RESEARCH ARTICLE ISOLATION and CHARACTERIZATION of MARINE ACTINOMYCETES from MUTHUPPETTAI MANGROVES , TAMILNADU, INDIA Prabhahar.C*,K.Saleshrani** and Enbarasan.R *1

Available Online at http://www.recentscientific.com International Journal of Recent Scientific International Journal of Recent Scientific Research Research Vol. 5, Issue, 4, pp.906-910, April, 2014 ISSN: 0976-3031 RESEARCH ARTICLE ISOLATION AND CHARACTERIZATION OF MARINE ACTINOMYCETES FROM MUTHUPPETTAI MANGROVES , TAMILNADU, INDIA Prabhahar.C*,K.Saleshrani** and Enbarasan.R *1

*.Department of zoology, Annamalai University, Tamil Nadu, India ** ARTICLE INFO Department of zoology, ManonmaniamABSTRACT Sundaranar University, Thirunelveli, Tamil Nadu Article History: Microorganisms from Muthuppettai Mangrove areas are play an important role in Received 17th, March, 2014 biodegradation of dead plant material. This area is rich in Avicennia officinalis, Received in revised form 24th, March, 2014 Rhizophora mucaronata. Acanthus illicifolius and Excoecaria agallocha plants. Accepted 12th, April, 2014 Mangrove has a salty ecosystem and it is known to be large sources of organic matter Published online 28th, April, 2014 due to various microbial enzymatic and metabolic activities . Actinomycetes are all Gram-positive, filamentous and are facultatively anaerobic. All species grow best Key words: under anaerobic conditions. Actinomycetes play an important role among the Actinomycetes, Muthuppettai Mangrove sediments, mangroves bacterial communities, because of its diversity and ability to produce novel marine ecosystem, Antibacterial, antifungal activity chemical compounds of high commercial value A total ten isolates of Actinomycetes were collected from sediments of Muthuppettai mangroves These isolates were designated as PAct-1 to PAct-10. Out of ten isolates, eight isolates exhibited significant antibacterial activity. All the ten isolates were charecterised for biochemical and cultural aspects. These isolates were identified as Streptomyces sp. Among the ten isolates, PAct-1 exhibited the maximum zone of inhibition against bacterial activity. © Copy Right, IJRSR, 2014, Academic Journals. All rights reserved. INTRODUCTION carried out to sea in the form of resistant spores (Ravel et al., 1998; Ramya and Vijayakumar, 2008; Deepika and Marine environments are largely untapped source for the Kannabiran, 2010). Microorganisms found in marine isolation of new microorganisms with potentiality to produce environments have attracted a great deal of attention, due to active secondary metabolites. Among such microorganisms, the production of various natural compounds and their Actinomycetes are of special interest, since they are known to specialized mechanisms for adaptation to extreme environment produce chemically diverse compounds with a wide range of (Solingen et al., 2001). The pre-treatment including biological activities (Bredholt et al., 2008; Kalyani et al., enrichment, physical and selective media may be used to study 2012). The demand for new antibiotics continues to grow due the ecology of actinomycetes in natural habitats such as soil or to the rapid emerging of multiple antibiotic resistant pathogens water samples (Jensen et al., 2005; Gebreyohannes et al., causing life threatening infection. Although, considerable 2013). Since marine sediments represent an environment progress is being made within the fields of chemical synthesis which is markedly different from that associated with soil and engineered biosynthesis of antibacterial compounds, samples, it is not clear how effective the pre-treatment of such nature still remains the richest and the most versatile source for sediments would be for the recovery of bioactive new antibiotics (Baltz, 2006; Pelaez, 2006). Traditionally, actinomycetes. Marine sediment is an inexhaustible resource actinomycetes have been isolated