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Portrait of Louis Pasteur in his laboratory

Fermentation is the metabolic process which occurs with or without oxygen to produce desirable products in large scale. Many products like Ethanol, Lactic acid, Gases, Proteins etc are the products of fermentation. Various kinds of bioreactors can be used in different modes of fermentation like Batch, Fed-batch, Packed etc. The word "ferment" in latin means to boil and this word has been used since the 1600s. Advances in fermentation technology has made life easier. Antibiotics (drugs), amino acids and some other important biomolecules can also be produced in large scale for the world to use.

Antibiotic Production by Fermentation

Antibiotics are a range of drugs used to kill bacteria but they cannot kill viruses or are not useful for viral infection. They are produced by fermentation in large bioreactors. Organisms that serve as a source for antibiotics are grown in a growth medium. Some organisms are mutant or genetically modified to get greater yield. The process needs lot of chemicals, clean bioreactor, correct pH, Temperature etc. First, the media is prepared which involves the microbes and then along with the media other components are also sterilized. After inoculum preparation is completed fermentation starts in the bioreactor with controlled conditions. Usually, antibiotics are secondary metabolites so we go till the stationary phase and do not let the cells die. It could be done by surface culture or submerged culture. Antibiotics are then separated from the impurities present followed by extraction by various methods and drying. Penicillin was the first discovered antibiotic.

Penicillin Production by Fermentation

History

Alexander Fleming

Penicillin is the first discovered antibiotic by Scottish scientist Alexander Fleming in 1928. He was the first one to observe the effects of penicillin as it stopped the growth of bacteria in a petri dish containing Staphylococci. Penicillium rubens (fungus species from genus Penicillium) was the first species from which penicillin was made. By 1857 Pasture had already proved how fermentation of lactic acid is done by living organisms, by 1860 souring of milk was also studied. Penicillium notatum was the original strain used by fleming which produced Penicillin-F, known as 2-pentinyl penicillin. Fermentation process was already in research by that time and then in 1944 mass production of penicillin was done. Wartime was going and deep tank fermentation was used. Penicillin production gave those times the name of “Golden era of antibiotic”.

Introduction

Benzylpenicillin

Penicillin is a secondary metabolite produced when fungus growth is inhibited by stress. 6-Amino penicillanic acid (6-APA) is the natural structure which consists of thiazolidine ring with a condensed β-lactum ring. R-side chain is the primary difference among another penicillin present. P. chrysogenum was used more for submerged fermentation.

Classification of Penicillium chrysogenum

Kingdom:Fungi

Division: Ascomycota

Class: Eurotiomycetes

Order: Eurotiales

Family: Trichocomaceae

Genus: Penicillium

Species: Chrysogenum

Structure of Penicillium chrysogenum

Cell structure of eukaryotes with tubulin cytoskeleton for motility. Hyphae is filamentous and contains many conidia (blue to blue-green). These conidia are responsible of allergies and all pathogenic activity in humans also. Sterigmata is the one that give rise to conidia at the top. Hyphae is usually colourless and slender, conidia is fluffy like cotton.

The fermentation process of penicillin

The process is divided into steps like:

• Strain development
• Inoculation preparation
• Inoculation
• Extraction and purification

Strain development

Penicillium notatum

Strain is one of the variants of that particular microorganism which is required. “Higher yielding strain” will yield more amount of penicillin so some wild strains are mutated or there can be a combination of chemical mutagenesis and X-ray radiations, example: Penicillium chrysogenum. Ultraviolet radiations are also used either in combination or individual. Strain improvement is an important step but consumes time. To make better quality penicillin, side-chain precursors can be added. These are called semisynthetic if they are modified after we get natural penicillin from fermentation.

Inoculum preparation

Inoculum is basically the microorganism which is used in the fermentation process, here Penicillium chrysogenum. From the working stock which is heavily sporulated, spores are taken and suspended in a nontoxic solution of the wetting agent of substances like sodium sulfonate (1:10000) or sodium lauryl sulfate (1:10000). After this spores are added to bottles containing wheat bran and nutrient-solution. After incubation at 24 degrees for 5 to 7 days, heavy sporulation is seen and this process is repeated to get more spores. After the inoculation tank is inoculated with spores, aeration and agitation are maintained for 2 days for heavy mycelial growth.

Production fermentation (Inoculation)

Many methods like batch, Fed-batch or deep tank fermentation can be done. Three methods can be employed:

Dry spores used as inoculum: Spores used here are hydrophobic.

