yophilization (microbial cells or spores are conserved by quick removal of water by sublimation at a low temperature) or conservation by keeping cultures at a very low temperature (- 70 o C) in liquid nitrogen. In both cases cultures keep their properties for at least 10 years.
Laboratory cultivation. Cultivation in the laboratory, irrespective of the fact whether the microorganism will finally be used for inoculation of a production fermentor or in laboratory experiments, is carried out in test tubes or in 200-1000-ml bottles and flasks. The volume of the culture medium mostly represents about one tenth of the total volume of the flask. The flasks are sealed with stoppers allowing diffusion of the air into the flasks to ensure aerobic conditions for growth. At the same time, the stoppers prevent microorganisms from the environment to penetrate into the flasks (cotton-wool stoppers, etc.). Producers of antibiotics require a proper aeration, that is important for both the growth and production of the antibiotic. Therefore, the flask contents is well mixed by agitation on rotary or reciprocal shakers placed in thermostated rooms or boxes. Strictly sterile conditions have to be ensured for the cultivation of antibiotic producers since, in the case of contamination, the producing culture can be suppressed by more rapidly growing microorganisms. in fermentors. Microbial producers of antibiotics are cultivated in fermentors of various size. The lower limit of size of laboratory fermentors is about 1 litre. Owing to the use of complex media, foam is often formed during cultivation and, therefore, the fermentors are filled with the medium up to one half or two thirds of their maximal capacity. When the process of antibiotic production is scaled up from the laboratory conditions to those of true production, basic parameters can be established using several-litre, laboratory fermentors. However, they should be verified in pilot plant fermentors having a size of several cubic meters. The basic equipment of both laboratory and pilot plant fermentors is practically the same. They are made of inert materials such as glass and stainless steel, or their walls are at least lined with an inert material. The fermentors are equipped with a device keeping the cultivation temperature constant (mainly cooling device is important in large fermentors) and with an efficient aeration system, since antibiotic producers require a sufficient oxygen supply for both the growth and synthesis of the antibiotic. The aeration systems based on intensive stirring are not suitable for cultivation of antibiotic producers since a majority of them are filamentous microorganisms that can suffer damage when intensively stirred. The air flowing into the fermentor has to be sterile. It is sterilized by filtration; most often glass wool or mineral wool filters are used.
Most antibiotics are produced in a fed batch system, ie a certain amount of the culture medium is inoculated with the producing microorganism and, after a time interval, another dose of nutrients is added to the fermentor. Thus a prolonged cultivation can be accomplished that enables us to increase the yield of the antibiotic. The inflow of nutrients makes possible keep their optimal levels. In cultivations whose course is well known, the nutrient inflow is programmed in advance.
Solid-state fermentation .Solid-state, or substrate, fermentation is characterized by a fermentation process on a solid support, which has a low moistre content and occurs in a non-septic and natural state [24]. The use of solid-state technology for the production of antibiotics has some advantages. Due to the lack of free water, smaller fermentors are required and the mycelial microorganisms, used predominantely for antibiotic production are well suited to grow on a solid support. On the contrary, a liquid fermentation process allows greater control and monitoring of parameters, such pH, ??heat, nutrient condition etc [25].
1.3.2 Isolation, separation and purification of antibiotics
Isolation of an antibiotic from the fermentation medium depends on the fact whether the antibiotic is secreted into the medium or remains in the biomass, inside the cell or bound to the cell wall. If the antibiotic is bound to the biomass or, in contrast, present in the broth supernatant, the two phases are separated by filtration or centrifugation and extracted separately. If the antibiotic is present in both phases the whole broth is used for extraction. Another isolation step usually includes an extraction with solvents of different polarities, followed by evaporation of the extracts to dryness. If the antibiotic is extractable by nonpolar solvents, the extraction is preceded by dehydration, most often using methanol ...
