Professional Documents
Culture Documents
Terminologies
Secondary metabolites are organic compounds that are not directly involved in
the normal growth, development, or reproduction of an organism. Unlike primary
metabolites, absence of secondary metabolities does not result in immediate
death, but rather in long-term impairment of the
organism's survivability, fecundity, or aesthetics, or perhaps in no significant
change at all. Secondary metabolites are often restricted to a narrow set of species
within a phylogenetic group. Secondary metabolites often play an important role
in plant defense against herbivory and other interspecies defenses. Humans use
secondary metabolites as medicines, flavorings, and recreational drugs. For
example : Small "small molecules"
Alkaloids(usually a small, heavily derivatized amino acid):
Hyoscyamine, present in Datura stramonium
Atropine, present in Atropa belladonna, Deadly nightshade
Cocaine, present in Erythroxylon coca the Coca plant.
Natural phenols:
Resveratrol, etc.
Big "small molecules", produced by large, modular, "molecular factories"
Polyketides:
Erythromycin
Discodermolide
Nonribosomal peptides:
Vancomycin
Antibiotic Development :
The latest progressive trend in the logistic aspects of antibiotic development may be
observed vividly by the under mentioned sequence of goals and objectives, such
as :
To screen and evaluate different types of sources of microorganisms for the
detection of purposeful antagonism.
To identify and select modified versions of microbial mutants, establish optimal
environmental and nutritional conditions, and to develop suitable technique(s) for
the recovery of antibiotics from cultures,
To induce the production of particular desired metabolites,
To improve upon and modify the fermentative metabolites either by the aid of
biological and chemical manipulations to accomplish more useful antibiotic
substances,
To develop an elaborated methods for the total synthesis of antibiotics from ab
initio for a feasible economic advantage, and
To make use of an adjunct agent to distinctly enhance the impact or availability of
an antibiotic.
Antibiotics
These are naturally occurring antimicrobial agents produced by
microorganisms that in small quantities inhibit the growth or kill
unrelated species of microorganisms.
Sources of antibiotics
Main sources of antibiotics are bacteria, fungi and
actinomycetes. Depending on the sources antibiotics are
classified as those obtained from fungi, those obtained from
bacteria, those synthesized in the laboratory and those
produced by the microorganisms and modified in the laboratory.
From Fungi Cephalosporins from Cephalosporium, Pencillin
from Pencillium.
From Bacteria Bacitracin from Bacillus subtilis, Chorophenicol
from Streptomyces venezuela .
Synthetic Etambutol, Nitrafurans
Semi-synthetic Rifampin from Rifampicin (Streptomyces
mediterranei), Amikacin (from Kanamycin).
Classification of Antibiotics
Classification of Antibiotics is based on different criteria:
a) Based on their spectrum of activity:Group-1: Active against Gram-positive bacteria and gram-negative cocci
Ex: Pencillins, Erythromycin
Group-2: Mainly active against Gram-negative bacilli.
a) For systemic infection: - Aminoglycosides, Polymyxins.
b) For urinary tract infections:- Nitrofurantoin, Nalidixic acid
Group-3: Broad spectrum antibiotics
Ex: Sulphanamides, Tetracycline
Group-4: Specific antibacterials
a) Active against anaerobic organisms.
Ex: metronidazole, lincomycin
b) For tuberculosis
Ex: Streptomycin, Isoniazid
c) for Chlamydia, Rickettsia, Mycoplasma infections
Ex: Erythromycin, Tetracyline
It may be defined as a
determination of the least amount of an antimicrobial chemotherapeutic agent
that will inhibit the growth of a microorganism in vitro, using a tube-dilution
method, agar-cup method, or disk-diffusion method.
OR
Dilution test
Various dilutions of antibiotic are prepared in agar or broth and the
microorganism is inoculated and incubated at 37C for 18 -24 hours.
The minimum concentration of the antibiotic required to inhibit the visible
growth is MIC (minimum inhibitory concentration) of the antibiotic.
The minimum amount of the antibiotic required to kill the microorganism
is the MBC (minimum bactericidal concentration) of the antibiotic. MBC is
obtained by sub culturing the organisms from tubes containing the
antibiotic and no visible growth onto an antibiotic free medium and
determining the number of survivors.
Advantages of dilution methods:
1) To determine the MIC and MBC of an antibiotic.
2) They permit a quantitative result to be reported, indicating the amount of
a given drug necessary to kill or inhibit the microorganism.
