Dissimilatory Arsenate Reduction and In Situ Microbial Activities and ...

Microb Ecol DOI 10.1007/s00248-015-0650-3

ENVIRONMENTAL MICROBIOLOGY

Dissimilatory Arsenate Reduction and In Situ Microbial Activities and Diversity in Arsenic-rich Groundwater of Chianan Plain, Southwestern Taiwan Suvendu Das 1 & Chia-Chuan Liu 1 & Jiin-Shuh Jean 1 & Tsunglin Liu 2

Received: 13 May 2015 / Accepted: 13 July 2015 # Springer Science+Business Media New York 2015

Abstract Although dissimilatory arsenic reduction (DAsR) has been recognized as an important process for groundwater arsenic (As) enrichment, its characterization and association with in situ microbial activities and diversity in As-rich groundwater is barely studied. In this work, we collected As-rich groundwater at depths of 23, 300, and 313 m, respectively, from Yenshui-3, Budai-Shinwen, and Budai-4 of Chianan plain, southwestern Taiwan, and conducted incubation experiments using different electron donors, acceptors, and sulfate-reducing bacterial inhibitor (tungstate) to characterize DAsR. Moreover, bacterial diversity was evaluated using 454-pyrosequencing targeting bacterial 16S rRNAs. MPN technique was used to enumerate microorganisms with different in situ metabolic functions. The results revealed that DAsR in groundwater of Chianan plain was a biotic phenomenon (as DAsR was totally inhibited by filter sterilization), enhanced by the type of electron donor (in this case, lactate enhanced DAsR but acetate and succinate did not), and limited by the availability of arsenate. In addition to oxidative recycling of As(III), dissolution of As(V)-saturated manganese and iron minerals by indigenous dissimilatory Mn(IV)- and Fe(III)-reducing bacteria, and abiotic oxidation of As(III) with Mn(IV) regenerated As(V) in the groundwater. Sulfate-respiring Electronic supplementary material The online version of this article (doi:10.1007/s00248-015-0650-3) contains supplementary material, which is available to authorized users. * Jiin-Shuh Jean [email protected] 1

Department of Earth Sciences, National Cheng Kung University, Tainan, Taiwan

2

Institute of Bioinformatics and Biosignal Transduction, National Cheng Kung University, Tainan, Taiwan

bacteria contributed 7.4 and 28.2 % to the observed DAsR in groundwater of Yinshui-3 and Budai-Shinwen, respectively, whereas their contribution was negligible in groundwater of Budai-4. A noticeable variation in dominant genera Acinetobacter and Bacillus was observed within the groundwater. Firmicutes dominated in highly As-rich groundwater of Yenshui-3, whereas Proteobacteria dominated in comparatively less As-rich groundwater of Budai-Shinwen and Budai 4. Keywords Arsenate respiration . Arsenic-rich groundwater . Bacterial diversity . 454 pyrosequencing . Alternate electron donor and acceptor . SRB inhibitor

Introduction The bioavailability and toxicological effects of arsenic (As) depend on its speciation and transformation within the environment [1]. Arsenic can occur in the environment in four oxidation states, i.e., arsenate [As (V)], arsenite [As (III)], elemental [As (0)], and arsine [As (-III)] [1]. In the aquatic environment, As is mostly found in inorganic forms, i.e., As(V) and As(III) [1, 2]. The reduction of less toxic and less bioavailable As(V) to more toxic and more bioavailable As(III) is the cause of As contamination and toxicity in environments [1]. Although abiotic (chemical) reduction of As(V) in the presence of sulfide is possible, which occurs mostly under strongly acidic conditions, biotic (biochemical) reduction of As(V) is more prevalent [3, 4]. The biotic reduction of As(V) occurred by two mechanisms: (1) detoxification of cells by cytosolic As(V) reductase–As(III) efflux systems encoded by ars-acr genes and (2) a dissimilatory (respiratory) As(V) reductase (encoded by arrA and arrB genes) present in certain obligate or facultative anaerobic bacteria that enable them to conserve energy derived from the oxidation of organic (e.g.,

