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INTRODUCTION:
Natural products are naturally derived metabolites and/or by-products from
microorganisms, plants, or animals.
These products have been exploited for human use for thousands of years, and
plants have been the chief source of compounds used for medicine. Even today the
largest users of traditional medicines are the Chinese, with more than 5,000 plants and
plant products in their pharmacopoeia In fact, the worlds best known and most
universally used medicine is aspirin (salicylic acid), which has its natural origins from the
glycoside salicin which is found in many species of the plant genera Salix and Populus.
Examples abound of natural-product use especially in small native populations in a
myriad of remote locations on Earth. For instance, certain tribal groups in the Amazon
basin, the highland peoples of Papua New Guinea, and the Aborigines of Australia each
has identified.1
More recently, the Benedictine monks (800 AD) began to apply Papaver
somniferum as an anesthetic and pain reliever as the Greeks had done for years before
Many people, in past times, realized that leaf, root, and stem concoctions had the
potential to help them. These plant products, in general, enhanced the quality of life,
reduced pain and suffering, and provided relief, even though an understanding of the
chemical nature of bioactive compounds in these complex mixtures and how they
functioned remained a mystery.
These plant products, in general, enhanced the quality of life, reduced pain,
suffering, and provided relief, even though an understanding of the chemical nature of
bioactive compounds in these complex mixtures and how they functioned remained a
mystery. It was not until Pasteur discovered that fermentation is caused by living cells
that people seriously began to investigate microbes as a source for bioactive natural
products. Then, scientific serendipity and the power of observation provided the impetus
to Fleming to push in the antibiotic era via the discovery of penicillin from the fungus
Penicillium notatum. Since then, people have been engaged in the discovery and
INTRODUCTION
application of microbial metabolites with activity against both plant and human
pathogens.
Soil Microbiology2
It is branch of science / microbiology which deals with study of soil
microorganisms and their activities in the soil.
Soil:
It is the outer, loose material of earths surface which is distinctly different from
the underlying bedrock and the region which support plant life. Agriculturally, soil is the
region which supports the plant life by providing mechanical support and nutrients
required for growth. From the microbiologist view point, soil is one of the most dynamic
sites of biological interactions in the nature. It is the region where most of the physical,
biological and biochemical reactions related to decomposition of organic weathering of
parent rock take place.
Components of Soil:3,4
Soil is an admixture of five major components viz. organic mater, mineral matter,
soil-air, soil water and soil microorganisms/living organisms. The amount/ proposition of
these components varies with locality and climate.
1. Mineral / Inorganic Matter:
It is derived from parent rocks/bed rocks through decomposition, disintegration
and weathering process. Different types of inorganic compounds containing various
minerals are present in soil. Amongst them the dominant minerals are Silicon, Aluminum
and iron and others like Carbon, Calcium Potassium, Manganese, Sodium, Sulphur,
Phosphorus etc. are in trace amount. The proportion of mineral matter in soil is slightly
less than half of the total volume of the soil.
INTRODUCTION
2. Organic matter/components:
Derived from organic residues of plants and animals added in the soil. Organic
matter serves not only as a source of food for microorganisms but also supplies energy
for the vital processes of metabolism which are characteristics of all living organisms.
Organic matter in the soil is the potential source of N, P and S for plant growth.
Microbial decomposition of organic matter releases the unavailable nutrients in available
from. The proportion of organic matter in the soil ranges from 3-6% of the total volume
of soil.
3. Soil Water:
The amount of water present in soil varies considerably. Soil water comes from
rain, snow, dew or irrigation. Soil water serves as a solvent and carrier of nutrients for the
plant growth. The microorganisms inhabiting in the soil also require water for their
metabolic activities. Soil water thus, indirectly affects plant growth through its effects on
soil and microorganisms. Percentage of soil-water is 25% total volume of soil.
4. Soil air (Soil gases):
A part of the soil volume which is not occupied by soil particles i.e. pore spaces
are filled partly with soil water and partly with soil air. These two components (water &
air) together only accounts for approximately half the soil's volume. Compared with
atmospheric air, soil is lower in oxygen and higher in carbon dioxide, because CO2 is
continuous recycled by the microorganisms during the process of decomposition of
organic matter. Soil air comes from external atmosphere and contains nitrogen, oxygen
CO2 and water vapour (CO2 > oxygen). CO2 in soil air (0.3-1.0%) is more than
atmospheric air (0.03%). Soil aeration plays important role in plant growth, microbial
population, and microbial activities in the soil.
INTRODUCTION
5. Soil microorganisms:
Soil is an excellent culture media for the growth and development of various
microorganisms. Soil is not an inert static material but a medium pulsating with life. Soil
is now believed to be dynamic or living system.
Soil contains several distinct groups of microorganisms and amongst them
bacteria, fungi, actinomycetes, algae, protozoa and viruses are the most important. But
bacteria are more numerous than any other kinds of microorganisms. Microorganisms
form a very small fraction of the soil mass and occupy a volume of less than one percent.
In the upper layer of soil (top soil up to 10-30 cm depth i.e. Horizon A), the microbial
population is very high which decreases with depth of soil. Each organisms or a group of
organisms are responsible for a specific change / transformation in the soil. The final
effect of various activities of microorganisms in the soil is to make the soil fit for growth
of higher plants.
Living organisms present in the soil are grouped into two categories as follows.
1. Soil flora (micro flora) e.g. Bacteria, fungi, Actinomycetes, Algae and
2. Soil fauna (micro fauna) animal like eg. Protozoa, Nematodes, earthworms, moles, ants,
rodents.
Relative proportion / percentage of various soil microorganisms are: Bacteriaaerobic (70%), anaerobic (13 %), Actinomycetes (13%), Fungi /molds (03 %) and others
(Algae Protozoa viruses) 0.2-0.8 %. Soil organisms play key role in the nutrient
transformations.
Scope and Importance of Soil Microbiology5
Living organisms both plant and animal types constitute an important component
of soil. Though these organisms form only a fraction (less than one percent) of the total
soil mass, but they play important role in supporting plant communities on the earth
surface. While studying the scope and importance of soil microbiology, soil-plant-animal
INTRODUCTION
ecosystem as such must be taken into account. Therefore, the scope and importance of
soil microbiology, can be understood in better way by studying aspects like
1. Soil as a living system
2. Soil microbes and plant growth
3. Soil microorganisms and soil structure
4. Organic matter decomposition
5. Humus formation
6. Biogeochemical cycling of elements
7. Soil microorganisms as bio-control agents
8. Soil microbes and seed germination
9. Biological N2 fixation
10. Degradation of pesticides in soil.
1. Soil as a living system:6
Soil inhabit diverse group of living organisms, both micro flora (fungi, bacteria,
algae and actinomycetes) and micro-fauna (protozoa, nematodes, earthworms, moles,
ants). The density of living organisms in soil is very high i.e. as much as billions / gm of
soil, usually density of organisms is less in cultivated soil than uncultivated / virgin land
and population decreases with soil acidity. Top soil, the surface layer contains greater
number of microorganisms because it is well supplied with Oxygen and nutrients. Lower
layer / subsoil is depleted with Oxygen and nutrients hence it contains fewer organisms.
Soil ecosystem comprises of organisms which are both, autotrophs (Algae, BOA) and
heterotrophs (fungi, bacteria). Autotrophs use inorganic carbon from CO 2 and are
"primary producers" of organic matter, whereas heterotrophs use organic carbon and are
decomposers/consumers.
