Environ Sci Pollut Res DOI 10.1007/s11356-014-2759-1

RESEARCH ARTICLE

Studies on arsenic transforming groundwater bacteria and their role in arsenic release from subsurface sediment Angana Sarkar & Sufia K Kazy & Pinaki Sar

Received: 7 August 2013 / Accepted: 10 March 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Ten different Gram-negative arsenic (As)-resistant and As-transforming bacteria isolated from As-rich groundwater of West Bengal were characterized to assess their role in As mobilization. 16S rRNA gene analysis confirmed the affiliation of these bacteria to genera Achromobacter, Brevu ndimonas, Rhizobium , Ochrobac tr um, a nd Pseudoxanthomonas. Along with superior As-resistance and As-transformation abilities, the isolates showed broad metabolic capacity in terms of utilizing a variety of electron donors and acceptors (including As) under aerobic and anaerobic conditions, respectively. Arsenic transformation studies performed under various conditions indicated highly efficient As3+ oxidation or As5+ reduction kinetics. Genes encoding As3+ oxidase (aioA), cytosolic As5+ reductase (arsC), and As3+ efflux pump (arsB and acr3) were detected within the test isolates. Sequence analyses suggested that As homeostasis genes (particularly arsC, arsB, and acr3) were acquired by most of the bacteria through horizontal gene transfer. A strong correlation between As resistance phenotype and the presence of As3+ transporter genes was observed. Microcosm study showed that bacterial strain having cytosolic As5+ reductase

Responsible editor: Robert Duran Electronic supplementary material The online version of this article (doi:10.1007/s11356-014-2759-1) contains supplementary material, which is available to authorized users. A. Sarkar : P. Sar (*) Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur 721302, India e-mail: [email protected] P. Sar e-mail: [email protected] S. K. Kazy Department of Biotechnology, National Institute of Technology Durgapur, Durgapur 713209, India

property could play important role in mobilizing As (as As3+) from subsurface sediment. Keywords Arsenic . Bacteria . Groundwater . As5+ reduction . As3+ oxidation . As mobilization . HGT

Introduction Arsenic (As) mobilization in groundwater from natural sources threatens the health of over 100 millions of people worldwide, particularly in Bengal Delta Plain (BDP) of Bangladesh and India (Cavalca et al. 2013). Deciphering the geochemical behavior of As and possible cause of its release remain a subject of great interest (Islam et al. 2004). Considerable research in the past decade has confirmed that along with abiotic processes, activities of indigenous microorganisms in subsurface aquifers play critical role in mobilization of As from its natural sources (Islam et al. 2004; Drewniak et al. 2008; Hery et al. 2010). Mobility and toxicity of As in aqueous environment is strongly regulated by its oxidation state. While the oxidized arsenate (As5+) shows greater affinity to get adsorbed on to the minerals like iron oxyhydroxide, the reduced form arsenite (As3+) with lesser affinity to get adsorbed remains in aqueous phase (Mukhopadhyay et al. 2002). Though both of the forms of As are toxic to life, the degree of toxicity is 100 times higher in case of As3+ exposure (Silver and Phung 2005). Geomicrobiological studies on As-rich environments have confirmed that inhabitant bacteria interact with As with an array of mechanisms. These organisms are found to be taxonomically diverse and metabolically well equipped to carry out redox transformations of As (Jackson and Dugas 2003; Macur et al. 2004; Delavat et al. 2012). With respect to nutrient limiting As-rich aquifer environments, such As transformation abilities not only allow the inhabitant bacteria to

Environ Sci Pollut Res

utilize As and other inorganic elements as metabolic resource but also impart significant impact on local geochemistry facilitating As release into groundwater (Islam et al. 2004; Sutton et al. 2009; Liao et al. 2011). Arsenite can serve as an energy source to certain chemolithotrophic bacteria having respiratory As3+ oxidation system, whereas some heterotrophic organisms can oxidize As3+ only for detoxification without gaining any energy (Santini et al. 2000; Gihring et al. 2001; Muller et al. 2003; Silver and Phung 2005). Microbial reduction of As5+ to As3+ may be carried out as a part of detoxification mechanism (ars system), involving cytosolic reduction of As5+ (by As5+ reductase, ArsC), followed by efflux of reduced As3+ species via ArsB or Acr3p efflux system (Rosen 1999; Silver and Phung 2005; Achour et al. 2007). Alternatively, some bacteria can utilize As5+ as terminal electron acceptor, thus reducing As5+ during anaerobic respiration using Arr system (Malasarn et al. 2004). In last few years, several studies have reported abundance of As-transforming bacteria from diverse contaminated habitats (Drewniak et al. 2008; Fan et al. 2008; Cai et al. 2009; Liao et al. 2011; Drewniak and Sklodowska 2013). Although taxonomic identity and metabolic properties of such bacteria have been well documented, the specific involvement of oxidative and reductive transformations in As mobilization has not yet been explored. A number of molecular and ecological studies have shown the role of Fe3+-reducing anaerobic bacteria in releasing sediment-bound As through a “cascade of redox process” (Sutton et al. 2009; Hery et al. 2010). At the same time, simultaneous presence of both the As3+oxidizing and As5+-reducing bacteria, devoid of Fe3+ reduction capacity, has been reported within the bacteria isolated from As-contaminated groundwater (Liao et al. 2011; Sarkar et al. 2013). Abilities to reduce As5+ using cytosolic As5+ reductase and grow under chemolithotrophic condition are observed in many of these organisms (Sarkar et al. 2013; Ghosh and Sar 2013). Since most of the As-contaminated aquifers are low in available nutrients, As mobilization due to microbial activities toward acquisition of essential nutrients like phosphate and iron by producing phosphate solubilizing enzymes or siderophores, respectively, has also been observed for bacteria inhabitant to such environment (Drewniak et al. 2008; Mailloux et al. 2009). Considering the overall ecophysiological and molecular properties of indigenous bacterial populations and prevailing geochemical conditions therein, synergistic microbial activities leading toward As mobilization have been proposed (Sarkar et al. 2013). While the coexistence of both As3+-oxidizing and As5+-reducing bacteria became conspicuous at several locations including the BDP, their potential role toward release of As remains highly enigmatic. Therefore, to elucidate the role of As3+-oxidizing and As5+-reducing bacteria on mobilization of sedimentbound As, the present study aimed to (i) characterize the metabolic properties of ten As-resistant and As-transforming

strains indigenous to contaminated groundwater, (ii) study the correlation between the presence of various genetic determinants conferring As-homeostasis and As-transformation process, and (iii) assess the potential of As3+-oxidizing and As5+reducing bacteria in release of As from subsurface sediment.

Materials and methods Microorganisms, media, and growth conditions Ten bacterial strains previously isolated from a highly Ascontaminated groundwater (5 μM As) of West Bengal were used in this study (Sarkar et al. 2013). These strains were selected based on their superior metabolic properties, particularly the As-transformation and As-resistance abilities. All the strains were routinely grown in either R2A medium or minimal salt medium (MSM) (Reasoner and Geldreich 1985; Kazy et al. 1999). Anaerobic growth was performed using anaerobic agar incubated in anaerobic jar containing anero-gaspak (HiMedia, India) at 30 °C for 7 days. PCR amplification and analyses of 16S rRNA and As homeostasis genes Genomic DNA of test bacterial strains was extracted using QIAamp DNA Mini Kit (Qiagen, Germany) following the manufacturer’s instructions. Polymerase chain reaction (PCR) amplification of 16S rRNA gene was carried out using bacteria-specific and universal primers (Table 1). Amplification of As homeostasis genes including As3+ oxidase (aio), As5+ reductase (arsC), and As3+ transporter [arsB, acr3(1), and acr3(2)] was done using sets of degenerate and specific primers (Table 1). Amplified fragments of desired gene products were gel-eluted, cloned in pGEM-T easy vector (Promega), and sequenced using an ABI 3100 DNA sequencer (Eurofin, Bangaluru, India). For each of our sequences, most similar sequences were retrieved from NCBI GenBank (http://www.ncbi.nlm.nih.gov) using BLAST search program. 16S rRNA gene-based phylogenetic analysis of our strains was performed using the closest sequences as retrieved from GenBank, sequences of type strains of respective genera detected in our study, and sequences of bacterial strains previously isolated from the same or other As-contaminated groundwater/sediment or posses As-resistance and/or Astransformation properties. Distance analysis and phylogenetic tree construction were performed using the Jukes-cantor algorithm of neighbor-joining (NJ) method available in Molecular Evolutionary Genetics Analysis (MEGA 4) package (Tamura et al. 2008). Nucleotide sequences of As-resistance and Astransformation genes were translated using the ExPASy tools (http://www.expasy.org/tools/dna.html), and appropriate reading frame for each gene was selected. Protein homology

