There is a large number of microorganisms found in a variety of habitats such as soil, water, air, volcano, arctic water and hot spring. Each microbe has some peculiar feature. All are not supposed to have novel and useful products.
Hence, they are first isolated from their natural habitat. Then the strains are further improved using physico-chemical or molecular biological techniques. So that products could be produced at commercial scale.
For example, Thermus aquaticus is a hyperthermophilic bacterium which grows on volcano. An enzyme, Taq polymerase, is isolated from this bacterium. It is a unique enzyme which is used in PCR for polymerisation of DNA synthesis at high temperature.
1. Isolation of Strains :
For isolation of microorganisms, samples are collected from several sites of a habitat. Accordingly, nutrient media are prepared. Such specially designed media for culture of specific microorganisms are called enrichment culture technique. Equal amount of medium is poured into sterilised Petri plates. Known amount of serially diluted samples is poured onto the surface of agar medium.
The inoculated plates are put into a BOD incubator at optimum temperature desired by the microbe (e.g. 25-35°C for mesophilic bacteria). The Petri plates are incubated for different period such as 24 hours for bacteria, 5-7 days for fungi and 14-21 days for actinomycetes. Because growth rate of different microorganisms differ. A flow diagram for isolation of microorganisms is given in.
After incubation mixture of microbial colonies grows on surface of nutrient medium. Each colony is identified. The desired microbe is then isolated and purified through sub-culturing.
A small amount of inoculum of desired microorganism is put onto the surface of fresh medium either in agar slants or Petri plates. These are further incubated as described above. Thus growth of the microorganism is further improved.
The microorganisms isolated as above is further characterised to find out its specific properties such as production of desired proteins, vitamins, antibiotics, etc. For example, testing of antibiotic producing strains is done by dual culture method, where growth of one bacterium is inhibited by the metabolic product (e.g. antibiotic of the other bacterium).
Further testing of purified product is also carried out in laboratory by the microbiologists. Then effect of antibiotics on certain metabolic activities e.g. respiration, protein synthesis, control systems, etc. are done. Using antibodies the microbial products are also assayed.
Many probes have been developed through molecular biology which is used in diagnostics of microorganisms in the group and detection of their products.
2. Strain Improvement of Microorganisms :
A microbe isolated as above does not ensure that the product produced by it would be in sufficient quantity. Therefore, the strain of such organisms is improved by using classical (mutation and selection) and modern recombinant DNA technology to get the desired product in sufficient quantity.
(a) Mutation and Mutant Selection:
A cell divides and produces two identical cells of parental types. There is least probability of changes in inheritable characters. A strain that shows changed characteristics is termed as mutant.
When the microbial culture is exposed to mutagenic agents such as ionising radiation, ultra violet light and various chemicals (e.g. NTG i.e. nitrosoguanidine, nitrous acid, etc.) the probability of mutation is increased. When mutagenic dose is high, many of cells die. The survivors may contain some mutants. A small portion of cells may be good metabolite producers also.
Geneticists make selection of superior producers from the inferior producers among the survivors.
(i) In 1957, Kinoshita isolated Corynebacterium glutamicum which was biotin auxotroph (i.e. biotin mutant) and excreted glutamic acid. But glutamic acid could be produced in high amount through biotin limitation and blocking the metabolic route that converts into TCA cycle intermediates.
Besides the production of glutamic acid, mutants of C. glutamicum were also developed which produced high amount of other amino acids also.
(ii) In 1961, Nakayama and co-workers succeeded in isolating a home-serine auxotroph of C. glutamicum stain for the production of lysine which was auxotroph of homo-serine and leucine.
Recombination is defined as any process which helps to generate new combinations of genes that were originally present in different individuals. Recombination system may or may not be associated with sexual reproduction among the organisms.
There are two approaches which have been made to produce recombinants (organisms having new combination of genes), protoplast fusion and recombinant DNA technology.
(i) Protoplast Fusion:
Protoplasts are the cells devoid of cell walls. You can produce protoplasts using lysosome (cell wall degrading enzyme) in isotonic solution. Methods have been developed to fuse protoplast of two cells of different microorganisms.
In 1982, Tosaka produced high lysine producing strain of Brevibacterium flavum by fusing protoplasts with another B. flavum strain (a non-lysine producer but high rate glucose consumer).
Hamega and Ball (1979) fused protoplast of two different strains of Cephalosporium acremonium and produced a recombinant which had a high growth rate and synthesised a higher level of cephalosporin.
In 1982, Chang and co-workers used protoplast fusion method in two strains of penicillin V-synthesising strains of Penicillium chrysogenum. They selected a recombinant of P. chrysogenum capable of synthesising only penicillin V with desired morphological type.
(ii) Recombinant DNA Technology (= Genetic Engineering Technique):
The first commercial genetically engineered protein (human insulin called Humulin) was produced in 1982. Humulin is used by the persons suffering from diabetes mellitus.
The efficiency of ammonia metabolism of Methylophilus methylotrophus (a bacterium used as single cell protein) has been improved by incorporation of a plasmid containing glutamate dehydrogenase gene from E. coli.
The improvement in production of commercially important enzymes has also been done by rDNA technology. In 1981, Colson and co-workers cloned a Bacillus coagulans gene (coding for a thermos table a-amylase) into E. coli where it was replicated and maintained at a high copy number resulting in high enzyme production.
Similarly, penicillin acylase production in an E. coli strain has also been improved by introducing the relevant gene into a plasmid which was then incorporated into the original strain.