This document discusses the production of penicillin. It begins with an introduction to antibiotics and penicillin. It then covers the history of penicillin's discovery. The structure and mechanism of action are described. The document outlines the biosynthesis process and commercial production process, including preparation of inoculum and medium, fermentation, removal of mycelium, extraction, purification and drying. Advantages and disadvantages of penicillins as antibiotics are discussed. Finally, the clinical applications of penicillin are presented.

1. UNIVERSITY COLLEGE OF ENGINEERING
BHARATHIDASAN INSTITUTE OF TECHNOLOGY
ANNA UNIVERSITY, TIRUCHIRAPPALLI-24
DEPARTMENT OF BIOTECHNOLOGY
SUBJECT CODE/ TITLE: BY4201/ BIOSEPARATION
TECHNOLOGY
Presented by
Name: U.Aathilakshmi
Reg no: 810021509001

2. INTRODUCTION
• Antibiotics are antimicrobial agents produced naturally by other microbes (usually fungi
or bacteria). The first antibiotic was discovered in 1928 by Alexander Fleming from the
filamentous fungus Penicilium .
• 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.
• Many derivatives of penicillin are available to kill the majority of bacteria than penicillin.
Although, it is useful in killing different types of bacteria like Streptococcus pneumoniae,
Listeria species, etc.

3. HISTORY
• 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 chrysogenum.
• 1939: Howard Florey and Ernst Chain managed to purify
penicillin in a powdered form.
• 1941: They successfully treated to 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. STRUCTURE OF PENICILLIN
• The basic chemical structure of all penicillin consists of a beta-lactam ring, a thiazolidine
ring, and a side chain (6-aminopenicillanic acid).
• Formula of penicillin G : C16H18N2O4S
• β-lactam ring contains an amide bond that is broken in an acidic and alkaline medium and
that bond is hydrolyzed by beta-lactamase which is synthesized by many bacteria.
• Naturally occurring penicillin have different structure which is separated by R groups.

5. THERAPEUTIC CLASSIFICATION OF PENICILLINS

6. MECHANISM OF ACTION
• Penicillin kills bacteria by inhibiting the completion of the synthesis of
peptidoglycans, the structural component of bacterial cell wall.
• It specifically inhibits the activity of enzymes that are needed for the cross-
linking of peptidoglycans during the final step in cell wall biosynthesis. It does this
by binding to penicillin binding proteins with the β-lactam ring, a structure found on
penicillin molecules.
• This causes the cell wall to weaken due to fewer cross-links and means water
uncontrollably flows into the cell because it cannot maintain the correct osmotic
gradient. This results in cell lysis and death.

7. BIOSYNTHESIS PROCESS OF PENICILLIN
Penicillin yield is done commercially by using
P.chrysogenum. Although the fungus was found
earlier in 1928, these biosynthesis processes were
concluded later.
Penicillin biosynthesis is described into three
main steps;
 Catalytic step
 Oxidative
 Exchange of different chains.

8. PRODUCTION OF PENICILLIN
The major steps in the commercial production of penicillin are as follows:
1. Preparation of inoculum.
2. Preparation of medium.
3. Inoculation of the medium in the fermenter.
4. Removal of mold mycelium after fermentation.
5. Extraction and purification of the penicillin.

9. Production process of penicillin

10. PREPARATION OF INOCULUM
• A strain of the fungus is sub-cultured from stock culture for inoculum development.
• Spores from primary source are suspended in water or in a dilute solution of a nontoxic
wetting agent such as 1:10000 sodium lauryl sulfate.
• The spores are then added to flasks or bottles of wheat bran plus nutrient solution and these
are incubated for five to seven days at 24°C so as to provide heavy sporulation.
• The resulting spores are used directly to inoculate inoculum tanks or stirred fermenters.

11. PREPARATION OF MEDIUM
• The medium employed for penicillin production should be suitable to achieve:
1. An abundant growth of the mycelium.
2. Maximum accumulation of the antibiotic.
3. Easy and inexpensive extraction and purification of the antibiotic.
• Penicillin-F and penicillin-K are the naturally produced penicillins synthesized by P.
notatum and P. chrysogenum, respectively, in the absence of precursor.
• But, if phenylacetic acid is supplied in the medium P. chrysogenum produces penicillin-G
instead of penicillin-K. Similarly, desired synthetic penicillins can be obtained by adding
the medium with suitable precursor.

