In Microbial Technology Microorganisms holds the key to the success or failure of a fermentation process. It is therefore important to select the most suitable microorganisms to carry out the desired industrial process.
The most important factor for the success of any fermentation industry is of a production strain. It is highly desirable to use a production strain possessing the following four characteristics:
It should be high-yielding strain.
It should have stable biochemical/ genetical characteristics.
It should not produce undesirable substances.
It should be easily cultivated on large-scale.
Detection and isolation of high-yielding species form the natural sources material, such as soil, containing a heterogeneous microbial population is called Screening
Screening may be defined as the use of highly selective procedures to allow the detection and isolation of only those microorganisms of interest from among a large microbial population.
Thus to be effective, screening must, in one or a few steps allow the discarding of many valueless microorganisms, while at the same time allowing the easy detection of the small percentage of useful microorganisms that are present in the population.
The concept of screening will be illustrated by citing specific examples of screening procedures that are or have been commonly employed in industrial research programs.
During screening programs except crowded plate technique a natural source such as soil is diluted to provide a cell concentration such that aliquots spread, sprayed or applied in some manner to the surface of the agar plates will yield well isolated colonies (30-300).
Primary screening of Organic acid/ amine producer:-
For primary screening of organic acid or organic amine producers, soil sample is taken as a source of microorganism.
· It is diluted serially to an extent to get well-isolated colonies on the plate when spread or applied in some form.
· After preparation of dilution these dilutions are applied on a media incorporated with a pH indicating dye such as Neutral red (Pink to yellow)or Bromothymol blue (Yellow -blue), into a poorly buffered agar nutrient medium. The production of these compounds is indicated by a change in the color of the indicating dye in the close vicinity of the colony to a color representing an acidic or alkaline reaction.
· The usefulness of this procedure is increased if media of greater buffer capacity are utilized so that only those microorganisms that produce considerable quantities of the acid or amine can induce changes in the color of the dye.
An alternative procedure for detecting organic acid production involves the incorporation of calcium carbonate (1-2 %) in the medium so that organic acid production is indicated by a cleared zone of dissolved calcium carbonate around the colony. These procedures are not foolproof, however, since inorganic acids or bases also are potential products of microbial growth. For instance, if the nitrogen source of the medium is the nitrogen of ammonium sulfate the organism may utilize the ammonium ion, leaving behind the sulfate ion as sulfuric acid, a condition indistinguishable form organic acid production. Thus cultures yielding positive reactions require further testing to be sure that an organic acid or base actually has been produced.
Primary screening of antibiotic producer (Crowded plate technique):
· The crowded plate technique is the simplest screening technique employed in detecting and isolating antibiotic producers.
· It consists of preparing a series of dilution of the source material for the antibiotic producing microorganisms, followed by spreading the dilution on the agar plates.
· The agar plates having 300- 400 or more colonies per plate after incubation for 2-4 days are observed since they are helpful in locating the colonies producing antibiotic activity.
· Colonies showing antibiotic activity is indicated by the presence of a zone of inhibition (arrow in fig) surrounding the colony.
· Such a colony is sub- cultured to a similar medium and purified.
· It is necessary to carry on further testing to confirm the antibiotic activity associated with a microorganism since zone of inhibition surrounding the colony may sometimes be due to other causes. Notable among these are a marked change in the pH value of the medium resulting from the metabolism of the colony, or rapid utilization of critical nutrients in the immediate vicinity of the colony.
· Thus, further testing again is required to prove that the inhibitory activity associated with a microorganism can really be attributed to the presence of an antibiotic.
The crowded plate technique has limited application, since usually we are interested in finding a microorganism producing antibiotic activity against specific microorganism and not against the unknown microorganism that were by chance on the plate in the vicinity of an antibiotic producing organism. Antibiotic screening is improved, therefore by the incorporation into the procedure of a “Test organism” that is an organism used as an indicator for the presence of specific antibiotic activity.
