Isolation of industrially important microorganisms, methods of isolation, screening of microorganisms, strain selection

1. BIOPROCESS TECHNOLOGY
INTRODUCTION TO INDUSTRIAL MICROORGANISMS
R. NITHYA, M. Sc., M. Phil.,(Ph. D)
ASSISTANT PROFESSOR
DEPARTMENT OF BIOTECHNOLOGY
SRI ADI CHUNCHNAGIRI WOMENS COLLEGE, CUMBUM, THENI DT,
TAMIL NADU, INDIA

2. What are industrial microorganisms?
Industrial microbiology includes the use
of microorganisms to manufacture food
or industrial products in large quantities.
Numerous microorganisms are used
within industrial microbiology; these include
naturally occurring organisms,
laboratory selected mutants, or even
genetically modified organisms (GMOs)

3. Microorganisms and its uses
 Production of dairy products: Bacteria are the key players
here.
 Bread Baking: A species of Streptococcus is added to the
dough before making bread to bring about the
required fermentation.
 Alcoholic Drinks
 Organic acids
 Enzymes
 Steroid production
 Help in sewage treatment
 Used as insecticides

4. WHAT IS A CULTURE?
Population of microorganisms grown under well defined conditions.
WHAT IS MIXED CULTURE?
When a particular species of microbe is present in a very small
number in comparison to the total number of microorganisms , such
culture is called as mixed culture.
WHAT IS PURE CULTURE?
A culture containing only one species of microbe is called pure
culture.
SPECIES- a collection of bacterial cells which share an overall similar
pattern of traits in contrast to other bacteria whose pattern differs
significantly.
STRAIN- A strain is a subset of a bacterial species differing from
other bacteria of the same species by some minor but identifiable
difference.

5. Strain selection
Important characteristics for strain
• The selection of strains resistant to infection
• The selection of non-foaming strains
• 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

6. Choosing microorganisms for Industrial
microbiology and Biotechnology
The characteristics of microbes that are
desirable to the industrial microbiologist are:
genetic stability,
 easy maintenance and growth, and
amenability to procedures for extraction and
purification of desired product

7. Finding microorganisms in nature
 major sources of microorganisms for use in
industrial processes are soil, water, and
spoiled bread and fruits;
 only a minor portion of microbial species in
most environments have been identified

8. COMMON METHODS OF ISOLATION OF PURE CULTURE
• The process of screening a pure culture by
separating one type of microbes from a mixture is
called Isolation.
Some common isolation methods are;
Streak plate method
Pour plate method-
a) Loop dilution technique
b) Serial Dilution technique
Spread plate method
Micromanipulator method
Roll tube method

10. Streak plate method
 Prepare nutrient agar or any required medium and
poured into the petri plates.
 Allow the plates to solidify.
 Sterilize the inoculation loop using flaming technique.
 Transfer microbial mixture from a tube to the edge of
an agar plate with an inoculation loop as per
illustration.
 Incubate plates at 370C for 24hours.

11. Streak plate method

12.  TYPES
Different types of streaking techniques are available. They are T - streak,
Quadrant streak, simple streak, radiant streak and continuous streak. These
techniques are named based on the types of line of streak drawn on the
surface of the medium.

13. Pour plate method
To isolate and get pure culture of microorganisms
 Prepare nutrient agar and PDA potato Dextrose Agar medium (one plate needs approximately
15 - 20 ml) and sterilize at 1210 C for 15 minutes.
 Dilute the sample up to 1: 10000000 (10-7) using diluents.
 Add 1 ml of samples from 1:1000 (10-3).
 Pour the medium into sample added petriplates.
 Rotate the petriplates clockwise and anticlockwise direction.
 Allow the plates to solidify.
 Similarly perform pour plating for other dilutions like 10-4, 10-5 and 10-6.
 Incubate all nutrient agar plates at 370C for 24 hours and PDA plates at 25 – 300C for 48 hours
and record the results.

14. Pour plate method

15. Spread plate method
The method is performed for the assay of chemicals
like antibiotics, vitamins etc.
This method is called spread because L rod or cotton
swab is used to spread the sample.
This techniques also used for the isolation and
enumeration of microorganisms from samples with lower
populations of bacteria and other microorganisms.

16. Spread plate method
 Prepare nutrient agar medium, sterilize at 1210 C and pour in to the petri plates.
 Dilute the sample up to 1:100000 (10-5).
 Add 1 ml of sample from 10-3 on to the centre of an agar medium using sterile pipette.
 Perform similar procedure for 10-4 and 10-5 dilutions.
 Dip the glass spreader (L- rod) in to a beaker of ethanol/spirit.
 Briefly flame ethanol soaked spreader on Bunsen burner and allowed it to cool.
 Spread the sample evenly over the agar surface.
 Incubate all the plates at appropriate temperature (for bacteria 370C and for fungus 25 – 300 C)
for 24 to 48 hours.

17. SERIAL DILUTION

19. MICROMANIPULATOR METHOD
 Micromanipulators
 • Micromanipulators have been built, which permit one to pick out
 a single cell from a mixed culture. This instrument is used in
 conjunction with a microscope to pick a single cell (particularly
 bacterial cell) from a hanging drop preparation.
ADVANTAGES OF MICROMANIPULATOR METHOD
 • The advantages of this method are that one can be reasonably
 sure that the cultures come from a single cell and one can obtain
 strains with in the species.
DISADVANTAGES
 • Disadvantages are that the equipment is expensive,
 • Its manipulation is very tedious, and it requires a skilled operator

21. ROLL TUBE METHOD
 Prepare ten-fold dilution and add 0.1 ml
of diluted culture to molten agar cooled to
500C poured in test tube.
 Now tilt the tube and roll so that
medium is formed as a thin film
around the wall of tube.
 Incubate and count the no of colonies
on next day.

