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Module – 31: Biological Assay of Fermentation Product

Introduction

Assay of Product means to check the quality and quantity of the product produced. There are
many types of assays but here we are discussing Biological assay.

For biological assay, sensitive test organism or enzyme is required. The product tested
against sensitive organism, which may depress or stimulates the growth of the sensitive test
organisms.

On solid media, decrease or increase in growth of sensitive organism can project as zone of
exhibition or inhibition. In liquid media growth of test organism measured as turbidity of the
media.

If biological assay employs enzyme then quality and quantity of the product measured as
increase in the enzymatic activity. Enzymatic activity means utilization of reactant or the
production of product by enzyme.

As every coin has two sides, biological assay also has some limitations. Despite this
limitation, this assay plays an important role in the assay process in industry.

Certain limitations of Bioassay outlined below

• It is more difficult to perform this bioassay

• It provides greater error
• It is less reproducible

As said earlier this assay requires test organism, but we cannot use any organism but it
requires specific organism.

Test organisms should have following characteristics

• It should be genetically stable

• It should respond in a graded manner only to test compound and not to other materials
that may be present in the solution for assay
• It should grow relatively quickly on simple media
• It should not be a pathogen
• The cell should not clump for turbidimetric assay or swarm across the agar for
diffusion assay
• It should, if possible aerobic or facultative aerobic
• It should grow well at pH that does not affect the stability or toxicity of the material
under assay

Biological Assays divided into four categories

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• Diffusion Assay
• Turbidimetric Assay
• Metabolic Response Assay
• Enzymatic Assay

Diffusion Assay

Diffusion assays carried out on solid media, usually an agar medium, which is suitable for the
growth of the test organisms. The compound to be tested is allowed to diffuse through the
medium in a radial fashion from a pad or cup so that the adjacent growth of the test organism
is depressed either as with an antibiotic, or stimulated, as with growth factor. The diameter of
zone of inhibition or zone of exhibition reflects the concentration of the compound used for
assay, and it compared with similar zones produced by various unknown concentrations of
standard or reference compound. The zone diameters of the standard plotted against the
logarithms of the concentration used, and the linear portion of this graph used for the
determination of the actual concentration of the sample used for assay.

There are two types of Diffusion Assay

 Cylinder Method
 Paper Disc Method

1. Cylinder Method

In cylinder method, 10 ml of molten agar poured in sterile petri plate. As soon as this base
layer is hard, 5 ml of the same or a different agar medium inoculated with a test organism
added above the base layer and allowed to harden to form the „seeded agar‟ layer. Then
numbers of cylinders placed per plate depending on the expecting size of the zone. The
cylinders filled with appropriate dilutions of the solutions used for assay or with solutions
containing known concentrations of the reference compound, and the plates incubated for a
specified period at constant temperature. The diameters of the zones of stimulated or reduced
growth then measured in milliliters, and the concentration in the solutions under assay is
determined by comparing with standard curved.

2. Paper Disc Method

In the paper disc method, plates of seeded agar medium are prepared and inoculated as for the
cylinder assay. However, solution to use for assay or solutions of reference compound added
at a volume of 0.1 ml to disc of sterile filter paper, usually 12.8 mm in diameters, laid on the
surface of the seeded agar. The incubation and calculation of assay are similar to the cylinder
method.

Turbidimetric Assay

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Turbidimetric assay performed in liquid medium. The compound use for assay used in liquid
medium and growth rate or total growth of the organism used for test measured in terms of
turbidity of the medium. Growth may increase or decreases depend on the compound used for
test. A suitable sterile medium dispensed in series of tubes, and graded amount of the sterile
material to use for assay added. All the tubes inoculated with a small and constant amount of
vigorous, young culture of the test organism, and then incubated for specific period at
constant temperature. The length of the incubation period depends on whether the turbidity of
the culture is to be determined at some point during logarithmic growth, the total growth of
the organisms, which can occur in the particular medium. Turbidity measured visually or
spectrophotometrically or as optical densities or absorbance. The optical densities plotted
against the concentration of standard to obtain a standard curve.

End-point Determination Assay

Microbial growth does not always yield dispersed cells for determination of turbidity.
Sometimes, the growth occurs as a pellicle on the surface of the medium, or as a clump of the
cells at the bottom, which cannot be suitably broken up and dispersed for turbidity
determinations. A bioassay is still possible, however, by employing as end-point assay. Thus
for the fermentation product that inhibits growth, is inoculated with the test organism. The
tubes incubated for a defined period at constant temperature, and each tube observed to
determine the presence or absence of growth. The relative amount of the fermentation
product in the original fermentation broth determined by the amount of dilution, which the
fermentation product can withstand in the assay tubes and still inhibit growth of the test
organism.

Metabolic Response Assay

Metabolic Response Assays are similar to turbidimetric assay except that, instead of
measuring the effect of the fermentation product on the rate of growth or the total growth of
the test organism, we measure the effect on some metabolic reaction that the test organism
carries out during growth. Organism may evolve carbon dioxide gas or uptake oxygen gas
during particular metabolic process when certain compound is present. Thus, the compound
types, its concentration, and its uptake measured indirectly by measuring the amount of gas
evolved by the organism under standard conditions.

Enzymatic Assay

Enzymatic assays are highly specific, and they will quantitatively detect minute amount of
fermentation product, as well as differentiate biologically active and inactive compound. An
enzyme preparation incubated with a sample of culture broth to cause some enzyme-mediated
change in the fermentation product, such as partial decomposition with consequent formation
of a measurable product.

For example….

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L-glutamic acid production assayed by adding washed cells of certain strains of Escherichia
coli that contain enzyme “glutamic acid decarboxylase.” Assay carried out at a pH of five.
One mole of CO2 liberated from each mole of glutamic acid.

Enzymatic assay tested carefully to determine whether they are actually functioning under
specific conditions employed. A known amount of the pure chemical product added as an
internal standard to one sample of a typical fermentation broth, but not to another sample.
The assay results should quantitatively reflect the amount of added chemical when the assay
values for the two samples compared. If values are not same then several possibilities need to
be check.

 The pH or temperature may not be optimum for the enzymatic reaction.

 Compound in the sample like metal or inhibitor may inhibit the enzymatic reaction.
 The enzyme many be unstable under assay conditions.
 Only small quantity or no enzyme produced.

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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)
 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/
 http://www.soyinfocenter.com/HSS/fermentation.php
 http://www.ensymm.com/pdf/ensymm_fermentation_abstract.pdf
 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_b809d1c59ba2a6e2abfdJtWiJOiFDm02.att
ach.pdf
 http://bioprocess-maulik.blogspot.in/2007/07/design-of-industrial-fermentation.html

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 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%205301/Lect6%28In
oculum%20Preparation%20and%20Development%29.pdf
 http://www.biotechresources.com/services-strain.shtml
 http://www.idosi.org/wjc/4%281%2909/14.pdf
 http://cheserver.ent.ohiou.edu/Paper-gu/DualFeed.pdf

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