Production of PenicillinProduction of Penicillin

Downstream Processing

• Products in a fermenter are impure and dilute, so Products in a fermenter are impure and dilute, so need to be purified by downstream processing.need to be purified by downstream processing.

• This usually involves filtration to separate the This usually involves filtration to separate the microbial cells from the liquid medium, followed by microbial cells from the liquid medium, followed by chemical purification and concentration of the chemical purification and concentration of the product product

• Downstream processing can account for 50% of Downstream processing can account for 50% of the cost of a process.the cost of a process.

• Antibiotics are antimicrobial agents Antibiotics are antimicrobial agents produced naturally by other microbes produced naturally by other microbes (usually fungi or bacteria).(usually fungi or bacteria).

• The first antibiotic was discovered in The first antibiotic was discovered in 1896 by Ernest Duchesne and 1896 by Ernest Duchesne and "rediscovered" by Alexander Flemming "rediscovered" by Alexander Flemming in 1928 from the filamentous fungus in 1928 from the filamentous fungus Penicilium notatuPenicilium notatum. m.

• The antibiotic substance, named penicillin, The antibiotic substance, named penicillin, was not purified until the 1940s (by Florey was not purified until the 1940s (by Florey and Chain), just in time to be used at the end and Chain), just in time to be used at the end of the second world war.of the second world war.

• Penicillin was the first important commercial Penicillin was the first important commercial product produced by an aerobic, product produced by an aerobic, submerged submerged fermentationfermentation

• When penicillin was first made at the end of When penicillin was first made at the end of the second world war using the fungus the second world war using the fungus Penicilium notatumPenicilium notatum, the process made 1 mg, the process made 1 mg

• Today, using a different species (Today, using a different species (P. P.

chrysogenumchrysogenum) and a better extraction ) and a better extraction procedures the yield is 50 gprocedures the yield is 50 g

• There is a constant search to improve the There is a constant search to improve the yield. yield.

• Antibiotics can be selectively toxic by Antibiotics can be selectively toxic by targeting such features as the targeting such features as the bacterial bacterial cell wall, 70S ribosomes , and enzymes cell wall, 70S ribosomes , and enzymes that are specific to bacteria. that are specific to bacteria.

• In this way the human eukaryotic cells In this way the human eukaryotic cells are unaffected. are unaffected.

For example:For example:

• penicillin, ampicillin, amoxycillin, methicillinpenicillin, ampicillin, amoxycillin, methicillin• Inhibits enzymes involved in synthesis of Inhibits enzymes involved in synthesis of

peptidoglycan for bacterial cell wall, causing peptidoglycan for bacterial cell wall, causing cell lysis.cell lysis.

• BacteriocidalBacteriocidal• Narrow spectrum- little effect on Gram Narrow spectrum- little effect on Gram

negative cells.negative cells.

Other antibiotics may affect:Other antibiotics may affect:

• Cell membraneCell membrane

• DNA replicationDNA replication

• TranscriptionTranscription

• TranslationTranslation

Antibiotic productionAntibiotic production

• There are over 10 000 different antibiotics There are over 10 000 different antibiotics known, but only about 200 in commercial known, but only about 200 in commercial use, since most new antibiotics are no better use, since most new antibiotics are no better than existing ones. than existing ones.

• There is a constant search for new There is a constant search for new antibiotics. Antibiotics are the most-antibiotics. Antibiotics are the most-prescribed drugs and are big business. prescribed drugs and are big business.

• Finding a new antibiotic and getting it on to Finding a new antibiotic and getting it on to the market is a very long process and can the market is a very long process and can take 15 years.take 15 years.

• Figure shows the kinetics of the penicillin fermentation with Penicillium chrysogenum.

• If the penicillin fermentation is carried out without addition of side-chain precursors, the natural penicillins are produced. The fermentation can be more directed by adding to the broth a side-chain precursor so that only one desired penicillin is produced.

• The product formed under these conditions is referred to as a biosynthetic penicillin. To produce the most useful penicillins, those with activity against gram-negative Bacteria, a combined fermentation and chemical approach is used that leads to the production of semisynthetic penicillins.

•All of these antibiotics are typical secondary metabolites, and their industrial production is well worked out despite the fact that the biochemistry and genetics of their biosynthesis are only partially understood.

Antibiotic Production MethodsAntibiotic Production Methods

• Antibiotics are produced on an industrial scale Antibiotics are produced on an industrial scale using a variety of fungi and bacteria. using a variety of fungi and bacteria.

• Penicillin is produced by the fungus Penicillin is produced by the fungus Penicillium Penicillium chrysogenum chrysogenum which requires lactose, other sugars, which requires lactose, other sugars, and a source of nitrogen (in this case a yeast and a source of nitrogen (in this case a yeast extract) in the medium to grow well.extract) in the medium to grow well.