Disadvantages: Laborious and time consuming, costly.
http://www.medschool.lsuhsc.edu/micro
biology/Flash/MICMBC.htm
Diffusion Test-Principle
information
Mller-Hinton
agar
is
an microbiological growth medium that is
commonly
used
for
antibiotic
susceptibility testing. It is also used to
isolate
and
maintain
Neisseria
and Moraxella species.
It typically contains (w/v)
30.0% beef infusion
1.75% casein hydrolysate
0.15% starch
1.7% agar
pH adjusted to neutral at 25 C.
Five percent sheep blood may also be
added when susceptibility testing is done
on Streptococcus species. This type is also
commonly used for susceptibility testing
of Campylobacter.
Serological Tests :
It is a common practice to use blood for carrying out the serological tests,
but quite rarely for virus isolation.
However, it is absolutely important and vital that both acute and
convalescent-phase blood specimens should be examined thoroughly in
parallel to estimate precisely whether antibodies have appeared,
lowered or enhanced in the titer value in the span of the disease.
Examples : A few typical examples of human viral infections are as
enumerated under :
Respiratory infections (e.g. Adenovirus group)
Diseases of the nervous system (e.g., Polio and Coxsackie viruses of the
picornavirus group)
Small pox (Poxvirus group)
Measles (Paramyxovirus group)
Chicken pox (Herpesvirus group)
Influenza (Myxovirus group)
Clinical Parasitology :
Year Event
1938 : Federal Food, Drug and Cosmetic Act Introduction in stages of the
batch certification of antibiotics meant for human or verternary applications.
1945 : Penicillin
1948 : Streptomycin
1949 : Aureomycin, Bacitracin and Chloramphenicol.
1962 : Kefauver-Harris Amendments as part of these amendments it was
mandatory for the batch certification of all antibiotics intended for human
use.
1982 : Federal Drug Authority (FDA)-USA issued regulations which totally
exempted the antibiotics from the batch certification requirements so long
as the articles complied with standards ; however, section 507 (i.e., related to
certification of Antibiotics) remains intact and hence applicable.
2. Turbidimetric Method
Soil is the best available source from which one may obtain ultimately a
broad spectrum of viable microorganisms.
Soil comprises of various kinds of microorganisms among which many of
them exhibit the biosynthetic abilities which are of genuine interest.
why is soil invariably regarded to be the ideal source from which to obtain
diverse types of microorganisms ?
1. A sizable quantum, of the debris of the world finds its normal passage
either onto or into the soil ; and ultimately gets adequately decomposed
by one microorganism or the other.
2. Soil may be thought of as being of a specified kind of huge natural
fermentation vat wherein a plethora of organisms are actively engaged
not only in the actual decomposition and resynthesis of simple to complex
organic materials, but also in carrying out effectively the process of
oxidation, reduction and other chemical changes pertaining to inorganic
materials.
3. It has been duly demonstrated and established that more than one type, and often
many types, of soil microorganisms are invariably capable of performing each of
these individual chemical or biochemical transformation.
4. Though a large volume of different types of microorganisms do occur in the soil ;
however, it is not yet so clear and evident that actually upto what extent of these
organisms, as on date, been pinned down and isolated in the form of purest
laboratory culture.
5. The availability of nutrients in soil is invariably found to be relatively at low ebb ;
and, therefore, the prevailing microbial competition for these nutrients is quite
prevalent. In case, a highly desired and specific nutrient is timely incorporated to
the moistened soil, and the treated soil is duly incubated then a relatively much
appreciable larger growth response takes place amongst the ensuing soil
microorganisms that are capable of attacking this specific nutrient thereby
rendering the isolation of these particular organisms much simpler and
convenient. In other words, one may accomplish judiciously the enrichment in
soil for specific microorganisms of our interest.
Screening
Screening may be defined as the application of
highly selective, specific and sophisticated sequential
procedures to make the detection and isolation of
only such microorganisms that are of genuine
interest out of a large microbial population.
Various underlying concepts of screening essentially
include:
1. Segregation of Viable Microorganisms : It should be highly effective in the
sense that either a few steps or a single step would be able to discard a
major portion of the relatively not-so-useful microorganisms ; whereas,
simultaneously allowing the rapid and fast detection of the small
percentage of viable and useful microorganisms which are usually present
in the population.
Crowded-plate Technique :
It is one of the simplest screening techniques invariably
employed by the antibiotic producers.
Many of the most useful antibiotics are derived from
compounds originally isolated from microorganisms.
Penicillin, as is well known, was first discovered in mold, and
various other antibiotics were isolated from soil bacteria in
the 1950s and 1960s.
