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CHAPTER 5
DISCUSSION

Actinomycetes population has been identified as one of the major

groups of the soil population. They are widespread in nature and can be
separated into two sub-groups, oxidative forms, mostly found in soil and
fermentative forms, living in natural cavities of human and animals
(Lechevalier and Pine 1977). It has transitional characters between bacteria and
fungi and is sometimes called as fungi-like bacteria. They are phylogenetically
and chemo taxonomically related with gram-positive bacteria with a high G+C
content in their genome (Locci 1994). They are capable of producing spores
(Cross, 1981), which facilitate their rapid dispersal in aquatic habits
(Actinoplane zoospores), their dispersal in air and survival in soil
(Streptomycete arthrospores) and may even ensure viability over many
decades.
The actinomycetes are nutritionally versatile being able to grow both
on rich substrates and on those containing a minimum or even an apparent lack
of nutrients (Lechevalier 1964). Actinomycetes have characteristic biological
aspects such as mycelial forms of growth that accumulates in sporulation and
the ability to produce an array of secondary metabolites, many of which have
antibacterial or antifungal properties. In fact, most antibiotics developed for
human pharmaceutical use are actinomycete metabolites, many being derived
from Streptomyces spp. (Goodfellow et al 1984). Complex morphological

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development in the genera is phenotypically related to secondary metabolism

(Horinouchi et al 1992).
5.1

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

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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

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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

The most widely accepted theory is that antibiotics are used to

compete with other organism growth in terms of nutrient depleting
environments including well established environmental conditions. Although
other organisms are capable of producing antibiotics, they remain susceptible
to Streptomycete production due to optimum biotic and abiotic factors which
are essential for enhancing growth metabolism (Madigan et al 2000).
Streptomyces isolates are important for the ecology of soils as they degrade
recalcitrant substrates such as chitin and lignin and produce a variety of
antibiotics. Actinomycetes strains showed a prominent growth at a pH of 6, 30
C temperature, 80% relative humidity, 2% NaCl concentration and 12D:12L
photo periods (Figure 4.5 and 4.6).
Among the carbon and nitrogen sources tested to enhance the growth
of S. sannanensis strains, monosaccharides followed by disaccharides and
sulphate followed by ammonium nitrate; respectively were found to be
good.Actinomycetes are aerobic, Gram positive bacteria that form branching
filaments or hyphae that may persist as a stable mycelium or may break up into
rod shaped or coccoid elements. Motility when present is due to flagellation.
For certain features, such as the determination of the nature of the surface of
spores, the use of an electron microscope is essential (Prescott and Klein 1993).
Hyphae and spores of most actinomycetes strain readily with giemsa solution,
crystal violet, methyl-violet, hematoxylin and methylene blue. The media for
the cultivation of actinomycetes are ESPY, containing frames of nutrients like
casein hydrolysate, yeast extract, or starch (Kuster and Williams 1964). It has
been observed that actinomycetes occur in soil, water, plant and animal tissues
as saprophytes and/or parasites and cause important diseases of animals and
human.
Similarly, of various amino acids and vitamins tested to enhance the
growth of S. sannanensis strains methionine followed by alanine and biotin

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).

Different plants produce different type of secondary

metabolites and some of these chemical compounds are found to be toxic to

soil microorganism including actinomycetes. However, adaptation has in turn
lead the actinomycetes to produce their own secondary metabolites under
aseptic condition with optimum nutrient supplements line carbon and nitrogen
sources (Ho et al 2000).
It has been observed that a huge amount of pesticides, fungicides and
weedicides are being applied in the soil to control a wide variety of pests and
phytopathogens which in turn affect the soil properties in the sustainable
agriculture. Among the agrochemicals tested,

pesticide

like

propargite,

fungicides like hexaconazole and paraquat dichloride weedicide were inhibited

the growth of S. sannanensis (Table 4.19). Bibb (1996) used Streptomyces as

129

growth promoters, agents for plant protection, anti-parasitic agents and

fungicides / pesticides / herbicides. Long-term antibiotic treatment and surgical
management are necessary. In vitro, S. somaliensis is sensitive to rifampicin,
erythromycin, tobramycin, fusidic acid and streptomycin and some groups of
fungicides and pesticides. Most of the strains strains tested were resistant to
broad spectrum of antibiotics and fungicides / pesticides (Vining 1992, Bibb
1996).
5.3

