INDUSTRIAL MICROBIOLOGY AND SCREENING

INDUSTRIAL MICROBIOLOGY
Definition:
Industrial Microbiology deals with all forms of microbiology that have an economic aspect. It deals with those areas of microbiology on which, in some manner, a monetary value can be placed, regardless of whether the microbiology involves a fermentation product or some form of deterioration, disease or waste disposal.
OR
Use of Microbes to obtain a product or service of economic value constitutes Industrial Microbiology.

Fermentation: Any process mediated by or involving Microorganisms in which a product of economic value is obtained is called Fermentation
Industrial microbiology is an important branch of microbiology dealing with those areas of microbiology involving economic aspects, where valuable products are prepared from cheaper and often disposable substrates.
· Hence it has become possible for the industrial microbiologist to compare with the industrial chemist. e.g. fermentative production costs of all antibiotics, except one or two are appreciably less than the synthetic production costs of the same.
Scope of Industrial Microbiology:
Industrial microbiology is a very broad area for study. In fact, many nonindustrial areas of microbiology are important to industrial microbiology and should be taken into consideration in understanding the concepts and practice of Industrial Microbiology. These areas include: soil and Agricultural Microbiology, Medical Microbiology, Microbial Physiology, Cytology and Morphology, Virology, Genetics, Marine Microbiology, Food and Dairy Microbiology and Immunology.
Disciplines not normally considered to be included in microbiology are also important to industrial microbiology and include organic, inorganic and physical chemistry, biochemistry, engineering, medicine, economics, sales and law, particularly patent law and labor law, governmental regulations on the use of certain substrates and the sale of certain products also are relevant to industrial microbiology, as a consideration of space and marine exploration.
Further more, areas not presently considered to have any relationship to industrial microbiology, under the proper conditions easily can become a matter for consideration e.g. An industrial concern producing fiberglass may have no apparent need for a knowledge of industrial microbiology. But, if a change or improvement in some processing step suddenly allows microbial growth on the sizing applied to the glass fibers, then industrial microbiology immediately assumes importance. That is to say that many different branches of microbiology and non-microbiological fields are directly or indirectly involved in the study of industrial microbiology.
The industrial microbiologist is always dependent upon a biochemical engineer and vice versa. Apart form the cultural responsibilities; the remaining bioparameters of the fermentation process are to be controlled by the biochemical engineer. Thus economization of a fermentation process requires both a biochemical engineer and a microbiologist.
FERMENTOR: DESIGN AND ROLE OF DIFFERENT PARTS OF FERMENTOR
In fermentation industries, microbes are to be grown in specially designed vessels loaded with particular type of nutritive media. These vessels are referred to as Fermentor or Bioreactors.
Bioreactors or fermentors are complicated in design, because they must provide for the control and observation of many facts of microbial growth and biosynthesis. The design of fermentor depends upon the purpose for which it is to be utilized. Industrial fermentors are designed to provide the best possible growth and biosynthesis conditions for industrially important microorganisms and allows ease of manipulation for all operations associated with the use of the fermentors. The fermentor used for a particular process should possess following characters:

