World J Microbiol Biotechnol (2009) 25:21032111 DOI 10.

1007/s11274-009-0113-4

ORIGINAL PAPER

Screening of marine actinomycetes isolated from the Bay of Bengal, India for antimicrobial activity and industrial enzymes
Subramani Ramesh Narayanasamy Mathivanan

Received: 27 March 2009 / Accepted: 2 July 2009 / Published online: 16 July 2009 Springer Science+Business Media B.V. 2009

Abstract A total of 288 marine samples were collected from different locations of the Bay of Bengal starting from Pulicat lake to Kanyakumari, and 208 isolates of marine actinomycetes were isolated using starch casein agar medium. The growth pattern, mycelial coloration, production of exopolysaccharides and diffusible pigment and abundance of Streptomyces spp. were documented. Among marine actinomycetes, Streptomyces spp. were present in large proportion (88%). Among 208 marine actinomycetes, 111 isolates exhibited antimicrobial activity against human pathogens, and 151 showed antifungal activity against two plant pathogens. Among 208 isolates, 183, 157, 116, 72 and 68 isolates produced lipase, caseinase, gelatinase, cellulase and amylase, respectively. The results of diversity, antimicrobial activity and enzymes production have increased the scope of nding industrially important marine actinomycetes from the Bay of Bengal and these organisms could be vital sources for the discovery of industrially useful molecules/enzymes. Keywords Bay of Bengal Actinomycetes Diversity Antimicrobial activity Extracellular enzymes

Introduction Marine microorganisms are increasingly becoming an important source in the search for industrially important molecules. Today both academic and industrial interest in

S. Ramesh N. Mathivanan (&) Biocontrol and Microbial Metabolites Lab, Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai, Tamil Nadu 600025, India e-mail: prabhamathi@yahoo.com

marine microorganisms are on the rise, because unique and biologically active metabolites have been reported from marine organisms (Jensen and William 1994; Imada 2004; Zhang et al. 2005). Actinomycetes are present in various ecological habitats such as soil, fresh water, back water, lake, compost, sewage and marine environment (Goodfellow and Williams 1983). They are considered highly valuable as they produce various antibiotics and other therapeutically useful compounds with diverse biological activities. The vast majority of these metabolites (70%) have been isolated from actinomycetes with the remaining 20% from fungi, 7% from Bacillus and 12% from Pseudomonas. Hence, it is known that the actinomycetes are perhaps the most important group of organisms studied extensively for the discovery of drugs and other bioactive metabolites programme (Lange and Lopez 1996; Prabavathy et al. 2006). Marine environment contains a wide range of distinct microorganisms that are not present in the terrestrial environment. Though some reports are available on antibiotic and enzyme production by marine actinomycetes, the marine environment is still a potential source for new actinomycetes, which can yield novel bioactive compounds and industrially important enzymes (Sharma and Pant 2001). Since late 1980s, the number of novel compounds isolated from terrestrial microorganisms has steadily decreased. To cope up with the demand for new pharmaceutical compounds and to combat the antibiotic resistant pathogens, researchers have been forced to look for novel microorganisms in unusual environment. Relatively, the Bay of Bengal, an arm of the Indian Ocean has rarely been explored for microbial diversity and microbial metabolites. Hence, there is an immense possibility to identify new marine actinomycetes in the Bay of Bengal to discover novel bioactive compounds. Accordingly, the present study

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was aimed to investigate the diversity of industrially important marine actinomycetes in the Bay of Bengal with the ultimate objective of discovering novel bioactive compounds.

