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Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesa20

Characterization of arsenite-oxidizing bacteria isolated from arsenic-contaminated groundwater of West Bengal a

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Dhiraj Paul , Soumya Poddar & Pinaki Sar

a

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Department of Biotechnology, Indian Institute of Technology, Kharagpur, India Published online: 19 Aug 2014.

To cite this article: Dhiraj Paul, Soumya Poddar & Pinaki Sar (2014) Characterization of arsenite-oxidizing bacteria isolated from arsenic-contaminated groundwater of West Bengal, Journal of Environmental Science and Health, Part A: Toxic/ Hazardous Substances and Environmental Engineering, 49:13, 1481-1492, DOI: 10.1080/10934529.2014.937162 To link to this article: http://dx.doi.org/10.1080/10934529.2014.937162

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Journal of Environmental Science and Health, Part A (2014) 49, 1481–1492 Copyright © Taylor & Francis Group, LLC ISSN: 1093-4529 (Print); 1532-4117 (Online) DOI: 10.1080/10934529.2014.937162

Characterization of arsenite-oxidizing bacteria isolated from arsenic-contaminated groundwater of West Bengal DHIRAJ PAUL, SOUMYA PODDAR, and PINAKI SAR

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Department of Biotechnology, Indian Institute of Technology, Kharagpur, India

Nine arsenic (As)-resistant bacterial strains isolated from As-rich groundwater samples of West Bengal were characterized to elucidate their potential in geomicrobial transformation and bioremediation aspects. The 16S rRNA gene-based phylogenetic analysis revealed that the strains were affiliated with genera Actinobacteria, Microbacterium, Pseudomonas and Rhizobium. The strains exhibited high resistance to As [Minimum inhibitory concentration (MIC)
10 mM As3C and MIC
450 mM As5C] and other heavy metals, e.g., Cu2C, Cr2C, Ni2C, etc. (MIC
2 mM) as well as As transformation (As3C oxidation and As5C reduction) capabilities. Their ability to utilize diverse carbon source(s) including hydrocarbons and different alternative electron acceptor(s) (As5C, SO42¡, S2O32¡, etc.) during anaerobic growth was noted. Growth at wide range of pH, temperature and salinity, production of siderophore and biofilm were observed. Together with these, growth pattern and transformation kinetics indicated a high As3C oxidation activity of the isolates Rhizobium sp. CAS934i, Microbacterium sp. CAS905i and Pseudomonas sp. CAS912i. A positive relation between high As3C resistance and As3C oxidation and the supportive role of As3C in bacterial growth was noted. The results highlighted As3C oxidation process and metabolic repertory of strains indigenous to contaminated groundwater and indicates their potential in As3C detoxification. Thus, such metabolically well equipped bacterial strains with highest As3C oxidation activities may be used for bioremediation of As contaminated water and effluents in the near future. Keywords: Arsenic, groundwater, indigenous bacteria, arsenate reduction, arsenite oxidation.

Introduction Geogenic arsenic (As) in groundwater of the Bengal delta plain (BDP) covering large part of West Bengal and Bangladesh, poses a severe threat to drinking water resources of this region affecting tens of millions people.[1,2] Arsenic is a ubiquitous toxic metalloid and is widely distributed within the terrestrial and subsurface ecosystems and in many regions of the world through natural geochemical and anthropogenic activities.[3] Understanding the geochemical characteristics of As in subsurface environment and mechanisms underlying its release into groundwater is a subject of significant interest for developing sustainable strategies for drinking water supply in the affected regions.[4,5] Address correspondence to Pinaki Sar, Department of Biotechnology, Indian Institute of Technology Kharagpur, West Bengal 721302, India; E-mail: [email protected] Present address for Soumya Poddar is Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, University of California, Los Angeles, CA, USA Received April 5, 2014. Color versions of one or more of the figures in this article can be found online at www.tandfonline.com/lesa.

In aqueous environment soluble As is mostly represented by its two forms, arsenate [As5C as H2AsO4¡ and HAsO42¡] and arsenite [As3C as H3AsO30 and H2AsO3¡]; having different levels of toxicity to living system. The latter species is more soluble and highly toxic than As5C. Arsenite (As3C), that adsorbs poorly on solid phases of Fe/Mn oxide-, hydroxide- mineral surface represents the predominate species in anoxic subsurface system, while under oxic condition, As5C which strongly adsorbs on minerals is the major form.[1] Studies on several affected sites at Bangladesh, West Bengal and other parts of southeast Asia have confirmed that along with geochemical processes, the catalytic function of indigenous microbes are strongly responsible for mobilization of As within the groundwater.[6] It is well appreciated that inhabitant bacteria play a critical role in As biogeochemistry by their metabolic activities (e.g., redox transformation and methylation) which ultimately affect speciation, mobility and toxicity of the metalloid. Interestingly though As5C is considerably less toxic than As3C, resistance to As5C required its reduction to the latter through cytosolic detoxification system (ars), which allows efflux of As3C in cell exterior. Alternatively, As5C is reduced by dissimilatory reductase system (arr) that facilitates its use as terminal electron acceptor (TEA) during anaerobic growth of the bacteria. On the other

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1482 hand, toxic As3C is oxidized by oxidase (aio) system during chemolithoautotrophic/heterotrophic metabolism. The latter groups use energy and reducing power from As3C oxidation during CO2 fixation or other anaerobic metabolism and cell growth under aerobic or anaerobic (nitrate reduction) condition.[1,7] Recent investigations on geomicrobial activities within As rich groundwater have noted simultaneous presence of both As-oxidizing and -reducing abilities. Presence and abundance of required enzymes (e.g., As3C oxidase and As5C reductases) and genes for redox transformation of As are widely reported within the bacteria isolated from several locations within the contaminated aquifers.[4,7–9] Particularly, the potential role of As5C-reducing processes (dissimilatory or detoxification based) in mobilizing As from subsurface sediment has been highlighted in several studies.[4,6,10] It is interesting that irrespective of the nature of the reduction system (ars or arr), toxic As3C concentration eventually builds up in the groundwater and consequently, inhabitant bacteria must be able to oxidize As3C to less toxic As5C. Therefore it is imperative to characterize such As3C-oxidizing bacteria and their oxidizing process in order to understand the geomicrobiology of As-contaminated aquifers. Oxidation of As3C generates less toxic As5C, which can either precipitate with iron (Fe3C) or be adsorbed by ferrihydride and then creates opportunities for developing systems for remediation of contaminated water. Several bioprocess for treatment of As-contaminated water have been developed based on such microbial catalytic systems.[11] In order to characterized the bacterial As3C oxidation a number of aerobic and anaerobic bacterial strains namely Herminimonas arsenicoxydans, Rhizobium sp. strain NT26, Alkallimnicola ehrlichii, Bacillus arsenoxydans, Pseudomonas sp., Alcaligenes sp., Hydrogenophaga sp. and Thiomonas sp. have been isolated from different contaminated niches including groundwater.[12–15] In recent time a strain of Bacillus and Geobacillus have been isolated from As contaminated soils in West Bengal, India.[16] Though there are reports on isolation, identification and characterization of As3C oxidizing bacteria from different habitats, similar study on As-rich groundwater of eastern part of West Bengal are very limited. In this study we have described the detail metabolic characters of As-resistant and oxidizing strains isolated from high As-contaminated groundwater of West Bengal. Their ability to utilize different organic compounds including hydrocarbons as sole source of carbon during growth, or different electron acceptors, to grow over a wide range of temperature, pH and salinity, to produce siderophore, to tolerate various heavy metals during growth, to transform As mainly from As3C to less toxic As5C and to form biofilm were revealed. The results will be helpful for further study on exploring bioremediation method for As- contaminated aquifers or removing arsenic from drinking water by utilizing such potential As transforming indigenous bacteria.