S. Das et al.

lactate, acetate, formate, and aromatic compounds) or inorganic (e.g., hydrogen and sulfide) electron donors [5, 6]. In contrast to the detoxification pathway, dissimilatory As(V) reduction (DAsR) plays a major role in the release of As in anoxic environments [7, 8]. Notably, dissimilatory As(V)-respiring bacteria (DARB) can reduce both sorbed and dissolved As(V) to As(III) because the enzymes responsible for As(V) reduction are membrane-bound and linked to electron transport chains involved in energy generation [9]. These bacteria are phylogenetically diverse, including members of Firmicutes, Gamma-, Delta-, and Epsilonproteobacteria [6, 9]. Despite the pivotal role of DAsR in groundwater As contamination, DAsR has not been characterized toward As mobilization and enrichment in groundwater of several Ascontaminated sites worldwide. Nonetheless, the ability of indigenous microorganisms to reduce As at lower concentrations is elusive, especially when As is associated with liquid phase rather than solid phase in an aquifer [10]. In addition, no detailed studies have been conducted in groundwater of Chianan plain to evaluate the role of microbes for carrying out DAsR in the As-, Fe-, and Mn-rich oligotrophic environments. The Chianan plain in southwestern Taiwan was associated with the endemic blackfoot disease (BFD), a peripheral vascular disease caused by long-term consumption of high As (>0.35 mg L−1) water [11]. The solid-phase As in this aquifer is mostly As(V), whereas aqueous As is dominated by As(III) [12–14]. Several studies have described the possible mechanisms of As release from solid phase into the groundwater [13, 15, 16]. The most widely accepted mechanism is the reductive dissolution of host minerals (Fe/Mn oxyhydroxides) by complex geo-microbiological activities [15, 16]. Noteworthy, the high concentration of As(III) in the groundwater of Chianan plain emphasizes the importance of DAsR in As enrichment. This motivates deciphering the role of DAsR in groundwater As contamination in this aquifer using groundwater samples, thereby circumventing the more problematic conditions encountered in experiments with the sediment. Arsenic-rich aquifers of Chianan plain and other areas of similar hydrogeology are often nutrient limited, i.e., with a low concentration of total organic carbon in aquifer sediment and dissolved organic carbon in groundwater [13, 17–20]. In such oligotrophic environment, the nutrition provided by the inorganic electron donors/acceptors (e.g., As, Fe, Mn) to the indigenous bacteria could influence As biotransformation and enrichment within the aquifers [10, 15, 20]. In the last few years, microbial communities showing diverse respiratory processes have been reported in As-rich environments [10, 21, 22]. Microbial respiration of Fe(III), SO42−, and NO3− have been reported to influence the biogeochemical cycling of As, thereby the As mobilization and enrichment in the aquifers [21]. Despite considerable research interests, microbial diversity and in situ microbial activities influencing As

mobilization and enrichment in groundwater are largely unknown and even not yet studied in Chianan plain. The aim of this work was to advance our understanding of bacterial DAsR, in situ microbial activities, and diversity in As-rich groundwater of Chianan plain. More precisely, we addressed five questions: (1) is the reduction of As(V) to As(III) a biotic or abiotic phenomenon? (2) Is DAsR limited by in situ electron donor(s)? (2) Is DAsR limited by availability of arsenate? (3) What is the source of regeneration of As(V)? (4) Which are the important bacterial processes responsible for the observed DAsR? (5) How do indigenous bacterial communities change with As enrichment and hydrology?

Materials and Methods Groundwater Sampling and Analysis Groundwater samples were collected from Yenshu-3 (N 23° 18′ 6.7″/E 120° 15′ 11.1″), Budai-Shinwen (N 23° 20′ 22″/E 120° 7′ 57.9″), and Budai-4 (N 23° 19′ 37.8″/E 120° 9′ 3.2″) at depths of 23, 313, and 300 m, respectively. Water from wells was purged for 15–30 min to discharge the standing volume of groundwater in wells and obtain representative groundwater from the aquifer with stable levels of dissolved O2 and Eh. Temperature, pH, Eh, electrical conductivity (EC), and total dissolved solids (TDS) were measured at the field sites. The groundwater samples were filtered in the field (0.45 μm cellulose nitrate filter), acidified with 1 % HNO3 to pH 2, shipped to the laboratory, and stored at 4 °C in the dark until analysis. The anion and cation concentrations of groundwater were measured with an ion chromatography (Dionex, CA, USA) using external calibration (accuracy and precision was within ±5 %). Trace elements were measured with an inductively coupled plasma mass spectrometer (ICP-MS; Hewlett– Packard, Yamanashi-Ken, Japan). Accuracy was checked in each analytical batch by measuring certified reference material ‘Trace Metal in Drinking Water’ (TMDW; Lot # 623609) from High Purity Standards (HPS, USA) and was within ±5 % of certified values. Total As and As speciation were measured with ICP-MS. Dissolved organic carbon (DOC) was measured by TOC 5000 A analyzer (Shimadzu). The geochemical program PHREEQC was used to calculate speciation and mineral saturation indices in the groundwater [17, 23]. Fifteen observed chemical components (e.g., pH, Eh, temperature, alkalinity, As, Fe, Mn, SO4, Cl, Mg, Ca, Na, K, NH4, and NO3) in groundwater were used in the calculations. Water Sample Incubation for DAsR For incubation experiment, groundwater samples were collected in a 1-L glass bottle, drained to overflowing, sealed

