Isolation of bioactive marine actinomycetes from sediments isolated from Goa and Maharashtra coastlines (west coast of India)

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1 Indian Journal of Marine Sciences Vol. 33(3), September 2004, pp Isolation of bioactive marine actinomycetes from sediments isolated from Goa and Maharashtra coastlines (west coast of India) C R Kokare, K R Mahadik & S S Kadam Bharati Vidyapeeth Deemed University, Poona College of Pharmacy, Erandwane, Pune , India and B A Chopade* Department of Microbiology and Institute of Bioinformatics and Biotechnology, University of Pune, Pune , India *[ chopade@unipune.ernet.in] Received 20 May 2003; revised 23 March 2004 Twenty marine sediment samples were collected at the time of low tide from Maharashtra and Goa coasts in July Heat pretreatment at 41 C for 1 to 2 months was found to be most effective for the isolation of marine actinomycetes. Eight different selective media were used for isolation of actinomycetes and starch casein agar and glucose asparagine agar media prepared in natural seawater showed good growth of actinomycetes. Best growth of actinomycetes was observed at 28 C after incubation for three weeks. Significant differences were found between the counts of actinomycetes on different media. Out of 80 actinomycetes, 59 actinomycetes showed fast growth, 21 showed slow growth and 53 actinomycetes showed pigmentation of brown (5), gray (30), orange (2), black (8), yellow (4), red (2) and greenish (2) colors. Antibacterial and antifungal activities were tested using cross streak and cylinder plate technique. Out of 80 strains of actinomycetes, 35 showed bioactive activities. Out of 35 strains, 20 showed strong antimicrobial activity against different types of organisms and remaining 15 showed very weak antimicrobial activity. From these twenty strongly bioactive strains, 16 showed activity against Gram-positive, 2 against Gram-negative microorganisms and 7 showed antifungal activities. Out of these actinomycetes, 16 belonged to genus Streptomyces and one each to Streptoverticillium, Catellatospora, Nocardia and Actinopolyspora. Interestingly, Actinopolyspora and Catellatospora species were found in sediments and it shows good antimicrobial activity. Present study clearly indicates that sediment samples from Alibag, Janjira and Goa are potent sources for the isolation of bioactive actinomycetes. [Key words: Marine sediment, pretreatment, selective media, pigmentation, antimicrobial activity, Maharashtra coast, Goa coast, Alibag, Janjira] [IPC Code: Int. Cl. 7 C12N 1/02, C12Q 1/02 ] Introduction Marine microorganisms have become important in the search for novel microbial products. Many marine microorganisms contain substances that have antimicrobial 1, antiviral 1, anticoagulant 2 and cardioactive properties 3. Some of these substances have chemical structures that are unlike any compounds found in terrestrial species 3,4. These active compounds may serve as models in the discovery of new drugs. Marine habitats may yield strains having new genetic elements such as plasmids, which may produce new metabolites 4. Actinomycetes are a group of bacteria which possess many important and interesting features 4. The isolation of actinomycetes from mixed microflora present in nature is complicated by their characteristic slow growth relative to that of other bacteria 4,5. They are of considerable value as antibiotic producers and of other therapeutically useful compounds with diverse biological activites 6,7. There are few reports on antibiotic production from marine actinomycetes. The marine environment is a potential source for new actinomycetes and novel antibiotics 7. Streptomyces, Micromonospora and Nocardia are the three most common genera in the marine environment 7,8. In the last decade a few novel bioactive substances have been isolated from marine strains of streptomycetes and other actinomycetes 8,9. There are very few reports on antimicrobial activity from marine actinomycetes from west coast of India 9,10. This paper deals with the isolation and characterization of bioactive actinomycetes from marine sediments from west coast of India.

