Arsenic Dissolution from Japanese Paddy Soil by a Dissimilatory

May 13, 2013 - ABSTRACT: Dissimilatory As(V) (arsenate)-reducing bacteria may play an important role in arsenic release from anoxic sediments in the f...
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Arsenic Dissolution from Japanese Paddy Soil by a Dissimilatory Arsenate-Reducing Bacterium Geobacter sp. OR‑1 Toshihiko Ohtsuka,† Noriko Yamaguchi,‡ Tomoyuki Makino,‡ Kazuhiro Sakurai,† Kenta Kimura,† Keitaro Kudo,† Eri Homma,† Dian Tao Dong,† and Seigo Amachi*,† †

Graduate School of Horticulture, Chiba University, 648 Matsudo, Matsudo-city, Chiba 271-8510, Japan National Institute for Agro-Environmental Sciences, 3-1-3, Kan-nondai, Tsukuba-city, Ibaraki 305-8604, Japan

‡

S Supporting Information *

ABSTRACT: Dissimilatory As(V) (arsenate)-reducing bacteria may play an important role in arsenic release from anoxic sediments in the form of As(III) (arsenite). Although respiratory arsenate reductase genes (arrA) closely related to Geobacter species have been frequently detected in arsenic-rich sediments, it is still unclear whether they directly participate in arsenic release, mainly due to lack of pure cultures capable of arsenate reduction. In this study, we isolated a novel dissimilatory arsenate-reducing bacterium, strain OR-1, from Japanese paddy soil, and found that it was phylogenetically closely related to Geobacter pelophilus. OR-1 also utilized soluble Fe(III), ferrihydrite, nitrate, and fumarate as electron acceptors. OR-1 catalyzed dissolution of arsenic from arsenate-adsorbed ferrihydrite, while Geobacter metallireducens GS-15 did not. Furthermore, inoculation of washed cells of OR-1 into sterilized paddy soil successfully restored arsenic release. Arsenic K-edge X-ray absorption near-edge structure analysis revealed that strain OR-1 reduced arsenate directly on the soil solid phase. Analysis of putative ArrA sequences from paddy soils suggested that Geobacter-related bacteria, including those closely related to OR-1, play an important role in arsenic release from paddy soils. Our results provide direct evidence for arsenic dissolution by Geobacter species and support the hypothesis that Geobacter species play a significant role in reduction and mobilization of arsenic in flooded soils and anoxic sediments.

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observed in flooded rice paddies.9,10 Rice is thus the greatest contributor to uptake of inorganic arsenic through agricultural crops.11 In Japan, arsenic contamination has been a problem in rice paddy fields located downstream of mining areas,12 and the Japanese criterion for arsenic pollution of paddy soils is now set at 15 mg kg−1. Therefore, it is of great importance to understand the mechanism of arsenic release from flooded paddy soils. Microorganisms, such as metal-reducing bacteria, may play an important role in arsenic release from flooded soils and anoxic sediments.13 Reductive dissolution of arsenic-bearing iron (hydr)oxides by dissimilatory iron-reducing bacteria and the subsequent mobilization of arsenic are one of the possible mechanisms by which arsenic can be released from these environments.14,15 In addition, direct reduction of arsenate adsorbed on iron (hydr)oxides by dissimilatory arsenatereducing bacteria is now considered to be another important mechanism of arsenic release.16,17 Dissimilatory arsenatereducing bacteria are capable of utilizing arsenate as a terminal electron acceptor for respiration and are phylogenetically

INTRODUCTION The predominant forms of arsenic in soils and aquifers are inorganic arsenate [As(V)] and arsenite [As(III)], and the latter form is considered to be much more toxic than the former.1,2 Arsenate exists in anionic forms (H2AsO4− and HAsO42−) at circumneutral pH and is thermodynamically stable under oxic conditions. However, arsenite takes a nonionic form at the pH conditions found in most natural environments (H3AsO3, pKa = 9.22) and is stable under reducing conditions. Arsenic sorption on metal oxide minerals, especially on iron (hydr)oxides, is an important process controlling the dissolved concentration of arsenic in various environments. Generally, arsenate is strongly associated with soil minerals, including iron, aluminum, and manganese (hydr)oxides, whereas arsenite predominantly adsorbs to iron (hydr)oxides and is more mobile than arsenate.3−5 Elevated levels of arsenic in groundwater threaten the health of people worldwide. Bangladesh and West Bengal have the most serious groundwater arsenic problem, and approximately 60−100 million people are exposed to more than 10 μg L−1 of arsenic in drinking water, which is the health standard set by the World Health Organization.2 Direct consumption of rice irrigated with arsenic-contaminated water is another significant source of arsenic.6−8 Under reducing conditions, elevated concentrations of arsenite in solution have commonly been © XXXX American Chemical Society