• Like all antibiotics, penicillin is a secondary Like all antibiotics, penicillin is a secondary metabolite, so is only produced in the stationary metabolite, so is only produced in the stationary phase. phase.

• It requires a batch fermenter, and a fed batch It requires a batch fermenter, and a fed batch process is normally used to prolong the stationary process is normally used to prolong the stationary period and so increase production.period and so increase production.

• Downstream processing is relatively easy Downstream processing is relatively easy since penicillin is secreted into the medium since penicillin is secreted into the medium (to kill other cells), so there is no need to (to kill other cells), so there is no need to break open the fungal cells.break open the fungal cells.

• However, the product needs to be very pure, However, the product needs to be very pure, since it being used as a therapeutic medical since it being used as a therapeutic medical drug, so it is dissolved and then precipitated drug, so it is dissolved and then precipitated as a potassium salt to separate it from other as a potassium salt to separate it from other substances in the medium. substances in the medium.

• The resulting penicillin (called penicillin G) The resulting penicillin (called penicillin G) can be chemically and enzymatically can be chemically and enzymatically modified to make a variety of penicillins with modified to make a variety of penicillins with slightly different properties. slightly different properties.

• These semi-synthetic penicillins include These semi-synthetic penicillins include penicillin V, penicillin O, ampicillin and penicillin V, penicillin O, ampicillin and amoxycillin.amoxycillin.

PRODUCTION OF STREOPTMYCIN

Streptomycin and various other antibiotics are produced using strains of Streptomyces griseus. Spores of this actinomycete are inoculated into a medium to establish a culture with a high mycelial biomass for introduction into an inoculum tank, with subsequent use of the mycelial inoculum to initiate the fermentation process in the production tank. 

The medium contains soybean meal (N- source), glucose (C- source) and NaCl. Temperature: 28°C pH: 7.6-8.0. High agitation and aeration are needed. Fermentation Period: 10 days. The classic fermentation process involves three phases.

During the first phase there is rapid growth of the microbe with production of mycelial biomass. 

Proteolytic activity of the microbe releases NH3 to the medium from the soybean meal, causing a rise in pH. During this initial fermentation phase there is little production of streptomycin. During second phase there is little additional production of mycelium, but the secondary metabolite, streptomycin accumulates in the medium. The glucose and NH3 released are consumed during this phase.

The pH remains fairly constant-between 7.6 and 8.0. In the third and final phase, when carbohydrates become depleted, streptomycin production ceases and the microbial cells begin to lyse pH increases and process normally ends by this time. 

After the process is complete, mycelium is separated from the broth by filtration and the antibiotic recovered. In one method of recovery and purification, streptomycin is adsorbed onto activated charcol and eluted with acid alcohol. It is then precipitated with acetone and further purified by use of column chromatography

PRODUCTION OF CHLOREMPHENICOL

Isolated from Streptomyces venezuelae in 1947

Inhibitory activity against both gram positive and gram negative bacteria

Also effective against actinomycetes, mycobacteria and anaerobic bacteria

Quite effective but having severe side effects

It affects bone marrow 

Submerged Aerobic Fermentation

The medium contains Carbon source: Glycerol, molasses and dried distillers grain slops

Nitrogen source: Yeast fermentation solubles, hog residues, tryptone, etc.

Mineral Nutrients: Sodium chloride, zinc and iron in traces

pH: 7.5

Temperature: 25 degree centigrade

Period of Fermentation: 10-15 days

Isolation and Purification:

Extracted from clarified broth

Extracts are concentrated, diluted with kerosene and washed with diluted acids, alkalis and water.

Lipids with petroleum ether are removed

Passed through column of charcoal or alumina

The product is then crystallized

PRODUCTION OF GRISEOFULVIN

Isolated from Penicillium patulum, P. raistrickii, P. urticase

Inhibitory activity against fungi

Treatment and control of fungal diseases of skin, hair, nails caused by the dermatophytes.

Strain with conidia yields maximum products

It is less toxic and systemic 

Submerged Aerobic Fed Batch Fermentation

The medium contains Carbon source: Malted cereal extracts, glucose, soluble starch

Nitrogen source: Whey powder nitrogen, protopeptone, corn steep liquor

Mineral Nutrients: Calcium carbonate, Potassium chloride, Potassium Dihydrogen phosphate

pH: 6.8-7.2

Temperature: 25 degree centigrade

Period of Fermentation: 10 days

Phosphate and chlorine are required in sufficient amount otherwise the obtained product is devoid of antifungal property.

Carbohydrate glucose syrup (50%) is added stepwise.

Air flow is required

Yield: 6-8g/Litre

To increase the yield methyl donors may be added to the medium

Extraction and Purification:

Treated with organic solvents and removed by filtration

Wet mycelia treated with butyl acetate followed by subsequent evaporation

Crude griseofulvin is washed with chloroform and crystallized

For higher production extraction from mycelium is done with methylene chloride and recrystallization with acetone.