Characteristics of and Ideal Fermentor or bioreactor:
There cannot be a fermentor ideal for all most all fermentation processes, but if there is then it should following characteristics:
1. Material used in the fabrication of fermentor should be strong enough to withstand the interior pressure due to the fermentation media, it should be resistant to corrosion and free form any toxic effect for the microbial culture and the product formed by the microbial culture.
2. A fermentor should permit easy control of contaminating microbes.
3. It should be provided with the inoculation point for aseptic transfer of inoculum.
4. Should be equipped with the aerating device (Spargers).
5. Should be equipped with a stirring device for uniform distribution of air, nutrients and microbes (Impellers).
6. There should be provision of baffles to avoid vortex formation.
7. Fermentor should be provided with a sampling valve for aseptic withdrawing of sample for different laboratory tests.
8. Fermentor should possess a device for controlling temperature (Temperature sensor and water jacket internally fitted with heating coil).
9. Fermentor should be provided with pH controlling device for monitoring and maintaining pH of media during fermentation process (pH probe and Acid base reservior).
10. Should be provided with a facility for intermittent addition of antifoam agents for controlling foam formation (Reservior of sterile Antifoming agents or mechanical foam breakers).
11. There should be provision fro feeding certain media components during the progress of fermentation (Precursors).
12. A drain at the bottom is essential for the removal of the completed fermentation broth for further processing.
13. A man hole should be provided at the top of fermentor for acess inside the fermentor for different purposes like repairing and thorough cleaning of feermentors between runs.
14. A exit valve should be provided at the top for the exit of metabolic gases produced during fermentation processes.
TYPES OF FERMENTORS:
Batch fermentors are used to carry out microbiological processes on batch basis. They are available with varying capacities. The capacity of the fermentor may range form a few hundred to several thousand gallons. The capacity of the fermentor is usrally stated on the basis of the total volume capacity of the same. Thus, based on total volume capacity the fermentors are of following types:
i) Small Laboratory fermentors (ii) Pilot plant fermentors (iii) Large industrial fermentors (iv) Horton spheres.
The small laboratory fermentor ranges from 1-2 liters with a maximum up to of 12 –15 liters.
Pilot plant fermentors have a total volume of 25 –100 gallons upto 2000 gallons total volume.
Larger fermentors range form 5,000 or 10,000 gallons total volume to approximately 1,00,000 gallons.
Horton spheres are rarely employed with a size range of 2,50,000 to 5,00,000 gallons total capacity.
Actually the working volume in a fermentor is always less than that of the total volume. In other words, a ‘head space’ is left at the top of the fermentor above the level of fermentation media. The reason for keeping a head space is to allow aeration, splashing and foaming of the aqueous medium. This head space usually occupies a fifth to a quarter or more of the volume of the fermentor.
pH Control:
pH control is achieved by acid or alkali addition, which is controlled by an auto- titrator. The autotitrator in turn is connected to a pH probe.
Temperature control:
Temperature control is achieved by a water jacket around the vessel. This is often supplemented by the use of internal coils, in order to provide sufficient heat-transfer surface.
Agitation:
The agitating device consists of a strong and straight shaft to which impellors are fitted. An impeller, in turn consists of a circular disc to which blades are fitted with bolts. Different types of blades are available and are used according to the requirements. The shaft passes through a bearing in the lid of the fermentation tank. It is rotated with the help of an electric motor mounted externally at the top of the tank. The liquid medium is thrown up towards the walls of the fermentor while rotating the impeller blades at a high speed. This results in the formation of a vortex, which is eliminated, usually by four equally spaced baffles attached to the walls of the fermentor.
Aeration:
Usually, the aerating device consists of a pipe with minute holes, through which pressurized air escapes into the aqueous medium in the form of tiny air bubbles. This aeration device is called a “SPARGER”. The size of the holes in a sparger ranges from 1/64 to 1/32 of an inch or larger. Holes smaller than this requires too high air pressure for economical bubble formation. One should always remember that the smaller the air bubbles, the greater is the bubble surface area. It is desirable to adjust the size of the air bubbles to give the greatest possible aeration without greatly increasing the overall cost of the fermentation process. The reason for this is that sterile air is a costly item for large-scale fermentation. The cheapest means of sterilization of air is to pass it through a sterile filter composed of glass wool, carbon particles or some other finely divided material that will trap microorganisms present in the air.
Spargers in fermentors for growth of mycelium forming organisms often utilize 1/4 inch holes to prevent plugging of the holes by hyphal growth. Pipes crimped at the end or with a single small hole to produce a stream of air bubbles also are employed in some instances. The air bubbles from the sparger are picked up and dispersed through the medium by the action of the impeller blade mounted above the sparger.
In some very large fermentation tanks, an impeller is not utilized. The medium is stirred by the directed rush of air bubbles from a sparger at the bottom of the tank. These tanks are specially designed and usually do not contain baffles.
Foam Control:
Aeration and agitation of a liquid medium can cause the production of foam. This is particularly true for the media containing high levels of proteins or peptides. If the foam is not controlled, it will rise in the head space of the tank and be forced from the tank along with the exit valve. This condition often causes contamination of the fermentation from organisms picked up by breaking of some of the foam which then drains back into the tank. Excessive foaming also causes other problems for fermentation.
The usual procedure for controlling foam is to add an antifoaming agent, although a supplementary impeller blade mounted high in the tank may at times be effective. An antifoam agent lowers surface tension and in the process decreases the stability of the foam bubbles so that they burst. The antifoam may be added at media makeup or may be added after sterilization or as called for during the fermentation process.
There are two types of antifoam agents:
(i) Inert Antifoam agents
(ii) Antifoam agents made from crude organic materials.

e.g. Animal and vegetable oils , lard oil, corn and soybean oil, long chain alcohols such as octadecanol. In addition mixtures of oils and alcohols are effective in controlling foam. Silicone compounds are ideal inert antifoam agents but are too expensive.
Antifoam agents are often difficult to sterilize, particularly if they are of an oily nature, because of poor heat penetration and transport through the oil.
The use of inert antifoam agents, such as various silicone compounds is the ideal way to control foam, but these agents generally are too expensive for use in large scale industrial fermentations.
When antifoam is required in a tank, it is added either manually or electrically. Obviously, manually addition requires that some one continuously observe the tank so that the antifoam can be added as required. Electrical addition of antifoam is usually preferred. To accomplish this automatic addition, a sensing mechanism is employed to determine when the foam has risen into the head space of bioreactor. Such a device is provided with two electrodes mounted in the top of the fermentor. These electrodes are connected to a pump associated with a reservoir of sterile antifoam and as the foam rises in the reactor it touches the two electrodes in the process allowing current to flow between them so as to activate the pump for addition of antifoam. The foam then collapses away from the electrode thus breaking the electrical connection between them and stopping further addition of antifoam agent.
SCREENING
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.
Def:
Detection and isolation of high-yielding species form the natural sources material, such as soil, containing a heterogeneous microbial population is called Screening
OR
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:
Step I
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.
Step II
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.
OR
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
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:
1. 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.