22. Enrichment methods for isolation of microorganisms
Enrichment methods are useful for quick isolation of specific types of
organisms.
Types of organisms Enrichment method
Thermophiles High temp (42-1000C)
Psychrotrophs low temp (5-150C)
Acidophiles Low pH (2-4)
Halophiles High NaCl concentration
Anaerobes N2 atmosphere
Actinoplanes Pollen grains
Myxobacteria Wood bark

23. Strains of microorganisms from unusual environments
The unusual environments such as cold habitats, high altitudes, deserts, deep
sea and petroleum fields are constantly being tried for this purpose.
Such strains may be capable of producing new products of industrial
importance

24. Screening of metabolites for isolation of microorganisms
The microorganisms can be tested directly for the product
formation, and isolated.
For example – if the product is an antibiotic, then the test system
consists of the strains of organisms which inhibit zones, on the
agar plates.
Another example is the isolation of microorganisms producing
amylases. When grown on agar plates containing starch, and
then stained with iodine, amylase producing organisms can be
identified and isolated.

25. Screening of microbes
 Although there are many screening techniques, all of
them are generally grouped into two broad categories.
 They are:
 1. Primary screening, and
 2. Secondary screening.

27. Primary Screening of Microorganisms:
Primary screening may be defined as detection and isolation of the
desired microorganism based on its qualitative ability to produce the desired
product like antibiotic or amino acid or an enzyme etc.
The following are some of the important primary screening techniques:
Indicator dye technique
The crowded plate technique
Enrichment culture technique
Auxanographic technique

28. PRIMARY SCREENING OF ORGANIC ACID PRODUCING
MICROORGANISMS
 The ph indicating dyes may be used for detecting microorganism that are
capable of producing organic acids.
 These dyes undergo colour changes according to its ph.
 Dyes such as Neutral red, Bromothymol blue are added to
the poorly buffered nutrient agar media .
 Colonies are sub cultured to make stock culture.
 Further testing is needed since inorganic acids, bases are
also metabolic products of microbial growth

29.  Incorporation of CaCO3 in medium is also used to screen
 organic acid producing microbes on basis of formation of clear zone of
dissolved CaCO3 around the colony.

30. PRIMARY SCREENING OF ANTIBIOTIC PRODUCING MICROORGANISMS
 Crowded plate technique is used for screening of antibiotic
producing microorganisms.
 Does not give information about the sensitivity of antibiotics
towards other microorganisms.
 Dilutions are made and then pouring and spreading of soil
samples that give 300 to 400 or more colonies per plate.
 Colonies showing antibiotic activity are indicated by zone of
inhibition around the colony .
 Such colonies are sub cultured and purified by streak before
making stock cultures.

32. Antibiotic producing Amylase producing
microbe microbes

33. Antibiotic activity against microbe

34. AUXANOGRAPHY TECHNIQUE
This technique is largely employed for detecting microorganisms
are able to produce growth factors (e.g. Amino acid and vitamins) extracellular.
ENRICHMENT CULTURE TECHNIQUE
This technique was used to isolate the desired microorganisms form
a heterogeneous microbial population present in the sample.
Either medium or incubation conditions are adjusted so as to favor
the growth of the desired microorganism.

35. SECONDARY SCREENING
 It’s a systematic screening programme intended to isolate industrially important or
useful microorganisms .
SOME IMPORTANT POINTS ASSOCIATED WITH SECONDARY SCREENING ARE
 It is useful in sorting of microorganisms that have real commercial value. The
microorganisms having poor applicability in fermentation process are discarded.
 Provides the information whether the product formed by microorganisms is new or
not.
 This may be accomplished by paper , thin layer, chromatographic technique.

36. Screening for new metabolites, and isolation of microorganisms
Research work is particularly directed for identifying
chemotherapeutically important products for the
treatment of tumors,
bacterial diseases (newer antibiotics against resistant
strains) and
viral diseases,
besides several other substances (e.g. hormones, enzyme
inhibitors).
In addition, isolation of microorganisms for improvement of
food industry, and for efficient degradation of the environmental
pollutants and hazardous chemicals also assumes significance.

37. An isolated producer strain should possess the following
characters:
 1. It should be able to grow on relatively cheaper substrates.
 2. It should grow well in an ambient temperature preferably at 30-40°C. This
reduces the cooling costs.
 3. It should yield high quantity of the end product.
 4. It should possess minimum reaction time with the equipment used in a
fermentation process.
 5. It should possess stable biochemical characteristics.
 6. It should yield only the desired substance without producing undesirable
substances.
 7. It should possess optimum growth rate so that it can be easily cultivated on a large
scale.

38. Microbes in Industrial Products
 Beverages.
Yeasts are the widely used microorganism for the production of beverages
like beer, brandy, rum, wine, whiskey, etc.
 Organic acids.
Microbes are also used for the industrial production of certain organic acids.
 Enzymes.
 Antibiotic.
 Vitamins.

39. A List of important microorganisms and their products
Microorganism Product
Algae
Chlorella sorokiniana Single cell protein
Spirulina maxima Single cell protein
Bacteria
Acetobacter aceti Acetic acid
Bacillus subtilis Bacitracin
Actinomycetes
Streptomyces aureofaciens Tetracycline
Fungi
Aspergillus niger Citric acid