INTRODUCTION
2. Soil microbes and plant growth:6
Microorganisms being minute and microscopic, they are universally present in
soil, water and air. Besides supporting the growth of various biological systems, soil and
soil microbes serve as a best medium for plant growth. Soil fauna & flora convert
complex organic nutrients into simpler inorganic forms which are readily absorbed by the
plant for growth. Further, they produce variety of substances like IAA, gibberellins,
antibiotics etc. which directly or indirectly promote the plant growth
3. Soil microbes and soil structure:
Soil structure is dependent on stable aggregates of soil particles-Soil organisms
play important role in soil aggregation. Constituents of soil are viz. organic matter,
polysaccharides, lignins and gums, synthesized by soil microbes plays important role in
cementing / binding of soil particles. Further, cells and mycelial strands of fungi and
actinomycetes, Vormicasts from earthworm is also found to play important role in soil
aggregation. Different soil microorganisms, having soil aggregation / soil binding
properties are graded in the order as fungi > actinomycetes > gum producing bacteria >
yeasts.
Examples
are:
Fungi
like Rhizopus,
Mucor,
Chaetomium,
Fusarium,
INTRODUCTION
substances include cellulose, lignins and proteins (in cell wall of plants), glycogen
(animal tissues), proteins and fats (plants, animals). Cellulose is degraded by bacteria,
especially those of genus Cytophagaand
other
genera (Bacillus,
Pseudomonas,
INTRODUCTION
7. Soil microbes and biological N2 fixation:
Conversion of atmospheric nitrogen in to ammonia and nitrate by microorganisms
is known as biological nitrogen fixation.
Fixation of atmospheric nitrogen is essential because of the reasons:
1. Fixed nitrogen is lost through the process of nitrogen cycle through de-nitrification.
2. Demand for fixed nitrogen by the biosphere always exceeds its availability.
3. The amount of nitrogen fixed chemically and lightning process is very less (i.e. 0.5%) as
compared to biologically fixed nitrogen.
4. Nitrogenous fertilizers contribute only 25% of the total world requirement while
biological nitrogen fixation contributes about 60% of the earth's fixed nitrogen.
5. Manufacture of nitrogenous fertilizers by "Haber" process is costly and time consuming.
The numbers of soil microorganisms carry out the process of biological nitrogen
fixation at normal atmospheric pressure (1 atmosphere) and temp (around 20C).
Two groups of microorganisms are involved in the process of BNF.
A. Non-symbiotic (free living) and B. Symbiotic (Associative)
Non-symbiotic (free living):
Depending upon the presence or absence of oxygen, non symbiotic N2 fixation
prokaryotic organisms may be aerobic heterotrophs (Azotobacter, Pseudomonas,
Achromobacter) or aerobic autotrophs (Nostoc, Anabena, Calothrix, BGA)and anaerobic
heterotrophs
Clostridium,
Kelbsiella.
Desulfovibrio
) oranaerobic
Autotrophs
organisms
involved
are Rhizobium,
Bratfyrhizobium in
legumes
INTRODUCTION
8. Soil microbes as biocontrol agents:
Several ecofriendly bioformulations of microbial origin are used in agriculture for
the effective management of plant diseases, insect pests, weeds etc. eg: Trichoderma sp
and Gleocladium sp are used for biological control of seed and soil borne diseases.
Fungal genera Entomophthora, Beauveria, Metarrhizium and protozoa Maltesia grandis.
Malameba locustiae etc are used in the management of insect pests. Nuclear polyhydrosis
virus (NPV) is used for the control of Heliothis / American boll worm. Bacteria
like Bacillus thuringiensis, Pseudomonas are used in cotton against Angular leaf spot and
boll worms.
9. Degradation of pesticides in soil by microorganisms:
Soil receives different toxic chemicals in various forms and causes adverse effects
on beneficial soil micro flora / micro fauna, plants, animals and human beings. Various
microbes present in soil act as the scavengers of these harmful chemicals in soil. The
pesticides/chemicals reaching the soil are acted upon by several physical, chemical and
biological forces exerted by microbes in the soil and they are degraded into non-toxic
substances and thereby minimize the damage caused by the pesticides to the ecosystem.
For example, bacterial genera likePseudomonas, Clostridium, Bacillus, Thiobacillus,
Achromobacter etc. and fungal genera like Trichoderma, Penicillium, Aspergillus,
Rhizopus, and Fusarium are playing important role in the degradation of the toxic
chemicals / pesticides in soil.
10.Biodegradation of hydrocarbons:
Natural hydrocarbons in soil like waxes, paraffins, oils etc are degraded by
fungi, bacteria and actinomycetes. E.g. ethane (C2 H6) a paraffin hydrocarbon is
metabolized and degraded by Mycobacteria, Nocardia, Streptomyces Pseudomonas,
Flavobacterium and several fungi.
INTRODUCTION
History of Soil Microbiology (1600 - 1920)5,7
There is enough evidence in the literature to believe that microorganisms were the
earliest of the living things that existed on this planet. Man depends on crop plants for his
existence and crop plants in turn depend on soil and soil microorganisms for their
nutrition. Scientists form the beginning studied the microorganisms from water, air, soil
etc. and recognized the role of microorganisms in natural processes and realized the
importance of soil microorganisms in growth and development of plants.
Thus, we see that microorganisms have been playing a significant role long before
they were discovered by man. Today, soil is considered to be the main source of
scavenging the organic wastes through microbial action and is also a rich store house for
industrial micro flora of great economic importance.
Unlike soil science whose origin can be traced back to Roman & Aryan times,
soil microbiology is emerged as a distinct branch of soil science during first half of the
19th century. Some of the notable contributions made by several scientists in field of soil
microbiology are highlighted in the following paragraphs.
minute, moving objects which he called animalcules" (small animals) which are now
known as protozoa, fungi and bacteria. He for the first time made the authentic drawings
of microorganisms (protozoa, bacteria, fungi).
Robert Hook (1635-1703) developed a compound microscope with multiple lenses and
described the fascinating world of the microbes.
J. B. Boussingault (1838) showed that leguminous plants can fix atmospheric nitrogen
and increase nitrogen content in the soil.
10
INTRODUCTION
J. Von Liebig ( 1856 ) showed that nitrates were formed in soil due to addition of
nitrogenous fertilizers in soil.
Jodin (1862, France) gave the first experimental evidence of elemental nitrogen fixation
by microorganisms.
Robert Koch (1882) developed gelatin plate / streak plate technique for isolation of
specific type of bacteria in soil , formulated Koch 's postulates to establish causal
relationship between host - pathogen and disease.
H. Hellriegel and H. Wilfarth (1886) showed that the growth of non-legume plant was
directly proportional to the amount of nitrogen supplied, whereas, in legumes there was
no relationship between the quantity of nitrogen supplied and extent of plant growth.
11
INTRODUCTION
They also suggested that bacteria in the root nodules of legumes accumulate atmospheric
nitrogen and made it available to plants. Showed that a mutually beneficial association
exists between bacteria ( Rhizobia ) and legume root and legumes could utilize
atmospheric nitrogen (1988).
M. W. Beijerinck (1888) isolated root nodule bacteria in pure culture from nodules in
legumes a nd named t hem as Bacillus radicola Considered as father of "Microbial
ecology". He was the first Director of the Delft School of microbiology (Netherland).
Beijerinck and Winogradsky ( 1890) developed the enrichment culture technique for
isolation of soil organisms, proved independently that transformation of nitrogen in
nature is largely due to the activities of various groups of soil microorganisms (1891).
Therefore, they are considered as "Pioneer's in soil bacteriology.
Hiltner (Germany, 1904) coined the term "Rhizosphere" to denote that region of soil
which is subjected to the influence of plant roots. Rhizosphere is the region where soil
and plant roots make contact.
Russel and Hutchinson (1909, England), proved the importance of protozoa controlling
/ maintaining bacterial population and their activity in soil.
12
INTRODUCTION
Conn ( 1918 ) developed Direct soil examination technique for studying soil
microorganisms.