Received: January 16, 2013 Revised: May 3, 2013 Accepted: May 13, 2013

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Enrichment and Isolation. Strict anaerobic technique26 was used in the preparation of the minimal medium and the manipulation of the enrichments. The medium used for enrichment contained the following (per liter): NH4Cl (0.535 g), KH2PO4 (0.136 g), MgCl2·6H2O (0.204 g), CaCl2·2H2O (0.147 g), trace mineral element solution (1 mL), vitamin solution (1 mL), 1 g L−1 of resazurin solution (1 mL), and NaHCO3 (2.52 g). The medium was dispensed into 60-mL serum bottles under N2/CO2 (80:20) atmosphere and autoclaved. Acetate (2 mM), arsenate (5 mM), and cysteineHCl (1 mM) were added separately from sterile anaerobic stock solutions. Enrichment was prepared by inoculating 1 mL of soil slurry (20 g wet weight of soil A and 60 mL of distilled water) into 19 mL of the minimal medium, and incubated at 30 °C in the dark. Enrichment was analyzed for arsenate consumption and arsenite production by high-performance liquid chromatography (HPLC; L-7000, Hitachi, Tokyo, Japan) with an Aminex HPX-87H ion exclusion column (Bio-Rad Laboratories, Hercules, CA, USA) under UV detection at 212 nm. The mobile phase was 0.01 N H2SO4 at a flow rate of 0.6 mL min−1, and the column was maintained at 35 °C. After complete reduction of arsenate was confirmed, 1 mL of the enrichment was transferred to 19 mL of fresh liquid medium. After several rounds of subculturing, the enrichment was serially diluted and inoculated into anaerobic shake tubes prepared with the minimal medium and 2% Bacto Agar (Difco, Sparks, MD, USA). After incubation at 30 °C, a single colony was picked, serially diluted, and inoculated into new shake tubes to ensure purity. The pure culture was then grown anaerobically on acetate and arsenate in liquid medium. Culture purity was determined by microscopy and PCR-denaturing gradient gel electrophoresis (DGGE) (see below). Growth Experiments. In most cases, strain OR-1 was grown on 2 mM acetate and 5 mM arsenate in the minimal medium. When potential electron donors were tested, 5 mM arsenate was used as the sole electron acceptor, and the electron donors being tested were added to the medium at 5 mM. The strain was considered positive for electron donor utilization if more than 3 mM of arsenate consumption as well as arsenite production was confirmed by HPLC. Fe(III) chelated with nitrilotriacetic acid [Fe(III)-NTA], ferrihydrite, nitrate, nitrite, manganese oxide (MnO2), selenate, fumarate, malate, sulfate, thiosulfate, sulfite, and elemental sulfur were tested as potential electron acceptors. Acetate (5 mM) was used as the electron donor and carbon source, and the electron acceptors being tested were added to the medium at 5 mM or 5 mmol L−1. The strain was considered positive for electron acceptor utilization if more than 1 mM of acetate consumption was confirmed by HPLC. In cases of mineral reduction, utilization was assessed by observation of color changes from black to white (MnO2), from clear to red [Se(VI)], and from dark brown to clear [Fe(III)-NTA]. Twoline ferrihydrite was synthesized using the method of Schwertmann and Cornell.27 The structure of ferrihydrite was confirmed by X-ray diffraction analysis. Reduction of ferrihydrite was confirmed by colorimetric detection of dissolved Fe(II) by the ferrozine method.28 Dicumarol (Acros Organics, Geel, Belgium) and 2-heptyl-4-hydroxyquinoline Noxide (HQNO; Enzo Life Sciences, Farmingdale, NY, USA) were used as potential inhibitors of growth on arsenate. Sequencing and Phylogenetic Analysis of 16S rRNA Genes. Genomic DNA of strain OR-1 was isolated using the

