Chinese Journal of Oceanology and Limnology Vol. 26 No. 2, P. 166-177, 2008 DOI: 10.1007/s00343-008-0166-5
Distribution and generic composition of culturable marine actinomycetes from the sediments of Indian continental slope of Bay of Bengal Surajit DAS , P. S. LYLA and S. AJMAL KHAN Centre of Advanced Study in Marine Biology, Annamalai University, Parangipettai-608 502, Tamil Nadu, India
Received May 23, 2007; revision accepted Aug. 6, 2007 Abstract Actinomycetes population from continental slope sediment of the Bay of Bengal was studied. Samples were collected during two voyages of FORV Sagar Sampada in 2004 (May–June) and 2005 (July) respectively from 11 transects (each transect had ca. 200 m, 500 m, and 1 000 m depth stations). The physicochemical parameters of overlying water, and sediment samples were also recorded. The actinomycete population ranged from 5.17 to 51.94 CFU/g dry sediment weight and 9.38 to 45.22 CFU/g dry sediment weight during the two cruises respectively. No actinomycete colony was isolated from stations in 1 000 m depth. Two-way analysis of variance showed significant variation among stations (ANOVA two-way, P55 mol %), which are phylogenetically related from the evidence of 16S ribosomal cataloguing and DNA:rRNA pairing studies (Goodfellow and Williams, 1983). The name “Actinomycetes” was derived from Greek “atkis” (a ray) and “mykes” (fungus), and has features of both bacteria and fungi. However, they are now generally considered to be more closely related to bacteria. The chemical composition of their cell wall is similar to that of gram-positive bacteria but because of their well-developed morphological (hyphae) and cultural characteristics, actinomycetes have been
considered as a group, well separated from other common bacteria. Actinomycetes are a group of bacteria with many important and interesting features. They are of considerable value as producers of antibiotics and of other therapeutically useful compounds, exhibit a range of life cycles unique among the prokaryotes, and appear to play a major role in the cycling of organic matter in soil ecosystem (Veiga et al., 1983). Therefore, actinomycetes hold a prominent position due to their diversity and proven ability to produce new compounds. Actinomycetes population has been initially considered as one of the major group of soil population; however it has been increasingly isolated from various marine samples from very *Corresponding author:
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deep waters and near hydrothermal vents (Murphy and Hill, 1998). It is now accepted that actinomycetes can be indigenous to the marine environment and that this environment is likely to yield many unusual actinomycetes potential of producing novel antibiotics and other compounds. Furthermore, similar to their saprophytic relatives in soils these marine actinomycetes may play important ecological roles and substantially impact the cycling of complex carbon substrates in benthic ocean habitats (Mincer et al., 2002). However, a well-defined biodiversity and taxonomy of actinomycetes are important to understand actinomycetes from the continental slope of Bay of Bengal. Additional to previous reports on aerobic culturable heterotrophic bacterial population (Das et al., 2007a) and the role in biogeochemical cycles (Das et al., 2007b), this study is the first report of actinomycetes from the slope sediment of Bay of Bengal.
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duplicate and kept for 7 to 15 days at 27°C, and then the number of powdery actinomycetes colonies formed on the plates was counted. Because the number was small, counts from the duplicate plates and two media were summed up following Jensen et al. (1991). All the colonies were subsequently collected, sub-cultured and maintained in corresponding media slanted at 4°C temperature for further studies (Kokare et al., 2004a, b).
2 MATERIAL AND METHOD 2.1 Sampling area The Bay of Bengal is a unique basin receiving considerable amount of freshwater and sediment discharges from major rivers viz. Ganges, Brahamaputra, Mahanadi, Godavari, Krishna and Cauvery. Among these rivers, the GangaBrahamaputra riverine system transports about 1.4×109 tons of suspended sediments to the bay, atop all the global rivers (Madhupratap et al., 2003). 2.2 Samples collection and actinomycetes enumeration The sediment samples were collected using Smith McIntyre Grab during Cruise No. 225 and 236 of FORV Sagar Sampada in 2004 (from 11 May to 03 June, 2004) and 2005 (from 13 to 30 July, 2005) covering 33 stations over 11 transects in the bay (10°36–20°01'N, 79°59–87°30'E) (Fig.1). After the collection, the central portion of the sediment were collected aseptically for enumeration of actinomycetes population onto Starch Casein Agar (Hi-Media, India) and Yeast Extract-Malt Extract Agar (ISP-2) media supplemented with nystatin and cyclohexamide at 25 and 10 μg/ml respectively to minimize contamination with fungi and 10 μg/ml nalidixic acid to minimize bacterial growth (Ravel et al., 1998). An inoculum of 0.5 ml was inoculated in spread plate method from undiluted samples (50% w/v). All the sample aliquots were analyzed in
Fig.1 Study area-Indian continental slope of Bay of Bengal
2.3 Identification and systematic The isolates were identified to generic level according to Shirling and Gottlieb (1966), Lechevalier and Lechevalier (1970), and Nonomura (1974). 2.3.1 Phenotypic characters a) Aerial mass colour: The color of mature sporulating aerial mycelium was recorded by visual observation (white, gray, red, green, blue, and violet). When the aerial mass colour fell between two colours series, both colours were recorded. The media used were Yeast Extract–Malt Extract Agar (ISP2), Inorganic Salt Starch Agar (ISP4), and Starch Casein Agar (Hi-Media, India). b) Spore chain morphology: The species with spore bearing hyphae had three types (Shirling and Gottlieb, 1966; Nonomura, 1974)-Flexible-Rectiflexibiles (RF), Open loops-Retinaculiaperti (RA) and Spira-Spirales (S). Characteristics of the spore bearing hyphae and spore chains were observed
