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1. INTRODUCTION

[“The role of the infinitely small in nature is infinitely large”- Louis Pasteur.]

1.1 Marine environment

Nearly three quarters of the earth’s surface is comprised of the marine

environment and it can be considered as a storehouse of basically all conceivable types

of microbes (Konig & Wright, 1999). They may present in suspended form on

inanimate or animate surfaces as epibionts or as symbionts. Microorganisms are

intimately involved in ecological phenomena, e.g. settlement, biofouling and

metamorphosis as they play important roles in all the foremost elemental cycles which

occur in the oceans (Hawksworth, 1991). The marine environment is radically

distinctive in terms of its unique composition for both organic and inorganic

substances, as well as pressure conditions and temperature ranges. Ecological niches

like mangrove forests, deep-sea hydrothermal vents, sponge, algae, and fish supply

habitats for the assessment of specific microorganisms (Kohlmeyer, 1979). Many

research groups were motivated by the difficulties coupled with the collection of

marine macroorganisms and the insufficient amount of the isolated bioactive substance

(Edrada et al., 2000) to investigate the microbes linked with them, or those constituted

in the marine sediments or water columns (Konig & Wright, 1996). There are some

noticeable advantages while looking into microbes comparing with macroorganisms.

These comprise isolation of the compounds by large scale cultivation of the

microorganisms biotechnological fermentations with different parameters without

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ecological exploitation and microorganisms being easily genetically manipulated.

Based on this, marine microbes have emerged as a central topic for many groups

investigating natural products intending to find pharmaceutical drugs or compounds

valuable for agriculture (Osterhage, 2001).

1.2 Mangrove environment

Mangrove ecosystem is one of the world’s most productive ecosystem that

yields commercial forest products, enriches coastal waters, support coastal fisheries and

protect coastlines. Nevertheless, mangroves survive under extreme tides, condition of

high salinity, high temperature, strong winds, and muddy and anaerobic soils. No other

group of plants has been reported with such highly evolved ecological, morphological,

physiological and biological adaptations to extreme conditions.

Mangroves are the coastal wet land forests mainly found in the intertidal zone

of creeks, estuaries, back waters, marshes, deltas, lagoons and also mud flats of the

tropical and subtropical latitudes (Sahoo et al., 2009). The ecosystems where the

mangrove plants grow are termed as “Mangrove Ecosystem” which occupies millions

of hectors across the world coastal areas (Spalding et al., 1997; Alongi, 2002).

Mangrove marine ecosystems are largely unwrap source for screening and isolation of

new microbes with rich potential to produce the important active secondary bioactive

metabolites. The environment of the mangrove ecosystem is saline and highly rich in an

organic matter because of its various microbial enzymatic and metabolic activities

(Kizhekkedathu and Parukuttyamma, 2005). The products of natural origin remain to

be the most important source of antibiotics (Bull and Starch, 2007). Marine derived

compounds are more efficient in action against the pathogens that are resistant to the

existing antibiotics (Donia and Hamman, 2003). The risk undermining the health care

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system is because of the relentless and rapid spread of the multiple antibiotic resistant

pathogens causing life threatening infection (Talbot et al., 2006) and therefore the

demand for new antibiotics grows continually. Though considerable progress has been

made within the fields of chemical synthesis and engineered biosynthesis of

antibacterial compounds, nature remains the richest and the most versatile source for

new antibiotics (Baskaran et al., 2011). Since actinomycetes have an important proven

capacity to produce novel antibiotics (Bentley et al., 2002), the practice in screening

such organisms for the new bioactive compounds is continued (Berdy, 2005). However,

difficulty to discover the commercially potent secondary metabolites from well-known

Actinomycetes is becoming increasingly difficult due to the practice of wasteful

screening that is leading to rediscovery of the known bioactive compounds (Kui et al.,

2009). This stringent condition emphasizes the need to screen and isolate the

undiscovered representatives of the unexplored actinomycetes taxa. It is a clear object

that the mangrove ecosystem is a rich source of novel actinomycetes that have the

capacity to produce interesting new bioactive compounds including antibiotics.

Screening for the microbial species is an important aspect as there is a remarkable

source for the production of structurally diverse secondary metabolites that possess

pharmaceutically relevant biological activities (Berdy, 2005). It has long been an

observed fact that the search for the new secondary metabolites from microorganisms

in general has been confounded because different strains belonging to the same species

produce different types of secondary metabolites (Waksman and Bugie, 1943) but

identical secondary metabolites are produced by taxonomically diverse strains (Larsen

et al., 2005).

To differentiate among closely related actinomycetes contribute to the former

research. Sequence based approaches can now tackle the challenges that determine the

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taxonomic arrangements and provides opportunities to extract the relationship between

the groups of related strains and the secondary metabolites they produce. In addition,

this method is the tool to probe the evolutionary history of the metabolic pathways and

thus deduce the root and action of the LGT (lateral gene transfer) responsible for the

unrelated organisms to produce similar compounds (Paul et al.,) In view of the above

discussion, the present study is taken up to isolate, screen and characterize the

biologically diverse strains of actinomycetes from the mangrove sediment samples for

bioactive secondary metabolites. Taxonomic characterization was carried out based on

16S rRNA sequence analysis in combination with morphological, biochemical and

physiological data. The ability to produce antibacterial and antifungal compounds was

also investigated.

1.3 The Actinomycetes

Actinomycetes are Gram-positive bacteria with DNA rich in guanine and cytosine

(Urakawa, et al., 1999). They are unicellular filamentous microorganisms that branch

monopodially, more rarely dichotomously. These filaments can be either of a single

type called substrate or vegetative, or of two types, substrate and aerial. Some

Actinomycetes, like Mycobacterium, do not form mycelia and grow as pleomorphic or

coccoid elements. Due to their filamentous aspect, actinomycetes were thought to be

fungi, explaining the origin of the name actinomycetes which in Greek means “radiant

fungi”. Actinomycetes used to form a group on their own between the bacteria and the

fungi but in the 1950s, after investigation of their chemical composition and fine

structure, they were confirmed as prokaryotes and joined the bacterial domain.

Actinomycetes belong to the class Actinobacteria (Stackebrandt et al., 1997), order

Actinomycetales which includes 10 suborders and 30 families. This relatively recent

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Actinobacteria class was proposed based on the 16S rRNA analysis of hundreds of

actinomycete sequences.

1.3.1 Some of the general characters of Actinomycetes

1. Most of the Actinomycetes are chemoorganotrophic although some of them

grow on simple mineral media.

2. Cell wall has some peptidoglycan as in gram negative bacteria, but there is

variety in the peptidoglycan composition more than in gram negative bacteria.

3. Majority of them have a branched mycelia body.

4. The filaments is made up of several cells but in some, septa are absent and the

members of coenocytes.

5. Reproduction is by conidia borne on conidiophores. Endospore formation is

generally not seen.

6. Except for some (Actinomycetaceae), majority are aerobic.

7. They are not sheathed, stalked or photosynthetic.

8. They are prokaryotic and gram positive.

9. In certain families, filaments tend to break and fragmentation leads to coccoid

or elongate cells which develop into new individuals.

10. Some species are motile but most of them are not.

11. Cells have a diameter 0.5µ to 5µ. In some branched members the filaments may

be as long as several millimetres.

12. Most of them are saprophytic, widely distributed in organic matter in soil, dung

and marine and fresh waters. Some are pathogenic parasites (e.g.

Mycobacterium).

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1.3.2 Actinomycetes differ from true bacteria and fungi in the following aspects

1.3.2.1 Difference between Bacteria and Actinomycetes

1. Actinomycetes are not sheathed or photosynthetic while bacteria (at least some)

are.

2. Actinomycetes do not accumulate irons, sulphur or other free elements in or on

the cells while bacteria do.

3. Actinomycetes do not form endospores.

4. Actinomycetes have true branching, bacteria do not.

1.3.2.2 Differences between Actinomycetes and Fungi

1. Cell walls of Actinomycetes contain mucopolysaccharides and both muramic

and diaminopimelic acid as in bacteria while fungal cell walls are chitinous.

2. Actinomycetes are prokaryotic.

3. Actinomycetes are much smaller (1-5µ in diameter and not more than few µm

in length) than fungi (10-20 µ in diameter; mycelial length varies).

4. Sexual reproduction seen in fungi is absent in Actinomycetes.

1.3.3 Habitat of Actinomycetes

Actinomycetes are found in a wide range of habitats. They are present in the

frozen soils of polar regions and in the dry soils of deserts. They can be found in crude

oil, heavily metal contaminated soil and sediments and fresh and salt water

environments. They are not extremophiles and seem to be absent in highly acidic

(pH<1) and extremely hot (hot spring) environments. Actinomycetes are mostly

saprophytes though some can form parasitic or symbiotic associations with animals and

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plants. Selman Waksman in the early part of the 20th century contributed greatly to the

understanding of actinomycete ecology by publishing more than two hundred papers

and many books on the subject and established the predominance of actinomycetes in

soil (Waksman & Curtis, 1916, 1918). The techniques described in these studies along

with those from Stanley Williams (Williams et al., 1983, 1984) another major

contributor to the field of actinomycete ecology, are still in use in today’s laboratories.

In the last quarter of the 20th century, investigation of the marine environment such as

near-shore and deep-sea sediments, have revealed the presence of actinomycetes

(Jensen et al., 1991; Weyland, 1969, 1981). It is worth mentioning that despite the fact

that oceans cover 70% of the Earth surface and contain the most diverse ecosystems on

the planet, they have not been widely recognized as an important source for novel

actinomycetes. The distributions of actinomycetes in the marine environment and their

ecological roles remain largely undescribed. For a long time, the existence of

indigenous populations of marine actinomycetes was challenged. Actinomycetes

produce resistant spores that can remain viable but dormant for many years and it was

argued that the actinomycetes recovered from the marine environment were in fact the

result of spores from soil actinomycetes that had washed into the oceans.

This theory persisted despite evidence that actinomycetes can be recovered from

deep-sea sediments (Weyland, 1969) and that marine actinomycetes can be

metabolically active (Moran et al., 1995) and physiologically adapted to the salt

concentration encountered in the sea (Jensen et al., 1991; Mincer et al., 2002).

Rhodococcus marinonascens was the first actinomycete species that was described and

accepted as an autochthonous marine species. Mincer et al., (2002) studied 212

actinomycete isolates from a group called Mar 1. These bacteria were isolated from

geographically distant sediments collected from tropical or subtropical locations. The

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strains were differentiated by morphological characteristics like small-subunit rRNA

gene signature nucleotides and by an obligate requirement for sea water for growth.

Phylogenetic analysis of 16S rRNA gene sequences of seven strains showed that they

formed a monophyletic clade within the family Micromonosporaceae suggesting

novelty at the genus level. The Mar 1 strains were provisionally called ‘Salinospora’.

Later the taxon was formally named Salinispora, belonging to the family

Micromonosporaceae (Maldonado et al., 2005).

Actinomycetes are capable of producing several types of secondary metabolites and

being gram-positive bacteria they grow extensively in soils having profuse organic

matter (Henis, 1986 and Demain, 1999). The dispersion and presence of Actinomycetes

have been exhibited to be related with their different ecological habitats, including

seawater (Takizawa et al., 1993) and beach sand (Suzuki et al., 1994). Actinomycetes

capable of yielding antimicrobial compounds have been isolated from terrestrial

habitats as well as marine environments (Grein and Meyers, 1958.; Zobell and Upham,

1944) which suggests that Actinomycetes from marine sediments are a rich source of

natural bioactive compounds. Lately, new species and genera of marine Actinomycetes

species have been reported (Fenical and Jensen, 2006; Maldonado et al., 2005). The

present study was designed to evaluate various types of samples from different marine

environments as sources of Actinomycetes in order to screen for bioactive compounds.