Paul et al. Materials and methods Microorganisms, media and growth conditions Nine bacterial strains were isolated from As-rich groundwater samples collected from Barasat and Chakdaha areas of West Bengal following a dilution plate technique with R2A agar medium.[17] All the strains were routinely grown on R2A medium or minimal salt media (MSM).[18]

16S rRNA gene-based identification of test bacterial strains Genomic DNA of the bacterial strains was prepared by the sodium dodecyl sulfate-proteinase K-cetyltrimethyl ammonium bromide (CTAB) method.[19] PCR amplification of 16S rRNA gene from the isolated genomic DNA was done using bacteria specific primers 27F and 1492R.[20] Each 100 mL PCR reaction contained: 100 ng template DNA, 10 mL 10£ reaction buffer, 5 mL MgCl2 (25 mM), 2 mL dNTP mix (10 mM), 10 mole of each primer, and 2U of Taq polymerase (Promega, USA). The PCR amplification was performed with an initial denaturation step of 94 C for 5 min; followed by 30 cycles of denaturation at 94 C for 60 sec, annealing at 58 C for 45 sec, elongation at 68 C for 90 sec and a final elongation step at 68 C for 7 min. Amplified fragments were separated by 1% agarose gel electrophoresis and ˷1.5 kb bands were purified, cloned in pGEM-T easy vector (Promega, Madison, WI, USA) and sequencing was done with vector specific primers on an ABI 3100 automated sequencer.

Phylogenetic analysis Sequence data were compared with 16S rRNA gene sequences deposited in public databases by BLAST (NCBI) program and classification was made using classifier program in the Ribosomal Database Project. The 16S rRNA gene sequences of various bacteria showing high similarity to amplified 16S rRNA sequences of test bacterial strains were retrieved from the GenBank database, aligned with ClustalW. The resulting alignment was used to construct phylogenetic tree incorporating neighbor joining method and Jukes–Cantor distance matrix by MEGA 5.1 software package.[21] Bootstrap percentages (1000 bootstrap replications) were used to test the robustness of phylogenetic relationships within the tree.

Physiological characterization Bacterial shape and Gram’s nature were determined by bright field microscopy using gram staining procedure. Swimming motility was assessed following the procedure described previously by Lee et al. [22] where swim plates were prepared by adding 0.3% agar to R2A broth and inoculated with bacteria from an overnight culture. The

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Characterization of arsenite-oxidizing bacteria plates were incubated at 30 C and bacterial migration was observed after 24 h. Temperature sensitivity was monitored following bacterial growth at temperatures 10, 20, 25, 30, 37, 45 and 50 C in minimal salt medium (MSM). Ability to survive at varying pH (pH 3.0_12.0) was tested by using minimal medium buffered with Tris (for pH range 7.0_12.0) or sodium citrate (for pH range 3.0–6.0). Salt tolerance was observed using 0 to 10% salt (NaCl) containing MSM. To check the ability of carbon source utilization, MSM without any addition of carbon source except the test organic compound [casein, glycerol, glucose, acetate, pyruvate, lactate, citrate, starch and ascorbic acid at 10 mM concentration each and hydrocarbons BTEX (50 mg mL¡1) (mixture of benzene, toluene, ethyl benzene and xylene in 1:1:1:1), nonadecane, docosane, dodecane, pentadecane, cycloanthrene, naphthalene, pyrene, flurone, anthracene, hexane] was used. Fresh colonies were inoculated on agar plates followed by incubation at 30 C for 7 days. Control plate was made with MSM agar without any electron donor and incubated with bacterial strains under same condition. Multiple electron acceptor utilizing ability of the isolates was tested following growth in anaerobic agar plates of MSM supplemented with different electron acceptors (sodium nitrate, sodium sulphate, sodium thiosulphate, sodium sulphite, sodium selenate and ferric chloride; 10 mM of each) and donors (glucose 0.05%).[23] The pH of the medium was adjusted to 7.2 after adding 1 g L¡1 of Lcysteine-HCl as a reducing reagent. Control plates were kept without addition of any electron acceptor and incubated with test strains under the same anaerobic environment. All experiments were conducted in an anaerobic workstation (Coy Laboratories, model 1025 S/N) filled with 95% N2, 5% H2, and 5% CO2 gases. Enzymatic (oxidase and catalase) and IMVIC (Indole Methyl red VogesProskauer Citrate) tests were performed using Himedia Kit.

Effect of As on bacterial growth Growth kinetics of all the bacterial strains was studied in R2A medium with or without supplementation of 250 mg L¡1 As3C or As5C. Each strain was inoculated (1% inoculum) in both As free R2A and As (As3C and As5C) containing medium and incubated at 30 C for 48 h in an incubator shaker at 120 rpm. Growth was monitored at various time intervals by measuring the optical density (OD) of cultures at 660 nm with UV/Vis spectrophotometer (Varian Cary 50Bio UV/Vis, Agilent Technologies, North America). Arsenite oxidase, arsenate reductase and siderophore assay Arsenite oxidase and arsenate reductase activities of the bacterial strains were tested following the procedure of Drewniak et al.[10] MSM agar containing 10 mM sodium arsenite as electron donor was used for the determination of As3C oxidation while the same medium containing 5 mM lactate as a carbon source and 5 mM sodium arsenate as electron acceptor was used for arsenate reductase assay. For arsenite oxidase and arsenate reductase assay plates were incubated at 30 C either aerobically for 5 days or anaerobically for 7 days, respectively. Following bacterial growth, plates were flooded with 0.1 M AgNO3 to allow formation of a coloured precipitate upon its reaction with As3C or As5C. A brownish precipitate revealed the presence of silver arsenate (Ag3AsO4) in the plate as a result of arsenite oxidase activity, while yellow precipitate indicated silver arsenite (Ag3AsO3) formation due to arsenate reductase activity. Production of siderophore was studied using Chrome Azurol S (CAS) agar media.[24] CAS agar plates were inoculated with bacterial strains and incubated at 30 C for 7 days. Colonies showing orange hollow zone following incubation were recognized as siderophore positive. Quantitative estimation of As3C oxidation