Dissimilatory Arsenate Reduction and In Situ Microbial Activities

with a rubber septum, and tightly capped. To minimize indigenous microbial activity before use, groundwater samples were shipped on ice to laboratory and stored at ~4 °C in the dark. Bottles were thoroughly shaken and opened under N2; triplicate subsamples (8 mL each) were dispensed into a vacuumnator tube (10 mL) and tightly capped with rubber septum. Electron acceptors, electron donors, and sulfurreducing bacterial (SRB) inhibitor were added from anaerobic stock solutions with the help of a syringe and were at their final concentrations: Na2HAsO4 (1 and 5 mM), NaH2AsO3 (1 mM), NaNO3 (5 mM), sodium lactate (1 mM), sodium acetate (1 mM), sodium succinate (1 mM), Na 2 WO 4 (30 mM), MnO2 (5 mM), and Fe(III)-nitrilotriacetic acid (5 mM). Triplicate samples were incubated in the dark at 24 °C. Abiotic controls were the groundwater samples filtered through a sterile cellulose acetate membrane syringe filter (0.20 μm pore size). Discrete samples were withdrawn by syringe at various time intervals and As speciation was measured by ICP-MS.

Most Probable Number Estimates of Cells for Metabolic Functions Most probable number (MPN) technique was used to enumerate microorganisms with different in situ metabolic functions (i.e., As(V) reduction, As(III) oxidation, Fe(III) reduction, anaerobic nitrate-reducing Fe(II) oxidation (anFeOx), microaerophilic Fe(II) oxidation, and sulfur reduction and oxidation). Cell numbers were estimated from standard MPN table [24]. An anaerobic (gas atmosphere of N2/CO2: 80 %/ 20 % by volume) modified liquid medium (MLM) (29.5 mM NaHCO3, 2.8 mM NH4Cl, 5.0 mM NaH2PO4, 1.3 mM KCl, 10 mM lactate, and 10 ml each of Wolfe’s vitamin solution and modified Wolfe’s mineral solution) was used for the reduction of 5 mM As(V) (Na2HAsO4.7H2O), 10 mM amorphous Fe(III) oxyhydroxide (FeOOH) [25], and anaerobic Fe(II) oxidation of 20 mM FeSO4 ·7H2O+10 mM KNO3 [26]. The oxygen gradient tubes for enumeration of microaerophilic Fe(II) oxidizers were prepared as described by Emerson and Floyd [27]. The reduction and oxidation of Fe in culture media were determined by the ferrozine method [25]. Iron reduction and anaerobic Fe(II) oxidation were also detected by the transformation of FeOOH from orange to dark brown precipitate and the changes of Fe(II)/NO3− medium from green to orange, respectively. The microaerophilic Fe(II) oxidation in FeS gradient tubes was indicated by a visible horizon of cell growth and orange ferric precipitate. The aerobic MLM medium supplemented with 5 mM As(III) (Na3AsO2) and the medium adjusted to pH 6.8–7.0 was used for enumeration of As(III)-oxidizing population. Sulfur reducing and oxidizing bacterial population were enumerated as reported by Sievert et al. [28].