Environ Sci Pollut Res Table 1 Details of the genes targeted for PCR and their respective primers Gene

Description

Primer sequence

Reference

16S rRNA

Ribosomal RNA gene

27 F: 5-AGAGTTTGATCMTGGCTCAG-3 1492R: 5-GGTTACCTTGTTACGACTT-3

Islam and Sar (2011)

arsC

Cytosolic arsenate reductase

amlt-42 F: 5-TCG CGT AAT ACG CTG GAG AT-3 amlt-376 R: 5-ACT TTCTCG CCG TCT TCC TT-3

Sun et al. (2004)

arsC

Cytosolic arsenate reductase

smrc-42 F: 5-TCA CGC AAT ACC CTT GAA ATG ATC-3 smrc-376 R: 5-ACC TTT TCA CCG TCC TCT TTC GT-3

Sun et al. (2004)

aioB

Periplasmic arsenite oxidase

AoxA-F: 5-ACV TTCAAS TGY CCH KGY CAY TTC-3 AoxB-R1: 5-TGRTTN AGR AAR TAR TTN GTY TG-3

Inskeep et al. (2008)

AoxB-F: 5-TGYCAY TTY TGY ATH GTN GGN TG-3 AoxB-R2:5-TAN GCN GGN CGR TTR TGD AT-3 arsB

Arsenite efflux pump

dacr1F 5:-GCCATCGGCCTGATCGTNATGATGTAYCC-3 darsB1R: 5-CAGGCCGTACACCACCAGRTACATNCC-3

Achour et al. (2007)

acr3(1)

Arsenite efflux pump

dacr1F 5:-GCCATCGGCCTGATCGTNATGATGTAYCC-3 dacr1R 5:-CGGCGATGGCCAGCTCYAAYTTYTT-3

Achour et al. (2007)

acr3(2)

Arsenite efflux pump

dacr5F 5:-TGATCTGGGTCATGATCTTCCCVATGMTGVT-3 dacr4R 5:-CGGCCACGGCCAGYTCRAARAARTT-3

Achour et al. (2007)

B G, T, or C; M A or C; N A, C, G, or T; R A or G; S G or C; V A, C, or G; Y C or T

of translated products was determined using BLASTP of NCBI database. Phylogenetic trees were constructed using MEGA 4 with NJ method (Tamura et al. 2008). Bootstrap percentages (1,000 bootstrap replications) were used to test the robustness of phylogenetic relationships within the trees. GC content of all the As homeostasis genes was calculated using the online DNA/RNA GC content calculator (http:// www.endmemo.com/bio/gc.php). The characteristic ranges of GC% of genomes of respective genera was obtained from Dworkin et al. (2007) and validated further with recent reports from Genome ONLine Databases (GOLD) (http:// genomesonline.org/cgi-bin/GOLD/index.cgi) (Table 2). Physiological characterization of the strains Ability of bacterial strains to metabolize vairious carbon sources was tested using standard carbohydrate discs (25 μg sugar/disk) (HiMedia, India) in phenol red agar plates. Temperature and pH sensitivity were monitored following bacterial growth in MSM with varying pH (pH 3–10) and incubation temperature (10–60 °C). General biochemical properties (viz. nitrite reduction, catalase and oxidase activities, etc.) were characterized using standard protocols (Smibert and Krieg 1994). Antibiotic sensitivity of the test bacteria was determined by the standard antibiotic impregnated disk-agar diffusion method (Bauer et al. 1966).

screening method (Simeonova et al. 2004). Quantitative estimation of As transformation (oxidation/reduction) was studied following the spectrophotometric measurement of the As5+ molybdenum blue complex as described by Johnson (1971). Arsenic transformation study was carried out under three conditions viz. (i) during active cell growth, (ii) during growth decoupled resting state, and (iii) with cell free extract. In the first condition, As-transformation process was observed by incubating each bacterial strain with As3+ or As5+ in MSM for 16–18 h at 30 °C in a rotary shaker. Samples were collected for estimation of As concentration at specific time intervals. In second condition, 300 mL of bacterial cultures was grown in MSM up to log phase and then harvested by centrifugation at 12,000 rpm for 10 min. The collected pellets were then washed twice with reaction buffer (10 mM Tris pH 7.5, 1 mM EDTA, 1 mM MgCl2) and resuspended in 5 mL of same reaction buffer. After addition of 10 μM As5+ or As3+, 100-μL samples were collected in specific time intervals. In the third condition, cells were grown up to log phase in 300 mL of MSM containing 10 mM As5+ or 2.5 mM As3+. Cells were harvested by centrifugation (12,000 rpm, 20 min, 4 °C) and washed twice in 50 mL of ice-cold reaction buffer and finally disrupted by sonication (600 W, 5–10×3 s, depending on the isolate). The unbroken cells were removed by centrifugation as mentioned above. As-transformation assay was carried out by adding 10 μM of As5+ or As3+ to the extracted supernatant (crude cell-free extract).

Arsenic transformation assay Electron donor and acceptor utilization assay Abilities of the test isolates to oxidize As3+ and reduce As5+ were tested using both qualitative and quantitative approaches. Qualitative assay was performed using AgNO3

Abilities of test bacterial strains to utilize different electron donors and acceptors were studied by growing the cells in a

Environ Sci Pollut Res Table 2 Details of GC% values for As homeostasis genes and reference genomes Strains

As homeostasis genes aioB arsC G+C contents (%)

Achromobacter sp. strain KAs3-1 Achromobacter sp. strain KAs3-5

61.19 60.92

– 52

Genome reference arsB

acr3(1)

acr3(2)

62.75 61.75

62.99 62.64

60.26 60.18

60–68a

Achromobacter sp. strain KAs5-7

60.95



61.75

62.64

60.52

Achromobacter sp. strain KAs5-12

60.49



61.84

62.64



Ochrobactrum sp. strain KAs3-4 Ochrobactrum sp. strain KAs3-7

– –

52 52

– 63

62.64 62.64

– 59.43

56–59a

Brevundimonas sp. strain KAs5-6



52



62.82

60.06

62–68a

Rhizobium sp. strain KAs5-8 Rhizobium sp. strain KAs5-22

61.82 –

52 52

54.89 54.14

62.45 –

– –

60–68a

Pseudoxanthomonas sp. strain KAs5-3



52



62.99



68–71a

– absence of the respective gene a

Indicates characteristic range of GC% for genome of respective genus obtained from Dworkin et al. (2007) and Genome ONLine Database (GOLD)

modified MSM (composition L−1: 0.225 g K2HPO4, 0.225 g KH2PO4, 0.46 g NaCl, 0.225 g NH4Cl, 0.117 MgCl2, 0.03 g CaCl2, 2 mM glycerol phosphate, pH 7.4±0.2). For electron donor assay, MMS was amended with 10 mM of either of the following compounds (acetate, arsenite, citrate, ethanol, formate, galactose, glucose, glycerol, lactate, methanol, malate, pyruvate, and succinate), and upon inoculation, plates were incubated aerobically at 30 °C. Control plate was made with MMS agar without any electron donor and incubated with bacterial strains under the same condition. For electron acceptor assay, bacterial strains were inoculated on MMS agar plates with addition of 10 mM Na-lactate as electron donor and 10 mM of any one of the test electron acceptors [arsenate (As5+), selenate (SeO42−), sulfur (S0), sulfate (SO42−), sulfite (S2O32−), nitrate (NO3−), nitrite (NO2−), iron (Fe3+), and manganese (Mn4+)] and incubated anaerobically in an anaerobic jar (HiMedia, India) at 30 °C for 7 days. To make the medium dissolved oxygen free, 1 % cystine hydrochloride was added and pH was adjusted to 7.4 with filter-sterilized 1 M NaOH. Control plates were kept without addition of any electron acceptor and incubated with test strains under the same anaerobic environment.