2 KOKARE et al.: ISOLATION OF BIOACTIVE MARINE ACTINOMYCETES 249 Materials and Methods Twenty marine sediment samples were collected from different sites of Alibag (17 52 N; E) and Janjira (17 12 N; E) coast of Maharashtra and Goa (Arambol coast, Madgaon, N; E) during low tide in July 1999 (salinity 35 o / oo, temperature 21 C). Samples were collected 0.4 km from coastal area and at 4 cm depth. The surface layers of sediments were removed and central portions of sediments, approximately 0.5 kg each were aseptically transferred into sterile plastic bags. The samples were kept in cold box containing ice and transferred to laboratory. Ten gram of each sediment sample was air dried in laminar airflow for hr. These samples were transferred to a sterile Petri plate and kept at 41 C for 10, 30 and 60 days. These preheat-treated samples were used for isolation of actinomycetes 11. Ten gram of each sediment sample and heat-treated sample were suspended in 100 ml of sterile natural seawater. Flasks were rotary shaken at 150 rpm for 30 min and from this suspension dilutions were prepared as 1:10, 1:100, 1:1000 and 1: About 200 µl of each suspension were spread over the surface of different agar media prepared by using natural seawater and the plates incubated at 28 C for 2 to 3 weeks. Eight different media namely modified starch casein agar(sca), glucose asparagine agar(gaa), glycerol asparagine agar(glaa), tyrosine agar(ta), yeast malt extract agar(ymea), nutrient agar(na), maltose yeast extract agar(mye) and glycerol glycine medium(mgg) were employed for the isolation of marine actinomycetes 12. Preheated samples (0.5 g) were directly sprinkled on different agar media. After 3 to 7 days, total number of bacteria, fungi and actinomycetes present on each plate were counted. Plates were observed for 28 days. Actinomycetes were purified on the respective selective medium by using separate Petri plates and then transferred on slants for storage at 4 C temperature. Morphological and cultural characteristics were studied as described by Shirling & Gottlieb 11. Morphological characteristics were observed by using the slide culture technique. Growth of actinomycetes was observed on different media and characteristic growth of aerial mycelium and substrate mycelium was studied. Spore chain structure was studied by scanning electron microscopy (SEM, Cambridge, UK). The growth on slide cultures was fixed by using 2% (v/v) glutaraldehyde and dehydrated for 15 minutes at refrigerator by using different concentrations of alcohol (10 to 90% v/v). After dehydration the upper surface of each coverslip was gold coated, under vacuum, with a film, about 500 Å thick. The material was scanned using Cambridge, UK scanning electron microscope at a magnification of Suitable subjects were photographed using Ilford H.P.3 film. Biochemical tests were performed as described by Shirling & Gottlieb 11. Carbon utilization was performed by using basal medium and by using 1% (w/v) carbon sources which was sterilized by membrane filtration. Nitrogen utilization was performed by using 0.1% (w/v) nitrogen sources. Starch hydrolysis, proteolytic activity, melanin pigmentation, H 2 S production, nitrate reduction and gelatin liquefaction were tested 9. Growth of different isolates was observed by using 0, 3.5, 7 and 14% (w/v) concentration of sodium chloride. Tolerance of ph was studied at 3.5, 5, 7 and 9 ph. Maximum temperature for growth of different species 13,14 was tested at 4, 28, 37 and 41 C. The results were recorded after 1, 2 and 3 weeks. Actinomycetes identification was made, based on characteristic growth on different media, pigmentation, growth and arrangement of spores by slide cultures technique, requirements of sodium chloride, temperature and ph for growth, cultural and physiological characteristics 15,16. Eighty actinomycetes, isolated from twenty marine sediment samples were screened for their antimicrobial activity by two methods: i) Cross streak method: The actinomycete isolates were inoculated as a single streak onto dried plates of starch casein agar medium and incubated at room temperature (28±1 C). After seven days growth, the test organisms were streaked at right angles to the original streak of the isolates. The plates were then incubated at 37 C for bacterial test organisms and at 25 C for fungal organisms. The zone of inhibition was measured after an incubation period of 24 to 48 hr for bacteria and yeast and after 48 to 72 hr for filamentous fungi. Control plates of the same medium without actinomycetes growth were also simultaneously streaked with test organisms to assess their normal growth. ii) Cylinder plate method: The isolates were grown on different media at 28 C for 7 days and then transferred to plates containing test organisms for antimicrobial activity. Plates were incubated at standard conditions for bacteria and fungi, after which the zone of inhibition was recorded. The standard test organisms were obtained from national collection of industrial microorganisms (NCIM) at National Chemical Laboratory, Pune. These were:

3 250 INDIAN J. MAR. SCI., VOL. 33, NO. 3, SEPTEMBER 2004 Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Aspergillus niger, Aspergillus fumigatus, Candida albicans, Penicillium sp. and Cryptococcus sp Results Enrichment of marine actinomycetes with preheat treatment Alibag, Janjira and Goa coast sediment samples were found to contain , and CFU/g of actinomycetes on starch casein agar respectively. By heat treatment at 41 C for 30 days counts observed were , and CFU/g on starch casein agar of Alibag, Janjira and Goa samples respectively (Table 1). Goa sample contained more number of actinomycetes as compared to Alibag and Janjira. Preheat-treatment reduced the other contaminating organisms like bacteria and fungi and increased the actinomycete counts (Table 1). Preheat-treatment at 41 C for 60 days was found to contain much less number of bacteria and no fungi. Isolation of marine actinomycetes on various media Starch casein agar and glucose asparagine agar media prepared in natural seawater yielded good growth and more number of actinomycetes as compared to other media in all samples. Total counts of actinomycetes, other bacteria and fungi on different media are shown in Table 1. Yeast malt extract agar and nutrient agar favoured the growth of fungi and bacteria respectively and showed less number of actinomycetes. Effect of sodium chloride, ph and temperature on growth Out of 80 actinomycete isolates, 20 were selected Table 1 Total count of actinomycete, bacteria and fungi on different media employing preheat treatment Sample Treatment Microrganisms Media place in days ( 10 3 /g) SCA GAA GlAA TA YMEA NA MYE MGG Alibag Nil Actinomycetes Bacteria Fungi Actinomycetes Bacteria Fungi Actinomycetes Bacteria Fungi Janjira Nil Actinomycetes Bacteria Fungi Actinomycetes Bacteria Fungi Actinomycetes Bacteria Fungi Goa Nil Actinomycetes Bacteria Fungi Actinomycetes Bacteria Fungi Actinomycetes Bacteria Fungi SCA- Starch casein agar, GAA- Glycerol asparagine agar, GIAA- Glucose aspargine agar, TA- Tyrosine agar, YMEA-Yeast malt extract agar, NA- Nutrient agar, MYE- Maltose yeast extract agar, MGG- Medium glycerol glycine

4 KOKARE et al.: ISOLATION OF BIOACTIVE MARINE ACTINOMYCETES 251 for further studies on the basis of antibacterial activity and good growth characteristics. All the isolates were able to grow in the presence of 3.5% (w/v) NaCl. Three isolates were able to tolerate 14% (w/v) NaCl. However, they showed poor growth. Five species showed good growth in absence of sodium chloride and seven species showed good growth even at 7% (w/v) sodium chloride concentration (Fig. 1A). All species of actinomycetes showed best growth at ph 7. Nine actinomycetes showed growth at ph 5 but only two isolates showed growth at ph 3.5. Five isolates showed growth at alkaline ph 9 (Fig. 1B). Many species of actinomycetes showed good growth at 37 C and same species of actinomycetes also showed good growth at 28 and 41 C. Two actinomycetes (S1 and C2) showed growth even at 4 C (Fig. 1C). Identification of actinomycetes Most of the marine actinomycete isolates produced grey and white colonies without pigmentation. Out of 80 actinomycetes, 59 showed fast growth within two days and 21 showed slow growth after three days. Fifty three actinomycetes showed pigmentation of brown (5), gray(30), orange(2), black(8), yellow(4), red(2), greenish(2) and other 27 showed white colouration. Grey and white colour series were found to be predominant as compared to others. Finally 20 actinomycetes were selected on the basis of broadspectrum antimicrobial activity for carbon utilization and biochemical activity for identification up to genus level. Maximum actinomycetes were observed from Streptomyces group, identified by slide cultures (Fig. 2), morphological characteristics, carbon utilization (Table 2), nitrogen utilization (Table 3) and biochemical characteristics (Table 4). Based on these characteristics, one each was identified as Actinopolyspora (AH1), Streptoverticillium (BH5), Catellatospora (AH4) and Nocardia (S7). The other 16 were identified as belonging to Streptomyces group. Fig. 1 Utilization of sodium chloride (Fig. A), ph (Fig. B) and temperature (Fig. C) for growth of marine bioactive actinomycetes. Antimicrobial activity of marine actinomycetes Out of 80 actinomycetes, 35 showed antimicrobial activity. Out of these 20 showed strong activity against different types of organisms (Table 5). Sixteen of these showed strong antimicrobial activity against Gram-positive bacteria, 2 against Gram-negative bacteria and 7 against fungi. Antimicrobial spectrum analysis showed that the antibiotics produced by the isolates were more active against Gram-positive bacteria than Gram-negative bacteria and fungi. Antimicrobial spectrum may change depending on media used for the growth of actinomycetes species. Many species showed good antimicrobial activity on maltose yeast extract agar and tyrosine agar but not on yeast malt extract agar and nutrient agar. Some species showed good activity on starch casein agar, glucose aspargine agar, glycerol glycine medium as compare to other media. Many species showed antifungal activity against Aspergillus niger but not against Candida albicans (Fig. 3).

