Choice of Organism
The organism should give high yields of antibiotic from economic sources of nutrients and should not produce excessive amounts of substances chemically related to the antibiotic because separation may be difficult. Cephalosporins are β-lactam antibiotics which are originally derived from the fungus named Acremonium, which was previously known as “Cephalosporium.”
Cephalosporins are indicated for the prophylaxis and treatment of infections caused by bacteria susceptible to this particular form of antibiotic. They interfere with bacterial cell wall synthesis. More reliable strains are obtained by subjecting cultures to physical or chemical mutagens such as X rays ultraviolet light or nitrogen mustard. Variation of the new strains must be prevented usually by freeze-drying the spores followed by storage in the dark (Sreeju, Michael Babu, Mariappan, & Selvamohan, 2011).
For experimentation, the microorganism is grown in agar plates. A study revealed that recombinant DNA modified strain of the fungus Cephalosporium acremonium, strain LU4-79-6, produced more of the antibiotic than a non-recombinant strain, from which it was derived by transformation (Skatrud et al., 1989). Thus recombinant Cephalosporium could be used to obtain impressive yields.
Choice of Medium
The basis of most fermentation media is corn steep liquor, the fluid obtained from the steeping of maize prior to starch extraction. It provides a variety of nutrients and has good buffering capacity. For most fermentations, particularly those involving fungi, additional sugar (glucose or lactose) is needed as a source of energy for mycelium production. Occasionally, when the nitrogen demand is high, soya bean, or peanut, meal may be added. Buffering salts may be required when the antibiotic is pH sensitive. To improve the yield, a precursor may be added. E.g. L ValineCultivation of the Organism: A slope is inoculated with spores from the master (e.g. freeze-dried) culture.
After incubation, the growth is checked for free from contamination and variation. A spore suspension made from the slope is transferred to culture flasks, several liters in capacity, which are incubated to obtain a large crop of spores. The flasks are shaken during incubation to ensure that enough air is available to the aerobic mould. The suspension in the flasks is used to inoculate a 200dm3 tank, known as the seed vessel; this is a small version of the fermenter. After further incubation, sterile compressed air is used to blow the contents through sterile pipes into huge fermentation tanks (fermenters) with a capacity of 50000dm3 or more. The growth of the mould is allowed to continue until a satisfactory level of antibiotic has been reached.
Because of the moulds used in antibiotic production are aerobic they will not grow well deep in a tank of medium unless they are supplied with plenty of oxygen. This is done by pumping in large volumes of air through an inlet (sparger) at the bottom of the fermenter. The sparger introduces the air as streams of bubbles that are further broken down and distributed by a high powered stirrer (agitator).
The resulting tiny bubbles dissolve readily to replace the oxygen absorbed from the liquid by the mould. Because of the protein content, culture media froth readily, particularly when aerated, and this is reduced by antifoaming agents such as silicon fluids, vegetable oils or octadecanol.
The culture medium, vessels, pipelines and air must be sterile to prevent contamination by extraneous organisms that might reduce yields and produce unwanted metabolites.They are usually sterilized by steam under pressure. Sterilization of the medium may be carried out in the fermenter or in a separate vessel. High pressure steam is passed in the fermenter into a heating jacket or through heating coils inside the tank. External sterilization maybe done by a high temperature short time method. Afterwards the hot medium is reduced to fermentation temperature, by passing cooling water through the internal coils. Because the demand for oxygen is high, continuous method air sterilization is appropriate, a compressor of high capacity and capability of high compression is used because the pressure necessary to provide the sterilization temperature is also adequate to force the air through the medium against the head of pressure in the fermentation tank. As an added precaution, the air is passed through a sterilizing filter immediately before entry to the tank.
Part of the energy produced by carbohydrate breakdown during metabolism of mould is liberated as heat. This must be controlled because high temperatures inhibit the mould and destroy the antibiotic. An optimum temperature (24 degrees Celsius) is obtained by passing cooling water through coils or a jacket, or simply by running cold water over the surface of the fermenter. Isolation of the Product: After the fermentation process, the mycelium is removed by a rotary filter. The antibiotic is therefore obtained from the filtrate by adjustment to a pH which the acid or base (depending on the nature of the antibiotic) is liberated, followed by removal of unwanted substances and extraction of the active material with a solvent system in, e.g. a Podbielniak counter current machine. Where the free acid or base is unstable, impurities are separated from the salt by chromatographic or ion exchange techniques. The crude product of extraction is purified.The final drying is done under vacuum and may involve freeze drying.