Penicillin- Harvest & Recovery

 Media components have a great effect on yields in Penicillin fermentation.

 CSL- provides some carbon nutrients, precursor phenyl acetic acid, and peptides, amino acids and amines which are deaminated to produce ammonia required to bring the pH to 7-7.5

Glucose- rapid utilization of this simple sugar allows mycelial growth, but no penicillin production

Lactose- slow degradation to glucose and galactose, ensures slow glucose availability and starvation condition to induce penicillin production

Lipids/fatty oils/edible oil/lard oil/ soybean oil/linseed oil – antifoam and also, increase mycelium growth and give better yields

CaCO₃ – maintain pH

Harvest & Recovery

After sufficient amount of penicillin is produced during fermentation process, it is extracted and then purified. Extreme care is taken to avoid contaminating microorganisms which produce penicillinase enzyme, degrading the penicillin.

The recovery of penicillin is carried out in three successive stages:

1. Removal of mycelium

2. Extraction of penicillin

3. Treatment of crude extracts 

(a) Separation of Mycelium: The penicillin is excreted into the medium and less than 1% remains as mycelium bound. The fermentation broth is filtered on a rotatory vacuum filter to remove the mycelium and other solids.

(b) Extraction of Penicillin:

·         Extraction of penicillin is carried out by 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. This treatment converts penicillin into anionic form.

·         The liquid is immediately extracted with an organic solvent such as amyl acetate or butyl acetate or methyl isobutyl ketone. This step is done quickly because penicillin is quite unstable at low pH values. Podbielniak counter current extractor is used for this purpose.

·         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.

(c) Treatment of Crude Extract: The sodium salt of penicillin obtained is treated with charcoal to remove colour producing substances and pyrogens (fever causing substances). It may be sterilized to remove bacteria by using Seitz filter. Then, the sodium penicillin is prepared in crystalline form by crystallization.

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 penicillins

Ø  Waste Disposal

•      Spent broth/mycelium, wash waters, residual organic solvents etc are wastes with high BOD
• Ø   Mycelium dried and used as feed supplement/buried
• Ø   Solvents recovered by distillation for reuse
• Ø   Aqueous fractions are treated and discharged to sanitary sewage systems

 Uses of Penicillin:

1. Most of the penicillins are active against Gram-positive bacteria, in which they inhibit the cell wall synthesis leading to the death of bacteria.

2. Used therapeutically in the treatment of infectious diseases of humans caused by Gram (+) positive bacteria.

3. Synthetic penicillins are active against Gram-negative bacteria, even Pseudomonas aeruginosa. 

Natural Penicillins - Narrow range of activity and not stable in the presence of acid (acid-labile), so must be taken by intramuscular. Many of the semi-synthetic Penicillins can be taken orally.

Biosynthetic penicillin is natural penicillin that is harvested from the mould itself through targeted fermentation, by adding several precursors to produce desired penicillin.

Semi synthetic penicillins includes Ampicillin, Methicillin, Carbenicillin, Oxacillin, Penicillin V, etc.  They are produced by modifying natural penicillins by removing the acyl group from 6-APA using penicillin acylase enzyme, and then adding new acyl groups having different properties like,

 i resistance to stomach acids so it can be taken orally

ii  resistance to penicillinase/ β-lactamase (penicillin degrading enzyme)

iii extended range of activity against Gram-negative bacteria

 

Penicillin acylase

• Ø Enzyme used to produce semi-synthetic Penicillins by chemically modifying natural pencillins/pencillin nucleus (6-APA).
• Ø  Penicillin acylase cleaves 6-aminopenicillanic acid and removes its acyl group.
• Ø  Later other acyl groups can be added by chemical treatments to produce pencillins with new and desired properties.

 

Penicillinase

Ø  Penicillinase is an extracellular enzyme adaptively produced by Staphylococcus aureus, coliforms, Bacillus sp. etc

Ø  Major factor for penicillin resistance during infection.

Ø  It hydrolyses penicillin to penicilloic acid.

 

6-amino penicillanic acid

Ø  Sensitive to Penicillinase enzyme.

Ø  Can be recovered from fermentation broth.

Ø  Chemically converted to penicillin by chemical addition of various R side chains. Yield of this material is low.

Ø  Penicillin acylase enzyme cleaves side chains of penicillin so resulted in commercial production 6-Amino penicillanic acid.

 

References

Principles of Fermentation Technology:  (2nd  edition,  by Peter F. Stanbury, Allan Whitaker and Stephen J. Hall, Butterworth-Heinemann, An  imprint of Elsevier Science.)

Industrial Microbiology:  (By Casida L. E.New Age international (P) ltd publications)

A Text Book of Industrial Microbiology: (2nd edition By Wulf Crueger & Anneliese Crueger)