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:
- 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
Cont…
 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 Micro-
organism
 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
 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