Ungerminated spore suspension: This is completed when fermented is fed by spray guns pipette.

Pre-germinated spores: Mycelial pellet and pre-germinated spores are formed by germination of spores and fed into the fermenter.

The capacity of fermenters used for penicillin production could be from 35000 to 2 lakh litres.

The medium used

The medium which is used should be able to give maximum mycelium growth and antibiotic accumulation. Carbon source can be Sucrose, lactose, sorbitol, glucose and glycerol. Nitrogen supply is in the form of ammonium nitrate, ammonium sulphate or ammonium acetate. Corn steep liquor can be used for maximum mycelium growth as it contains essential amino acids required. Salts of Potassium, Zinc, Sulphur, Phosphorus, Magnesium etc are supplied and some of them are present in corn-steep liquor. Penicillin -K and Penicillin-F are produced naturally in the absence of precursors by P. chrysogenum and P. notatum respectively. But in the presence of phenylacetic acid, Penicillin-G is produced instead of Penicillin-K.

Fermenter: Stirred tank or Air-lift fermenter can be used.

pH: Usually kept at 5.5 to 6. When NH3 gas is released then pH is raised to 7-7.5 as lactic acid is used up. Addition of CaCO3 or MgCO3 buffer is there if pH is reaching Temperature: 25-27 C.

Agitation: 120-15- rpm

Aeration: 0.5-1 vvm

Days: 3 to 5

Antifoam: edible oil (0.25%)

Penicillin yield will depend on the quantity of carbohydrate consumed which will be equal to equal to 1500 international units per millilitre approximately. Contamination problems should not be there.

Extraction and purification of Penicillin

After production of penicillin is over the last step is to extract and purify it. This process could be divided into three steps:

Separation of mycelium: Rotatory vacuum filter can be used for separation and any kind of contamination should be avoided or penicillin could be degraded. This step separates the broth also.

Extraction of penicillin: Penicillin is converted to anionic form by adding acids like phosphoric or sulphuric acid. Less than 1% of penicillin was bound to mycelium. Since penicillin is unstable the extraction step is quickly processed by adding an organic solvent such as amylacetate or butylacetate or methyl isobutyl ketone. It is back-extracted from organic solvent into the water by adding sodium or Potassium hydroxide which raises the pH. But this aqueous solution is again acidified to re-extract penicillin into the organic medium. “Penicillin is finally obtained in the form of sodium-penicillin”.

Treatment of extract: This extract is treated by charcoal so that pyrogens (fever causing substances) are removed from sodium-penicillin. Bacteria can be removed (if any) by sterilization. Crystallization is the process by which penicillin is obtained in crystal forms. After this it could be made into powder or tablets in bottles for oral usage. Even syrups are made.

Modern methods of Production

In the modern dyas submerged culture tanks are mostly used with constant aeration and agitation using Penicillium chrysogenum strain. 50000 gallons stainless steel tanks are there. Some strains used earlier had low recovery rates compared to this strain with higher recovery rate and 90% production. With the latest techniques of recombinant DNA technology and gene therapy many new modifications are made in host cell or wild strains. Like the gene responsible for penicillin biosynthesis can be amplified or peroxisome level could be increased. With such advancements production has increased many-fold.

Applications and side effects

Penicillin V

Uses: Work against bacterial infections and not viral infections (like treating common cold). They are used for skin and soft tissue infection, upper respiratory tract infection, scarlet fever, erysipelas infections, for preventing chorea and rheumatic fever.

Side effects: Common and mild effects could be vomiting, nausea, diarrhoea, upset stomach etc. but some serious ones are allergic reaction with skin rashes, swelling of throat, watery diarrhoea with or without stomach cramps sometimes with blood.

Penicillin G

Uses: Antimicrobial properties which work against Gram-positive bacteria and some of the Gram-negative bacteria like Neisseria gonorrhoeae and Leptospira weilii are also susceptible.

Side effects: common ones are mild skin rash, diarrhoea, headache, vomiting, upset stomach, nausea, fever and sometimes serious allergic reactions depending on the person’s body.

AminoPenicillin (Ampicillin)

Uses: In diseases like bacterial meningitis, for gastrointestinal infections, heart diseases, wooping cough etc.

Side effects: If taken in high doses may cause serious effects. People allergic to penicillin should not consume otherwise side effects like anaphylaxis may occur.

Many different members of penicillin are available like Aminopenicillin, Carboxypenicillin, Natural penicillins, Beta-lactamase resistant, Beta-lactamase inhibitors etc.