Determination of minimum inhibitory concentration (MIC) of As and other heavy metals Minimum inhibitory concentrations of As and other heavy metal were tested for all the isolates along with multiple metal resistant bacterium Cupriavidus metallidurans (DSMZ 2839). MSM agar plates were supplemented with graded concentrations of As5C (Na2HAsO4.7H2O) or As3C (NaAsO2) or other metals [Cu(NO3)2. 3H2O, Cd (NO3)2. 4H2O, NiCl2. 6H2O, ZnCl2, Pb(NO3)2, HgCl2 and K2Cr2O7; Merck, Germany] and inoculated with mid log phase cells, incubated at 30 C for 7 days and checked bacterial growth in every 24 h. In this context, minimum inhibitory concentration (MIC) refers to the lowest concentration of metal that completely inhibited bacterial growth in minimal medium agar.

Arsenite oxidation kinetics of the test bacteria was studied using growth decoupled resting cells suspended in artificial groundwater (AGW) medium (Composition g L¡1: CaCl2. 2H2O 0.148; NaCl 0.315; KCl 0.1067; MgSO4. H2O 0.9399; NaNO3 0.2488 and NaHCO3 0.11; to pH 7). For each strain, overnight grown cultures were centrifuged (10,000 rpm, 10 min); pellet was suspended and washed thoroughly with AGW medium. Washed pellet was finally re-suspended in AGW medium and supplemented with NaAsO₂ to a final concentration of 250 mg L¡1. The assay was monitored for next 120 min by sampling at every 20min interval. To determine the amount of As3C transformation, a blank test without bacterial inoculation was performed using the same condition. Total As content was analyzed using hydride generated- atomic absorbance spectra (HG-AAS) (AAnalyst 200, PerkinElmer, USA).

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The As speciation (As5C and As3C) was determined as described by Drewniak et al.[10]

Results Identification of bacterial strains isolated from As-contaminated groundwater

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Biofilm formation assay Biofilm formation was assayed in microtiter plates as described previously Gallant et al.[25], with some modifications. In brief, cultures (O.D 660 nm »0.2) were inoculated in R2A broth with/without As3C or As5C (final concentration of 10 mM) and incubated for 48 h at 30 C in static condition. After incubation, planktonic cells were removed by washing with phosphate buffer saline (PBS). The formed biofilm was stained with 0.5% crystal violet (CV) for 15 min and excess CV was removed by washing with water. Finally, the retained CV was solubilised with absolute ethanol and absorbance was measured at 660 nm. Statistical analysis Relationship among the bacterial strains was estimated by comparing their As3C, As5C and other heavy metal resistance (MTC) values by two components Principal Component Analysis (PCA). An Unweighted Pair Group Method with Arithmetic Mean (UPGMA) analysis was performed on the basis of the metabolic properties of the strains considering particularly their ability towards the utilization of different C source and inorganic electron acceptors during anaerobic growth, activity of As3C oxidase and As5C reductase, and formation of sedirophore and biofilm. All statistical analyses were done using the Multi-Variate Statistical Package (MVSP, Version 3.1).

Taxonomic identity and phylogenetic lineage of the isolated strains were deduced by 16S rRNA gene sequence analysis (Table 1, Fig. 1). Similarity search in NCBI and RDP database revealed that most of the strains (BAS310i, BAS316i, BAS325i, BAS335i and CAS934i) were affiliated to the high GC, Gram negative genus Rhizobium, followed by Pseudomonas (strains CAS908i and CAS912i) and high GC containing Microbacterium (strain CAS905i) and Actinobacteria (BAS123i). Phylogenetic analysis as ascertained by neighbour-joining tree revealed close lineages of strains BAS310i, BAS316i, BAS325i, BAS335i and CAS934i with members of Rhizobiaceae family including Rhizobium spp.; previously reported from As contaminated sites of West Bengal and Assam, India and R. selenitireducens (Fig. 1). Bacterial strains CAS912i and CAS908i shared high sequence identity among themselves and branched together showing lineage to Pseudomonas spp., isolated earlier from subsurface environment. A distant lineage of CAS912i and CAS908i with several Pseudomonas spp. previously retrieved from As-contaminated groundwater was observed.[9] Sequence related to Acinetobacter was represented by only one isolate, namely BAS123i. A close lineage of BAS123i with A. lwoffi and other related species retrieved from different As-contaminated sites was noted. Sequence of the strain CAS905i showed close relatedness with Microbacterium phyllosphaerae and M. favescens at 95% bootstrap support.

Nucleotide sequence accession numbers The sequences obtained in this study have been deposited in the GenBank database under accession no. KJ40077KJ40084 and KF442762.

Physiological characterization of bacterial isolates A range of physiological properties (including utilization of different organic carbohydrate compounds, hydrocarbons

Table 1. Closest match for 16S rRNA gene sequences of arsenic contaminated groundwater isolates and their taxonomic affiliation. Strains BAS123i CAS905i CAS908i CAS912i BAS310i BAS316i BAS325i BAS335i CAS934i

Closest NCBI Match (Accession no.) (% identity) Acinetobacter lwoffii (KF228924.1) (99) Microbacterium sp. iso-74 (KC768764.1) (99) Pseudomonas sp. 01WB02.2-37 (FM161393.1) (99) Pseudomonas sp. 01WB02.2-37 (FM161393.1) (100) Rhizobiaceae bacterium KAs3-R11 (JX110539.1) (99) Rhizobiaceae bacterium KAs5-R14 (JX110527.1) (99) Rhizobium sp. BZ6 (HQ588848.1) (99) Rhizobiaceae bacterium BAS325i (KF442762.1) (100) Rhizobiaceae bacterium KAs5-R14 (JX110527.1) (99)

*Genbank accession no. KJ140084 KJ140082 KJ140080 KJ140081 KJ140078 KJ140079 KF442762 KJ140077 KJ140083

Genus assigned Acinetobacter sp. Microbacterium sp. Pseudomonas sp. Pseudomonas sp. Rhizobium sp. Rhizobium sp. Rhizobium sp. Rhizobium sp. Rhizobium sp.

*GenBank accession number assigned for 16S rRNA gene sequences of arsenic contaminated groundwater isolates submitted in GeneBank (NCBI database). #Genus assigned to each isolate based on 16S rRNA gene sequence and phylogenetic analysis.