from terrestrial sources that has not been properly exploited. Reports from the East although, the first report of mycelium forming Actinomycetes Coast of India, suggests that soil is a major source of being recovered from marine sediments appeared several actinomycetes (Sivakumar et al., 2005; Vijayakumar et al., decades ago (Weyland, 1969). Recently, the marine derived 2007; Dhanasekaran et al., 2008; Vijayakumar et al., 2009; actinomycetes have become recognized as a source of novel Deepika and Kannabiran, 2010; Kalyani et al., 2012; antibiotic and anticancer agent with unusual structure and Gebreyohannes et al., 2013). Correspondingly, the Muthuppettai properties (Jensen et al., 2005; Gebreyohannes et al., 2013). mangroves ecosystem is largely unexplored, and may provide Actinomycetes represent a ubiquitous group of microbes a rich source of the microorganisms producing novel and widely distributed in natural ecosystems around the world and efficient antimicrobial compounds. Hence, the present study especially significant for their role on the recycling of organic was undertaken to isolate the Actinomycetes from marine matter (Srinivasan et al., 1991). The literatures suggested that, sediments of Muthuppettai mangroves region. marine sediment sources are voluble for the isolation of novel actinomycetes with the potential to yield useful new products MATERIALS AND METHODS (Goodfellow and Haynes, 1984). However, it has been Collection of sample resolved whether Actinomycetes form part of the autochthonous marine microbial community of sediment The sediments sample were collected from Muthuppettai samples originated from terrestrial habitats and were simply mangroves area Muthupet mangrove environment (Lat.10’

* Corresponding author: Prabhahar.C Department of zoology, Annamalai University, Tamil Nadu, India International Journal of Recent Scientific Research, Vol. 5, Issue, 4, pp.906-910, 2014 20’N and 79’ 35’E) is known to be very rich microbial of oxygen. Type of carbon source utilized by microorganism diversity due to high amount of dissolved and particulate was identified by change in pH of the carbon utilization agar organic matter and therefore, different types of medium. Positive assimilation of growth indicated by color microorganisms are found in this type of environment .For the change from purple to yellow induced by bromocresol purple isolation of actinomycetes from different places at a depth of dye present in the medium. Sterile carbohydrate utilization 10 m using a core sampler. The central portions of the agar with bromocresol purple dye was prepared. It was, then, sediments were ascetically transferred to the sterile bottles. inoculated with 1 ml of soil isolates and poured into sterile The sediment sample was blackish brown color and of a sandy petri dishes. After solidification, sterile discs containing 3% of texture. different carbon sources such as dextrose, sucrose, starch, lactose, and maltose were placed aseptically on the surface of Isolation of Actinomycetes colonies from the marine the medium and incubated at 27°C for 8-10 days. sediments Starch hydrolysis Isolation and enumeration of Actinomycetes from sediments were performed by the soil dilution plate technique (Ellaiah et The soil isolates were streaked across starch – peptone agar al., 1996) using starch casein agar medium (g/;L: starch-10, plates using loopful of spores and incubated for 5 days at 28°C casein- 0.3, KNO3- 2, NaCl- 2, K2HPO4-2, MgSO4.7H2O- 0.05, (Kokare et al., 2003). At the end of incubation, the plates were CaCO3- 0.02, FeSO4.7H2O- 0.01 and agar- 18). 50 ml of starch flooded with weak iodine solution. The width of the casein agar media in 250 ml flask were sterilized at 121°C for hydrolysed zone around the growth and the width of the 20 min by autoclaving. The media was prepared by using 50% growth were measured. (v/v) sea water. 