DNA Extraction, 16S rRNA Gene Amplification, and Analysis of Pyrosequencing Data Total genomic DNAs were extracted from groundwater samples using water master DNA extraction kit (Epicenter, USA) following the manufacturer’s instruction. Bacterial 16S rRNA genes were amplified by a primer pair (forward: 5′TA C G G R A G G C A G C A G - 3 ′ a n d r e v e r s e : 5 ′ CCGTCAATTYYTTTRAGTTT-3′), which targeted the hypervariable regions V3–V5 [29]. To assess primer bias, four additional primers were used to amplify 16S rRNA genes for all three samples (three additional primers for the Yenshui-3 sample) (supplementary Table S1). The 25 μL PCR product contained 50 ng of genomic DNA, 22 μL SuperTherm Gold Master Mix (BIONOVAS Biotechnology Co., USA), 1 μL forward primer (10 uM), and 1 μL reverse primer (10 uM). PCR reactions were carried out using the protocols: an initial denaturation at 95 °C for 5 min followed by 25 cycles of denaturation (95 °C, 1 min), annealing (50 °C, 1 min) and extension (72 °C, 1 min), and a final elongation of 10 min at 72 °C. DNA libraries from different locations were labeled with different barcodes and further amplified according to Roche 454 em-PCR amplification manual–Lib L (454 Life Sciences, USA). Pyrosequencing was done on the GS Junior system at National Cheng Kung University, Taiwan, following standard protocols. Because samples were pooled for sequencing, we first split the reads by barcode and removed the barcode sequences using SFF tools (v2.7) (commands: sfffile and sffinfo). Only reads longer than 200 bp were retained for taxonomy assignments. RDP Classifier (v2.5, with default parameters) was used to assign taxonomy to each read. An assigned taxonomy was considered uncertain if the confidence score was less than 0.8. Results of the primer targeting V3–V5 region were used for calculating bacterial compositions. The percentage of a taxonomy was calculated as the ratio in number of the associated confident reads to total reads in a sample. Data Accessibility The pyrosequencing-derived 16S-rRNA gene sequences have been deposited in the GenBank short-read archive under accession number SRP044066.

Results Physiochemical Properties of Groundwater Physicochemical parameters of groundwater collected from Yenshui-3, Budai-Shinwen, and Budai-4 sites were shown in

S. Das et al. Table 1 Physicochemical parameters of collected groundwater samples

Parameters

Well location Yenshui 3

Latitude/longitude

Budai-Shinwen

Budai 4 N 23° 19′ 37.8″/

N 23° 18′ 6.7″/

N 23° 20′ 22″/

E 120° 15′ 11.1″

E 120° 7′ 57.9″

E 120° 9′ 3.2″

Depth (m)

23

313

300

Temperature (°C) ORP (mV) DO (mg L−1)

24.7±1.26 −158±13 0.4±0.2

23.7±1.52 −140±8 0.5±0.3

23.9±1.52 −100±11 0.8±0.2

pH EC (μS cm−1) TDS (mg L−1)a Salinity (%) Alkalinity (mg L−1) As(III) (μg L−1) As(V) (μg L−1) Fe (μg L−1) Mn (μg L−1) SO4 (mg L−1) NO3 (mg L−1) Cl (mg L−1) NH4 (mg L−1) Na (mg L−1)

7.92±0.82 1564±113 751±12.6

7.63±0.46 1154±116 554±7.22

7.34±0.52 355±21 1704±17.06

0.6±0.004 670±12.8 954.07±57.84 177.08±25.31 139. 8±3.22 122.6±13.8 86.8±2.56 3.96±0.55 76.8±2.56 4.45±1.26 193±8.6

0.4±0.002 700±8.6 604.48±198.68 100.25±3.26 76.8±1.16 96.8±1.16 287±6.2 3.51±0.26 171±6.2 2.23±0.8 421±12.8

1.8±0.008 860±18.2 24.51±1.33 285.29±29.36 48.6±1.47 63.4±2.28 3.36±0.28 BDL 70.2±1.2 BDL 222.61±10.6

20.02±0.86 77.2±0.96 101.8±3.01 2.7±0.88

14.56±0.53 24.81±6.56 34.82±4.8 1.5±0.67

31.50±2.6 12.78±0.56 16.20±2.2 0.8±0.26

K (mg L−1) Mg (mg L−1) Ca (mg L−1) TOC (mg L−1)