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Characterization of arsenite-oxidizing bacteria

Fig. 1. 16S rRNA gene sequence-based neighbor-joining phylogenetic tree. The tree is constructed using Jukes–Cantor distances. Then 1000 bootstraps analyses are conducted and more than 50% are denoted in nodes. Sequences retrieved from As-contaminated environment are suffixed as ‘A’. Sequences represented in bold red font are derived from isolated strains of this study.

and inorganic compounds as carbon and electron donors/ acceptors as well as growth at different pH, temperature and salinity ranges) was studied using the test bacterial isolates (Table 2). All the isolates recovered from As rich groundwater and used in this study were gram negative (except Microbacterium sp. CAS905i) in nature and except isolate Acinetobacter sp. BAS123i cells of all strains were rod shaped. All the bacterial strains were able to utilize carbon sources with varying carbon content [C3 viz., (glycerol, pyruvate and lactate), C6 (glucose), C12 (sucrose) and polysacceride (starch)]. Acetone (C3) and ascorbic acid (C6) were least utilized while C2 compound acetate was metabolized by all the strains except Rhizobium sp. CAS934i. Apart from these sugars the As-contaminated groundwater isolates were capable of utilizing several aliphatic and aromatic hydrocarbons (dodecane, docosane, pyrene, anthracene, BTEX, naphthalene, etc.) as their sole carbon source. Out of the nine isolates, Microbacterium sp.

CAS905i, Pseudomonas sp. CAS912i, Rhizobium sp. CAS934i were highly efficient in utilizing most of the test hydrocarbons (except one or two hydrocarbon). Utilization of alternative electron acceptor(s) during anaerobic growth was studied. It was noted that all the Rhizobium strains and Acinetobacter sp. BAS123i were capable to use As5C as TEA followed by Se6C. Utilization of S2O32¡, SO42¡ was detected among fewer (2-4) strains affiliated to Pseudomonas sp. CAS908i, Acinetobacter sp. BAS123i and Rhizobium (strains BAS310i and BAS325i) while NO3¡ was used by one Rhizobium (strain BAS325i). Iron (Fe3C) was not preferred as TEA by any of the test bacteria. Microbacterium sp. CAS905i and Pseudomonas sp. CAS912i were unable to utilize any of the electron acceptors during anaerobic growth. Most of the isolates showed their ability to grow over a broad range of pH (pH 5.0_10.0), temperature range (20_37 C) and salinity (1–9%). Noticeably, most of the Rhizobium spp. and Acinetobacter sp. were able to grow even at 50 C temperature. Both the Pseudomonas strains showed positive result in motility test. All the isolates were able to produce catalase and oxidase except strain Acinetobacter sp. BAS123i, which can produce catalase enzyme only. All the bacterial isolates were found to be positive for indole and methyl red tests and negative for Simmon’s citrate and Voges Prausker tests. Arsenic transformation (As3C oxidase and As5C reductase) and siderophore production abilities were tested within the isolates (Table 2). Acinetobacter sp. BAS123i, Microbacterium sp. CAS905i, Rhizobium sp. BAS316i and Rhizobium sp. BAS325i showed both As3-oxidizing as well as As5C-reducing abilities. Although strains Pseudomonas sp. CAS912i, Rhizobium sp. CAS934i, Rhizobium sp. BAS310i and Rhizobium sp. BAS335i demonstrated only As3C oxidase activity. Siderophore production was detected only within the Pseudomonas sp. CAS908i.

Determination of MIC values for the metals tested Arsenic (As3C and As5C) resistance properties were determined by monitoring bacterial growth in minimal medium supplemented with varying concentrations of As3C or As5C (Table 3). All the test bacterial strains showed very high resistance against As5C (MIC
600 mM), and resistance to As3C (MIC
10 mM) was found in fewer strains only. Several bacterial strains (viz., Pseudomonas sp. CAS912i, Rhizobium sp. CAS934i, etc.) showed elevated resistance to both As3C and As5C (Table 3). Resistance to other heavy metals (Ni, Cu, Cd, Zn, Pb, Cr and Hg) was also ascertained together with C. metallidurans (Table 3) by growing bacterial isolates on amended medium. It was observed that most of the isolates were resistant to metals (Cu, Cr, Zn and Pb) but were sensitive to Hg. For Cu, Cd and Cr, MIC values obtained for several isolates were found to be considerably high compared

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Table 2. Biochemical characterization of the nine bacterial isolates. Strain ID

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Biochemical tests Shape Gram character pH Temp ( C) Salinity (%) Motility Indole Methyl red Voges prausker Citrate utilization Oxidase Catalase Arsenate reductase Arsenite oxidase Siderophores Carbon sources Acetate Glycerol Pyruvate Lactate Acetone Glucose Citrate Ascorbic acid Sucrose Starch Dodecane Pentadecane Nonadecane Docosane Cyclohexane Naphthalene Fluorene Anthracene Phenanthrene Pyrene BTEX Electron donors As5C Se6C Fe3C SO42¡ S2O32¡ SO32¡ NO3¡

CAS905i

CAS908i

CAS912i

CAS934i

BAS123i

BAS310i

BAS325i

BAS316i

BAS335i

Rod C 4–11 25–37 1–9 ¡ C C ¡ ¡ C C C C ¡

Rod ¡ 4–11 20–37 1–3 C C C ¡ ¡ C C C C C

Rod ¡ 5–10 25–37 1–11 C C C ¡ ¡ C C ¡ C ¡

Rod ¡ 5–11 20–37 1–6 ¡ C C ¡ ¡ C C ¡ C ¡

Coccobacilli ¡ 5–11 20–50 1–3 ¡ C C ¡ ¡ ¡ C C C ¡

Rod ¡ 6–11 20–50 1–8 ¡ C C ¡ ¡ C C ¡ C ¡

Rod ¡ 5–10 20–50 1–6 ¡ C C ¡ ¡ C C C C ¡

Rod ¡ 5–9 20–50 1–6 ¡ C C ¡ ¡ C C C C ¡

Rod ¡ 4–10 20–50 1–6 ¡ C C ¡ ¡ C C ¡ C ¡

C C C C C C C ¡ C C ¡ ¡ ¡ C C ¡ C C C C C

C C C C C C C ¡ C C C ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ C

C C C C C C C C C C C C C C C C C C C C C

¡ C C C C C C ¡ C C C C C C C C ¡ C ¡ C C

C C C C ¡ C C ¡ C C ¡ ¡ ¡ ¡ ¡ C ¡ ¡ C ¡ C

C C C C C C C C C C C C ¡ C ¡ C ¡ C ¡ ¡ C

C C C C C C C ¡ C C C C C C C ¡ C C C ¡ C

C C C C ¡ C C C C C C C ¡ ¡ ¡ ¡ ¡ ¡ ¡ C C

C C C C ¡ C C ¡ C C C C ¡ ¡ ¡ ¡ ¡ ¡ ¡ C C

¡ ¡ ¡ ¡ ¡ ¡ ¡

¡ ¡ ¡ ¡ C C ¡

¡ ¡ ¡ ¡ ¡ ¡ ¡

C C ¡ ¡ C ¡ ¡

C ¡ ¡ ¡ ¡ C ¡

C C ¡ C C ¡ ¡

C ¡ ¡ C C ¡ C

C C ¡ ¡ ¡ ¡ ¡

C C ¡ ¡ ¡ ¡ ¡

C ve character, ¡ ve character

to that of C. Metallidurans, indicating their superior metal resistance. Among the test bacteria Microbacterium sp. CAS905i, Pseudomonas sp. CAS912i, Rhizobium sp. BAS310i and Rhizobium sp. BAS335i were endowed with high resistance to multiple metals. Noticeably resistance to Cu and Cd (MIC
6 mM) was present in all nine strains.

