Effect of iron redox transformations on arsenic solid-phase associations in an arsenic-rich, ferruginous hydrothermal sediment

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Geochimica et Cosmochimica Acta 102 (2013) 124–142 www.elsevier.com/locate/gca

Effect of iron redox transformations on arsenic solid-phase associations in an arsenic-rich, ferruginous hydrothermal sediment Kim M. Handley 1, Joyce M. McBeth, John M. Charnock, David J. Vaughan, Paul L. Wincott, David A. Polya, Jonathan R. Lloyd ⇑ Williamson Research Centre for Molecular Environmental Science, and School of Earth, Atmospheric and Environmental Sciences, The University of Manchester, Manchester M13 9PL, UK Received 17 July 2012; accepted in revised form 15 October 2012; available online 23 October 2012

Abstract Well-constrained laboratory incubations of a ferruginous marine hydrothermal sediment from Santorini, Greece, were used to elucidate the effect of microbially induced redox transformations on arsenic speciation and mobility. Despite naturally high arsenic concentrations (400 mg/kg), the sediment has a low As:Fe ratio (1:1000 wt/wt). Acetate-amendment of sediment, extracted from the naturally-occurring suboxic–anoxic (Eh 60 to 138 mV) transition zone, promoted Fe(III) reduction, and increased the concentration of Fe(II) from 40% to 60% in the bulk sediment. Sulfate, which was present at lower concentrations, was also reduced. Phylogenetic 16S rRNA and dsr gene analysis suggested that Fe(III) and sulfate were reduced by bacteria related to Malonomonas rubra and Desulfosarcina variabilis, respectively. Arsenic remained predominantly as arsenic trioxide (As2O3) throughout the amendment experiment. However, the percentage of total arsenic present within poorly-crystalline iron oxides decreased from 69% to 32%, while the percentage incorporated within crystalline iron-containing minerals or sorbed to surfaces via inner-sphere complexes increased significantly (to 22% and 30%, respectively). Re-oxidation of the system with nitrate resulted in incomplete reduction of the nitrate pool, and partial re-association of arsenic with the poorly-crystalline iron fraction. Exposure to air led to virtually complete reversal of the arsenic partitioning, and oxidation of 71% As(III) to As(V). During aeration, oxidation of sediment-bound sulfur/sulfide occurred, alongside an observed 63% decrease in arsenic bound to this minor component. Analogous trends in arsenic-sediment associations were observed in the natural, unamended sediment depth-profile, whereby a greater proportion of arsenic (34% As(III), 66% As(V)) was bound within poorly-crystalline iron oxides at the sediment–water interface. Arsenic (96% As(III)) was increasingly incorporated within well-crystallized forms of iron with depth and decreasing Eh values. At the greatest depth sampled (35 cm) arsenic increased substantially within the sulfide/organic fraction. Results here contribute to existing evidence that arsenic is not necessarily released from iron-rich sediment systems under conditions of anoxia, but that Fe(II)-bearing minerals forming concomitantly can immobilize arsenic in the solid-phase. Such results may have implications for other systems with high Fe:As ratios. Ó 2012 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Address: School of Earth, Atmospheric and Environmental Sciences, University of Manchester, Williamson

Building, Oxford Road, Manchester M13 9PL, UK. Tel.: +44 161 275 7155; fax: +44 161 306 9361. E-mail addresses: [email protected] (K.M. Handley), [email protected] (J.R. Lloyd). 1 Present addresses: Computation Institute, University of Chicago, Searle Chemistry Laboratory, 5735 South Ellis Avenue, Chicago, IL 60637, USA; Computing, Environment and Life Sciences, Argonne National Laboratory, 9700 South Cass Avenue, Lemont, IL 60439, USA. Tel.: +1 630 252 2873; fax: +1 773 834 6818. 0016-7037/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.gca.2012.10.024

