Assessment of mercury pollution through mercury resistant...

Indian Journal of Marine SciencesVol. 43(6), June 2014, pp. 1l03-1115

Assessment of mercury pollution through mercury resistant marinebacteria in Bhitarkanika mangrove ecosystem, Odisha, India

Hirak Ranjan Dash & Surajit Das*

Department of Life Science, National Institute of Technology, Rourkela- 769 008, Odisha, India

[E-mail: [email protected]@myself.com]

Received 17 December 2012; revised 3 May 2014

Level of mercury pollution by estimating the number of mercury resistant marine bacteria is examined in this study.Total mercury levels in the water and sediment samples were estimated between 0.14 and 0.66 ppb (0.00014 and 0.00066ppm) and from 0.23 to 0.97 ppb (0.00023 to 0.00097 ppm) respectively. pH, temperature and salinity level of the watersamples were in the range of 7.13-7.16, 27.35-36.1°C and 14.25-15.6 ppt respectively during this period. Mercuryresistant marine bacteria followed the trend of mercury level and ranged from 2.60x 103 to 7.05x 104 CFU/mL and from0.16x 103 to 7.4x 104 CFU/g in water and sediment respectively. Percentage of mercury resistant bacteria varied from0.12 to 89.28 in water and 0.14 to 88.88 in sediment. Some of the resistant isolates (positive for mer operon mediatedmercury resistance) were characterized further. They were found to be under the genera Vibrio and Bacillus which canresist other toxic metals (Cd, Zn, Pb, As) as well as antibiotics (AM, VA, NX, AZM, A, AC) confirming the pollutionlevel in the ecosystem.

[Keywords: Mercury resistant bacteria, Mangrove, merA, Bhitarkanika mangrove ecosystem, Mercury pollution]

Introduction

Heavy metals have the most adverse effects onthe floral and faunal diversity of the wetlands as itcan get accumulated III the process ofbiomagnifications causing havoc at all trophic levelsof the food chain 1. Due to rapid rate ofindustrialization and urbanization, India iscontributing major amounts of mercury to the globalbudget at a rate of 149.9 mg of mercury per year mostof which are coming from the coal fired power plants,chloro-alkali industries, agricultural wastes, wildfires,disinfectants and many more-.

Mercury can be accumulated in the animals thatlive on mangroves like fish, shrimp and molluscs. Itcauses physiological stress and affects negatively oncrab production. Methylmercury, the organic form ofmercury, possess the main threat to the live stocks bycausing harmful effects on adult survival,reproduction, behaviour, cell development andteratogenic effects',

The most studied mechanism is the mer operonmediated mechanism of mercury resistance. Meroperon is a positively induced operon which maycontain certain structural genes like merA, merB,merD, merG, merT, merP apart from the operator andpromoter regions". In the present study, BhitarkanikaMangrove Ecosystem has been used as a model ofmarine ecosystems to assess mercury pollution interms of mercury resistant marine bacteria. Geneticmechanism of mercury resistance has been confirmedin some potential isolates.

Materials and Methods

Bhitarkanika Mangrove Ecosystem, Odisha, India(20030'-20048'N & 86°45'-87°03'E) situated at thedelta of two rivers Brahmani and Baitarani coveringan area of about 65,000 hectors, is a Ramsar site. Asthe two river components carry a huge load ofpollutants to this mangrove wetland, it possesses ahuge threat from mercury contamination on thewetland communities. During the study period (2010-2012) sampling was conducted in monsoon and

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Indian Journal of Geo-Marine Sciences

1104 INDIAN J MAR set. VOL 43(6), JUNE 2014

summer season. Both water and sediment sampleswere collected from ten stations (St. 1-10) located inthe ecosystem (Fig. 1).

G--00 ••••••.....••

••• ..., ••• ..., ., .,'"Fig. i-Study site: Bhitarkanika mangrove ecosystem, Odisha,

India

Both water and sediment samples were analyzedfor various physico-chemical parameters like pH,salinity, temperature and total mercury contentfollowing the standard methods". For mercuryanalysis, samples of water and sediment were treatedaccording to the protocol of the EnvironmentalProtection Agency" and analyzed afteracid digestionby cold vapour Atomic Absorption Spectrophotometer (Perkin Elmer; AAnalyst™ 200). All thesamples were analyzed in duplicate and representedas Mean±SD.