1 g each of the marine sediment sample was Coagulation and peptonization of milk taken in 250 ml Erlenmeyer flask containing 50 ml of sterile Skimmed milk was prepared by centrifuging the milk water. The flasks were shaken on rotary shaker for 30 min for contained in 50 ml plastic conical centrifuge tubes (Tarsons, the detachment of spore chains. The flasks were kept aside for Calcutta) at 2000 rpm for 45 min (Kokare et al., 2003). It was 15 min to settle down the particulate matter. The suspension transferred in 10 ml portions aseptically into the sterile boiling was serially diluted up to 6 fold. 1 ml each of these dilutions tubes. These boiling tubes were tyndallized at 80°C for 1 h for were added to each of 50 ml of the aforementioned sterile 3 consecutive days. The skimmed milk tubes were inoculated molten media maintained between 40 to 45°C, thoroughly with test cultures and incubated at 28°C. The extent of mixed and poured into Petri plates and incubated at 28°C. The coagulation and peptonization were recorded after 5 days of incubated Petri plates were observed from one week onwards incubation. for three weeks or colonies of Actinomycetes. The starch casein agar media was supplemented with 2.5 μg/ml of Nitrate reduction test rifampicin and 75 μg/ml of flucanozole to minimize bacterial Organic nitrate broth of 10 ml was inoculated with 50 µl of and fungal contaminations, respectively. Actinomycetes spore suspension and incubated at 28°C for 7 days (Kokare et colonies were marked, thus identical colonies were scored out al., 2003). Controls were run also without inoculation. On 7th and the selected colonies were sub cultured on SCA slants and day, the clear broth was tested for the presence of nitrite. To 1 incubated at 28°C for one week. ml of broth under examination and 1ml of control, two drops of sulphanilic acid solution followed by 2 drops of α - Characterization of Actinomycetes napthylamine solution were added. The presence of nitrite was Biochemical characterization indicated by a pink, red or orange colour and absence of colour Melanin production change was considered as nitrite negative. In the later case, the presence or absence of nitrate in the broth under examination The production of melanin pigment on agar slants of peptone – was confirmed by adding a pinch of zinc dust after the addition yeast extract Iron was determined and further tested the of the reagents; the unreduced nitrate, if present, would give a production in tryptone–yeast extract broth (Homans et al., pink, red or orange colour. 1970; James et al., 1991). The inoculated tubes were observed after 2 days and 4 days. Appearance of deep brown, greenish Morphological characterization brown, greenish black or black colours were recorded as Cultural characterization melanin positive. Absence of brown to black colour, or total Morphological and cultural characters of the selected absence of diffusible pigment, was considered as negative for actinomycetes strain was studied by inoculating into sterile melanin production. International Streptomycetes Project ISP 1,3,4, 5 and 6media Gelatin hydrolysis (Matson et al., 1989; Ohshima et al., 1991). The media were Gelatin agar plates were streaked across with test culture and sterilized and poured into sterile petridishes. After incubated for 5 days at 28°C (Homans et al., 1970; James et solidification of the media, culture of the selected strain was al., 1991). At the end of incubation period, the plates were streaked on the media surface by simple method aseptically flooded with 1ml of mercuric chloride solution. The widths of and incubated at 28°C for 7 days (Salleetal.,1948;Shomura et the hydrolyzed zone and growth zone were measured. al., 1987). Morphological characters such as colony characteristics, type of aerial hyphae, growth of vegetative Carbohydrate assimilation test hyphae, fragmentation pattern