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1.4 Rare Actinomycetes

Actinomycetes are widely distributed in natural and manmade environments

where they play an important role in the degradation of organic matter. Being well

known as a rich source of antibiotics and bioactive molecules they are of considerable

importance in industries. While applying conventional isolation techniques, most of the

isolates recovered on agar plates have been identified as genus Streptomyces, the

dominant actinomycetes found in soil. Different factors must be considered for the

function of screening novel bioactive molecules: choice of source of screening,

pretreatment procedure, selective media, the culture condition and identification of

candidate colonies on a primary isolation plate. Re-isolation of previously known

antibiotics strains is a major problem in new drug discovery. Less well studied

organisms such as non-streptomycete species (rare actinomycetes) provide attractive

opportunities for developing new antibiotics (Nareeluk N et al., 2009). The successful

discovery of novel rare actinomycetes needs ecological study of their distribution. New

methods for isolating them from diverse habitats and culturing them in the laboratory

are needed for such studies because complicated procedures for isolation and

cultivation are currently required (Lazzarini et al., 2000).

The role of rare actinomycetes as bioactive molecule sources became apparent

as these organisms provided about 25% of the antibiotics of actinomycete origin

reported during 1975 to 1980. Usually rare actinomycetes have been considered as

strains of actinomycetes whose isolation frequency by conventional methods is much

lower than that of streptomycete strains. Subsequently, employing pretreatments of soil

by drying and heating stimulated the isolation of rare actinomycetes. An alternative

approach was to make the isolation procedure more selective by adding chemicals such

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as phenol to the soil suspension. Many actinomycetes have shown multiple resistances

to wide ranges of antibiotics. Different antibiotic molecules were used in selective

medium to inhibit the competing bacteria including fast-growing actinomycetes.

Macromolecules such as hair hydrolysate, casein, humic acid and chitin and were

chosen as carbon and nitrogen sources for isolation of rare actinomycetes.

After isolating an actinomycete, it was initially identified on the basis of

morphological characters so as to have a preliminary determination of the genus.

Actinomycetes can be observed under the light microscope using coverslip culture

(Arifuzzaman et al., 2010, Khan et al., 2008), and slide culture techniques (Kavitha &

Vijayalakshmi, 2007). Strains are observed for several characters such as presence or

absence of aerial mycelium, fragmentation or non fragmentation of substrate and aerial

mycelium, presence of sclerotia, spore chain morphology and color of spore mass

(Kavitha &Vijayalakshmi, 2007). Genera of purified isolates can be identified based on

morphological comparisons to the existing description of known genera as given in

Bergey's Manual of Determinative Bacteriology. It is important to avoid strain

duplication by an accurate identification of isolates. However taxonomic

characterization based only on morphological and biochemical characteristics, is

tedious (Singh et al., 2009). There is a need to develop molecular methods that are used

in conjunction with the earlier techniques would help in differentiating between the rare

and common genera of Actinomycetes (Valenzuela-Tovar et al., 2005).

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Table 1. Summary of methods developed for the selective isolation of rare -actinomycetes from soil (1987-2007), (Masayuki Hayakawa 2008).

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1.5 Antimicrobial property of Actinomycetes

1.5.1 Antibacterial property of Actinomycetes

Infectious diseases are the leading cause of death worldwide which accounts for

13.3 billion deaths constituting about 25% of all deaths. Presently, resistance capacity

to the drugs used in the treatment and cure of many infectious diseases is increasing,

however microbial infections are being found to be responsible for more number of life

threatening diseases than previously thought. The reasons for the addition in incidence

of infectious disease are not properly understood. One of the reasons is the emergence

of multidrug resistant pathogens (Cassell and Mekalanos, 2001). Among the different

drug resistant pathogens, methicillin-resistant Staphylococcus aureus (MRSA),

vancomycin-resistant Staphylococcus aureus (VRSA), extended spectrum β-lactamases

(ESBL) producing bacteria such as E. coli, Klebsiella sp. and Pseudomonas aeruginosa

and multi drug resistant Mycobacterium tuberculosis (MDR-MTB) are of major

concern.

The demand for new antibiotics continues to grow due to the rapid emergence

of antibiotic resistant pathogens causing life threatening infections in spite of

considerable progress in the fields of chemical synthesis and engineered biosynthesis of

antimicrobial compounds. The changing pattern of diseases as well as the emergence of

resistant bacterial strains to currently used antibiotics continuously put demand on the

drug discovery scientists to search for novel antibiotics (Baltz, 2007).

Actinomycetes make important biogeochemical functions in terrestrial soils and

are highly assessed for their unique ability to produce biologically dynamic secondary

metabolites. Altogether 22,500 bioactive secondary metabolites have been reported and

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out of which 16,500 compounds show antibiotic activities. Out of the total 22,500

bioactive secondary metabolites, 45% (10,100) are reported to be produced by

actinomycetes in which 7630 from Streptomycetes and 2470 from rare actinomycetes.

A search for recent literature brought out that at least 4607 patents have been issued on

actinomycetes related products and processes (Berdy J. 2005).

1.5.2 Antifungal properties of Actinomycetes

Plant pathogens are estimated to cause yield reductions in crops of almost 20%

worldwide. Fungal diseases are among the main causes of low yields, viz. the effect of

a disease caused by Pyricularia grisea Sacc, reaches up to 80% due to its destructive

capacity (Fabregat, 1984; Cárdenas, 1999) under favorable conditions. Each year it is

estimated to destroy enough rice to feed more than 60 million people. The fungus is

known to occur in approximately 85 countries worldwide. The extensive use of

chemicals viz. fungicides can not be considered as an optimum solution because it

enhances the risk of the chemical pollution of the environment and agricultural

production.

An important study published by the US environment protection agency

indicates that in the US alone 3000-6000 cancer cases are induced annually by pesticide

residues on foods and another 50-150 by exposure to pesticides during application

(Goud, 2004).This type of findings increasingly put emphasis on drawbacks of many

chemical fungicides, pesticides in terms of their effect on the environment as well as on

the grower and consumer of agriculture products (Cool, 1993).Large demands for

fungicides exist in agriculture, food protection and medicine. In order to cope with the

needs of the fast-growing world population, yields must be improved by optimizing

inputs, including fungicides (Knight et al., 1997).

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Actinomycetes are known to have the capacity to synthesize bioactive

secondary metabolites which include enzymes, herbicides, pesticides, and antibiotics.

Almost 80% of the world’s antibiotics are known to come from actinomycetes, mostly

from the genera Micromonospora and Streptomyces (Pandey et al., 2004).

Actinomycetes are also used to control plant diseases. For example, Streptomyces strain

5406 has been used in China for more than 30 years now to protect cotton crops against

soilborne pathogens (Yin et al., 1965). One decade ago, Kemira Oy has developed a

biofungicide that contains living cells of S. griseoviridis Anderson, Erlich, Sun and

Burkholder to protect crops against Fusarium and Alternaria infections (Lahdenpera et

al., 1991).

Soil inoculation with specific streptomycete strains could significantly reduce

damages caused by Pythium or Phytophthora species in ornamental (Bolton 1980;

Malajczuk, 1983), legume (Filnow and Lockwood, 1985) and horticultural productions

(Crawford et al., 1993; Turhan and Turhan, 1989). Moreover, food quality has to be

guaranteed by controlling fungi that produce mycotoxins. Filamentous fungi can also

cause opportunistic systemic mycoses, associated primarily with patients with AIDS or

those receiving treatment with immunosuppressive agents.

Antifungal chemotherapy relies heavily on fungicides and many efforts have

been made to standardize test procedures in order to increase reproducibility between

laboratories (Cormican and Pfaller, 1996).

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Table 2. Some of the metabolites of Actinomycetes

Sl. No

Name References

1 Abamectin T. W. Miller et al.: Antimicrob. Agents Chemother. 15: 368 (1979) 2 G. Alber-Schoenberg et al.: J. Am. Chem. Soc. 103: 4216 (1981) 3 Aclarubicin T. Oki et al.: J. Antibiot. 28: 830 (1975) 4 Actaplanin M. Debono et al.: J. Antibiot. 37: 85 (1984) A. H. Hunt et al.: J. Org. Chem. 49: 635, 641 (1984) 5 Actinobolin T. H. Haskell et al.: Antibiot. Ann. 1958-1959: 505 6 Actithiazic acid E. Tejera et al.: Antibiot. Chemother. 2: 233 (1952) 7 Albomycin G. F. Gauze et al.: Novosti Med. Akad. Med. Nauk. (USSR) 23: 3

(1951) 8 Amdinocillin D. S. Reeves et al.: J. Antimicrob. Chemother. 3 (Suppl. B): 5 (1977) J. W. Krajewski et al.: J. Antibiot. 34: 282 (1981) 9 Amicetin A C. DeBoer et al.: J. Am. Chem. Soc. 75: 499, 5864 (1953) M. H. McKormich et al.: Antibiot. Chemother. 3: 718 (1953)

10 Amidinomycin S. Nakamura et al.: J. Antibiot. 14A: 103, 193 (1961) S. Nakamura et al.: Chem. Pharm. Bull. 9: 641 (1961)

11 6-Aminopenicillanic acid F. R. Batchelor et al.: Nature 183: 257 (1959) 12 Amoxicillin Beecham, series: Antimicrob. Agents Chemother. 1970: 407-430

Long et al.: J. Chem. Soc. (C), 1020 (1971) R. Sutherland et al.: Antimicrob. Agents Chemother. 1971: 411

13 Amphomycin B. Heinemann et al.: Antibiot. Chemother. 3: 1239 (1953) Bodanszky et al.: J. Am. Chem. Soc. 95: 2352 (1973) (Glumamycin)

14 Ampicillin G. N. Rolinson, S. Stevens: Brit. Med. J. 2: 191 (1961) Beecham, series: Brit. Med. J. 2: 193 (1961) Doyle et al.: J. Chem.Soc. 1440 (1962)

15 Ansamitocin E. Higashide et al.: Nature 270: 721 (1977) 16 Anthramycin M. D. Tendler et al.: Nature 199: 501 (1963)

W. Leimgruber et al.: J. Am. Chem. Soc. 87: 5791, 5793 (1965) N. Komatsu et al.: J. Antibiot. 33: 54 (1980)

17 Aplasmomycin Y. Okami et al.: J. Antibiot. 29: 1019 (1976) H. Nakamura et al.: J. Antibiot. 30: 714 (1977)

18 Augmentin C. P. Robinson et al.: Med. Actual. 18: 213 (1982) D. J. Weber et al.: Pharmacotherapy (Carlisle, Mass.) 4: 122 (1984)

19 Aureothricin H. Umezawa et al.: Japan Med. J. 1: 512 (1948) 20 Avoparcin M. P. Kunstmann et al.: Antimicrob. Agents Chemother. 8: 242 (1968) 21 Azalomycin F M. Arai et al.: J. Antibiot. 13A: 46 (1960)

M. Meguro et al.: Antibiot. Chemother. 12: 554 (1962) 22 Azithromycin S. C. Aronoff et al.: Antimicrob. Chemother. 19: 275 (1987) 23 Benzylpenicillin A. Fleming: Br. J. Exp. Pathol. 10: 226 (1929)

E. B. Chain et al.: Lancet II: 226 (1940) 24 Bicozamycin T. Miyoshi et al.: J. Antibiot. 25: 569 (1972)

T. Kamiya et al.: J. Antibiot. 25: 576 (1972) M. Nishida et al.: J. Antibiot. 25: 582, 594 (1972)

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25 Butirosin Woo et al.: Tetrahedron Lett. 2617, 2621, 2625 (1971) Dion et al.: Antimicrob. Agents Chemother. 2: 84 (1972)

