Penicllin production

2. History:Discovery &Production
● 1928: Scottish biologist, Alexander Fleming
discovered that the Staphylococcus culture he
had mistakenly left growing in open was
contaminated with a mould which had
destroyed the bacteria.
● After isolating a sample and testing it, he
found that it belonged to the Penicillium
family.
Later the mould was classified as Penicillium
notanum.
● At first, it was difficult to convince people
about its potential uses.

3. History:Discovery &Production
● But later (1939), using Fleming’s work, two medical
researchers, Howard Florey and Ernst Chain
managed to purify penicillin in a powdered form.
● 1941: They successfully treated a human.
● 1943: They produced penicillin on a large scale.
This helped immensely to treat casualties during the
WWII that had bacterial infections due to their
wounds.

4. General Structure ofPenicillins

5. General Structure of Penicillins
● Have β-Lactam functional group, thus they belong to
the β-Lactam antibiotic group.
● They all have a basic ring-like structure (a β-Lactam)
derived from two amino acids (valine and cysteine)
via a tripeptide intermediate. The third amino acid of
this tripeptide is replaced by an acyl group (R).
The nature of this acyl group produces specific
properties on different types of penicillin.

6. Penicillin Derivatives
● Derivatives produced to deal with the problem of
resistance to resistance to penicillin.
● All penicillin or penicillin
derivative have a constant core
region which is the 6-APA.
● The only region that is different
from different types of penicillin
derivative is its R group.

7. How Does PenicillinWork?
●Inhibits the synthesis of peptidoglycan in cell walls.
○ β-Lactam of penicillin binds to the enzyme -
transpeptidase, that is used in the formation of
peptidoglycan cross linking.
○ The enzyme is inhibited, thus inability to form cross
linking.
○ Cell wall is weakened causing osmotic imbalance in the
cell. This leads to cell death.
● As human cells do not have cell walls, penicillin does not affect
them.

8. • Penicillin is produced by the fungus
Penicilium chrysogenum which requires
lactose, other sugars, and a source of
nitrogen (in this case a yeast extract) in the
medium to grow well.
• Like all antibiotics, penicillin is a secondary
metabolite, so is only produced in the
stationary phase.

9. • It exhibits the properties of a typical secondary metabolites.
• It active against certain Gram-positive bacteria in presence
of blood, pus and body fluids.
• It is soluble in water. It is very soluble in acetone, ethyl
alcohol and ether and it is less soluble in benzene,
chloroform.
• Aqueoussolution of penicillin are unstable and must be
stored under refrigeration.
• Penicillinis most stable in the pH range of 6.0 to 6.5 and
reasonably stable over the pH range of 5.5 to 7.5 .
Properties of penicillin :

10. • The Inoculum : Master stock (spores) is the source of
inoculum. Spores (P. chrysogenum) from working stocks cultured
are suspended in water or non toxic lauryl sulphonate, then added
to the flask containing wheat bran and nutrient soln. A shake flask
culture of 4 day old is inoculated into a seed tank for 3 days.
• The medium : A typical media for penicillin production is
given below . The major constituent of typical medium includes,
Commercial production of penicillin :
1) Fermentable carbohydrate
2) Potassium dihydrogen phosphate
3) Organic nitrogen source
4) Phenyl acetic acid precursor
5) Edible oil
6) Calcium carbonate (act as buffer)
7) pH after sterilization
Corn steep liquor(3.5%),
lactose(3.5%),glucose(1%).
0.4%
0.25%
1%
5.5 to 6.5

11. ● pH 6.5
● Temperature 20-24 °C
● Oxygen
● Nitrogen: corn steep liquor 8.5 %
● Glucose 1%
● 80% ethanol
● Phenylacetic acid
● Lactose 1%
● Calcium Carbonate 1%
● Sodium hydrogen phosphate 0.4%
● Antifoaming agent: vegetable oil

12. • Procedure :

13. ★ Fermentation
It is done in a fed-batch mode as glucose must not be added
in high amounts at the beginning of growth (which will result
in low yield of penicillin production as excessive glucose
inhibit penicillin production).
The fermentation conditions for the Penicillium mold, usually
requires temperatures at 20-24°C while pH conditions are
kept at 6.5. The pressure in the bioreactor is much higher than
the atmospheric pressure (1.02atm). This is to prevent
contamination from occurring as it prevents external
contaminants from entering.

14. It is necessary to mix the culture evenly throughout the
culture medium. Fungal cells are able to handle rotation
speed of around 200 rpm.
Fermentors

15. • Aeration (oxygen supply) : supply of oxygen in a
bioreactor is the limiting factor in penicillin biosynthesis.
Aeration speed is between 150 to 250 rpm.
• Temperature : Temperature plays an important role in
penicillin production it should be maintained at 25° C.
• Biomass production : production of penicillin depends
upon biomass production therefore, it is describle to
have a high biomass concentration in the vessel. It is
achieved by increasing the agitation rate and power.
• pH : It is maintained early neutrality by calcium and
magnesium carbonate in the medium by phosphate
buffer. It is also controlled by adding sodium hydroxide
or sulphuric acid in the medium.

16. Important Process Parameters
● Mass Transfer: Good transfer of oxygen across the liquid
interface-the Sparger delivers this oxygen efficiently.
● Heat Transfer: metabolism as a process tends to give off heat-
achieved through cooling jacket whereby cool water is passed
through.
● Bulk Flow and Mixing: Impellers, bubble columns or loop
reactors.
● Batch, Fed-Batch and Continuous Culture: how nutrients
and substrate will be delivered to a culture in a reactor.
● Steam: Used to keep the reactor running aseptically.
(temperature/pressure of 121°C/15 psi for 15-30min).

17. Batch fermenter

18. Harvested culture broth includes penicillin G
along with a variety of other metabolites .
Vacuum filter is used for separation of mycelium from
the broth on a rotary .
Conversion of penicillin to the anionic form occurs at
low pH (2.0 to 2.5). The lowering of pH is done by
adding phosphoric acid or sulphuric acid.

19. For removal of pigment and other impurities
from solvent containing penicillin it is treated
with active charcoal.
The product is back extracted into water from
solvent by adding potassium or sodium
hydroxide to form its sail.
The product of penicillin is then crystallized into
sodium or potassium penicillin.

20. • Antibiotics are probably the most important group of
Conclusion :
• compounds synthesized by industrial microorganisms.
• The best known and probably the most medically
important antibiotics are the β- lactam, penicillin and
cephalosporin's.
• Penicillin exhibits the properties of a typical secondary
metabolites.
• Penicillin is commerciallyproduced usually via a fed batch
• process carried out aseptically in stirred tank fermenters.