and spore formation were Type of carbohydrate source utilized by actinomycetes is an observed. important biochemical property for their identification Microscopical characterization (Homans et al., 1970; James et al., 1991). Assimilation is the Gram staining utilization of carbon source by microorganisms in the presence A smear of isolate was prepared on a clean glass slide and 907 | P a g e International Journal of Recent Scientific Research, Vol. 5, Issue, 4, pp.906-910, 2014 smear was allowed to air dry and then heat fixed. Heat fixed degraded form across the cytoplasmic membrane of the cell. smear was flooded with crystal violet and after one minute, Actinomycetes possess the ability to produce amylase that it was washed with water and flooded with mordant Gram’s breaks starch into maltose. The amylase is an extracellular iodine. Smear was decolorized with 95% ethyl alcohol, and enzyme, which is released from the cell of microorganisms. then washed with water. The smear was counter stained with The isolates produced amylase and hence, they failed to safranin for 45 seconds. After washing with water, smear was produce blue colour upon treatment with iodine solution. dried with tissue paper and examined under oil immersion (100 x). Screening for antimicrobial activity The antimicrobial activity was studied preliminarily by cup plate method (Haque et al., 1992) against bacteria. The test organisms were used are Salmonella typhi, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Bacillus subtilis. After preliminary testing of the isolates for their antimicrobial activities the most active isolates was selected for further study. RESULTS AND DISCUSSION During the course of our survey, a total of 10 actinomycetes were isolated in different area of from Muthuppettai Figure-1 Isolated culture of Actinomycetes on SCA medium mangroves area and designated as PAct-1, PAct-2, PAct-3, PAct-4, PAct-5, PAct-6, PAct-7, PAct-8, PAct-9 and PAct-10. In the nitrate reduction test, PAct-1, PAct-4, PAct-6, PAct-9 and the actinomycetes were recorded in sample -1 (28.22 x10-4 PAct-10 gave positive results and PAct-2, PAct-3, PAct-5, PAct-7 cfu per ml) and the minimum population was recorded and PAct-8 gave the negative results (Table 2). The reduction of maximum samples-1 (8.66 x10-4 cfu per ml) (Table-1 & Fig- nitrate to nitrite is used as criteria for species differentiation. Either 1;Fig-2). Further these isolates were taken for biochemical and NaNO3 or KNO3 are used as electron acceptor by some cultural characterization. organisms. NO3 and NO2 serve as sources of nitrogen for the synthesis of organic nitrogenous compounds or they may function Table 1 Enumeration of Actinomycetes isolates from the + as H acceptors. Nitrate converts NO3 to NO2. All the isolates marine sediments of Pichavaram coastal area showed positive results in melanin production test (Table 2). Actinomycetes population Name of the location Isolate code (1x 104 cfu g-1 on oven dry weight) Sample -1 PAct-1 28.22 Sample -2 PAct-2 14.22 Sample -3 PAct-3 10.88 Sample -4 PAct-4 18.66 Sample -5 PAct-5 8.66 Sample -6 PAct-6 12.66 Sample -7 PAct-7 16.44 Pichavaram Sample -8 PAct-8 25.88 Sample -9 PAct-9 19.33 Sample -10 PAct-10 24.88 Biochemical tests were conducted for melanin formation, nitrate reduction, coagulation and peptonization of milk, gelatin hydrolysis, starch hydrolysis, and carbon assimilation. All the isolates namely PAct-1, PAct-2, PAct-3, PAct-4, PAct- 5, PAct-6, PAct-7, PAct-8, PAct-9 and PAct-10 showed Figure -2 isolated culture of Actinomycetes PAct-1 positive results in starch hydrolysis (Table 2). Table 2 Biochemical characterization of Actinomycetes isolates Nitrate Milk coagulation and Gelatin Starch Carbon Isolates Melanin formation reduction peptonization liquefication hydrolysis assimilation PAct-1 Brown + Clear with acidic reaction + + Glucose PAct-2 Light