INTRODUCTION:

Bay of Bengal is unique for its salinity in the world. As some large rivers and fresh water bodies of the world have fallen into the bay so the salinity of the surface water of the bay is less saline as compared to other seas of the world. Except this seasonal variation in salinity is seen in the bay, this causes mainly due to the variation in rainfall seasonally and the discharge of water through the Buckingham canal forming the brackish water. In rainy season when rainfall is highest then the water discharge from Brackish water increases also and due to this huge amount of discharge in the monsoon sometimes at the interphase the salinity tends to move towards zero. The surface salinity in the open part of the Bay oscillates from 32% to 34.5% (parts per thousand, i.e. grams per kilogram of sea water) and in the coastal region varies from 10% to 25% [1]. The coastal water is significantly diluted throughout the year, although the brackish water uptake is greatly reduced during winter. Salinity gradually increases from the coast towards the open part of the Bay and near the coast the seasonal variation in salinity is greatest when in the deep sea this variation is very less [2]. Swells observed at the ocean surface can propagate thousands of kilometers [3]. Identification of low-frequency swells propagating large distances from the generation region is vital in wave hind cast studies [4]. Site 12.8663° N latitude and 80.2447° E longitude is intertidal zone between the Bay of Bengal and brackish water, Chennai, India is untapped resource for exploration of microbial diversity. The marine environment includes a vast array of diverse habitats ranging from tropical, shallow-water coral reefs to subzero and oceanic trenches. These habitats are ecological niche for majority of microbial and aquatic flora and fauna. A unique oceanographic setting and being one of the most vulnerable regions in the world due to climate change, Bay of Bengal is one of the hotspots for climate change research. Thus, it is pertinent to investigate how marine productivity of such intertidal zones.

MATERIALS AND METHODS:

Sampling

Site 12.8663° N, 80.2447° E site is intertidal zone between the Bay of Bengal and brackish water. The physiochemical parameters of seawater were estimated in and ex-situ. 5 water Samples were collected from Kanathur beach during the period of swell (high tides). The samples were collected in sterile containers. The samples were immediately moved to the laboratory and processed [5].

Media preparation and Isolation of Halo-tolerant Marine Actinomycetes:

1 ml of each water sample was serially diluted to 10^-5, 10^-6 and 10^-7 in sterile aseptic conditions. Diluted water samples were plated on modified-Actinomycetes isolation agar (AIA) (Sodium caseinate-2.00 gm/L, L-Asparagine 0.10 gm/L, Sodium propionate 4.00 gm/L, Di-potassium phosphate 0.50 gm/L, Magnesium sulphate 0.10 gm/L, Ferrous sulphate 0.001 gm/L, Agar 15.00 gm/L, Glycerol 0.5 ml and final pH (at 28°C) 8.7±0.2 and prepared in sterile seawater (SSW) spread plate technique [6]. The plates were incubated at 28°C for 14 days and observed at intervals of 24 hours for the presence of microbial growth. HTMA colonies were streaked on AIA obtain and maintain axenic culture. Strians were named according to AMET repository coding regulations as AMET_YTC01 to AMET_YTC24. Potential 15 HTMA strains were selected on basis of their growth and further analyzed for their biochemical potential.

Biochemical characterization of HTMA:

Biochemical tests were performed to detect utilization of the myo-Inositol, D-Arabinose, L-Arabinose, Cellobiose, Erythritol, Fructose, Galactose, Glycerol, Glycogen, Inositol, Inulin, Lactose, Maltose, Mannitol, Mannose, Raffinose, Rhamnose, Salicin, Sorbitol, Starch, Sucrose, Trehalose, Xylose compounds as C sources[7]. Degradation of Aesculin, Casein, Chitin, Tyrosine, Elastin, Gelatin, Guanine, Hypoxanthine, Keratin, Starch, Urea and Xanthine by inoculating the HTMA in modified 15ml ISP2 broth Supplemented with respective compounds 1% w/v and incubated for 7 days at 28°C and respective negative controls were also incubated[8]. Based on above characteristics 5 strains are selected for primary screening and proceeded.

Screening for Halo-tolerant Marine Actinomycetes:

Tolerance of selected 5 Marine Actinomycetes against different parameters pH, temperature and salt concentrations were studied by inoculating loopful of AMETYTC_07, AMET_YTC14, AMET_YTC18, AMET_YTC19 and AMET_YTC21 strain in medium specified below with respective controls. Tolerance range of various pH (5, 6, 7, 8, 9,10) was tested using Zobell Marine Broth (ZMB) (Peptic digest of animal tissue 5.00 g/L, Yeast extract 1.00 g/L, Ferric citrate 0.10g/L, Magnesium chloride 8.80 g/L, Sodium sulphate 3.24 g/L, Sodium chloride 19.45g/L, Calcium chloride 1.80 g/L, Potassium chloride 0.55 g/L, Sodium bicarbonate 0.16 g/L, Potassium bromide 0.08 g/L, Strontium chloride 0.034 g/L, Boric acid 0.022 g/L,Sodium silicate 0.004 g/L, Sodium fluorate 0.0024 g/L, Ammonium nitrate 0.0016 g/L, Disodium phosphate 0.008 g/L).Temperature variation tolerance (15,20,28,37,48 and 55°C)was determined using Zobell Marine Broth[9]. Salt tolerance was determined using various concentrations of NaCl (0, 0.5, 1.0, 1.5, 2.0, 3.5, 4.0, 4.5 and 5.0 % w/v ) incorporated in modified Zobell Marine Broth(ZMB)( Peptic digest of animal tissue 5.00 g/L, Yeast extract 1.00 g/L, Ferric citrate 0.10g/L, Sodium chloride 19.45g/L, Magnesium chloride 8.80 g/L, Sodium sulphate 3.24 g/L, Calcium chloride 1.80 g/L, Potassium chloride 0.55 g/L, Sodium bicarbonate 0.16 g/L, Potassium bromide 0.08 g/L, Strontium chloride 0.034 g/L, Boric acid 0.022 g/L,Sodium silicate 0.004 g/L, Sodium fluorate 0.0024 g/L, Ammonium nitrate 0.0016 g/L, Disodium phosphate 0.008 g/L) at 28°C for 7 days and their optical density is measured at 600nm.

DNA Extraction from HTMA AMET_YTC21 strain and Polymerase chain reaction (PCR) protocol 16S rRNA amplification

Bacterial Genomic DNA was isolated using the Insta Gene TM Matrix Genomic DNA isolation kit. An isolated bacterial colony was picked and suspend in 1ml of sterile water in a microfuge tube. Centrifuge it for 1 minute at 10,000–12,000 rpm to remove the supernatant. Add 200 μl of Insta Gene matrix to the pellet and incubate at 56 °C for 15 minutes. Vortex at high speed for 10 seconds. Place the tube in a 100 °C boiling water bath for 8 minutes. Centrifuge the tube at 10,000– 12,000 rpm for 2 minutes. In result, 20μl of the supernatant was used per 50 μl PCR reaction[10].16S rRNA primers gene fragment was amplified using MJ Research Peltier Thermal Cycler. Forward primer-27F (AGAGTTTGATCMTGGCTCAG) with 20 bases and reverse primer-1492R (TACGGYTACCTTGTTACGACTT) with 22 bases. Add 1μL of template DNA in 20 μL of PCR reaction solution. Initial denaturation 94℃ for 2 min and then 35 amplification cycles at 94℃ for 45 sec, 55℃ for 60 sec, and 72℃ for 60 sec. Final Extension at 72℃ for 10 min. DNA fragments are amplified about 1,400bp. A positive control (E.coli genomic DNA) and a negative control are incorporated in PCR [11].

Purification of PCR products and Sanger Dideoxy Sequencing protocol:

Removed unincorporated PCR primers and dNTPs from PCR products by using Montage PCR Clean up kit (Millipore).The PCR product was sequenced using the 518F/800R primers. Sequencing reactions were performed using a ABI PRISM® Big Dye TM Terminator Cycle Sequencing Kits with AmpliTaq® DNA polymerase (FS enzyme) (Applied Biosystems). Single-pass sequencing was performed on each template using below 16s rRNA universal primers. The fluorescent-labeled fragments were purified from the unincorporated terminators with an ethanol precipitation protocol. The samples were resuspended in distilled water and subjected to electrophoresis in an ABI 3730xl sequencer (Applied Biosystems).

Bioinformatics Tool protocol:

The 16s r RNA sequence was blast using NCBI blast similarity search tool. The phylogeny analysis of our sequence with the closely related sequence of blast results was performed followed by multiple sequence alignment. The program MUSCLE 3.7 was used for multiple alignments of sequences [12]. The resulting aligned sequences were cured using the program Gblocks 0.91b. Forward Primer 785F (GGATTAGATACCCTGGTA) no of bases 18 and Reverse Primer 907R (CCGTCAATTCMTTTRAGTTT) no of bases 20. This Gblocks eliminates poorly aligned positions and divergent regions (removes alignment noise) [13]. Finally, the program PhyML 3.0 aLRT was used for phylogeny analysis and HKY85 as Substitution model. PhyML was shown to be at least as accurate as other existing phylogeny programs using simulated data, while being one order of magnitude faster [14]. PhyML was shown to be at least as accurate as other existing phylogeny programs using simulated data, while being one order of magnitude faster. The program Tree Dyn 198.3 was used for tree rendering [15].

RESULTS AND DISCUSSION:

Physiochemical parameters of 5 seawater samples

Randomized Seawater samples were aseptically collected at Kanathur beach, Chennai, India during Swell (High Tide). Physiochemical parameters of samples were estimated (Mean±Standard Deviation) as Temperature Seawater (28.00±0.50)°C, Salinity (34.96±0.05) ppt, Electrical Conductivity (34.96±0.05) μs/cm, pH (8.88±0.084), Dissolved Oxygen (11.94±0.11) mg/L, NO3 (0.25±0.021) mg/L, Total Dissolved Solids (27850. 2±0.44) mg/L and Total PO4 (0.013±0.00) mg/L [16] and their results were follows indicated in Table-1. The water samples were alkaline in nature and slight to moderately turbid with suspended organic matter.

