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C h a p t e r 4 :

MICROBIAL CELL CULTURE AND ITS APPLICATIONS

(Chapter 4 in NCERT Book)

“It is possible to store the mind with a million facts and still be entirely uneducated.”

— Alec Brourne

WHAT WOULD YOU LEARN IN THIS CHAPTER?

In this chapter, you would learn about the topics given below in brief and would also be

able to answer the questions based on them:

4.1 INTRODUCTION

4.1 MICROBIAL CULTURE TECHNIQUES

4.1 MEASUREMENT AND KINETICS OF MICROBIAL GROWTH

4.1 SCALE UP OF MICROBIAL PROCESS

4.1 ISOLATION OF MICROBIAL PROCESS

4.1 STRAIN ISOLATION AND IMPROVEMENT

4.1 APPLICATIONS OF MICROBIAL CULTURE TECHNOLOGY

4.1 BIOETHICS IN MICROBIAL TECHNOLOGY

QUICK RECAP; STUDY EXERCISE - QUESTIONS & ANSWERS; PRACTICE QUESTIONS

(## additional information provided for more conceptual clarity)

(The above topics have been explained in brief in the pages ahead. For a detailed explanation of the chapter please

enquire and order for FUNDAMENTALS of BIOTECHNOLGY for Class XII)

Chapter 4: Microbial Cell Culture and its Applications · Chapter at a Glance

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CHAPTER AT A GLANCE

The importance of microorganisms is known to mankind since time immemorial. The

production of vinegar, alcohol, antibodies and various other microbial products for the

benefit of mankind is already known to us. This chapter deals with the microbial culture

techniques including the ideal conditions under which the maximum growth of the

microorganisms take place. When the microbes are cultured in the laboratory, the various

elaborate procedures e.g. sterilization etc. are followed hence the culture procedures have

been discussed. The specific procedure of measurement of microbial growth, growth

kinetics and specific growth rate have been introduced along with the commercial

production of microbial culture. Proper procedures have to be followed for the isolation of

the microbial products. The newly developed strains or the useful strains are preserved for

further use and investigations. There are specific culture collection centers for this purpose.

Also the applications of microbial culture technology along with their uses are discussed.

(For a detailed explanation of the above topics please enquire and order for

FUNDAMENTALS of BIOTECHNOLGY for Class XII)

IMPORTANT TERMS

M

ICROBIAL

GROWTH

Microbial growth is defined as an orderly increase in all chemical components in the

presence of suitable medium and environment.

D

OUBLIN G

TIME

It is the time required for the cell mass or number to double its original value during the

balanced growth of the organism.

MTCC

Microbial Type Culture Collection

ATCC

American Type Culture Collection

NCIB

National Collection of Industrial Bacteria, British
Chemical equation for the production of products from reactants:

The rate of increase in cell mass with time dX/dt is equivalent to the product of specific

growth rate u and the biomass concentration:

where X is the cell concentration in gm/L, t is time in hour and u is specific

growth rate in . The specific growth rate is an index of rate of growth
of the cells in that particular environment.

B

AFFLE

F

LASK

The flasks with a v-shaped notch or indentation in the side of the flask are called baffle

flask.

F

ERMENTORS

These are vessels which are used for large scale growth of microorganisms under a

controlled environment.

B

ATCH

C

ULTURE

Batch culture is a closed culture system which contains limited amount of nutrients.

C

w

H

x

O

y

N

z

+ aO

2

+ bH

g

O

h

N cCH

a

O

b

N

d

+ dCO

2

+ eH

2

O

dX dt

⁄

uX

=

hour 1

–

u

1

X

---- dX

dt

-------

⋅

=

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F

ED

-B

ATCH

C

ULTURE

If a batch culture described above is continuously or sequentially fed with fresh medium

without removing the growing culture, it is called fed-batch culture.

C

HEMOSTAT

In a chemostat, constant chemical environment is maintained.

T

URBIDOTSTAT

In a turbidostat constant cell concentration is maintained.

STUDY EXERCISE – QUESTIONS AND ANSWERS

Q. 1 :

What are the differences between: (a) Batch and Fed Batch culture

Ans :

Batch culture

•

Batch culture is a closed culture system, which contains limited amount of nutrients.

•

The medium is inoculated with the bacterial inoculum and the organism follows normal growth

phases i.e. lag, log, stationary and decline phase.

•

Due to the growth the nutrients are consumed and microbial products are excreted.

•

At stationary phases, the growth declines to zero.

•

E.g. culturing microbes in the laboratory, in an ordinary flask, is basically a batch culture.

Fed-Batch Culture

•

If a batch culture described above is continuously or sequentially fed with fresh medium with-

out removing the growing culture, it is called fed-batch culture.

•

Over a period of time, the volume in the culture vessel goes on increasing.

•

Fed-batch culture is preferred when high substrate concentration causes growth inhibition. To

overcome this problem, substrate is fed at concentrations below its toxic level to achieve cell

growth.

•

By using fed-batch culture, high cell densities can be achieved in the same reactor volume in

comparison to the normal batch culture.

•

E.g. Fed batch culture is an ideal process for maximum production of intracellular metabolites

from the same volume of the reactor.

Q. 2 :

How is continuous culture better than batch or fed batch cultures?

Ans :

Continuous culture is better than batch or fed batch culture because we can get a continuous supply of
microbial growth and/or by products. In this the growth medium is designed in such a way that one of
the nutrients is in limited quantitiy and during the exponential growth, as this nutrient is exhausted the
growth will stop. However, just before the nutrient is fully exhausted, fresh medium containing the
limited nutrient is added. Hence in continuous culture, cells can be grown at a particular growth rate for
an extended period of time.
Continuous culture is most widely used for the treatment of liquid waste wherein waste organic materials
are converted in to microbial biomass.