diverse, including members of Firmicutes, gamma-, delta-, and epsilon-Proteobacteria.13,18 In these bacteria, arsenate reduction is thought to be catalyzed by the dissimilatory arsenate reductase complex, which consists of a large molybdenumcontaining subunit (ArrA) and a small iron−sulfur-containing subunit (ArrB).19,20 Islam et al.15 showed significant release of arsenite and Fe(II) from a microcosm containing arsenic-rich West Bengali sediments amended with acetate. Analysis of 16S rRNA gene clone libraries revealed that members of the family Geobacteraceae were dominant (70% of the total clones) in the microcosm. In addition, Héry et al.21 reported that a functional gene for arsenate respiratory reductase, arrA, amplified from West Bengali sediments showed high amino acid sequence identity to sequences of putative arrA genes found in genomes of Geobacter uraniireducens and G. lovleyi. Similar results were also observed from Cambodian sediments that host arsenic-rich groundwater.22 These results suggest that Geobacter species may play an important role in reduction and mobilization of arsenic in these anoxic sediments. However, to date, attempts to grow Geobacter species with arsenate as the sole electron acceptor have been unsuccessful.23,24 Therefore, it is still unclear whether Geobacter species directly participate in arsenic release from flooded soils and anoxic sediments. In a previous study, we prepared soil slurries from Japanese paddy soils containing approximately 40 mg kg−1 of arsenic and incubated them under anaerobic conditions.25 Release of arsenic and Fe(II) occurred simultaneously when the Eh of the slurries decreased from +500 mV to +100 mV, and the dominant form of arsenic released was arsenite. Interestingly, arsenic K-edge X-ray absorption near-edge structure (XANES) analysis revealed that the proportion of arsenite in the soil solid phase increased to 80% of the total arsenic after anaerobic incubation, whereas before the incubation the proportion was only 20%. In addition, the proportion of arsenite in the solid phase did not change in the gamma-irradiated slurries. These results strongly suggest that microbial reduction of arsenate is necessary for release of arsenite from paddy soils.25 In this study, we isolated a novel dissimilatory arsenatereducing bacterium, strain OR-1, from Japanese paddy soil. It was phylogenetically closely related with Geobacter pelophilus and could utilize both iron (soluble iron and ferrihydrite) and arsenate as the electron acceptor for growth. To confirm if Geobacter species could dissolve arsenic adsorbed on soil solid phase, dissolution of arsenic from arsenate-adsorbed ferrihydrite as well as from sterile paddy soil by strain OR-1 was determined. In addition, XANES analysis of the soil solid phase was performed to understand if strain OR-1 could reduce arsenate directly on the soil solid phase. The potential impact of OR-1 and related microorganisms on the mobilization of arsenic from flooded soils and anoxic sediments is also discussed.

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MATERIALS AND METHODS Soils Samples. Soils were collected from the plowed layer of a paddy field (soil A) and from the surface layer of a fallow paddy field (soil B). Detailed properties of the soils have been reported by Yamaguchi et al.25 The soils were passed through a 2-mm sieve and stored in a refrigerator at 4 °C until use. Both soils were classified as Aeric Epiaquents according to U.S. soil taxonomy and contained approximately 40 mg kg−1 of total arsenic.25 B

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method of Hiraishi.29 The 16S rRNA gene was amplified by PCR using the bacterial consensus primers 8F (5′-AGAGTTTGATCCTGGCTCAG-3′, Escherichia coli positions 8−27) and 1491R (5′-GGTTACCTTGTTACGACTT-3′, Escherichia coli positions 1509−1491). PCR products were purified using a QIAquick PCR Purification kit (Qiagen, Hilden, Germany), and sequenced using a BigDye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) and an ABI Prism 3100 Genetic Analyzer (Applied Biosystems) using appropriate sequencing primers.30 The obtained 16S rRNA gene sequences were subjected to BLAST search (http://www. ncbi.nlm.nih.gov/BLAST/) to determine sequence identity. The retrieved sequences were aligned using the Clustal X program, version 2.0. The phylogenetic tree was constructed using the neighbor-joining method.31 Bootstrap values were obtained for 1000 replicates to estimate the confidence of tree topologies. PCR-DGGE. DNA was extracted from the enrichments using a FastDNA Spin kit (MP Biomedicals, Morgan Irvine, CA, USA) according to the manufacturer’s instructions. PCRDGGE was performed according to the method reported by Muyzer et al.32 The variable V3 region of the 16S rRNA gene was amplified using the primers 341fGC (a 40-bp GC-clamp was linked at the 5′ end of the primer 341f)32 and 534r.32 DGGE was performed using a DCode Universal Mutation Detection System (Bio-Rad Laboratories). The major bands were excised and used for reamplification with the primers 341f and 534r; the products obtained were sequenced as described previously.33 ArrA Analysis. The dissimilatory arsenate reductase gene (arrA) of strain OR-1 was amplified from genomic DNA using primers AS1F and AS1R.34 For analysis of ArrA in paddy soils, DNA was extracted from soil slurries prepared from soils A and B, which had been incubated anaerobically for 100 and 60 days, respectively. From these slurries, 2250−5430 nM of arsenite had been released.25 A FastDNA Spin kit (MP Biomedicals) was used for DNA extraction. The putative arrA gene was amplified by nested PCR according to the PCR protocols described by Song et al.,34 in which the first PCR was performed with primers AS1F and AS1R, and the second PCR was performed with AS2F and AS1R. The amplified products (approximately 640 bp) were confirmed on 2% agarose gels by electrophoresis and purified with a QIAquick PCR Purification kit (Qiagen). When nonspecific amplification was observed on the gels, DNA fragments of interest were excised from the gels with a razor blade. The purified products were cloned into pCR4-TOPO plasmid vectors using a TOPO-TA Cloning kit (Invitrogen, Carlsbad, CA, USA) and transformed into One Shot TOP10 chemically competent Escherichia coli (Invitrogen) according to the manufacturer’s instructions. Twenty-eight and 10 clones were randomly selected for sequencing from soils A and B, respectively. The obtained gene sequences were translated to amino acid sequences, and phylogenetic analysis was carried out as described above. Arsenic Release from Arsenate-Adsorbed Ferrihydrite. Arsenate-adsorbed ferrihydrite was prepared by adding 3 g of ferrihydrite to 50 mL of KH2AsO4 solution (500 mM) and stirring for 24 h. After centrifugation, the precipitate was washed twice with distilled water and freeze-dried. Strain OR-1 was pregrown either on arsenate or on Fe(III)-NTA as the electron acceptor. After the culture (1 mL) was centrifuged (15000g, 10 min), the cells were washed with sterile 0.8% NaCl and resuspended in 1 mL of 0.8% NaCl. The cell suspension (1