Table 1. The dissolved As in groundwaters from Yenshui-3 and Budai-Shinwen was predominantly As(III), whereas it was As(V) from Budai-4. The As concentrations in groundwater were much above both World Health Organization (WHO) standard and Taiwan drinking water standard (TDWS) of 10 μg L−1. The groundwaters were near neutral to mildly alkaline and reduced as evidenced from the pH and ORP values. In addition, the DO concentrations of less than 1 mg L−1 in the groundwater indicate that the groundwater collected at depths of 23, 313, and 300 m from Yenshui 3, Budai-Shinwen, and Budai 4, respectively, were mostly anoxic [30]. The groundwaters were rich in Fe and Mn but poor in TOC concentrations. The sulfate concentration of groundwater from BudaiShinwen (287 mg L−1) was much higher compared to those of Yenshui-3 (86.8 mg L − 1 ) and Budai-4 (3.36 mg L−1). The mineral saturation indices of groundwater calculated from PHREEQC revealed there may be abundances of Fe- and Mn-bearing oxides and carbonate minerals in the groundwater. The minerals siderite, magnetite, maghemite, hematite, goethite, and rhodochrosite may be in precipitated form, whereas scorodite,

melanterite, hausmannite, bixbyte, manganite, pyrolusite, and birnessite may be in dissolved form (Fig. 1).

Fig. 1 Saturation indices of certain mineral phase in groundwater of the three study sites


Influences of Electron Donors, Acceptors, and SRB Inhibitors on As(V) Reduction Addition of lactate to As(V)-amended groundwater stimulated DAsR with most rapid reduction observed after 50 h compared with lactate devoid controls, whereas addition of acetate had negligible effects on DAsR (Fig. 2). Succinate had similar effects as acetate on DAsR (results not shown). The increase in rate of DAsR with increase in As(V) addition was observed in with-lactate and without-lactate groundwater from all the three sites. However, the rate of increase was higher in Yenshui-3 (10.76 μM h − 1 for without-lactate and 20.39 μM h−1 for with-lactate in 5 mM As(V)-amended groundwater) than in Budai-Shinwen (9.69 μM h−1 for without-lactate and 14.77 μM h−1 for with-lactate in 5 mM As(V)-amended groundwater) and Budai-4 (5.72 μM h−1 for without-lactate and 8.10 μM h−1 for with-lactate in 5 mM As(V)-amended groundwater).

Fig. 2 Reduction of As(V) in groundwater samples amended with 5 mM As(V) (open symbols) and 1 mM As(V) (solid symbols) as influenced by different electron donors. The mean of three replicate values was plotted, bars/half-bars indicate the standard deviation. a Yenshui-3; b BudaiShinwen; c Budai-4

Addition of Fe(III)-NTA to 1 mM As(III)-amended groundwater noticeably increased As(III) oxidation in live as well as sterilized samples (Fig. 3). The As(V) concentration in live groundwater samples steadily increased and reached 0.33, 30, and 19 mM at 200 h in Yenshui-3, Budai-Shinwen, and Budai-4, respectively, followed by a decline. At the end of the incubation (500 h), 0.16, 19, and 17 mM As(V) in live samples of Yenshui-3, Budai-Shinwen, and Budai-4, respectively, were recorded. In case of abiotic (sterilized) samples, As(V) concentration gradually increased up to 400 h followed by a slight decline. However, the initial As(V) production in live samples exceeded the abiotic As(III) oxidation. The abiotic oxidation of As(III) by Mn(IV) was immediately apparent as evidenced by the occurrence of 186, 172, and 154 μM As(V) in groundwaters of Yenshui-3, Budai-Shinwen, and Budai-4, respectively, at the start of the experiment (Fig. 3) and the continuous increase in As(V) concentration in sterile samples followed by a slight decline at the end of the incubation. In contrast, a sharp decline in As(V) concentration was observed in live samples. Addition of 25 mM tungstate achieved 7.4 and 28.2 % inhibition of DAsR in groundwaters of Yinshui-3

Fig. 3 Oxidation of 1 mM added As(III) in groundwater samples. The mean of three replicate values was plotted, bars/half-bars indicate the standard deviation. a Yenshui-3; b Budai-Shinwen; c Budai-4

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and Budai-Shinwen, respectively, whereas the inhibition was negligible (0.83 %) in case of Budai-4 (Fig. 4)

groundwater of Budai-Shinwen compared to Yenshui-3 and Budai-4, respectively.