Effect of As on bacterial growth Bacterial isolates were grown in medium supplemented with either 10 mM As3Cor As5 C to ascertain effect of these toxic metal species in cell growth (Fig. 2). All the isolates followed a sigmoid pattern of growth. As evident from the

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Characterization of arsenite-oxidizing bacteria Table 3. Minimum inhibitory concentration (MIC) values of heavy metals.

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*MIC (mM) Strains

As5C

As3C

Cu

Cd

Ni

Cr

Hg

Pb

Zn

Acinetobacter sp. BAS123i Microbacterium sp. CAS905i Pseudomonas sp. CAS908i Pseudomonas sp. CAS912i Rhizobium sp. BAS310i Rhizobium sp. BAS316i Rhizobium sp. BAS325i Rhizobium sp. BAS335i Rhizobium sp. CAS934i C. metallidurans (DSMZ2839)

600 650 450 650 600 600 600 600 600 200

19 10 9 40 9 13 9 9 40 5

6 10 10 10 10 6 9 6 10 2.5

7 6 10 10 10 10 10 10 10 2

3 5 5 5 4 3 2 5 4 6

5 2 5 5 5 0.05 5 0.05 0.1 0.1

0.05 0 0.05 0.02 0.05 0.01 0.02 0.05 0.01 0.08

6 9 9 8 10 9 10 10 8 10

5 5 4.5 4 5 5 5 5 5 10

*MICs were tested in minimal medium agar plates supplemented with respective metals.

kinetics, except Pseudomonas sp. CAS908i, all the strains had a near similar kind of growth with or without As (As3C or As5C). To study the further effect of As on the growth of the test bacteria, both doubling time and growth rate constant were calculated in the presence and absence of As (Table 4). The bacterial cells growing in the presence of As3C or As5C showed relatively slower growth rate in comparison with bacterial cells growing without metalloids as evident from the increasing doubling time (maximum 1.0-fold for As3C or 1.9-fold for As5C). A few strain such as Pseudomonas sp. CAS912i showed faster growth in the presence of both As3C and As5C, where Rhizobium sp. BAS335i, Acinetobacter sp. BAS123i and other Rhizobium strain showed similar effects only in presence of As5C. The growth rate constant and doubling time of all the nine Asresistant bacterial isolates were shown in Table 4.

Biofilm formation Formation of biofilm of the test bacterial isolates during their growth with and without As was tested (Fig. 4). All the strains showed biofilm formation and interestingly presence of As3C or As5C lead to a change in their characteristic property in most of the cases. It was noted that except Rhizobium sp. BAS310i, in all other strains level of biofilm produces was either improved or remain unchanged in presence of As5C. In Pseudomonas sp. CAS912i and Rhizobium sp. BAS335i, presence of As5C caused nearly 2-fold increase in biofilm formation, while for the same strains As3C had a negative effect. Microbacterium sp. CAS905i showed increased biofilm in presence of both As5C and As3C. Presence of As3C in general causes a lowering in biofilm; however, strains Rhizobium sp. CAS934i and Acinetobacter sp. BAS123i showed some level of increase formation of biofilm in presence of As3C.

Statistical analysis Arsenic oxidation studies Arsenic oxidation efficiency of all the bacterial isolates were studied using growth decoupled resting cells exposed to 250 mg L¡1 As3C (Fig. 3). All the test strains showed their ability to oxidized As3C, although the efficiency of transformation varied within the individual strains. Four strains (Microbacterium sp. CAS905i, Pseudomonas sp. CAS908i, Pseudomonas sp. CAS912i and Rhizobium sp. CAS934i) showed highest As3C oxidase efficiency with
50% of total As3C transformed within 1 h. Particularly, the Rhizobium sp. CAS934i strain possesses only As3C oxidase but no As5C reduction activity. Except strains Acinetobacter sp. BAS123i, Microbacterium sp. CAS905i and Pseudomonas sp. CAS912i, all other stains indicated rapid oxidase kinetics during the initial 20 min, suggesting towards possible constitutive nature of As3C oxidase in their strains.

To understand the relationship between bacterial strains isolated from different sampling sites, a set of multivariate statistical analyses were performed. Bacterial ability to withstand As and other metals was subjected to two-component PCA, while other metabolic characters were used for an UPGMA-based analysis. From the graphic representation (score-plot) of PCA (Fig. 5), it was clear that first (PC 1) and second principal components (PC 2) were sufficient to explain the total variance i.e., 99.6%. Three completely different groups could be distinguished clearly from the graph. Group I was located on the right side of the score-plot, consisted of isolates Rhizobium sp. CAS934i, Pseudomonas sp. CAS912i and Acinetobacter sp. BAS123i. Group II included strains Rhizobium sp. BAS316i, Rhizobium sp. BAS325i, Rhizobium sp. BAS335i, Rhizobium sp. BAS310i and Microbacterium sp. CAS905i whereas

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Paul et al.