Measurement of pH The pH of water samples was measured directly. Ten grams of each marine sediment sample was suspended in 20 ml of distilled water. It was allowed to stand for 20 min with intermittent stirring to reach equilibrium. After being left to settle, the pH was measured. Isolation of marine actinomycetes All the marine sediment and seawater samples were subjected to pre-heat treatment prior to serial dilution. Pre-heat treatment was performed by incubating the seawater and sediment samples in a water bath at 50C for 60 min (Takizawa et al. 1993). Ten grams of sediment samples were suspended in 95 ml of sterile aged seawater and these suspensions were considered as 10-1 dilution. Ten millilitre of seawater sample was suspended in 90 ml of sterile aged seawater and these suspensions were considered as 10-1 dilution. Starch casein agar (SCA) medium contained soluble starch 10 g, vitamin free casein 0.3 g, KNO3 2 g, NaCl 2 g, K2HPO4 2 g, MgSO4. 7H2O 0.05 g, CaCO3 0.02 g, FeSO47H2O 0.01 g, agar 20 g, natural aged seawater 1,000 ml, pH 7.0 0.2 was used for isolation of marine actinomycetes. Serial dilutions were done and overlaid on the surface of SCA. The medium also contained cycloheximide at 50 lg/ml to minimize fungal contamination. All the plates were incubated at room temperature (28 2C) for 21 days. The appearance and growth of marine actinomycetes were observed everyday on SCA plates and the colonies were recognized by their characteristic chalky to leathery appearance. Further, they were observed using a light microscope for their lamentous nature, spores, width of hyphae and spiral sporophores. Individual colonies were picked up, and subcultured on SCA and International Streptomyces Project medium 2 (ISP2) to ascertain their purity. Various colony characteristics such as size, shape, mycelial colour, exopolysaccharide (EPS) and diffusible pigment production were recorded. The pure cultures of marine actinomycetes were sub-cultured in SCA slants; incubated at room temperature for 57 days to achieve good sporulation; and then preserved in 20% glycerol vials at -80C (Williams and Cross 1971). Identication of actinomycetes The cover slip culture technique (Williams and Wilkins 1994) was used to study the morphological characteristics such as substrate and aerial mycelia, spores in chain and chain with coil formation, formation of rectiexibiles, retinaculipetri and spiral spores for all the isolates. In addition, nature of Grams staining, motility and cell wall

Materials and methods Study area The study area covered the Bay of Bengal coast of Tamil Nadu starting from Pulicat lagoon in the north to Kanyakumari in the south. This vast area has a variety of niches such as Pulicat lake, Ennore creek, Chennai harbour and several estuaries viz., Coovum, Adyar, Palar, Vellar, etc. Pulicat lake is the second largest brackish water lagoon in India which runs parallel to the Bay of Bengal. It is located at 60 km north-east of Chennai and is separated from the Bay of Bengal by Sriharikota island in Andhra Pradesh state. The lake is about 360 km2 in size and its depth (water column) varies from 1 to 6 m. To our knowledge, there is no report from this lake for microbial diversity. Pichavaram mangrove is located along the coast of Bay of Bengal with 11220 N to 11300 N wide and 79450 E to 79520 E long. The total area of this mangrove is about 1,470 ha consisting of about 50 small islands. Collection of samples A total of 80 seashore sediments, 43 seawater samples, 6 marine animals and 5 marine algal samples were collected from different locations in Tamil Nadu coast of the Bay of Bengal. Totally 21 mangrove sediments, 6 mangrove rhizosphere sediments and 10 water samples were collected from Pichavaram and Ennore mangroves in Tamil Nadu. At least half a kilometer of distance was maintained between the sampling stations. The sediment samples were collected at 23 m depth using grab sampler. Forty-one sediments and two brackish water samples were collected from Pulicat lake, Tamil Nadu at 57 m depth by grab sampler. In addition, 26 deep sea sediment samples, 4 corer sediments and 39 deep seawater samples were collected from the Bay of Bengal. The deep sea marine samples were collected during the cruise programme organized by the National Institute of Ocean Technology (NIOT), Chennai, India. A total of four sediments and one water samples were collected from the estuary of Adayar river and Marina beach, Chennai, Tamil Nadu. The sediments and water samples were collected in sterile polypropylene bags and screw cap bottles, respectively. The collected samples were brought to the laboratory for isolation of marine actinomycetes and the location, nature of sample and pH were documented.

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amino acid analysis (Becker et al. 1964) were also determined to identify the marine actinomycetes isolates up to genus level. Growth characteristics of marine actinomycetes All the isolates of marine actinomycetes were grown on SCA at room temperature and the growth rate was monitored every day up to 21 days. The isolates, which showed good growth in 4 days were considered as fast growers and those that showed good growth between 4 and 7 days were classied as moderate growers and the slow growers took more than 7 days for their growth. In addition to growth, mycelial colour was monitored in all the isolates of marine actinomycetes and documented. Screening of marine actinomycetes for antimicrobial activity All the 208 marine actinomycete isolates were screened for antibacterial and antifungal activity by cross streak and dual culture, respectively. In cross streak method, the marine isolates were streaked on modied nutrient agar (NA) (50% NA ? 50% SCA) as a straight line in the left side corner of the Petriplate and were incubated at room temperature for 5 days. After incubation, the test human bacterial pathogens (Staphylococcus epidermidis MTCC3615, Bacillus subtilis MTCC441, Pseudomonas aeruginosa MTCC1688, Escherichia coli MTCC1687 and Candida albicans MTCC227) were streaked at right angle to the original streak of the actinomycetes isolates. The zone of inhibition (ZOI) against human bacterial pathogens was measured after 48 h of incubation. Plates with the same medium without inoculation of actinomycetes but with simultaneous streaking of test organisms were maintained for controls. Four marine actinomycete isolates were streaked on modied potato dextrose agar (PDA) (50% PDA ? 50% SCA) as straight lines in four corners of the Petriplate and incubated at room temperature for 5 days. After incubation, a fresh mycelial disc of fungal phytopathogens (Rhizoctonia solani and Alternaria alternata) was placed in the center of each Petriplate and the ZOI against phytopathogens was measured after 5 days of incubation. The mycelial discs of the test phytopathogens were also kept in control plates where no actinomycetes were inoculated. Screening of marine actinomycetes for extracellular enzymes production All the isolated marine actinomycetes were screened qualitatively for the production of ve important enzymes such as lipase, caseinase, gelatinase, cellulase and amylase.

