Distribution of Cultivable Actinobacteria from the Marine Sediments along the Andaman Coast of Eastern Indian Ocean

| Andaman and Nicobar Islands is one of the important biodiversity hot spots in the world which is situated in the Eastern Indian Ocean. The combination of mangrove, rocky and coral reef habitats make it an interesting area for studying microbial population. The present study discusses the distribution status of 643 isolates of Actinobacteria isolated from the marine sediments along the coast of Andaman group of Islands at a depth from 0 to 10m. Spatially, the coast of South Andaman harbours most number of isolates (290) than the other zones and among these, the highest (7.5%) was isolated from Marina Park. Bathymetrically, the highest number of isolates (269) was recorded from 0 – 1 m depth zone and the most number was isolated from Burmanallah. Among these, Streptomyces spp. was found to be highly dominant (83.4%) spatially as well as bathymetrically. Other genera identified were Streptoverticillium spp., Streptosporangia spp., Nocardia spp., Micromonospora spp., Actinoplanes spp. and Actinomadura spp. Highest percentage of Streptomyces spp. was recorded from Burmanallah (7.3 %). Marina Park and Science Centre stations recorded highest number of Streptoverticillium spp. (11.1% each) and Nocardia spp. was found more in Carbyn’s Cove. Micromonospora spp., Streptosporangium spp. and Actinoplanes spp. were recorded only from few stations and Actinomadhura spp. was found only from two stations. In general, the number of isolates decreased from the shore to deeper areas. The mangrove and rocky habitats harbours had more number of Actinobacteria than the sandy and reef ecosystems. The present study have confirmed the potential of this region in terms of actinobacterial diversity and can form a baseline for further research in marine pharmacology.


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organic load in turn supports a variety of microbial population in the sediment and water (Maria et al., 2006;Farooqui et al., 2012).The coral reef habitats are furthermore diverse and extend to more than 30 m depth towards the sea from the shore.Habitat associations like mangrove and reef habitats with rocky, sandy and muddy coastal regions also support a higher diversity along the coast (Qasim and Wafar, 1990).The disastrous earthquake of 26 December 2004 has caused submergence of some of the Islands and upheaval of some other Islands.Consequently, virgin land areas were exposed to wave attack (Roy et al., 2009).Andaman and Nicobar Islands, considered as the richest source for microbial community.The marine microbial taxa and actinomycetes diversity are not been fully explored (Abirami et al., 2013).So in the post tsunami scenario it is very imperative to evaluate the microbial wealth especially marine Actinobacteria in the modified coastal environment along the Bay Island.These microbes are considered to be terrestrial in origin and believed to occur in the ocean largely as dormant spores that were washed into the sea ( Jensen et al., 2005;Goodfellow and Haynes, 1984).According to Takizawa et al. (1993) marine Actinomycetes are distributed throughout the marine environment from shallow to deep sediments.They are isolated from different depths, but it is found that littoral inshore zone is most favourable for their survival (Mincer et al., 2002).
Actinomycetes are well known as potent producers of a variety of secondary metabolites with distinct biological activities (Berdy, 2005;Ben et al., 2006), including AMSs active against both pathogenic (Sun et al., 2011;Xiong et al., 2012) and phytopathogenic microorganisms (Xiong et al., 2012;Jain and Jain, 2007).The exploration of soils and other habitats for microbes of biotechnological interest has led to the isolation of novel actinomycete strains (Ouhdouch et al., 2001).Secondary metabolites of Actinomycetes are therapeutically important compounds, especially antiviral, anti-cancerous, antibacterial compounds and around 70% of the antibiotics used in the world were identified and extracted from these microorganisms (Sivakumar et al., 2007).The isolation of actinomycetes from mixed micro flora present in nature is complicated by their characteristic slow growth (Kerkar, 1994).So the present study conducted with an aim to study the distribution of marine Actinobacteria species from the marine sediments of various hab-itats along the coasts of Andaman group of Islands.

MATERIALS AND METHODS
The entire Andaman coast (Figure 1) was divided into 3 zones viz.South Andaman (Chidiyatapu to Baratang), Middle Andaman (Baratang to Mayabunder) and North Andaman (Mayabunder to Diglipur).Sediment samples were collected aseptically from 0-1m, 5-6m and 10 -11m depth at each station by skin and scuba diving using PVC corer with 2.5cm diameter during the period January 2011-May 2013.Various physiochemical factors like Sea surface Temperature, Salinity, water pH and sediment pH were recorded from each station.Total Organic Carbon of the sediment was estimated following Walkley and Black (1934) and Udotong et al. (2008).Sediment texture was analysed using Pippet analysis ( Jayaraj et al., 2007) to understand the habitat structure.
The sediment samples were pre-treated because some of the Actinobacteria may not appear by normal plating techniques.In order to isolate these Actinobacteria, three pre-treatment techniques were applied: 1. Dry heat method: The sediment samples were heated at 120 o C for 1 hour and plated for isolation of temperature tolerant species like Thermoactinomycetes ( Jensen et al., 1991).