In fact, this technique has an added advantage for exclusively
looking for microorganisms which produce on antibiotic
without any specific consideration whatsoever about the
types of microorganisms that may be sensitive to the
antibiotic.
Methodology
Various steps involved in the crowded plate technique:
1. First, a sample of organisms from soil or some other source is diluted in
water, then spread onto Petri dishes containing agar gel rich in the
nutrients the bacteria will need to grow. Scientists select plates that have
a large number of colonies, then look for microorganisms that have
inhibited the growth of other microorganisms in their vicinity. These
microbes are possibly secreting some kind of compound that is killing or
inhibiting their neighbors.
2. It is a common practice to subculture such a colony (colonies that may be
producing antibiotics) further in an identical medium, and purified
subsequently be streaking, just prior to making stock cultures. The
purified culture thus obtained is now almost ready for testing to
establish precisely the types of microorganisms that are sensitive to the
antibiotic under investigation, by, means of the minimum inhibition
concentration (MIC) or the microbial inhibition spectrum (MIS).
It's entirely possible, of course, that the colony was really just altering the
pH of its environment or making some other change that killed other
bacteria, rather than secreting an antibiotic, so further tests are needed to
confirm that it is indeed an antibiotic-producing strain. Nonetheless, the
crowded plate technique was sometimes helpful in identifying
microorganisms that could serve as sources of new antibiotics.
Advantages
The crowded plate technique is fairly simple -- indeed, the simplest
method to find antibiotic-producing microorganisms in soil samples. It's
also fairly rapid, taking only a couple of days to produce results.
Introducing "test organisms" can help to determine whether a specific
kind of microorganism (e.g., a disease-causing germ) is susceptible to the
antibiotic compound. If it does indeed prove useful for this purpose, the
compound can be isolated for further study.
Drawbacks
The crowded plate technique only detects microorganisms that produce
compounds to kill bacteria found in their immediate environment. These
compounds could potentially be toxic to humans, and they may be lethal
only to certain types of bacteria (e.g., soil bacteria), as opposed to the
bacteria that actually cause disease in humans. Moreover, they will only
detect microorganisms that start to produce antibiotic compounds within
a couple of days of being cultured and incubated, so they might well miss
other compounds that could potentially be of interest.
5.
6.
7.
Secondary screening
Primary screening (or preliminary screening) solely enables not only the
detection, but also the isolation of such viable microorganisms that
essentially possess potentially interesting and commercially feasible
applications. Nevertheless, this screening is invariably followed by a
secondary screening so as to ascertain more useful information about
these organisms, besides their actual inherent capabilities.
Primary screening establishes exclusively the capability of microorganisms
that are responsible for producing a compound without giving enough
idea either with respect to the yield or production potential for the
organisms.
On the contrary, the secondary screening categorically enables the further
sorting out of those specific microorganisms that essentially possess the
real inherent value for feasible and gainful industrial processes ; and
distinctly eliminating those devoid of such a potential.
Fermentors (Bioreactors)
Fermentor vs bioreactor
While mammalian cells are fragile having shear sensitive cell membranes,
bacterial cells are robust as they have strong cell walls.
Mammalian cells are slow in their growth (they have 24 hour doubling
time). On the other hand, bacterial cells are fast growing and double in just 20
minutes.
Insect and mammalian cells have low oxygen demand whereas bacterial
cells have a very high oxygen demand
There is no viral threat in case of bacterial cells whereas this threat is
present in case of mammalian cells and has to be either inactivated or
removed.
There are differences in sterilization process also. While in the case of a
fermentor, it has to be sterilized full, a bioreactor is so designed so as to be
sterilized empty.
Bioreactors are large in comparison to fermentors and range in size from a
few liters to cubic meters whereas fermentors are typically small having a
capacity of up to 2 liters. Bioreactors are cylindrical vessels made of stainless
steel.
It can be supported with the help of an equation that describes the
substrates used in the fermentation process to give the desired products. One
of its examples is alcoholic fermentation whose equation is given below.
Acetaldehyde + NADH + H+
alcohol
Alcohol + NAD+
-------------->
dehydrogenase
fermentation, with the help of the fungus yeast, which is not in contact with
the atmosphere or oxygen. Acetaldehyde and NADH are the substrates of the
reaction which are fermented along with one hydrogen ion to form the
product which is alcohol. This reaction takes place in presence of the active
enzyme alcohol dehydrogenase to give the alcohol and a cofactor which is
one ion of NAD. This reaction follows the decarbaxylation of pyruvate with
the help of the enzyme pyruvate decarboxylase, Thiamine pyrophosphate
(TPP) and two Mg ions. After this acetyldehyde and CO are formed to give the
above
mentioned
reaction.