In Situ Microbial Activities

Bacterial Diversity

In situ metabolic functions in As-rich groundwater expressed as MPN cell numbers were presented in Table 2. Arsenatereducing bacterial populations in the groundwater of Yenshui3 were 2.8 and 13.0 orders of magnitude more abundant than that of Budai-Shinwen and Budai-4, respectively, whereas As(III)-oxidizer were dominated in the groundwater of Budai-4. Iron-reducing bacterial populations were 2.0 and 14.0 orders of magnitude more abundant in the groundwater of Yenshui-3 compared to Budai-Shinwen and Budai-4, respectively. Microaerophilic Fe(II)-oxidation dominated over anaerobic Fe(II)-oxidation and the highest and lowest values were recorded at Yenshui-3 and Budai-4, respectively. The population of sulfate-reducing bacteria (SRB) in groundwater of Budai-Shinwen was 5.0- and 17.0-folds higher than the SRB population in the groundwaters of Yenshui-3 and Budai-4, respectively. Sulfate-oxidizing bacteria populations were 1.5 and 3.4 orders of magnitude more abundant in the

Bacterial 16S rRNA genes were extracted by a primer targeting the hypervariable regions V3–V5, followed by 454 sequencing. A total of 2374 16S rRNA reads (≥200 bp) were obtained from As-rich groundwater (supplementary Table S2). Read classification revealed nine different phyla, 20 classes, 16 orders, 23 families, and 15 genera (supplementary Tables S3) with more than one confidently classified read in at least one of the samples. At the phylum level, Firmicutes was the most abundant (42.2 %) in highly As-enriched groundwater of Yenshui-3, whereas Proteobacteria was the most abundant in comparatively less As-enriched groundwaters of Budai-Shinwen and Budai-4, comprising 94.0 and 47.2 % of the bacteria, respectively (Fig. 5). In the highly As-rich groundwater of Yenshui-3, Bacilli were the most dominant class within Firmicutes. In the groundwater of BudaiShinwen, most Proteobacteria were Gammaproteobacteria. In contrast, both Betaproteobacteria and Gammaproteobacteria constituted a significant portion of Proteobacteria in the groundwater of Budai-4. At the genus level, Bacillus was the most abundant in the groundwater of Yenshui-3. In the groundwaters of Budai-Shinwen and Budai4, Acinetobacter was the most abundant genus. Notably, at the genus level, the taxonomy of 66, 6, and 69 % of reads were uncertain in the groundwaters of Yenshui-3, Budai-Shinwen, and Budai-4, respectively. To assess whether the bacterial compositions based on one single primer could be biased, we repeated the above procedure with four additional primers. At the phylum level, the results of all six primers were consistent for all three groundwater samples (supplementary Figure S1–S3) except that greater percentages of reads from primer E were not classified with confidence. In the Budai-Shinwen and Budai-4 samples, the results of primer D and F started to deviate from those of other primers at the class level. Specifically, primers D and F captured more Betaproteobacteria sequences than other primers. Despite these differences, the above statements remained valid to the genus level (additional details are provided in supplementary information).

Discussion

Fig. 4 Reduction of 1 mM added As(V) in groundwater samples. The mean of three replicate values plotted, bars/half-bars indicate the standard deviation. a Yenshui-3; b Budai-Shinwen; c Budai-4

Dissimilatory reduction of As has been recognized as an important process for As mobilization and enrichment within As-rich aquifers [5]. In the groundwater from the Chianan plain, we evaluated the poorly understood processes of DAsR and in situ microbial activities and diversity influencing the As enrichment.

Dissimilatory Arsenate Reduction and In Situ Microbial Activities Table 2

MPN estimates of cells in As-rich groundwater determined according to the metabolic functions Yenshui-3

As(V)-red.