Fig. 2. Growth curves of bacterial strains in presence of As3C, As5C and without As in R2A medium (a: Acinetobacter sp. BAS123i; b: Rhizobium sp. BAS310i; c: Rhizobium sp. BAS316i; d: Rhizobium sp. BAS325i; e: Rhizobium sp. BAS335i; f: Microbacterium sp. CAS905i; g: CAS908i; h: Pseudomonas sp. CAS912i and i:Rhizobium sp. CAS934i). Growth was monitored by measurement of the optical density at 660 nm. Error bars indicate standard deviations (n D 3). —&— R2A — — As3C —~— As5C



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Characterization of arsenite-oxidizing bacteria Table 4. Doubling time and growth rate constant of the isolates in presence and absence of arsenic. Growth rate constant (h¡1)

Isolates

R2A

As3C

As5C

R2A

As3C

As5C

Acinetobacter sp. BAS123i Microbacterium sp. CAS905i Pseudomonas sp. CAS908i Pseudomonas sp. CAS912i Rhizobium sp. BAS310i Rhizobium sp. BAS316i Rhizobium sp. BAS325i Rhizobium sp. BAS335i Rhizobium sp. CAS934i

5.327 3.086 2.94 2.577 1.824 1.78 2.48 7.092 2.35

5 3.597 2.86 1.31 2.481 1.869 3.2 11.11 2.739

4.784 4.149 4.5 1.865 1.786 1.394 4.716 5.668 2.58

0.1877 0.324 0.339 0.388 0.548 0.56 0.4023 0.141 0.424

0.2 0.278 0.349 0.762 0.403 0.535 0312 0.09 0.365

0.209 0.241 0.22 0.536 0.559 0.717 0.212 0.176 0.387

group III included only strain Pseudomonas sp. CAS908i. All the parameters had clustered the isolates and described their non-taxonomic relationship. The UPGMA-based dendogram, on the basis metabolic properties, demonstrated diversification of isolates with respect to these selected traits (Fig. 6). The dendogram revealed formation of a number of distinguishable subgroups in 0.6% level. It was noted that strains affiliated to different taxonomic groups and/or isolated from different places shared common traits.

Discussion

work were closely related to the genera Pseudomonas, Rhizobium, Acinetobacter and Microbacterium. Members of these genera have previously been reported from and identified as the As3C¡oxidizing population within As-rich groundwater samples of West Bengal and China.[7,14] Particularly, the presence of Pseudomonas, Rhizobium and Acinetobacter bacteria within our isolates corroborated well with similar findings in vast majority of the bacterial isolates from arsenic-contaminated Bengal delta. Members of the genera Pseudomonas, Rhizobium and Acinetobacter isolated earlier from various As-contaminated sites, have been shown to utilize multiple electron donors and/or acceptors.[4,9,26]

Molecular phylogenetic analysis revealed that As3C-resistant and -oxidizing bacterial strains used in the present

1.0

260

Relative bioflim, O.D 660 nm

220 200 180 160 140 120 100 80

0.8

R2A 3+ As 5+ As

0.6

0.4

0.2

0.0

60 0

20

40

60

80

100

120

Time (min)

Control BAS123i BAS310i

BAS316i BAS325i

CAS912i CAS934i

BAS335i CAS905i

Fig. 3. Arsenite oxidation kinetics of bacterial isolates during growth free resting state. Error bars indicates standard deviations (n D 3).

BA S1 23 i BA S3 10 i BA S3 16 i BA S3 25 i BA S3 35 i CA S9 05 i CA S9 08 i CA S9 12 i CA S9 34 +v i ec on tro _v l ec on tro l

3+ (mg/L)

240

Concentration of As

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Doubling time (hrs)

Fig. 4. Quantitative analysis of biofilm formation in presence of As3C, As5C and without As in R2A medium. Experiments were repeated at least six times for each strain and results represent mean values with standard deviation bars from a representative experiment. Positive control: Pseudomonas, negative control: Escherichia coli.

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Fig. 5. Plot of Principal Component Analysis (PCA) of isolates based on their heavy metal resistance property.

During the present study we have observed that bacterial strains isolated from As-contaminated groundwater were capable of utilizing four or more carbon sources. Noticeably they were also capable of utilizing hydrocarbon (long-chain alkenes as well as aromatic compounds) as their sole source of carbon and energy. Their ability to use various electron acceptors in the absence of oxygen was noticeable. Arsenic-rich aquifer systems of BDP are considered to be oligotrophic with scarcity of easily accessible nutrients.[27] Recent studies have indicated that Holocene aquifer groundwater in the BDP, particularly in its western part, have low OC and dissolved oxygen content and show seasonal variations between oxic and anoxic conditions.[7,28] Presence of a dynamic geochemical behaviour of groundwater in the As-affected regions of the deltaic plain is also linked to the acquisition of both Asreducing as well as As-oxidizing mechanisms. Although the overall organic carbon content of the groundwater is considerably low, nevertheless some carbohydrate residues and hydrocarbon compound are present within the aquifer supporting heterotrophic bacterial

Paul et al. metabolism. Presence of low concentrations of hydrocarbon fractions containing n-alkenes derived from thermally mature petroleum-type hydrocarbons has been noted.[6,27] The abilities of many of our isolated strains to utilize more than three sugar molecules, different hydrocarbon compounds cyclohexane, nonadecane, docosane, BTEX, etc.—as their energy and/or carbon source and use of alternate electron acceptor(s) during anaerobic condition, clearly indicates that the indigenous organisms are well equipped to survive and flourish under As-rich oligotrophic condition. Assemblages of these characteristics possibly favour their distribution in As-rich subsurface habitats. It is also noteworthy that such abilities not only allow these organisms to play an important role in As biogeochemistry but also create anoxic/reducing conditions within the As-rich environments.[29, 30] Bacterial resistance towards As or other heavy metals is widely found in the environment, including contaminated aquifers. Bacterial genera that can withstand the concentration of 10 mM As3C or 100 mM As5C are considered to be highly resistant.[31] A number of studies on As resistance property of indigenous bacteria isolated from parts of the Bengal basin have found that resistance to As5C remains more abundant compared to that of As3C. The same was noted in our study as well, where isolates were found to tolerate 100 mM As5C, and more than 50% of the isolates could withstand 10 mM As3C concentration or more than that. In line with resistance to As, all the isolates showed their abilities to resist different other heavy metals and metalloids, particularly higher resistance to Ni, Zn, Pb, Cu, Cr, etc. was observed. It is known that genetic determinants for heavy metal resistance including those for As are widespread in microbes from contaminated sites.[10] Arsenic transformation by bacteria represents an important component of the As biogeochemical cycle in natural environment. Arsenite oxidase activity was found to be present in all the test bacteria, while As5C reductase property was present in fewer strains. Presence of As3C oxidase activity facilitates the bacteria either to grow

Fig. 6. UPGMA based on metabolic properties of bacterial strains. Characters: 1 acetate; 2 glycerol; 3 pyruvate; 4 lactate; 5 acetone; 6 glucose; 7 citrate; 8 ascorbic acid; 9 sucrose; 10 starch; 11 dodecane; 12 pentadecane; 13 nonadecane; 14 docosane; 15 cyclohexane; 16 naphthalene; 17 fluorene; 18 anthracene; 19 Phenanthrene; 20 pyrene; 21 BTEX; 22 As5C; 23 Se6C; 24 SO 42_; 25 S 2O 32_; 26 SO32_; 27 NO3_; 28 Fe3C; 29 Arsenate reductase; 30 Arsenite oxidase; 31 Siderophores; 32 Bioflim (only R2A); 33 Bioflim (R2A C As3C); 34 Biofilm (R2AC As5C).