Rayner (192I) and Melin (1927) carried out the intensive study on Mycorrhiza.
13
INTRODUCTION
History of Soil Microbiology (1921 20th Century)5,7
S. A. Waksman published the book Principles of soil Microbiology" and thereby
encouraged the research in soil microbiology (1927). Studied the role of soil as the source
of antagonistic organisms with special reference to soil actinomycetes (1942) and
discovered the antibiotic "Streptomycin" produced by Streptomyces griseus, a soil
actinomycetes(1944).
nitrogen fixation.
Ruinen (1956) Dutch microbiologist coined the term "Phyllosphere" to denote the
region of leaf influenced by microorganisms.
Jensen (1942) developed the method of studying nodulation on agar media in test tubes.
Barbara Mosse and J. W. Gerdemann (1944) reported occurrence of VAM (vesiculararbuscular Mycorrhiza) fungi (Glomus, Aculopora genera) in the roots of agricultural
crop
plants
which
helps
in
the
mobilization
of
phosphate.
14
INTRODUCTION
Burris and Wilson (1957) developed the "Isotope technique" to quantify the amount of
nitrogen fixed and further isolated and characterized the enzyme "Nitrogenase".
Foog and Stewart (1970, UK) intensified the work on N2 fixing blue-green algae.
Dommergues & associates (France and Senegal) had discovered / reported nodules on
stem of Sesbania rostrata which could fix nitrogen and therefore this legume can be used
as an excellent green manure crop in low land rice cultivation. Similarly they also
discovered N2 fixing stem nodules on Casurina sp caused by Frankia, an actinomycete.
the
Gerretsen
nature
&
of
Mulder
organic
matter
and
environmental
conditions.
mobilization" by
soil
15
INTRODUCTION
W. S. Cook, G. C. Papavizas, J. Baker and N.S. Kerr contributed to the field of
biological control of plant pathogens using antagonistic organisms from soil. From the
beginning of 20th century emphasis was given to the study of microorganisms in soil in
relation to their physiology, ecology, interrelationship, role in soil processes and soil
fertility. Further role of fungi and actinomycetes in cellulose decomposition was better
understood and cellulose decomposing, sulphur oxidizing, iron bacteria etc were isolated
from soil and studied in detail.
History of Soil Microbiology in India
During last few decades greater emphasis has been given on some of the important
aspects in soil microbiology in India which are:
1. Characterization of N2 fixing Azotobacter, Rhizobium, Beijerinckia, BGA etc.
2. Studies on P- solubilizing bacteria and fungi, celluloytic microorganisms, silage
production role of humic acid etc.
3. Establishment (1979) of All-India Coordinated Project (AICP) on BNF at IARI and field
oriented work on BNF.
4. Standardization of methods of bio-inoculants application to seed and soil.
5. Seed bacterization and response of crops to bio-inoculants.
Some of the most important contributions made on the different aspects in the field of
soil microbiology by the scientists and research institutes in the country are highlighted in
thefollowing paragraphs:
Sundara Rao (1962) established the "Division of Microbiology" at IARI New Delhi.
Sanyasi Raju & Rajagopalan (Coimbatore) initiated the research work on root
nodulation in legumes at Madras, Agil. College.
16
INTRODUCTION
P. K. Dey (West Bengal) worked on free living N2 fixing organisms viz Azotobacter,
Beijerinckia and BGA in rice fields and discovered N2 fixation by BGA in paddy.
M.O.P. lyengar (Madras Univ.) laid foundation stone of algal research in India.
17
INTRODUCTION
Modi, Sushil Kumar, Das and Thomas carried research on "Genetics of "Nif" gene in
relation to BNF by Rhizobium, Azospirillum and Kelbsiella.
Gaur (IARI) and Mishra (Hissar) studied the role of celluloytic microorganisms in
accelerating the process of composting and compost making.
Karla and Garcha ( Ludhiana ) studied the phenomenon of cellulose degradation and
legume bacteriology.
N. V. Joshi (1920) reported first isolation and identification of Rhizobium from different
cultivated legumes
A. Sankaran (1958) standardized quality of legume inoculants for first time in India.
P. K. Dey and R. Bhattacharya isolated for the first time a new, non-symbiotic N2
fixing bacterium Derixa gummosa in the world.
V. Iswaran (1959) reported the use of Indian peat as carrier for Biofertilizers production.
Dube J. N. (1975) reported coal (wood-coal), an alternative to peat as carrier material for
biofertilizer production.
18
INTRODUCTION
Types of Microorganisms in Soil
Living organisms both plants and animals, constitute an important component of
soil. The pioneering investigations of a number of early microbiologists showed for the
first time that the soil was not an inert static material but a medium pulsating with life.
The soil is now believed to be a dynamic or rather a living system, containing a dynamic
population of organisms/microorganisms. Cultivated soil has relatively more population
of microorganisms than the fallow land, and the soils rich in organic matter contain much
more population than sandy and eroded soils. Microbes in the soil are important to us in
maintaining soil fertility / productivity, cycling of nutrient elements in the biosphere and
sources of industrial products such as enzymes, antibiotics, vitamins, hormones, organic
acids etc. At the same time certain soil microbes are the causal agents of human and plant
diseases.
The soil organisms are broadly classified in to two groups viz soil flora and soil
fauna, the detailed classification of which is as follows.
Soil Organisms
A. Soil Flora
a) Microflora:
1. Bacteria
2. Fungi, Molds, Yeast, Mushroom
3. Actinomycetes, Stretomyces
4. Algae eg. BGA, Yellow Green Algae, Golden Brown Algae.
1. Bacteria is again classified in
I) Heterotrophic eg. symbiotic & non - symbiotic N2 fixers, Ammonifier, Cellulose
Decomposers, Denitrifiers
Institute of Pharmaceutical Sciences and Research Center, Bhagwant University
19
INTRODUCTION
II) Autrotrophic eg. Nitrosomonas, Nitrobacter, Sulphur oxidizers, etc.
b) Macroflora: Roots of higher plants
B. Soil Fauna
a) Microfauna: Protozoa, Nematodes
b) Macrofauna: Earthworms. moles, ants & others.
As soil inhabit several diverse groups of microorganisms, but the most important
amongst them are: bacteria, actinomycetes, fungi, algae and protozoa. The characteristics
and their functions / role in the soil are described in the next topics.
Soil Microorganism Actinomycetes
These are the organisms with characteristics
but
yet possessing distinctive features to delimit them into a distinct category. In the
strict taxonomic sense, actinomycetes are clubbed with bacteria the same class of
Schizomycetes and confined belonging to the important order of Actinomycetales.
They are unicellular like bacteria, but produce a mycelium which is non-septate
(coenocytic) and more slender, tike true bacteria they do not have distinct cell-wall and
their cell wall is without chitin and cellulose (commonly found in the cell wall of fungi).
On culture media unlike slimy distinct colonies of true bacteria which grow quickly,
actinomycetes colonies grow slowly, show powdery consistency and stick firmly to agar
surface. They produce hyphae and conidia / sporangia like fungi. Certain actinomycetes
whose hyphae undergo segmentation resemble bacteria, both morphologically and
physiologically.
Actinomycetes1,8 are numerous and widely distributed in soil and are next to
bacteria in abundance. They are widely distributed in the soil, compost etc. Plate count
estimates give values ranging from 10^4 to 10^8 per gram of soil. They are sensitive to
20
INTRODUCTION
acidity / low PH (optimum PH range 6.5 to 8.0) and waterlogged soil conditions. The
population of actinomycetes increases with depth of soil even up to horizon C of a soil
profiler They are heterotrophic, aerobic and mesophilic (25-30 ^c) organisms and some
species are commonly present in compost and manures are thermophilic growing at 5565C temperature (eg. Thermoatinomycetes, Streptomyces).