Selection of these bacteria was based on their superior ability to transform (oxidize or reduce) As, the presence of different As-related genes, and overall metabolic properties. Microcosm experiments were carried out in triplicate sets, each containing 20 g of orange sand and 50 mL 0.1× MSM medium in 250 mL conical flask. The sand was pre-sterilized to remove inhabitant bacteria. The abiotic control experiment was performed using only sterilized sand without any bacterial amendment. Bacterial cultures grown in MSM medium up to their log phase were harvested by centrifugation (10,000 rpm for 5 min), washed with 0.1× MSM twice, and resuspended in the same medium. Resuspended cells (105 CFU mL−1) were used as inocula for microcosm experiments. From each microcosm, samples were withdrawn at 24 h interval; concentrations of the dissolved As3+, As5+, and viable cells were monitored. Samples recovered from each microcosm were centrifuged, and As species in the supernatant was quantified using molybdenum blue spectrophotometric assay as described previously (Johnson 1971). Number of viable cells was measured by counting colony forming units (CFU) on MSM agar medium. SEM–EDX analysis of microcosm sediments

Microcosm study on arsenic mobilization Role of As-transforming bacteria in release of As from As-rich orange sand obtained from contaminated aquifer was investigated by microcosm experiment. Arsenic-bearing orange sand (containing ~3.5 μM of As3+ and ~10 μM of As5+) obtained from previous geological drilling in the studied region of BDP (by Department of Geology and Geophysics, IIT Kharagpur) was sterilized and incubated with either As3+-oxidizing Achromobacter sp. strain KAs3-5 or with As5+-reducing Ochrobactrum sp. strain KAs3-7 at 26 °C for 15 days.

Scanning electron microscope equipped with an energy dispersive X-ray spectrometer (SEM–EDX) (SEM: JEOL JSM5200LV and EDX: PHILLIPS EDAX PV9800EX) was used in order to observe the micro-morphological structures of the As-bearing orange sand samples obtained before and after 7 days of incubation with the As5+-reducing strain. Sand samples were fixed onto the 0.22-μm membrane filter with the aid of 2.5 % glutaraldehyde (v/v). Further, the samples were washed and dehydrated through successive alcohol washing and dried up. Dried samples were transferred onto

Environ Sci Pollut Res

the double-sided carbon tape-pasted bronze stub. The bronze stub was allowed to coat with carbon for SEM–EDX analysis. Electron microscopic analyses were carried out in 15 kV with different magnifications using SEM–EDX facility available at Central Research Facility, IIT Kharagpur. Statistical analysis All data represent mean of three independent experiments to avoid the error in reproducibility among measurements. Significance of difference among the datasets was analyzed by Student’s t test at 95 % confidence level (P value 0.5) As5+ reduction could occur only after the cells enter their exponential growth phase. Ochrobactrum sp. strain KAs3-7 was found to be the most efficient in reducing almost 100 % of added As5+ steadily within 12 h of growth, while other strains exhibited relatively slower and biphasic kinetics. Arsenate reduction by resting cells or cell free cytosolic extracts showed nearly similar trends, and

Genes encoding As3+ oxidase (aioB), As5+ reductase (arsC), and As3+ efflux pump [(arsB), acr3(1), and acr3(2)] were detected within the bacterial strains (Fig. 5). The aioB gene was present in four As3+-oxidizing strains (Achromobacter spp. strains: KAs3-1, KAs3-5, KAs5-7, and KAs5-12) as well as in Rhizobium sp. strain KAs5-8. All these AioB sequences showed high (96–100 %) identity (at nucleotide as well as protein levels) and phylogenetic relatedness to As3+ oxidase gene from Achromobacter spp. (WP_006221920, ABR04340) and Alcaligenes sp. S46 (ADF47197) (Fig. 5 a). Cytoplasmic As5+ reductase gene arsC was found in six As5+reducing bacteria (Ochrobactrum spp. strains KAs3-4 and KAs3-7, Pseudoxanthomonas sp. strain KAs5-3, Brevundimonas sp. strain KAs5-6, Rhizobium spp. strains KAs5-8 and KAs5-22) as well as in Achromobacter sp. strain KAs3-5. All ArsC sequences showed high identity (≥99 %) among themselves and showed close lineage with ArsC gene from several γ-proteobacterial members including Escherichia coli K-12 (NP_417960) and Vibrio sp. Maj4 (ABO29820) (Fig. 5 b). Genes encoding As3+ efflux pump (arsB and acr3) were found to be abundant within the test isolates. The arsB gene from Achromobacter spp. strains KAs3-1, KAs3-5, KAs5-7, and KAs5-12, and Ochrobactrum sp. strain KAs3-7 showed high identity (97–100 %) among themselves. A close phylogenetic proximity of these sequences to ArsB of Achromobacter piechaudii (WP_006221942) and Achromobacter arsenitoxydans (WP_008163580) was observed (Fig. 5 c). Sequences of arsB gene from Rhizobium spp. strains KAs5-8 and KAs5-22 showed relatedness with the same gene of several γ- and α- proteobacterial members

Environ Sci Pollut Res Fig. 3 Plot of PCA scores on utilization of alternate electron acceptors/donors of the test strains and related type strains

Group-1 Achromobacter sp. strain KAs3-1 Achromobacter sp. strain KAs3-5 Achromobacter sp. strain KAs5-12 Achromobacter sp. strain KAs5-7 Brevundimonas sp. strain KAs5-6 Ochobactrum sp. strain KAs3-4 Ochrobactrum sp. strain KAs3-7 Pseudoxanthomonas sp. strain KAs5-3 Rhizobium sp. strain KAs5-8 Rhizobium sp. strain KAs5-22 Group-2 Achromobacter xylosoxidans Alcaligenes faecalis Agrobacterium tumifacience Agrobacterium vitis Brevundimonas vesicularis Ochrobactrum anthropi Ochrobactrum tritici Pseudoxanthomonas brogbernensis Rhizobium leguminosarum

[Escherichia, Shigella, and Agrobacterium spp. (ZP07124993, YP 001882112, ACB05974)] (Fig. 5 c). The presence of acr3(1) gene was found in all the test bacteria except Rhizobium sp. strain KAs5-22. Acr3(1) sequence derived from Achromobacter sp. strain KAs5-7 showed its strong lineage with Ochrobactrum tritici Acr3(1) (ABF48394). Sequences of the same gene derived from rest of the strains, however, showed their phylogenetic proximity to Acr3(1) gene obtained from Brevundimonas spp. (ZP_05034581, ZP_08268056, CAY64629) (Fig. 5 d). Another As3+ transporter gene, acr3(2), was detected in five strains (Achromobacter spp. strains KAs3-1, KAs5-7, and KAs5-12; Ochrobactrum sp. strain KAs3-7; and Brevundimonas sp. strain KAs5-6). Acr3(2) gene sequences obtained from Achromobacter strains showed lineage with the same gene of Paracoccus sp. TRP (WP_010398864) and Nitrobacter winogradskyi Nb-255 (YP_319725) (Fig. 5 e). Sequences derived from Ochrobactrum and Brevundimonas strains showed their lineages with Acr3(2) sequences of diverse Ochrobactrum spp. (ZP_04680200, WP_007881796, WP_010660310) and Brevundimonas spp. (ZP_05034581, CAY64629), respectively. Interestingly, Achromobacter spp. strains KAs3-1 and KAs5-7 and Ochrobactrum sp. strain KAs3-7 showed the presence of all the three As3+ transporter genes [arsB, acr3(1), and acr3(2)]. The discrepancy between 16S rRNA gene-based evolutionary relationship and phylogeny of As homeostasis genes [aioB, arsC, arsB, acr3(1), and acr3(2)] as observed in most of the test bacteria suggests that these As-related genes are

Group-2

Group-1

most likely acquired by horizontal gene transfer (HGT) (Fig. 5). GC content of As homeostasis genes was compared with the characteristic GC% range for reference genomes of closest taxa (Table 2). The comparison further supports our phylogenetic interpretation with respect to occurrence of horizontal transfer of arsC, arsB, and acr3 genes among the members of the community. For instance, the GC% values calculated for arsC genes detected in our strains (affiliated to genera Brevundimonas, Ochrobactrum, and Rhizobium of αProteobacteria, Achromobacter of β-Proteobacteria, and Pseudoxanthomonas of γ-Proteobacteria) were close to the characteristic GC% (45–55 %) of γ-proteobacterial members E. coli or Vibrio sp. Similarly, discrepancy in GC content of reference genome average and that of either arsB gene from Rhizobium spp. strains KAs5-8 and KAs5-22 and Ochrobactrum sp. strain KAs3-7 or acr3(1) gene from all the strains except Brevundimonas sp. strain KAs5-6 also supported the incidence of HGT (Table 2). Regarding the acr3(2) gene, phylogenetic incongruence was observed only in case of Achromobacter spp. which corresponded with the discrepancy among GC content of this gene and the reference genome average for the genus Achromobacter (Table 2). Similarity between aioB gene from Rhizobium sp. strain KAs5-8 with Achromobacter sp. in terms of sequences homology as well as GC content also indicated possible transfer of aioB gene from Achromobacter sp. to Rhizobium sp. (Table 2). In order to ascertain the correlation between the presence of As homeostasis genes in our isolates and their As transformation abilities, a UPGMA-based analysis was performed