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Characterization of arsenite-oxidizing bacteria chemolithotrophically using As3C as an electron donor or heterotrophically using As3C oxidation as a means of detoxification mechanism. Presence of As5C reduction activity is often related to the detoxification mechanism, which allows the bacteria to change As5C intracellularly to more mobile As3C and efflux out within the aquifer. Presence of As3C-oxidizing and As5C-reducing bacteria in the same geological environment is ecologically very important because it controls the biogeochemical cycle within the aquifer environment. In this study it was noted that in the presence of As3C and/or As5C cell growth for the strains Rhizobium sp. BAS335i, Rhizobium sp. BAS316i and Pseudomonas sp. CAS912i was increased, whereas for the remainder of the other strains, it remained almost unaffected. The data, in line with the earlier report, suggest a possible role of As, supporting the growth of bacterial strains indigenous to As-rich groundwater.[7] Prolonged association of microorganisms with lethal heavy metal(s) within metal rich sites often acts in support of microbial metabolism.[32] The same was found true in this case, as most of the test bacteria showed shorter generation time and higher cell yield when grown in the presence of As. Within the two species of As, presence of As5C supported most of the bacterial growth, which might be due to its significantly less toxicity (100-fold) compared to As3C.[33] Apart from the abilities towards resistance/transformation of As or other heavy metal or utilization of diverse carbon source and alternative electron donors, abundance of siderophore formation and ability to form biofilm by the isolates were noted. According to earlier studies, it was reported that there may be a link between As mobilization as by-products and iron acquisition through siderophores from minerals such as iron oxyhydroxides and hydroxyapatite present in Bengal basin.[34,35] But in our study only one strain have siderophore forming ability. Since we have selectively used As3C oxidase bacteria for this study, it is likely that the presence of siderophores is linked with As5C-reducing bacteria rather than As3C-oxidizing species. The reason for the same could be a possible necessity for reducing the As5C released during siderophores-based Fe acquisition from the sediments, however, which is not required by the As3C oxidizing bacteria. Most of the test isolates showed biofilm formation and the same showed enhancement during growth with As (mostly in the presence of As5C). It is well known that bacteria produce more biofilm during metal-stressed conditions. Biofilm is an extracellular polymeric substance, responsible for protecting bacterial cells from heavy metal and other environmental stresses by binding with the heavy metals and retarding their diffusion within the biofilm.[36] Predominance of this property within the isolates allows their attachment on mineral surfaces and thereby facilitates their physiological and metabolic success to survive and play a role in As cycling within the contaminated aquifer.

Conclusions In this study we observed that nine bacterial strains isolated from As-contaminated groundwater of West Bengal were metabolically well equipped with multifaceted abilities. Along with As and other heavy metal-resistant properties, utilization of diverse carbon/electron donor and electron acceptor, As transformation, siderophore and biofilm formation was noted. Ability of the strains to grow at broad pH and temperature ranges, salinity and both under aerobic and anaerobic conditions was observed. The result suggested that bacterial strains with such metabolic versatility could play a critical role in As biogeochemical cycling within aquifer environment. Along with such diverse metabolic ability, bacterial strains Rhizobium sp. CAS934i, Microbacterium sp. CAS905i and Pseudomonas sp. CAS912i have the capability to oxidize rapidly toxic As3C to less toxic and highly sportive As5C. Therefore, this bacterial isolates may be used for bioremediation of As-contaminated drinking water as well as other Ascontaminated sites in the future.

Funding The authors acknowledge Council of Scientific and Industrial Research (Scheme No. 38/1314/11/EMR II) and Department of Biotechnology, Govt. of India (RGYI Scheme No. BT/PR8933/GBD/27/41/2006) for financial support. Dhiraj Paul acknowledges the University Grants Commission (UGC), India, for providing a fellowship.

References [1] Oremland, R.S.; Stolz, J.F. Arsenic, microbes and contaminated aquifers. Trends Microbiol. 2005, 13(2), 45–49. [2] Fendorf, S.; Michael, H.A.; van Geen, A. Spatial and temporal variations of groundwater arsenic in south and southeast Asia. Science 2010, 328(5982), 1123–1127. [3] Islam, F.S.; Gault, A.G.; Boothman, C.; Polya, D.A.; Charnock, J. M.; Chatterjee, D.; Lloyd, J. R. Role of metal-reducing bacteria in arsenic release from Bengal delta sediments. Nature 2004, 430(6995), 68–71. [4] Sarkar, A.; Kazy, K.S.; Sar, P. Studies on arsenic transforming groundwater bacteria and their role in arsenic release from subsurface sediment, Environ. Sci. Pollut. Res. 2014, 21(14), 8645–8662. [5] Cavalca, L.; Corsini, A.; Zaccheo, P.; Andreoni, V.; Muyzer, G. Microbial transformations of arsenic: perspectives for biological removal of arsenic from water. Fut. Microbiol. 2013, 8(6), 753–768. [6] Hery, M.; Van Dongen, B.E.; Gill, F.; Mondal, D.; Vaughan, D.J.; Pancost, R.D.; Polya, D.A.; Lloyd, J.R. Arsenic release and attenuation in low organic carbon aquifer sediments from West Bengal. Geobiol. 2010, 8(2), 155–168. [7] Sarkar, A.; Kazy, S.K.; Sar, P. Characterization of arsenic resistant bacteria from arsenic rich groundwater of West Bengal, India. Ecotox. 2013, 22(2), 363–376. [8] Liao, V.H.C.; Chu, Y.J.; Su, Y.C.; Hsiao, S.Y.; Wei, C.C.; Liu, C. W.; Liao, C.M.; Shen, W.C.; Chang, F.J. Arsenite-oxidizing and arsenate-reducing bacteria associated with arsenic-rich groundwater in Taiwan. J Contam. Hydrol. 2011, 123(1), 20–29.