Actinomycetes belonging to the order of Actinomycetales are grouped four
families viz Mycobacteriaceae, Actinomycetaceae, Streptomycetaceae and
Actinoplanaceae.
Actinomycetous genera which are agriculturally and industrially important are
present in only two families of Actinomycetaceae and Strepotmycetaceae.
2. Organic residues / substances added soil are first attacked by bacteria and fungi
and later by actinomycetes, because they are slow in activity and growth than bacteria
and fungi.
3. They decompose / degrade the more resistant and indecomposable organic substance
/matter and produce a number of dark black to brown pigments which contribute to the
dark colour of soil humus.
4. They are also responsible for subsequent further decomposition of humus (resistant
material) in soil.
5. They are responsible for earthy / musty odor / smell of freshly ploughed soils.
21
INTRODUCTION
6. Many genera species and strains (eg. Streptomyces if actinomycetes produce
/synthesize number of antibiotics like Streptomycin, Terramycin, Aureomycin etc.
7. One of the species of actinomycetes Streptomyces scabies causes disease "Potato scab"
in potato.
Soil Microorganisms in Cycling of Elements or Plant Nutrient
Soil microorganisms are the most important agents in the cycling / transformation
of various elements (N, P, K, S, Iron etc.) in the biosphere; where the essential elements
undergo cyclic alterations between the inorganic state as free elements in nature and the
combined state in living organisms. Life on earth is dependent on the cycling of nutrient
elements from their elemental states to inorganic compounds to organic compounds and
back into their elemental states.
The microbes through the process of biochemical reactions convert / breakdown
complex organic compounds into simple inorganic compounds and finally into their
constituent elements. This process is known as "Mineralization".
Mineralization of organic carbon, nitrogen, phosphorus, sulphur and iron by soil
microorganisms makes these elements available for reuse by plants. In the following
paragraphs the cycling / transformations of some of the important elements are discussed.
The four most important cycles are mention below
A. Nitrogen Cycle
B. Sulphur Cycle / Sulphur Transformation
C. Phosphorus Cycle / Transformation
D. Iron Cycle / Transformation
22
INTRODUCTION
Actinomycetes9
Actinomycetes are best known for their ability to produce antibiotics and are gram
positive bacteria which comprise a group of branching unicellular microorganisms. They
produce branching mycelium which may be of two kinds viz., substrate mycelium and
aerial mycelium. Among actinomycetes, the streptomycetes are the dominant. The
nonstreptomycetes are called rare actinomycetes,comprising approximately 100
genera. Members of the actinomycetes, which live in marine environment, are poorly
understood and only few reports are available
Various approaches for the identification of actinomycets are given briefly below:
a) Molecular Approach
The most powerful approaches to taxonomy are through the study of nucleic
acids. Because these are either direct gene products or the genes themselves and
comparisons of nucleic aids yield considerable information about true relatedness.
Molecular systematics, which includes both classification and identification, has
its origin in the early nucleic acid hybridization studies, but has achieved a new status
following the introduction of nucleic acid sequencing techniques (ODonnell et al.,
1993). Significance of phylogenetic studies based on 16S rDNA sequences is increasing
in the systematics of bacteria and actinomycetes (Yokota, 1997). Sequences of 16S
ribosomal DNA have provided actinomycetologists with a phylogenetic tree that allows
the investigation of evolution of actinomycetes and also provides the basis for
identification.
b) Chemotaxonomical Approach
Chemotaxonomy is the study of chemical variation in organisms and the use of
chemical characters in the classification and identification. It is one of the valuable
methods to identify the genera of actinomycetes.
Studies of Cummins and Harris (1956) established that actinomycetes have a cell
wall composition akin to that of grampositive bacteria, and also indicated that the
23
INTRODUCTION
chemical composition of the cell wall might furnish practical methods of differentiating
various types of actinomycetes.
This is because of the fact that chemical components of the organisms that satisfy
the following conditions, have significant meaning in systematics.K. Sivakumar
i. They should be distributed universally among the microorganisms studied; and,
ii. The components should be homologous among the strains within a taxon, while
significant differences exist between the taxa to be differentiated.
Presence of Diaminopimelic Acid (DAP) isomers is one of the most important
cellwall properties of grampositive bacteria and actinomycetes. Most bacteria have a
characteristic wall envelope, composed of peptidoglycan. The 2, 6 Diaminopimelic
Acid (DAP) is widely distributed as a key aminoacid and it has optical isomers. The
systematic significance lies mostly in the key aminoacid with two amino bases, and
determination of the key aminoacid is usually sufficient for characterisation. If DAP is
present, bacteria generally contain one of the isomers, the LLform or the mesoform,
mostly located in the peptidoglycan.
Actinomycete cells contain some kinds of sugars, in addition to the glucosamine
and muramic acid of peptidoglycan. The sugar pattern plays a key role in the
identification of sporulating actinomycetes which have mesoDAP in their cell walls.
However, the actinomycetes which have LLDAP along with glycine (wall chemo typeI)
have no characteristic pattern of sugars
(Lechevalier and Lechevalier, 1970) and hence the whole cell sugar test has not
received much attention here.
c) Classical Approach
Classical approaches for classification make use of morphological, physiological,
and biochemical characters. The classical method200 Actinomycetes described in the
identification key by Nonomura (1974) and Bergeys
24
INTRODUCTION
Manual of Determinative Bacteriology (Buchanan & Gibbons, 1974) is very
much useful in the identification of streptomycetes. These characteristics have been
commonly employed in taxonomy of streptomycetes for many years. They are quite
useful in routine identification. They are as follows.
1. Aerial Mass Colour
The colour of the mature sporulating aerial mycelium is recorded in a simple way
(White, grey, red, green, blue and violet). When the aerial mass colour falls between two
colour series, both the colours are recorded. If the aerial mass colour of a strain to be
studied shows intermediate tints, then also, both the colour series are noted.
2. Melanoid Pigments
The grouping is made on the production of melanoid pigments (i.e.greenish
brown, brownish black or distinct brown, pigment modified by other colours) on the
medium. The strains are grouped as melanoid pigment produced (+) and not produced
().
3. Reverse Side Pigments
The strains were divided into two groups, according to their ability to produce
characteristic pigments on the reverse side of the colony, namely, distinctive (+) and not
distinctive or none (). In case, a colour with low chroma such as pale yellow, olive or
yellowish brown occurs, it is included in the latter group ().
4. Soluble Pigments
The strains are divided into two groups by their ability to produce soluble
pigments other than melanin: namely, produced (+) and not produced (). The colour is
recorded (red, orange, green, yellow, blue and violet).
5. Spore Chain Morphology
With regard to spore chains, the strains can be grouped into sections.The species
belonging to the genus Streptomyces are divided into three sections (Shirling & Gottlieb,
25
INTRODUCTION
1966), namely rectiflexibiles (RF), retinaculiaperti (RA) and Spirales (S). When a strain
forms two types of spore chains, both are noted (e.g. SRA).
Characteristics of the sporebearing hyphae and spore chains should be
determined by using direct microscopic examination of the culture surface. Adequate
magnification (400x) could be used to establish the presence or absence of spore chains
and to observe the nature of sporophores.
Spore morphological characters of the strains can be studied by inoculating a
loopful of one week old cultures into 1.5% agar medium contained in test tubes
6. Spore Surface
Spore morphology and its surface features should be observed under the scanning
electron microscope. The cross hatched cultures prepared for observation under the light
microscope can be used for this purpose.
RF Spore chains (400X)
RA Spore chains (400X)
Spiral Spore chains (400X)202 Actinomycetes
The electron grid should be cleaned and adhesive tape should be placed on the
surface of the grid. The mature spores of the strain should be carefully placed on the
surface of the adhesive tape and gold coating should be applied for half an hour and the
specimen can be examined under the electron microscope at different magnifications. The
spore silhouettes can be characterized as smooth, spiny, hairy and warty.