5 252 INDIAN J. MAR. SCI., VOL. 33, NO. 3, SEPTEMBER 2004 Table 2 Utilization of carbon sources by marine bioactive actinomycetes* Actinomycetes species Glucose Arabinose Sucrose Xylose Carbon sources M- Inositol Mannitol Fructose L- Rhamnose Raffinose Streptomyces sp.a ± ± Streptomyces sp. A Streptomyces sp.ah ± Streptomyces sp.bh4 + + ± ± + + Streptomyces sp.ch ± Streptomyces sp.bn Streptomyces spbn ± Streptomyces sp.en ± Streptomyces sp.c ± ± Streptomyces sp.c Streptomyces sp.c Streptomyces sp.c Streptomyces sp.s Streptomyces sp.s3 ++ ± ± + + Streptomyces sp.s Streptomyces sp.s Actinopolyspora sp.ah1 ++ ± ± Catellatospora sp. AH ± ± + + Streptoverticillium sp.bh5 + + ± + ± ± Nocardia sp.s strongly positive utilization, + positive utilization, ± utilization doubtful, -- utilization negative. *Carbon utilization performed by using basal medium with 1%(w/v) carbon source and incubated at 28 C for 15 days. Table 3 Utilization of nitrogen sources by marine bioactive actinomycetes* Actinomycetes species L- Aspargine DL- Alanine L- Leucine L- Cysteine Nitrogen sources L- L- Histidine Tyrosine DL-Phenyl alanine Potassium nitrate DL-2- Aminobutyrate Streptomyces sp.a Streptomyces sp. A ± ++ + Streptomyces sp.ah Streptomyces sp.bh Streptomyces sp.ch Streptomyces sp.bn Streptomyces spbn Streptomyces sp.en ± Streptomyces sp.c Streptomyces sp.c ± Streptomyces sp.c Streptomyces sp.c Streptomyces sp.s Streptomyces sp.s ± + + Streptomyces sp.s Streptomyces sp.s Actinopolyspora sp.ah Catellatospora sp. AH Streptoverticillium sp.bh Nocardia sp.s strongly positive utilization, + positive utilization, ± utilization doubtful, -- utilization negative. *Nitrogen utilization was performed by using basal medium with 0.1% (w/v) nitrogen source and incubated at 28 C for 15 days.

6 KOKARE et al.: ISOLATION OF BIOACTIVE MARINE ACTINOMYCETES 253 Table 4 Physiological and enzymatic properties of marine bioactive actinomycetes Actinomycetes species Properties Amylase Protease Gelatinase Lipase Cellulase Melanin pigmentation H 2 S production Nitrate reduction Streptomyces sp.a Streptomyces sp. A Streptomyces sp.ah Streptomyces sp.bh Streptomyces sp.ch Streptomyces sp.bn Streptomyces spbn Streptomyces sp.en Streptomyces sp.c Streptomyces sp.c Streptomyces sp.c Streptomyces sp.c Streptomyces sp.s Streptomyces sp.s Streptomyces sp.s Streptomyces sp.s Actinopolyspora sp.ah Catellatospora sp. AH Streptoverticillium sp.bh Nocardia sp.s positive; -- negative. Table 5 Antimicrobial activities of marine actinomycetes Actinomycetes species Bacillus subtilis Escherichia coli Test organisms and zone of inhibition in mm Pseudomonas Stphylococcus Aspergillus Aspergillus aeruginosa aureus niger fumigatus Candida albicans Penicillium species Cryptococcus species Streptomyces sp.a Streptomyces sp. A Streptomyces sp.ah Streptomyces sp.bh Streptomyces sp.ch Streptomyces sp.bn Streptomyces spbn Streptomyces sp.en Streptomyces sp.c Streptomyces sp.c Streptomyces sp.c Streptomyces sp.c Streptomyces sp.s Streptomyces sp.s Streptomyces sp.s Streptomyces sp.s Actinopolyspora sp.ah Catellatospora sp. AH Streptoverticillium sp.bh Nocardia sp.s Indicates no activity, Zone of inhibition: 10 to 14 mm less activity, Zone of inhibition: above 14 mm good activity. *Antibacterial activity was performed by using cylinder plate technique and by using modified starch casein agar. This result is mean of three experiments

