile vials are used for packaging of an antibiotic either as a powder or suspension.
For oral use it is tableted usually with a film coating.
Yield: 0.4 gallons from 1 gallon