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directly with a microscope of 400X magnification. Spore morphological characters of the strains were studied by coverslip culture technique. c) Spore surface morphology: Spore surface features were observed with a scanning electron microscope (Oxford-UK). The crosshatched cultures prepared for observation under a light microscope were used for this purpose. Four types of the spore structures in actinomycetes were reported previously as: smooth (sm), spiny (sp), warty (wa) and hairy (ha) (Nonomura, 1974). 2.3.2 Chemotaxonomical characteristics Chemotaxonomy is the study of chemical variation in organisms and the use of chemical characters for classification and identification. Therefore, it is generally considered as one of the essential milestones in the development of modern classification of bacteria and actinomycetes. Cummins and Harris (1956) stated that actinomycetes have cell wall composition akin to that of gram-positive bacteria and indicated that the chemical composition of the cell wall might furnish practical methods of differentiating various types of actinomycetes. The 2,6-Diaminopimelic Acid (DAP) in actinomycetes is widely distributed as a key amino acid and it has optical isomers. The systematic significance lies mostly in the key amino acid with two amino bases, and determination of the key amino acid is usually sufficient for characterization. If DAP is present, bacteria generally would contain one of the isomers, the LL-form or the meso-form, mostly located in the peptidoglycan. The sugar composition often provides valuable information on the classification and identification of actinomycetes (Lechevalier and Lechevalier, 1970). Actinomycete cells contain some kinds of sugars, in addition to the glucosamine and muramic acid of peptidoglycan. The sugar pattern plays a key role in the identification of sporulating actinomycetes that have meso-DAP in their cell walls. Considering the importance of chemotaxonomical characteristics in the identification of actinomycetes, an attempt was made to identify the actinomycetes, by analyzing their cell components. Whole-cell sugars were analyzed according to the method of Becker et al. (1965). a) Hydrolysis: Hydrolysis was made for releasing amino acids. Harvested cells of each strain weighing 20 mg were placed in an ampo bottle, and 1 ml of 6 mol/L HCl was added and sealed with alcohol blast
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burner. The samples were kept at 121°C for 20 h in a sand bath. The bottles were cooled by keeping them at a room temperature of 28 ± 2°C. For releasing sugars, harvested cells of each strain weighing 50 mg were placed in an ampo bottle, and 1 ml of 0.5 mol/L HCl was added and sealed with alcohol blast burner. The samples were kept at 110°C for 2 h. The bottles were then cooled by keeping them at a room temperature of 28 ± 2°C. b) Thin Layer Chromatography (TLC): Spotting of the whole cell hydrolysates were made carefully on TLC plate using a microliter pipette. Spots were of 5–10 mm in diameter. This was done with multiple applications on the same spot of very small portions of the sample that dried by a hand drier. Each sample (3 μl) was applied on the base lines of cellulose TLC plate (20 × 20 cm). Adjacent to this, 1 μl of DL-diaminopimelic acid (an authentic material mixture of DAP isomers) and 1 μl of amino acetic acid (glycine) were spotted as standards. TLC plate was developed with a solvent system containing methanol: pyridine: glacial acetic acid: H2O at 5: 0.5: 0.125: 2.5 v/v. The spots were visualized by spraying with 0.4% ninhydrin solution in water-saturated n-butanol, followed by heating at 100°C for 5 min. Spots of amino acetic acid ran faster than DAP. The sample spots were immediately compared with the spots of the standards otherwise the spots gradually disappeared in few hours. On a cellulose TLC plate (procedure as done for DAP), 5 μl of samples was spotted, along with 3 μl of sugar solutions as standards on the same plates. Galactose, arabinose, xylose and mannose were sugars that were used as standards. TLC plate was developed with the solvent mixture containing ethyl acetate: pyridine: acetic acid: distilled water at 8: 5: 1: 1.5 v/v. The developing time was more than 4 h. Spots were visualized by spraying with aniline phthalate reagent. The sprayed plate was heated at 100°C for 4 min. Hexoses appeared as yellowish brown spots and pentoses, as maroon coloured spots. 2.4 Physico-chemical parameter Physico-chemical parameters of the sediment overlying water were examined against appropriate datasets obtained through sensors on a CTD (Sea-Bird 911 Plus). Sediment temperature and pH were estimated by centigrade thermometer and pH meter (Soil pH cone, Hanna Instruments, Mauritius), respectively. Water contents of sediment samples
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were estimated by weight loss of wet sediment samples dried at 60°C and were expressed in percentage. Total organic carbon (TOC) was estimated following El Wakeel and Riley (1956) and expressed in mg/g dry sediment. Texture of the sediments was observed as per Krumbein and Pettijohn (1938). 2.5 Statistical analysis F-Values (ANOVA, two-way) were calculated and compared with tabulated values to determine the significant differences in actinomycetes population between stations and cruises, and station depths and cruises. Multiple regression analysis (Statistica®, Ver 5, StatSoft Inc.) was performed in order to determine the influence of physicochemical variables on total actinomycetes populations.
3 RESULT 3.1 Actinomycetes population Actinomycetes population in Cr#225 and Cr#236 ranged from 5.17 (Stn. 63, off Singarayakonda, 200 m) to 51.94 CFU/g dry sediment weight (Stn. 67, off Divipoint, 500 m) and 9.38 CFU/g (Stn. 54, off Cheyyur, 200 m) to 45.22 CFU/g dry sediment weight (Stn. 76, off Barua, 500 m) respectively (Table 1). Two-way analysis of variance showed significant variation in actinomycetes population among the stations (ANOVA two-way, F=2.5681, Fcrit=1.8044, P
