The below mentioned article provides an overview on the production of antibiotics:- 1. Introduction to Antibiotics 2. Use of Inoculum in Production of Antibiotics 3. Use of Fermentor 4. Medium used 5. Biotransformation.
Introduction to Antibiotics:
Antibiotics are metabolites having preferential antimicrobial activity. Therefore, they are widely used for curing of human ailments caused by microorganisms. But some of the antibiotics have anti-tumour activity, as well (Table 40.1). The antibiotic penicillin was discovered by Fleming in 1929, but its commercial production could commence only during early 1940s.
Although more than 7,000 antibiotic compounds have been isolated, only 100 or so are being used to treat human, animal and plant diseases.
Antibiotic compounds are used either in their natural form or as semisynthetic derivatives; the latter arc usually produced by isolating the antibiotic nucleus and subjecting it to chemical modification. Antibiotics are produced by both fungi and bacteria but over 50% of them arc obtained from Streptomyces alone.
Use of Inoculum in Production of Antibiotics:
A high yielding strain is a prerequisite for antibiotic production. Therefore, constant strain improvement is an integral part of commercial production activities. The improved strain/strains are kept in long-term storage, from which the smallest quantity is taken to initiate inoculum development.
Inoculum development begins on solid media, and subsequently liquid media are used; the media used are specific for inoculum development. The inoculum is prepared usually in form of a spore suspension, which is transferred into the fermentor by placing it in a metal vessel that is attached to the fermenter.
The spore suspension may be blown into the fermentor by using sterile air or it may be allowed to run in under gravity. As a rule, the number of stages between the preserved material and the final inoculum stage is kept to the minimum to minimise the risk of the organism losing its high yield potential.
Use of Fermentor in Production of Antibiotics:
Antibiotics are generally produced in stainless steel fermentors (30,000-200,000 1 medium volume) used in the batch or fed-batch mode. Agitation is mostly by impellers, but air-lift system is also used. Water cooling is often used to maintain the temperature between 24-26°C for most antibiotic producers.
Generally, the fermentor is maintained at above atmospheric pressure; this reduces contamination risk and enhances O2 supply in the medium. Sterile air is supplied as per need, and for some processes, e.g., penicillin production, materials need to be added throughout the process. In most cases, it is critical to prevent contamination, and suitable cleaning procedures between fermentor runs must be adhered to.
The final stage fermentor is preferably used for antibiotic production for the longest possible period. But the initial stages of fermentation are designed for considerable microbial growth; typically these are carried out in seed-stage fermentors of smaller size.
One or more seed-stages may be used, depending on the process and the strain, to produce the maximum amount of biomass in the correct physiological state for high antibiotic production when introduced in the final stage fermentor.
Medium used for Antibiotic Production:
Antibiotic production employs a variety of media, a different one for each stage of operation (Table 40.2). A considerable research effort is directed at developing seed-stage and production media to reduce costs and to enhance yields. A typical production medium has about 10% (w/v) solids.
Generally, yields are much higher on complex media. In some cases, a suitable precursor for the antibiotic is also provided, e.g., for penicillin G production, phenylacetic acid or phenoxyacetic acid is used as precursor.
Since the antibiotics are secondary metabolites, the production medium is so designed that a key nutrient becomes limiting at a critical stage to initiate the secondary metabolism in the organism. The nutrient to be made limiting depends on the process, e.g., glucose for penicillin production and phosphate for several antibiotics produced by Streptomyces.
In contrast, the seed-stage medium is devised for rapid growth, and to prevent antibiotic production and sporulation. When the seed culture reaches the correct stage, i.e..the stage suitable for optimal production in the final production stage (in many cases, it is the log phase), about 0.1-20% of the final stage volume is transferred into the production fermentor. Rigorous contamination control is essential at this stage.
In most production processes, the production fermenter is run in a fed-batch mode in which a nutrient, e.g., glucose, is added continuously throughout the fermentation to enhance the duration of antibiotic production. In such a case, small volumes of broth are usually withdrawn to check increase in volume of the broth in the fermenter.
Antifoaming agents are added at the appropriate stage of fermentation to minimise foaming; silicons, vegetable oils, etc. are used for this purpose. Selected microbiological, physical and chemical features are monitored during the fermentation in order to achieve a proper control on the process.
The nutritional status of broth is monitored regularly to adjust nutrient and precursor (if any) feeding in fed-batch systems. Microprocessors and computers are commonly used to record all the parameters and process changes, and to control various operations like sterilization, etc.
At the end of final production stage incubation, the broth contains only a low concentration (3-35% of the total solutes in the broth) of the antibiotic.
The initial step in antibiotic recovery is separation of cells from the broth; this is usually achieved by filtration or centrifugation. But in some cases, the whole, broth is used for extraction. Antibiotic isolation and purification employs solvent extraction, ion exchange, ultrafiltration, reverse osmosis, precipitation and crystallization.
Biotransformation of Antibiotics:
Biotransformation is routinely used for commercial production of several useful antibiotics. Semisynthetic antibiotics, e.g., semisynthetic penicillin and cephalosporins, are produced by chemical modification of the penicillin nucleus, e.g., 6-aminopenicillanic acid. The penicillin nucleus is produced by microbial deacylation of naturally produced penicillin G and penicillin V (Fig. 40.8).