brown - Clear with acidic reaction + + Glucose Not clear with slightly alkaline PAct-3 Light brown - + + Fructose reaction PAct-4 Brown + Clear with acidic reaction + + Glucose PAct-5 Light brown - - + + Lactose PAct-6 Brown pigmentation + - + + Maltose PAct-7 Light brown - Clear with acidic reaction + + Glucose PAct-8 Light brown - - + + Lactose PAct-9 Brown + Not clear with acidic reaction + + Lactose PAct-10 Brown + Clear with acidic reaction + + Glucose Starch is an insoluble polymer of glucose which acts as a This test is done to observe the production of pigments in the source of carbon for microorganisms which have an ability to mycelia of organisms and also the excretion of pigments into degrade them. Starch degrading microorganisms transport the the media. Pigment production is one of the most significant

908 | P a g e International Journal of Recent Scientific Research, Vol. 5, Issue, 4, pp.906-910, 2014 properties of actinomycetes. These pigments vary greatly in source utilized by microorganism was identified by change in nature. It depends on the composition of different media, pH of the carbon utilization agar medium. condition of growth and age of culture. Thus, pigment Positive assimilation of growth indicated by color change from production is one of the easily recognizable characteristics of purple to yellow induced by bromocresol purple dye present in actinomycetes, when media of known composition and definite the medium (Table 2). PAct-1, PAct-2, PAct-4, PAct-7 and conditions of culture are used. PAct-10 assimilated glucose while PAct-5, PAct-8 and PAct-9 Table 3 Sensitivity of different pathogenic bacteria against assimilated lactose. Fructose was assimilated by PAct-3 and Actinomycetes isolates maltose by PAct-6. Zone of inhibition (in mm) S. P. S. E. B. Praveen and Jain, (2007) have isolated Streptomyces sampsonii Isolates typhi aeruginosa aureus coli subtilis GS 1322 from local garden soil, Sagar, India and reported their PAct-1 15 18 23 17 20 antifungal secondary metabolite production. Streptomyces PAct-2 10 7 15 12 9 cheonanensis sp. nov., a novel Streptomyces with antifungal PAct-3 6 - - 10 5 PAct-4 9 12 - 8 14 activity was isolated from the soil sample of Cheonan, Korea PAct-5 13 10 16 10 9 (Kim et al., 2006). Debananda et al. (2009) have isolated a PAct-6 6 - 5 3 - broad spectrum actinobacteria Streptomyces sindenensis strain PAct-7 - - - - - LS1-128 having both antibacterial and antifungal activity from PAct-8 - - - - - PAct-9 12 9 10 - 7 Loktak Lake in Eastern India. Prabhahar et.,,al.(2011) have PAct-10 5 7 6 - 4 reported that the minimum inhibiting concentrartion (MIC) value of Gracilaria grassa against fungi was ranged between 1ug/ml to 16ug/ml . Among the ten isolates, eight isolates namely PAct-1, PAct-2, PAct-3, PAct-4, PAct-5, PAct-6, PAct-9 and PAct-10 showed significant antibacterial activity (Table 3). However the two isolates namely PAct-7 and PAct-8 of the isolates exhibited antibacterial activity. The isolates PAct-1 actinomycetes strain recorded the maximum antibacterial activity which was selected through the sensitivity test were further taken for morphological and cultural characteristics. The morphological and cultural characteristics of the most active isolate PAct-1 were studied on International Figure 3 Gram staining of PAct-1 on 100x. Streptomyces Project (ISP) media such as ISP-1, ISP-3, ISP-4, ISP-5 and ISP-6. The growth characteristics, presence of All the ten isolates showed positive result in gelatin mycelium and soluble pigments were observed (Table 4). The liquefication (Table 2). The solid character of the medium morphological characters of the active isolates were also depends upon the gelatin remaining in the gel state. Many studied microscopically under oil-immersion (100 x) after microorganisms including actinomycetes produce exoenzymes Gram-staining. The observations