Each actinomycete strain was streaked on the four corners of the respective substrates such as starch, carboxyl methyl cellulose, gelatin, casein and tween 20 amended agar plates separately and was incubated for 5 days at room temperature. Then the plate was ooded with relevant indicator solution and the development of clear zone around the growth of organism was considered positive for enzyme activity.

Results and discussion Actinomycetes constantly hold a special signicance in the research arena for the past 60 years as the members of this group, especially streptomycetes, are known to produce a vast array of compounds with diverse biological properties. The discovery of new bioactive compounds is a never ending process to meet the everlasting demand for novel drug and other biomolecules with antimicrobial and therapeutic properties in order to combat human and plant pathogens and also to treat other human ailments. In this scenario, it is more important to identify newer or rare actinomycetes because they are the pivotal sources of potent molecules. Marine environment is the biggest reservoir of chemical and biological diversity. Therefore, research focus on marine environment has been gaining importance in recent years. However, still it has not been fully explored and there is tremendous potential to identify novel organisms with various biological properties. In line with this view, the present research has been initiated to identify novel actinomycetes from Indian marine environment, because its rich microbial diversity has been studied only to a limited extent. Totally 288 different marine samples were collected from various locations of the Bay of Bengal, India (Fig. 1). Among them, a total of 98 actinomycetes were isolated from marine sediments, ve from seawater and nine from marine animals (star shes, mollusks and sea urchins), but no actinomycetes were isolated from marine algae. From Pulicat lake samples, 30 actinomycetes were isolated from sediments and 13 from brackish water. From mangrove, 15 actinomycetes were isolated from sediments, 7 from mangrove rhizospheres and none of the strains were isolated from mangrove water. In addition, 18 actinomycete strains were isolated from deep sea sediments and one strain was isolated from deep seawater and corer sediments, respectively. A total of seven actinomycetes were isolated from estuary sediments and four from estuary water samples (Table 1). Although soils are considered excellent sources for the isolation of actinomycetes with diverse potential (Ouhdouch et al. 2001; Lee and Hwang 2002; Prabavathy 2005; Malarvizhi 2006), several actinomycetes have been isolated from marine samples (Sujatha et al. 2005; Maldonado et al.

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Fig. 1 Sampling locations in the Bay of Bengal, India Table 1 Details of the isolation of marine actinomycetes from the Bay of Bengal, India Location Nature of sample No. of sample 80 43 5 6 26 39 4 6 21 10 4 1 41 2 288 No. of actinomycetes isolate(s) 98 5 0 9 18 1 1 7 15 0 7 4 30 13 208

Coastal

Sediments Seawater Macro algae Animals

Deep sea

Sediments Seawater Corer sediments

Mangrove

Rhizosphere sediments Sediments Water

Estuary Brackish lake Total

Sediments Water Sediments Brackish water

2005; Fenical and Jensen 2006; Ramesh et al. 2006, 2009). The isolation of actinomycetes from marine sediments was well documented, yet the proportion of these lamentous bacteria which represents the indigenous marine microora remains unclear. This question persists, in part, because there is little published information describing the distribution, growth and ecological role of actinomycetes in

