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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5472_Библиотеки_им_академика_М_И_Перельмана.pdf
Microbial Culture and Fermentation Process
105
concentration becomes too high, the amount of cells that are removed from the reactor
cannot be replenished by growth as the addition of the limiting nutrient is insufficient. It
results in an equilibrium situation ( steady state), where the rate of cell growth is equal to
that of cell removal (Fig. 7.3).
2. Turbidostat bioreactor: In this case, turbidity measurement is used to monitor the biomass
concentration.
Plug flow bioreactor
In this bioreactor, the culture solution is fed through a tubular reaction vessel without back mixing.
The composition of medium, the quantity of cells, O
2
supply and product formation vary at different
locations in the bioreactor.
Continuous fermentation process is used for the production of antibiotics, organic solvents,
single-cell proteins, beer and ethanol, besides its use in wastewater treatment.
Advantage of continuous cultures
1. Continuous fermentation can be run in a cost-effective manner.
2. The size of the bioreactor and other equipment are smaller as compared to batch
fermentation.
3. The yield of the product is more consistent.
4. The time required for cleaning and preparing the bioreactor for reuse between two successive
fermentations can be avoided.
Limitation of continuous cultures
1. Maintenance of sterile condition for such a long period is difficult.
2. It is difficult to maintain the same quality of the culture medium for all the additions.
3. Recombinant cells with plasmid construct cannot function continuously.
Choice of Fermenter
The selection of the type, size, mode of operation and the pattern of the fermenter to be used in a
particular biological process is based on the following characteristics:
Figure 7.3 Chemostat bioreactor.
Pump
Pump
Air in
Air out
Out
In
Textbook of Pharmaceutical Biotechnology
106
1. Characteristics of the microorganism in use: The mode of operation depends substantially
on the type and the stability of the microbial strain. The operational conditions are determined
based on aerobic or anaerobic nature of microbial strain. The size and the shape of the
microbial cells also have considerable effect on the design of the fermenter. The spherical
cells are usually smaller in size and less sensitive to shear than the filamentous organisms.
The spherical cells need a higher degree of dispersion of the air as compared to filamentous
mycelia. Small dimensions of cells ensure a high surface to volume ratio and a high rate of
uptake of substrate. Therefore, a rapid growth is possible.
2. Characteristics predetermined by properties of medium: The choice of the strain generally
not only determines the culture medium, but also it exerts a pronounced influence on the
choice of the bioreactor. The characteristics of the medium such as physical and biokinetics,
difficulty in sterilization and rheological behaviour are the most important properties to be
considered for the selection of the reactor. When the substrate or product is responsible for the
high viscosity, the medium frequently has a new toning behaviour. A high apparent viscosity
due to the morphology of the organism is almost always associated with a non-Newtonian
behaviour. The physical properties of the substrate used generally differ like gaseous, liquid
and water soluble, solid and water soluble, liquid and water insoluble and solid and sparingly
soluble or insoluble in water.
3. Characteristics predetermined by parameters of biomedical process: The important factor
influencing choice of the fermenter in cultivation of aerobic organism is specific oxygen
transfer rate in the medium. The rate of growth and rate of product formation are temperature
dependent. Consequently, the cultivation and the formation of the product are usually carried
out at controlled temperature.
Some important fermentation-based commercial products are given in Table 7.1.
Table 7.1 Some products produced by microorganisms in fermenter
Products Microorganisms
Industrial chemicals
Ethanol
Saccharomyces cerevisiae
Citric acid
Aspergillus niger
Gluconic acid
A. niger
Acetic acid
Acetobacter spp.