revealed that all the isolates that are capable of hydrolyzing gelatin and liquefying the are gram positive (Fig.3). Thus, morphological, cultural and nutrient gelatin medium. Some organisms ferment lactose, biochemical characterization indicated that the isolates belong others hydrolyze casein, and other groups simultaneously to Streptomyces genus of actinomycetes. ferment and hydrolyze. The fermentation of lactose produces an acid, which forms an insoluble complex of calcium and CONCLUSION casein resulting in coagulation of milk. The digestion of The study has showed that Starch-casein agar supplemented coagulated mass by the action of proteolytic enzymes is termed with nystatin and nalidixic acid was found to be suitable for peptonization. Some species peptonize milk without isolating Actinomycetes from Mangrove sediments, of coagulation and if caseinase is not present in the Muthuppettai Tamilnadu .Of ten isolates, only eight microorganisms, peptonization however, does not follow exhibited significant antibacterial activity. Further the precipitation. All the isolates were able to cause milk biochemical test and cultural characterization showed the coagulation but no peptonization as precipitate persisted at the positive for the Streptomyces sp. of Actinomycetes. Among end of the test (Table 2). the ten actinomycetes isolates, the isolate Pact-1 showed the Table 4 Cultural characterization of Actinomycetes PAct-1 Medium Growth Aerial mycelium Substrate mycelium Pigment (ISP-1) Good White Yellowish-brown None (ISP-3) Moderate to good White Colourless to white Creemish yellow (ISP-4) Poor to moderate White Yellowish to light yellow None (ISP-5) Poor White Yellowish-white None (ISP-6) Moderate to good Yellowish- white Yellowish-white Light blue Type of carbohydrate source utilized by actinomycete is an maximum antibacterial activity, which can be further studied important biochemical property for the identification of for the antibiotic production. actinomycetes. Assimilation is the utilization of carbon source Acknowledgements by microorganisms in the presence of oxygen. Type of carbon The authors acknowledged the Financial assistance provided 909 | P a g e International Journal of Recent Scientific Research, Vol. 5, Issue, 4, pp.906-910, 2014 by the University Grants Commission (UGC) New Delhi and B.A. (2003). Isolation, characterization and antimicrobial also to the authorities of the Annamalai university to carry activi-ty of marine halophilic Actinomonospora species out the research work AH1 from the West Coast of India. Curr. Sci. 86: 593- 597. References 15. Matson, J.A., Claridge, C., Bush, J.A., Titers, J., Bradner, 1. Baltz RH (2006). Marcel Faber Roundtable, Is our W.T., Terrence, W. (1989). Novel antitumor antibiotic antibiotic pipeline unproductive because of starvation, com-pounds produced by Actinomadura melliaura. constitution or lack of inspiration? J. Ind. Microbiol. Taxonomy, fulmentation, isolation and biological Biotechnol. 33:507-513. properties. J. Anti-biot., pp1547-1555. 2. Bredholt H, Fjaervik E, Jhonsen G, Zotechev SB (2008). 16. Ohshima, T., Takada, H., Yoshimura, T., Esaki, N., Soda, Actinomycetes from sediments in the Trondhein Fjrod, K.(1991). Distribution, Purification, and Characterization Norway: Diversity and biological activity. J. Mar. Drugs of Thermostable Phenyalanine Dehydrogenase from 6:12-24. Thermophillic actinomycetes. J. Bacteriol., 173: 3943- 3. Deepika L, Kannabiran K (2010) Isolation and 3948. Characterization of Antagonistic Actinomycetes from 17. Pelaez F (2006). The historical derive of antibiotic from Marine Soil. J Microbial Biochem Technol 2: 001-006. microbial natural product – can history repeat? J. doi:10.4172/1948-5948.1000015. Biochem. Pharmacol. 71:981-990. 4. Dhanasekaran D, Panneerselvam A, Thajuddin N (2008). 