Isolation of Total Heterotrophic Bacteria (THB) andMercury Resistant Marine Bacteria (MRMB)

To enumerate total heterotrophic bacteria (THB),both sediment (1 g) and water (1 mL) samples wereserially diluted and aliquots of 100 III of 10 and 100fold dilutions were spread on petri-plates containingZobell's Marine Agar medium (Hi-Media, India). Forenumeration of mercury resistant marine bacteria(MRMB), the same procedure was followed with SeaWater Nutrient (SWN) agar medium (Peptone-S g,Yeast Extract-3.0 g, Agar-IS g, Aged Sea Water-SOOml, deionised water-SOO ml; pH-7.S± 0.1)supplemented with 10 ppm HgC12 followingJaysankar and Ramaiah". Plates were incubated at2SoC for 48 h and the number of colony forming unitswas calculated. THB and MRMB were calculated and

expressed as CFU/g and CFU/mL for sediment andwater samples respectively. Based upon theobservable difference in colony morphology, theisolated colonies were selected and re-streaked onHgC12 supplemented (10 ppm) SWN agar plates andanalysed further. Percentage of MRMB wascalculated using the formula: % of MRMB =

(MRMB/THB) x 100.

Genetic aspects of mercury resistant marine bacteria

The conserved region of merA gene was amplifiedby the following primers: FlmerA-S'TCGTGATGTTCGACCGCT3'; F2 merA-S'TACTCCCGCCGTTTCCAAT3 '8. The amplificationreactions were performed in a total volume of 20 ul,by using a thermal cycler (BioRad). The PCR mixturecontained 1U/IlL Taq polymerase (Sigma), IXEnzyme buffer, 200 11Mof each dNTP (Sigma), 1.2SmM MgC12, O.S 11Mof each primer. Optimizedamplification conditions included a pre denaturationstep at 94°C for 1min followed by 30 cycles of 94°Cfor 1 min, SsoC for 1 min and an extension step atnoc for 1 min and final extension at noc for 7minutes. PCR products were analyzed using gelelectrophoresis (l. S%) and visualized in GelDocumentation System (BioRad). A sensitive strainof E. coli (DHSa) was used as the negative control.

Biochemical characterization of the positive isolates

The merA positive isolates were characterizedfurther by various tests. The biochemical tests wereconducted using standard protocols by Methyl Red,Voges Proskauer's, ONPG, Lysine, Ornithine, Urease,Phenyl alanine, Nitrate Reduction, H

2S Production,

Citrate Utilization, Indole production, Oxidase test,Motility Test, OIF Test, sugar fermentation i.e.Lactose, Xylose, Maltose, Fructose, Dextrose,Galactose, Trehalose, Sucrose, Mannose, Inulin,Inositol, Sorbitol, Mannitol, Arabitol, Cellobiose,Rhamnose and Salicin.

Identification of the isolates

The more resistant isolate was subjected to l6SrRNA gene sequencing based identification. Briefly,genomic DNA was extracted from the bacterial

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1105DASH & DAS et al: MERCURY RESISTANT MARINE BACTERIA

culture using Bacterial Genomic DNA extraction kit(Genomic DNA extraction kit, QiaGen) and theamplification reaction was performed using theuniversal 16S rDNA primers of 27F (5’-AGAGTTTGATCMTGGCTCAG-3’) and 1492R (5’-ACGGCTACCTTGTTACGA-3’) (Sigma) in athermal cycler (BioRad). Polymerase Chain Reactionwas performed in 50 µl volumes containing 2 mMMgCl

2, 2.5 U Taq polymerase (Sigma), 100 µM of

each dNTP, 0.2 µM of each primer and 3 µl templateDNA. PCR programme used was an initialdenaturation at 96°C for 5 min followed by 30 cyclesof 95°C for 15s, 49°C for 30s and 72°C for 1 min anda final extension at 72°C for 10 min. Amplified DNAwere purified by Sigma PCR purification kit followingmanufacturer’s instructions and finally quantified byNano drop (Eppendroff). The sequencing reaction wascarried out by Chromous Biotech, India. Sequencedata was compiled and consensus sequence wasobtained by using BioEdit (7.0.5.3) programme andexamined for sequence homology with the archived16S rDNA sequences from GenBank atwww.ncbi.nlm.nih.gov/nucleotide, employing theBLAST. In other two isolates identification procedurewas carried out by using the biochemical test resultsfollowed by PIBWin software9.

