The following points highlight the five main steps for choosing micro-organisms for industrial microbiology. The steps are: 1. By the Process of Genetic Manipulation 2. By the Production of Transgenic Microbes 3. By Modifying Gene Expression 4. Preservation of Selected Strains of Micro-Organisms 5. Growth of Micro-Organisms.
Step # 1. By the Process of Genetic Manipulation:
The fundamental idea behind this technique is to change the genetic makeup of the microbe in order to develop new genetic traits which will be profitable to us.
This can be achieved by following ways:
(a) Protoplast Fusion:
Protoplast fusions now widely used for the improvement of fungal organisms like yeasts and moulds. To achieve protoplast fusion protoplasts of these microbes are obtained by growing the cells in an isotonic solution followed by treating them with enzymes such as cellulose and beta-galacturonidase.
This step is followed by the regeneration of protoplasts by using sucrose. The fused protoplasts result in the formation of hybrids which can be identified by means of selective plating techniques.
Mutations, especially the one which are achieved with the help of chemical mutagens and ultraviolet light, have been very useful in developing new strains of microbes. For example, ordinary cultures of Penicilliumnotatum yields low concentrations of penicillin.
Later on a strain of Penicilliumchrysogenum (NRRL 1951) was isolated by repetitive selection procedure which was further improved through the process of mutation. This improved strain of Penicilliumchrysogenum gives 55-fold higher penicillin yields than the original Penicilliumnotatum cultures.
(c) Site directed Mutagenesis:
In the process of site directed mutagenesis short lengths of chemically synthesized DNA sequences, having the genetic information of novel traits, can be inserted into the genome of the target microorganisms. This process results in the genetic alterations of the microbe that leads to a change of one or several amino acids in the target protein.
As a consequence of this there are many changes in protein characteristics, and results in new products. These approaches are part of the field of protein engineering.
Step # 2. By the Production of Transgenic Microbes:
New varieties of microbes can be developed by encouraging the transfer of nucleic acids between them. This process involves the transfer of genes, having the genetic information for the synthesis of a specific product, from one organism into another. The recipient organism becomes a transgenic organism. Example of such a microbe is “Superbug” developed by A. M. Chakarabarty in 1974.
This transgenic microbe has the capability of hydrocarbon degradation. For the production of superbug he used the microbe Pseudomonas putida. It is capable of utilizing complex chemical compounds like hydrocarbons.
It is also called oil eating bug. Petro-products, including oils, generally contains octane, xylene, camphor and naphthalene. No single strain of P. putida is able to digest all these four petrochemical. Dr. Chakarabarty introduced four plasmid DNA’s from four different strains into one single cell of Pseudomonas putida and generate a superbug.
Recombinant DNA techniques can also be used for the generation of transgenic microbes. This technique has been widely exploited for the production of vaccines; for example the vaccine for the foot and mouth disease of livestock.
Genetic information for a foot-and-mouth disease virus antigen can be inserted into E. coli. This is followed by the expression of this genetic information and synthesis of the gene product for use in vaccine production.
Step # 3. By Modifying Gene Expression:
In addition to inserting new genes in organisms, it is also possible to filter the pattern of gene regulation. This is possible by means of changing gene transcription, fusing proteins, creating hybrid promoters, and by removing feedback regulation controls.
Step # 4. Preservation of Selected Strains of Micro-Organisms:
Once we have selected the microorganism or virus catering to the industrial interest, then it must be preserved in its original form for any further use and research. There are different methods for microbial preservation.
Suitable methods are selected based on the:
(a) Type of micro-organism,
(b) Effect of the preservation method on the viability of micro-organism,
(c) Frequency at which the cultures are withdrawn,
(d) Size of the microbial population to be preserved,
(e) Availability of resources, and
(f) Cost of the preservation method.
Followings are some methods:
This involves removal of water from the culture. Desiccation is used to preserve actinomycetes (a form of fungi-like bacteria) for very long period of time. The microorganisms can be preserved by desiccating on sand, silica gel, or paper strips.
(b) Agar Slopes:
Microorganisms are grown on agar slopes in test tubes and stored at 5 to -20 °C for six months. If the surface area for growth is covered with mineral oil the micro-organisms can be stored for one year.
(c) Liquid Nitrogen:
This is the most commonly used technique to store micro-organisms for a long period. Storage takes place at temperatures of less than -196 °C and even less in vapour phase. Microorganisms are made stationary and suspended in a cryoprotective agent before storing in liquid nitrogen.
This method is especially used for sporulating microorganisms (organisms that produce spores). They Eire sterilized, inoculated, and incubated to allow microbial growth, then dried at room temperature. The resultant dry soil is stored at 4° to 5 °C.
This process is also known as freeze-drying. The microbial culture is first dried under vacuum, filled in ampoules (glass vessels) then frozen. This is a most convenient technique, since it is cheap to store and easy to ship. The disadvantage is that it is difficult to open the freeze dried ampoules; also, several subcultures have to be done to restore the original characteristics of the micro-organisms.
Step # 5. Growth of Micro-Organisms:
In the industrial processes the selected micro-organisms are grown using specifically designed culture media under very carefully controlled conditions such as temperature, aeration, etc.