26 Cactinomycin H. Brockmann, Grubhofer: Naturwiss. 36: 376 (1949) 27 Candicidin D Lechevalier et al.: Mycologia 45: 155 (1953) 28 Carbenicillin E. T. Kundsen et al.: Br. Med. J. 3: 75 (1967) 29 Carbomycin F. W. Tanner et al.: Antibiot. Chemother. 2: 441 (1952)

F. A. Hochstein, K. Murai: J. Am. Chem. Soc. 76: 5080 (1954) 30 Carumonam R. L. Then et al.: Chemotherapy 30: 398 (1984) 31 Cefetamet Pivoxil Takeda: Ger. Pat. (1977)

US Pat. (1987) 32 Cefotaxime R. Heymes et al.: Infection (Munich) 5: 529 (1977)

R. Weise et al.: Antimicrob. Agents Chemother. 14: 807 (1978) 33 Ceftezole T. Noto et al.: J. Antibiot. 29: 1058 (1976)

Fujisawa, series: Chemotherapy (Tokyo) 24: 619, 655, 671, 722, 1006 (1976)

34 Ceftriaxone R. Reiner et al.: J. Antibiot. 33: 783 (1980) M. Seddon et al.: Antimicrob. Agents Chemother. 18: 240 (1980) P. Angehrn et al.: Antimicrob. Agents Chemother. 18: 913 (1980)

35 Cephalexin Muggeleton et al.: Antimicrob. Agents Chemother. 353 (1968) Kind et al.: Antimicrob. Agents Chemother. 361 (1968)

36 Cephaloglycin Kurita et al.: J. Antibiot. 19A: 243 (1966) J. L. Spencer et al.: J. Med. Chem. 9: 746 (1966)

37 Cephalosporin C G. Brotzu: Lay. Ist. Igiene Cagliari (1948) G. G. F. Newton, E. Abraham: Nature 175: 548 (1955)

38 Chloramphenicol J. Ehrlich et al.: Science 106: 417 (1947) G. Keiser: Dtsch. Med. Wochensch. 96: 1544 (1971)

39 Coumermycin H. Kawaguchi et al.: J. Antibiot. 18A: 1, 11, 220 (1965) 40 Cycloheximide A. J. Whiffen et al.: J. Bacteriol. 52: 610 (1946)

B. E. Leach et al.: J. Am. Chem. Soc. 69: 474 (1947) 41 Cyclosporin A A. Ruegger et al.: Helv. Chim. Acta 59: 1075 (1976) 42 Dactinomycin S. A. Waksman et al.: Proc. Soc. Exp. Biol. Med. 45: 609 (1940)

Manaker et al.: Antibiot. Ann. 1954-55: 853 43 Dermostatin M. J. Thirumalachar, S. K. Menon: Hindustan Antibiot. Bull. 4: 106

(1962) D. S. Bhate et al.: Hindustan Antibiot. Bull. 4: 159 (1962) R. C. Pandey et al.: J. Antibiot. 26: 475 (1973) revised str.

44 Detoxin H. Yonehara et al.: J. Antibiot. 21: 369 (1968) 45 Dicloxacillin C. Gloxhuger et al.: Arzneimittel-Forsch. 15: 322 (1965)

H. Yoshioka et al.: J. Antibiot. B 20: 34 (1967) 46 Dirithromycin P. Luger, R. Maier: J. Cryst. Mol. Struct. 9: 329 (1979)

F. T. Counter et al.: Antimicrob. Agents Chemother. 15: 1116 (1991) 47 Doxorubicin F. Arcamone et al.: Tetrahedron Lett. 1007 (1969) 48 Erythromycin

Estolate V. C. Stephens et al.: J. Am. Pharm. Assoc. Sci. Ed. 48: 620 (1959)

49 Erythromycin J. M. McGuire et al.: Antibiot. Chemother. 2: 281 (1952) 50 Floxacillin R. Sutherland et al.: Br. Med. J. 4: 455 (1970) 51 Fungichromin M. C. McCowen et al.: Science 113: 292 (1951)

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52 Fusidic acid W.O. Godtfredsen et al.: Nature 193: 987 (1962) 53 Gentamicin M. J. Weinstein et al.: Antimicrob. Agents Chemother. 1 (1963)

J. Black et al.: Antimicrob. Agents Chemother. 138 (1964) D. J. Cooper et al.: J. Chem. Soc. (C), 960, 2876, 3126 (1971)

54 Gibberelic acid P. J. Curtis, B. E. Cross: Chem. & Ind. 1066 (1954) B. E. Cross: J. Chem. Soc. 4670 (1954)

55 Gusperimus H. Iwasawa et al.: J. Antibiot. 35: 1665 (1982) Y. Umeda et al.: J. Antibiot. 38: 886 (1985)

56 Griseofulvin A. E. Oxford et al.: Biochem. J. 33: 240 (1939) J. F. Grove et al.: J. Chem. Soc. 3977 (1952)

57 Herbimycin S. Omura et al.: J. Antibiot. 32: 255 (1979) S. Omura et al.: Tetrahedron Lett. 4323 (1979) A. Furusaki et al.: J. Antibiot. 33: 781 (1980)

58 Hetacillin Hardcastle et al.: J. Org. Chem. 31: 897 (1966) Ueda et al.: J. Antibiot. 20B: 206 (1967)

59 Idarubicin F. Arcamone et al.: Cancer Treat. Rep. 60: 829 (1976) (Carminomycin I)

M. J. Broadhurst et al.: J. Chem. Soc, Chem. Commun. 158 (1982) 60 Imipenem-Cirastatin F. M. Kahan et al.: J. Antimicrob. Chemother. 12: Suppl. D, 1-35

(1983) Merck, series: J. Antimicrob. Chemother. 12: Suppl. D, 1-155 (1983) J. Birnbaum et al.: Am. J. Med. 78: Suppl. 6A, 3-21 (1985)

61 Isepamicin T. L. Nagabhushan et al.: J. Antibiot. 31: 681 (1978) 62 Josamycin T. Osono et al.: J. Antibiot. 20: 174 (1967) 63 Kanamycin H. Umezawaet al.: J. Antibiot. A10: 181 (1957) 64 Kasugamycin H. Umezawa et al.: J. Antibiot. 18A: 101 (1965) 65 Kitasamycin T. Hata et al.: J. Antibiot. 6A: 87 (1953)

Y. Sano et al.: J. Antibiot. 7A: 93 (1954) 66 Lactacystin S. Omura et al.: J. Antibiot. 44: 113, 117 (1991) 67 Laidlomycin F. Kitame et al.: J. Antibiot. 27: 884 (1974)

F. Kitame et al.: J. Antibiot. 29: 759 (1976) R. D. Clark et al.: J. Antibiot. 35: 1527 (1982) R. D. Clark et al.: J. Antibiot. 39: 1765 (1986)

68 Lenampicillin F. Sakamoto et al.: Chem. Pharm. Bull. 32: 2241 (1984) 69 Leptomycin T. Hamamoto et al.: J. Antibiot. 36: 639, 646 (1983) 70 Lincomycin D. J. Mason et al.: Antimicrob. Agents Chemother. 555 (1962) 71 Lividomycin T. Mori et al.: J. A antibiot. 24: 339 (1971) 72 Macbecin S. Tanida et al.: J. Antibiot. 33: 199 (1980) 73 Maduramicin C. M. Liu et al.: J. Aantibiot. 36: 343 (1983) 74 Maridomycin H. Ono et al.: J. Antibiot. 26: 191 (1973) 75 Micronomicin R. Okachi et al.: J. Antibiot. 27: 793 (1974)

R. S. Egan et al.: J. Antibiot. 28: 29 (1975) 76 Mycobactin P Francis et al.: Nature 163: 365 (1949)

Francis et al.: Biochem. J. 55: 596 (1953) 77 Nanaomycin S. Omura et al.: J. Antibiot. 27: 363 (1974)

H.Tanaka et al.: J. Antibiot. 28: 860, 868, 925 (1975)

18

78 Nocardicin H. Aoki et al.: J. Antibiot. 29: 492 (1976) M. Hashimoto et al.: J. Antibiot. 29: 890 (1976)

79 Nogalamycin B. K. Bhuyan et al.: Antimicrob. Agents Chemother. 836 (1965) 80 Paromomycin Parke Davis: US Pat. 2,916,485 (1959)

Pfizer: US Pat. 2.895,876 (1959) 81 Pecilocin S. Takeuchi et al.: J. Antibiot. 12A: 109, 195 (1959)

S. Takeuchi et al.: J. Antibiot. 17A: 267 (1964) 82 Piperacillin T. Saito et al.: Jap. J. Antibiot. 30: 835 (1977)

K. Ueo et al.: Antimicrob. Agents Chemother. 12: 455 (1977) 83 Pivampicillin von Daehne et al.: J. Med. Chem. 13: 607 (1970)

von Daehne et al.: Antimicrob. Agents Chemother. 1970: 430 84 Quatrimycin McCormick et al.: J. Am. Chem. Soc. 79: 2849 (1957)

Kaplan et al.: Antibiot. Chemother. (Basel) 7: 569 (1957) 85 Quinacillin Richards et al.: Nature 199: 354 (1963) 86 Rifamycin Maggi et al.: Chemotherapia 11: 285 (1966)

P. Sensi et al.: Antimicrob. Agents Chemother. 699 (1967) 87 Roxithromycin R. N. Jones et al.: Antimicrob. Agents Chemother. 24: 209 (1983) 88 Salinomycin H. Kinashi et al.: Tetrahedron Lett. 4955 (1973)

Y. Miyazaki et al.: J. Antibiot. 27: 814 (1974) 89 Streptolydigin T. E. Eble et al.: Antibiot. Ann. 1955-1956: 893

K. L. Rinehart et al.: J. Am. Chem. Soc. 85: 4083 (1963) 90 Streptomycin A. Schatz, E. Bugie, S. A. Waksman: Proc. Soc. Exp. Biol. Med. 55: 66

(1944) S. A. Waksman: Streptomycin. The Williams and Wikins Co.

Baltimore (1949) 91 Streptovaricin P. Siminoff et al.: Am. Rev. Tuberc. Pulm. Dis. 75: 576 (1957) 92 Tazobactam M. R. Jacobs et al.: Antimicrob. Agents Chemother. 29: 980 (1986) 93 Tetracycline J. H. Boothe et al.: J. Am. Chem. Soc. 75: 4621 (1953)

L. H. Conover et al.: J. Am. Chem. Soc. 75: 4622 (1953) 94 Tetracycline,

Demethylchlor- J. R. D. McCormick et al.: J. Am. Chem. Soc. 79: 4561 (1957)

95 Thienamycin J. S. Kahan et al.: Abstracts Papers of 16th Intersci. Conf. Antimicrob. Agents

96 Tobramycin W. M. Stark et al.: Antimicrob. Agents Chemother. 314-348 (1967) 97 Tunicamycin A. Takatsuki et al.: J. Antibiot. 24: 215 (1971) 98 Ubenimex H. Umezawa et al.: J. Antibiot. 29: 97 (1976)

H. Suda et al.: J. Antibiot. 29: 100 (1976) 100 Vernamycin B F. Benazet et al.: Therapeutique 38: 13 (1962) (Pristinamycin) 101 Viridin Brian, McGowan: Nature 156: 144 (1945) 102 WF-11899 T. Iwamoto et al.: J. Antibiot. 47: 1084, 1092 (1994) 103 WS-6897 M. Iwami et al.: J. Antibiot. 40 (5): 589 (1987)

S. Kiyoto et al.: J. Antibiot. 40 (5): 594 (1987) 104 Zearalenone Stob et al.: Nature 196: 1318 (1962)

Urry et al.: Tetrahedron Lett. 3109 (1966) 105 Zearanol, Zearalanol Sharp et al.: J. Anim. Sci. 34: 176 (1972) 106 Zorubicin R. Maral et al.: Comt. Rend. Ser. D. 273: 301 (1972)

19

2. AIMS AND OBJECTIVES

Isolation of Actinomycetes from Marine environment.