Antibiotic susceptibility test of the isolates

The antibiotic susceptibility testing of the selectedisolates were performed by Kirby Bauer’s discdiffusion technique using commercially availablediscs. Antibiotics tested were Amoxycillin (AM, 30µg), Vancomycin (VA, 30 µg), Ampicillin (AMP, 10µg), Chloramphenicol (C, 30 µg), Tetracyclin (TE,30 µg), Norfloxacin (NX, 10 µg), Ciprofloxacin (CF,5 µg), Azithromycin (AZM, 15 µg), Neomycin (N,30 µg), Cefotaxime (CTX, 5 µg), Clavulanic acid(AC, 30 µg) and Methicillin (MET, 5 µg).

Minimum Inhibitory Concentration (MIC) formercury and other heavy metals

Minimum inhibitory concentration of thebacterial pure cultures from the study sites werecarried out for five heavy metals i.e. Hg, Cd, Zn, Pband As following CLSI guidelines by broth micro-

dilution technique10. Briefly, bacterial isolates wereinoculated in to Luria-Bertani Broth medium (Caseinenzymic hydrolysate-10 g, yeast extract-0.5g, NaCl-10 g and Distilled water- 1000 ml, pH- 7.5 ± 0.2).Stock solutions of the metals were prepared bydissolving the compound in autoclaved MuellerHinton (Beef infusion-300 g, Casein acid hydrolysate-17.50, Starch-1.50g, Distilled Water-1000 ml, pH-7.4±0.2) broth (MHB). MIC was determined by using2 fold serial dilutions in MHB medium containingvarious dilutions of the metals

in a micro-titre plate.

To each dilution of the compound 10 µl of 0.5McFarland culture was added and the plate wasincubated at 37°C for 24 h. Un-inoculated MHB wastaken as negative control and MHB without mercuryinoculated with the isolate was taken as positivecontrol. After incubation, the bacterial growth wasmonitored by measuring the turbidity of the cultureby a micro-titre plate reader (OD

630). MIC was

determined as the lowest concentration of compoundat which the visible growth of the organisms wascompletely inhibited.

Statistical analyses

F-value (two-way ANOVA, P<0.05) werecalculated and compared with tabulated values todetermine the significant variation in physico-chemical parameters studied i.e. pH, salinity,temperature and total mercury content as well as totalheterotrophic bacteria and mercury resistant bacteriabetween two seasons i.e. summer and monsoon. Toexamine the relationship of bacterial population withphysico-chemical parameters correlation coefficientswere calculated. F value (ANOVA) was checked tofind out the differences in bacterial populations aswell as the physico-chemical parameters between thestudy periods.

Results

There not much variation observed in pH of thewater samples analysed during the study period whichranged between 6.80 and 7.38 during monsoon andfrom 6.84 to 7.50 during summer. However, thesalinity level in the water of the ecosystem in all thestations was found to vary between 12 and 18 ppt

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6.85xl03 CFU/mL). Percentage of mercury resistant. bacteria ranged between 0.11 and 16.38% during

monsoon whereas the value was between 0.90 and89.28% during summer season (Table 1).

A total of fifteen isolates with visiblydistinguished colony morphology on mercurysupplemented plates were selected for the analysis oftheir mercury resistant genotype. When the conservedregion for the inorganic mercury resistant genotype(merA) was amplified in the isolates, three of themshowed a clear distinct band of 431bp confirming themer mediated mercury resistance mechanism in theseisolates (Fig. 3). Though other isolates did not showany banding pattern, phenotypically they wereresistant to inorganic mercury and they may possessnon-mer mediated mercury resistance mechanisms .

INDIAN J MAR SCI VOL 43 (6), JUNE 2014

during monsoon and from 13 to 19 ppt in summer.Highest temperature observed during summer was39°C (range 34-39°C) and the lowest temperature(25°C) was observed in monsoon (range 25-29°C).Total mercury content of both water and sedimentsample was determined higher during summer (0.45± 0.03 ppb; 0.63 ± 0.03 ppb) (0.00045 and 0.00063ppm) and lower during monsoon (0.32 ± 0.009 ppb;0.54 ± 0.02 ppb) (0.00032 and 0.00054 ppm). In allthe stations studied, total mercury content was foundto be within the permissible limit!' except in onestation (St. 6) (1.03 ppb or 0.00103 ppm). The valuesof physico-chemical parameters and total mercurycontent have been provided in Fig. 2.

