Fermentation process results in the production of antibiotics. Any large-scale microbial procedure taking place with or without air is called Fermentation. This process may take a few days to get an extractable amount of product. Production of antibiotics can also be performed by the batch process.
What are Antibiotics?
Antibiotics are compounds that can kill or retard the growth of other organisms. Filamentous fungi or Actinomycetes produce majority of the antibiotics.
Antibiotics can be derived from the certain microorganisms or many other living organisms. Scientists produce antibiotics on an industrial scale by using a fermentation process. Today, more than 10,000 antibiotic substances have been reported.
Properties of the Microorganism
Properties of useful industrial microbe include:
- Amenable to genetic modification
- Grow rapidly on large scale in a low cost medium
- Should produce desired product in less time
- Should be Non-pathogenic
A wide range of products produced by microorganisms include microbial cells, enzymes, antibodies, steroids, alkaloids, food additives, ethanol, and citric acid.
Metabolites are the intermediates or the final products of metabolism process. The metabolites which are essential for the growth and maintenance of all the cellular functions are primary metabolites. These metabolites are formed during the growth phase, such as amino acids, carbohydrates, vitamins, nucleic acids, lipids, and enzymes.
These are the metabolites not required for the growth and maintenance of functions of cells. These may be the end products of primary metabolites, hence, are called secondary metabolites.
Secondary metabolites are more likely to form near the stationary phase of growth. Major examples of secondary metabolites include drugs, toxins, steroids, and polymeric substances like rubber.
Production of Antibiotics
When did the production of antibiotics start? Its mass production began during World War II with streptomycin and penicillin. Now, industrialists produce most of the antibiotics with the help of staged fermentations in which strains of microorganisms producing high yields are grown under optimum conditions.
Primarily, production of antibiotics involves 3 methods:
- Natural microbial production using Fermentation technology, like Penicillin
- Semi synthetic production (post production modification of natural occuring antibiotics) like Ampicillin
- Synthetic production of antibiotics in the lab, such as Quinoline
The microorganism (taken as a source) grown in large containers (maybe 100,000–150,000 liters or more) contains a liquid growth medium. Oxygen concentration, pH, temperature, and nutrient levels must be at optimum level.
Since antibiotics are secondary metabolites, their population size must be in control carefully. It will ensure that maximum yield will obtain before the cells die.
The fermentation process requires the following
1. A pure culture of the selected organism, in sufficient amount.
2. Sterilized, carefully designed medium for the growth of organism
3. A seed fermenter which is a mini-model of production fermenter to create inoculums to start the process in the main fermenter.
4. A production fermenter which is the large functional model.
5. Equipment for:
- Cell separation
- Drawing the culture medium smoothly
- Collection of cell free supernatant
- Product purification, and
- Effluent treatment
Step 1 to 3 encodes the upstream and step 5 encodes the downstream of the fermentation process.
Use of Strains for antibiotics production
Primarily, species can be modified genetically to yield maximum amounts of antibiotics. Mutation introduces that often uses introducing mutagens such as ultraviolet radiation or x-rays. Selection and further growth of the higher acquired strains can increase yields by 20 times or more. Another technique to raise yields is gene amplification in which copies of genes coding for enzymes help in the antibiotic production.
Primary raw materials are the compounds that make the fermentation broth for antibiotic production. The broth is an aqueous solution consists of all of the ingredients essential for the reproduction of the microorganisms.
Typically, it contains;
- Carbon source: molasses, soy meal, acetic acid, alcohols, or hydrocarbons. These materials are important as a food source for the organisms.
- Nitrogen Source: Nitrogen is another important compound in the metabolic cycles of the microorganisms. Ammonia salt is of utmost importance in such a case.
Trace elements essential for the proper proliferation of antibiotic producing microorganisms such as:
- Anti-foaming agents to avoid foaming during fermentation such as, lard oil or octadecanol.
Steps in Production
First, it involves identification of the organism to makes the antibiotic. Desired microorganism must then be isolated. Then the organism grows on a large scale enough to ensure the purification and chemical analysis of the antibiotic. It’s important to test the antibiotic against a wide variety of bacterial species. Industrialists maintain the sterile conditions throughout the manufacturing process as contamination by foreign microbes can ruin the fermentation process.