Isolation of Marine rare Actinomycetes by Pre treatment of sediment samples.

Morphological and Biochemical studies of isolated Actinomycetes.

To study Antimicrobial activity of Marine Actinomycetes.

Isolation and partial screening of bioactive molecules from selected

Actinomycetes.

Molecular studies and Identification of selected Marine Actinomycetes by

16S rRNA sequencing.

20

3. REVIEW OF LITERATURE

3.1 Natural habitat and Isolation of Actinomycetes

Weyland. H. (1969) opined that Bacteria belonging to the family

Actinomycetaceae are well known for their ability to produce secondary metabolites,

some of which are active against pathogenic microorganisms. Traditionally, these

bacteria have been isolated from terrestrial sources although the first report of

mycelium-forming actinomycetes being recovered from marine sediments.

A study was done by Paul R. Jensen et al., (2005) on natural product discovery

to characterize marine actinomycete diversity and how adaptations to the marine

environment affect secondary metabolite production. It would create a better

understanding of the possible utility of these bacteria as a source of useful products for

biotechnology. In Marinophilus and Salinospora strains, there appears to be a

correlation between phylogeny and biosynthetic capacity.

Abou-elela et al., (2005) worked on phenotypic characterization and numerical

taxonomy of some actinomycetes strains isolated from Burullos Lake. Twenty nine

actinomycetes isolates randomly selected of 130 from Burullos Lake were investigated.

These isolates were characterized taxonomically for 63 phenotypic characters including

morphological, biochemical, nutritional, substrate utilization and anti-microbial

analyses. A representative strain from sample site was chosen, they were identified as

21

Streptoverticillum morookaense, Nocardiabrasiliensis, Streptomyces alanosinicus,

Streptomyces globosus and Streptomyces gancidicus.

Jensen et al., (2005) reported that major populations of marine Actinomycetes

reside in ocean sediments and that these bacteria display highly evolved marine

adaptations including the requirement of seawater for growth. These findings will

hopefully encourage additional studies addressing the ecological roles of

Actinomycetes in the marine environment, their diversity, distributions, culture

requirements, and evolutionary responses to life in the sea.

A study was conducted by Crawford et al., (1993) on the use of selective media

where they included 267 actinomycete strains that were isolated from rhizosphere-

associated and non-rhizosphere-associated British soils. For isolating diverse group of

actinomycetes the organic media which has low nutrient concentrations were found to

be very good by avoiding contamination and overgrowth of isolation media by

eubacteria and fungi. All isolates grew well at the pH range of 6.5 to 8.0 and only some

strains were unable to grow at pH 6.0. A significant number of isolated strains failed to

grow at the pH 5.5.

Luis A. Maldonado and Fragoso-Yanez (2009) Reported Seventeen different

media known to support the growth and isolation of members of the class

Actinobacteria were evaluated as selective isolation media for the recovery of this

microbial group from marine sediments samples collected in the Gulf of California and

the Gulf of Mexico. Complete 16S rRNA gene sequencing revealed that the isolates

belonged to several actinobacterial taxa, notably to the genera Actinomadura, Dietzia,

Gordonia, Micromonospora, Nonomuraea, Rhodococcus, Saccharomonospora,

Saccharopolyspora, Salinispora, Streptomyces, ‘‘Solwaraspora’’ and Verrucosispora.

22

Previous works on marine sediments have been restricted to the isolation of members

of the genera Micromonospora, Rhodococcus and Streptomyces. This study provides

further evidence that Actinobacteria present in marine habitats are not restricted to the

Micromonospora- Rhodococcus-Streptomyces grouping. Indeed, this first systematic

study shows the extent of actinobacterial diversity that can be found in marine

sediments collected in Mexico and probably worldwide.

Streptomycete growth in nonsaline media was reduced by 39% compared with

that in seawater. The actinoplanetes had a near obligate requirement of seawater for

growth, and this is presented as evidence that actinomycetes can be physiologically

active in the marine environment Jensen, (1991).

A study was conducted by Vijayakumar et al., (2007) by the collection of 192

actinomycetes. Colonies were isolated from 18 marine sediment samples of Palk Strait

region of Bay of Bengal, India. Among those colonies, 68 isolates were

morphologically distinct on the basis of colour of spore mass, aerial and substrate

mycelia formation, production of diffusible pigment, sporophore morphology and

reverse side colour. Thirty-nine isolates were assigned to the genus Streptomyces (1),

Actinopolyspora (10), Saccharopolyspora (7), Actinomadura (4), Nocardiopsis (3),

Micromonospora (2), Actinomyces (1), Actinoplanes (1) and Microbispora (1). From

these, 64 isolates with aerial mycelia, 65 isolates with substrate mycelia and 61 isolates

had both aerial and substrate mycelia.

Actinomycetes population from continental slope sediment of the Bay of Bengal

was studied by Das et al., (2008). The diverged range of actinomycete population is

from 5.17 to 51.94 CFU/g and 9.38 to 45.22 CFU/g dry sediment weight. From stations

in 1000 m depth no actinomycete colony was isolated. Populations in stations in 500 m

23

depth in were higher than that of 200 m depth stations. Three actinomycetes genera

were identified. The found Streptomyces was the dominating one which was followed

by Micromonospora and Actinomyces. Spiral spore chain showed the maximum

abundance and the spore surface was smooth.

Actinomycetes population has been identified as one of the major groups of the

soil population Kuster, (1968). They are gram-positive organisms and assumed to be

the transition group between fungi and bacteria. They belong to the order

Actinomycetales (Super kingdom: Bacteria, Phylum: Firmicutes, Class:

Actinobacteria, Subclass: Actinobacteridae). According to Bergey's Manual

actinomycetes are divided into eight diverse families: Actinomycetaceae,

Mycobacteriaceae, Actinoplanaceae, Frankiaceae, Dermatophilaceae, Nocardiaceae,

Streptomycetaceae, Micromonosporaceae Holt, (1989) and they comprise 63 genera

Nisbet and Fox, (1991). Based on 16s rRNA classification system they have recently

been grouped in ten suborders: Actinomycineae, Corynebacterineae, Frankineae,

Glycomycineae, Micrococineae, Micromonosporineae, Propionibacterineae,

Pseudonocardineae, Streptomycineae and a large members of actinomycetes are still

remained to be grouped (www.ncbi.nlm.nih.gov).

Vijayakumar and Remya. (2008) studied 173 actinomycetes colonies which

were isolated from near shore marine environment and mangrove ecosystem at 8

different locations of Kerala, West Coast of India. Among them, 64 isolates were

morphologically distinct on the basis of spore mass colour, reverse side colour, aerial

and substrate mycelia formation and production of diffusible pigment. The majority

(47%; n=30) of these isolates were assigned to the genus Streptomyces.

24

The concept of biocontrol of plant diseases includes disease reduction or

decrease in inoculums potential of a pathogen brought about directly or indirectly by

other biological agencies Johnson and Carl, (1972). Outside the host, the biocontrol

agent may be antagonistic and thereby reduce the activity, efficiency and inoculums

density of the pathogen through antibiosis, competition and predation/hyper parasitism.

This leads to a reduction in inoculum potential of the pathogens Baker, (1977). The

biocontrol agent may operate primarily in the host tissue, there by indicating a

resistance response in the host, by transmitting factors that render the pathogens

avirulent Cook and Baker, (1983). These interactions are mediated by environment and

may have an overriding impact in determining whether biocontrol operates in a system

or not.

3.2 Antimicrobial properties and Identification of Actinomycetes

Kathiresan et al., (2005) Reported 160 isolates of marine Actinomycetes were

isolated from the sediment sample drawn from mangroves, estuary, sand dune, and

industrially polluted coast. Of these, mangrove sediments were rich sources of marine

Actinomycetes. Each isolate was tested against four phytopathogenic fungi, viz.

Pyriculariaoryzae, Rhizoctoniasolani, Helminthosporiumoryzae (causing blast, sheath

blight and leaf spot diseases of rice) and Colletotrichum falcatum (causing red rot

disease of sugar cane). The isolates appeared to produce high antifungal compounds at

120 hrs of incubation period of production medium culture. Glucose and soybean meal

were the best carbon and nitrogen sources, respectively and 17.5 ppt was the best

salinity level for maximum antibiotic production. Cylinder plate method was better for

antifungal assay than the disc diffusion method. Based on the morphological and

culture characteristics, the potent strains were identified as the species belonged to the

25

genus Strepomyces. These strains may prove to be the potent source for isolation of

agro based fungicides.

Lam (2006) reported that marine actinomycetes produce different types of new

secondary metabolites. Many of these metabolites possess biological activities and have

the potential to be developed as therapeutic agents. Marine actinomycetes are a prolific

but underexploited source for the discovery of novel secondary metabolites.

Hong et al. (2009) conclude that actinomycetes isolated from mangrove habitats

are a potentially rich source for the discovery of anti-infection and anti-tumor

compounds, and of agents for treating neurodegenerative diseases and diabetes.

A study was done by Debananda S. Ningthoujam et al. (2009) on screening of

Actinomycete isolates. From niche habitats in Manipur for Antibiotic Activity from 172

lake sediment (SCNA, LS1series), 35 lake sediment (CA, LSCH series), 120 river

(NRP, NRB and….Series), 39 forest (AML series), 35 cave (KC1 series), 101 salt

spring (NH, N3S and …..Series), 46 Shirui jungle (SJ series) and 66 Shirui hill (SH

series) actinomycetes isolates were obtained. About 18 potent antibacterial, 1 anti

pseudomonas, 1exclusively antifungal and 3 broad-spectrum antimicrobial

actinomycetes were chosen for further studies.

A study was done by Jeffrey et al., (2008) on actinomycetes and screening

results indicate that 48, 46 and 41 numbered isolates showed the ability to secrete the

enzyme cellulase, lipase and protease respectively. By the selection of phytopathogens

as test strains, antimicrobial test was done and it was observed that four isolates 3, 25,

35 and 37 showed antagonistic reaction with Fusarium palmivora, Pantoae dipersa,

26

Bacillus subtilis and Ralstonia solanacearum respectively. The most promising six

isolates were selected and identified by their 16SrRNA sequence.

Vellar Estuary was investigated by Dhanasekaran et al., (2009) as a source of

actinomycetes to screen for the production of novel bioactive compounds. Insignificant

variation was shown by the physiochemical characteristics of soils samples in

temperature, pH and dissolved phosphate, and total variation was shown by nitrogen

compounds and organic matter. Among the 20 actinomycetes isolate, some of the very

putative antibiotics producing actinomycetes were isolated which were strongly

inhibiting the growth of both Gram positive and Gram-negative bacteria including yeast

like fungi. Only 4 isolates exhibited the antimicrobial activity and only the strain

DPTD-5 showed broad-spectrum activity and was further characterized and identified.

Earlier studies done by Joe D' Souza and Nelson De Souza (2000) proved that estuarine

soils are rich in actinomycetes and they can produce antibiotics. The samples from

estuary were reported as very rich habitat for the microbial diversity. After this, the

morphological, physiological characterization and its DNA homology suggested that

the strain DPTD-5 was very similar to Streptomyces bikiniensis.

A study was done by Nathan et al., (2004) which recommended a unique

selective enrichment procedure resulted in the identification and isolation of two new

genera which were marine-derived actinobacteria. By this study it was revealed that

approximately 90% of the microorganisms were cultured by using the presented

method which was from the prospective new genera, it indicated as a result of its high

selectivity. From the Bismarck Sea and the Solomon Sea off coast of Papua New

Guinea, 102 actinomycetes were isolated from the subtidal marine sediment. Among

the isolated actinomycetes two new genera (represented by strains of the PNG1 clade

27

and strain UMM518) within the family Micromonosporaceae, showed activities against

multidrug-resistant gram-positive pathogens, vaccinia virus replication and malignant

cells.