MOLECULAR ANALYSIS OF S. SANNANENSIS STRAINS

The genomic DNA isolated from all the strains of S. sannanensis

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

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of different Streptomyces have been investigated for plasmids supposed to

involve in the genetic control of the production of antibiotics (Hopwood 1995).
However, more detailed analysis has shown that antibiotic biosynthetic
structural genes reside mostly on the chromosome.
Many alkaline lysis procedures for the isolation and purification of
CCC-DNA have been described (Kieser et al 2000, Daniel and Tiraby 2008)
which can be adopted well for actinomycetes. Based on the plasmid profile, the
actinomycete strains are being segregated and correlated with its secondary
metabolite production. On the other hand, plasmid profile of actinomycete
strains may be correlated with its occurrence and gene expression under
specific condition. Plasmids are found in different species of streptomycetes
where they can affect various characteristics (Hopwood et al 1993). Isolation
and investigation of new plasmids are important both for ascertaining the
effects on the hosts as well as utilising those with potential in bioengineering
(Pernodetet al 1984).
5.4

EVALUATION

OF

BIOACTIVE

SECONDARY

METABOLITES OF STREPTOMYCES SANNANENSIS

From the inception of civilisation, human being struggles for
existence against the affliction of disease, decay and death. It is the eternal
want of human to remain healthy and cure from his surroundings. But in the
ancient era, millions of people died from various infectious diseases like
plague, cholera, diarrhoea, tuberculosis etc., in epidemic form, which has
instigated the man to endeavour for remedy from their sufferings. In fact in the
way of long struggle for relieving from such infectious diseases as well as
illness, physical discomforts, injuries, pain and wounds, man would achieve the
mastery over the diseases. Streptomyces in particular, produce a vast array of

131

clinically important secondary metabolites that include antibiotic, anti-viral,

anti-cancer or as an immunosuppressive agent (Demain 1998).
Streptomyces are used to produce the majority of antibiotics that are
applied in veterinary medicine and agriculture. As well as several enzymes
were synthesized and used in food and other industries. It is also unique
amongst bacteria in their mycelia, sporulating life cycle, which involves
complex regulation of gene expression (Zahner et al 1979). The crude extracts
obtained from S. sannanensis strains in the present study were evaluated for
their antibacterial, antifungal and anticancerous activities (Figures 4.17 and
4.18).

Zakir Sultan et al (2002) reported that, the bioactive secondary

metabolites produced by the actinomycetes exhibited significant antibacterial

activity against five gram positive and nine gram negative bacteria when
compared with standard Kanamycin antibiotic with the zone of inhibition of
17-20 mm dia. And with the MIC value of 32-64g.mL-1. Kavitha and
Vijayalakshmi (2007) reported that, metabolites produced by the Streptomyces
rochei showed good antimicrobial activity against gram positive and gram
negative bacteria particularly Bacillus subtilis, Bacillus cereus and Escherichia
coli.
Among

the

bacterial

pathogens,

Escherichia coli,

Serratia

marcescens, Proteus vulgaris, Salmonella paratyphi A and Pseudomonas

aeuroginosaexhibited a least resistance against S.sannanensis strains.
Similarly, among the fungal pathogens such as Aspergillus fumigatus,
Aspergillus

flavus,

Stachybotrys

chartarum,

Histoplasma

capsulatum,

Pneumocystis jirovecii, Candida albicans, Scizophyllum commune and

Ustilago maydis exhibited a least resistance against S.sannanensis strains
(Table 4.26). Of the various types of microorganisms that produce antibiotics,
actinomycetes in general Streptomyces spp. in particular is effective against
Gram-positive bacteria and also inhibits Mycobacterium tuberculosis.