Environ Sci Pollut Res Achromobacter sp. strain KAs3-1 Achromobacter sp. strain KAs3-5 Achromobacter sp. strain Kas5-7 Achromobacter sp. strain KAs5-12

0.5

B

0.4

0.20

0.3

0.15

0.2

0.1

0.10

0.05

a 0

2

4

6

8

10

12

14

16

0

18

2

4

As ( M)

5+

60

As

40

14

16

18

60

40

b

b 0

0 0

2

4

6

8

10

12

14

16

0

18

2

4

6

8

10

12

14

16

18

Time (h)

Time (h) 10

10

8

6

6

5+

8

4

As

3+

12

20

20

As

10

80

80

4

2

2

c

c 0

0 0

20

40

60

80

0

100

20

40

60

80

Time (min)

Time (min) 10

8

8

6

6

5+

10

4

As

3+

8

100

100

As

6

Time(h)

Time (h)

( M)

a

0.00

0.0

3+

Ochrobactrum sp. strain KAs3-4 Ochrobactrum sp. strain KAs3-7 Pseudoxanthomonas sp. strain KAs5-3 Brevundimonas sp.strain KAs5-6 Rhizobium sp. strain KAs5-8 Rhizobium sp. strain KAs5-22

0.25

OD 600nm

A OD 600nm

Fig. 4 Growth and As transformation kinetics of bacterial strains. a Bacterial growth in the presence of As3+ and As3+ oxidation during growth (b), at resting cell condition (c), and with cell free extract (d). b Bacterial growth in the presence of As5+ and As5+ reduction during growth (b), at resting cell condition (c), and with cell free extracts (d). Values are mean± standard deviation (n=3)

2

4

2

d

d 0

0 0

50

100

150

Time (min)

(Fig. 6). The dendrogram demonstrated diversification of isolates with respect to these selected traits. It was observed that strains affiliated to different taxonomic groups shared common clade while strains belonging to same taxonomic groups were distributed in different clades on the basis of their different traits.

200

250

0

20

40

60

80

Time (min)

Microcosm study on arsenic mobilization by bacteria Role of As-transforming bacteria in release of this metalloid from sediment sample was evaluated using microcosm-based experiment (Fig. 7). Arsenite-oxidizing Achromobacter sp. strain KAs3-5 and As5+-reducing Ochrobactrum sp. strain

Environ Sci Pollut Res Ochrobactrum sp. strain KAs3-7 [Acr3(1)] Achromobacter sp. strain KAs3-5 [Acr3(1)] Achromobacter sp. strain KAs5-12 [Acr3(1)] Brevundimonas sp. strain KAs5-6 [Acr3(1)] Ochrobactrum sp. strain KAs3-4 [Acr3(1)] Pseudoxanthomonas sp.strain KAs5-3 [Acr3(1)] Achromobacter sp. strain KAs3-1 [Acr3(1)] Rhizobium sp. strain KAs5-8 [Acr3(1)] Brevundimonas sp. BAL3 Acr3(1) (ZP_05034581) Brevundimonas diminuta Acr3 (ZP_08268056) Brevundimonas intermedia Acr3(1) (CAY64629) Sinorhizobium sp. M14 Acr3(1) (ADO95166) Pseudomonas aeruginosa Acr3(1)(ACD39063)

100

a

86

Achromobacter sp. strain KAs3-5 (AioB) Alcaligenes sp. S46, AoxB (ADF47197) Achromobacter sp. strain KAs5-12 (AioB) Achromobacter sp. strain KAs3-1 (AioB) 92

70 92

Achromobacter piechaudii AoxB (WP_006221920) uncultured Achromobacter sp AoxB (ABR04340)

76 86

Alcaligenes sp. YI13H AoxB (ABY19322) Achromobacter sp. 40AGIII AoxB (AEL22195)

Paracoccus denitrificans SD1Acr3(1) (AEJ27330)

100

Alcaligenes sp. T12RB AoxB (ABY19321) Arsenite-oxidising beta proteobacterium (ABD72610) Achromobacter sp. strain KAs5-7 (AioB)

98

Achromobacter sp. strain KAs5-7 [Acr3(1)] Ochrobactrum tritici Acr3(1) (ABF48394) 100

0.2

Rhizobium sp. strain KAs5-8 (AioB) Achromobacter spanius AoxB (BAK39658) 90 88

84

Achromobacter arsenitoxydans AoxB (ABP63660) Ralstonia sp. 22 AoxB (ABY19329) Achromobacter arsenitoxydans AoxB(WP_008163524)

73

Alcaligenes faecalis NCIB 8687 AoxB (AAQ19838) Burkholderia sp. S232 AoxB (ADF47194)|

85

93

100

0.2

b

100

Rhizobium sp. strain KAs5-8 (ArsC)

100

Escherichia coli H299 ArsC (ZP_0762599)

0.2

Escherichia coli O111 ArsC (YP_0032366)

c

Vibrio sp. Maj4 ArsC (ABO29820) Pseudomonas aeruginosa ArsC (ABO28445) Escherichia coli ArsC (WP_001691205) Pseudoxanthomonas sp.strain KAs5-3 (ArsC)

100

Pseudomonas sp. AFP12 ArsC (ABO28442)

99

Pseudomonas stutzeri ArsC (ABO28452) Escherichia coli str. K-12 ArsC (NP_417960)

Achromobacter sp. strain KAs3-5 (ArsB) Achromobacter sp. strain KAs3-1 (ArsB) Ochrobactrum sp. strain KAs3-7 (ArsB) Achromobacter sp. strain KAs5-12 (ArsB) Achromobacter sp. strain KAs5-7 (ArsB) Achromobacter piechaudii ArsB (WP_006221942)

100

Achromobacter sp. strain KAs3-5 (ArsC)

Acidovorax ebreus ArsB (YP_002553590) Alcaligenes faecalis ArsB (AAS45119)

Ochrobactrum sp. strain KAs3-7 (ArsC) 99

Vibrio aestuarianus ArsC (ABO29822)

60

94

e

Achromobacter sp. strain KAs5-12 [Acr3(2)] Achromobacter sp. strain KAs5-7 [Acr3(2)] Achromobacter sp. strain KAs3-1 [Acr3(2)] Paracoccus sp. TRP Acr3 (WP_010398864) Nitrobacter winogradskyi Nb-255 Acr3 (YP_319725) Methylobacterium extorquens Acr3 (YP_003066984) Agrobacterium vitis S4Acr3(2) (YP_002547789) Ochrobactrum tritici Acr3(2) (ABF48394) Ochrobactrum sp. strain KAs3-7 [Acr3(2)] Ochrobactrum intermedium Acr3 (ZP_04680200) Ochrobactrum sp. CDB2 Acr3 (WP_007881796) Ochrobactrum anthropi Acr3 (WP_010660310) Agrobacterium radiobacter Acr3(2) (YP_002543904) Ralstonia eutropha H16ACR3(2) (YP_726641) Agrobacterium tumefaciens Acr3 (WP_020810061) Rhizobium sp. Acr3 (CCF22144) Pseudomonas pseudoalcaligenes Acr3 (WP_004420839) Brevundimonas sp. strain KAs5-6 [Acr3(2)] Brevundimonas sp. BAL3Acr3(2) (ZP_05034581) Brevundimonas intermedia Acr3 (CAY64629)

Achromobacter arsenitoxydans ArsB (WP_008163580)

Rhizobium sp. strain KAs5-22 (ArsC)

60

d

99

Comamonas testosteroni ArsB (ZP_03544311) Comamonas testosteroni ArsB (WP_003081409)

Escherichia coli B7A ArsC (ZP_03027080)

Alcaligenes sp. ArsB (WP_003799898)

Escherichia coli ArsC (WP_001719018)

Nitrosomonas sp.ArsB (ZP_05314443)

Shigella dysenteriae ArsC (YP_405024)

Escherichia coli ArsB (ZP_07620915)

Escherichia coli MS 84-1 ArsC (ZP_07124992)

Rhizobium sp. strain KAs5-22 (ArsB)

Brevundimonas sp. strain KAs5-6 (ArsC)

100

Rhizobium sp. strain KAs5-8 (ArsB)

Ochrobactrum sp. strain KAs3-4 (ArsC)