A study conducted by Arora et al., (2005) on actinomycete strain Streptomyces

griseus B1, isolated from soil showed that when it was grown on cellulose powder as

submerged culture it produced high levels of all the three components i.e. filter paper

lyase (FPase), CM-Cellulase and β-glucosidase of the cellulolytic enzyme system.

Extracellular activity was shown by FP activity and CMCellulase whereas

β-glucosidase was both intra- and extra-cellular. When grown on hardwood powder

under submerged culture it showed highest FPase activity. It was not able to use lignin

monomers (ferulic acid, syringic acid and vanillic acid) as carbon source. When it

grows on hardwood and softwood powders under solid-state conditions, it depleted the

cellulose (36.3% in the case of softwood and 14.4% in the case of hardwood). And it

also caused partial loss of lignin content in both the substrates by solubilizing them.

Mangamuri et al. (2012) reported that Pseudonocardia species VUK-10 strain checked

against the pathogens of Staphylococcus aureus MTCC 3160, Streptococcus mutans

MTCC 497, Bacillus subtilis ATCC 6633, E.coli ATCC 35218, Enterococcus faecalis

MTCC 439, Pseudomonas aeruginosa ATCC 9027, Candida albicans ATCC 10231,

Fusarium oxysporum MTCC 3075, Aspergillus niger and reported that this strain

inhibited growth of Gram positive and Gram negative bacteria, yeast and fungi

suggesting a broad spectrum nature of the active compound.

28

Oskay et al., (2004) worked on antibacterial activity of some Actinomycetes

isolated from farming soils of Turkey worked on 50 different actinomycete strains were

recovered from farming soil samples collected from Manisa Province and its

surrounding. These were then assessed for their antibacterial activity against four

phytopathogenic and six pathogenic bacteria. Results indicated that 34% of all isolates

are active against, at least, one of the test organisms; Agrobacterium tumefaciens,

Erwiniaamylovora, Pseudomonas viridiflova, Clavibactermichiganensis subsp.

michiganensis, Bacillus subtilis ATTC 6633, Klebsiellapneumoniae ATTC 10031,

Enterococcus feacalis ATCC 10541, Staphylococcus aureus ATCC 6538, Esherichia

coli ATCC 29998 and Sarcinalutea ATCC 9341. According to antibacterial activity

and spectrum broadness, seven of the isolates were selected and characterized by

conventional methods. The unusual antibiotic profile of these isolates underlined their

potential as a source of novel antibiotics.

Nakashima et al., (2005) worked on the Actinomycetes as host cells for

production of recombinant proteins and reported that the host-vector systems of

Actinomycetes are suitable for expressing proteins of Actinomycetes and proteins from

closely related organisms as well as from higher eukaryotes. However, further

development of host-vector system in Actinomycetes is required, particularly with

respect to the modification of host cells.

In a study performed by Baskaran et al., (2011), various pre-treatment methods

and three different media were employed for the isolation of bioactive actinomycetes

from mangrove sediments of Andaman and Nicobar Islands, India. Collection of

sediment sample was done from four different sites of mangrove forest and pre-treated

by dry heat method, after that the media were supplemented with nalidixic acid 75

29

µg/mL and cycloheximide 80 µg/mL. In sediment samples, the mean actinomycetes

population density were recorded as 22 CFU-10-6/gm in KUA medium followed by 12

CFU-10-6/gm in AIA medium and 8 CFU-10-6/gm in SCA medium. Total 42

actinomycetes strains were isolated. All the isolates were evaluated for their

antibacterial activity against pathogenic bacteria on two different media. Among all the

tested isolates, antibacterial metabolite production was shown by 22 species. They were

tested against test bacteria namely, Bacillus subtilis, Staphylococcus aureus, Klebsiella

pneumoniae and Salmonella typhi. The strain A107 was identified as Streptomyces spp.

which had shown the maximum activity against all used pathogens.

Several antibiotics were tested by Williams and Davies, (1964) against a range

of actinomycetes, fungi and bacteria representing types found in soil. From these tests

four antibiotics polymyxin-B sulphate (5.0 pg/ml), actidione (50 pg/ml), nystatin (50

pg/ml.) and sodium penicillin (1.0 µg/ml) were selected for incorporation into a starch

casein medium to achieve selective growth of actinomycetes on soil dilution plates. The

antifungal ones (nystatin, actidione) did not inhibit any strains not even at the highest

concentration of 100µg/ml among all the seven antibiotics tested against a range of

actinomycetes. Same types of results were obtained by Porter et al. (1960). Polymyxin-

B sulphate has shown the least inhibition by antibacterial antibiotics. Most appropriate

mixture for the enumeration of soil actinomycete colonies was starch casein medium

with the two antifungal antibiotics (nystatin, actidione). And for isolation of

actinomycete colonies the use of same medium with all four antibiotics was most

satisfactory.

Actinobacteria producing bioactive compounds were isolated by Kumar et al.,

(2011) by the serial dilution method from marine sediments collected from Bay of

30

Bengal at a depth of 10-40m near pudimadaka coast of Andhra pradesh. During the

study total 78 isolates were obtained and among all the isolates Streptomycetes is

predominant. Among all the 78 isolates antibacterial and antifungal activity exhibited

by 22 isolates exhibited antibacterial and antifungal activity, respectively. Promising

activities were shown by the active isolates viz. BTS-112, BTS-314 and BTS-401.

After this the strains were further characterized and identified to be belonging to the

genus Rhodococcus and Streptomyces.

A total of 55 actinomycetes isolated from soil sample of Karanjal region in

Sundarbans were characterized by Arifuzzaman et al., (2010) for morphological

recognition as well as antimicrobial activity. The total numbers of isolates were 14, 11,

27, and 3 which belong to Nocardia, Streptomyces, Actinomyces and Micromonospora

respectively, as they were identified from the sample. Against one or more gram

negative pathogenic bacteria such as Shigella dysenterriae type-1, Shigella boydii,

Shigella sonnei, Pseudomonas, Vibrio cholerae-0139, Salmonella typhi-Ao-12014,

Shigella flexneri-AN-31153,Plesiomonas, Hafnia spp., Escherichia coli- 186LT and

Vibrio cholerae-OGET, twenty actinomycetes isolates were found which could produce

antibiotics. A varied group of actinomycetes were found in sundarbans soil and among

them three of the tested isolates had a broader spectrum antibacterial activity that

showed there potential as a source of antibiotics for pharmaceutical interest.

A study was performed by the Santhi et al., 2010 by the assortment of total of

two marine actinomycetes isolated from different locations of the Manakudi Estuary in

Arabian Sea of Tamilnadu, India. All the isolated strains exhibits higher antagonistic

activity against the Gram positive bacteria; methicillin resistant Staphylococcus aureus,

Salmonella typhi, Enterobacter sp, Bacillus subtilis, Proteus vulgaris and Klebsiella

31

pneumoniae. Intermediate activity was shown by them against Gram negative organism

Pseudomas auregionosa and it shows no antagonistic effect towards yeast like Candida

albicans. Pink colored actinomycetes (PJS) with white aerial mycelium and pink

substrate mycelium and black colonies (BJS) of white aerial mycelium and yellowish

white substrate mycelium shows potent inhibiting effect of other microorganisms. And

after that 16S rDNA phylogenetic typing gave ~1500 bp amplified product and it was

cloned in pGEMT easy vector. The sequencing of amplified product will give the

phylogeny of isolated actinomycetes and the further study on this organism may

provide a new antibiotic for the welfare of human being.

A total of 94 actinomycete strains were isolated by the You et al., 2005 from the

marine sediments of a shrimp farm, 87.2% belonged to the genus Streptomyces, others

were Micromonospora spp. Fifty-one percent of the actinomycete strains among them

showed activity against the pathogenic Vibrio spp. strains. Almost thirty-eight percent

of marine Streptomyces strains produced siderophores on chrome azurol S (CAS) agar

plates. From the total strains seven strains of Streptomyces were found to produce

siderophores and they inhibit the growth of Vibrio spp. in vitro. Two strains belonged

to the Cinerogriseus group, which was the most frequently isolated group of

Streptomyces. From the obtained results it could be assumed that in aquaculture it

could be use as biocontrol agent.

A study was performed by Imeda (2005) on several enzyme-inhibitor-producing

actinomycetes isolated from various samples collected from the marine environment

and were characterized. Among them it was found that they could produce novel

compounds which were useful in medicine and agriculture. From neurotic sea water a

strain of actinomycetes was isolated and characterized which produced antibiotics

32

against gram positive bacteria only in the presence of sea water. And the production of

antibiotics was observed at seawater concentrations ranging from 60 to 110% (v/v).

Hence, the production was seawater-dependent. They showed the production of

tetrodotoxin (TTX), it was known otherwise as puffer fish toxin, and it was investigated

in various actinomycetes collected from the marine environment. Among the 10

isolates from various regions of sea, 9 produced TTX as judged by their retention times

on high performance liquid chromatography (HPLC).

A study was done by Thenmozhi and Kannabiran (2010) to screen the

antifungal activity of the crude extract prepared from the 8 strain of Streptomyces spp.

isolated from the Puducherry coast of India. Primarily, eight strains were screened

against three species of Aspergillus namely A. fumigatus, A. flavus and A. niger for

antifungal activity. This search resulted in the isolation of a potential strain VITSTK7.

The optimization was done by the production media for the maximum yield of

secondary metabolites. And the metabolites were extracted using ethyl acetate; it is

than lyophilized and screened for antifungal activity against the three Aspergillus

species by well diffusion method. Maximum zone of inhibition observed was 21mm for

A. fumigatus in comparison with the standard antifungal antibiotic Nystatin which

shows 20 mm. The molecular taxonomy and phylogeny revealed that the strain

belonged to the genus Streptomyces. The sequence of 16s rRNA of the strain

Streptomyces spp.VITSTK7 was submitted to the database of GenBank under the

accession number GQ 499369.

Seventy-nine Actinomycetes were isolated from soils of Kalapatthar (5545m),

Mount Everest region by Gurung et al., 2009. Among all the isolates twenty

seven(34.18%) of the isolates showed an antibacterial activity against at least one test

33

bacteria among two Gram positive and nine Gram negative bacteria in primary

screening by the technique of perpendicular streak method. In secondary screening

thirteen (48.15%) showed antibacterial activity. After that the MIC test was done and

the minimum inhibitory concentration (MIC) of antibacterial metabolites of the isolate

K.6.3 was 1mg/ml whereas that of isolates K.14.2 and K.58.5 was 2mg/ml. Each of the

metabolites showed two spots on thin layer chromatography plate which was

completely different from the spot produced by vancomycin. And the active isolates

from primary screening were heterogeneous in their overall biochemical, macroscopic

and physiological characteristics.

A study was carried by Harald Bredholt et al., (2007) on Actinomycetes from

Norway for Diversity and Biological Activity. Approximately 3,200 actinomycete

bacteria were isolated using four different agar media from the sediment samples

collected at different locations and depths (4.5 to 450 m). Grouping of the isolates first

according to the morphology followed by characterization of isolates chosen as group

representatives by molecular taxonomy revealed that Micromonospora was the

dominating actinomycete genus isolated from the sediments. Micromonospora was

found at higher relative amount in the deep water sediments compared to the shallow

water samples. The nine percent of the isolates clearly required sea water for normal

growth which suggests that these strains represent obligate marine organisms.