Budai-Shinwen

Budai-4

MPN (cells mL−1)

≥95 % CI

≥99 % CI

MPN (cells mL−1)

≥95 % CI

≥99 % CI

MPN (cells mL−1)

≥95 % CI

≥99 % CI

22.0×105

7.0–44.0

4.0–57.0

7.9×105

2.3–22.0

1.5–27.0

17.0×104

6.0–39.0

4.0–51.0

0.4–4.4 2.1–28.0 0.7–9.4 0.20–2.8 4.0–51.0 0.04–2.1

2

0.6–3.4 1.4–11.3 0.7–4.1 1.4–11.3 3.4–22.0 0.19–1.7

0.4–4.4 0.9–14.7 0.5–5.3 0.9–14.7 2.1–27.0 0.10–2.3

0.93×103 6.3×103 1.4×102 3.3×102 4.9×102 0.20×102

0.34–2.2 2.1–14.9 0.6–3.4 1.0–10.0 1.5–14.9 0.02–0.99

0.20–2.8 1.4–20.0 0.4–4.4 0.7–14.7 0.9–20.0 0.01–1.4

2

As(III)-ox.

1.7×10

FeOOH-red. An-Fe(II)-ox. FeS ox. Sulfur-red. Sulfur-ox.

9.4×104 2.7×102 0.93×103 17.0×102 0.45×102

0.6–3.4 3.4–22.0 1.0–6.6 0.34–2.2 6.0–39.0 0.08–1.4

1.3×10 4.7×104 2.1×102 4.7×102 8.4×103 0.68×102

CI confidence interval

Undetectable changes in As(V) reduction in filter-sterilized controls (Fig. 2) strongly indicated that As(V) reduction in the groundwater of Chianan plain is biological. It also rests on the observation that prominent stimulation of DAsR occurred in the presence of electron donor (i.e., lactate) during incubation of the groundwater samples (Fig. 2). In addition, a degree of

Fig. 5 Bacterial compositions in the three groundwater samples at phylum, class, order, family, and genus level. The relative abundance is presented in terms of percentage in total bacterial sequences per sample. a Yenshui3; b Budai-Shinwen; c Budai-4

substrate specificity was observed as evidenced by As(V) reduction with lactate but not with acetate and succinate (Fig. 2). This also implied that the type of the electron donor was important to promote DAsR in the groundwater. It is noteworthy that, the energy yield of lactate oxidation by As(V) is more than that for acetate and succinate [3, 31]. For this reason,

S. Das et al.

though bacteria can use these electron donors for As(V) reduction, lactate is expected to enhance the rate of As(V) reduction. Although lactate can stimulate DAsR in the Chianan plain groundwater, a rapid stimulation of DAsR was favored by more reducing nature of the groundwater. This was evidenced by the higher rate of reaction for DAsR in the groundwater of Yenshui-3, which is comparatively more reduced than Budai-Shinwen and Budai-4. The high DAsR rate along with 100–285 μg L−1 As(V) in the groundwater further suggests that biological or chemical phenomenon must also exist to resupply As(V). The biological means of As(III) oxidation, in which oxygen acts as electron acceptor, could occur prevalently in suboxic water [2]. But in reduced groundwater with low organic carbon, other available oxidants (i.e., Mn and Fe) would play a vital role in the regeneration of As(V). Given the abundance of Mn and Fe in the groundwater of Chianan plain, they could have a role in the oxidation of As(III) to As(V) in the reduced groundwater and indeed we observed abiotic oxidation of As(III) by Mn (Fig. 3). This was evidenced by the generation of 154 to 186 μM As(V) when the experiment began and the continuous increase of As(V) concentration in sterilized samples (Fig. 3). The efficient abiotic oxidation of As(III) with Mn(IV) has been reported in the aquatic environment [32–34]. In contrast, the rapid decrease in As(V) concentration during incubation in live samples indicated its removal by dissimilatory As(V) reduction. In case of Fe(III), a potential electron acceptor for As(III) oxidation, we detected As(V) production in both live and sterile samples (Fig. 3). However, As(V) concentration never exceeded 0.33 mM in the presence of Fe(III), indicating adsorption of As(V) to the precipitated Fe(III). The rapid decline in As(V) during incubation in live samples would be attributed to dissimilatory reduction at the expense of other available electron donors. In addition to the regeneration of As(V) in the groundwater, a likely source of As(V) is from dissolution of As(V)-saturated iron minerals or As-bearing minerals by indigenous dissimilatory Fe(III)-reducing bacteria. To support this view, saturation states of Mn- and Fe-bearing oxides and carbonates (Fig. 1), to which As can be adsorbed, were predicted from the groundwater. In addition, some genera (Acinetobacter and Bacillus) that can achieve dissimilatory Mn(IV)- and Fe(III)reduction [35–40] were dominated in the groundwater (Fig. 5). The increase in rate of DAsR with the increase in As(V) concentration (Fig. 2) further suggested that DAsR activity in the groundwater of Chianan plain was limited by the availability of As(V). Attributing DAsR to the most prominent microbial activity in the groundwater of Chianan plain raises a question of whether it was by As(V) reducers or sulfate reducers. The ability of sulfate-reducing bacteria from diverse phylogenies to achieve growth via DAsR [3, 41] urges its importance in DAsR in sulfate-rich anoxic groundwater. In our experiment, we had disrupted the activity of sulfate-reducing bacteria in