.-•.•.....

. -.. ••.•..c,.-.,J_...--.-.-...-.(c:) su su: su su sas su: 5&.7 St.I ,sruSt.••

.(d)su su su SlA SLS SU 81.7 SLI SU SU.

Fig. 2-Physiochemical parameters (values are in mean±S.D)of the study site during study periods (a) pH of water (b)

Temperature of water (c) Salinity of water (d) Total mercurycontent of water and sediment

The number of Total Heterotrophic Bacteria(THB) and mercury resistant marine bacteria wereenumerated during the two years of study period inboth water and sediment samples. THB count in thewater samples during monsoon ranged between3.4xl04 and 7.5x107 CFU/ml, whereas in summer itwas in the range of 9.5xl02-8.4xl04 CFU/ml.However, THB in sediment varied from 3.6xl05 to9.8xl07 CFU/g during monsoon and 4.5xl03 to8.9x 104 CFU/g during summer season. Mercuryresistant marine bacteria also followed the same trendand found to be higher in sediments (Summer: 7.4x 104

CFU/g; Monsoon: 5.5x 104CFU/g) than that of watersamples (Summer: 7.05 <I 04 CFU/ml; Monsoon:

431bp

Fig. 3-Amplification of conserved regions of merA gene in theisolates; Lane 1: 100 bp ladder, Lane 2: BW-03, Lane 3: BW-

101, Lane 4: BS-202T, Lane 5: Negative control (E. coliDH5a).

Isolates (BW-03, BW-I0l and BS-202T) showingpositive result for merA amplification were furthercharacterised biochemically. Out of these threeisolates, two (BW-03 and BW-I0l) were Gram-positive which share almost same biochemical patternand the other one (BS-202T) was Gram-negativewhich was further identified as Vibrio hepatarius by16S rRNA gene amplification and sequencing with16S Universal primer (27F and 1492R). BW-03 and

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Table 1–Heterotrophic bacterial population and Mercury resistant marine bacterial population from the water andsediment samples collected from Bhitarkanika mangrove ecosystem during 2010-2012

Sl. Geogra Total Heterotrophic Bacterial Mercury Resistant Marine % of Mercury ResistantNo. phical population Bacterial Population Bacteria

position Sediment Water Sediment Water Sediment WaterMons Summer Mons Summer Mons Summer Mons Summer Mons Sum Mons Sumoon (×103 oon (×105 oon (×104 oon (×104 oon mer oon mer

(×106 CFU/ (×105 CFU/ (×103 CFU/g) (×103 CFU/ml)CFU/ g)±SD CFU/ ml)±SD CFU/ ±SD CFU/ ±SDg)±SD ml)±SD g)±SD ml)±SD

1 20°74.3 0.57± 48.6± 4.85± 4.20± 55.5± 6.20± 2.60± 2.85± 9.73 12.75 20.46 14.323’N & 0.05 40.4 0.01 0.07 7.5 0.2 0.02 0.02086°86.06’E

2 20°74.2 5.25± 61±25 4.32± 4.50± 7.1±0.3 4.6± 3.05± 3.45± 0.13 7.54 0.19 6.7056’N & 0.65 0.01 0.03 0.1 0.08 0.06086°86.670’E

3 20°74.2 64.5± 232.2± 7.92± 4.40± - 0.69± - 4.85± - 0.30 - 0.3035’N & 33.5 227.75 0.03 0.02 0.05 0.07086° 86.076’E

4 20°74. 65.5± 48.2± 5.92± 4.65± 0.26±0 2.06± 5.90±0 6.15± - 4.27 - 0.73102’N 8.5 38.8 0.05 0.09 1.74 0.02&086°86.009’E

5 20°74. 4.95± 6.05± 3.22± 5.05± 6.75± 4.4± 5.45± 5.55± 0.14 72.72 - 9.69105’N & 0.65 1.55 0.04 0.07 1.45 0.4 0.15 0.02086° 86.086’E

6 20°74. 0.39± 42.75± 2.85± 6.20± 61.5± 6.5± 3.35± 3.65± 15.76 15.20 3.51 7.4535’N & 0.03 33.25 0.04 0.05 2.5 1.1 0.05 0.05086° 86.71’E

