Production of Penicillin
Penicillin is an antibiotic active against Gram positive bacteria, Nocardia, and Actinomycetes, but not against most Gram-negative bacteria. It interferes with cell wall synthesis by inhibiting the cross-linking steps of peptidoglycan synthesis in the cell wall.
Penicillium species used in the Penicillin fermentation are Penicillium notatum and Penicillium chrysogenum. P notatum was used initially in stationary mat culture, however the strain was highly unstable and the yield variable. Later on, stationary fermentation was replaced by submerged fermentation methods and P. notatum was found unsuitable. P chrysogenum was isolated and was found as a high yielding strain and better suited for submerged fermentation.
Over the years, extensive strain improvement programs involving mutation and selection have led to significant increases in penicillin yields.
Fermentation Process
Inoculum Preparation - The process begins with the preparation of an inoculum, which involves growing the Penicillium chrysogenum. Inoculum is built up sequentially from small flasks to small fermenters. This ensures a sufficient quantity of healthy fungi for the production fermenter.
Fermentation Medium - The fermentation medium is carefully formulated to provide the necessary nutrients for Penicillium growth and penicillin production. Key components include Carbon sources (e.g., lactose, glucose), Nitrogen sources (e.g., corn steep liquor), Other essential minerals and precursors. Various types of precursors are added into production medium to produce specific type of penicillin. 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. Edible oil is added as antifoam agent.
Fermentation - Industrial penicillin production is typically carried out in large, stainless-steel fermenters under controlled conditions. The fermentation process is a fed-batch process. Key parameters that are carefully monitored and controlled include Temperature, pH, Oxygen levels, Nutrient concentrations, etc. Penicillin is a secondary metabolite, it is produced during the stationary phase of growth.
Downstream Processing – This includes cell separation, Extraction and purification.
Cell Separation - After fermentation, the mold cells are separated from the fermentation broth through filtration or centrifugation.
Extraction - Penicillin is extracted from the clarified broth using solvent extraction which typically involves acidification of the broth and extraction with an organic solvent.
Purification - The extracted penicillin is further purified through a series of steps, such as Back-extraction into an aqueous solution, crystallization and drying to obtain the final product as highly purified penicillin crystals, which are then formulated for pharmaceutical use.
Production of Vitamin-B12
Vitamin B12, also called Cobalamin, is a water- soluble vitamin, that has a key role in the normal functioning of the brain and nervous system, and the formation of red blood cells.
Vitamin B12 is naturally present in foods of animal origin, including fish, meat, poultry, eggs, and dairy products. Those who consume only small amounts of animal products or vegetarians must compensate for this. Unlike most vitamins, vitamin B12 is not synthesized by plants or animals. It is exclusively produced by certain bacteria. Therefore, industrial production relies on microbial fermentation.
Industrial Production
Strain Selection - High-yielding strains of the chosen bacteria are selected. Commonly used microorganisms include Propionibacterium freudenreichii, Pseudomonas denitrificans, Streptomyces griseus, Propionibacterium shermanii, Bacillus coagulans, Bacillus megaterium, Streptomyces olivaceus, etc.
Medium Preparation - A specialized fermentation medium is prepared, containing Carbon sources (e.g., glucose, molasses), Nitrogen sources, Cobalt salts and other essential nutrients. Cobalt is a crucial component of the vitamin B12 molecule. Therefore, the fermentation medium must contain an adequate supply of cobalt.
Fermentation
The selected bacterial strain is cultured in large fermenters under controlled conditions (temperature, pH, oxygen levels). Fermentation can be either Anaerobic fermentation (e.g., using Propionibacterium) or Aerobic fermentation (e.g., using Pseudomonas) or sometimes there is a combination of anaerobic and aerobic steps.
Recovery and Purification:
After fermentation, the vitamin B12 is extracted from the fermentation broth. This involves Cell separation (by centrifugation or filtration), Cell lysis to release the intracellular vitamin and Purification steps, including Adsorption, Solvent extraction, Chromatography and Crystallization. The vitamin is often converted to cyanocobalamin, a stable form of vitamin B12.
The Final Product is the purified cyanocobalamin which is then formulated into various products, such as Dietary supplements, Food additives, Pharmaceutical preparations, etc