marine habitats. As actinomycetes represent a small component of the total bacterial population in marine sediments (Goodfellow and Williams 1983), their role in the marine environment is difcult to assess. Nevertheless, these marine actinomycetes are considered economically important as often they are reported to produce valuable bioactive molecules and industrially important enzymes (Jensen et al. 2005b; Ramesh et al. 2009). In this study, 208 different actinomycetes were isolated from 288 marine samples using SCA selective medium prepared in aged seawater. It has already been reported that the aged seawater amended media were used to isolate and maintain the marine microorganisms. Although, a number of selective media (Kuster and Williams 1964; Hayakawa and Nonomura 1987; Crawford et al. 1993; Duangmal et al. 2005; Jensen et al. 2005a) were developed for isolation of actinomycetes, SCA was selected, because in this medium the development of bacterial and fungal colony was very much suppressed, allowing only the actinomycetes to grow. Distribution of actinomycetes is inuenced by the pH of the respective environment. In the present study, among the 208 marine actinomycetes, 99 isolates were isolated in the pH between 8.1 and 8.5, which was followed by the pH range of 7.68.0 from which 92 isolates were obtained. However, only eight marine actinomycetes were isolated from the pH range of 7.07.5 (Table 2). This is in agreement with the ndings of Taber (1960) who demonstrated that most actinomycetes prefer neutral or slightly alkaline soils for their growth. Similar to the present results,

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World J Microbiol Biotechnol (2009) 25:21032111 Table 2 Potential of marine actinomycetes isolated from different pH on antimicrobial activity and extracellular enzyme production

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pH range

Total isolates 8 92 99 199

No. of isolates with antimicrobial activity Against human pathogens 3 52 50 105 Against plant pathogens 6 76 88 170

No. of isolates with extracellular enzymes 8 88 96 192

7.07.5 7.68.0 8.18.5 Total

250

200

150

100

50

0 Total isolates Fast Moderate Slow

Growth rate

Fig. 2 Growth rate of marine actinomycetes isolated from the Bay of Bengal

Malarvizhi (2006) obtained 33% of the actinomycetes from soils with pH between 7.6 and 8.5. In contrast, Lee and Hwang (2002) isolated many streptomycetes even from acidic soil with pH less than 5.0. They also reported the distribution of other actinomycetes genera such as Micromonospora, Dactylosporangium, Streptosporangium, Actinomadura and Nocardioformis in soils at pH 4.05.0. Further, they observed that Streptomyces were predominant in soils with a pH range of 5.16.5. All the isolated marine actinomycetes were screened for growth rate on SCA. Surprisingly, among 208 isolates, 151
Fig. 3 Mycelial colouration of marine actinomycetes isolated from the Bay of Bengal
79

isolates (72.59%) showed fast growth, 47 (22.59%) exhibited slow growth and the remaining of 10 isolates (4.8%) showed moderate growth (Fig. 2). The results clearly revealed that all the marine actinomycetes are not slow growing microorganisms and most of their growth is comparable with lamentous fungi. Among 208 isolates, 115, 79, 6, 7 and 1 were grey, white, blue, pink and orange pigmented, respectively. Interestingly, grey and white mycelial pigmented marine actinomycetes were prominent in the Bay of Bengal. Further, out of 208 isolates, 6 produced diffusible pigment on SCA agar and 58 isolates produced EPS (Fig. 3). These pigments and EPS production could be protective mechanisms for actinomycetes to survive in the hostile marine environment. Ramesh et al. (2006) isolated many EPS producing and pigmented actinomycetes in the post-Tsunami periods from the Bay of Bengal and most of them were able to survive for a long period in marine environment compared to non-pigmented and non-EPS producing actinomycetes. The adaptation of marine microorganisms to the diverse marine habitats provides seemingly limitless evolutionary opportunities for the production of unique secondary metabolites. The colonies of actinomycetes were elevated, convex and powdery in nature. Many of such morphological characteristics are common in most of the streptomycetes (Anderson and Wellington 2001; Lo et al. 2002; Fguira et al. 2005; Sujatha et al. 2005). Most of the marine

Number of isolates

58 7

208

6 115 1

Total isolates White

Orange Pink

Grey EPS

Blue Diffusible pigment

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25

183

208

Total isolates

Streptomycetes

Others

Fig. 4 Abundance of streptomycetes in the Bay of Bengal

actinomycetes exhibited different mycelial colourations. The spore morphology is considered as one of the important characteristics in the identication of Streptomyces and it greatly varies among the species (Tresner et al. 1961). It has been found that the majority of the marine isolates produced aerial coiled mycelia and the spores arranged in chains as already reported by Mukherjee and Sen (2004) and Roes and Meyer (2005). Further, results of the tests as outlined in the Bergeys Manual of Determinative Bacteriology (Williams et al. 1989) and the Laboratory Manual for Identication of Actinomycetes (IMTECH 1998) showed that among the 208 actinomycetes isolated from the Bay of Bengal, 183 isolates were identied as Streptomyces spp. and the remaining 25 isolates belonged to other genera. Remarkably, majority of the marine actinomycetes (87.98%) were Streptomyces spp. (Fig. 4). The cell wall composition is an important criterion for the identication of Streptomyces (Sujatha et al. 2005) and chemotaxonomic investigation using isomeric diaminopimelic acid (DAP) conguration was already established (Becker et al. 1964; Lechevalier and Lechevalier 1970). It