7. Assimilation of Carbon Source
The ability of different actinomycete strains in utilizing various carbon
compounds as source of energy should be studied following the method recommended by
International Streptomyces Project (Shirling and Gottlieb, 1966). Chemically, pure
carbon sources, certified to be free of admixture with other carbohydrates or
26
INTRODUCTION
contaminating materials, should be used for this purpose. Carbon sources for this test
could be arabinose, xylose, inositol, mannitol, fructose, rhamnose, sucrose and raffinose.
These carbon sources should be sterilized by ether sterilization without heating.
Comparing the properties of the isolated strain with the representative species found in
the key of Nonomura (1974) and
Bergeys Manual of Determinative Bacteriology (Buchanan and Gibbons, 1974)
can help in the species level identification. If the isolated strain could not be assigned to
any of the valid representatives listed in the key of Nonomura (1974) and Bergeys
Manual of Determinative Bacteriology (Buchanan & Gibbons, 1974), then it can be
identified based on the numerical taxonomic studies.
d) Numerical Taxonomic Approach
Numerical taxonomy involves examining many strains for a large number of
characters prior to assigning the test organism to a cluster based on shared features. The
numerically defined taxa are polythetic; so, no single property is either indispensable or
sufficient to entitle an organism for membership of a group. Once classification has been
achieved, clusterspecific or predictive characters can be selected for identification
(Williams et al, 1983).
Silvestri et al. (1962). The numerical taxonomic study of the genus. Streptomyces by
Williams et al. (1983) involves determination of 139 unit characters for 394 type cultures
of Streptomyces; clusters were defined at 77.5% or 81% Ssm and 63% Sj similarity
levels, and the former coeffieientK. Sivakumar 203 is being used to define the clusters.
His study includes 23 major, 20 minor and 25 single member clusters.
The numerical classification of the genus Streptomyces by Kampfer et al. (1991)
involves determination of 329 physiological tests.
His study includes 15 major clusters, 34 minor clusters and 40 single member
clusters which are defined at 81.5% similarity level Ssm using the simple matching
coefficient (Sokal and Michener, 1958) and 59.6 to 64.6% similarity level Sj using
27
INTRODUCTION
Jaccard coefficient (Sneath, 1957). Thus, numerical taxonomy provides us with an
invaluable framework for Streptomyces taxonomy, including identification of species.
28
INTRODUCTION
branching threads or rods, frequently giving rise to a typical mycelium which is
unicellular, especially during the early stages of growth. Actinomycetes are heterotrophic
group in nature. Most of them are strict saprophytes, while some parasitic or mutualistic
association with plants and animals. They are aerobic and most of them readily grow on
the common bacteriological media like
Nutrient Agar
Trypticase Agar
Blood Agar
Starch Casein Agar
Albumin Agar etc
It is noteworthy that, of the nearly 300,000 plant species that exist on the earth,
each individual plant is host to one or more Endophytes. Endophytic Actinomycetes have
been defined as that can be isolated from the disinfected surfaces of plant tissues or that
can be extracted from within the plant that do not cause visible harm to the host.
Endophytes have been isolated from flowers, fruits, leaves, stem roots and seeds of
various plant species.
ANTIMICROBIAL COMPOUND:10,11,12,13
An anti-microbial is
substance
that
kills
or
inhibits
the
growth
(microbiocidal)
or
prevent
the
growth
of
microbes
29
INTRODUCTION
microorganism. Of course, in today's common usage, the term antibiotic is used to refer
to almost any drug that attempts to rid your body of a bacterial infection. Antimicrobials
include not just antibiotics, but synthetically formed compounds as well.
The discovery of antimicrobials like penicillin and tetracycline paved the way for
better health for millions around the world. Before penicillin became a viable medical
treatment
in
the
early
1940s,
no
true
cure
throat,
or pneumonia existed. Patients with infected wounds often had to have a wounded limb
removed, or face death from infection. Now, most of these infections can be cured easily
with a short course of antimicrobials.
However, with the development of antimicrobials, microorganisms have adapted
and become resistant to previous antimicrobial agents. The old antimicrobial technology
was based either on poisons or heavy metals, which may not have killed the microbe
completely, allowing the microbe survive, change, and become resistant to the poisons
and/or heavy metals.
Antimicrobial nanotechnology is a recent addition to the fight against disease
causing organisms, replacing heavy metals and toxins and may some day be a viable
alternative.
It was not until Pasteur discovered that fermentation is caused by living cells that
people seriously began to investigate microbes as a source for bioactive natural products.
Then, scientific serendipity and the power of observation provided the impetus to
Fleming to pusher in the antibiotic era via the discovery of penicillin from the fungus
Penicillium notatum. Since then, people have been engaged in the discovery and
application of microbial metabolites with activity against both plant and human
pathogens. Furthermore, the discovery of a plethora of microbes for applications that
span a broad spectrum of utility in medicine (e.g., anticancer and immunosuppressant
functions), agriculture and industry is now practical because of the development of novel
and sophisticated screening processes in both medicine and agriculture.
30
INTRODUCTION
Natural-product-based compounds have had an immense impact on modern
medicine since about 40% of prescription drugs are based on them. Furthermore, 49% of
the new chemical products registered by the U.S. Food and Drug Administration are
natural products .
ANTIBIOTIC10,11,12
The word
antibiotic comes from the Greek meaning 'against' and bios meaning
'life' (a bacterium is a life form).' Antibiotics are also known as antibacterials and they are
drugs used to treat infections caused by bacteria.
31
INTRODUCTION
Actually, Fleming was not the first person to recognize the antibacterial properties
of mold. As far back as 2,500 years ago, the Chinese were using treatments made of
moldy soybean curd to treat infections. The ancient Egyptians rubbed moldy bread on
wounds to cure them, and moldy cheese was used for the same purpose in parts of
Europe.
HISTORY OF DEVELOPMENT OF RESISTANCE
Resistant bacteria have always been around and existed long before humans
began using antibiotics therapeutically. What is new in the world of resistance is how
quickly new resistant strains arise. The widespread use and misuse of antibiotics
contribute to the problem. For the first time in decades, people in the United States are
dying of bacterial infections that cannot be treated.
Right after we began using penicillin, some Staphylococcus strains were identified as
resistant to it.
In the 1940s and early 1950s, streptomycin, chloramphenicol, and tetracycline were
discovered.
By 1953, a strain of Shigella was found that resisted these antibiotics and sulfanilamides.
The 1990s saw the development of true superbugs, bacteria that resist all known
antibiotics.
One antibiotic of last resort is Vancomycin, a powerful antibiotic that attacks bacteria on
many fronts.
By the 1940s and 1950s, a single antibiotic, such as Streptomycin, no longer cured
tuberculosis, as it had in the past.
32
INTRODUCTION
WHERE ANTIBIOTICS COME FROM
Below are listed some common antibiotics and their natural sources.
SOURCES OF ANTIBIOTICS
Source
Examples
molds
penicillium
penicillin
cephalosporium
cephalosporins
actinomycetes
tetracycline
aminoglycosides
(streptomycin)
macrolides
(erythromycin)
chloramphenicol
ivermectin
rifamycins
bacteria
bacilli
B. cereus
Zwittermicin
synthetic
oxazolidinones
33
INTRODUCTION
MECHANISMS OF ANTIBIOTIC ACTION
The many modes of antibiotic action are shown schematically in the diagram below.
Beta-Lactam antibiotics
The b-lactam ring mimics a component of the cell wall to which transpeptidase binds.
The affected bacterium will eventually lyse (rupture) because the unsupported cell wall
cannot withstand its growth.