mL) was inoculated into the minimal medium (19 mL) containing 50 mg of arsenate-adsorbed ferrihydrite as the sole electron acceptor. Acetate (2 mM) was also included in the minimal medium as the electron donor. G. metallireducens GS15 (ATCC53774)54 was pregrown in the minimal medium supplemented with 0.1 g L−1 casamino acids, with 20 mM acetate and 5 mM Fe(III)-NTA as the electron donor and acceptor, respectively. This bacterium is a representative dissimilatory iron-reducing bacterium, but neither reduces arsenate nor possesses the arrA gene in its genome.55 Washed cells of GS-15 were prepared and inoculated in the same manner as strain OR-1. The atomic ratio of As to Fe in arsenate-adsorbed ferrihydrite was 0.4. If it dissolved completely in the minimal medium, As and Fe concentrations in the liquid phase theoretically equaled 7.2 and 17.8 mM, respectively. Arsenic Release from Sterile Paddy Soil. In the previous study, we incubated soils A and B under reducing conditions and found that a much higher amount of arsenic was released from soil B than from soil A.25 This was probably due to the formation of secondary Fe mineral (siderite) in soil A, which may adsorb arsenite and prevent its dissolution. Thus, we used soil B for the arsenic release experiment in this study. Soil B (20 g wet weight) was mixed with 40 mL of distilled water to prepare the soil slurry. The slurry was dispensed into 60-mL serum bottles, and they were flushed with a N2 gas stream and sealed with butyl rubber stoppers and aluminum caps. The bottles were then sterilized with gamma-ray irradiation at 50 kGy. A previous study has shown that 50 kGy of gamma-ray irradiation destroyed all culturable microorganisms in the slurry, and that the Eh of the irradiated slurry did not decrease for at least 60 days.25 After irradiation, 3 mM acetate was added to the slurry as the electron donor, and it was flushed with H2 gas aseptically to maintain an Eh value of less than −200 mV. Strain OR-1 was pregrown either on arsenate or on Fe(III)NTA as the electron acceptor. After the culture (13.3 mL) was centrifuged (10000g, 20 min), the cells were washed three times with sterile 0.8% NaCl and resuspended in 1 mL of 0.8% NaCl. The cell suspension (1 mL) was inoculated into the sterile soil slurry. Sterile (autoclaved cells inoculated) and background (no cells inoculated) controls were also prepared. The slurries were incubated at 30 °C in the dark without shaking. After incubation for 7−28 days, the slurries were centrifuged (5000g, 10 min), and the supernatant was filtered through a 0.22-μm filter unit (Vivaspin 20, Sartorius Stedim, Göttingen, Germany). To prevent precipitation of Fe hydroxide, 1 mL of 1.5 M HNO3 was added to 9 mL of the filtrate. Conversion of arsenite to arsenate was negligible in the solution. Arsenic speciation in the solution phase was analyzed with an HPLC (PU 712i, GL Science, Tokyo, Japan) connected with a quadrupole inductively coupled plasma mass spectrometer (ICP-MS, ELAN DRC-e, Perkin-Elmer, Waltham, MA, USA) as described previously.25 A SuperIC anion column (TOSOH, Tokyo, Japan) was used for the separation of arsenite and arsenate. Fe concentration was determined using an inductively coupled plasma optical emission spectrometer (ICP-OES, Vista-Pro, Varian, Palo Alto, CA, USA). For analysis of arsenic speciation in the soil solid phase, arsenic K-edge (11,867 eV) XANES spectra were acquired at BL12C at the Photon Factory (High-Energy Accelerator Research Organization, KEK, Tsukuba, Japan) as described previously.9,25 Briefly, the XANES spectra of soil samples were C

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collected in the fluorescence detection mode using a 19element Ge semiconductor detector, whereas those of the reference materials (Na2AsO3 and NaHAsO4) were collected in the transmission mode. The soil samples were analyzed as a wet paste at an ambient temperature. Compositions of arsenic species in the soil solid phases were evaluated by linear combination fitting (LCF) of XANES spectra with reference compounds. The REX 2000 ver. 2.5 program packages (Rigaku, Tokyo, Japan) were used to subtract the pre-edge background and normalize the spectra and LCFs. Nucleotide Sequence Accession Numbers. The 16S rRNA gene and arrA gene sequences identified in this study have been deposited in the DDBJ/EMBL/GenBank databases under accession numbers AB769234−AB769238, AB769384− AB769400, and AB769875.