A) Starting a Culture
Before the fermentation process the desired microbe needs to isolate. Its number must increase by many times. A starter culture comes from a sample of previously isolated organisms created in the lab.
A sample of the organism can be transfer to an agar-containing plate. The initial culture then transfers back to shake flask containing nutrients necessary for growth. Suspension forms, which may, then, transfer to seed tanks for further growth.
The seed tanks are steel tanks that maintain an ideal environment for the growth of microorganisms. The seed tanks equip with mixers, which mix the growth medium with microbes, and a pump to deliver sterilized, filtered air. After about 24-28 hours, the material in the seed tanks transfer to the primary fermentation tank.
The fermentation tank is able to hold about 30,000 gallons. Microorganisms allow to grow and multiply. During this process, they excrete large quantities of the desired antibiotic.
These tanks then cool to maintain the temperature between 73-81° F (23-27.2 ° C). It agitates constantly, and a continuous stream of sterilized air pumps into it. Anti- foaming agents periodically enter.
Since pH control is compulsory for optimal growth, acids or bases may add to the tank as required.
C) Isolation & Purification
After 3-5days, the maximum amount of antibiotic will produce. The isolation process can begin. The isolation depend on the specific antibiotic produced, the fermentation broth processes by various purification methods.
Water soluble Antibiotics
For antibiotic compounds that are water soluble, an ion-exchange method process for purification. The compound first separates from the waste organic materials in the broth. Then sent through equipment and separates the other water-soluble compounds from the desired one.
Oil soluble Antibiotics
Industrialists use solvent extraction method for the isolation of oil soluble or organic antibiotics. They treat broth with organic solvents, such as butyl acetate or methyl isobutyl ketone, which can dissolve the antibiotic.
The dissolved antibiotic needs to recover using various organic chemical means. At the end of this step, a purified powdered form of the antibiotic is available which can further refine into different product types.
Refining or Packaging
Antibiotic products can take on several different forms. People may sell in solutions for intravenous bags or syringes, powders, or in pill or gel capsule form. These all forms, no doubt includes wholly incorporation of the topical ointments.
After the initial isolation, various refining steps may include, such as
- For intravenous bags, the solution contains a dissolve crystalline antibiotic putting in the bag. It may then hermetically sealed.
- For gel capsules, the powdered antibiotic may physically fill into the bottom half of a capsule then the top half can mechanically put in place.
- When used in topical ointments, it’s good to mix antibiotic into the ointment.
Is it important to control quality?
Quality control is highly important in the production of antibiotics. Since it involves a fermentation process, it ensures that absolutely no contamination is present at any point during antibiotic production.
During manufacturing or processing, the quality of all the compounds must be checked on a regular basis. There must be frequent checks of the condition of the microorganism culture during fermentation. Various physical and chemical properties of the finished product observe such as pH, melting point, and moisture content.
Industrial production of Penicillin
Penicillin was the first important commercial antibiotic product. By an aerobic, submerged fermentation, it produced.
The fungus Penicillium chrysogenum produces Penicillin. The process requires lactose, other sugars, and a source of nitrogen (in this case a yeast extract) in the medium to grow well. Like all antibiotics, penicillin is a secondary metabolite, so will only produce in the stationary phase.
Batch fermentor is a sort of fermenter it requires. A fed batch process is a growth limiting nutrient that can prolong the stationary phase and, so increase production.
The downstream processing is comparatively easy since the secretion of penicillin into the medium. There is no requirement to open the fungal cells. However, the product must be pure, since it being used as a therapeutic medical drug. The antibiotics, then, need to be dissolved and then precipitated by potassium salt to separate it from other substances in the medium.
Industrialists modify, chemically and enzymatically, the resulting penicillin (called penicillin G) to make a variety of penicillin containing slightly different properties. This semi-synthetic penicillin may include penicillin V, penicillin O, ampicillin and amoxicillin.
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