Shigella boydii ArsB (YP_001882112)

Halobacterium sp. NRC-1 ArsC (AAC82910)

Escherichia coli ArsB (ZP_07124993) Agrobacterium sp. ArsB (ACB05974)

0.2

Shigella sonnei ArsB (YP_312518) Achromobacter xylosoxidans ArsB (YP_003977995) Bordetella bronchiseptica ArsB (NP_887740)

82

0.2

Fig. 5 Phylogenetic tree of deduced amino acid sequences of genes encoding As5+ reductase (arsC), As3+ oxidase (aioB), and As3+ efflux [arsB, acr3(1), and acr3(2)]. Genes detected in our study are shown in red font. Percentage values on each branch represent the corresponding

bootstrap probability values obtained in 100 replications. Significant bootstrap values (≥50 %) of major branch points are shown in the tree. Sequence suffixed with asterisk indicates discrepancy with 16S rRNA gene phylogeny

Environ Sci Pollut Res Fig. 6 UPGMA analysis of As homeostasis genes and As transformation abilities of the isolates

As (μ μM)

CFU mL-1

KAs3-7 were used in two different sets of microcosms to understand specific involvement of either of these transformation processes. CFU counts obtained at different time intervals indicated viability and growth of both the organisms within respective microcosms. Increasing cell counts in initial 72 h indicated active growth of the bioaugmented bacteria during their incubation with the As-rich sediment, which, however, stabilized thereafter. Concomitant to their survival, both the As3+-oxidizing as well as As5+-reducing strains showed release of considerable amount of As in the aqueous phase. In the presence of As 5 + -reducing Ochrobactrum sp. strain KAs3-7, release of As 3 + (~0.37 μM) was detected from second day onward, and in fourth day, the value reached to its maximum (~1 μM). Increase in soluble As5+ concentration within the same set of microcosms occurred at much lower extent. Microcosms

Time

Fig. 7 Microcosm study on release of sediment-bound As by selected As3+-oxidizing and As5+-reducing strains. The data reported are from days 1 to 15. Arsenic concentration at day 1 is to be considered as the initial concentration. Values are mean±standard deviation (n=3)

with As3+-oxidizing Achromobacter sp. strain KAs3-5 showed a reverse trend with more than 7-fold higher concentration of As5+ (than As3+) detected in the aqueous phase up to 2 days of incubation. Control set devoid of any bacterial augmentation did not show any appreciable release of soluble As. Since the As5+-reducing strain was able to release arsenic as more toxic As3+ form, the sediment sample from the same microcosm was recovered and subjected to SEM and EDX analyses (Fig. 8). The micrographs showed bacterial colonization with apparent change in sediment morphology (compared to uninoculated sediment) (Fig. 8a, b). Energy dispersive X-ray analysis further revealed the presence of As along with and other metals (Al, Fe, Si, Ca, etc.) at initial day of the incubation and showed a vivid release of As following incubation with the test bacterium (Fig. 8c, d). Analysis of elemental composition as obtained from EDX study indicated that along with As, considerable amount of Fe, Ca, and Al was lost from the sediment following incubation with As5+reducing strain (Supplementary Table 1).

Discussion Arsenic-contaminated aquifers of BDP are found to be occupied by diverse bacteria capable of playing important role in As biogeochemistry. Arsenic is metabolized by indigenous bacteria using broad array of metabolic functions that result into alteration in its environmental fate and toxicity. Several studies have confirmed that inhabitant microbes play critical role in releasing sediment-bound As within the subsurface environment. However, the catalytic role of inhabiting microorganisms toward mobilization of sediment As in the affected region on West Bengal and Bangladesh is still to be resolved (Islam et al. 2004; Sutton et al. 2009; Sarkar et al. 2013). While the role of dissimilatory As/Fe-reducing bacteria has been noticed by some researchers, several others have not found their presence, rather observed the predominance of heterotrophic and chemolithotrophic genera with cytoplasmic As5+ reductase activity. As a whole, the metabolic activity and role of As-transforming bacteria, particularly those surviving

Environ Sci Pollut Res Fig. 8 SEM photograph of Asbearing sediment: a before and b after incubation with As5+reducing Ochrobactrum sp. strain KAs3-7. EDX analysis of sediment: c before and d after incubation with As5+-reducing Ochrobactrum sp. strain KAs3-7

a

b

in both oxic and anoxic environment and devoid of dissimilatory metal reduction ability, remain enigmatic. The present study provides new insights in understanding microbial metabolic capabilities and role toward As mobilization from subsurface sediment. Among the test bacterial genera, except Pseudoxanthomonas, others have been previously reported from diverse As-rich environments and known for their As-resistance and/or Astransformation abilities (Jackson and Dugas 2003; Achour et al. 2007; Branco et al. 2008; Drewniak et al. 2008; Cavalca et al. 2010; Liao et al. 2011; Sarkar et al. 2013). Ability of Rhizobium, Ochrobactrum, Brevundimonas, and Achromobacter strains to grow chemolithotrophically utilizing As3+ or other inorganic compounds as source of electron or as a mechanism of As-resistance or both remained an interesting phenomenon to gain better insight on microbial role in As mobilization/ immobilization (Cai et al. 2009; Hery et al. 2010). Lineage of our strains with members of same genera but having ability to metabolize hydrocarbon and nitrate reduction was noteworthy. Arsenic-rich sediments of BDP are generally oligotrophic in nature but often reported to contain low concentration of petroleum-derived hydrocarbon compounds. Inhabiting microbes, therefore, might have potential to metabolize such organic matters as carbon source, while nitrate is used a terminal electron acceptor under the prevailing anoxic/reducing environment (Islam et al. 2004; Hery et al. 2010). Arsenic resistance and transformation by resident microorganisms may be considered as essential properties for their survival and growth under subsurface conditions. Particularly, the abundance of both As3+-oxidizing and As5+-reducing

c

d

organisms in the same environment indicates that both the processes possibly have important role in the community. Results from the present as well as from several other studies are in favor of the hypothesis that redox transformations of As occur in phylogenetically diverse bacteria via mechanisms that are not only associated with respiration or chemolithotrophic metabolism but may have additional survival value (Silver and Phung 2005). In this respect, cooccurrence of As3+-oxidizing and As5+-reducing bacteria could be considered as an evolutionary outcome to allow aerobic/facultative anaerobic metabolism within the shallow aquifer system (Liao et al. 2011). Although most of the shallow aquifers of West Bengal have aqueous As in its reduced form, certain oxidative conditions might be established at later stage (after digging) through transportation of aerobic groundwater and diffusion of oxygen through this zone (Hery et al. 2010). The later events may facilitate emergence of bacterial population with both the abilities. Arsenicoxidizing bacteria may grow heterotrophically with sediment organic carbon or chemolithoautotrophically using inorganic (arsenic) electron donor and available carbon dioxide/ bicarbonate. During either of the two metabolisms, these organisms eventually oxidize As3+ to As5+ facilitating the immobilization of As5+ onto the solid phases, such as Fe oxide, goethite, and clay minerals. On the other hand, As5+reducing bacteria in the same environment may transform As5+ to soluble As3+, thereby releasing As from the aquifer sediment (Drewniak et al. 2008). In the present study, we have observed that along with Asresistance and As-transformation abilities, the test bacteria were