2. Anaerobic Fermentation
3. Aerobic Fermentation
Constant adequate aeration is required(supplied).
It has been observed that in certain specific instances
the actual quantum of air required per hour is almost 60
folds in comparison to the prevailing medium volume.
Hence, bioreactors employed invariably for carrying out
the aerobic fermentation have an essential provision for
the constant, adequate and compressed (pressurized
supply of sterile air that is usually sparged into the liquid
culture medium.
Besides, such biorectors (fermentors) should possess a
befitting device and mechanism for efficient stirring and
mixing of the liquid culture medium and the cells.
Advantages : The various vital advantages of stirredtank type fermentors are as stated below :
(1) Several heteroploid cell-lines may be grown
successfully in such vessels.
(2) Small scale reactors (cap. 2-50 L) fulfil the need for
research biochemicals from cells.
(3) Large scale reactors (cap. 100-5000 L) are largely
employed for growing hybridoma cells for the
production of monoclonal antibodies (MABs) ;
whereas, their yields from the cultured cells ranges
meagrely between 1-2% of those obtained by passing
the cells via peritoneal cavity of mice.
2. Entrapment Cultures:
In this particular instance, the cells are very
much retained within an open matrix via
which the medium flows freely.
6. Cylindro-conical vessels:
Brewing of lager and beers
22-25 m.ht6m. Dia
Adv:
Reduced process times
Primary fermentation & maturity in the same
vessel
Ease of removal of sediment
Maturity time reduction-by washing with
CO2
7.Air-lift fermentor:
For SCP production from MethanolContinuous
production.
Single-cell protein (SCP) typically refers to sources of mixed protein extracted from pure
or mixed cultures of algae, yeasts, fungi or bacteria (grown on agricultural wastes) used
as a substitute for protein-rich foods, in human and animal feeds.
Continuous Sterilization:
1. It offers more flexibility in the temperature conditions.
2. It involves passing of production medium through a heat exchanger, a
holding coil and a cooler.
3.The medium is finally cooled by counter circulating it against cool input
medium and then against cool water.
4. It allows the sterilization at higher temp. without affecting nutritional
values.
Advantages:
1. Saves production time and plant space
2. Improves quality of medium
3. Economical
4. Allows the use of lower sterilizing temp.
Disadvantages:
Media containing heat resistant bacterial spores,
enzymes etc may not be sterilized.
vitamins,
3. Electrostatic precipitation
4. U.V. light and
5. Chemical agents.
Sterilization of air in fermentation industries is widely done by
filtration methods.
Batch process
Fed-batch process
Continuous process
Semi-continuous process
Fermentation Process:
Batch Process: All the nutrients needed for cell
growth will only be added once at the beginning of
fermentation.
Fed-batch process: During the fermentation,
additional nutrients will be added in a batch way to
promote the cell growth or product formation and
to avoid nutrient deficiency.
Semi-continuous process:Similar to continuous
process, with the addition of nutrients and outflow
of fermentation broth in a continuous and batch
way.
Chemostat
Turbidostat
Laboratory
Process
Development
Fermentor
Experiments
Agitation
Cooling and
heating (Temp.
control)
Air inlet and
outlet (Aeration)
pH control
Nutrient addition
Inoculation
Viewing port
Imp. Components:
A) Impellers (agitators): Typesdisc turbine,
vaned disc,
open turbine,
marine propeller
Genetic stability
Good aeration and agitation
Easy product recovery
Precursor provision
Allow or prevent sporulation
Rapidly available components
throughout the year
Economical/cheap
Consistent quality
Disadvantages
Highly expensive
Low yields
pH
Cell Dry Weight
Sugar consumption
Temperature
Scale Up:
Isolation of mutants
In molecular biology and genetics, mutations are changes in
a genomic sequence: the DNA sequence of a cell's genome or
the DNA or RNA sequence of virus. They can be defined as
sudden and spontaneous changes in the cell. Mutations are
caused by radiation, viruses, transposons and mutagenic
chemicals, as well as errors that occur during meiosis or DNA
replication.
Mutants may be defined as variations of genetic structures
that eventually breed true. OR
An organism that has characteristics resulting from
chromosomal alteration or an organism that results due to
mutation.
Somaclonal variation
Screening
It is solely based upon the observation of a substantial
number of cells or regenerated plants for the ultimate
detection of variant individuals.