7 20°74. 88±7 191.5±1 6.45± 6.80± 0.21± 3.1± 6.60± 6.80± - 1.62 - 0.8323’N & 48.5 0.05 0.05 0 0.1 0 0.05086° 86.07’E

8 20°74. 0.62± 80±6 4.40± 5.40± 69±15 7.4± 4.55± 4.85± 11.12 9.25 2.38 6.86224’N& 0.24 0.22 0.07 0.2 0.05 0.06086° 86.089’E

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1108 INDIAN J MAR SCI. VOL 43(6), JUNE 2014

9 20°74. 72±13 191.5±1 5.60± 7.30± 0.16±0 1.94± 6.85±0 7.05± 1.01 1.00

218'N & 18.5 0.08 0.05 1.26 0.09086°86.076'E

10 20°74. 7.85± 6.9±0.6 5.87± 5.50± 4.53± 5.3±0.1 6.05± 6.45± 0.06 76.81 0.02 10.7321'N & 1.75 0.01 0.06 4.07 0.08 0.01086°86.06'E

Table 2-Biochemical characteristics of three isolates showing more resistant to mercury

Tests BW-03 BW-101 BS-202T

Methyl Red +ve +ve +ve

Voges Proskauer 's -ve -ve -ve

ONPG -ve -ve +ve

Lysine -ve -ve +ve

Ornithine -ve -ve +ve

Urease -ve -ve -ve

Phenyl alanine -ve -ve -ve

Nitrate Reduction +ve +ve +ve

H2S Production -ve -ve -ve

Citrate Utilization +ve +ve +ve

Indole +ve +ve +ve

Oxidase -ve -ve +ve

Motility Test +ve +ve +ve

OfF Test Fermentative Fermentative Oxidative

Lactose +ve +ve +ve

Xylose +ve +ve -ve

Maltose +ve +ve +ve

Fructose +ve +ve +ve

Dextrose +ve +ve +ve

Galactose +ve +ve +ve

Trehalose +ve +ve +ve

Sucrose +ve +ve +ve

Mannose +ve +ve +ve

Inulin -ve +ve -ve

Inositol -ve -ve -ve

Sorbitol +ve +ve -ve

Mannitol +ve +ve +ve

Arabitol -ve -ve +ve

Cellobiose +ve +ve -ve

Rhamnose -ve -ve -ve

Salicin +ve +ve -ve

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BW-101 were having the same biochemical test resultsexcept inulin utilization, whereas, BS-202T was positivefor ONPG test, lysine and ornithine utilization andoxidase test (Table 2). Considering the biochemical testsinto account, BW-03 was identified as Bacillus Cereusand BW-101 as Bacillus circulans.

The partial sequence of the 16S rRNA gene ofBS-202T was submitted to NCBI GenBank and theassigned accession number is JX273780 which has beenidentified to be Vibrio hepatarius.

BW-03 (Bacillus cereus) showed resistance towardssix antibiotics (AM, VA, NX, AZM, N, AC) out of twelveantibiotics tested, whereas BW-101 (Bacillus circulans)was also resistant towards six antibiotics (AM, VA, NX,AZM, A, AC). BS-202T (Vibrio hepatarius) was resistantto four antibiotics (VA, NX, AZM, AC).

When the tolerance limit for mercury and other heavymetals were tested for the three selected isolates theywere observed to resist upto 50 ppm of Hg and higherlevel of other heavy metals, viz. Cadmium (109.9-219.9ppm), Zinc (100.1-403.6 ppm), Lead (625-1250 ppm)and Arsenate (1860.1-3720.2 ppm) (Fig. 4). Moreresistant toward all the metals were was observed in BS-202T (Vibrio hepatarius).

In order to obtain the variation in the results obtainedin the studied parameters of the sample two-way ANOVAwas performed (P<0.05). The physicochemicalparameters of the samples collected did not showsignificant variation for pH (F=3.354), salinity (F=2.866)

and total mercury content (Fwater

=2.345 andF

sediment=0.476) during summer and monsoon

season. However, the temperature during the studyperiod showed significant variation (F=137.14).During the summer season mercury resistantmarine bacterial population was higher than thoseof monsoon season and the value showedsignificant variation in sediment samples(F=32.529). In both water (F=31.014) andsediment samples (F=39.866) total heterotrophicbacterial load showed significant variation insummer and monsoon. Similarly, the percentageof mercury resistant marine bacterial populationdid not show significant variation (P>0.05).