Q. 3 :

Explain what is meant by steady state in relation to the growth of microbial cultures?

Ans :

At steady state, the cell growth and substrate consumption takes place at a fixed rate. Growth rate of cells
remain constant during steady state operation. In other words, at steady state the concentration of cells,
metabolites and other nutrients inside the reactor remain constant. That is the new biomass by the
culture is balanced by the loss of the culture from the vessel.

Q. 4 :

Suggest two methods of preserving microbial strains.

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Ans :

Two methods of preserving microbial strains are:
(a)

Storage on Agar: Cultures are grown on agar stabs or plates/slants and stored at 5 to -20

o

C. These

must be sub-cultured at approximately 6 month intervals. The time of subculture may be extended
to 1 year if cultures are covered with sterile mineral oil.

(b)

Storage in Liquid Nitrogen: The culture is grown and a cryoprotective agent like glycerol (10-30%)
is added. These are dispensed into sealed ampoules and frozen in liquid nitrogen (-176 to - 196

o

C).

Q. 5 :

What is lyophilization?

Ans :

Lyophilization or freeze-drying involves freezing of a culture followed by dry under vacuum. This results
in sublimation of cell water. Lyophilised cultures can remain viable for 10 years or more.

Q. 6 :

Why are antifoaming agents added in the microbiological cultures?

Ans :

The antifoam agents are added because in most microbiological processes foaming is produced may be
due to the components of the medium or some times the microbes themselves produce it. The most
common cause of foaming is the presence of proteins in the culture medium. Commonly used antifoams
are fatty acids such as olive oil or sunflower oil and silicones.

Q. 7 :

What is the role of water in the culture media?

Ans :

Water is the major component of the culture media. Single or double distilled water is used for culturing
the microbes. For large-scale microbial culture in industry, the pH and dissolved salts of the water source
has to be considered. Water is also required for the ancillary services such as heating, cooling and
rinsing. Clean water of consistent composition is therefore a prerequisite for large scale culturing of
microbes.

Q. 8 :

What is the importance of pH of the medium during the microbial culture?

Ans :

The pH of the medium influences the growth of the organism therefore before autoclaving the medium,
the pH is balanced according to the need. E.g. bacteria grow at neutral pH whereas fungi and yeast grow
at acidic pH. The rapid growth of the microorganisms in the fermenter quickly alters the pH of the
medium so the pH of the growing culture is continuously monitored and maintained by adding acid or
alkali as required.

Q. 9 :

How can we measure the microbial growth?

Ans :

The microbial growth can be measured by
(a)

By Measuring the Dry Cell Mass: It is carried out by measuring the dry weight of the cell material
in a fixed volume of the culture by removing the cells from the medium and drying them till constant
weight is obtained.

(b)

By Measuring the Absorbance of Cell Suspensions in a Spectrophotometer: This principle is
based on the fact that small molecules scatter light proportionate to their concentration. When light
passes through a suspension of bacteria, there is a reduction in light transmitted as a consequence of
scattering. If we have a standard graph plotted with absorbance versus cell concentration, the cell
concentration of the unknown microbial sample can be calculated by measuring the absorbance at
the same wavelength.

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(c)

By Counting the Cell Number: Such counting is usually done with a special microscope slide
known as a counting chamber. These counting chambers have fixed volumes and by counting the
number of cells one can calculate the number of cells per unit volume of the culture.

(d) Besides these other methods are- measurement of wet weight of cells, turbidity measurements, ATP

measurements, viable plate count, and use of Coulter counter.

Q. 10 :

Give two examples of application of microbes for human use?

Ans :

Expression of human insulin in E. Coli and Hepatitis B vaccine in yeast are the most notable examples
of application of microbes for human use.

PRACTICE QUESTIONS

Q. 1 :

What are the factors required for the growth of microbes in the cell culture?

Q. 2 :

Define Synthetic media and semisynthetic media?

Q. 3 :

What sterilization procedures are normally used during cell culture procedures?

Q. 4 :

Why the flasks are put on shakers during the cultivation of microorganisms?

Q. 5 :

What is a fermenter? Describe the various parts of a fermenter ?

Q. 6 :

Give examples of carbon sources and nitrogen sources for the growth of microbial culture.

Q. 7 :

Why are sometimes growth factors added in the medium used for the culture of microbes?

Q. 8 :

Fill in the blanks
(a) In the stirred tank bioreactor the culture medium is stirred with an ___________.
(b) ___________ are used for large scale growth of microbes in the biotechnology industry.
(c) ___________ is a closed culture system, which contains limited amount of nutrients.
(d) At ___________ phase, the growth declines to zero.
(e) If a batch culture is continuously or sequentially fed with fresh medium without removing the growing culture, it
is called ___________ culture.
(f) In a ___________, constant chemical environment is maintained.
(g) In a ___________, constant cell concentration is maintained.
(h) At ___________, the cell growth and substrate consumption takes place at a fixed rate.
(i) ___________ is an electronic instrument used for direct counting of microbial cells in suspension.
(j) ___________ or freeze drying involves freezing of a culture followed by drying under vacuum.

Q. 9 :

What do you mean by downstream processing?

Q. 10 :

What are the steps involved in the isolation of microbial metabolite?

Q. 11 :

Name two microbial species used for producing commercial products.

Q. 12 :

Why are safety measures of paramount importance while dealing with biotechnological processes?

Q. 13 :

Draw the basic design of a fermenter?

Q. 14 :

How can we improve strains?

Q. 15 :

What do you mean by scale up?