Downloaded by [Univ Of Tenn Grad Sch Of Med] at 00:46 02 October 2014

1492 [9] Ghosh, S.; Sar, P. Identification and characterization of metabolic properties of bacterial populations recovered from arsenic contaminated ground water of North East India (Assam). Water Res. 2013, 47(19), 6992–7005. [10] Drewniak, L.; Styczek, A.; Lopatka, M.M.; Sklodowska, A. Bacteria, hypertolerant to arsenic in the rocks of an ancient gold mine, and their potential role in dissemination of arsenic pollution. Environ. Pollut. 2008, 156(3), 1069–1074. [11] Heinrich-Salmeron, A.; Cordi, A.; Brochier-Armanet, C.; Halter, D.; Pagnout, C.; Abbaszadeh-Fard, E.; Montaut, D.; Fabienne, S.; Bertin, P.N.; Bauda, P.; Ars
ene-Ploetze, F. Unsuspected diversity of arsenite-oxidizing bacteria as revealed by widespread distribution of the aoxB gene in prokaryotes. J Appl. Microbiol. 2011, 77(13), 4685–4692. [12] Santini, J.M.; Sly, L.I.; Schnagl, R.D.; Macy, J.M. A new chemolithoautotrophic arsenite-oxidizing bacterium isolated from a gold mine: phylogenetic, physiological, and preliminary biochemical studies. Appl. Environ. Microbiol. 2000, 66(1), 92–97. [13] Salmassi, T.M.; Venkateswaren, K.; Satomi, M.; Newman, D.K.; Hering, J. G. Oxidation of arsenite by Agrobacterium albertimagni, AOL15, sp. nov., isolated from Hot Creek, California. Geomicrobiol J. 2002, 19(1), 53–66. [14] Fan, H.; Su, C.; Wang, Y.; Yao, J.; Zhao, K.; Wang, G. Sedimentary arsenite-oxidizing and arsenate-reducing bacteria associated with high arsenic groundwater from Shanyin, Northwestern China. J. Appl. Microbiol. 2008, 105(2), 529–539. [15] Valenzuela, C.; Campos, V.L.; Ya~ nez, J.; Zaror, C.A.; Mondaca, M.A. Isolation of arsenite-oxidizing bacteria from arsenic-enriched sediments from Camarones River, Northern Chile. Bull. Environ. Contam. 2009, 82(5), 593–596. [16] Majumder, A.; Bhattacharyya, K.; Bhattacharyya, S.; Kole, S.C. Arsenic-tolerant, arsenite-oxidising bacterial strains in the contaminated soils of West Bengal, India. Sci. Total. Environ. 2013, 463, 1006–1014. [17] Reasoner, D.J.; Geldreich, E.E. A new medium for the enumeration and subculture of bacteria from potable water. Appl. Environ. Microbiol. 1985, 49(1), 1–7. [18] Kazy, S.K.; Sar, P.; Asthana, R.K.; Singh, S.P. Copper uptake and its compartmentalization in Pseudomonas aeruginosa strains: chemical nature of cellular metal. World J. Microb. Biotechnol. 1999, 15(5), 599–605. [19] Sambrook, J.; Fritsch, E.F.; Maniatis, T. Molecular Cloning; Cold Spring Harbor Laboratory Press: New York, 1989, 2, 14–19. [20] Reardon, C.L.; Cummings, D.E.; Petzke, L.M.; Kinsall, B.L.; Watson, D.B.; Peyton, B.M.; Geesey, G.G. Composition and diversity of microbial communities recovered from surrogate minerals incubated in an acidic uranium-contaminated aquifer. Appl. Environ. Microbiol. 2004, 70(10), 6037–6046. [21] Tamura, K.; Dudley, J.; Nei, M.; Kumar, S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 2007, 24(8), 1596–1599.

Paul et al. [22] Lee, J.; Jayaraman, A.; Wood, T.K. Indole is an inter-species biofilm signal mediated by SdiA. BMC Microbiol. 2007, 7(1), 42. [23] Yan, Z.; Song, N.; Cai, H.; Tay, J.H.; Jiang, H. Enhanced degradation of phenanthrene and pyrene in freshwater sediments by combined employment of sediment microbial fuel cell and amorphous ferric hydroxide. J. Hazard. Mater. 2012, 199, 217–225. [24] Schwyn, B.; Neilands, J.B. Universal chemical assay for the detection and determination of siderophores. Anal. Biochem. 1987, 160(1), 47–56. [25] Gallant, C.V.; Daniels, C.; Leung, J.M.; Ghosh, A.S.; Young, K. D.; Kotra, L.P.; Burrows, L.L. Common b-lactamases inhibit bacterial biofilm formation. Mol. Microbiol. 2005, 58, 1012–1024. [26] Gihring, T.M.; Banfield, J.F. Arsenite oxidation and arsenate respiration by a new Thermus isolate. FEMS Microbiol. Lett. 2001, 204(4), 335–340. [27] Rowland, H.A.L.; Pederick, R.L.; Polya, D.A.; Pancost, R.D.; Van Dongen, B.E.; Gault, A.G.; Vaughan, D.J.; Bryant, C.; Anderson, B.; Lloyd, J.R. The control of organic matter on microbially mediated iron reduction and arsenic release in shallow alluvial aquifers, Cambodia. Geobiology 2007, 5(3), 281–292. [28] Kar, S.; Maity, J.P.; Jean, J.S.; Liu, C.C.; Nath, B.; Yang, H.J.; Bundschuh, J. Arsenic-enriched aquifers: Occurrences and mobilization of arsenic in groundwater of Ganges Delta Plain, Barasat, West Bengal, India. Appl. Geochem. 2010, 25(12), 1805–1814. [29] Freikowski, D.; Neidhardt, H.; Winter, J.; Berner, Z.; Gallert, C. Effect of carbon sources and of sulfate on microbial arsenic mobilization in sediments of West Bengal, India. Ecotox. Environ. Safe. 2013, 91, 139–146. [30] Jiang, Z.; Ping, L.; Yanhong, W.; Bing, L.; Yamin, D.; Yanxin, W. Vertical distribution of bacterial populations associated with arsenic mobilization in aquifer sediments from the Hetao plain, Inner Mongolia. Environ. Earth Sci. 2013, 71, 311–318. [31] Jackson, C.R.; Dugas, S.L. Phylogenetic analysis of bacterial and archaeal arsC gene sequences suggests an ancient, common origin for arsenate reductase. BMC Evol. Biol. 2003, 3(1), 3–18. [32] Piotrowska-Seget, Z.; Kozdroj, J. Changes in culturable bacterial community of soil treated with high dosages of Cu or Cd. Plant Soil Environ. 2008, 54, 520–528. [33] Osborne, F.H.; Ehrlich, H.L. Oxidation of arsenite by a soil isolate of Alcaligenes. J. Appl. Microbiol. 1976, 41(2), 295–305. [34] Mailloux, B.J.; Alexandrova, E.; Keimowitz, A.R.; Wovkulich, K.; Freyer, G.A.; Herron, M.; Stolz, J.F.; Kenna, T.C.; Pichler, T.; Polizzotto, M.L.; Dong, H.; Bishop, M.; Knappett, P.S.K. Microbial mineral weathering for nutrient acquisition releases arsenic. Appl. Environ. Microbiol. 2009, 75(8), 2558–2565. [35] Pal, T.; Mukherjee, P.K. Study of subsurface geology in locating arsenic-free groundwater in Bengal delta, West Bengal, India. Environ. Geol. 2009, 56(6), 1211–1225. [36] Teitzel, G.M.; Parsek, M.R. Heavy metal resistance of biofilm and planktonic Pseudomonas aeruginosa. Appl. Environ. Microbiol. 2003, 69(4), 2313–2320.