Lactic acid
Lactobacillus delbrueckii
Amino acids
L-lysine
Corynebacterium glutamicum
MSG
C. glutamicum
Glutamic acid
C. glutamicum
Vitamins
Riboflavin
Ashbya gossypii
Vitamin B
12
Pseudomonas denitrifi cans
Ascorbic acid
Gluconobacter oxydans
(Continued)
Microbial Culture and Fermentation Process
107
Products Microorganisms
Enzymes
Amylase
Asper
gillus oryzae
Cellulose
Trichoderma reesei
Invertase
Saccharomyces cerevisiae
Lipase
Saccharomycopsis lipolytica
Polysaccharides
Dextran
Leuconostoc mesenteroides
Xanthan gum
Xanthomonas campestris
Pharmaceuticals
Penicillin
Penicillium notatum
Cephalosporins
Cephalosporium acremonium
Amphotericin B
Streptomyces nodosus
Kanamycin
Streptomyces kanamyceticus
Neomycin
Streptomyces fradiae
Streptomycin
Streptomyces griseus
QUALITY CONTROL OF MICROBIAL CULTURE MEDIA
The culture media are widely employed for isolation, identification and sensitivity testing of
different pathogenic microorganisms. Most of the laboratories usually prepare their own media for
routine diagnostics, as well as research purposes. However, to ensure that the media prepared are of
good quality and capable of giving satisfactory results, proper quality management system should
be in place. For this purpose, following parameters of media preparation should be thoroughly
checked and then taken up for laboratory use.
Raw Materials
The quality of the medium depends directly upon the quality of the raw materials used for
its preparation. Water is the most important raw material used for the preparation of culture
medium. The parameters to be checked for water are presence of copper ions, conductivity and
pH. Ideally, there should be no copper ions present in water, because they are inhibitory to the
growth of microorganisms. The quality of Petri dishes used is also an important factor. Normally
Petri dishes are ethylene oxide sterilized or gamma irradiated. The maximum permissible limit
for residual ethylene oxide is 1 g/g, and it can be measured by standard gas chromatographic
method. Only borosilicate glassware should be used, because soda glass can leach alkali into the
medium.
For blood-containing culture medium, sterility, homogeneity, viscosity and colour of the
blood should be scrupulously checked. For other additives, the certificate of analysis and sterility
conditions should be taken into consideration.
Textbook of Pharmaceutical Biotechnology
108
Sterilization Parameter
Sterilization of the medium plays an important role in maintaining the quality of the culture
medium. Sterilization by autoclaving is the usual practice in preparation of medium. However, the
time of autoclaving and the quantity of medium sterilized should be closely monitored. Excessive
heat treatment of complex culture medium may result in destruction of its nutrients either by direct
thermal degradation or by reactions between the components. Therefore, it is very important to
optimize the heating process to minimize the loss of nutrients.
Physical Parameter
The gross physical appearance of medium often suggests the quality. The culture medium prepared
should be screened for physical characteristics, such as excessive bubbles or pits, unequal filling
of plates (uniform levelling), cracked medium in plate and effect on freezing or crystallization.
All these characters can be checked visually by naked eye. However, in order to ascertain unequal
filling of plates, thickness of medium can be checked at two ends of two diameters of the plate,
which are at right angles to each other. Thus, all the four sides can be simultaneously checked. The
thickness at these four points is noted down, and the mean thickness is determined and reported
as mean thickness of the medium in the plate, which must be 4.0 ± 0.2 mm. The pH value can be
measured during preparation of the medium, before and after autoclaving, by using the standard pH
metre after proper calibration with standard buffers.
Microbiological Parameter
Growth-supporting characteristics are the most important parameters in quality control of culture
medium. Standard inoculating procedures should be used. Both qualitative and quantitative
results should be examined. While testing new lots, both previous batch and new batch should be
simultaneously grown.
Econometric Method
This is a simple and numerical method. Both absolute growth index (AGI) and relative growth
index (RGI) can be determined. The method is based on streaking of inoculum to extinction. This
method can be used to compare results with previous batches of same medium or between selective
and nonselective media. In this method, 5 mL of brain heart infusion broth is inoculated with
loopful of chosen test organism and incubated for 4 h.
Productivity Ratio
The productivity ratio (PR) of a culture medium is related to control medium, which is usually a
nutritious agar-like tryptone soy agar (TSA). The inoculum used should be the same for both media
and PR, and it is calculated by counting the colonies on test and control medium.
Microbial Culture and Fermentation Process
109
Total productivity = Output quantity/Input quantity
PR =
No. of colonies on test × dilution factor
No. of colonies on control × dilution factor
The method employed is modified Miles and Misra technique. A 10-fold dilution of an
overnight culture of test organism is prepared in buffered peptone water. The test plates are divided
into four quadrants, and each quadrant is marked with the dilution to be used. Starting with highest
dilution, each drop of the dilution is placed on the relevant quadrant. Same steps are repeated for
control plate. Each drop is spread in the corresponding quadrant and incubated at 37°C for 18 h.