When the physico-chemical parameters wereanalysed for correlation with the THB and MRMBpopulation only, mercury content was found tohave significant correlation with the MRMBpopulation throughout the study period (Table 3).Though other parameters like pH, salinity andtemperature also influence the THB as well asMRMB both positively and negatively, however,mercury content is found to be the only parameterhaving positive correlation with MRMBpopulation during two years of study period at both95% and 99% of confidence limit (Monsoon: 0.94,Summer: 0.93).

Discussion

The physico-chemical parameters of thesamples analysed were very similar to each otherin the two seasons studied (summer and monsoon)among the sampling stations and there is nosignificant variation observed. An earlier study12

reported that the pH of Bhitarkanika water variedfrom 7.5 to 7.6 and in the nearby water bodiesfrom 6.95 to 7.36. Hence, the decrease of pH inthe water of the ecosystem by 0.2-0.3 in less thantwo years may be attributed to the seasonalfluctuations and/or anthropogenic pressure ofpollutants. Though heavy metal pollution does notcontribute to the lowering of pH in the ecosystem,however, it increases the toxicity level of themetals towards inhabitant organisms. Largestmangrove ecosystem Sundarban is also facing the

Fig. 4–Minimum Inhibitory Concentration (MIC in ppm) of threeisolates towards different heavy metals

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Table 3-A comparative account of mercury contamination in various wetlands in the world

decreasing trend of pH 13 • The decreasing trend of pHin the ecosystem may cause many harmful effects inthe future like extinction of species, increase of BODand decrease of fish population in mangroveecosystem".

Significant variation was observed in thetemperature of water in this ecosystem during thestudy period which is quiet natural when thegeographical location of the ecosystem is taken intoconsideration. Average value of salinity level in theecosystem is 14.25 ppt during monsoon and 15.6 pptduring summer season signifying the brackishenvironment ofthe ecosystem". There are reports ofchanging pattern of uptake of heavy metals bysubmerged plants like Elodea and Potamogeton due .to variation in physical parameters like temperatureand salinity. In these two plants, lower temperatureand salinity level increases the accumulation of heavymetals by 50%16. Mercury as well as other heavymetals can be accumulated by the mangrove plantsand the mangrove sediments which may be influencedby the above two physical factors. As two favourablefactors (low temperature and salinity) have beenobserved in Bhitarkanika ecosystem, the uptake ofmercury by the mangrove is supposed to be increasedwhich will create a great problem as mercury will begradually accumulating in the animals throughmangrove leaf litters.

An increasing level of mercury in both monsoonand summer season was observed during the two yearsof study period. The increasing trend of mercury level

WetlandsMangrove wetlands of French GuianaSunderban Mangrove Wetland, India

in summer season is usual as the water sample getsdiluted during monsoon season due to various dilutionfactors like increase in river water, rain fall etc. Sameresult of lower concentration of heavy metals inmonsoon than that of summer was also reported fromRamgarh Lake, India 17.However, the greatest concernis the level of mercury (1.03 ppb) at St. 6 duringsummer-2012. This was above the permissible limitof mercury (1 ppb or 0.001 ppm)". During monsoonseason the national park remains closed for the touristdue to the breeding season of the inhabitingcrocodiles, hence no human interference. However,Stn 6 is present well within the Dangrnal forest resthouse permises and during summer due to humaninterference as well as tourist activities the mercurylevel might have raised beyond the permissible level".If the concentration of mercury is in this increasingtrend, it will definitely cause havoc on the inhabitantsof the ecosystem. However, the mercury level in theBhitarkanika mangrove ecosystem is less than that ofthe mangrove ecosystem of Brazil (60-184 ppb)19,China (0.01-2.39 ppb )20, French Guiana (0.15-2.57nmol/g)". Seasonal variation of mercury content i.e.higher in summer season than that of the monsoonseason has also been established in Sunderbanmangrove wetlands/", Though the mercurycontamination in Bhitarkanika mangrove wetland isquiet less in comparison to the various other mangroveand other wetlands in other parts of the globe(Table 3), it is in an increasing trend when the twoyears data were compared in the present study.Though the highly protected core area ofBhitarkanika

Total Hg content0.41 nmol/g43.32ng/g

(Post monsoon)196.45 ng/g900 ng/kg

Detected inMangrove sediments"

Core sediments" 17.2 ng/g(Pre monsoon)

Surface sediments"Wetland soils?