34
INTRODUCTION
Disrupters of nucleic acid synthesis
The antibiotic rifampin interferes with prokaryotic RNA polymerase and thus, interferes
with transcription.
Both of these disruptions prevent bacteria from dividing to make more bacteria.
Disrupters of protein synthesis
They are L-shaped molecules that fit into L-shaped pockets of bacterial ribosomal RNA.
When they insert themselves into rRNA, they disrupt ribosomal structure.
Aminoglycosides dont have this effect on human cells because the L-shaped pocket is
specific to bacteria.
Inhibitors of metabolism
35
INTRODUCTION
Mechanism
Pennicillin
Inhibits
transpeptidase
enzymes.
Activates
lytic
Inhibits
transpeptidation
in
cross-linking
Rifampin
Erthromycin
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INTRODUCTION
Fusidic acid
Blocks translocation.
Linezolid
Streptomycin
Tetracyclines
Metabolic inhibitors
Dapsone
Sulfonamides
Trimethoprim
Inhibits
dihydrofolate
reductase,
blocking
tetrahydrofolate synthesis.
37
INTRODUCTION
MECHANISMS OF RESISTANCE
Bacteria either have preexisting resistance to drugs, or they develop resistance.
Human activity has contributed greatly to the increase in resistant strains of bacteria.
Often, when bacteria acquire resistance to a certain drug from a particular class (e.g., the
penicillins), the bacteria also acquire resistance to all other drugs in that class.
Some of the many mechanisms of resistance are indicated schematically in the following
diagram:
38
INTRODUCTION
39
INTRODUCTION
This type of acquisition allows resistance to spread among a population of bacterial cells
much
faster
than
simple
mutation
and
vertical
evolution
would
permit.
Transduction
A virus serves as the agent of transfer between bacterial strains.
Transformation
DNA released from a bacterium is picked up by a new cell.
After the new DNA is introducedwhether via conjugation, transduction, or
transformationit is incorporated into the cell and results in the emergence of a new,
resistant genotype.
40
INTRODUCTION
Resistance to
Gram-negative bacteria
Penicillin
and
other b-lactam
antibiotics
Proteus
mirabilis (rheumatoid
Tetracycline
arthritis,
urinary tract infections)
Klebsiella pneumoniae (ankylosing
Ampicillin
Methicillin
Resist b-lactam antibiotics through modifications in the genetic code for the proteins that
bind penicillin.
Genes for enzymes that can destroy or disable antibiotics are acquired or arise through
mutation. For example, a b-lactamase enzyme can destroy the b-lactam ring of penicillins
41
INTRODUCTION
through hydrolysis, and without a b-lactam ring, penicillins are ineffective against the
bacteria.
42
INTRODUCTION
Ribosomal modifications
The ribosome can be methylated so that an antibiotic cannot bind to it.
Protein modifications
For antibiotics that target DNA gyrase, the enzyme that unwinds DNA for
replication, random mutations in the bacterial DNA may alter the gyrase and make it
unrecognizable to antibiotics while still leaving it functional.
Metabolic resistance
In the case of sulfonamides, which operate by mimicking PABA and competing
for an enzyme that synthesizes folic acid, an increase in the amount of PABA can
outcompete the sulfonamide and render it ineffective; or an alteration in the code for the
enzyme itself can prevent its sulfonamide binding.
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INTRODUCTION
Effluxing the toxin
One particularly active way a bacterium may deal with an antibiotic is to pump it
out, perhaps using proteins encoded by acquired genes. For example, a strain
of enterococcal bacteria can pump out tetracycline. This type of pumping is called an
efflux phenomenon.
Note: Bacteria without inherent antibiotic resistance can acquirethrough
conjugation, transduction, or transformationthe genes that encode proteins that confer
resistance.
MECHANISM OF ACTION OF ANTIBIOTICS12,13
Aminoglycoside
Inhibit protein synthesis by binding to a portion of the bacterial ribosome. Most of
them are bactericidal (i.e., cause bacterial cell death)
Neomycin
44
INTRODUCTION
Bacitracin
Inhibits cell wall production by blocking the step in the process (recycling of the
membrane lipid carrier) which is needed to add on new cell wall subunits.
Bacitracin
Beta-lactam antibiotics
A name for the group of antibiotics which contain a specific chemical structure
(i.e., a beta-lactam ring)
cephalosporins, etc.
( i ) Penicillins
Inhibits formation of the bacterial cell wall by blocking cross-linking of the cell
wall structure. The cell wall is a needed protective casing for the bacterial cell.
45
INTRODUCTION
(ii) Cephalosporins
Similar to penicillins in their mode of action but they treat a broader range of
bacterial infections. They have structural similarities to penicillins and many people with
allergies to penicillins also have allergic reactions to cephalosporins.
Chloramphenicol
Inhibits protein synthesis by binding to a subunit of bacterial ribosomes (50S).
Erythromycin.
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INTRODUCTION
Quinolones
Blocks DNA synthesis by inhibiting one of the enzymes (DNA gyrase) needed
this process.
Nalidixic acid
Rifampin
Inhibits RNA synthesis by inhibiting one of the enzymes (DNA-dependent RNA
polymerase) needed in this process. RNA is needed to make proteins.
Rifampin
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INTRODUCTION
Tetracyclines
Inhibit protein synthesis by binding to the subunit of the bacterial ribosome
(30S subunit ).
48
INTRODUCTION
IMPORTANT MICROBES PRODUCING ANTIBIOTICS 14
S. No.
NAMEOF
NAME OF ANTIBIOTICS
MICROORGANISM
P. notatum
Penicillin
P. griseofulvu
Griseofulvin
P. chrysogenum
Penicillin
S. griseus
Streptomycin
S. venezuelae
Chloramphenicol
S. aureofaciens
Chlortetracycline
S. virdofaciens
Aureomycin
S. rimosus
Oxytetracycline
S. texas
Tetracycline
10
S. aureofaciens
Dimethyl-chlortetracycline
11
S. erythricas
Erythromycin
12
S. halstedii
Carbamycin
13
S. ambofaciens
Ravomycin
14
S. noursei
Nystatin
15
S. griseus
Cycloheximide
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INTRODUCTION
NUMBER OF ANTIBIOTICS PRODUCED BY MAJOR GROUP OF
MICROORGANISMS 15
TAXONOMIC GROUPS
NUMBER OF ANTIBIOTICS
Bacteria
950
other
than
actinomycetes
Actinomycetes
Fungi
4600
1600
Table No.5. Represents that most of the drug mainly produced by Actinomycetes.
50
INTRODUCTION
Antibiotics
Total
(with
Bioactive metabolites
other
activity)
no
antibiotic
(antibiotics +
other
Total bioactive
bioactives)
activity
metabolites
Bacteria
2900
(780)
900
(1680)
3800
Actinomycetales
8700
(2400)
1400
(3800)
10100
Fungi
4900
(2300)
3700
(6000)
8600
Total Microbial
16500
(5500)
6000
(11500)
22500
derived antibiotics.
The two terms are usually used synonymously and that practice will continue
throughout this presentation.
The word antibiotic will be used to describe: a chemical substance derivable
from a microorganism or produced by chemical synthesis that kills or inhibits
microorganisms and cures infections.
51
INTRODUCTION
BIOACTIVITY AND INDUSTRIAL POTENTIAL1
1. ANTIBIOTICS
More than 50% of the known natural antibiotics produced by actinomycetes. Two
thirds of the present day antibiotics are of actinomycetes origin like Aminoglycosides,
Chloramphenicol, Macrolides, Tetracyclines, Nucleosides, Peptides and Polyenes. Till
1974 antibiotics of actinomycetes origin were almost exclusively confirmed to
Streptomyces. Nowadays efforts are being made to explore rare actinomycetes like
Actinomadura
Actinoplanes
Ampullariella
Actinosynnema
Dactylosporangium
Kibdilosporangium etc.
