The Isolation, Preservation & Improvement of Industrially Important Micro-organisms

Introduction
 The first stage in the screening for microorganisms of

potential industrial application is their isolation.  Isolation involves obtaining either pure or mixed cultures followed by their assessment to determine which carry out the desired reaction or produce the desired product.  The isolate must eventually carry out the process economically  Therefore, the selection of the culture to be used is a compromise between the productivity of the organism and the economic constraints of the process

Cont…
 Criteria as being important in the choice of organism:  the nutritional characteristics of the organism: using a     

very cheap medium or a pre-determined one the optimum temperature of the organism the reaction of the organism with the equipment to be employed and the suitability of the organism to the type of process to be used the stability of the organism and its amenability to genetic manipulation the productivity of the organism, measured in its ability to convert substrate into product and to give a high yield of product per unit time the ease of product recovery from the culture

The Isolation of Industrially Important Micro-organism
 Isolation methods utilizing selection of the desired

characteristic:  enrichment liquid culture  enrichment cultures using solidified media • Isolation methods not utilizing selection of the desired characteristic • Screening methods

Enrichment Liquid Culture
 Frequently carried out in shake flask  By inoculating the enriched culture into identical fresh

medium (sub-culture)  Sub-culturing may be repeated several times before the dominant organism is isolated by spreading a small inoculum of the enriched culture into solid medium  The time of sub-culture in an enrichment process is critical and should correspond to the point at which the desired organism is dominant.  The prevalence of an organism in a batch enrichment culture will depend on its maximum specific growth rate compared with the maximum specific growth rates of the other organisms capable of growth in the inoculum

Enrichment Cultures Using Solidified Media
 The isolation of certain enzyme producers  The use of a selective medium incorporating the substrate of

the enzyme which encourages the growth of the producing types  Example: - isolated species of Bacillus producing alkaline protease - soils of various pHs were used as the initial inoculum - the number of producers isolated correlated with the alkalinity of the soil sample - spread onto the surface of media agar at pH 9-10, containing a dispersion of an insoluble protein - Colonies which produced a clear zone due to the digestion of the insoluble protein were taken to be alkaline protease producers

Isolation Methods Not Utilizing of The Desired Characteristic
 Production of antibiotics and growth promoters  Therefore, a pool of organisms has to be isolated and   

subsequently tested for the desired characteristic The major problem- reisolation of strains which have already been screened many times before An environmental sample contains a vast variety of organism Design of isolation media based on knowledge of only one taxon may inadvertently result in the preferential isolation of undesirable types Example: used the statistical stepwise discrimination analysis (SDA) technique to design media for the positive selection of antibiotic producing soil isolates - by nutritional test

Screening Methods
 More precisely targeted to identify the desired activity  The evolution of antibiotic screens serves as an

excellent illustration of the development of more precise, targeted systems  Antibiotics were initially detected by growing the potential producer on an agar plate in the presence of an organism (or organisms) against which antimicrobial action was required  Alternatively, the microbial isolate could be grown in liquid culture and the cell-free broth tested for activity

The Preservation of Industrially Important Micro-organisms
 The culture used to initiate an industrial fermentation

must be viable and free from contamination  Cultures must be stored in such way as to eliminate genetic change, protect against contamination and retain viability  Storage at reduced temperature  storage on agar slopes  storage under liquid nitrogen  Storage in a dehydrated form:  dried culture  lyophilization • Quality control of preserved stock culture

Storage at Reduced Temperature
 Storage at agar slopes:
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Store in a refrigerator (5°C) or freezer (-20°) and sub-cultured at approximately 6-monthly intervals The time of sub-culture may be extended to 1 year if the slopes are covered with sterile medicinal grade mineral oil

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 Storage under liquid nitrogen

- the metabolic activities of micro-organism may be reduced considerably by storage at the very low temperatures (-150°C to -196°C) which may be achieved using a liquid nitrogen refrigerator - this approach is the most universally applicable of all preservation methods - technique: growing a culture to the maximum stationary phase, resuspending the cells in a cryoprotective agent (10% ethanol) and freezing the suspension in a sealed ampoules before storage under liquid nitrogen - viability maybe predictable even after a period of many years - The liquid nitrogen evaporates and must be replenished regularly

Storage in a Dehydrated Form
 Dried culture:

- Dried soil culture have been used widely for culture preservation - Moist, sterile soil may be inoculated with a culture and incubated for several days for some growth to occur and then allowed to dry at room temperature for approximately 2 weeks - The dried soil may be stored in a dry atmosphere or preferably, in a refrigerator

Cont…
 Lyophilization:

- Lyophilization/freeze-drying involves the freezing of a culture followed by its drying under vacuum, which result in the sublimation of the cell water - Growing the culture to the maximum stationary phase and resuspending the cells in a protective medium (milk, serum, sodium glutamate) - a few drops of the suspension are transferred to an ampoule, which is then frozen and subjected to a high vacuum until sublimation is complete, after which the ampoule is sealed - the ampoule may be stored in a refrigerator and the cell may be remain viable for 10 years or more

Quality Control of Preserved Stock Cultures
 Each batch of newly preserved cultures should be routinely     

checked to ensure their quality A single colony of the culture to be preserved is inoculated into a shake flask and the growth of the culture observed to ensure a typical growth pattern After a further shake flask sub-culture the broth is used to prepare a large number of storage ampoules At least 3% of the ampoules are reconstituted and the cultures assessed for purity, viability and productivity If the samples fail any one of these tests the entire batch should be destroyed Therefore, stock cultures may be retained and used with confidence

The Improvement of Industrial Microorganism
 The selection of induced mutants synthesizing

improved levels of primary metabolites  modification of the permeability  the isolation of mutants which do not produce feedback inhibitors or repressors  the isolation of mutants that do not recognize the presence of inhibitors and repressors

The Selection of Induced Mutants Synthesizing Improved Levels of Primary Metabolites
 The levels of primary metabolites in micro-organisms are

regulated by feedback systems  The major systems: feedback inhibition and feedback repression  Feedback inhibition: the end product of a biochemical pathway inhibits the activity of an enzyme catalyzing one of the reactions of the pathway - Inhibition acts by the end product binding to the enzyme at an allosteric site which result in interference with the attachment of the enzyme to its substrate

Cont..
 Feedback repression: the end of the product (or

derivative of the end product) of a biochemical pathway prevents the synthesis of an enzymes catalyzing a reactions of the pathway - repression occurs at the gene level by a derivative of the end product combining with the genome in such a way as to prevent the transcription of the gene into messenger RNA, resulting in the prevention of enzymes synthesis

The use of recombinant systems
 the application of the parasexual cycle  the application of protoplast fusion techniques  the application of recombinant DNA techniques

The improvement of industrial strains by modifying properties other than the yield of product
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the selection of stable strains the selection of strains resistant to infection the selection of non-foaming strain the selection of strains which are resistant to components in the medium the selection of morphologically favourable strains the selection of strains which are tolerant of low oxygen tension the elimination of undesirable products from a production strain the development of strains producing new fermentation products