Environ Sci Pollut Res

endowed with high “respiratory diversity” in terms of utilizing a wide variety of carbon and/or electron donors and acceptors under aerobic and anaerobic conditions, respectively. The broad metabolic diversity of inhabitant microorganisms is considered to be advantageous, particularly at times of nutrient scarcity and can be correlated with local geochemistry of shallow aquifers (Islam et al. 2004; Liao et al. 2011). Capability of utilizing multiple electron donors and acceptors has been reported previously in several bacteria like Thermus spp., Agrobacterium sp., etc. and in strain SES-3, Desulfitobacterium spp., Bacillus arsenicoselenatis, and Bacillus selenitireducens, respectively, from As-rich sites (Laverman et al. 1995; Niggemyer et al. 2001; Salmassi et al. 2002; Yamamura et al. 2007). However, ability to utilize more than three different compounds as electron source by a single strain is rare, and in such comparison, few of our isolates may be considered truly versatile. It has been reported that the depth distribution of groundwater within the aquifer varies seasonally and diurnally creating a dynamic nature of the water table in terms of oxic– anoxic states and hence availability of electron acceptors as well (Fenchel 2002). Therefore, it is highly likely that inhabiting microorganisms equipped themselves with ability to utilize diverse electron donors and acceptors to survive and flourish under such changing environments. PCA analysis further indicated that the versatile metabolic abilities of the test microorganisms are not related to their taxonomy, rather possibly evolved as a result of their long term association within the As-contaminated aquifer. Antibiotic resistance property as observed in the test strains seems an interesting finding. Previously, Shivaji et al. (2005) have reported resistance to antibiotics by a Bacillus arsenicus strain isolated from As-contaminated water of West Bengal. In addition to As resistance, ability to resist other antimicrobial compounds have been noticed in bacteria from different Asrich sites (Dib et al. 2008; Shakoori et al. 2010). Genes conferring resistance to ampicillin (ampC), tetracycline (tetA), sulfonamide (sul1), vancomycin (vanA and vanB), and chloramphenicol (catI) have been reported in natural as well as in drinking water (Soge et al. 2009; Zhang et al. 2009). The presence of antibiotic resistance within the bacterial population from apparently antibiotic-free environment often indicates either co-evolution of such property with other (e.g., As) resistance systems or synergy in mechanisms those conferring resistance phenotypes. In recent time, considerable evidence supports prevalence of antibiotic resistance in bacteria from diverse aquatic systems (Kümmerer 2009). Evolution of these antibiotics and antibiotic resistance in nature occurred through intra and inter-domain communication in various ecosystems (Aminov 2009). Abundance of antibiotic resistance property pose significant health risk to human and animals when transferred via various direct and indirect means; as a result, there is a considerable research interest in environmental antibioticresistant bacteria and genes encoding antibiotic resistance.

In our study, four strains affiliated to the genus Achromobacter showed As3+ oxidase activity. Members of this genus are well-known for their As3+ oxidase activity (Fan et al. 2008; Cai et al. 2009; Bachate et al. 2012). The high rate of As3+ oxidation by these strains as observed during their logarithmic growth phase corroborated well with earlier reports, which could be attributed to the involvement of As3+ induced quorum-sensing signal transduction mechanism (Gihring et al. 2001). Study on regulatory control of microbial As3+ oxidation has confirmed that expression of As3+ oxidase operon (aioAB) is “controlled in a fashion consistent with quorum-sensing,” wherein expression of aioAB is observed only during log phase (Gihring et al. 2001). The decrease of oxidase activity in crude extract possibly due to the loss of membrane associated subunits of As3+ oxidase enzyme as well as of additional TAT leader sequences essential for oxidase activity during cell lysis (Slyemi and Bonnefoy 2011). With respect to aioB gene, phylogenetic incongruence was noted only in Rhizobium sp. strain KAs5-8, indicating the possible incidence of HGT. Interestingly, this particular strain did not show As3+ oxidase activity; rather, it showed As5+ reductase activity. In contrast to previous finding on the presence of aioAB gene in As3+-oxidizing bacteria and its potential to be used as functional marker (Inskeep et al. 2008; Quéméneur et al. 2008; Hamamura et al. 2009), the present study supports more recent observation indicating that phylogeny of aioAB gene might not always be correlated strictly with that of the 16S rRNA genes, due to HGT (HeinrichSalmeron et al. 2011; Li et al. 2013). All the As5+ reductase-positive strains showed rapid 5+ As reduction under different conditions. Arsenate reduction as observed in our Agrobacterium–Rhizobium strains or in Pseudoxanthomonas sp. strain KAs5-3 is noteworthy. Members of the former group are well-known for As3+ oxidation, while for Pseudoxanthomonas, there is no report on redox transformation of arsenic (Salmassi et al. 2002). The present data, therefore, certainly expand our understanding on microbial As geocycling/transformation. Arsenate reduction rates of our strain are comparable with that of other strains reported earlier, though it may be noted that the rate of As transformation is regulated by in vitro assay condition (Supplementary Table 2). Arsenate reduction can be achieved either via the cytoplasmic As5+ reductase mediated detoxification mechanism or periplasmic respiratory reductase system facilitating anaerobic metabolism (Malasarn et al. 2004). During this study, we could detect the arsC gene encoding cytosolic As5+ reductase from all the six strains, and the sequences showed close similarity with arsC of γ-proteobacterial members. The phylogenetic incongruence between 16S rRNA and arsC genes as evident in our strains is an important observation with respect to evolution of molecular mechanism related to As homeostasis within the aquifer community.

Environ Sci Pollut Res

Genetic determinants for As resistance and transformations were widely detected within the isolates. Among these acr3p genotype was predominant over arsB, while arsC was more abundant than aioB. In general, a strong correlation between bacterial As resistance phenotype and the presence of As3+ transporter genes was observed. With respect to As3+/As5+ resistance, it was observed that except few cases, higher resistance was coupled with the presence of periplasmic As3+ oxidase or cytosolic As5+ reductase gene, respectively. Noticeably, simultaneous presence of three types of As3+ transporter genes [arsB, acr3(1), and acr3(2)] in the same strain (as found in our Achromobacter spp. strains KAs3-1 and KAs5-7 and Ochrobactrum sp. strain KAs3-7) is rare. However, the presence of multiple sets of As resistance genes was reported earlier in several bacteria including Bacillus subtilis, Pseudomonas putida, Acidithiobacillus caldus, Corynebacterium glutamicum, Herminiimonas arsenicoxydans and O. tritici SCII24 (Rosen 1999; Mukhopadhyay et al. 2002; Achour et al. 2007; Branco et al. 2008). The observed phylogenetic incongruence between 16S rRNA and arsC or arsB/acr3(1)/acr3(2)/aioB genes within the test strains indicated the possible occurrence of HGT within the bacterial populations under long-term As stress (Jackson and Dugas 2003; Cai et al. 2009). Microorganisms acquire multiple As resistance determinants either via chromosomal duplication or in most cases due to HGT (Achour et al. 2007). The HGT is facilitated by a number of mechanisms including involvement of self-transferable, broad-hostrange plasmid that contribute natural competence within the selected organisms (Drewniak et al. 2013). During the present study, phylogenetic analysis as well as GC% values indicated a possible transfer of these genes among the different genera of the same phylum (Proteobacteria). Recent reports, however, hypothesized that HGT may not be limited to the closely related genomes only but can occur between distantly related bacteria as well (Maria et al. 2011; Davolos and Pietrangeli 2013). During the HGT, transfer of different As homeostasis genes might occur individually or together as an operon or its part. It is not surprising as most of the bacteria in contaminated niche harbor a number of DNA mobilization-related genes (i.e., transposases, integrases, resolvases, etc.) that facilitate acquisition of relevant functional genes and promoting bacterial adaptation under environmental stresses (Huang et al. 2012; Li et al. 2013; Andres et al. 2013). Noticeably, sequence analysis of arsC and arsB genes of Rhizobium sp. strain KAs5-8 showed their relatedness with arsC and arsB genes from E. coli. ClustalW multiple alignment with published ars operon (containing genes arsR, arsB, arsC) of E. coli (X80057) clearly showed that arsB gene of the test strain (KAs5-8) aligned entirely with E. coli ars gene from nucleotide position 1228 to 2004 whereas arsC gene showed alignment with the same ars operon from nucleotide position 2477 to 2813 (Supplementary Fig. 1). The present study provided

further evidence in support of the same, indicating that these two genes might have moved together as complete operon or its part from a common ancestor. The overall finding is in line with earlier report on HGT of whole ars operon from E. coli to Pseudomonas aeruginosa and Acidiphilium sp. (Saltikov and Olson 2002). This type of genetic event is considered to be an important mechanism of microbial genome evolution and shaping the structure and function of microbial communities in various metal-contaminated niches, including the As-rich aquifer system (Achour et al. 2007; Fondi et al. 2013). In contrast to the dissimilatory As5+ reduction mediated release of sediment-bound As, the role of detoxificationdriven cytosolic As5+ reduction, particularly in low organic carbon aquifers, has gained recent attention (Islam et al. 2004; Drewniak et al. 2008; Sutton et al. 2009; Hery et al. 2010; Sarkar et al. 2013). During the present study, microcosm experiment revealed that the As5+-reducing Ochrobactrum strain was efficient in releasing arsenic as more toxic and mobile As3+ form than the oxidizing strain, which, however, acted in reverse to yield high concentration of As5+ under similar condition. The enhanced As3+ mobilization by As5+reducing organisms is likely to be a result of redox transformation of sediment associated As5+ to As3+ by cytosolic As5+ reductase. Previously, we have observed that indigenous bacteria (affiliated to the same genera Ochrobactrum, Rhizobium, etc.) from As-rich groundwater are capable of producing siderophore and phosphatase enzyme, which might act synergistically to release the sorbed As5+ (Sarkar et al. 2013). The present data, therefore, support such hypothesis confirming the consorted role of bacterial cytosolic As5+ reductase activity in transforming sediment-bound As5+ to more soluble As3+. Transcriptomic analysis further revealed significant upregulation of genes encoding siderophore, cytosolic As5+ reductase, and As3+ efflux systems in the test Ochrobactrum sp. strain KAs3-7 during its incubation with As-rich sediment (data not shown). These results corroborate well with other related studies, wherein reduction of As5+ to As3+ was described mainly by means of ars detoxification mechanism followed by release of sediment-bound As5+ during nutrient acquisition by bacteria (Drewniak et al. 2008; Sarkar et al. 2013). The observed change in sediment morphology following incubation with bacteria revealed by SEM–EDX analysis may be due to the change in mineralogical composition during bacterial activities. Concomitant to the release of As in the aqueous phase, loss of substantial Fe, Al, and Ca could possibly indicate toward dissolution of As-bearing minerals or mineral phases (e.g., Fe oxide/hydroxide-coated sand, phyllosilicates, Mn oxides, Al oxides, and hydroxides) from the sediment due to the activity of As 5+ -reducing Ochrobactrum sp. strain KAs3-7. Similar morphological change was documented earlier during As mobilization from As-bearing rock or sediment (Campos et al. 2011, Islam et al. 2012).