A search for novel antibiotic producing actinomycetes in new ecological niches
with a target directed approach is currently being pursued.
2. ENZYMES
Actinomycetes have been found to be a promising source of wide range of
important enzymes, some of which are produced on an industrial scale, Presently
enzymes of microbial origin are widely used in food processing, detergent,
manufacturing, textile and pharmaceutical, industries, medical, bioorganic chemistry and
molecular biology.
52
INTRODUCTION
3. TRANSFORMATION OF XENOBIOTICS.
Xenobiotics are defined as the structural modification of compounds foreign to an
organisms metabolite which occur in its chemical environment. Members of Nocardia
and Streptomyces have ability to perform highly selective chemical modification of
complicated compounds of natural and synthetic origin.
DDT(1,1,1-trichloro-2,2,di-4chlorophenyl-ethane)-dechlorinated by
S. aureofaciens, S.cinnamoneus.
4. ENZYME INHIBITORS.
In 1972 Umezawa reported a low mol.wt. enzyme inhibitors from Streptomyces
strain. More than 60 inhibitors have been reported e.q. Inhibitors for papain, plasmin,
trypsin, chymotrypsin etc.
5. IMMUNOMODIFIERS.
Low Mol. wt. compounds have been isolated from culture filtrates of
actinomycetes which enhance immune response such agents called immunomodifiers.
53
INTRODUCTION
6. AGRICULTURE.
Actinomycetes carry out numerous activities in soil including degradation of
organic matter, inhibition or stimulation of other microorganisms and
plants,
Lignocellulose-humus (Streptomyces)
Actinomycetes isolated from Rhizosphere of pine produced vitamin and are known to
enhance the growth.
7. BIOTECHNOLOGY.
Biotechnological application is a natural result of the greatest metabolite diversity
of these organisms and their long association with environment and needs of human.
Other beneficial activity of actinomycetes related to biotechnology is their gene
expression activity which is becoming an active research area. Several different sps. of
RNA polymerase have been isolated from Streptomyces coelicolor.
Genome size in Streptomyces is thought to be 1.5-2 time of that E.coli,
Streptomyces
advantageous in nature.
FERMENTATION 11
The term fermentation is derived from Latin word fevere, to boil, thus
describing the appearance of the action of yeast on extracts of fruit or matted grain. The
boiling appearance is due to the production of carbon dioxide bubbles caused by the
anaerobic catabolism of the sugars present in the extract. According to industrial
microbiologist, fermentation is any process for the production of product by the mass
culture of microorganism.
54
INTRODUCTION
THE RANGE OF FERMENTATION PROCESSES
There are five major groups of commercially important fermentations.
It will produce the maximum conc. of product or biomass per gram of substrate used.
It will cause minimal problems in other aspects or the production process particularly
aeration, agitation, extraction, purification and waste treatment.
55
INTRODUCTION
PRODUCT RECOVERY 11
One of the most critical aspects of an industrial fermentation process is the
recovery and purification of the product.
In biological processes after the production of the product, extraction and purification
remain the next most important steps. The steps involved are most difficult and costly.
Therefore, in biological processes the down streaming processes should be efficient and
cost effective to make the process economically viable. But, unfortunately, in general the
recovery cost in microbial processes ranges from 15% to 75% of the total manufacturing
cost. The recovery cost depends on the nature and purity of the product and the methods
chosen for the recovery purpose. On the other hand, production parameters such as
temperature, pH, process handling and aseptic conditions also affect the down streaming
process. It is known that production cost is less in the case of extracellular and simple
products; on the other hand intracellular products cost more for the recovery. Therefore,
the following criteria are taken into consideration while designing a recovery process:
1. Localization of the product ( i.e. whether intracellular or extracellular)
2. Product concentration in the fermented broth
3. Physical and chemical nature of the product which may serve as an aid for the
selection separation process
4. Acceptable purity of the product by the competent authority (like FDA)
5. Marketable price of the product Along with these, bio-safety consideration of the
production process has also to be taken care of. The first step for the recovery of an
extracellular product is to remove the cellular parts and media components. The preferred
method that is used for the separation of cellular parts is centrifugation. The next steps
are fractionation of the broth for the extraction of the product. Ultrafiltration, reverse
osmosis, adsorption, ion exchange, gel filtration or affinity chromatography, liquid-liquid
extraction, two phase aqueous extraction or precipitation are the methods used for
isolation or purification of the desired product. The preference for the method depends on
56
INTRODUCTION
the nature of the product. Afterwards the product is crystallized to attain the required
purity. By products may be isolated by the modification of this system. Product recovery
may be made easier by taking care of the following steps in the upstream or
bioconversion process.
1. By selecting microorganisms that do not produce any pigment during the fermentation
or do not produce any undesirable metabolites.
2. By modifying the production conditions so that least amount of undesirable secondary
metabolites are formed.
The following process parameters should be checked and maintained precisely:
1. Harvesting time
2. pH control during fermentation and harvesting
3. Temperature control and monitoring
4. Addition of appropriate reagents for flocculation and separation
Purification of many of the compounds can be made by using a number of alternative
routes. Ultimate choice of a particular process can be made by considering various
criteria such as capital cost, processing cost, throughput requirement, yield potential,
product quality and technical expertise available.
The choice of the recovery process is based on the following criteria
The physical and chemical properties of the desired products (All aid to selecting
separating procedures).
57
INTRODUCTION
Diagram No.9. Shows removal of microbial cells and other solid matters
58
INTRODUCTION
59
INTRODUCTION
flocculating characteristics and the use of magnetic field. But these techniques suffer
from high cost and difficulties of scale up.
Two phase liquid extraction technique, in the more recent time, has been of much
interest because of its easy scalability and cost effectiveness and can be used for the
products that needs gentle conditions.
Foam Separation
Foam separation of solid materials like microbial cells or proteins and colloidal
materials is mainly based on the surface properties of the materials. Cells or molecules
are absorbed on surfaces of the gas bubbles arising with the bowling air and finally
separated by skimming.
Surfactants used generally are called collector and the material that is made
surface active and collected are termed as colligends. For a process to be developed on
the basis of this technique, the following parameters should be taken care of: pH,
temperature, and air flow rate and surfactant and colligend collection ratio.
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INTRODUCTION
Precipitation
Precipitation is mainly a product recovery process and can be applied at any stage
of downstream processing. It is helpful to separate some product at one step or remove
some impurities in the other. Sometimes the process allows enrichment and concentration
of the product and reduces the volume to be handled. The following techniques are used
for the precipitation of various materials:
1. Change in the pH of the solution to reach the isoelectric point of the compound. The
molecules precipitate due to a decrease in the solubility.
2. Ammonium and sodium salts (sulphates) are mainly used to precipitate the proteins.
Salts remove the water molecules from the surface of protein molecules to facilitate the
exposure of the non-polar surfaces of the molecules. Due to the non-polar interaction of
the molecules that aggregate and precipitate.
3. Solvents (chilled ethanol, acetone) are used for the precipitation of proteins from the
broth by changing the dielectric properties of the molecules. Non ionic polymers such as
polyethylene glycol also precipitate proteins in the same way.
4. Poly-electrolytes are also used for the aggregation and precipitation of proteins.
Certain newer methods, such as, affinity precipitation or the selective precipitation of
compounds and for the protein precipitation dye binding and aggregation are of particular
interest.
61
INTRODUCTION
Filtration
Filtration is one of the most widely used and efficient processes for solid-liquid
separation with different types of filtration systems. The process uses a porous medium
that allows the flow of gas or liquids but not the solid material. It is amenable to scale up.