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RESULTS Enrichment of Arsenate-Reducing Bacteria from Paddy Soil. To enrich dissimilatory arsenate-reducing bacteria, 1 mL of soil slurry was inoculated into 19 mL of minimal medium containing 2 mM acetate and 5 mM arsenate, and arsenate reduction and arsenite production were observed. After complete reduction of arsenate was confirmed, a portion of the enrichment was transferred to fresh liquid medium. After three rounds of subculturing, the microbial community in the enrichment was analyzed by PCR-DGGE. As shown in Figure S1 and Table S1, Supporting Information, bacteria closely related to Geobacter spp. were predominant in the enrichment, although putative fermenting bacteria closely related to Caloramator spp. were also present. Isolation of Arsenate-Reducing Bacterial Strain OR-1. After repeated picking of single colony, an anaerobic microorganism, designated strain OR-1, was obtained from the enrichment. Cells of strain OR-1 were Gram-negative, motile, regular rods, 1.5−3 μm long, and 0.5 μm in diameter (Figure S2A). Cells had at least two flagella (Figure S2B). No spore formation was observed. Colonies grown on the solid minimal medium containing acetate and arsenate were pink, probably due to the high c-type cytochrome content of the cells.42 Strain OR-1 grew by the oxidation of acetate coupled to the reduction of arsenate (Figure 1). Neither arsenate reduction nor cell growth was observed in the absence of acetate (OD660 of less than 0.002). In addition, no cell growth occurred in the absence of arsenate (OD660 of less than 0.003). The ratios of arsenate consumed and arsenite produced to acetate consumed were 3.96 and 3.72, respectively. The reduction of arsenate by strain OR-1 was almost completely inhibited by respiratory inhibitors such as dicumarol (100 μM) and HQNO (10 μM) (Figure S3). These results suggest that arsenate reduction by strain OR-1 is a respiratory process and that strain OR-1 oxidizes acetate according to the following equation:

Figure 1. Growth of strain OR-1 on arsenate as the sole electron acceptor. The strain was grown on 2 mM acetate as the electron donor and 6 mM arsenate as the electron acceptor. Symbols represent the mean values obtained for triplicate determinations, and bars indicate standard deviations.

sulfite, or elemental sulfur did not serve as the electron acceptor. Phylogenetic Analysis of the 16S rRNA Gene and the arrA Gene. BLAST analysis of 16S rRNA gene sequence showed that strain OR-1 was most similar to G. pelophilus (sequence identity of 98.7%) and G. argillaceus (sequence identity of 94.8%) in the class Deltaproteobacteria. Phylogenetic comparison of the 16S rRNA gene sequence of strain OR-1 with those of selected representatives was performed using approximately 1500 bases (Figure S4). The result indicated that strain OR-1 is most closely related to G. pelophilus. It also showed close relationships with uncultured bacterial clones recovered from arsenic-contaminated sediments in Cambodia (GenBank accession number EF014912) and West Bengal (DQ369324) (data not shown). A putative respiratory arsenate reductase gene (arrA) was amplified from the genomic DNA of strain OR-1. The method of Song et al.34 yielded a 637-bp PCR product which shared 90.4% and 86.4% amino acid sequence identity with the arrA genes identified by the genome sequencing projects for G. uraniireducens Rf4 and G. lovleyi SZ, respectively (Figure 2). Other related sequences included putative arrA genes found in a biofilm reactor used to remove arsenic from drinking water (AEK98615, amino acid identity of 90%), low organic carbon aquifer sediments from West Bengal (CAZ04936, 87%), arsenic-rich glauconitic sediment from New Jersey (AFG33225, 82%), and sediment from Chesapeake Bay (ACN22107, 80%). Dissolution of Arsenic Adsorbed on Ferrihydrite. Bacteria were cultured with arsenate-adsorbed ferrihydrite as the sole electron acceptor. At time zero, approximately 350− 450 μM arsenate was detected in the liquid phase, indicating that partial dissolution−desorption of arsenate had occurred.14,16,17 However, in the absence of bacterial cells, the arsenate concentration did not increase (Figure 3A). In the culture inoculated with G. metallireducens GS-15, a representative dissimilatory iron-reducing bacterium without the capacity for arsenate reduction, soluble Fe(II) increased to 11 mM during 30 days of incubation (Figure 3B). Arsenate disappeared from the liquid phase within 7 days, but no arsenite