132

Actinomycetes,

mainly

Streptomyces

species

produce

oxytetracycline

(Zygmunt 1961), amino glycosides (Streptomycin and its relatives) macrolids

(erythromycin and its relatives) which are very active against a large number of
human pathogens (Weber 1985). Several species from the Streptomyces genus
produce bioactive molecules, including enzymatic inhibitors with antibiotic
activity and many commercially valuable enzymes such as lipases, cellulases
and proteases (Goodfellow et al 1983). These types of proteins have been
found to have application in the pharmaceutics, detergent and food industries as
well as in laboratory practice (Lyons 1988).
Biosynthesis of secondary metabolites during adverse condition is
very interesting phenomenon, since these are metabolically expensive and their
biosynthesis takes a significant amount of energy (Beppu et al 1992). Many of
the extra cellular proteinases obtained from Streptomycetes have been
characterized as metallo and serine proteinases (Kim et al 1996). It appears that
they involved in the nutrition, degradation of aerial mycelia and sporulation
processes. In addition, this group of actinomycetes is also able to synthesize
and excrete dark pigments, the melanin or melanoid pigments, which are
considered as useful tool in taxonomic studies (Mikami et al 1972). Therefore
the characterization of components secreted by actinomycetes may be useful in
many Biotechnological fields.
Cancer still represents one of the most serious human health
problems despite the great in understanding its biology and pharmacology. The
usual therapeutics methods for cancer treatment are surgery, radiotherapy,
immunotherapy and chemotherapy (Cocco et al 2003). These techniques are
individually useful in particular situations and when combined, they offer a
more efficient treatment for tumor (Izbika et al 1998). An analysis of the
number of chemotherapeutic drugs and their sources indicates that, over 60%
of approved drug are derived from natural compounds (Newman et al 2005)

133

and many have been extracted from actinomycetes. Anti-tumor antibiotics

produced by

actinomycetes

are

among

the

most

important

cancer

chemotherapeutics agents including members of the anthracycline, bleomycin,

actinomycin, mitomycin and aureolic acid families (Rocha et al 2001, Newman
and Cragg 2003).
Cancer is considered a public health problem in developed and
developing countries.

Despite the intense efforts to develop treatments,

effective agents are still not available. Therefore, it is of seminal importance to

provide new drug leads that may be developed into new cancer medicines with
antitumor activity. In the present study, after studying antifungal and
antibacterial activities, the crude extracts obtained from S.sannanensis strains
were subjected to evaluate their efficacy using human breast cancer cell lines
viz., SKBR3, MCF7 and MDA-MB231 (Figures 4.20 4.22). The results
clearly indicated that inhibition of cell growth of all the tested cell lines was
prominent when compared with standard Nolvadex drug. Mario Luiz Conte da
Frotaet al (2009) reported that, bioactive secondary metabolites obtained from
Streptomyces strains induces apoptic cell death in human U318MG glioma cell
line at a concentration of 10 and 50 g mL-1. Hood et al (2001) reported that,
the mycalamide A and pateamine metabolites induced the apoptosis even at
low concentration between 10-20

g mL-1 and the Ecteinascidin-743 an

alkaloid isolated from marine actinomyctes showed potential activity (Izbicka

et al 1998) but limited research have been focused with the anticancer property
of secondary metabolites from marine actinomycetes.
Noemi Antal et al (2005) reported that the retymicin, Galtamycin,
Saquayamycin Z and Ribofuranosyllumichrome from Micromonospora spp.
showed antitumor property against cancer cell lines. Liyan song et al (2008)
reported that, two different molecular weight proteins (8.2 kDa and 16.0 kDa)
identified from terrestrial actinomycetes exhibited antitumor activity against 7

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human cancer cell lines. Jorn Piel et al (2004) identified an antitumor

polyketide biosynthesis by an uncultivated bacterial symbiont of the terrestrial
actinomycetes.