Isolation of Halo-tolerant Marine Actinomycetes

Figure 1-HTMA colonies isolated on modified AIA medium

There were 5 (A, B, C, D and E) sets of triplicate plates of modified AIA per dilution 10^-5, 10^-6 and 10^-7 were inoculated with 1 ml of respective dilution of Seawater sample by spread plate technique. The plates were incubated at 28 °C to maintain their ecological niche temperature. Total of 45 plates were inoculated and plate with 10^-7 dilution showed very promising results post day 7 to day 10. Distinct pin point colonies of HTMA were observed on day 7 and on Day 10 there were varying demarcations of aerial and substrate mycelium on colonies. 15 Colonies were isolated and streaked on modified AIA to maintain axenic culture as shown in fig.1 .and phenotypic characteristics of 15 HTMA were as follows as given in table-2.

Table 2-Phenotypic characteristics of HTMA strains

Colony Morphology	Elevation	Margin	Aerial mycelium	Substrate mycelium	Diffusible pigments
AMET_YTC 02	raised	undulate	white	white	Absent
AMET_YTC 04	raised	undulate	Creamy yellow	white	present
AMET_YTC 05	convex	concentric	grey	yellow	absent
AMET_YTC 07	convex	smooth	Pale yellow	grey	present
AMET_YTC 10	raised	concentric	brown	white	absent
AMET_YTC 12	raised	smooth	grey	grey	present
AMET_YTC 14	raised	smooth	grey	white	absent
AMET_YTC 15	convex	rugose	Pale yellow	brown	present
AMET_YTC 16	raised	smooth	grey	white	absent
AMET_YTC 17	raised	concentric	brown	Bluish grey	absent
AMET_YTC 18	raised	concentric	grey	Light orange	absent
AMET_YTC 19	convex	concentric	brown	white	absent
AMET_YTC 21	raised	rugose	grey	Light pink	absent
AMET_YTC 22	raised	concentric	Light green	grey	present
AMET_YTC 23	convex	concentric	yellow	grey	absent

Table 3-Biochemical characterization of HTMA strains by Degradation of compounds

Degradation of

AMET_YTC 02

AMET_YTC 04

AMET_YTC 05

AMET_YTC 07

AMET_YTC 10

AMET_YTC 12

AMET_YTC 14

AMET_YTC 15

AMET_YTC 16

AMET_YTC 17

AMET_YTC 18

AMET_YTC 19

AMET_YTC 21

AMET_YTC 22

AMET_YTC 23

Aesculin

Casein

Chitin

Tyrosine

Elastin

Gelatin

Guanine

Hypoxanthine

Keratin

Starch

Urea

Xanthine

+high growth,-no growth ,w weak positive

Table 4-Biochemical characterization of HTMA strains by utilization of different C-sources.

Growth on sole

carbon source (1 %, w/v)

AMET_YTC 02

AMET_YTC 04

AMET_YTC 05

AMET_YTC 07

AMET_YTC 10

AMET_YTC 12

AMET_YTC 14

AMET_YTC 15

AMET_YTC 16

AMET_YTC 17

AMET_YTC 18

AMET_YTC 19

AMET_YTC 21

AMET_YTC 22

AMET_YTC 23

Inositol

D-Arabinose

L- Arabinose

Cellobiose

Erythritol

Fructose

Galactose

Glycerol

Glycogen

Inositol

Inulin

Lactose

Maltose

Mannitol

Mannose

Raffinose

Rhamnose

Salicin

Sorbitol

Starch

Sucrose

Trehalose

Xylose

+high growth,-no growth ,w weak positive

Sample	A	B	C	D	E	Mean±SD
Temperature Seawater(°C)	27.5	28	27.7	28.8	28	28.00±0.50
Salinity( ppt)	34.9	35	34.9	35	35	34.96±0.05
Electrical Conductivity(μs/cm)	53200	53200	53200	53200	53200	53200±0.00
pH	8.8	8.8	8.9	8.9	9	8.88±0.084
Dissolved Oxygen(DO)(mg/L)	12	11.8	12.1	11.9	11.9	11.94±0.11
NO₃(mg/L)	0.27	0.28	0.24	0.23	0.25	0.25±0.021
Total Dissolved Solids(TDS)(mg/L)	27850	27850	27851	27850	27850	27850.2±0.44
Total PO₄(mg/L)	0.014	0.013	0.011	0.014	0.013	0.013±0.00

% NaCl	0	0.5	1	1.5	2	2.5	3	3.5	4	4.5	5
AMET_YTC07	-1	-1	-1	-1	-1	0	1	1	0	-1	-1
AMET_YTC14	-1	-1	-1	-1	0	1	1	1	0	-1	-1
AMET_YTC18	-1	-1	-1	-1	-1	0	1	0	-1	-1	-1
AMET_YTC19	-1	-1	0	0	0	1	1	1	0	-1	-1
AMET_YTC21	-1	0	0	0	0	1	1	1	1	1	1