Professional Documents
Culture Documents
10 Chapter 5
10 Chapter 5
CHAPTER 5
DISCUSSION
124
NUTRIENT
AND
MICROBIAL
ANALYSIS
IN
SOIL
SAMPLES
Soil nutrient analysis of Yercaud and Kolli hills belonging to eastern
Ghats of Tamil Nadu, India indicated that the soil physical properties like pH
ranges, electrical conductivity ratio, water holding capacity and the nutrient
elements such as total organic carbon, nitrogen, potassium, phosphorous,
calcium, magnesium and sodium contents were estimated which were varied in
varying levels. It has been reported that actinomycetes usually prefer neutral or
slightly alkaline soil few are able to grow in acidic soil pH more than 5 (Lacey
1971). Water content and aeration of soils are also closely related. Most soil
actinomycetes is aerobic and intolerant of water logging (Kouyeas et al 1964)
and are often said to be more numerous in plant rhizospheres than away from
the roots (Nithya et al 2012). Actinomycete populations were positively
correlated with the organic matter and soil water contents, and negatively with
pH value (Rao and Subramanyan 1929).
Actinomycetes population was enumerated from rhizosphere as well
as non-rhizosphere regions of various medicinal plants using starch casein
nitrate (SCN) agar medium by following serial dilution technique which
revealed that it was found to be more in rhizosphere than in non-rhizosphere
soils revealed that it was found to be higher in the rhizosphere than in nonrhizosphere regions (Table 4.5 and Table 4.6). Environment is the ultimate
source of many different types of microorganisms. Soil samples have been a
very common substrate for the isolation of microbes for many research works.
Generally the plants release some of the soluble and easily diffusible
compounds (e.g., sugars, amino acids and organic acids) and many insoluble
125
compounds (e.g., cellulose, lignin and protein). So the soil samples were
collected from the specified areas of both rhizosphere and non- rhizosphere
regions of fertile crops (Webley 1952, Ponmurugan and Nithya 2008).
The distribution pattern of actinomycetes population of different
medicinal plants was studied which revealed that the population density was
decreased when the distance as well as depth of soil sampling from the plants
was increased. In order to study the characteristics of soil samples used, various
parameters of the soil were estimated which includes pH of the soil and the
percentage of organic content most of the isolates tend to grow in alkaline soils
which is an important features of Streptomyces sp., (Stackebrandt et al 1991)
and with adequate source of carbon and nitrogen present in the sample (Tien
et al 1987). It has been documented that Streptomyces are of special interest
because of their morphological complexity and their ability to produce
antibiotics in rhizosphere soils of medicinal plants. Typically Streptomyces
populations in soil are in the range of 105-107 colony forming units/gram of
soil (Gray and Williams 1971). Many types of microbial activities are possible
under certain soil conditions. The distribution of Streptomyces in soil often
reflects their activities on these substrates or their responses to chemical or
physical conditions (Ponmurugan et al 2007) and most widely used for the
isolation of actinomycetes.
5.2
CHARACTERIZATION
AND
IDENTIFICATION
OF
ACTINOMYCETES
A total of 168 strains were isolated from soil samples and screened
based on their potential of secondary metabolite production in which Yer11
and Yer28 strains obtained from Yercaud hills and similarly, Kol35 and Kol44
strains isolated from Kolli hills for further studies. All the strains were
identified by following the method of Bergeys manual, wherein, polyphasic
126
taxonomic approach was adopted. All the actinomycete strains were identified
as Streptomyces sannanensis.Most of the isolates were presumed to be the
genera of Streptomyces spp., as they showed sporulation with compact, chalk
like dry colonies of different colour varying from pink to white (Holt et al
1989).