12. Media formulation
• Method: fed-batch or batch reactor.
• Substrate: Corn steep liquor, glucose, phenoxyacetic acid (fed component used for
production of side chain),Additional nitrogen source , carbon source (lactose).
• Fermenter: stirred tank or air lift tank.
• pH: set at 5.5 t0 6.0 which increased up to 7-7.5 (optimum) due to liberation of NH3 gas and
consumption of lactic acid. If pH is 8 or more, CaCO3 or MgCO3 or phosphate buffer is
added.
• Temperature : 25-27 °C
• Agitation : 120-150 rpm.
• Time : 3-5 days.
• Antifoam : edible oil (0.25%).

13. INOCULATION OF THE MEDIUM IN THE
FERMENTER
• The medium surface is inoculated with dry spores. Since the spores of P. chrysogenum are
hydrophobic, either spores are blown deep into the medium or a wetting agent such as
sodium lauryl sulphate is used.
• Fermenters with a capacity of 40,000 to 2 lakhs liters are generally employed for the
production of penicillin. Due to difficulties with the oxygen supply larger tanks are not
employed.
• Depending upon the production strain, the operational temperature is maintained
between 25°-27°C.
• Penicillin yields with time are linear from approximately 48 to 96 hours. The final penicillin
yield is in the range of 3 to 5% which largely depends upon the amount of carbohydrate
consumed during fermentation process, which is approximately equal to 1500 international
units per milliliter

14. Separation of Mycelium:
• The first step in harvesting penicillin is to
remove the cells by filtration.
• Mycelium is separated from the medium by
employing rotatory vacuum filter.
• This stage is done under conditions that avoid
contamination of the filtrate with enzymes,
which may destroy the penicillin.
REMOVAL OF MOLD MYCELIUM AFTER
FERMENTATION

15. • Extraction of penicillin is carried out by employing counter current extraction method.
The pH of the liquid after separation of the mycelium is adjusted to 2.0 to 2.5 by adding
phosphoric or sulphuric acid.
• The liquid is immediately extracted with an organic solvent such as amyl acetate or butyl
acetate.
• The penicillin is then back extracted into water from the organic solvent by adding enough
potassium or sodium hydroxide which also results in the elevation of pH to 7.0 to 7.5.
• The resulting aqueous solution is again acidified and re-extracted with organic solvent.
These shifts between the water and the solvent help in the purification of the penicillin.
• Finally, the penicillin is obtained in the form of sodium penicillin. The spent solvent is
recovered by distillation for reuse.
EXTRACTION AND PURIFICATION

16. DRYING
• Drying completely removes the moisture content from the penicillin salt.
• This is done through drying method-fluid bed dryer where gas(hot) is pumped into
chamber base (where penicillin is present) passing to a vacuum chamber resulting in
obtaining a dried form of Penicillin.
• It may be packed as powder in sterile vials or prepared in the form of tablets or in the
form of syrups for oral usage.
• The pharmaceutical grade may be used in the production of semi synthetic penicillin.

17. ADVANTAGES AND DISADVANTAGE OF
PENICILLINS AS ANTIBIOTICS
Advantages
• Bactericidal against sensitive strains.
• Excellent tissue penetration. Nontoxic.
• Effective in the treatment of bacterial
infections.
• Inexpensive compared to other antibiotics.
• Newer penicillin are resistant to the
acidic pH of the stomach, e.g. penicillin V
and broader-
• Spectrum penicillin like ampicillin and
amoxicillin.
Disadvantage
• Most are destroyed by gastric acid.
• Lack of activity against Gram-
negative bacteria.
• Intramuscular administration of drugs
is painful.
• Short duration of action.
• Many patients experience
gastrointestinal (GI) tract upsets.

18. CLINICALAPPLICATION
• Urinary tract infections (UTIs).
• Respiratory tract infections (RTIs).
• Meningitis.
• Gonorrhea and syphilis.
• Typhoid and bacillary dysentery.
• Bone and joint infections
• Bronchitis and pneumonia.
• Skin and soft-tissue infections.

19. REFERENCES
• Sahoo, B.M. and Banik, B.K., 2020. Therapeutic Potentials of β‐Lactam: A Scaffold for New
Drug Development. Synthetic Approaches to Nonaromatic Nitrogen Heterocycles, pp.59-88.
• https://biologyreader.com/production-of-penicillin.html.
• https://www.wikiwand.com/en/Penicillin.