18. Prabhahar.C,. Saleshrani.K,Tharmaraj .K,and Saranraj.P An antifungal compound: 4' phenyl-1-napthyl–phenyl (2012). Investigation on antibacterial activity of acetamide from Streptomyces spp. DPTB16. Facta Gracilaria grassa extracts against pathogenic micro Universitatis Series: Med. Biol. 15:7-12. organisms,. International Journal of Recent Scientific 5. Ellaiah P, Kalyan D, Rao VS, Rao BV (1996). Isolation Research3:31 pp 166-171., and characterization of bioactive Actinomycetes from 19. Ravel J, Amoroso MJ, Colwell RR, Hill RT (1998). marine sediments. Hindustan Antibiot. Bull. 38:48-52. Mercury resistant Actinomycetes from the Chesapeake 6. Gebreyohannes, G, Moges. F, Sahile. S, Raja, N (2013) Bay. FEMS Microbiol. Lett. p. 162. Isolation and characterization of potential antibiotic 20. Remya,M and Vijayakumar, R (2008) Isolation and producing actinomycetes from water and sediments of characterization of marine antagonistic actinomycetes Lake Tana, Ethiopia. Asian Pac J Trop Biomed, 3(6): from West coast of India. Medicine and Biology, 15 (1): 426-435. 13 – 7. Goodfellow M, Haynes JA (1984). Actinomycetes in 21. Salle, A.J. Laboratory manual on Fundamental principles marine sediments. In: Biological, Biochemical and of Bacteriology ( 3rd edn.) New York: McGraw-Hill Biomedical Aspects of Actinomycetes. Oritz-Oritz, L., Book Company; 1948. Bojali, C. F. and Yakoleff, V. (eds.). Academic Press. 22. Shomura, T., Gomi, S., Ito, M., Yoshida, J., Tanaka, E., New York, London. pp. 453-463. Amono, S. (1987). Studies on new antibiotics SF2415 I. 8. Haque, S.K., Sen, S.K., Pal, S.C. (1992). Screening and taxonomy, fermentation, isolation, physico-chemical identification of antibiotic producing strains of Strepto- properties and biological activities. J. Antibiot., 11: 732- myces. Hind. Antibiot. Bull., 4: 76-83. 739. 9. Homans, A.L., Fuchs, A. (1970). Direct bioautography on 23. Sivakumar K, Sahu M, Kathiresan K (2005). Isolation thin-layer chromatograms as a method for detecting and characterization of Streptomycetes producing fungitoxic substances. J. Chromatogr., 51: 327-329. antibiotic from mangrove environment. Asian J. Microb. 10. James, P.D., Edwards, C., Dawson, M. (1991). The Biotechnol. Environ. Sci. 7:457-764. effects of temperature, pH and growth rate on secondary 24. Solingen P, Dean VM, Wilhelmus AHK, Christopher B, metabol-ism in Streptomyces thermoviolaceus grown in a Robertus B, Scott DP, Brian EJ (2001). From a novel chemostat. J. Gen. Microbiol., 137:1715-1720. Streptomycetes isolated from an East African Soda Lake. 11. Jensen PR, Gontang E, Mafnas C, Mincer TJ, Fenical W Extremophiles 5:333-341. (2005). Culturable marine Actinomycetes diversity from 25. Srinivasan MC, Laxman RS, Deshpande MV (1991). tropical Pacific Ocean sediments. Appl. Environ. Physiology and nutrition aspects of Actinomycetes – An Microbiol. 7:1039-1048. overview. World J. Microb. Biotechnol. 7:171-184. 12. Kalyani A.L.T, Ramya Sravani K. M, Annapurna J 26. Vijayakumar R, Muthukumar C, Thajuddin N, (2012) Isolation and characterization of antibiotic Pannerselvam A, Saravanamuthu R (2007). Studies on producing Actinomycetes from marine soil samples. the diversity of Actinomycetes in the PalkStrait region of International Journal of Current Pharmaceutical Research, Bay of Bengal, India. Actinomycetologica 21:59-65. 4(2): 109-112. 27. Vijayakumar R, Seethalakshmi V, Anitha S, 13. Kim HJ, Lee SC, Hwang BK (2006) Streptomyces Saravanamuthu R (2009). Isolation andCharacterization cheonanensis sp. nov., a novel streptomycete with of antagonistic Actinomycetes from Coimbatore soils, antifungal activity. Int J Syst Evol Microbiol 56: 471- Tamil Nadu, India. J. Sci. Trance Environ. Technol. 475. 2:191-201. 14. Kokare, C.R., Mahadik, K.R., Kadam, S.S., Chopade, 28. Weyland H (1969). Actinomycetes in North Sea and Atlantic Ocean sediments. Nature 223:858. *******

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