Environ Sci Pollut Res

Conclusion The present paper provides experimental evidence on metabolic versatility of indigenous bacteria in As-contaminated groundwater and the role of cytosolic As5+ reduction in releasing As within the aqueous phase. Arsenic transformation abilities, utilization of diverse electron acceptors and donors, and the presence of genetic determinants for As resistance and transformation provide important insights on the capabilities of such bacteria in controlling subsurface arsenic geochemistry. The result shows that phylogenetically diverse As3+-oxidizing and As5+-reducing bacteria can co-exist in the same habitat and play critical role(s) in As transformation. Overall observations may be helpful for understanding hydrogeomicrobial processes responsible for the great environmental problem of As poisoning. Acknowledgments The authors acknowledge financial support from the Department of Biotechnology (RGYI scheme) and Council of Scientific and Industrial Research (CSIR), Government of India. Kind help of Prof. Anindya Sarkar and Dr. Abhijit Mukherjee, Department of Geology, IIT Kharagpur in providing arsenic-bearing sand is greatly acknowledged. Angana Sarkar acknowledges IIT Kharagpur for her doctoral fellowship. The authors acknowledge the support extended by Mr. Buddha Deb Banerjee, Barasat, North 24 Parganas, West Bengal during field work. Authors express their thanks to the anonymous reviewer for critical comments in improving the manuscript.

References Achour AR, Bauda P, Billard P (2007) Diversity of arsenite transporter genes from arsenic-resistant soil bacteria. Res Microbiol 28:128– 137 Aminov RI (2009) The role of antibiotics and antibiotic resistance in nature. Environ Microbiol 11:2970–2988 Andres J et al (2013) Life in an arsenic-containing gold mine: genome and physiology of the autotrophic arsenite-oxidizing bacterium Rhizobium sp. NT-26. Genome Biol Evol 5:934–953 Bachate SP, Khapare RM, Kodam KM (2012) Oxidation of arsenite by two β-proteobacteria isolated from soil. Appl Microbiol Biotechnol 93:2136–2145 Bauer AW, Kirby WM, Sherris JC, Turck M (1966) Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 45:493–496 Branco R, Chung AP, Morais PV (2008) Sequencing and expression of two arsenic resistance operons with different functions in the highly arsenic-resistant strain Ochrobactrum tritici SCII24T. BMC Microbiol 8:95–104 Cai L, Liu G, Rensing C, Wang G (2009) Genes involved in arsenic transformation and resistance associated with different levels of arsenic-contaminated soils. BMC Microbiol 9:4–13 Campos VL, León C, Mondaca MA, Yañez J, Zaror C (2011) Arsenic mobilization by epilithic bacterial communities associated with volcanic rocks from Camarones river, Atacama Desert, Northern Chile. Arch Environ Contam Toxicol 61:185–192 Cavalca L, Corsini A, Andreoni V, Muyzer G (2013) Microbial transformations of arsenic: perspective for biological removal of arsenic from water. Future Microbiol 8:753–768

Cavalca L, Zanchi R, Corsini A, Colombo M, Romagnoli C, Canzi E, Andreoni V (2010) Arsenic resistant bacteria associated with roots of the wild Cirsium arvense (L) plant from an arsenic polluted soil, and screening of potential plant growth-promoting characteristics. Syst Appl Microbiol 33:124–164 Davolos D, Pietrangeli B (2013) A molecular study on bacterial resistance to arsenic-toxicity in surface and underground waters of Latium. Ecotoxicol Environ Saf 96:1–9 Delavat F, Lett MC, Lièvremont D (2012) Novel and unexpected bacterial diversity in an arsenic-rich ecosystem revealed by culturedependent approaches. Biol Direct 7:28–42 Dib AWJ, Motok J, Zenoff VF, Ordoñez O, Farías ME (2008) Occurrence of resistance to antibiotics, UV-B, and arsenic in bacteria isolated from extreme environments in high-altitude (above 4400 m). Curr Microbiol 56:510–517 Drewniak L, Dziewit L, Ciezkowska M, Gawor J, Gromadka R, Sklodowska A (2013) Structural and functional genomics of plasmid pSinA of Sinorhizobium sp. M14 encoding genes for the arsenite oxidation and arsenic resistance. J Bacteriol 164:479–488 Drewniak L, Sklodowska A (2013) Arsenic-transforming microbes and their role in biomining processes. Environ Sci Pollut Res 20:7728– 7739 Drewniak L, Styczek A, Lopatka MM, Sklodowska A (2008) Bacteria, hypertolerant to arsenic in the rocks of an ancient gold mine, and their potential role in dissemination of arsenic pollution. Environ Pollut 26:1069–1074 Dworkin M, Falkow S, Rosenberg E, Schleifer KH, Stackebrandt E, Dworkin M, Falkow S, Rosenberg E, Schleifer KH, Stackebrandt E (2007) The prokaryotes: a handbook on the biology of bacteria. In: Kersters K, De vos P, Gillis M, Swings J, Vandamme P, Stackebrandt E (eds) Introduction to the Proteobacteria, 3rd edn. Springer, New York, p 3 Fan H, Su C, Wang Y, Yao J, Zhao K, Wang Y, Wang G (2008) Sedimentary arsenite-oxidizing and arsenate-reducing bacteria associated with high arsenic groundwater from Shanyin, northwestern China. J Appl Microbiol 105:529–539 Fenchel T (2002) Microbial behavior in a heterogeneous world. Science 296:1068–1081 Fondi M, Rizzi E, Emiliani G, Orlandini V, Berna L, Papaleo MC, Perrin E, Maida I, Corti G, De Bellis G, Baldi F, Dijkshoorn L, Vaneechoutte M, Fani R (2013) The genome sequence of the hydrocarbon-degrading Acinetobacter venetianus VE-C3. Res Microbiol 165:439–449 Ghosh S, Sar P (2013) Identification and characterization of metabolic properties of bacterial populations recovered from arsenic contaminated ground water of North East India (Assam). Water Res 47: 6992–7005 Gihring TM, Druschel GK, McCleskey RB, Hamers RJ, Banfield JF (2001) Rapid arsenite oxidation by Thermus aquaticus and Thermus thermophilus: field and laboratory investigations. Environ Sci Technol 6:3858–3862 Hamamura N, Macur RE, Korf S, Ackerman GG, Taylor WP, Kozubal M, Reysenbach AL, Inskeep WP (2009) Linking microbial oxidation of arsenic with detection and phylogenetic analysis of arsenite oxidase genes in diverse geothermal environments. Environ Microbiol 11:421–431 Heinrich-Salmeron A, Cordi A, B-Armanet C, Halter D, Pagnout C, Abbaszadeh-F E, Montaut D, Seby F, Bertin PN, Bauda P, A-Ploetze F (2011) Unsuspected diversity of arsenite oxidizing bacteria as revealed by widespread distribution of the aoxB gene in prokaryotes. Appl Environ Microbiol 77:4685–4692 Hery M, Van Dongen BE, Gill F, Mondal D, Vaughan DJ, Pancost RD, Polya DA, Lloyd JR (2010) Arsenic release and attenuation in low organic carbon aquifer sediments from West Bengal. Geobiology 8:25–168 Huang Y, Li H, Rensing C, Zhao K, Johnstone L, Wang G (2012) Genome sequence of the facultative anaerobic arsenite-oxidizing

