Antibiotic - defined by Selman Waksman as
“an organic compound produced by one microorganism that, at great dilutions, inhibits the growth of or kills another or even group of other harmful microorganisms”
Antibiotics are available in various forms- ointment, powders, capsules, etc. For example, to treat bacterial infection on the surface, an antibiotic should be there in an ointment or cream form. But to treat internal infection it can be directly injected into the bloodstream which finally distributed throughout the body.
Antibiotics are produced primarily by bacteria, Streptomyces, Nocardia and fungi. Antibiotics produced by Streptomyces spp. (Streptomycin, Neomycin etc..) have the most commercial applications.
On the basis of mode of action antibiotics are divided into
i) Antibiotics affecting cell wall
ii) Antibiotics damaging cell membrane
iii) Antibiotics interfering with protein synthesis
iv) Antibiotics inhibiting nucleic acid synthesis
v) Antibiotics blocking cell metabolism
The first discovered natural antibiotic was Penicillin by Alexander Fleming. Penicillin was obtained from “Penicillium notatum”.
Penicillin
Penicillin is a group of compounds having common basic nucleus, 6-amino penicillanic acid (6-APA). 6-APA contains ring like structure termed as a β-lactam ring.
Penicillin
Penicillin was active against Gram positive bacteria, Nocardia, and Actinomycetes, but not against most Gram negative bacteria. It interferes with cell wall synthesis of actively growing sensitive organisms. It mainly inhibits the cross linking steps of peptidoglycan synthesis in the cell wall.
Structure of Penicillin
Penicillin is a group of compounds having common basic nucleus, 6-amino penicillanic acid (6-APA). 6-APA contains two rings- β-lactam & semithiazolidine ring.
Penicillin F (2-pentenyl penicillin) was the original Penicillin obtained in more amount with P. notatum. Later, when P. chrysogenum was isolated and employed in fermentation, Penicillin K was obtained majorly along with dihydropenicillin F in the fermentation broth. With the addition of Corn steep liquor (CSL) as precursor, Penicillin G or benzyl penicillin was the major product in the mixture. It had better pharmacological properties and better stability.
Thus penicillin is a group of compounds and the major compound depends on the microorganism and the media employed for fermentation.
Penicillins are of two different types,
i Natural Penicillin
ii Synthetic Penicillin
Natural penicillin is directly harvested from the Penicillium mold (P. notatum or P. Chrysogenum).
Synthetic penicillin consists of the basic Penicillin nucleus (6-APA), but with new side chains that provide altered properties to the natural penicillin. Eg., Ampicillin, Methicillin, Penicillin V, etc. These Penicillins could be produced by fermentation (biosynthetic) or chemical treatment (semi-synthetic).
Penicillin Fermentation -Microorganisms
Penicillium species are used in the Penicillin fermentation- P. notatum & P. chrysogenum.
P. notatum was used initially in stationary mat culture, however the strain was highly unstable and the yield variable. Stationary fermentation was replaced by submerged fermentation methods and P. notatum was found unsuitable.
P. chrysogenum P. notatum
In 1943, P. chrysogenum was isolated from moldy fruit and was found as a high yielding strain and better suited for submerged fermentation. Descendants of P. chrysogenum is now widely used as production strain.
Strain development methods such as mutagenesis (x-ray/uv/alkylating agents, nitrosoguanidine), genetic recombination, protoplast fusion technique were employed to obtain high yielding variety- - P. chrysogenum NRRl 1951 Wis Q176 strain.
Fleming’s isolate, P. notatum gave a yield of around 2 IU/ml whereas P. chrysogenum NRRl 1951 WIS Q176 strain and its descendants yielded around > 85000 IU/ml after strain improvement. Such is the power of strain improvement techniques.
After strain improvement the production strains are carefully maintained by different preservation techniques like,
1. A spore suspension may be mixed with a sterile, finely separated inert support like soil or sand and then desiccated.
2. The spore suspension can be stored under liquid nitrogen (-196°C) i.e. in a frozen state.
3. The spore suspension can be lyophilized in appropriate media.
Along with improved yield, other properties were also modified, such as pigment production. P. chrysogenum produced yellow water soluble pigment which gave a yellow tint to the final preparations. With mutation and selection, strains that do not produce the pigment but produced penicillin in high yields could be selected.
Biosynthesis, media preparation, fermentation
§ The basic structure of penicillin consists of a thiazolidine ring condensed with a B-lactam ring.
§ 6-amino-penicillanic acid (6-APA) is penicillin nucleus
Penicillin Biosynthesis
Penicillin is a modified tripeptide made from three amino acids aminoadipic acid, cysteine and valine. The principle enzyme involved in penicillin biosynthesis is ACV-synthetase. The β-lactam ring is formed by isopenicillin-N synthetase; finally, side chains are added
By fermentation technology penicillin is produced from Penicillium chrysogenum.
ü If penicillin fermentation is carried out without addition of side chain precursor, the natural Penicillins are produced.
ü But fermentation can be better controlled by adding a side chain precursor to obtain derived Penicillin.
ü The synthetic Penicillins are produced by enzymatic hydrolysis of 6 APA by penicillin acylase enzyme and then addition of desired side chain by chemical means
Penicillin Fermentation- Medium preparation
Raw materials are primary requirement to design the fermentation broth. Fermentation broth contains all the necessary elements required for the proliferation of the microorganisms. Generally, it contains a carbon source, nitrogen source, mineral source, precursors and antifoam agents if necessary.
