NMEICT-MHRD (Govt.

of India) Project on - Creation of e-Contents on Fermentation Technology

Module-6: Primary and Secondary Screening of Industrially Important

Microbes

Screening

“The use of highly selective procedures to allow the detection & isolation of only those
microorganisms which are of interest from among a large microbial population”

 Screening allows the discarding of many valueless microorganisms, at the same time it
allows the easy detection of the useful microorganisms that are present in the population
in very less number

Primary screening

“Primary screening allows the detection & isolation of microorganisms that possess
potentially interesting industrial application”

 Primary screening separate out only a few microorganisms having real commercial value.

 Primary screening determines which microorganisms are able to produce a compound

without providing much idea of the production or yield potential of the organisms

A. Primary screening of organic acid producing microorganisms

 Incorporation of a pH indicating dye such as neutral red or bromothymol blue into a

poorly buffered agar medium.

 Greater buffer capacity of medium screen microbes having capability to produce

considerable quantities of the acid

 Incorporation of calcium carbonate in the medium is also used to screen organic acid
producing microbes on the basis cleared zone of dissolved calcium carbonate around the
colony

 These screening approaches do not give idea that which organic acid has been produced

 Thus the colonies of microorganisms showing the potential to produce any fermentation
product should immediately be purified and sub-cultured into appropriate medium to be
maintained as stock cultures for further testing.

B. Primary screening of antibiotic producing microorganisms

 The simplest screening technique for antibiotic producers is :Crowded Plate” technique

 The technique is used to find out the microorganisms that produce an antibiotic without
giving much information of sensitivity towards other microorganisms.

 Procedure include dilution and spreading or pouring of soil samples that give 300 or 400
or more colonies per plate

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 Colonies producing antibiotic activity are indicated by an area of agar around the colony

 Such a colony is sub-cultured to a similar medium and purified by streaking, before

making stock cultures. The purified culture is then tested to find what types of
microorganisms are sensitive in the presence of these the antibiotics i.e. “Microbial
Inhibition Spectrum” (MIS).

 The crowded plate procedure also does not necessarily select an antibiotic producing
microorganism, because the inhibition area around the colony sometimes can be due to
other reason like….

(1) Marked change in the pH of the medium resulted due the metabolism of the colony.

(2) Rapid utilization of critical nutrients in the vicinity of the colony etc.

 Thus further testing is required to confirm the inhibitory activity associated with a
microorganisms is whether attributed to the presence of an antibiotic or not

 Screening of antibiotic producing microorganisms can be improved by using a “test

organism” and Wilkins method

C. Primary screening of extracellular metabolites (Vitamins, Amino acids and Growth

factors) producing microorganisms

D. Primary screening of microorganisms utilizing specific Carbon and Nitrogen sources

Secondary screening

Secondary screening allows further sorting out of microorganisms obtained from PS having
real value for industrial processes and discarding of those lacking this potential

1. SS is conducted on agar plates, in flasks or small fermenter containing liquid media

2. SS can be qualitative or quantitative in its approach

3. Secondary screening should give information about the evaluation of the true potential of
the microorganisms for industrial usage

4. SS should determine whether microorganisms are actually producing new chemical

compounds not previously described

5. SS should reveal whether there is pH, aeration or other critical requirements associated
with particular microorganisms, both for the growth of the organism and for the
formation of chemical products

6. SS should also detect gross genetic instability in microbial cultures

7. SS should show whether certain medium constituents are missing or possibly, are toxic to
the growth of the organisms or its ability to accumulate fermentation products

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 NMEICT-MHRD (Govt. of India) Project on - Creation of e-Contents on Fermentation Technology

8. SS should determine whether the product has a simple, complex, or even a

macromolecular structure, if this information is not already available

9. SS should show something of the chemical stability of the product and of the product’s
solubility picture on various organic solvents

10. SS should show whether the product possesses physical properties such as UV light
absorption or fluorescence or chemical properties that can be employed to detect the
compound during the use of paper chromatography or other analytical methods and
which also might be of value in predicting the structure of the compound

11. In some case, for certain kinds of fermentation product determinations should be made as
to whether gross animal, plant or human toxicity can be attributed to the fermentation
product, particularly if it is utilized (as are antibiotics) in disease treatment

12. SS should reveal whether a product resulting from a microbial fermentation occurs in the
culture broth in more than one chemical form and whether it is an optically or
biologically active material

13. SS should reveal whether the microorganisms are able to chemically alter or even destroy
their own fermentation products

14. Secondary screening helps in predicting the approaches to be utilized in conducting

further research on the microorganisms and its fermentation processes.

Strategies for isolation of industrially important microbes

 The diversity of microorganisms may be exploited still by searching for strains from the
neutral environment able to produce products of commercial value

 The first stage in the screening of microorganisms of potential industrial 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

 In some cases it is possible to design the isolation procedure in such a way that the
growth of producers is encouraged or that they may be recognized at the isolation stage,
whereas in other cases organisms must be isolated and producers recognized at a
subsequent stage

 It should be remembered that the isolate must carry out the process economically and
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.

Criteria used for choice of organisms

 The nutritional characteristics of the organism: Organism should be capable to utilize the
ingredients present in the medium to produce interested product.

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 NMEICT-MHRD (Govt. of India) Project on - Creation of e-Contents on Fermentation Technology

 The optimum temperature of the organisms: For instance, the use of an organism having
an optimistic temperature above 40o C considerably reduces the cooling costs of a large-
scale fermentation, and therefore, the use of such a temperature in the isolation procedure
may be beneficial

 The reaction of the organism with the equipment to be employed

 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 easy product recovery from the cultures.