As a common practice the specific R1 progeny (i.e., the
progeny of regenerated R0 plants) are invariably scored for
the identification of variant- plants, and their corresponding
R2 progeny lines are mostly evaluated for confirmation.
Advantages : Screening has been exploited both profitably and
extensively for the categorical isolation of cell clones which evidently
give rise to certain higher amounts of some biochemicals ; besides,
computer-aided automated cell sorting devices (CAACSDs) have also
been introduced overwhelmingly to aid the screening of upto 10002000 cells per second from the asorted cell-pool the desirable variant
cells were segregated via automatic means.
Cell selection
Rescue Method : In this particular instance, the wild type cells are
virtually killed by the corresponding selection agent ; whereas, the
variant cells do remain very much alive, but fail to undergo division
by virtue of the ensuing unfavourable environment.
Subsequently, attempt is made to remove the selection agent
specifically so as to recover the prevailing variant cells. The rescue
method has been employed frequently to recover the lowtemperature as well as aluminium resistant variant cells.
Stepwise Selection : In this specific instance, the ensuing selection
pressure viz., salt concentration, may be enhanced slowly from a
relatively low level to the Cytotoxic level ; and, thus, the resistant
clones isolated at each and every progressive state are
appropriately subjected to the higher selection pressure. In actual
practice, stepwise selection approach may invariably favour gene
amplification (an unstable phenomenon) or subsequent mutations in
the organelle DNA.
It has been observed that sometimes the mutation is strategically taking place in
such a genetic locus that under one particular experimental parameters the
organism tends to grow normally, whereas under an altogether different
experimental parameters, either the expected growth is far from being normal or
the organism fails to grow at all. Thus, such not-so-steady mutations are usually
termed as the conditional mutations.
In actual practice, however, the prevailing conditions that invariably permit the
normal growth are called the permissive conditions ; and the other conditions
are collectively referred to as either the non-permissive conditions or the
restrictive conditions. Now, if under the influence of restrictive conditions the
organism is totally unable to grow, the mutation is known as a conditional lethal
mutation.
Auxotrophic Mutation : In this case, the growth media and the metabolic
conditions are entirely responsible for the ensuing expression of mutation.
Examples : A few specific mutants have the capability to grow very conveniently in
the presence of glucose but a possible replacement of glucose with any other
sugar entity would virtually cause the growth to a complete stand-still (i.e., stop).
Mutants may be either temperature sensitive (hot or cold) or supressor sensitive. In
the latter instance the organism is found to be viable in the presence of a
supressor, whereas the mutation becomes lethal in the absence of a supressor.
State of cellular metabolism: Both the state of cellular metabolism and the
phase of cell-cycle do play a cognizable major role on the remarkable
effect of ionizing radiations. Example : In response to the given irradiation
to the plant Trillium, the observed mutations were 60 times more
prevalent specifically at the metaphase in comparison to the interphase of
the cellcycle.
Effect of UV radiation:
It acts as weak mutagen.
Normal strenghts of UV-radiation in the sun light are not strong enough to
initiate and produce mutation. Interestingly, any extent of damage caused
to DNA is repaired instantly by the cell.
Nevetheless, the exposed UV radiation gets adequately absorbed by both
the purine and the pyrimidine bases respectively ; and, thus, are
converted into their corresponding excitable state that eventually render
them more reactive ultimately.
Spontaneous mutation
Spontaneous mutation
Spontaneous mutations on the molecular level can be caused by:
Tautomerism A base is changed by the repositioning of a hydrogen atom,
altering the hydrogen bonding pattern of that base resulting in incorrect base
pairing during replication.
Depurination Loss of a purine base (A or G) to form an apurinic site (AP
site).
Deamination Hydrolysis changes a normal base to an atypical base
containing a keto group in place of the original amine group.
Slipped strand mispairing Denaturation of the new strand from the
template during replication, followed by renaturation in a different spot
("slipping"). This can lead to insertions or deletions.
Media Preparation
The media preparation is precisely the backbone of the entire
bioprocess operation ; and, therefore, must be carried out
with utmost care and precaution. Importantly, the improper
and inadequate media design may ultimately give rise to both
impaired efficiency of growth as well as significantly poor
product formation.
The design of fermentation processes may be categorized into
the following five techniques, namely :
(a) Solid substrate fermentation,
(b) Submerged fermentation,
(c) Downstream processing,
(d) Technology of mammalian and plant-cell culture, and
(e) Cell-recycle technique.
Downstream Processing