Mangrove wetland, ChinaKejimkujik NationalPark, Nova Scotia, CanadaGulf of MexicoAcadia National ParkCoastal estuary and Mangrove, Brazil

3.38 ng/l19.1 ng/g

2.04mglkg

Surface water"Water43

Upper sediment layers"

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National Park suffers less from the human interferencerelated problems, the non-point source pollutants maybe the sole cause of increasing trend of mercury levelin the study site. On 9th September, 2009 a shipcarrying tonnes of iron ores and oil shunk near shoreof Bay of Bengal of Paradeep region causing themercury containing oil spills move towards thenational park area in high tide, causing increasingpollution level of the same. Many huge industries arealso present and certainly emitting their wastematerials to the nearby stream water i.e. rive Brahmaniand Baitarani that constitute the delta of Bhitarkanika.These rivers carry huge amount of pollutants whichultimately deposits in the mangrove ecosystem dueto its sieving nature to add the pollution relatedproblems of the natural ecosystem.

The mangrove wetland itself has a role to play inthe fate of mercury due to the presence of mercuryvolatilizing and methylating bacterial population aswell as the floral diversity. The methylating bacteriacan readily convert the inorganic form of mercury tothe highly toxic organic form whereas the resistantbacterial population convert them back to the lesstoxic volatile form making the environment free frommercury pollution23. As a protected naturalenvironment, the wetland does not possess any directsource to the mercury contaminants and all theinorganic from of mercury that comes to theecosystem are from the non-point sources through therivers, high tides and the air pollutants which arereflected in terms of mercury resistant bacterialpopulations. Hence there is an increase in mercuryresistant bacterial population corresponding to thepollutants invading to the wetland.

We found a strong positive correlation betweenthe mercury resistant bacterial population with thetotal mercury content and a negative correlation withthe total heterotrophic bacterial population in thestations. There is an increase in the number of THBpopulation from summer to monsoon which may bedue to the dilution through freshwater inflow. Totalheterotrophic bacteria in the coastal environments ofAndaman islands of India was in the range of 103 to104 CFU/ml in water and 104 to 105 CFU/g in

sediment samples24 which is less than the number ofpopulations found in the present study. However, theTHB population in Muthupettai mangroves, Southeastcoast of India was estimated to be in the range of 105

CFU/mL in water samples and 107 CFU/g in thesediment samples25 which is well within the rangeobtained in the present study. Thus, the totalheterotrophic bacterial population of Bhitarkanikamangrove ecosystem are in the same range observedin mangrove ecosystems of other parts of the country.

Distribution of mercury resistant bacteria is quietubiquitous in nature and accounts for 1-10% ofaerobic total heterotrophic bacteria26. Though mercuryresistant bacteria have been isolated from theuncontaminated areas, mercury resistant bacterialpopulation is often correlated with the level ofmercury contamination of a particular environment27.However, there are a few reports available regardingthe mercury resistant bacterial population in themarine environment. Ramaiah and De27 predicted theunusual rise of their population in coastalenvironments. As per their report 96% of the CFU inwater and 71.4% of the CFU in sediment can toleratea mercury level of 10 ppm (50 µM). In the presentstudy, percentage of mercury resistant bacterialpopulation was found to form upto 89.28% in watersamples and 88.88% in sediment samples duringsummer season where as 16.5% in water and 15.2%in sediment during monsoon season. Hence, themercury resistant bacterial population in Bhitarkanikaecosystem is found to follow the range reported byRamaiah and De27 from Bay of Bengal. Manyprevious studies in Europe and North America havealso reported the occurrence of mercury resistantbacterial population in marine environments whichhave been emerged by anthropogenic activities28.

In addition, a huge fraction of mercury resistantbacterial population from Bay of Bengal, India hasbeen reported which ranged from 12 to 49% of thetotal heterotrophic bacteria18. This is far below theresult the present study (16.5% to 89.28% in waterand 15.2% to 88.88% in sediment) confirming theincreasing mercury pollution status in Bhitarkanikamangrove ecosystem. Resistant to mercury by bacteria