 It should be a high yielding strain

 It should have stable biochemical characteristics

 It should not produce undesirable substances

 It should be easily cultivated on a large scale

 The ideal isolation procedure commences with an environmental source (frequently soil),
which is highly profitable to be rich in the desired types

 Selective pressure may be used in the isolation of organism that will grow on particular
substrates in the presence of certain compounds or under agricultural conditions adverse
in their types

 If it is not possible to apply selective pressure for the desired character it may be possible
to design a procedure to select for a microbial taxon which is known to show the
characteristics at a relatively high frequency. E.g. the production of antibiotic by
Streptomycin.

 Alternately, the isolation procedure may be designed to exclude certain microbial

“weeds” and to encourage the growth of more novel types

 The advantages in the taxonomic description of taxa have allowed the rational design of
procedures for the isolation of strains that may have a high probability of being
productive or are representatives of unusual groups.

 The advances in pharmacology and molecular biology have also enabled the design of
more effective screening tests to identify productive strains amongst the isolated
organisms.

Project control No: RE-02091011297, Christ College, Rajkot, Gujarat, India

 NMEICT-MHRD (Govt. of India) Project on - Creation of e-Contents on Fermentation Technology

References

 Principles of Fermentation Technology: (2nd edition, by Peter F. Stanbury, Allan

Whitaker and Stephen J. Hall, Butterworth-Heinemann, An imprint of Elsevier Science.)
 Industrial Microbiology: (By Casida L. E.New Age international (P) ltd publications)
 A Text Book of Industrial Microbiology: (2nd edition By Wulf Crueger & Anneliese
Crueger)
 Biotechnology: Food Fermentation Microbiology, Biochemistry & Technology Vol. 1
& 2:(By V.K. Joshi & Ashok Pandey)
 Manual of Industrial Microbiology and Biotechnology: (2nd Edition by Arnold L.
Demain and Julian E. Davies, Ronald M. Atlas, Gerald Cohen, Charles L. Hershberger,
Wei-Shou Hu, David H. Sherman, Richard C. Willson and J. H. David Wu)
 Industrial Microbiology-An introduction: By Michael J. Waites, Neil L. Morgan,
John S. Rockey and Gary Higton)
 Comprehensive Biotechnology-The Principles, Applications and Rugulations of
Biotechnology in Industry, Agriculture and Medicine: (By Mrray Moo Young)
 Fermentation Technology : Up Stream Fermentation Technology- Vol-I: (By H. A.
Modi-Pointer Publications)
 Fermentation Technology : Down Stream Fermentation Technology- Vol-II: (By H. A.
Modi-Pointer Publications)
 Industrial Microbiology by Prescott and Dunn's: (4th edition, edited by Gerald Reed,
CBR publications)
 Fermentation Technology: (By M.L. Srivastava, NAROSA publications)
 Industrial Microbiology: (By A.H. Patel)
 International student edition: Microbiology- A laboratory Manual: (4th edition. By
James G. Chappuccino & Natalie Sherman)
 Bacteriological Techniques: (By F.J. Baker)
 Introduction to Microbial Techniques: (By Gunasekaran)

Project control No: RE-02091011297, Christ College, Rajkot, Gujarat, India

 NMEICT-MHRD (Govt. of India) Project on - Creation of e-Contents on Fermentation Technology

 Mannual of Industrial Microbiology and Biotechnology: (2nd Edition by Arnold L.

Demain and Julian E. Davies, Ronald M. Atlas, Gerald Cohen, Charles L. Hershberger,
Wei-Shou Hu, David H. Sherman, Richard C. Willson and J. H. David Wu)

Web references

 http://www.homebrew.net/ferment/
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 http://scialert.net/fulltext/?doi=jm.2007.201.208
 http://aem.asm.org/content/7/1/57.full.pdf
 http://www.slideshare.net/yongkangbirdnest/lecture-4-sterilization
 http://www.ars.usda.gov/research/publications/publications.htm?seq_no_115=140721
 http://www.scribd.com/doc/30706834/Fermentation-Design
 http://www.wiley-vch.de/books/sample/3527318194_c01.pdf
 http://www.engineersirelandcork.ie/downloads/Biopharmaceuticals%2020Jan09%20-
%202%20-%20Ian%20Marison%20DCU.pdf
 www.yobrew.co.uk/fermentation.php
 http://bioscipub.com/journals/bbb/pdf/19-24.pdf
 http://gertrude-old.case.edu/276/materials/web/immobilizedenzymereview.pdf
 http://download.bioon.com.cn/upload/month_0902/20090223_b809d1c59ba2a6e2abfdJt
WiJOiFDm02.attach.pdf
 http://bioprocess-maulik.blogspot.in/2007/07/design-of-industrial-fermentation.html
 http://hsc.csu.edu.au/biology/options/biotechnology/3051/biotechnologyPart3.html
 http://www.rsc.org/ebooks/archive/free/BK9780854046065/BK9780854046065-
00001.pdf
 http://www.biotech.upm.edu.my/academics/On%20Line%20Note/Bioprocess/BTK%20
5301/Lect6%28Inoculum%20Preparation%20and%20Development%29.pdf
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 http://www.idosi.org/wjc/4%281%2909/14.pdf
 http://cheserver.ent.ohiou.edu/Paper-gu/DualFeed.pdf

Project control No: RE-02091011297, Christ College, Rajkot, Gujarat, India