Phenolic Treatment:
Air dried sediment samples were inoculated in media which contains 1.5% phenol, Nalidixic acid and cycloheximide (50µg) against fungal contaminants.Colonies with a powdery appearance and leathery colonies oppressed to media were selected.The organisms were identified based on their morphological characters viz.types of substrate mycelia and aerial mycelia, temperature tolerance at 27-65 o C, colour of spores, aerial and substrate mycelia (Bredholt et al., 2008).
Protease activity was checked by spot inoculating loopful of culture along with the spores, into Nutrient Gelatin Agar (Peptone -5gm, Beef extract -3gm, Gelatin -120 gm pH 6.8) after 24 to 48hrs incu-bation plates were flooded with gelatine precipitating reagent which consequently liquefy Gelatine to amino acids.A clear zone is the indication for positive result (Williams et al., 1983;Gourdeau et al., 2008).
Clearing zone indicates the positive result.Amylase activity was determined by spot inoculating the cultures in Starch Agar Medium (Peptone-0.5gm,Beef extract-0.3gm,Starch -0.2g, Agar-2.0g,Sea water-100ml, pH-7.2) (Mincer et al., 2002).Degradation of casein was determined following Lindqvist and Storgards (1960).The growth at different temperatures, other physiological and biochemical characteristics were studied using the method described by Williams et al. (1983).All tests were performed at 28°C.For enzymes like chitinase and cellulose, cultures were inoculated in cellulose agar medium and chitinase agar  et al., 2007;Shirling and Gottlieb, 1966).
The susceptibilities of the organisms to various antibiotics were studied on nutrient agar plates containing various antibiotics such as triacylolandomycin, lincomycin and rifampicin.The antibiotics were aseptically mixed with sterile molten agar and the preparations were maintained at 45°C and poured into plates.After inoculation, the plates were incubated at 30°C for 1 week.Growth on the media was compared with growth of a control and was recorded as negative or positive (no growth or growth respectively) (Lechevalier and Lechevalier, 1967).
Thin layer Chromatography was done to determine cell wall composition.To detect the presence of meso Diamino pimelic acid, the sample were pelletized and treated with 6N HCL.Vacuum evaporation was done to evaporate HCL present in the sample and spotted in TLC Plates (Merck).After running for 5-6 hour in solvent system (methanol: distilled water: 6N HCL: Pyridine (80:26:4:10), plates were air dried, sprayed with 0.2% Ninhydrin in acetone.Developed in hot air oven at 100 o C for 3 minutes observed for maroon coloured spots (Becker et al., 1965).

RESULTS habitat Structure
The detailed survey have shown that the entire study area along the coast was dominated mostly by mangrove forests or sandy beaches along with a combination of other habitats like rocky and corals (Table 1).The sediment analysis by standard pipette analysis have shown that most of the stations were loamy followed by sandy, clayey and muddy.The isolates from most of the stations were found to be more salt tolerant especially in the deeper regions.Even though they were showing growth at 0ppt to 70ppt salt concentration, highest percentage of growth was observed in the range 30-40ppt of salt concentration (Figure 2).

General diStribution and abundance
During the present study, 643 isolates were isolated from the sediment samples collected along the coast of Andaman Islands (Table 2).The highest abundance of Actinobacteria population was recorded from the South Andaman Zone in total as well as in all the sampled depths (2.90 x 10 2 cfu/g) followed by North Andaman (2.05 x 10 2 cfu/g) and Middle Andaman (1.46 x 10 2 cfu/g).A depth wise analysis have shown that the depth 0-1m inhabits highest number (269 isolates) followed by 5-7m (195) and 10-12m (179).

diStribution and abundance in South andaman Zone
Maximum number of isolates ( 123) was recorded at 0-1m depth followed by 5-6m (90) and the least was recorded from 10-11m (77) (Table 2).Marina Park station was recorded most number of Actinomycetes contributing to 16.6% and the least was in Minnie Bay (0.3%).In 0-1m depth zone, Burmanallah recorded the highest (23 colonies) and in the depth 10-11m, highest number was recorded in Chidiyatappu (18).