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Table 4-Correlation co-efficient values of Mercury resistant marine bacterial population with the pooled data of thephysico-chemical parameters during the study periods (a) Monsoon season of two years; (b) Summer season

of the two years

(a)

pH' Salinity" Temperature' Hg Content' THB' MRMB'

pH 1

Salinity 0.24 I

Temperature 0.25 0.25 I

Hg Content 0.16 -0.02 -0.27 1

THB 0.12 -0.06 -0.49 0.32 1

MRMB 0.21 0.02 -0.37 0.94$@ 0.42

(b)

pH' Salinity" Temperature' Hg Content' . THB' MRMB'

pH 1

Salinity -0.27 1

Temperature 0.48 -0.23 1

Hg Content 0.50 -0.17 0.03 tTHB 0.38 0.24 -0.05 0.67$ 1

MRMB 0.65$ -0.16 0.04 0.93$@ 0.60

in the environment do not come naturally, but theygradually develop the phenomenon by continuousexposure to the metals in their surroundingenvironment". Contamination of the coastal zones isincreasing at an alarming rate in other Asian wetlandslike Southern Kerala, India", Gulf of Mannar, India",Jinzhou Bay, China", coastal mangroves of HongKong" and in many more countries. Hence, the factof mercury pollution in Bhitarkanika ecosystemshould be taken into consideration to conserve thebiodiversity of this ecosystem.

Though a little knowledge is available. regardingthe mechanism of mercury resistant marine bacteriain the coastal region of India; this study reports thepresence of mer operon mediated mechanism in theisolates of the Bhitarkanika mangrove ecosystem. Out

of fifteen, merA gene has been traced in three isolateswhich confirms the mechanisms of resistance(volatilization of mercury). Highly significant positivecorrelation was reported between the harbouring ofmer genes, phenotypic resistance to mercury andconcentration of mercury in the source environment".Thus, the amplification of merA gene in isolates fromthis ecosystem confirms the mercury contaminationin it. This mer mediated mechanism of mercuryresistance is due to selection or genetic exchange byhorizontal gene transfer which promotes the bacterialcommunity to adapt in the mercury contaminatedenvironment. Though other phenotypically mercuryresistant isolates did not show proper banding patternfor merA amplification, they may harbour some nonmer mediated mechanisms for their resistance towardsthe noxious metal".

Pooled data of the physico-chemical and biological parameters of both water and sediment samples during two years of studyperiod; #pooled data of the physico-chemical parameter of water sample during two years of study period; SSignificant positivecorrelation at P d~0.05; @Significant positive correlation at P ~0.01. THB= Total Heterotrophic Bacteria; MRMB= MercuryResistant Marine Bacteria.

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Mercury resistant isolates have been found toresist other heavy metals like cadmium, zinc, lead andarsenate and many commonly used antibiotics likeAM, VA, NX, AZM, A, AC. All the isolates studiedwere purely environmental in nature but theirpossession of antibiotic resistant characteristicsbecomes a huge threat to the natural inhabitants ofthe ecosystem. The resistance to antibiotics mayevolve due to the fact that the mobile genetic elementslike plasmids and transposons which carry multiplegenes encode both for metal and antibioticresistance36. Thus, exposure to any one of thesenoxious agents may render microorganisms to becomeresistant to several toxicants35. However, resistanceto multiple metals and antibiotics have been foundand characterized in several other bacterial systemsat molecular level37. These isolates are of interest asthey promise for bioremediation of toxic heavymetals.

In the present study, minimum inhibitoryconcentration (MIC) of the three isolates in mercurywas found higher than the standards mentioned byvarious research groups38. A strong positivecorrelation between mercury concentration andminimum inhibitory concentration value of mercurywas observed in the present study which is in linewith the report of Nasrazadani et al.39 Thus, this studyconfirms the increasing mercury level in the studysite. Bacillus sp. are common flora of theenvironments40 which have been isolated from thestudy sites and the environment being saline in natureVibrio sp. are also abundant in it as they prefer to livein the brackish environments41.

Conclusions

Present study inferred that mercury pollution inthe Bhitarkanika mangrove ecosystem is increasingat an alarming rate. Marine bacteria and theircharacterization for mercury resistant genotype andMIC value to other heavy metals are confirmedthorugh the present study.

Acknowledgements

Authors would like to acknowledge theauthorities of NIT, Rourkela for providing facilities.

H.R.D gratefully acknowledges the receipt of researchfellowship from Ministry of Human ResourceDevelopment, Government of India for his doctoralresearch. S.D. thanks the Department ofBiotechnology, Government of India for researchgrant on bioremediation through marine bacterialbiofilm.

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