Zhonghui Z. et al., (2000) screened the actinomycetes for antimicrobial or

antitumor activity, which were isolated sea plants and animals collected from the

Taiwan Strait, China. MTT assay was used to study the antitumor activity and DNA

target activity was studied by the biochemical induction assay while antimicrobial

activity was determined by observing bacterial and fungal growth inhibition. 20.6% of

34

marine actinomycete cultures displayed cytotoxic activity on P388 cells at dilutions at

and below 1:320 and 18.6% on KB cells. 2.96% of marine actinomycete cultures

showed inducing activity. Among isolated all marine actinomycetes, the genus

Micromonospora had the highest positive rate of inducing activity. Even though, most

antimicrobial activity was found in the genus Streptomyces. These results indicate that

actinomycetes from marine organism could be used as source for antitumor and

antimicrobial bioactive agents.

Li and Liu (2006) worked on the Marine sponge Craniella austrialiensis-

associated bacterial diversity revelation based on 16S rDNA library and biologically

active Actinomycetes screening, phylogenetic analysis and reported that bacterial

diversity associated with South China Sea sponge C. austrialiensis was assessed using a

16S rDNA clone library alongside restriction fragment length polymorphism and

phylogenetic analysis. It was found that the C. austrialiensis-associated bacterial

community consisted of alpha, beta and gamma-Proteobacteria, Firmicutes,

Bacteroidetes as well as Actinobacterium. Actinomycetes were isolated successfully

using seawater medium with sponge extracts. According to the BLAST and

phylogenetic analysis based on about 600-bp 16S rDNA sequences, 11 of the

representative 23 isolates closely matched the Streptomyces sp. while the remaining 12

matched the Actinomycetales. Twenty Actinomycetes have antimicrobial potentials, of

which 15 are found to possess broad-spectrum antimicrobial potentials and finally

conclude the sponge C. austrialiensis-associated bacterial community is very abundant

including Proteobacteria, Firmicutes, Bacteroidetes and Actinobacterium while

Actinomycetes is not predominant. Artificial seawater medium with sponge extracts is

suitable for Actinomycetes isolation. Most of the isolated C. austrialiensis-associated

Actinomycetes have a broad spectrum of antimicrobial activity.

35

4. MATERIALS AND METHODS

In the present work it has been envisaged to investigate the marine

Actinomycetes and their biological properties i.e. Isolation, Characterization,

Antimicrobial properties, partial isolation of bioactive compounds and Molecular

studies for identification.

4.1 Area of Study

Marine soil samples were collected from four different stations, seashore soils

covering Karwar (Uttar Kannada District), Karnataka, Central West Coast of India.

Name and Geographical position of the study cites are shown in the Table 1. Locations

of the study sites were fixed during the time of collection with the help of Global

Position System (GPS) and Google map with four study stations.

Table 3. Locations of Study area

Station Name Latitude Longitude

Station 1-Kinner. 140 51` 28.12``N. 740 10` 38.72``E.

Station 2-Sunkeri. 140 50` 32.33``N. 740 10` 06.16``E.

Station 3-Devbhag. 140 51` 22.49``N. 740 06` 40.52``E.

Station 4-Majaali. 140 51` 21.29``N. 740 06` 38.51``E.

36

Figure 1. Location of Study stations

4.1.1 Samples Collection

Samples were collected during the low tide from above four stations. Marine

sediment samples were collected with hand core and sterile polythene bags from 0-6

inch depth (15.24 cm) as upper surface of sediments consist more microbial load

Nevine B. G. et al., (2000) and transported to the laboratory for process as described by

Jensen et al., (1991). The samples were stored in refrigerator for further study.

4.2 Isolation and enumeration of Actinomycetes

Along with the collection of sediment samples, some Hydrological parameters

like Temperature, Salinity, Water pH (Standard Methods, APHA 20th edition) and

dissolved oxygen (winklers method) were also recorded on the site itself (Table 2).

In the present study, Starch Casein Agar, Starch Nitrite Agar and

Actinomycetes Isolation Agar (Hi Media) were used for the isolation of Actinomycetes

37

by spread plate technique. The samples were subjected to serial dilution (up to 10-6

dilution) by adding 1g of sediment sample in 10 mL of distilled water and an aliquot of

0.1 ml from these samples were spread on the agar media (Singh and Agrawal, 2002 &

2003). After incubation of 11 – 21days at 280C-300C, Actinomycetes were recognized

by their characteristic tough, leathery colonies, branched vegetative mycelia, aerial

mycelia and spore formation. On the bases of these criteria, only actinomycetes with

well-developed and branched hyphae were included in this study. Streak plate method

was used to purify cultures of actinomycetes (Williams and Cross, 1971, Singh and

Agrawal 2002; Agrawal 2003). The actinomycete colonies those developed on the

plates were enumerated and expressed as colony forming units (CFU), (Williams and

Cross, 1971, Singh and Agrawal 2002; Agrawal 2003) (Table 9).

4.2.1 Rare actinomycetes

The actinomycetes, especially Streptomyces, are remarkable producers of

antibiotics arising from their unlimited capacity to produce secondary metabolites with

diverse chemical structures and biological activities. Tens of thousands of such

compounds have been isolated and characterized, and many have been developed into

drugs for treatment of a wide range of human diseases (Bull et al., 1992; Franco &

Coutinho, 1991). Re-isolation of previously known antibiotics strains is a major

problem in new drug discovery. Less well studied organisms such as non-streptomycete

species (rare actinomycetes) provide attractive opportunities for developing new

antibiotics (Nareeluk et al., 2009). The successful discovery of novel rare

actinomycetes needs ecological study of their distribution. New methods for isolating

them from diverse habitats and culturing them in the laboratory are needed for such

studies because complicated procedures for isolation and cultivation are currently

required (Lazzarini et al., 2000).

38

4.2.2 Sampling and pretreatment of soil

Collected sediment samples were subjected to five different pretreatment

methods namely control (without any Treatment), antibiotics, Dry heat, Air dry, wet

heat and Phenol (Hayakawa et al., 1995) as described in Table 3, were carried out in

the first 24h after sampling.

4.2.3 Selective isolation of rare actinomycetes

Serially diluted soil suspensions were spread onto selective isolation medium,

and incubated for upto 4 weeks at 28°C. Starch casein agar (SCA), humic acid vitamin

agar (HVA), hair hydrolysate vitamin agar (HHVA), and Bennet's agar (BA) were used

for the selective isolation of rare actinomycetes agar (Hayakawa & Nonomura, 1987).

Preliminary designation of rare actinomycete colonies were done by microscopic

observation with a long working distance microscope.

4.3 Morphological and Biochemical characterization of actinomycetes

Morphological studies of isolates include microscopic and macroscopic

observations. Microscopic observations were made with a light microscope by using

the method of Shirling and Gottlieb (1966). Active purified isolates of actinomycetes

were identified by comparing their morphology of spore bearing hyphae with entire

spore chain and structure of spore chain in 1000x (Oil immersion) (Figure 3) and Gram

nature with the actinomycetes colony morphologies such as configuration, margin and

elevation (Figure 2) as described in Bergey’s manual of Determinative Bacteriology,

Ninth edition (2000) and the organism was identified.(Cross, 1989; Lechevalier, 1989;

Locci, 1989; Wendisch and Kutzner, 1991; Williams et al., 1989). Various biochemical

tests were performed for the identification of the potent isolates.

39

In the present study with respect to sampling station, Out of selected 55 isolates

first A1-A10 actinomycetes were from Kinner station, A11-A20 actinomycete isolates

from Sunkeri, A21-A30 isolates from Devbhag and A31-A42 actinomycetes from

Majaali station. Whereas in case of rare actinomycetes were obtained from four

sampling stations in following manner RA43-RA47 from Kinner, RA48-RA52 isolates

from Sunkeri, RA53 and RA54 isolates from Devbhag and RA55 isolate from Majaali.

Figure 2. Different Colony characteristics of Actinomycetes on Agar medium (Madigan et al., 1997).

40

Figure 3. Different types of spore arrangement on actinomycetes mycelia

4.3.1 Gram staining

Gram staining was performed for pure strain cultures to determine whether Gram

negative or Gram positive. Gram staining detects a fundamental difference in the wall

composition of bacteria. All the chemicals required for Gram staining were procured

from Hi media, Mumbai, India.

A. Bacterial smears were prepared by using following steps

1. A drop of distilled water was taken on a clean glass slide.

2. Isolated colony was transferred to the slide with a sterile loop or needle

touch and mixed in the water drop.

41

3. Mixed until just slightly turbid and made smear on glass slide.

4. Smears were air dried and heat fixed.

5. Slides preparation allowed cooling.

B. Slides were flooded with crystal violet, and allowed the slide for 60 seconds.

C. Washed off the crystal violet with running tap water.

D. Slides were flooded with Gram’s iodine, and allow the slide for 60 seconds.

E. Washed off with running tap water.

F. Decolorized with 95% alcohol and 5% water solution until the solvent flows

colorless from the slide (approximate 5-10 seconds).

G. Washed off the crystal violet and Gram’s iodine complex with running tap

water.

H. Slides were flooded with counter stain, safranine for 60 seconds.

I. Rinsed with water and allow to air dry.

J. Gram negative: Bacterial cells are decolorized by the 95% alcohol solution and

take on a red to pink color when counterstained with safranine. They appeared

pink in colour under microscope.

Gram positive: Bacterial cells retain the crystal violet and remained purple to

dark blue in colour under microscope.

4.3.2 Biochemical tests for actinomycetes

All isolates were tested using the Rapid Biochemical kit (Himedia) according to

the manufacturer’s instructions and were tested for esculin hydrolysis and for acid

production from arabinose, cellobiose, fructose, glycogen, inositol, lactose, mannitol,

ribose and trehalose (at 1% w/v). Isolates were also tested for the presence of

preformed glycosidic enzyme activities such as (N-acetyl-β-glucosaminidase, N-acetyl-

42

bgalactosaminidase, α-L-fucosidase, β-glucosidase, α-glucosidase, α-arabinosidase, α-

galactosidase and β-galactosidase).

Catalase test

The catalase activity and ability of each isolate to grow in air was also determined.

2-3 ml of the hydrogen peroxide solution was poured in to a small test tube. Using a

sterile inoculation loop, test organisms were removed from Starch casein agar plate and

immersed in the hydrogen peroxide solution. The test tube was observed for Bubbles

formation. This test is used to detect the soil bacteria.

Lactose and mannitol fermentation test

This test is used to differentiate the microorganisms fermenting carbohydrate (such as

lactose and mannitol).

Voges-Proskauer test

To detect the production of acetylmethylcarbinolacetoin, a natural product formed from

pyruvic acid in the course of glucose fermentation. The glucose broth together with the

organism was inoculated and incubated at 28°C for 3 days. Approximately 3 ml of

alpha naphthol was added followed by 1 ml of 40% KOH and mixed well for 30

minutes. Pink coloration of solution indicate VP (+) and no coloration indicate VP (-).

ONPG Test

This test determines the presence of β-galactosidase. A fermenter may produce slow

results if it lacks the enzyme is permease. If this enzyme is missing, but the

β-galactosidase is present within the cell, fermentation can take place. The ONPG test

uses the reagent, orthonitrophenyl- β -D-galactopyranoside. When added to a bacterial

suspension, this reagent will produce a yellow color if β-galactosidase is present.

43

Urease test

This is determination test for the hydrolysis of urea to ammonia and water. In this test a

dense suspension of the test organism was prepared 2 ml saline in a small tube. A

urease tablet was added into the tube and incubated at 28- 30oC for up to 4 h or

overnight. When the medium becomes alkaline and an indicator produces a pink colour.

Decarboxylation of Arginine

Many species of bacteria possess enzyme capable of de-carboxylating specific

aminoacids in the test medium. The decarboxylase enzymes remove a molecule CO2

from an aminoacid to form alkaline-reacting amines. The following are the amino acids

most commonly tested and their amine degradation products. Arginine-Citruline, the

amines resulting from the decarboxylation reaction can be detected with Ninhydrin

reagent after extraction from the broth culture with chloroform.