Actinomycete strains were found to be Gram-positive, well
established spore chains with different colour morphology of the colony which
were varied from greyish to pinkish in colour. They responded positive for
catalytic and oxidative enzymes such as catalase, lipase and urease and
negative for indole production, methyl red and vogesproskauer tests. The basic
and main characteristic feature is the musty odour and colour produced by
actinomycetes due to Geosmin and 2-methyl isoborate (MIB) including some
pigments are the compounds responsible for the odour and colour released
during the growth metabolism (Ponmurugan et al 2011). On the other hand,
most of the cultures of Streptomyces showed the formation of secondary
metabolite like pigment (Nielson, 1993) also hydrolyses starch and casein as
well (Ravel et al 1998). It has also been identified that these species also have
the ability to secrete exo enzymes in them.
One of the most important genera of Actinomycetes is Streptomyces.
Streptomyces are gram positive filamentous saprophytic soil bacteria that are
distinct from other bacteria (Lacey 1971).
Streptomyces is a genus of
actinomycetes and they are able to catabolize sugars, alcohols, amino acids,
organic acids and some aromatic compounds. This is accomplished by
producing extra cellular hydrolytic enzymes that allow the catabolism of
organic molecules (Madigan et al 2000). Streptomycetes are widely used in
industry due to their ability to produce numerous chemical compounds
including antibiotics, enzymes and anti-tumor agents (Berdy 1989).
127
128
followed by Vit A and Vit B5; respectively were found to be good. The various
isolation techniques were designed to allow the growth of a wide range of
actinomycetes and to discourage other bacteria and fungi. Isolation may
involve the prior treatment of samples before transferring to a selective
medium formulated to encourage the growth of actinomycetes; such media may
also contain amino acids and vitamins including inhibitors, which suppress the
growth of associated bacteria and fungi (Good Fellow et al 1987). The most
extensively used media are starch casein nitrate agar (Kuster and Williams,
1964), which contain colloidal chitin as the sole source of carbon and nitrogen
(Lingappa and Lockwood 1962) for cultivation of actinomycetes under in vitro
condition.
Most of the secondary metabolites identified were produced by
Streptomyces species (Sanglier et al 1993). This gram positive bacterium
produces the secondary metabolites and sporulates well under stress condition.
The time of starting the secondary metabolite production and sporulation
depends on the environmental condition such as nitrogen and carbon sources
(Onakaet al 1995).
pesticide
like
propargite,
129
showed a similar banding pattern which was 10kb 21kb molecular weight.
Jaccards similarity index after studying the RAPD pattern showed Yer11 and
Kol44 strains had the similarity value of 0.667 which revealed that they are
more similar in nature. Actinomycetes comprise of 63 genera. Based on 16s
rRNA classification system, they have recently been grouped in 10 sub orders
and a large number of actinomycetes still remain to be ungrouped (Prescott and
Klein 1993). Secondary metabolites production in Streptomyces sp. is
associated with the plasmids present in the cells. Some of the genes are
responsible for the regulation of secondary metabolite production, which are
present in the plasmid DNA. It has been found that the plasmid number and
size varies in different strains (Yagisawa et al 1978, Westpheling 1985).
Streptomyces synthesize about two-thirds of the currently known
antibiotically active substances (Crandall and Hamill 1986). The economic
importance has led to increased interest in the genetic aspects of antibiotic
biosynthesis by these organisms (Stutzman-Engwall et al 1992). Many
Streptomyces carry detectable extra-chromosomal elements (plasmids) and, in
most cases, plasmids are present in the form of covalently closed circular
(ccc)-DNA, but, occasionally, linear elements are also found. So far, a number
130
EVALUATION
OF
BIOACTIVE
SECONDARY
131
the
bacterial
pathogens,
Escherichia coli,
Serratia
flavus,
Stachybotrys
chartarum,
Histoplasma
capsulatum,
132
Actinomycetes,
mainly
Streptomyces
species
produce
oxytetracycline
133
actinomycetes
are
among
the
most
important
cancer
134
BIOSYNTHESIS
OF
NANOPARTICLES
USING
135
phytochemical
fraction
reduced
gold
nanoparticles
shows
maximum
fraction
reduced
gold
nanoparticles
shows
maximum
136