Environ Sci Pollut Res and nitrate-reducing bacterium Acidovorax sp. strain NO1. J Bacteriol 194:1635–1636 Inskeep WP, Macur RE, Hamamura N, Warelow TP, Ward SA, Santini JM (2008) Detection, diversity and expression of aerobic bacterial arsenite oxidase genes. Environ Microbiol 9:934–943 Islam ABMR, Maity JP, Bundschuh J, Chen CY, Bhowmik BK, Tazaki K (2012) Arsenic mineral dissolution and possible mobilization in mineral–microbe–groundwater environment. J Hazard Mater 15: 989–996 Islam E, Sar P (2011) Culture-dependent and -independent molecular analysis of bacterial community within uranium ore. J Basic Microbiol 4:1–13 Islam FS, Gault AG, Boothman C, Polya DA, Charnock JM, Chatterjee D, Lloyd JR (2004) Role of metal-reducing bacteria in arsenic release from Bengal delta sediments. Nature 43:68–71 Jackson CR, Dugas SL (2003) Phylogenetic analysis of bacterial and archaeal arsC gene sequences suggests an ancient, common origin for arsenate reductase. BMC Evol Biol 3:18–28 Johnson DL (1971) Simultaneous determination of arsenate and phosphate in natural waters. Environ Sci Technol 5:411–421 Kazy SK, Sar P, Asthana RK, Singh SP (1999) Copper uptake and its compartmentalization in Pseudomonas aeruginosa strains: chemical nature of cellular metal. W J Microbiol Biotechnol 2:599–605 Kümmerer K (2009) Antibiotics in the aquatic environment—a review— part II. Chemosphere 75:435–441 Laverman AM, Switzer BJ, Schaefer JK, Philips EJP, Lovley DR, Oremland RS (1995) Growth of strain SES-3 with arsenate and other diverse electron acceptors. Appl Environ Microbiol 6:656–661 Li H, Li M, Huang Y, Rensing C, Wang G (2013) In silico analysis of bacterial arsenic islands reveals remarkable synteny and functional relatedness between arsenate and phosphate. Front Microbiol 4:1–9 Liao V-HC, Chu YJ, Su YC, Lin PC, Hwang YH, Liu CW, Liao CM, Chang FJ, Yu CW (2011) Assessing the mechanisms controlling the mobilization of arsenic in the arsenic contaminated shallow alluvial aquifer in the blackfoot disease endemic area. J Hazard Mater 198:398–403 Macur RE, Jackson CR, Botero LM, Mcdermott TR, Inskeep WP (2004) Bacterial populations associated with the oxidation and reduction of arsenic in an unsaturated soil. Environ Sci Technol 38:104–111 Mailloux BJ, Alexandrova E, Keimowitz AR, Wovkulich K, Freyer GA, Herron M, Stolz JF, Kenna TC, Pichler T, Polizzotto ML, Dong H, Bishop M, Knappett PSK (2009) Microbial mineral weathering for nutrient acquisition releases arsenic. Appl Environ Microbiol 75: 2558–2565 Malasarn D, Saltikov CW, Campbell KM, Santini JM, Hering JG, Newman DK (2004) arrA is a reliable marker for As(V)-respiration in the environment. Science 306:455 Maria FVT, Oscar CRB, Camilo S, Martha JVF, Jenny D (2011) Horizontal arsC gene transfer among microorganisms isolated from arsenic polluted soil. Int Biodeterior Biodegrad 65:1–6 Mukhopadhyay R, Rosen BP, Phung LT, Silver S (2002) Microbial arsenic: from geocycles to genes and enzymes. FEMS Microbiol Rev 4:311–325 Muller D, Lièvremont D, Simeonova DD, Hubert JC, Lett MC (2003) Arsenite oxidase aox genes from a metal-resistant betaproteobacterium. J Bacteriol 185:116–141 Niggemyer A, Spring S, Stackebrandt E, Rosenzweig RF (2001) Isolation and characterization of a novel As(v)-reducing bacterium: implications for arsenic mobilization and the genus Desulfitobacteria. Appl Environ Microbiol 67:5568–5580 Quéméneur M, Heinrich-Salmeron D, Muller D, Lièvremont D, Jauzein M, Bertin PN, Garrido F, Joulian C (2008) Diversity surveys and evolutionary relationships of aoxB genes in

aerobic arsenite-oxidizing bacteria. Appl Environ Microbiol 74:4567–4573 Reasoner DJ, Geldreich EE (1985) A new medium for the enumeration and subculture of bacteria from potable water. Appl Environ Microbiol 49:1–8 Rosen BP (1999) Families of arsenic transporters. Trends Microbiol 7: 207–212 Salmassi TM, Venkateswaren K, Satomi M, Nealson KH, Newman DK, Hering G (2002) Oxidation of arsenite by Agrobacterium albertimagni, AOL2, sp. nov., isolated from hot creek, California. Geomicrobiol J 19:53–66 Saltikov CW, Olson BH (2002) Homology of Escherichia coli R773 arsA, arsB, and arsC genes in arsenic-resistant bacteria isolated from raw sewage and arsenic-enriched creek waters. Appl Environ Microbiol 68:280–288 Santini JM, Sly LI, Schnagl RD, Macy JM (2000) A new chemolithoautotrophic arsenite-oxidizing bacterium isolated from a gold mine: phylogenetic, physiological, and preliminary biochemical studies. Appl Environ Microbiol 66:92–98 Sarkar A, Kazy SK, Sar P (2013) Characterization of arsenic resistant bacteria from arsenic rich groundwater of West Bengal. Ecotoxicology 22:363–374 Shakoori FR, Aziz I, Rehman A, Shakoori AR (2010) Isolation and characterization of arsenic reducing bacteria from industrial effluents and their potential use in bioremediation of wastewater. Pakistan J Zool 42:331–338 Shivaji S, Suresh K, Chaturvedi P, Dube S, Sengupta S (2005) Bacillus arsenicus sp. nov., an arsenic-resistant bacterium isolated from a siderite concretion in West Bengal, India. Int J Syst Evol Microbiol 55:1123–1127 Silver S, Phung LT (2005) Genes and enzymes involved in bacterial oxidation and reduction of inorganic arsenic. Appl Environ Microbiol 71:599–608 Simeonova DD, Lièvremont D, Lagarde F, Muller DA, Groudeva VI, Lett MC (2004) Microplate screening assay for the detection of arsenite-oxidizing and arsenate-reducing bacteria. FEMS Microbiol Lett 38:249–253 Slyemi D, Bonnefoy V (2011) How prokaryotes deal with arsenic. Microbiol Rep 1:16–24 Smibert RM, Krieg NR (1994) Phenotypic characterization. In: Gerhardt P, Murray RGE, Wood WA, Krieg NR (eds) Methods for general and molecular bacteriology, 2nd edn. American Society for Microbiology, Washington DC, pp 608– 654 Soge OO, Giardino MA, Ivanova IC, Pearson AL, Meschke JS, Roberts MC (2009) Low prevalence of antibiotic-resistant gram-negative bacteria isolated from rural south-western Ugandan groundwater. Water SA 35:343–347 Sun Y, Polischuk EA, Radoja U, Cullen WR (2004) Identification and quantification of arsC genes in environmental samples by using real-time PCR. J Microbiol Methods 58:336–349 Sutton NB, van der Kraan GM, van Loosdrecht MC, Muyzer G, Bruining J, Schotting RJ (2009) Characterization of geochemical constituents and bacterial populations associated with As mobilization in deep and shallow tube wells in Bangladesh. Water Res 43:1820–1825 Tamura K, Dudley J, Nei M, Kumar S (2008) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:296–299 Yamamura S, Yamashita M, Fujimoto N, Kuroda M, Kashiwa M, Sei K, Fujita M, Ike M (2007) Bacillus selenatarsenatis sp. nov., a selenate- and arsenate-reducing bacterium isolated from the effluent drain of a glass-manufacturing plant. Int J Syst Evol Microbiol 57: 1060–1064 Zhang XX, Zhang T, Fang HHP (2009) Antibiotic resistance genes in water environment. Appl Microbiol Biotechnol 82:397–414

Studies on arsenic transforming groundwater bacteria and their role in arsenic release from subsurface sediment.

Ten different Gram-negative arsenic (As)-resistant and As-transforming bacteria isolated from As-rich groundwater of West Bengal were characterized to...
2MB Sizes 0 Downloads 3 Views