Dilutions of soil or of other microbial sources are applied to the surface of agar plates so that well isolated colonies will develop. The plates are incubated until the colonies are a few millimeters in diameter and so that antibiotic production will have occurred for those organisms having this potential. A suspension of test organism is then sprayed or applied in some manner to the surface of the agar and the plates are further incubated to allow growth of the test organism. Antibiotic activity is indicated by zones of inhibited growth of the organism around antibiotic producing colonies. In addition a rough approximation of the relative amount of antibiotic produced by barious colonies can be gained by measuring in mm the diameters of the zones of inhibited test organism growth. Antibiotic producing colonies again must be isolated and purified before further testing.
Primary screening of growth factor (Amino acid/ Vit) producer (Auxanography):
This technique is largely employed for detecting microorganisms able to produce growth factors (eg. Amino acid and Vitamins) extracellularly. The two major steps are as follows:
A filter paper strip is kept across the bottom of a petri dish in such a way that the two ends pass over the edge of the dish.
A filter paper disc of petri dish size is placed over paper strip on the bottom of the plate.
The nutrient agar is poured on the paper disc in the dish and allowed to solidify.
Microbial source material such as soil, is subjected to dilution such that aliquots on plating will produce well isolated colonies.
Plating of aliquots of properly diluted soil sample is done.
A minimal medium lacking the growth factor under consideration is seeded with the test organism.
The seeded medium is poured on the surface of a fresh petri dish and allowed to solidify.
The agar in the first plate as prepared in step- I is carefully and aseptically lifted out with the help of tweezers and a spatula and placed without inverting on the surface of the second plate as prepared in the second step.
The growth factor(s) produced by colonies present on the surface of the first layer of agar can diffuse into the lower layer of agar containing the test organism. The zone of stimulated growth of the test organism around the colonies is an indication that they produce growth factor(s) extracellularly. Productive colonies are sub cultured and are further tested.
A similar screening approach can be used to find microorganisms capable of synthesizing extracellular vitamins, amino acids or other metabolites. However, the medium at makeup must be totally lacking in the metabolite under consideration. Again the microbial source is diluted and plated to provide well-isolated colonies and the test organism is applied to the plates before further incubation. The choice of the particular test organism to be used is critical. It must possess a definite growth requirement for the particular metabolite and for that metabolite only, so that production of this compound will be indicated by zones of growth or at least increased growth of the test organism adjacent to colonies that have produced the metabolite.
Enrichment culture technique:
This technique was designed by a soul microbiologist, Beijerinck, to isolate the desired microorganisms form a heterogeneous microbial population present in soil. Either medium or incubation conditions are adjusted so as to favour the growth of the desired microorganism. On the other hand, unwanted microbes are eliminated or develop poorly since they do not find suitable growth conditions in the newly created environment. Today this technique has become a valuable tool in many screening program for isolating industrially important strains.
Secondary screening is strictly essential in any systematic screening programme intended to isolate industrially useful microorganisms, since primary screening merely allows the detection and isolation of microbes that possess potentially interesting industrial applications. Moreover, primary screening does not provide much information needed in setting up a new fermentation process. Secondary screening helps in detecting really useful microorganisms in fermentation processes. This can be realized by a careful understanding of the following points associated with secondary screening:
It is very useful in sorting our microorganisms that have real commercial value from many isolates obtained during primary screening. At the same time, microbes that have poor applicability in a fermentation process are discarded. It is advisable to discard poor cultures as soon as possible since such studies involve much labour and high expense.
2. It provides information whether the product produced by a microorganism is a new one or not. This may be accomplished by paper, thin layer or other chromatographic techniques.
3. It gives an idea about the economic position of the fermentation process involving the use of a newly discovered culture. Thus one may have a comparative study of this process with processes that are already known, so far as the economic status picture is concerned.
4. It helps in providing information regarding the product yield potentials of different isolates. Thus this is useful in selecting efficient cultures for the fermentation processes.
5. It determines the optimum conditions for growth or accumulation of a product associated with a particular culture.
6. It provides information pertaining to the effect of different components of a medium. This is valuable in designing the medium that may be attractive so far as economic consideration is concerned.
7. It detects gross genetic instability in microbial cultures. This type of information is very important, since microorganisms tending to undergo mutation or alteration is some way may lose their capability for maximum accumulation of the fermentation products.
8. It gives information about the number of products produced in a single fermentation. Additional major or minor products are of distinct value, since their recovery and sale as by-products can markedly improve the economic status of the prime fermentation.