The colonies of the lowest dilution are counted for both test and control plates.
Detection of Contamination
It is an important parameter for determination of the quality of the culture medium. It is recommended
that the whole batch of the prepared medium be checked for contamination by keeping the plates
with medium, at least for 3 days at room temperature. Alternatively, two plates from the test batch
can be placed into the incubator set at 37°C for 24 h. After required incubation, the plates are
checked for any growth. If the growth is observed, the process is repeated, with two more plates
from the same batch. If contamination occurs again, it is inferred that contamination has occurred
in the prepared batch, which is required to be discarded.
Gel Strength Parameters
Gel strength is an indication of level of solidification of agar in the culture medium. It is measured
by using a tripod stand with a central rod that is used to impart pressure on the agar. The lower end
of the rod has a spherical portion, which rests on the surface of the medium. The upper portion of
the rod has a platform on which standard weights are placed. The spherical portion of the central
rod is placed on the medium, and weights are placed on the upper platform one by one and observed
for some time. The process is continued until the agar breaks under the weight of the central rod.
While calculating the gel strength, the weight of the central rod should be deducted. The force
imparted by the rod on the agar surface can be calculated by the formula
w//r
2
, where w = weight kept on the platform, r = radius of the spherical portion at the lower end
of the central rod and / = 3.14.
A gel strength of about 300–500 dyne/cm
2
is usually considered to be satisfactory.
QUESTIONS
1. What are the factors that affect microculture?
2. Differentiate between shake flask and solid-state fermentation. Explain their advantages.
3. Describe the physical and chemical environment for microbial growth.
4. Give some examples of viscous fermentation products.
5. What do you mean by fed-batch fermentation?
6. Write a short note on batch culture.
7. Draw a neat labelled diagram of fermentor and write its uses.
8. Differentiate between batch and continuous culture.
9. Give design and operation of batch fermentation with diagram.
INTRODUCTION
Genetics refers to the study of inheritance and variability of the characteristics of an organism.
Heredity is concerned with exact transmission of genetic information from parents to their
progeny.
Classical genetics: It refers to the study of mutants—study of properties of mutants, mapping and
generating hypothesis. The main principle of classical genetics is the function of genes and their
products that are observed when the genes are muted.
Reverse genetics: It is the study of isolation of genes with the help of phenotype and its linkage
with molecular markers.
Gene comprises the genome of cell that may consist of single DNA molecule or some other
small DNA molecules. The genotype variation brought about by changes in genetic information
could be because of mutation or loss of plasmid. The genetic material and components,
particularly nucleic acid, were first isolated in 1868 by Friedrich Miescher. He established the
material isolated containing phosphorus, known as nucleoprotein. Hydrolysis of nucleic acid
from thymus yielded purine and pyrimidine bases, 2 deoxyribose and phosphoric acid. The
acid has been named as deoxyribonucleic acid (DNA). Another nucleic acid was isolated that
contained uracil instead of thymine and ribose in place of deoxyribose. This has been known
as ribonucleic acid ( RNA). A small portion of DNA that is localized outside the chromosomal
region is known as plasmid.
Genetics and Molecular
Biotechnology
8
CHAPTER
Introduction
DNA as Segment of Molecular Heredity
Genes
Mutations
Molecular Biotechnology
Questions
Textbook of Pharmaceutical Biotechnology
112
History of Genetics
The modern science of genetics originated with the discovery of hereditary characteristics by
Gregor Johann Mendel (1822–84). Each such unit of hereditary nature, which is called a genetic
unit or gene, is a substance that satisfies at least the following two essential requirements:
1. It is inherited between generations in such a fashion that each descendent has a physical copy
of this material.
2. It provides information to its carriers in respect to structure, function and other biological
attributes.
Although, the science of genetics began with the applied and theoretical work of Gregor Mendel
in the mid 1800s, other theories of inheritance preceded Mendel. A popular theory put forward by
Jean-Baptiste Lamarck during those days was the concept of blending inheritance—the concept that
individuals inherit smooth blend of traits from their parents. Mendel’s work disapproved this theory
proving that traits are composed of combinations of distinct genes rather than a continuous blend,
and the experiences of individuals do not affect the genes they pass to their children. Other theories
included the pangenesis of Charles Darwin (1809–82) related to both acquired and inherited aspects
and Francis Galton’s (1822–1911) reformulation of pangenesis as both particulate and inherited.