has been reported that the streptomycetes are common inhabitants of marine environments (Kokare et al. 2004a, b; Fiedler et al. 2005; Ramesh et al. 2006), though other actinomycetes are also present (Jensen et al. 1991; Mincer et al. 2002; Magarvey et al. 2004; Maldonado et al. 2008). The degree of antimicrobial activity varied greatly among the actinomycetes as shown in Figs. 5 and 6. Among 208 isolates, 111 isolates (53%) showed high antimicrobial activity against human pathogens (Fig. 5). Of which, 31, 18, 52, 81, 28 isolates exhibited antimicrobial activity against E. coli, P. aeruginosa, S. epidermidis, B. subtilis and C. albicans, respectively. On the other hand, four isolates showed antimicrobial activity against all of the ve pathogens, but 97 isolates did not show antimicrobial activity. Several researchers have already reported similar antimicrobial activity of actinomycetes against various human pathogens. Saadoun and Gharaibeh (2003) obtained 90 different Streptomyces isolates, of which, 54% exhibited remarkable antibacterial activity against B. subtilis, S. aureus, E. coli, Klebsiella sp. and Shigella sp. Deshmukh and Sridhar (2002) isolated several actinomycetes from freshwater coastal stream, of which, four isolates inhibited B. subtilis and E. coli. In addition, Imada et al. (2007) isolated 100 actinomycete strains from various locations of the Otsuchi Bay and found that 59 strains produced antibacterial activity. In the present study it was recorded that 151 isolates (72%) showed antifungal activity against plant pathogens. 107 and 98 isolates exhibited antifungal activity against R. solani and A. alternata, respectively. On the other hand, 54 isolates showed antifungal activity against both the fungal phytopathogens, but 57 isolates did not exhibit antifungal activity (Fig. 6). Similarly, Yuan and Crawford

Fig. 5 Antimicrobial activity of marine actinomycetes against human pathogens

97 28 4 208

81

52 18 31

111

Total isolates E. coli S. epidermidis C. albicans No activity

Total antimicrobial activity P. aeruginosa B. subtilis Activity against five pathogens

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57 54 98 208

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107

151
Total antifungal activity A. alternata No activity

Total isolates R. solani Activity against two pathogens

Fig. 6 Antifungal phytopathogens

activity

of

marine

actinomycetes

against

(1995) demonstrated in vitro antagonism of Streptomyces lydicus against various fungal phytopathogens in plate assay. Further, they observed the inhibition of mycelial growth in Pythium ultimum and R. solani when grown in liquid medium with S. lydicus. Kathiresan et al. (2005) have isolated 160 actinomycetes from various mangrove environments in India and demonstrated their antifungal activity against plant pathogenic fungi. Zaitlin et al. (2004) demonstrated the antagonistic activity of Streptomyces halstedi and Streptomyces rochei against many phytopathogenic fungi. Search for novel secondary metabolites with diverse biological activity in assorted environment has gained greater attention in recent years. Among 99 marine actinomycetes isolated from the pH between 8.1 and 8.5, 50 (50.5%) and 88 (88.9%) isolates exhibited antimicrobial activity against human and plant pathogens, respectively. Among 92 isolates from the pH between 7.6 and 8.0, 52 (56.5%) and 76 (82.6%) marine

actinomycetes exhibited antimicrobial activity against human and plant pathogens, respectively. Among eight isolates from the pH range 77.5, 3 (37.5%) and 8 (100%) marine actinomycetes showed antimicrobial activity against human and plant pathogens, respectively (Table 2). With the growing awareness on environmental protection, the use of enzymes, particularly from extremophiles, gained considerable attention in many industrial processes. In recent years, the microbial enzymes have been replacing chemical catalysts in manufacturing chemicals, textiles, pharmaceuticals, paper, food and agricultural chemicals. Enzyme-based industrial bioprocess now directly competes with established chemical-based process within the processed foods, pharmaceutical and allied fermentation industries. In the case of terrestrial actinomycetes, many researchers reported the production of various industrial enzymes (Mohamedin 1999; Azeredo et al. 2001; Pandhare et al. 2002; Stamford et al. 2002; Goshev et al. 2005; Sharma et al. 2005). But to date, it has been concluded that there are not many reports on the extracellular enzymes from the marine actinomycetes. However, in this study, among the 208 isolates, 183, 157, 116, 72, 68 isolates produced lipase, caseinase, gelatinase, cellulase and amylase, respectively (Fig. 7). Interestingly, 22 isolates produced all the ve enzymes. The majority of actinomycete strains isolated from the Bay of Bengal produced lipase followed by caseinase, gelatinase, cellulase and amylase. Similarly, Leon et al. (2007) isolated many actinomycetes from marine sediments of the central coast of Peru with multi-enzyme activity. Notably, a strain among 208 isolates, identied as Streptomyces fungicidicus MML1614, was able to produce a thermostable alkaline protease (Ramesh et al. 2009). These results indicated the potential of marine actinomycetes from the Bay of Bengal for the