Esculin hydrolysis

The purpose was to see if the microbe can hydrolyze the compound esculin as a carbon

source, Bile escullin agar medium was used. This is a nutrient agar-based medium

containing 0.1% esculin and 10% bile salts, and allowed to solidify at a slant. The bile

salts inhibit some bacteria, and so the ability to grow in the presence of bile salts

represents a second test use for the medium.

An inoculum from a pure culture is transferred aseptically to a sterile tube of

bile esculin agar and streaked along the slant. After incubation at 28-30oC for

appropriate time abundant growth on the slant indicates a positive test for growth in the

presence of bile. If growth is present, esculin hydrolysis can be observed if the medium

has taken on an intense, chocolate brown coloration due to the production of dark

brown compound esculetin.

44

4.4 Screening of actinomycetes and rare actinomycetes for antimicrobial activity

The synthesis level of secondary metabolites of microorganisms has been found

to be at its peak at the late stationary phase of their life cycle. The arrows head indicates

direction and increase in different parameter like time, number of viable microbes or

cell and number of metabolites synthesis (Figure 4). The antimicrobial screening

method consists of two steps, primary screening and secondary screening.

Figure 4. Primary and secondary metabolites levels in viable cell growth phase

4.4.1 Primary screening

In primary screening the antimicrobial activity of pure isolates were determined

by perpendicular streak method on Nutrient agar (NA). In vitro screening of isolates for

antagonism Nutrient agar plates were inoculated with Actinomycetes isolate by a single

streak of inoculum in the petri plate. After 4 days of incubation at 28°C the plates were

seeded with perpendicular test organism’s single streak. The microbial interaction were

analyzed by the determination of the size of the of the clear/inhibition zone (Madigan et

al., 1997).The test organisms used were; Bacillus subtilis (MTCC 441), Staphylococcus

aureus (MTCC 737), Enterobacter aerogens (MTCC 111), Escherichia coli (MTCC

1668), Klebsiella pneumonia (MTCC 109), Proteus vulgaris (MTCC 1771),

Pseudomonas aeruginosa (MTCC 2453), Salmonella typhi (MTCC 733) and Shigella

sp (MTCC 1457).

45

4.4.1.2 Actinomycetes as biocontrol against fungal pathogens

Actinomycetes isolates were tested for their inhibitory activity against five

fungal plant pathogens, viz., Alternariasp, Rhizoctonia bataticola, Sclerotium rolfsii,

Fusarium udum and Pyricularia species by dual culture technique (Ganesan and

Gnanamanickam, 1987) as described below. These plant pathogens were obtained from

Department of Agricultural microbiology, University of Agricultural Sciences (U.A.S),

Dharwad.

In primary screening each fungal pathogen was grown on a PDA plate till it

covered the whole surface of the agar. With the help of a sterile 5 mm cork borer, a disc

of fungal growth from this plate was taken and placed at the center of a fresh PDA

plate, which consist 4 day old culture of actinomycetes isolates were streaked parallel

on either side of the fungal disc 3 cm away from the disc. The plates were kept for

incubation at 30oC for 96 hours. Visual observations on the inhibition of growth of

fungal pathogen were recorded after 96 hours of incubation in comparison with the

control PDA plate which was simultaneously inoculated and incubated with respective

fungal pathogen.

Figure 5. Different steps of antimicrobial assay

Isolation of Actinomycetes and Rare actinomycetes from sediments

Actinomycetes Isolates on agar slants

Growth on agar plates Cultivation in different growth media

Liquid fermentation Ethyl acetate, Methanol, Petroleum ether

Crude extract

Assay for antimicrobial activity

46

4.4.2 Secondary screening

Secondary screening was performed by disk diffusion (Kirby-Bauer et al.,

1966) method against the above mentioned standard test organisms.

4.4.3 Fermentation and Isolation of antimicrobial metabolites

Fermentation and Isolation of antimicrobial metabolites consists different steps

as shown in (figure 5). Fermentation was carried out using Starch Casein Broth in an

Erlenmeyer flask. Antimicrobial compound was recovered from the filtrate by solvent

extraction method. Ethyl acetate was added to the filtrate in the ratio of 1:1(v/v) and

shaken vigorously for 1 hour for complete extraction. The ethyl acetate phase that

contains bioactive compounds was separated from the aqueous phase. It was evaporated

to dryness in water bath at 80°-90°C and the residue obtained was weighed. Thus

obtained extract was used to determine antimicrobial activity and to perform

Bioautography (Pandey et al., 2004). In the present study diffusion methods were used

for Antimicrobial susceptibility test.

4.4.4 Disc Diffusion method

1. Turbidity standard for inoculum preparation:

To standardize the inoculum density for a susceptibility test, a BaSO4 turbidity

standard, equivalent to a 0.5 McFarland standard or its optical equivalent (e.g. latex

particle suspension), was used. A BaSO4 0.5 McFarland standard prepared by 0.5 ml

aliquot of 0.048 mol/L BaCl2 (1.175% w/v BaCl2. 2H2O) was added to 99.5 ml of 0.18

mol/L H2SO4 (1% v/v) with constant stirring to maintain a suspension. The correct

density of the turbidity standard was verified by using a spectrophotometer. The

absorbance at 625 nm should be 0.008 to 0.10 for the 0.5 McFarland standard.

47

2. Inoculums Preparation, with the help of sterile loop pure colony culture was

transferred to a tube containing 5 ml of Starch casein broth medium.

3. The broth cultures were incubated at 28±2C until they achieves the turbidity of the

0.5 McFarland standard.

4. The turbidity of the actively growing broth culture was adjusted with sterile saline or

broth to obtain turbidity optically comparable to that of the 0.5 McFarland standard.

This results in a suspension containing approximately 1-2 x 10­8 CFU/ml.

5. Test Plates were inoculated, within 15 minutes after adjusting the turbidity of the

inoculum suspension, a sterile cotton swab was dipped into the adjusted suspension.

The swab should be pressed gently on inner wall of the tube to remove excess

inoculum from the swab.

6. The dried surface of a Mueller-Hinton agar (Himedia) for bacteria and Potato

Dextrose Agar (Himedia) for fungi, plates were inoculated with respective inoculums

by streaking the swab over the entire sterile agar surface and ensure for an even

distribution of inoculum.

7. The petriplate lid partially kept open for 5 to 7 minutes, in Laminar air flow hood, to

allow for any excess surface moisture to be absorbed before applying the antimicrobial

impregnated disks.

8. The predetermined series of crude extract contained discs from the secondary screening

were dispensed onto the surface of the inoculated agar plate. Each disc pressed gently

to ensure complete contact with the agar surface.

48

9. The petriplates with discs were incubated at 37C for 24 to 48 hours for bacteria and

ambient temperature for 48 to 72 hours for fungi. Solvent Ethyl Acetate and broth

media were used as negative control (10µl/disc). Each plate was examined for the

uniform growth and uniformly circular resulting inhibition zones. The diameters of the

complete inhibition zones were measured, including the diameter of the disc. Zones

are measured to the nearest whole millimeter, using a scale, which is held on the back

of the inverted Petri plate.

4.5 Thin Layer Chromatography

Riguera (1997) described various strategies used for isolation and structural

elucidation of pharmacologically active metabolites from marine organisms.

Procedures adapted to the physical and chemical characteristics of the compounds

isolated, particularly to their polarity and lipo- or hydrophilic character have been

mentioned. After biomass extraction with an adequate solvent system (usually

methanol or acetone), the first step in the isolation of a natural compound from the

main extract or broth usually consists of a sequential gradient partition with solvents

(petroleum ether, hexane, CCl4, CHCl3, CH2Cl2, Ethyl acetate, n-butanol and water).

The fractions so obtained contain compounds distributed according to their polarity. In

the case of a bioactive extract, this process can be guided by the appropriate assay to

localize the active component. In this way, water-soluble organic material represented

mainly by alkaloid salts, amino acids, polyhydroxysteroids, and saponins is found in

the n-Butanol fraction. The CH2Cl2, EA, fraction affords compounds of medium

polarity such as peptides and depsipeptides, while in the hexanes, PE and CCl4, only

low polarity metabolites (hydrocarbons, fatty acids, acetogenins, terpenes, etc.) are

found.

49

According to their increasing polarity different solvent systems and ratios were

checked, among in the Petroleum ether: ethyl acetate (1:1) solvent system

actinomycetes crude extracts showed distinctive and clear separation on chromatogram.

Thin Layer Chromatography of the crude extracts and control (SCB medium)

was done using Petroleum ether: ethyl acetate (1:1) solvent systems. The procedure was

as follows.

Procedure:

1. Commercial Thin Layer Chromatography plates using alumina, backed sheets

(Silica gel 60 F254, 0.25mm thick Merck, Darmstadt, Germany) were used.

2. Using a pencil and ruler, a line was drawn 1 cm from the bottom of the plate. Points

were marked and labeled at a distance of approximately 1 cm from each other

corresponding to the samples to be loaded.

3. The 20µl of extracts and control were spotted on their respective labeled spots.

Followed by dry and spotting method.

4. The spots were allowed to dry.

5. The chromatography chamber was rinsed clean and dried. 100 ml of the solvent

system was added into it.

6. The TLC plate loaded with sample was placed in it with the bottom edge just

touching the solvent system.

7. The chamber was closed with the lid to prevent solvent from evaporating and the

solvent was allowed to move till it reached approximate 2cm below the top edge.

8. Once the solvent had reached the solvent front, it is removed from the developing

chamber and air-dried. The separated components were visualized by the following

methods

50

i. Placing the plate in the UV chamber and observed at 254nm and 366nm.

ii. Placing the plate in the Iodine chamber till the bands were observed.

iii. By spraying 5% methanolic sulphuric acid and placing it in the oven.

4.5.1 Bioautography of Thin Layer Chromatogram

The Ten microliters of the ethyl acetate fractions and reference antibiotic

(Tetracycline and Nystatin in the concentration 10µg/ml, as antibacterial and antifungal

respectively) were applied on the plates and the chromatogram was developed using

Petroleum ether: ethyl acetate (1:1) as solvent system. The plates are run in triplicate;

one set was used as the reference chromatogram and the other two were used for

Bioautography (Pandey et al., 2004). The spots in the chromatogram are visualized in

the iodine vapor chamber and UV chamber.

In case of fungi, the organic solvent of chromatogram was evaporated by a

stream of air. TLC plates were homogeneously sprayed with about 10 mL potato

dextrose broth containing conidia. Plates were incubated for 3 days in a moist chamber

at 25°C in the dark and the appearance of clear zones in the mycelium layer indicated

antifungal activity. Exposure (15–30 min) of fungi with unpigmented mycelia and

spores to iodine vapors significantly enhanced contrast in order to detect inhibition

zones (Hadacek & Greger 2000).

4.6 Isolation of Genomic DNA from Actinomycetes and 16S rRNA sequencing

4.6.1 Isolation of Genomic DNA from Actinomycetes by HiPurATM Miniprep

Purification Spin kit

(All the chemicals required for molecular characterization were procured from Hi

media, Mumbai, India.)

51

Introduction

The DNA purification procedure using the miniprep spin columns comprises of three

steps viz,

Adsorption of DNA to the membrane,

Removal of residual contaminants and

Elution of pure genomic DNA.

4.6.1.2 Concentration, yield and purity of DNA

In the present study, Nano drop instrument (Quawell) UV Spectrophotometer

and agarose gel electrophoresis techniques were used to reveal the concentration and

the purity of the genomic DNA.

The calibration was done by the spectrophotometer and the absorbance was

measured at 260 nm and 320 nm. Absorbance reading at 260 nm falls between 1.0

corresponds to approximately 50 µg/ml of DNA. The A260-A320/A280-A320 ratio should

be 1.5-2.0. Purity is determined by calculating the ratio of absorbance at 260 nm to

absorbance at 280 nm (Table 15).