The modern science of genetics traces its roots to the observations made by Gregor Johann Mendel,
a German-Czech Augustinian monk and scientist who studied the nature of inheritance in plants. The
importance of Mendel’s work did not gain worldwide attention until 1890. Thomas Hunt Morgan
(1866–1945) argued that genes are on chromosomes, based on observations of a sex-linked white eye
mutation in fruit flies.
Alfred Sturtevant (1891–1970), a student of Thomas Hunt Morgan, used the
phenomenon of genetic linkage to show that genes are arranged linearly on the chromosome.
Important Contributions
Some of the contributions of modern genetics are as follows:
1. It has revolutionized the approaches of genetic analysis.
2. All possible mutations can be studied by simply changing cloned DNA before reintroducing
it into cells.
3. Method of transferring cloned DNA back into living cells has provided the key to producing
gene product (e.g. hormones and interferons) in large quantities in cells that are simple and
easy to cultivate.
4. Isolation and characterization of genes involved in cancer is possible.
5. Interspecific gene transfer to establish conservation is a reality today.
Problems and Limitations of Genetics
They can be briefly enlisted as follows:
1. Some mutations are lethal in homozygotes.
2. Only mutation that produces readily detectable phenotype can be studied.
Genetics and Molecular Biotechnology
113
3. The genetic analysis is not possible except by retrospective studies of family pedigrees.
4. In dipole organism, such mutation can be detected after appropriate breeding strategy to
generate homozygote.
Evaluation and Molecular Biology of Plants
The questions concerning the evaluation of organelles have been a key force driving studies of
organelle molecular biology. The endosymbiont origin of cellular organelles was proposed in 1882,
and this hypothesis was further developed in early 20th century. Molecular feature of organelle
genes and genome provided the confirming data for the endosymbiont hypothesis. Molecular
research over the subsequent decades reveals many prokaryotic features in the modern-day plant
organelles, including some aspects of organelles division, genome, coding content and organization,
transcription, translation, RNA processing and protein turnover. Present-day mitochondrial genomes
are extremely diverse, and the mitochondria and the land plants are uniquely expanded over the
ancestral type. The functional transfer of genes from organelle to nuclear genes has occurred
frequently and recently in the plant lineages.
DNA AS SEGMENT OF MOLECULAR HEREDITY
It has been proved experimentally that DNA is the molecule of heredity. The importance of nucleus
that contains DNA is established by the observation that nuclei of male and female germi cells fuse
during the process of fertilization.
Structure of DNA
The structure of DNA is the physical basis for inheritance. The replication of DNA duplicates the
genetic information by splitting the strands and using each splitted strand as a template for synthesis
of a new partner strand. The genes are arranged linearly along long chains of DNA sequence called
chromosomes. In bacteria, each cell usually contains a single circular chromosome, whereas DNA
arranged in multiple linear chromosomes are characteristics of eukaryotic organisms (including
plants and animals). These DNA strands are often extremely long. The largest human chromosome,
for example, is about 247 million base pairs in length.
The DNA of a chromosome is associated with structural proteins that organize, compact and
control access to the DNA, forming a material called chromatin. In eukaryotes, chromatin is usually
composed of nucleosomes, and segments of DNA wound around cores of histone proteins. The full
unit of hereditary material in an organism (usually combined DNA sequences of all chromosomes)
is called the genome (Fig. 8.1).
Nucleotide
The molecular basis for genes is DNA. It is composed of a chain of nucleotides, of which there are
four types (Fig. 8.2): (1) adenine (A), (2) cytosine (C), (3) guanine (G) and (4) thymine (T). Genetic
Textbook of Pharmaceutical Biotechnology
114
Figure 8.1 Structure of DNA.
DNA
Adenine Guanine Cytosine Thymine
Purines Pyrimidines
Sugar A base Phosphate
Nucleosides Nucleotide
Figure 8.2 Structures of adenine, guanine, cytosine and thymine.
N
C
C
C
N
C
N
C
N N
Adenine
N
N
N
Cytosine
Guanine
C
N
C
N
C
C
C
NNN
Thymine
P
C
N
C
N
C
C
C
C