Fig. 7 Extracellular enzymes production in marine actinomycetes

72 68

22 208

116 183 157

Total isolates Amylase

Lipase Cellulase

Caseinase Five enzymes

Gelatinase

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World J Microbiol Biotechnol (2009) 25:21032111 Duangmal K, Ward CA, Goodfellow M (2005) Selective isolation of members of the Streptomyces violaceoruber clad from soil. Microbiol Lett 245:321327 Fenical W, Jensen PR (2006) Developing a new resource for drug discovery: marine actinomycete bacteria. Nat Chem Biol 2: 666673 Fguira L, Fotso S, Ben Ameur-Mehdi R, Mellouli L, Laatsch H (2005) Purication and structure elucidation of antifungal and antibacterial activities of newly isolated Streptomyces sp. strain US80. Res Microbiol 156:341347 Fiedler HP, Bruntner C, Bull AT, Ward AC, Goodfellow M, Potterat O, Puder C, Mihm G (2005) Marine actinomycetes as a source of novel secondary metabolites. Antonie Van Leeuwenhoek 87: 3742 Goodfellow M, Williams ST (1983) Ecology of actinomycetes. Annu Rev Microbiol 37:189216 Goshev I, Gousterova A, Vasileva-Tonkova E, Nedkov P (2005) Characterization of the enzyme complexed produced by two newly isolated thermophilic actinomycete strains during growth on collagen rich materials. Process Biochem 40:16271631 Hayakawa M, Nonomura H (1987) Humic acid vitamin agar, a new medium for the selective isolation of soil actinomycetes. J Ferment Technol 65:501509 Imada C (2004) Enzyme inhibitors and other bioactive compounds from marine actinomycetes. Antonie von Leewenhoek 87: 5963 Imada C, Koseki N, Kamata M, Kobayashi T, Hamada-Sato N (2007) Isolation and characterization of antibacterial substances produced by marine actinomycetes in the presence of seawater. Actinomycetologica 21:2731 IMTECH (1998) Laboratory manual for identication of actinomycetes. Institute of Microbial Technology, Chandigarh, p 94 Jensen P, William F (1994) Strategies for the discovery of secondary metabolites from marine bacteria. Ecological perspectives. Annu Rev Microbiol 48:559584 Jensen P, Dwight R, Fenical W (1991) The distribution of actinomycetes in near-shore tropical marine sediments. Appl Environ Microbiol 57:11021108 Jensen PR, Gontang E, Mafnas C, Mincer TJ, Fenical W (2005a) Culturable marine actinomycetes diversity from tropical Pacic ocean sediments. Environ Microbiol 7:10391048 Jensen PR, Mincer TJ, Williams PG, Fenical W (2005b) Marine actinomycete diversity and natural product discovery. Antonie Van Leeuwenhoek 87:4348 Kathiresan K, Balagurunathan R, Masilamani Selvam M (2005) Fungicidal activity of marine actinomycetes against phytopathogenic fungi. Indian J Biotechnol 4:271276 Kokare CR, Mahadik KR, Kadam SS, Chopade BA (2004a) Isolation, characterization and antimicrobial activity of marine halophilic Actinopolyspora species AH1 from the west coast of India. Curr Sci 86:593597 Kokare CR, Mahadik KR, Kadam SS, Chopade BA (2004b) Isolation of bioactive marine actinomycetes from sediments isolated from Goa and Maharashtra coastlines (west coast of India). Indian J Mar Sci 33:248256 Kuster E, Williams S (1964) Selection of media for the isolation of Streptomyces. Nature 202:928929 Lange L, Lopez CS (1996) Microorganisms as a source of biologically active secondary metabolites. In: Copping LG (ed) Crop protection agents from nature: natural products, analogues. The royal society of chemistry, Cambridge Lechevalier HA, Lechevalier MP (1970) A critical evaluation of the genera of aerobic actinomycetes. In: Prauser H (ed) The actinomycetes. Gustav F Ischer-Verlag, Jena, pp 393405 Lee JY, Hwang BK (2002) Diversity of antifungal actinomycetes in various vegetative soils of Korea. Can J Microbiol 48:407417