CH3COO− + 2HAsO4 2 − + 2H 2AsO4 − + 5H+ → 4H3AsO3 + 2HCO3−

With arsenate (5 mM) as the electron acceptor, strain OR-1 was capable of oxidizing acetate, formate, and lactate. Butyrate, propionate, pyruvate, succinate, malate, glycerol, glucose, fructose, and yeast extract did not serve as electron donors. With acetate (5 mM) as the electron donor, the strain was capable of reducing the following electron acceptors: arsenate, Fe(III)-NTA, ferryhydrite, nitrate, nitrite, manganese oxide (MnO2), selenate, fumarate, malate, and thiosulfate. Sulfate, D

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Figure 2. Phylogenetic tree of putative ArrA of strain OR-1 and those recovered from Japanese paddy soils. The tree was constructed based on approximately 200 amino acid sequences using the neighbor-joining method. arrA clones were translated into amino acid sequences, and they were grouped into phylotypes based on a 98% amino acid sequence identity cutoff. The number of clones in each phylotype is indicated in parentheses. Numbers at nodes show bootstrap values obtained from 1000 resamplings, but bootstrap values below 500 were omitted. Scale bar indicates 5% estimated sequence divergence.

the soil solid phase after incubation was 42%; in the absence of cells the proportion of arsenate was only 20% (Table 1). The latter value was the same as that of the original soil before incubation.25 In the second experiment, the slurry was incubated for 4 weeks with washed cells of OR-1. In this case, more than 4100 nM of arsenite was detected in the slurry incubated with the cells, whereas only 0−31 nM of arsenite was observed when the slurry was incubated without the cells or with autoclaved cells of OR-1. The molar ratios of dissolved As to Fe in the above two experiments were 0.004−0.008. These values were similar to those observed in our previous study, in which soil slurries were incubated under anaerobic conditions for 60−100 days,25 suggesting that strain OR-1 dissolved arsenic in a manner similar to the native soil microbial community. The proportion of arsenite in the soil solid phase increased to 70% in the second experiment (Figure 4). We also performed a similar experiment with washed cells of G. metallireducens GS-15, but it was unsuccessful since no Fe(II) was produced, even after incubation for 4 weeks (data not shown).

accumulated during the duration of the experiment. In the cultures inoculated with strain OR-1, however, soluble Fe(II) production was only 3 mM, and arsenate disappeared from the liquid phase within 4−7 days (Figure 3C,D). Notably, in these cultures, 600−700 μM of arsenite accumulated on days 5−7, although arsenite levels decreased again afterward, probably due to reassociation with the solid phase.5,16,17 The maximum levels of arsenite in the liquid phase were significantly higher than the arsenate levels at time zero, indicating that strain OR-1 is capable of reducing and dissolving arsenic adsorbed on ferrihydrite. Arsenic Release from Sterile Paddy Soil Inoculated with Cells of Strain OR-1. In the first experiment, the gamma-ray irradiated sterile soil slurry was incubated for 1 week with acetate and washed cells of strain OR-1. As shown in Table 1, 904 nM and 229 μM of arsenite and Fe, respectively, were detected in the supernatant of the incubated slurry. When slurries were not incubated with live cells, only 78 nM and 29 μM of arsenite and Fe were observed, respectively. Arsenic Kedge XANES analysis revealed that the proportion of arsenite in E

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Figure 3. Bacterial dissolution of arsenic from arsenate-adsorbed ferrihydrite. The minimal medium containing arsenate-adsorbed ferrihydrite as the sole electron acceptor was prepared and inoculated with sterile water (a background control without active cells) (A), washed cells of G. metallireducens GS-15 (B), and washed cells of strain OR-1 pregrown with arsenate (C) or with Fe(III)-NTA (D).

Table 1. Arsenic Release from Sterile Paddy Soil Slurry Inoculated with Cells of Strain OR-1a first experimentc second experimentc

no cells inoculated OR-1d no cells inoculated autoclaved cells inoculated OR-1e

As(III) (nM)

As(V) (nM)

77.7 ± 5.62 904 ± 53.1 0 30.8 ± 1.21 4110 ± 1210

11.2 ± 0.334 36.2 ± 32.0 70.4 ± 7.90 30.0 ± 1.18 487 ± 144

Fe (μM)

As(III) (%) in the soil solid phaseb

± ± ± ± ±

20 42 18 ± 0 21 ± 6 70 ± 3

29.1 229 68.6 65.5 524

4.34 37.6 14.3 18.7 81.6

a All values, except for two data from XANES analysis, are expressed as mean values ± SD from triplicate determinations. bArsenic speciation in the soil solid phase was determined by XANES analysis. cThe soil slurries were incubated for 7 and 28 days in the first and second experiments, respectively. dCells grown on Fe(III)-NTA as the electron acceptor were inoculated into the sterile soil slurry. eCells grown on arsenate as the electron acceptor were inoculated into the sterile soil slurry.