diStribution and abundance in north andaman
In North Andaman, out of 146 Actinobacteria recorded, 0-1m showed the maximum (71 colonies) followed by 5-6m (46) and the least was recorded from 10-11m (29).Kalipur station recorded the most number of Actinobacteria (32 colonies) contributing to 21.9% from the zone.In 0-1m depth zone, Kalipur contributed the highest (16 colonies).Culbert Bay recorded maximum number of Actinobacteria from the depth 5-6m (12).While in the depth 10-11m, highest number was recorded in the station Kalipur (6).
Nocardia spp. was the third most abundant group with a total of 14 colonies recorded from all the three zones with a maximum representation from South Andamans (1.2%) (Table 6).Micromonospora spp. was recorded maximum from South Andaman (0.6%) and was absent in the 0-1m depth and observed mostly from the 5-6m depth (0.9%).Actinoplanes spp.also found to distribute in all the three zones with highest contribution from North Andamans (0.5%).It was absent in the depth 0-1m and highest concentration was found in 5-6m (0.8%).Streptosporangium spp.could not be recorded from Middle Andaman and only 5 numbers could be recorded during the present study.Whereas, Actinomadura spp.could be recorded only from Middle Andamans and was absent in the 0-1m depth.

DISCUSSION
During the present study it could be noticed that most of the stations were found to be a combination of either mangroves or sandy with other habitats.The domination of Actinobacteria was discernible in the South Andaman when compared to other zones.This zone is mainly dominated by mangrove habitats followed by sandy and rocky habitats.In stations like Wandoor, Burmanallah and Chidiyatappu, the corals are mostly a continuation of the mangrove ecosystem and the inhabitants are found interacting very closely.
The human interferences including tourism activities were found to be higher in areas like   (Sujatha et al., 2005;Sivakumar et al., 2007;Abirami et al., 2013).
While Balagurunathan et al. (1989) has reported some common species like Rhodococcos sp., Streptomyces spp., Salinospora sp. and Micromonospora sp. from the mangrove sediments.Similarly, the dominance of Streptomyces spp. was evident in the present study also and the abundance of the other genera was negligible.
In the present study the saccarolytic Actinomycetes like Streptomyces spp.load was high and most of them were isolated from depth ranges 0-1m and 5 -6m depth zones.According to Grein and Meyers (1958), Streptomyces spp.are not autochthonous marine flora, they may be terrestrial forms adapted to salinity of sea water and sediment.Some scientists considered Actinomycetes to be part of indigenous marine micro flora (Zobell, 1940;Okazaki and Okami, 1976;Weyland, 1981) where as others considered them as wash in components that merely survived in marine and littoral sediment as spores (Goodfellow and Haynes, 1984;Takizawa et al., 1993;Jensen et al., 2005).This view is supported by the observation that the numbers of Actinomycetes in marine habitat decreases with increasing distance from land (Weyland, 1969).The present study have shown a general trend that the 0-1m depth have recorded highest concentration of Actinomycetes followed by 5-6m and 10-11m.Mincer et al. (2002) has reported that littoral inshore zone is most favourable for the survival.This indicates that these isolates include true marine forms as well as the washed away forms from terrestrial environment which showed a wide salt tolerance.
Andaman and Nicobar coast is one among the virgin coasts in the world and a hot spot of biodiversity which is not explored systematically and thoroughly.So the present study have shown the importance and potential of this region in terms of actinobacterial diversity and the future prospects of exploration for strains of bacterial flora for novel drugs.This work can be a baseline for the future studies in the field of pharmacological research to be conducted in the country.

Table 1 :
Physico-chemical parameters of sea bed and water of various stations *S = Sandy; R = Rocky; M = Mangrove; C = Coral NE US Academic Publishers Advances in Animal and Veterinary Sciences December 2014 | Volume 2 | Issue 12 | Page 673

Table 2 :
Zone wise and Depth wise Distribution of Actinobacteria

Table 4 :
Depth-wise percentage distribution of Streptomyces spp.
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in Animal and Veterinary Sciences December 2014 | Volume 2 | Issue 12 | Page 679Table 6 :
Junglighat, Minnie Bay, Marina Park, Science Centre, Wandoor and Chidiyatapu.Rocky bottoms were found in the sta-Depth-wise distribution of other Actinobacteria genera tions viz.Burmanallah, Dagar and Singh, 1999;Roy, 2003;a Park, and SRoy et al., 2009;le in other zones, mangroves and other types of habitats co-exist in stations like Yerratta, Kadamthala, Rangat, Karma-tang, Panchavati, Nimbuthala, Bakulthala etc. where a lot of anthropogenic activities like fishing, fire wood collection, etc. taking place.Sandy beaches dominated in stations like RRO camp, Culbert Bay, Shivpur, Aamkunj.The rich biodiversity of the Andaman and Nicobar Islands is already been reported by many workers(Qasim and Wafar, 1990;Dagar and Singh, 1999;Roy, 2003; Ramya, 2008;Roy et al., 2009; Ramkumar et al., 2013).The dominance of Streptomyces spp. was already re NE US