9. Information about the solubility of the product in various organic solvents is made available. (useful in product recovery operation and purification).
10. Chemical, physical and biological properties of a product are also determined during secondary screening. Moreover, it reveals whether a product produced in the culture broth occurs in more than one chemical form.
11. It reveals whether the culture is homofermentative or heterofermentative.
12. Determination of the structure of product is done. The product may have a simple, complex or even a macromolecular structure.
13. With certain types of products (e.g. antibiotics) determination of the toxicity for animals, plants or man are made if they are to be used for therapeutic purpose.
14. It reveals whether microorganisms are capable of chemical change or of even destroying their own fermentation products. E.g. microorganism that produce the adaptive enzyme, decarboxylase can remove carbon dioxide from amino acid, leaving behind an organic amine.
15. It tells us something about the chemical stability of the fermentation product.
Thus, secondary screening gives answers to many questions that arise during final sorting out of industrially useful microorganisms. This is accomplished by performing experiments on agar plates, in flasks or small bioreactors containing liquid media, or a combination of these approaches. A specific example of antibiotic producing Streptomyces species may be taken for an understanding of the sequence of events during a screening programme.
Those streptomycetes able to produce antibiotics are detected and isolated in a primary screening programme. These streptomycetes exhibiting antimicrobial activity are subjected to an initial secondary screening where their inhibition spectra are determined. A simple “Giant – Colony technique” is used to do this. Each of the streptomycal isolates is streaked in a narrow band across the centers of the nutritious agar plates. Then, these plates are incubated until growth of a streptomycete occurs. Now, the test organisms are streaked from the edges of the plates upto bur not touching the streptomycete growth. Again, the plates are incubated. At the end of incubation, growth inhibitory zones for each test organism are measured in millimeters. Thus, the microbial inhibition spectrum study extensively helps in discarding poor cultures. Ultimately, streptomycete isolates that have exhibited interesting microbial inhibition spectra need further testing. With streptomycetes suspected to produce antibiotics with poor solubility in water, the initial secondary screening is done in some different way.
Further screening is carried our employing liquid media in flask, since such studies give more information than that which can be obtained on agar media. At the same time, it is advisable to use accurate assay technique (e.g. paper disc agar diffusion assay) to exactly determine the amounts of antibiotic present in samples of culture fluids. Thus , each of the streptomycete isolates is studied by using several different liquid media in Erlenmeyer flasks provided with baffles. These streptomycete cultures are inoculated into sterilized liquid media. Then , such seeded flasks are incubated at a constant temperature. Usually such cultures are incubated at near room temperature. Moreover, such flasks are aerated by keeping them on mechanical shaker, since the growth of streptomycetes and production of antibiotics occur better in aerated flasks than in stationary ones. Samples are withdrawn at regular intervals under aseptic conditions and are tested in a quality control laboratory. Important tests to be carried out include:
i. Checking for contamination,
ii. Checking of pH
iii. Estimation of critical nutrients
iv. Assaying of the antibiotic, and
v. Other determinations, if necessary
The result of the above test, points out the best medium for antibiotic formation and the stage at which the antibiotic yields are greatest during the growth of culture on different media. After performing all necessary routine tests in the screening of an actually useful streptomycete for the fermentation process, other additional determinations are made. They are:
i. Screening of fermentation media through the exploitation of which the highest antibiotic yields may be obtained.
ii. Determination of whether the antibiotic is new.
iii. Determination of the number of antibiotics accumulated in the culture broth is made.
iv. Effect of different bioparameters on the growth of streptomycete culture, fermentation process and accumulation of antibiotic.
v. Solubility picture of antibiotic in various organic solvents. Also, it is to be determined whether antibiotic is adsorbed by adsorbent materials.
vi. Toxicity tests are conducted on mice or other laboratory animals. An antibiotic is also tested for the adverse effects if any, on man, animal or plant.
vii. The streptomycete culture is characterized and is classified upto species.
viii. Further studies are made on a selected individual streptomycete culture. For example mutation and other genetic studies for strain improvement are carried out.
In conclusion, tests are designed and conducted in such a way that production streptomycete strains may be obtained with least expenses. Similar screening and analytical techniques could be employed for the isolation of microbial isolates important in the production of other industrial chemical substances.