7 254 INDIAN J. MAR. SCI., VOL. 33, NO. 3, SEPTEMBER 2004 Discussion The search for new antibiotics in laboratories throughout the world stimulated a variety of different approaches for the isolation of antibiotic producing strains. Nonomura & Ohara20 in Japan looked for new genera by employing pretreatment such as dry heating, specialized growth media and long incubation times to isolate new species of Microbispora, Microtetraspora, Streptosporangium, Thermomonospora, Thermoactinomyces and Actinomadura. An alternative approach was to make the isolation medium more selective by adding antibacterial and antifungal compounds. However, the main disadvantage of this technique is that these substances inhibit the competing bacteria as well as some species of actinomycetes. Preheat-treatment of marine sediment samples at 41 C for 60 days showed presence of fewer bacteria and no fungi. This treatment was found to be promising for the isolation of marine actinomycetes. The results obtained in the present investigation dem- Fig. 2 Spore chain structure of Streptomyces species. A) A3 species, light microscope (LM) 400, B) A3 species, scanning electron micrograph (SEM) 1010, C) S1 species, light microscope (LM) 400, D) S1 species, scanning electron micrograph (SEM) 1000

8 KOKARE et al.: ISOLATION OF BIOACTIVE MARINE ACTINOMYCETES 255 Fig. 3 Antimicrobial activity of 35 bioactive isolates using different media (abbreviations of media is given in text). A) against Bacillus subtilis and Staphylococcus aureus, B) against Aspergillus niger and Candida albicans onstrate that the kinds of pretreatment methods reviewed by Cross 21 as well as Williams & Wellington 22 may be successfully applied to marine sediments to facilitate the isolation of actinomycetes. Amongst the samples obtained from the different places of Alibag, Janjira and Goa, there was significant difference in total count of actinomycetes. Significant difference was found between the counts of actinomycetes on different media. This count was observed in rainy season (July 1999) and may vary in other seasons. Walker & Colwell 23 reported that total count depends on season, medium composition, freezing, salt concentration, incubation time and temperature. It was found that media with glucose or starch as the carbon source and asparagine or casein as a nitrogen source were suitable for isolation of actinomycetes from marine sediments. Kuster & Willams 24 made a similar study for isolation of Streptomyces from soil. The salt requirement or tolerance data suggested that some actinomycetes might be terrestrial forms that have adapted to the salinity of seawater and sediments. Kriss et al. 25 expressed same view for marine actinomycetes. Biochemical and morphological characteristics and slide cultures revealed that most of the isolates belonged to Streptomyces group. Streptomyces is known to be the most common genus in the marine environment especially in the shallow organic rich coastal areas. Ellaiah & Reddy 18 have recorded Streptomyces, Micromonospora, Nocardia, Streptosporangium, Micropolyspora and Streptoverticillium in marine sediments. Interestingly, in present study, Actinopolyspora and Catellatospora genera were found to be present in marine sediments. It should be noted that these genera are rarely found in the marine environment. In the present study, 43.75% actinomycetes isolated from west coast region exhibited antimicrobial properties. Swamy 26 reported that 27% of 518 actinomycetes isolated from the sediments showed antimicrobial activities from Porto Novo region, east coast of India. Similarly, Okami & Okazaki 3 reported that 20.5% of 123 actinomycetes were found to be antagonistic against microorganisms isolated from marine sediments of Sagami Bay. However, in present study, 43.75% of 80 actinomycetes isolated from Alibag, Janjira, Goa exhibited antimicrobial activity. In the present study maximum species of marine actinomycetes were found to be active against Gram-positive bacteria. Rangaswamy et al. 27 reported that terrestrial isolates were active against both. Previous studies also indicate that actinomycetes of marine sediments inhibited mostly the Gram-positive bacteria 26. Antimicrobial activity on tyrosine agar and maltose yeast extract agar showed that components of media like tyrosine and maltose were more suitable for production of antibiotics. This study clearly indicates that marine sediment samples from Alibag, Janjira and Goa are potent sources for the isolation of bioactive actinomycetes. Acknowledgement We thank Director, Agharkar Research Institute, Pune for providing scanning electron microscope facilities. References 1 Fenical W, New pharmaceuticals from marine organisms, Trends Biotechnol, 15 (1997) Molinski T F, Developments in marine natural products. Receptor-specific bioactive compounds, J Nat Prod, 58 (1993) Okami Y & Okazaki T, Studies on marine microorganisms (1) Isolation from the sea, J Antibiot, 25 (1972)

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