But the following factors influence the choice of the most suitable equipment to meet the
specific requirement with obvious cost effectiveness.
1. Viscosity and density of the filtrate.
2. Nature of the solid particles: shape, size, distribution and packing characteristics.
3. Solid: liquid ratio.
4. Scale of operation.
5. Material required to be recovered i.e. solid or liquid or both.
6. Mode of operation: continuous or batch.
7. Need for additional attachment for vacuum suction or need for low temperature.
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INTRODUCTION
centrifuge
when
filtration is not a
Centrifuge may be expensive when compared with a filter it may be essential when
filtration is slow and difficult. The cells or other suspended matter must be obtained free
or filter aids continuous separation to a high standard of hygiene is required.
Centrifugation is used not only for separating particles from the liquid phase
(fluid/particles separation) but also for fluid/fluid/ particle separations.
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INTRODUCTION
When solid separation is not satisfactory by filtration or a very high degree of separation
is required, centrifugation is the method of separation. The main principle of
centrifugation process is the sedimentation under centrifugation force.
For the industrial application different types of centrifuges are available with
different dimensions and modes of application. They differ mainly in their mode of
operation, capacities, speed, mode of loading and discharging. The ultimate choice of the
type of centrifuge depends on the type of application and effectiveness
64
INTRODUCTION
disintegration methods are available but the choice of the method depends on its
suitability for the particular substance. Though in some cases by the application of a
particular method, a specific product can be recovered from inside the cells with greater
yield and purity, most of the times these types of applications are not feasible.
On the other hand, gross disruption of the cell releases a huge number of products
and its down stream processing becomes difficult. Sometimes application of a particular
process is only feasible in the laboratory scale and therefore, choice of suitable methods
for the industrial application is a matter of investigation. In recent times, enzymes are the
most important intercellular products of interest. But the disruption methods applied for
the release of enzymes must keep them active and properly folded, at the same time
release yield must be high.can be obtained with minimum contamination.
65
INTRODUCTION
of the
microbial cell membrane and release the intracellular enzyme. The detergents used may
be anionic or cationic or nonionic. The widely used detergents are quaternary ammonium
salts, sodium dodecyl sulphate, Triton X-100 etc. While applying these substances for the
release of enzyme, it has to be kept in mind that the substances can cause protein
denaturation and have to be removed from the cell free extract during further purification
as soon as possible. The use of Triton X-100 is widely known to release membrane bound
enzymes. The application of Triton X-100 along with guanidineHCl is very effective for
the release of a number of proteins.
2. Alkali Treatment
This method is applied for the cell disruption in very limited cases only when the
enzyme is highly alkali stable and can tolerate pH up to at least 11.5. Only very few
applications of this method are found for the release of the enzyme and one of the
classical examples is for the release of L-asparaginase.
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INTRODUCTION
Physical Methods
1. Liquid Shear
Liquid shear force for cell disruption is used mostly for the large scale release of
enzymes or intracellular products from the microbial cells. The method is based on the
shear force generated by cavitation in the cell slurry due to a large pressure drop. The
machine consists of a hollow cylinder made up of stainless steel and a piston with an
appropriate system of adjustable valves. Cell slurry is loaded inside the cylinder and the
pressure inside the cylinder is increased to thousands of psi through a system of
hydraulics. When the cell slurry is allowed to pass through a small orifice, the cells
experience a sudden pressure drop. That pressure drop causes cavitation and the shock
waves so produced disrupt the cells. The amount of pressure drop experienced by the
cells has a direct influence on the disruptive release of the enzymes from the cells.
Therefore, it can be concluded that higher pressure drop is more effective for cell
disruption. Mechanical strength of the cell wall, shape and size of the microorganisms
also play an important role in achieving effective disruption. The process of cell
disruption is exothermic and to prevent heat denaturation of the enzyme, an effective
cooling system is always needed. The whole process is operated within 0-4oC. To make
the process less abrasive, it should be operated in multi-pass mode for a longer time. In
an industrial process, proper balance has to be made between the maximum release due to
effective breakage of the cells and the percentage of released enzyme to achieve cost
effectiveness.
2. Solid-liquid shear
This is a suitable method for laboratory and can also be operated in semi-continuous
mode in
small scale in the industry. Frozen samples of microorganisms are passed at very low
temperature (-25oC) through a small orifice at very high pressure. The breakage occurs
due to the liquid-shear and the presence of ice crystals. The process is especially
applicable for temperature labile enzymes.
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INTRODUCTION
3. Agitation with abrasives
In this method cells are disrupted with the help of mechanically resistant beads
made up of glass, alumina or titanium compounds. Inside a hollow chamber, cells are
agitated with beads 14 by a system of agitator shaft. The shear forces so generated cause
cell disintegration. The process is exothermic and an efficient cooling system must be
associated to prevent thermal denaturation of the enzyme. The process can be applied at a
large scale after suitable investigation.
4. Ultrasonication
Ultrasonic waves are used to break the cells at small scale. The process relies on
the cavitation generated due to the sonic waves and the shear force generated thereby. A
high electrical power is converted in to mechanical energy in terms of sonic wave and
propagates through a horn in the liquid generating cavitation. The system suffers from
several draw backs: high power consumption, heat generation, small operable volume,
etc.
5. Freeze-thawing
This is a very mild process. Due to repeated freezing and thawing of the microbial
cells, ice crystals generate, create pores in the cell wall and facilitate the release of
enzyme. The process is applicable in combination with other methods.
6. Osmotic Shock
Osmotic shock is applied for the mild release for the enzymes from the cells. A
sudden change in the salt concentration changes the osmotic balance within the cells and
the cell is disrupted. But the method is not very efficient for the microbial cells having
tough cell wall.
To apply this method for the disruption of microbial cells, generally the cell wall is first
made weak by some other method. The method has proved to very efficient and unique
for the release of luciferase enzyme from Photobacterium fischeri.
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INTRODUCTION
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INTRODUCTION
Many antibiotics purified by use of solvent extraction. A two phase system is set
up, using a solvent which is immiscible with the aqueous fermentation broth.
After desired product is concentrated in the solvent phase, it can be further
purified; solvent extraction has been widely used in the antibiotic industry, for
antibiotics.
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INTRODUCTION
Adsorption Chromatography
Affinity Chromatography
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INTRODUCTION
DRYING
For the biological materials, it is essential to dry the final product in such a way
that that its activity is not lost. Drying essentially involves transfer heat to the wet
product, and removal of the moisture in a stream of gas. Heat transfer can be either by
direct contact, by convection or by radiation.
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INTRODUCTION
STRUCTURE DETERMINATION 11
Structure Determination is necessary for the identification of the new compounds;
it is mainly performed by the following methods.
N.M.R SPECTROSCOPY
MASS SPECTROSCOPY
INFRARED SPECTROSCOPY
N.M.R SPECTROSCOPY
NMR Spectroscopy is a technique that permits exploration of a molecule at the
level of the individual atom and affords information concerning the environment of that
atom. Only about one half of known element isotopes when placed in a magnetic field,
absorb energy from the radiofrequency region of electromagnetic spectrum.
The precise frequency from which energy is absorbed gives an indication of how
an atom is bound to, or located spatially with respect to other atom. Thus NMR offers an
excellent investing molecular interaction; NMR spectra may also be used for compound
identification.
MASS SPECTROSCOPY
The mass spectrum is a line spectrum, each line corresponding to a positive ion of
specific mass. The potential of m ass spectra in study of organic compounds was quickly
revealed by mass spectrometer.
INFRA RED SPECTROSCOPY
The number of absorption bands in an I.R. spectrum may be considerable and
there in lies its value as a means of identification. Interpretation of absorption spectra is
considerably correlating the frequency at which a band occurs with molecular structure.
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