Diversity of ArrA in Paddy Soils. DNA was extracted from paddy soil slurries, which had been incubated anaerobically for 60−100 days and had released 2250−5430 nM of arsenite.25 A putative arrA gene was amplified successfully using the nested PCR technique described by Song et al.,34 in which the first PCR was performed with primers AS1F and AS1R and a second PCR was performed with AS2F and AS1R. We also tested other PCR primers, including ArrAfwd/ArrArev,35 ArrAUF1/ArrAUR3,36 and a combination of AS1F/2R and AS2F/1R,34 but the amplifications were unsuccessful. In order to understand the diversity of ArrA in paddy soils, 38 clones (28 clones from soil A and 10 clones from soil B) were

analyzed. As shown in Figure 2, 12 clones were affiliated with cluster 1, whose translated amino acid sequences showed 89.4− 100% similarities with the putative ArrA of strain OR-1. They also showed 81.2−90.4% and 78.7−86.4% similarities with the putative ArrA of G. uraniireducens Rf4 and G. lovleyi SZ, respectively. Cluster 2 consisted of 12 clones, whose amino acid sequences showed 71.2−77.8% similarities with ArrA of strain OR-1, G. uraniireducens Rf4, and G. lovleyi SZ. Cluster 3 included 14 clones, but did not show more than 50% amino acid sequence identity with known ArrA sequences. On the other hand, these clones showed 62−65% sequence similarities with molybdopterin oxidoreductase of Alkalilimnicola ehrlichii F

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directly on the soil solid phase. To date, S. barnesii SES-317 and Shewanella sp. ANA-343 have been found to dissolve arsenic adsorbed on ferrihydrite as arsenate. In addition, arsenic release from sterile sediment or the soil solid phase by S. arsenophilum MIT-1316 and Bacillus selenatarsenatis SF-144 has been reported. However, whether these bacteria could have reduced arsenate directly on the soil solid phase was unclear. In several Geobacter species, proteins possibly involved in extracellular electron transfer, especially those involved in electron transfer to solid iron (hydr)oxide, have been identified.39 These include electrically conductive pili (PilA), periplasmic or outer membrane cytochromes (PpcA, OmcB, and OmcS), and multicopper proteins (OmpB and OmpC). Whether or not such proteins or their homologues participate in electron transfer to arsenate adsorbed on the soil mineral phase is unclear. In particular, it is of great importance to understand how dissimilatory arsenate reductase (Arr), which is commonly located in the periplasmic space,19,45 transfers electrons to extracellular arsenate adsorbed on the soil mineral phase. To identify proteins possibly involved in extracellular electron transfer and to obtain insights into the genomic arrangement for putative arr operon, genome sequencing of strain OR-1 is underway in our laboratory. The representative iron-reducing bacterium G. metallireducens could not reduce and dissolve arsenic adsorbed on ferrihydrite, whereas it actively reduced ferrihydrite to form Fe(II) (Figure 3B). Cummings et al.14 reported that the ironreducing bacterium Shewanella alga BrY did not reduce but dissolved arsenic (as arsenate) from crystalline ferric arsenate as well as from arsenic-rich lake sediments. However, in this study, dissolved arsenate disappeared quickly from the liquid phase of the G. metallireducens culture without the formation of arsenite. This was probably because the dissolved arsenate had reassociated with the mineral phase. Since disappearance of dissolved arsenate was not observed in the absence of active bacterial cells (Figure 3A), it seems likely that arsenate had adsorbed on Fe(II)-bearing secondary minerals that had been formed as a result of iron reduction by G. metallireducens. Islam et al.23 reported that such minerals, including siderite (FeCO3), vivianite (iron phosphate), and magnetite, can all adsorb arsenate efficiently and removed arsenate from a culture of G. sulf urreducens. Since our growth medium was buffered with bicarbonate, siderite formation could have been very favorable. The inability of G. metallireducens to dissolve arsenic may suggest that arsenate reduction accelerates dissolution of arsenic adsorbed on the solid mineral phase. Phylogenetic analysis of the putative ArrA indicated that ArrA sequences closely related to that of strain OR-1 (clusters 1 and 2) are actually present at relatively high frequency in paddy soils (Figure 2). In particular, sequences in cluster 1 were very similar to the putative ArrA of strain OR-1, with sequence similarities of 89−100%, and they consisted of 32% of the total sequences analyzed. Thus, it is possible that Geobacter species closely related to strain OR-1 play a significant role in release of arsenic from paddy soils. Since ArrA sequences in cluster 1 also showed relatively high sequence similarities with those recovered from various arsenic-rich environments, such as sediments from West Bengal, they potentially impact mobilization of arsenic not only in paddy soils but also in various anoxic sediments. ArrA sequences in cluster 3, however, showed only limited relatedness with known ArrA sequences, but showed more than 60% sequence identity with the molybdopterin oxidoreductases of A. ehrlichii MLHE-1 and

Figure 4. Arsenic K-edge XANES spectra of reference materials and sterile paddy soil incubated with cells of strain OR-1. Dotted lines represent the linear combination of XANES spectra from reference compounds to reproduce the experimental spectra. The results of the second experiment are shown.