Basic composition of inoculation/fermentation media
Corn steep liquor (3.5%)
Lactose (3.5%)
Glucose (1%)
CaCO3 (1%)
KH2PO4 (0.4%)
Edible Oil (0.25%)
and Penicillin precursor in fermentation media (absent during inoculum preparation)
Inoculum preparation
Aim is to develop a pure inoculum in an adequate amounts. Inoculum is built up sequentially from small flasks to small fermenters. All the parameters like temperature, pH, aeration, agitation etc. should be properly maintained.
pH: near 5.5 to 6.5
Temperature: 26°C to 28°C
Aeration: a continuous stream of sterilized air is pumped into it.
Agitation: have baffles which allow constant agitation.
Carbon Source
Lactose acts as a very satisfactory carbon compound for fermentation. Other carbohydrates like glucose is used during inoculum preparation. Lactose is preferred in fermentation media to provide slow feeding rate and starvation conditions to induce antibiotic formation. Sucrose may also be used. Complex as well as cheap sources like molasses, or soy meal can also be used.
Nitrogen Source
Another essential compound for metabolism of organisms is nitrogen. Ammonium salts such as ammonium sulfate, ammonium acetate, ammonium lactate or ammonia gas are used. Corn steep liquor also may also be used.
Mineral Source
Additionally, some minerals are necessary for the proper growth of these organisms like phosphorus, sulfur, magnesium, zinc, iron, and copper which are 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. The most important naturally occurring penicillin is Penicillin-G. Depending upon the precursors added, the type of penicillin going to produced can be changed.
For example, if phenyl acetic acid is provided then 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.
Penicillin Production
Penicillin production was done previously by surface process ie. Solid state fermentation and surface liquid fermentation. Nowadays a commercial production is carried out by fed batch submerged process
Microorganism
Initially Penicillium notatum strain was used for fermentation of penicillin. Later, Penicillium chrysogenum was discovered and improved by using strain improvement program by genetic mutation This strain should be maintained properly by lyophilization technique or storage in liquid nitrogen in frozen state.
Penicillin Production
Steps
1) Inoculum preparation
2) Production fermentation
3) Product recovery
1) Inoculum preparation:
In the process of inoculum preparation the pure culture of Penicillium chrysogenum is prepared in adequate amount for production of penicillin.
The primary working stock is added in special sporulating agar which allow maximum sporulation of spores. The sporulated spores are suspended in water or non-toxic wetting agents like SLS - sodium lauryl sulphonate solution in a proportion of 1:10,000.
These spores are added in a nutrient medium of wheat bran plus nutrient for heavy sporulation and the flask are incubated for 5 to 7 days at 24°C. This is done in small tanks with aeration and agitation for sufficient mycelial growth
These mycelia from inoculum medium are used as a inoculum in fermentation tank.-at 10% levels
2) Fermentation:
§ Method: fed-batch or batch- Substrate: glucose, lactose, phenyl acetic acid (precursor), Corn steep liquor, Additional nitrogen source ie, soya meal, yeast extract, lactic acid, inorganic ions, growth factors
ü Precursors for penicillin G – phenylacetic acid.
Fermentation media used in penicillin fermentation should provide a sufficient amount of carbon source, nitrogen source, Nutrients, amino-acids, salts and precursors. The fermentation media should provide growth of mycelium, accumulation, extraction, purification of penicillin. Exact composition of media used in industries are not know as it’s a matter of trade secrets.
§ Fermenter: stirred tank or air lift tank
(which increase up to 7-7.5 (optimum) due to consumption of lactic acid and liberation of NH3 gas. If pH is 8 or more, CaCO3 or MgCO3 or phosphate buffer is added)
§ temperature: 25-28°C
§ aeration
§ agitation: 120-150 rpm
Fermentation of penicillin is carried out by surface fermentation in trays or by submerged culture method. The 10 % of inoculum is added in the fermenter aseptically. The temperature of about 25°C to 28°C is maintained. The sterile air supply is provided continuously as the fermentation is aerobic fermentation. The fermentation is carried out for about 3 to 5 days.
During this fermentation process the samples are withdrawn aseptically and checked for yield of penicillin, pH and contamination. The checking for contamination is very important if the fermentation media gets contaminated by organism producing penicillinase enzyme then it can result in a great economic loss to industry. The fermentation process is monitored for foam formation also and if foam is produced, it is controlled by antifoaming agent.
Initially the pH of fermentation media remain constant as the inoculum initially utilizes carbon as a source of energy but further when concentration of carbon is reduced the micro-organism starts utilization of nitrogen as a source of energy so at this point the pH of the fermentation media increases to 7.0 to 7.5 due to deamination and release of ammonia. Now at this point the micro-organism starts product synthesis by utilization of lactose and production of penicillin. After product formation the concentration of lactose is decreased and pH rises to 8 or even more which results in autolysis of mycelium. Here at this point the fermentation is stopped and recovery and harvesting of product is started.
Initially during 20 to 30 hours the fungal spores utilizes carbohydrates and corn steep liquor and fungal spores develop as mycelium and further in the duration of 48 to 96 hours the mycelium starts production of penicillin product.
The yield obtained is 3 % to 5 % and 1500 unit per milliliter of fermentation medium.
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)
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