production of various industrial enzymes. Importantly, majority of the actinomycetes (87.98%) obtained from the Bay of Bengal produced lipase. The population of lipase producing actinomycetes is relatively large in marine environment, because the ocean contains signicant amounts of polymers. Microbes have to produce lipase enzyme to degrade the polymers in order to adapt in the extreme environment. Among the 99 isolates obtained from the pH between 8.1 and 8.5, 96 (97%) marine actinomycetes exhibited extracellular enzyme production, which is of signicance. Among 92 isolates from the pH between 7.6 and 8.0, 88 (95.7%) showed extracellular enzyme production. However, all of the eight isolates (100%) obtained from the pH range of 77.5 produced extracellular enzyme (Table 2).

Conclusion Marine actinomycetes are metabolically active more vigorously in the marine environment, which leads to the production of various enzymes and bioactive compounds compared to terrestrial strains. Therefore, it is important to understand the marine-derived actinomycetes in ecological terms and also as a resource for biotechnology. Our present study and other reports from our lab (Ramesh 2009; Ramesh et al. 2009) evidently revealed that the Bay of Bengal is a potential source for a wide spectrum of antimicrobial and industrial enzyme producing actinomycetes. Moreover, it can be an imperative resource for bioprospecting novel/ rare Streptomyces spp., which could yield valuable bioactive molecules.
Acknowledgments We thank the Director, CAS in Botany, University of Madras and the Director, National Institute of Ocean Technology, Chennai for laboratory facilities and organizing the Cruise programmes, respectively.

References
Anderson AS, Wellington EMH (2001) The taxonomy of Streptomyces and related genera. Int J Syst Evol Microbiol 51:797814 Azeredo LAI, Leite SGF, Freire DMG, Benchetrit LC, Coelho RRR (2001) Proteases from actinomycetes interfere in solid media plate assays of hyaluronidase activity. J Microbiol Methods 45:207212 Becker B, Lechevalier MP, Gordon RE, Lechevalier HA (1964) Rapid differentiation between Nocardia and Streptomyces by paper chromatography of whole cell hydrolysate. Appl Microbiol 12:421424 Crawford DI, Lynch JM, Whipps JM, Ousley MA (1993) Isolation and characterization of actinomycetes antagonistic to a fungal root pathogen. Appl Environ Microbiol 59:38993909 Deshmukh MB, Sridhar KR (2002) Distribution and antimicrobial activity of actinomycetes of a fresh water coastal stream. Asian J Microbiol Biotech Environ Sci 4:335340

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World J Microbiol Biotechnol (2009) 25:21032111 Leon J, Liza L, Soto I, Cuadra D, Patino L, Zerpa R (2007) Bioactives actinomycetes of marine sediment from the central coast of Peru. Revi Peru Boil 14:259270 Lo CW, Lai NS, Cheah HY, Wong NKI, Ho CC (2002) Asian review of biodiversity and environmental conservation. http://www. arbc.com.my/pdf/art21julysep02.pdf Magarvey NA, Keller JM, Bernan V, Dworkin M, Sherman DH (2004) Isolation and characterization of novel marine-derived actinomycete taxa rich in bioactive metabolites. Appl Environ Microbiol 70:75207529 Malarvizhi K (2006) Biodiversity and antagonistic potential of soil actinomycetes from south India: isolation, purication and characterization of antimicrobial metabolites produced by Streptomyces sp. MML1042. Ph.D. Thesis, University of Madras, Chennai, India Maldonado LA, Fenical W, Jensen PR, Kauffman CA, Mincer TJ, Wrad AC, Bull AT, Goodfellow M (2005) Salinispora arenicola gen. nov., and Salinispora tropica nov., obligate marine actinomycetes belonging to the family Micromonosporaceae. Int J Syst Evol Microbiol 55:17591766 Maldonado LA, Fragoso-Yanez D, Perez-Garca A, Rosellon-Druker J, Quintana ET (2008) Actinobacterial diversity from marine sediments collected in Mexico. Antonie van Leeuwenhoek 95:111120. doi: 10.1007/s10482-008-9294-34 Mincer TJ, Jensen PR, Kauffman CA, Fenical W (2002) Wide spread and persistent populations of a major new marine actinomycete taxon in ocean sediments. Appl Environ Microbiol 68:5005 5011 Mohamedin AH (1999) Isolation and identication and some cultural conditions of a protease producing thermophilic Streptomyces strain grown on chicken feather as a substrate. Int J Biodeter Biodeg 43:1321 Mukherjee G, Sen SK (2004) Characterization and identication of chitinase producing Streptomyces venezulae P10. Indian J Exp Biol 42:541544 Ouhdouch Y, Barakate M, Finanse C (2001) Actinomycetes of Moroccan habitats: isolation and screening for antifungal activities. Eur J Soil Biol 37:6974 Pandhare J, Zog K, Deshpande VV (2002) Differential stabilities of alkaline protease inhibitors from actinomycetes: effect of various additives on thermostability. Bioresour Technol 84:165169 Prabavathy VR (2005) Isolation, purication and characterization of antimicrobial metabolites produced by Streptomyces sp. and evolution against blast and sheath diseases of rice. Ph.D Thesis, University of Madras, India Prabavathy VR, Mathivanan N, Murugesan K (2006) Control of blast and sheath blight diseases of rice using antifungal metabolites produced by Streptomyces sp. PM5. Biol Control 39:313319 Ramesh S (2009) Marine actinomycetes diversity in Bay of Bengal, India: Isolation and characterization of bioactive compounds from Streptomyces fungicidicus MML1614. Ph. D. thesis, University of Madras, Chennai, India

