Deals with various methods adapted for the Improvement in Microbial Cell Culture alongwith the procedure and instruments used to carry out the operation through illustrative diagrams

1. SAM HIGGINBOTTOM INSTITUTE OF AGRICULTURE,
TECHNOLOGY & SCIENCES
Allahabad, Uttar Pradesh (INDIA)
Improvement in Microbial Cultures
by
A REHMAN
B Tech Food Technology
Department of Food Process Engineering

2. MICROBIAL STRAIN IMPROVEMENT
The Science and technology of manipulating and improving microbial strains in order to enhance
their metabolic capacities for biotechnological applications are referred to as strain improvement.
Improvement of strains can therefore be put down in simple term as follows:
(i) regulating the activity of the enzymes secreted by the organisms;
(ii) in the case of metabolites secreted extra cellularly, increasing the permeability of the
organism so that the microbial products can find these way more easily outside the cell;
(iii) selecting suitable producing strains from a natural population;
(iv) manipulation of the existing genetic apparatus in a producing organism;
(v) introducing new genetic properties into the organism by recombinant DNA technology or
genetic engineering.
Benefits of strain improvement
 Rapid growth
 Genetic stability
 Non-toxicity to humans
 Large cell size, for easy removal from the culture fluid
 Ability to use cheaper substrates
 Elimination of the production of compounds that may interfere with downstream
processing
 Increase productivity
 To improve the use of carbon and nitrogen sources
 Reduction of cultivation cost
-lower price in nutrition.
-lower requirement for oxygen.
 Production of
-additional enzymes.
-compounds to inhibit contaminant microorganisms
Improvement in Microbial Cultures
Improvement in Microbial Cultures
Techniques for Improvement in Microbial Strains:
 Protoplast Fusion
 Mutation
 rDNA Technology

3. Methods for Improvement in Microbial Strains
 Protoplast Fusipn
 Mutation
 rDNA Technology
Protoplast fusion
Protoplast fusion is a type of genetic modification in plants by which two distinct species of
plants are fused together to form a new hybrid plant with the characteristics of both, a somatic
hybrid.
The following points highlight the two methods of protoplast fusion. They are:
(1) Spontaneous Fusion and
(2) Induced Fusion.
1. Spontaneous Fusion:
Protoplasts during isolation often fuse spontaneously and this phenomenon is called spontaneous
fusion. Simply physical contact is sufficient to bring about the spontaneous fusion among the
similar parental protoplasts.
2. Induced Fusion:
Fusion of freely isolated protoplasts from different sources with the help of fusion inducing
chemical agents is known as induced fusion.

4. Mutations
Genes are chemically the segments of DNA molecules except in some viruses, as some viruses
are found to contain RNA as genetic material. They are normally transmitted with great
exactness. But sometimes variations may be caused by physical or chemical agents resulting in
altered phenotype. The heritable changes in the genome of a cell are called mutations.
Those mutations which occur in the somatic cells are called somatic mutations. These are not
transmitted to next generation.
Mutations occurring in the germ cells are called germinal mutations. These mutations influence
the gametes and are passed to next generation, generating new variability and contributing to the
process of evolution.
Mutation
Spontaneous mutations Induced mutations
(i) Spontaneous mutations : Mutations that occur naturally are called spontaneous
mutations. Their origin is indeterminate and unknown. They are generally assumed to
be random changes in the nucleotide sequences of genes.
(ii) Induced mutations : The mutations resulting from the influence of any artificial factor
are considered to be induced mutations.
Mutagens
Mutations inducing agents are called mutagens. They create mutations in different ways.
Mutagens
Physical Mutagens Chemical Mutagens
Ionizing radiation Non-ionizing radiation Alkylating Intercalating Base
agents agents analogs
Depending on the nature of mutagens they are of two types:
 Physical mutagens
 Chemical mutagens
A. Physical mutagens : Mutations can be naturally or artificially induced by a variety of
physical mutagens. The physical agents are broadly divided into two types :
 Ionizing radiation
 Nonionizing radiation

5. (i) Ionizing radiation: X-rays and gamma (Y) rays are ionizing radiations. They have
short wavelength and high penetration power. They can penetrate into deeper tissues
causing ionization of the molecules along their way.
(ii) Non-ionizing radiation : Ultra Violet (UV) rays are nonionizing radiations. They have
long wavelength and low penetration power. This property has been useful in the
detection and analysis of nucleic acids. The dimers damage the DNA structure and
effects normal replication.
B. Chemical mutagens : Mutatins can also be induced due to certain chemicals. Chemical
mutagens can remove, replace or modify DNA bases. They can be:
 Alkalyting agent
 Intercalating agents
 Base analogs
(i) Alkylating agents : Alkylatin of nitrogenous bases by the alkylating agents either
removes the base or modifies it. Guanine residues can be alkylated by the methyl
methane sulfonate and ethyl methane sulfonate. These agents alkylates guanine at N7
and weakens the purine-deoxyribose linkage. This leads to deppurination creating gap
at that site. N-methyl-N1-nitro –N –nitrosoguanidine CH3-N(NO)-C(NH)-NH-NO2
is a powerful mutagen in E.coli. Some alkylating agents change the GC positin ina
nucleotide to AT.
(iii) Intercalating agents : Intercalating agents produces frame shift mutatinos in
bacteriophages like T4.
(i) Base analogs : Base analogs are structurally similar to normal nitrogenous bases and
can be incorporated into the growing polynucleotide chain during replication.
Mutations occurring in the germ cells are called germinal mutations.

6. rDNA (Recombinant DNA) Technology
The technology used for manipulation of DNA and transfer of DNA from one cell to other cell is
termed as recombinant DNA technology. Application of this technology is called genetic
engineering.
The recombinant DNA experiment always starts with isolation or synthesis of DNA. Isolation of
a gene from a large chromosome requires methods for cutting DNA and of ligation DNA
fragments to another DNA (vector). All these steps requires the enzymes like restriction
endonucleases, polymerases and ligases. They can be propagated to form clones, called cloning.
Advances in the techniques of rDNA technology are revolutionizing medicine, agriculture, and
other industries.
Gene transfer mechanisms in bacteria
Transfer of genetic information to a cell from a donor cell by direct contact or in a free DNA
form or through an agent is called gene transfer. It results in developing many new features to the
transformed cell like pathogenicity, antibiotic resistance etc.
Figure: Representing cell undergoing
manipulation through rDNA
technology

7. Based on the mechanism gene transfer is of three types:
(1) Conjugation
(2) Transformation
(3) Transduction
 The transfer of genetic information from a donor cell to a recipient cell through direct
physical contact between cells is called conjugation.
 Transfer of the cell free DNA into a recipient cell is called transformation.
 Transfer of bacterial genes from one cell to another through bacterial viruses
(bacteriopphages ) is called transduction.