Concentration of DNA sample (µg/ml) = 50 X A260 X dilution factor.

Materials and Reagents required

1. 370C water bath or heating block.

2. 550C water bath or heating block.

3. Tabletop Microcentrifuge (with rotor for 2.0 ml tubes).

4. Ethanol (96-100%).

5. Molecular Biology Grade water.

52

Procedure:

1. Actinomycetes were grown in a Luria medium till they reach log phase.

2. Lysozyme solution was prepared using lysozyme from chicken egg white,

which is provided in the kit. A 45 mg/ml stock solution of lysozyme was

prepared as described under general preparation (200 µl of lysozyme solution

was required per isolation procedure).

3. Cell Harvestment: 1.5 ml of actinomycetes broth culture was pelleted by

centrifuging for 2 minutes at 12,000-16,000Xg (=13,000-16,000 rpm), the

culture medium (Luria broth) was removed completely and discarded.

4. Cell Resuspension: the pellet was resuspended thoroughly in 200 µl of

lysozyme solution (prepared in step 1) and incubated for 30 minutes at 37°C.

5. Cell Lysate: 20 µl of the proteinase K solution (20 mg/ml) was added to the

sample. Residual RNA was removed by following methods:

Optional RNase A treatment: The RNA-free genomic DNA was obtained, by

adding 20 µl of RNaseA solution; it was mixed and incubated for 5 minutes at

room temperature (15-25°C) then step 6 was continued.

RNaseA enzyme treatment: RNase A is a type of RNase that is commonly

used in research. RNase A (e.g., bovine pancreatic ribonuclease A) is one of the

sturdiest enzymes in common laboratory usage. It cleaves 3' end of unpaired C

and U residues.

6. 200 µl of Lysis solution was added. It was thoroughly vortexed for few seconds

and incubated at 550C for 10 minutes. A homogeneous mixture is essential for

efficient cell lysis.

7. Preparation for binding:

200 µl of ethanol (95-100%) was added to the lysate and was mixed thoroughly

by vortexing for few seconds.

53

8. Lysate loading onto Spin Column :

The lysate obtained was transfered from step 6 onto spin column. It was

centrifuged at >6,500Xg (=10000 rpm) for 1 minute. The flow-through liquid

was discarded and the spin column was placed in same 2.0 ml collection tube.

9. Prewashing:

500 µl of Prewash Solution was added to the spin column and centrifuge at

>6500Xg (=10,000rpm) for 1 minute. The flow through liquid was discarded

and the same collection tube was re-used with the column.

10. Washing:

500 µl of wash solution (WS) was added to the spin column and was

centrifuged for 3 minutes at maximum speed (12,000-16,000 rpm). The spin

column was transfered to new collection tube; it was centrifuged again at same

speed for the additional 1 minute to dry, the column must be free of ethanol

before the elution of the DNA.

11. DNA Elution:

The spin column was transfered to new collection tube. 200 µl of the Elution

Buffer (ET) was directly pipetted into the column without spilling to the sides.

It was incubated for 1 minute at room temperature. It was centrifuged at

>6500Xg (=10,000rpm) for 1 minute to elute the DNA.

12. Storage of the elute with Purified DNA:

Elute contained pure genomic DNA. For short-term storage (24-48 hrs) of the

DNA, 2-8°C is recommended for long–term storage, 20°C or lower temperature

(-80°C) is recommended. The elution Buffer would help to stabilize the DNA at

these temperatures.

54

4.6.2 DNA quantitation and electrophoresis

Electrophoretic analysis of DNA using agarose gels can confirm DNA integrity.

Typically, intact genomic DNA would be up to 40KB in size, depending upon the

species. 0.8% agarose gel was prepared by adding required quantity of agarose to 1X

Tris-Acetate-EDTA (TAE) buffer and mixed well. The mixture was heated in

microwave oven until it became clear and care was taken to avoid over boiling and

evaporation. The mixture was cooled to ~500 C and ethidium bromide was added to

make a final concentration of 0.001µg/ml. The entire mixture was poured in to a tray in

which combs were fixed to make wells in the gel. It was cooled to form uniform gel.

After gel formation, the tray was placed in buffer tank containing 1X TAE buffer for

submerged gel electrophoresis and combs were removed with care to avoid rupture of

wells. 3µl of each DNA sample was mixed with 1µl of loading dye and the mixture was

loaded into the wells. Gel was subjected to electrophoresis at 100V for 30 minutes and

visualized using gel documentation system (Vilber Lourmet. Germany), (Plate).

Chemicals and buffers used for gel electrophoresis

1. Tris-Acetate-EDTA (TAE) buffer 20X

2. Agarose (Himedia)

3. Ethidium Bromide

4. Loading Dye (Stock)

Preparation of DNA working Dilutions

100µl of DNA working dilutions were prepared at a concentration of 50ng/µl by

dissolving required amount of stock DNA sample in MB grade water. After preparation

of working dilutions the uniformity of the samples were checked by performing

electrophoresis on a 1% agarose gel. Samples were stored at -200C.

55

Polymerase Chain Reaction

The target sequences amplified on a ABI Veriti thermal cycler (Applied

Biosystems, USA) with an initial denaturation at 960C for 5 minutes and later on for 35

cycles at 950C for 60 seconds, at estimated annealing temperature of the primer for 45

seconds, extension at 720 C for 2 minutes and a final extension at the end of 35th cycle

at 680C for 7 minutes in a final volume of 20 µl containing 1.5mM MgCl2, 5pm of each

primer(Sigma Aldrich), 200 µM deoxy-NTP and 1U Taq polymerase (NEB, UK) .

Primer Details:

Table 4. Oligonucleotides used in the present study

Sl.

No

Primers

name Sequence (5’-3’)

No.of

bp

Temperat

ure in ºC Reference Specificity

1 27f AGA GTT TGA

TCA TGG CTC AG

20 56 Lane 1991;

Magarvey A.

et al.,2004.

Bacterial 16S

rRNA gene

(Universal)

2 1492r TAC GGC TAC

CTT GTT ACG

ACT T

22 56 Lane 1991;

Magarvey A.

et al.,2004.

Bacterial 16S

rRNA gene

(Universal)

Amplicon Check by Agarose Gel Electrophoresis

After the completion of 35 cycles of polymerase chain reaction 5µl of the

amplicon was electrophorased on a 1.2% agarose gel containing ethidium bromide.

DNA bands were visualized using gel documentation system (Vilber Lourmet,

Germany). The samples which were amplified successfully were used for the

sequencing after post PCR cleanup (Plate).

56

Post PCR cleanup

DNA fragments should be purified prior to sequencing to remove proteins, salts,

left out primers and dNTPs which may have detrimental effects on the sequencing

reaction. A combination of exonuclease I and shrimp alkaline phosphatase enzymes

(ExoI/SAP) were used to clean the PCR product. PCR cleanup master mix was prepared

by adding 5U of ExoI, 0.5U of SAP in final volume of 8.5 µl MilliQ water (Millipor

lifesciences, USA) and this master mix was added to 8µl of PCR product.

ExoI/SAP enzymatic reaction was allowed to proceed by heating the samples up

to 37°C for 30 minutes using a thermal cycler and then denatured the enzymes by

heating to 80°C for 15 minutes. Further 0.1 volumes of 3M sodium acetate (1.65µl) and

2.0 volumes of chilled 100% ethanol (33µl) were added and mixed well. Samples were

centrifuged at 4,000rpm for 30 minutes at 4°C. The ethanol was decanted and folded

paper towels were used to remove the excess ethanol by blotting the plate. 100µl of

cold 70% ethanol was added and centrifuged at 4,000rpm for 10 minutes at 4°C. After

decanting the ethanol PCR plate was blotted on folded paper towels to remove the

excess ethanol and the plate was centrifuged inversely for 30 seconds at 180rpm to

remove all ethanol. The pellet was resuspended in 8µl of water and stored at 40C.

4.6.3 DNA Sequencing

BigDye-terminator sequencing is a modification of the Sanger’s dideoxy chain

termination method. It utilizes labeling of the chain terminator dNTPs, which permits

sequencing in a single reaction. In BigDye-terminator sequencing, each of the four

dideoxynucleotide chain terminators is labeled with fluorescent dyes, each of which

with different wavelengths of fluorescence and emission. The dye labeled DNA

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fragments will be capillary electrophoresed and a detection system would identify the

labelled bases when they pass through a laser that activated the dye.

Cycle sequencing

BigDye labeling and chain termination was carried out by cycle sequencing

method. To label each base PCR amplicon was subjected to cycle sequencing reaction

with single primer was done using ABI Prism® BigDyeTM terminator v3.1 cycle

sequencing ready reaction kits (Applied Biosystems, USA) following the manufacturer’s

guidelines.

Sequencing Cleanup

To remove the remnants of the above reaction, to the each sample 2µl of 3M

sodium acetate, 50µl of 100% ethyl alcohol were added and incubated at room

temperature for 15 minutes to precipitate the DNA. The samples were centrifuged at

4000rpm for 30 minutes at 40C. The supernatant was discarded and the reaction plate

was centrifuged inversely at 300 rpm for 20 seconds. 100µl of 75% alcohol was added

to each sample and centrifuged at 4000rpm for 15 minutes at 250C. The supernatant was

discarded and plate was inversely centrifuged at 300 rpm for 20 seconds to remove

alcohol completely. The plate was dried at room temperature until left out alcohol was

dripped off.

Denaturation and Snap chilling of labeled amplicon

10µl of Hi-Di formamide was added to each well of the sample plate. The

samples were heated to 960C for 5 minutes and immediately cooled to 40C to denature

and linearise the cycle sequencing products.

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Sequencing run

Sample information sheets which contain analysis protocol along with the

sample details were prepared and imported into the data collection software. Prepared

samples were analysed on ABI 3500 genetic analyzer (Applied Biosystems, USA) to

generate DNA sequences.

Sequence quality check

After completion of sequencing reaction, the quality of generated sequence was

checked by using Sequencing Analysis v1.0 software (Applied Biosystems, USA). The

Applied Biosystems Sequencing Analysis Software v1.0 is designed to analyse, display,

edit, save, and print sample files generated ABI genetic analyzers. The program has a

basecaller algorithm that performs basecalling for pure and mixed base calls also. It

provides quality values (QV) for every single base and sample scores for the assessment

of average quality value of the bases in the clear range sequence for the sample. The QV

is a per-base estimate of the basecaller accuracy. The QVs are calibrated on a scale

corresponding to:

QV= –10 log10 (Pe)

Where, Pe is the probability of error.

For this study, typical high-quality pure bases QVs were set to 20 to 50 and typical high-

quality mixed bases QVs were set to 10 to 50. The samples which didn’t follow the

above conditions were re-sequenced after fresh PCR amplification.

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Sequence Alignment

The generated sequences were aligned to their respective reference sequences

with the use of SeqScape v2.5 software (Applied Biosystems, USA). SeqScape is one of

the suits of Applied Biosystems designed for automated sequence data analysis. It

performs sequence comparisons for variant identifications, SNP discovery and

validation. It allows analysis of the re-sequenced data, comparing the consensus

sequences to a known reference sequence. The reference sequences for the gene studied

were obtained from NCBI Gen bank data base.

To set clear range of the sequence, a method that considers quality values of the bases

was used which removes bases from the ends of sequences until fewer than 4 bases out

of 20 have QVs <20. Filter setting values to filter the inappropriate sequences were set

as maximum mixed bases to 20 and minimum sample score to 25. Depending on the

sequence quality and the criteria specified for filtering the samples with low quality

were not assembled by the program. These unassembled samples were re-sequenced

until it satisfied the quality.