2111 Ramesh S, Jayaprakashvel M, Mathivanan N (2006) Microbial status in seawater and coastal sediments during pre- and post-tsunami periods in the Bay of Bengal, India. Mar Ecol 27:198203 Ramesh S, Rajesh M, Mathivanan N (2009) Characterization of a thermostable alkaline protease produced by marine Streptomyces fungicidicus MML1614. Bioprocess Biosyst Eng. doi: 10.1007/ s00449-009-0305-1 Roes LM, Meyer PR (2005) Streptomyces pharetrae sp. nov., isolated from soil from the semi-arid Karoo region. Syst Appl Microbiol 28:488493 Saadoun I, Gharaibeh R (2003) The Streptomyces ora of Badia region of Jordan and its potential as a source of antibiotics active against resistant bacteria. J Arid Environ 53:365371 Sharma SL, Pant A (2001) Crude oil degradation by marine actinomycetes Rhodococcus sp. Indian J Mar Sci 30:146150 Sharma AD, Kainth S, Gill PK (2005) Inulinase production using garlic (Allium sativum) powder as a potential substrate in Streptomyces sp. J Food Eng 77:16 Stamford TLM, Stamford NP, Coelho LCBB, Araujo JM (2002) Production and characterization of a thermostable glucoamylase from Streptosporangium endophyte of maize leaves. Bioresour Technol 83:105109 Sujatha P, Bapi Raju KVVSN, Ramana T (2005) Studies on a new marine Streptomycete BT-408 producing polyketide antibiotic SBR-22 effective against methicillin resistant Staphylococcus aureus. Microbiol Res 160:119126 Taber WA (1960) Evidence for the existence of acid-sensitive actinomycetes in soil. Can J Microbiol 6:503 Takizawa M, Colwell RR, Hill RT (1993) Isolation and diversity of actinomycetes in the Chesapeake Bay. Appl Environ Microbiol 59:9971002 Tresner HD, Davies MC, Backus EJ (1961) Electron microscopy of Streptomyces spore morphology and its role in species differentiation. J Bacteriol 81:7080 Williams ST, Wilkins (1994) Bergeys manual of determinative bacteriology, 9th edn. Williams and Wilkins, Baltimore Williams ST, Cross T (1971) Actinomycetes, methods in microbiology, vol 4. Academic Press, New York Williams ST, Goodfellow M, Alderson G (1989) Genus Streptomyces Waksman and Henrici 1943, 399AL. In: Williams ST, Sharpe ME, Holt JG (eds) Bergeys manual of systematic bacteriology, vol 4. Williams and Wilkins, Baltimore, pp 24522492 Yuan W, Crawford DL (1995) Characterization of Streptomyces lydicus WYEC 108 as a potential biocontrol agent against fungal root and seed rots. Appl Environ Microbiol 61:31193128 Zaitlin B, Turkington K, Parkinson D, Clayton G (2004) Effects of tillage and inorganic fertilizers on culturable soil actinomycetes communities and inhibitors of fungi by specic actinomycetes. Appl Soil Ecol 26:5362 Zhang L, An R, Wang J, Sun N, Zhang S, Hu J, Kuai J (2005) Exploring novel bioactive compounds from marine microbes. Curr Opinion Microbiol 8:276281

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