Popov et al (2002) reported the cytotoxic and antitumor

activity of the imidazole alkaloid Polycarpin from the actinomycetes strains

and its synthetic analogues.
5.5

BIOSYNTHESIS

OF

NANOPARTICLES

USING

STREPTOMYCES SANNANENSIS ISOLATED FROM KOLLI

HILLS FOR BIOMEDICAL APPLICATIONS
Biosynthesis of gold, silver and copper nanoparticles was performed
using the efficient strain of S. sannanensis in the basal medium supplemented
with chloroauric acid (HAuCl4-), silver nitrate (AgNO3) and copper sulphate
(Cu (I) SO4.5H2O) as substrates; respectively. The characterization of the
silver, gold and copper nanoparticles synthesized was performed using
UV-Visible light spectroscopy in which the peak was observed at a wavelength
of 410 440 nm in case of AgNO3 treated samples, 520 550 nm in the case of
HAuCl4- treated samples and 530 580 nm for copper sulphate treated S.
sannanensis. The synthesis of nanoparticles was confirmed by monitoring the
formation of brownish yellow colour for silver and pink to purple colour for
gold nanoparticles and in the case of copper nanoparticles, pale yellow to
brownish colour was observed. UV-Vis spectroscopy is an important technique
to determine the formation and stability of metal nanoparticles in aqueous
solution. It is well known that nanoparticles exhibit different colours and size
due to excitation of surface plasmon resonance (SPR).
Similar to that of UV-Vis spectroscopy, FT-IR study is very much
essential and wide used technique to determine the chemical functional groups
present in the samples and the spectral analyses of silver, gold and copper
nanoparticles synthesized by S. sannanensis strains.The diethyl ether

135

phytochemical

fraction

reduced

gold

nanoparticles

shows

maximum

absorbance wavelength at 536 nm with different morphologies such as

spherical, triangle, truncated triangle and hexagonal shapes. The diethyl ether
phytochemical

fraction

reduced

gold

nanoparticles

shows

maximum

absorbance wavelength at 536 nm with different morphologies such as

spherical, triangle, truncated triangle and hexagonal shapes. Similar peak
values were observed correspondingly for S. sannanensis samples at 543nm,
520nm and 543 nm; respectively. FTIR analysis established the occurrence of
the amide (I) and (II) bands of the protein as capping and stabilizing agent on
the surface of the nanoparticles (Krishnasamy et al 2012) and it has been
analysed the proteins released into water by the S. sannanensis in terms of the
number of different proteins secreted and their molecular weights (Monica and
Ponmurugan 2012).
Baudot et al (2010), have successfully demonstrated the use of the
combination of an experimental FTIR analysis and a theoretical FTIR model to
characterize the functionalization of nano-sized objects. In the present FT-IR
study determination of the chemical functional groups present in the samples
and the spectral analyses of silver and gold nanoparticles synthesized by the
different antagonists were illustrated as well (Baudot et al 2010). Zhang et al
(2011), observed the absorption peak at 1650 cm-1 in gold nanoparticles
synthesized using chloroplasts there was N-H stretching of amide I, which was
characteristic of the stretch mode of the carbonyl group coupled to the amide
linkage. This suggested that proteins bind to GNPs through free amine groups.
Hence, the observation confirmed the secondary structure of the protein in the
sample which did not affect and got stabilized due to the presence silver and
gold nano particles. Bands showed the presence of various types of stretches
like amide C=O stretch and amide NH stretch which reveals the stabilization
of the structure of proteins.

136

The antibacterial activity of different nanoparticles biosynthesized

S. sannanensis by such as silver, gold and copper nanoparticles was carried out
using the different human bacterial pathogens such as S. paratyphi A,
S. paratyphi B, K. pneumonia, E. coli, P. aeuroginosa, S. marcescensand
S. aureus. The results revealed that among the different nanoparticles tested,
silver nanoparticles was found to be the best in terms of inhibition of growth of
human pathogens followed by gold and copper nanoparticles (Table 4.28).
Similarly, the biosynthesized silver, gold and copper nanoparticles from S.
sannanensis were also tested against the human breast cancer cell lines such as
SKBR3, MCF7 and MDA-MB231. The results indicated that growth inhibition
was the highest with gold followed by silver and copper nanoparticles in terms
of growth inhibition of cell lines (Table 4.29). It has been reported that
nanoparticle has been well known for its antibacterial and anticancerous
properties (Monica and Ponmurugan 2012).