MLHE-1 (YP_741061), anaerobic arsenite oxidase of Ectothiorhodospira sp. PHS-1 (ZP_09695308), and formate dehydrogenase of Halorhodospira halophila SL1 (YP_001001949).

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DISCUSSION 16S rRNA genes and putative arrA genes closely related to those of Geobacter species have frequently been detected in arsenic-rich sediments.15,21,22,37,38 Since Geobacter species are known to catalyze the dissimilatory reduction of iron in the environments,39 they may cause arsenic release by means of reductive dissolution of arsenic-bearing iron (hydr)oxides. In addition, Geobacter species may also function as dissimilatory arsenate-reducing bacteria and play a direct role in arsenic reduction and mobilization.40,41 At least two Geobacter species, G. uraniireducens Rf4 and G. lovleyi SZ, are known to possess arrA genes, but arsenate reduction by these bacteria has not yet been reported.39−41 Very recently, Giloteaux et al.24 reported that resting cells of G. lovleyi SZ reduced arsenate in the presence of acetate. However, attempts to grow this bacterium with arsenate as the sole electron acceptor were unsuccessful.24 Thus, it is still unclear if Geobacter species directly participate in arsenic release from flooded soils and anoxic sediments. In this study, we isolated a novel dissimilatory arsenate-reducing bacterium strain OR-1, which was closely related to G. pelophilus. To the best of our knowledge, strain OR-1 is the first Geobacter species capable of growth using arsenate as the electron acceptor. Strain OR-1 was capable of reducing and dissolving arsenic adsorbed on ferrihydrite (Figure 3) and was able to release arsenic from the sterile soil solid phase into the solution phase (Table 1). Thus, our results indicate that strain OR-1 can reduce not only dissolved arsenate but also arsenate adsorbed on the soil mineral phase, such as ferrihydrite. Furthermore, solid state speciation by XANES analysis revealed that the proportion of arsenite in the soil solid phase actually increased after incubation with washed cells of OR-1. These results strongly suggest that strain OR-1 is able to reduce arsenate G

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Ectothiorhodospira sp. PHS1. Both bacteria are known as anaerobic arsenite oxidizer,46,47 and neither has been found to reduce arsenate. Recently, these oxidoreductases (designated as ArxA) have been proposed as a novel clade of arsenite oxidase.48,49 Our results indicate that arxA-containing bacteria are present in flooded paddy soils, although their function with regard to arsenic is not clear. It should be noted that, if the ArxA-like sequences in cluster 3 are eliminated as arsenite oxidases, all of the putative ArrA sequences identified in this study were from bacteria related to Geobacter, with amino acid sequence similarities of more than 70%. In conclusion, our results provide direct evidence for arsenic dissolution by Geobacter species and support the hypothesis that Geobacter species may play an important role in reduction and mobilization of arsenic in nature. Since strain OR-1 is the first Geobacter species shown to be capable of growing with arsenate as the sole electron acceptor, it may be useful as a model microorganism that potentially impacts the mobilization of arsenic in flooded soils and anoxic sediments. To date, the detailed biochemical and molecular mechanisms of respiratory arsenate reduction have only been studied in Shewanella sp. ANA-3.20,45,50,51 However, this bacterium was originally isolated from a marine environment,52 and few putative ArrA sequences closely related to that of Shewanella sp. ANA-3 have been detected in arsenic-rich environments. Considering that Geobacter species play a pivotal role in dissimilatory iron reduction in terrestrial and freshwater environments,39,53 and that arsenic is commonly associated with soil minerals such as iron (hydr)oxide,1 it will not be surprising if Geobacter species with the capacity for dissimilatory arsenate reduction are shown to be key players in the release of arsenic from various flooded soils and anoxic sediments.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Figure S1. PCR-DGGE analysis of the arsenate-reducing enrichment prepared from Japanese paddy soil. Figure S2. (A) Scanning electron micrograph of strain OR-1. (B) Morphology of negatively stained cells of strain OR-1. Figure S3. Dicumarol and HQNO inhibit arsenate reduction by strain OR-1. Figure S4. Phylogenetic tree showing the relationship between strain OR-1 and related Geobacter species on the basis of 16S rRNA gene sequences. Table S1. Sequence analysis of 16S rRNA genes recovered from DGGE bands. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +81-47-308-8867; fax: +81-47-308-8867; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Prof. Y. Takahashi (Hiroshima University) for XANES measurements. This work was supported by grants from the Ministry of Agriculture, Forestry and Fisheries of Japan (Research Project for Ensuring Food Safety from Farm to Table AC-1122) and from JSPS KAKENHI (Grant Number 23580103). The XANES measurements were performed under approval of the High-Energy Accelerator Research Organization, KEK (Proposal Nos. 2011G016 and 2012G578). H

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