the groundwater by adding tungstate, a potential SRB inhibitor [3, 7, 34]. Addition of this SRB inhibitor to the inhibition of DAsR was negligible in the groundwater of Budai-4, whereas it inhibited 7.4 and 28.2 % of DAsR in the groundwaters of Yinshui-3 and Budai-Shinwen, respectively (Fig. 4). This indicates that sulfate-respiring bacteria have negligible contribution or a substantial contribution of the observed DAsR activity depending upon sulfate concentration and redox status of the groundwater [26]. To further support this point, the population density of sulfate-reducing bacteria (SRB) in the groundwater of Budai-Shinwen was 3.2-folds and 4.6-folds higher than the SRB population in the groundwaters of Yenshui-3 and Budai-4, respectively. Interestingly, the population of As-respiring bacteria (AsRB) outnumbered SRB (Table 2), indicating a major role played by AsRB in DAsR in the groundwater of Chianan plain. Arsenic (As) enrichment has frequently been implicated in altering microbial activities and diversity [9]. In the study, we found dominance of Firmicutes in highly As-rich groundwater of Yenshu-3, whereas Proteobacteria were the dominant phylum in comparatively less As-rich groundwaters of BudaiShinwen and Budai-4 (Fig. 5). It is possible that the endospore-forming capability of Firmicutes enabled them to survive under unfavorable (As-contaminated) conditions. Bacterial populations in As-rich groundwater were characterized not only by their potential abilities of As reduction, oxidation, and resistance but also by their potential abilities for mineral weathering, chemolithotrophic or chemolithoautotrophic mode of metabolism, and potential to grow as heterotrophically utilizing multiple electron donors and acceptors [5, 42–44]. Members of the genera Acinetobacter, Herbaspirillum, Bacillus, Nitrospira, Arthrobacter, and Sphingomonas detected in the As-rich groundwater (Fig. 5, supplementary Table S3) are well known for their chemolithoautotrophic metabolism [5, 10]. Although often isolated as heterotrophs, these bacteria can grow under aerobic or anaerobic oligotrophic environments using energy and reducing power from oxidation of various inorganic elements during CO2 fixation or other anaerobic reactions [5, 10, 45]. Several bacterial strains of these genera have been reported in As-contaminated environments [5, 9, 10, 19, 44]; most of them exhibit weathering activity, which in turn influences As mobility [43]. The genera Acinetobacter and Bacillus dominated in the groundwater have been reported as potential dissimilatory As reducers in As-contaminated environments [9, 10, 46]. In addition, bacterial genera like Acinetobacter, Herbaspirillum, Bacillus, and Massilia isolated from groundwater were reported to resist elevated concentration of As [44]. Metabolic robustness of these genera to withstand elevated concentration of As and to grow aerobically and anaerobically utilizing diverse electron donors and acceptors signified their potential in As biogeochemical cycling [44]. Enumerations of different microbial activities in As-rich


groundwater were achieved by MPN methods. Although MPNs are subjected to media biases and may underestimate population density, the results revealed that along with microbial As(V) reduction, a cascade of microbial processes (mainly iron reduction and oxidation, sulfate reduction and oxidation, and As(III) oxidation) also occurred in the groundwater of Chianan plain that mostly depends on the physicochemical parameters of the groundwater. These processes could influence biogeochemical cycling of As therein. It is not necessary that these processes should be mutually exclusive but may incorporate each other.

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15. Acknowledgments This work was supported by the National Science Council of Taiwan (NSC 100-2116-M-006-009). We thank the Molecular Medicine Core Lab, Research Center of Clinical Medicine, National Cheng Kung University Hospital, for providing services in 454 GS Junior Next Generation Sequencing.

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