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1. INTRODUCTION Arsenic occurs naturally at elevated levels in a range of environments, including aquifer groundwater and sediment (e.g. Polya et al., 2005; also see reviews by Smedley and Kinniburgh, 2002; Welch et al., 2006), lake sediments (e.g. Belzile and Tessier, 1990), terrestrial geothermal or geothermally affected systems (Anderson and Bruland, 1991; Kneebone and Hering, 2000; Planer-Friedrich et al., 2007) and marine hydrothermal sediments (e.g. Pichler and Veizer, 1999; Schaller et al., 2000; Rancourt et al., 2001; McCarthy et al., 2005), as well as in systems involving anthropogenic contamination (e.g. Sa´nchez-Rodas et al., 2005; Wilkin and Ford, 2006). The predominant forms of arsenic in the environment are inorganic arsenate, As(V), and arsenite, As(III), and, to a lesser extent, organic methylated arsenic (Smedley and Kinniburgh, 2002; Vaughan, 2006). In general, As(III) is considered to be more mobile in sediments than As(V); however, As(III) also has the potential to sorb effectively onto hydrous ferric oxides (HFO), and sorption can be similar to, or greater than, that of As(V) at circum-neutral pH, and even greater at more alkaline pH levels (Manning et al., 1998; Raven et al., 1998; Goldberg and Johnston, 2001; Bostick and Fendorf, 2003). The range of microbially and abiotically mediated processes affecting arsenic retention or release from sediments is complex (Smedley and Kinniburgh, 2002; Welch et al., 2006). Both field and laboratory studies to date have indicated that the processes relating to microbially-mediated and inorganic arsenic capture and release are highly dependent upon a number of factors, such as: Eh, pH, arsenic speciation, and host mineral crystallinity, surface area or concentration (Moore et al., 1988; Roden and Zachara, 1996; Raven et al., 1998; Farquhar et al., 2002; Gencß-Fuhrman et al., 2004; Richmond et al., 2004; Islam et al., 2005b; Burnol et al., 2007). In particular, the nature of As(V) and As(III) co-precipitation/sorption response is heterogeneous over a range of iron and sulfide mineralogies (e.g. Farquhar et al., 2002; Bostick and Fendorf, 2003; O’Day et al., 2004; Islam et al., 2005b). The mechanisms of the binding of arsenic to iron minerals and of its release have received a great deal of attention, owing to the prevalence of HFO in the natural environment and their strong affinity and uptake capacity for arsenic (Ferguson and Anderson, 1974; Fuller et al., 1993; Raven et al., 1998). In circumneutral oxidizing environments, where HFO are stable, they provide an important sink for arsenic immobilisation (e.g. Price and Pichler, 2005). Conversely, under reducing conditions the microbial respiration and dissolution of Fe(III) to aqueous Fe(II) can liberate arsenic from sediments, or more specifically from iron minerals (e.g. Cummings et al., 1999; Islam et al., 2004). Numerous studies have also demonstrated that ecologically and phylogenetically diverse bacteria and archaea are capable of enzymatically transforming arsenic oxyanions from As(V) to As(III) and vice versa (reviewed by Oremland and Stolz, 2003; Lloyd and Oremland, 2006; Stolz et al., 2006). Arsenate-respiring bacteria are also capable of promoting the release of arsenic as As(III) sorbed to sediment

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even in the absence of host mineral dissolution (Ahmann et al., 1997; Zobrist et al., 2000). Nonetheless, oxidation state alone cannot be relied upon to determine arsenic mobility or retention. The idealized trend of arsenic capture and release can also operate in reverse; for example, via the oxidative dissolution of arsenicbearing sulfide minerals, which can be responsible for arsenic discharge from sediments (e.g. Nickson et al., 2000). Alternatively, the transition between oxidizing and reducing sediments can be accompanied by a solid-phase change in the minerals carrying arsenic. Accordingly, arsenic may be adsorbed onto, or co-precipitated with, a variety of Fe(II)bearing minerals or sulfides where conditions become reducing (e.g. Bostick and Fendorf, 2003; Kirk et al., 2004; O’Day et al., 2004; Islam et al., 2005a,b; Wolthers et al., 2005; Kocar et al., 2006; Root et al., 2007). Further to this, the trend of arsenic solubility with depth in sediments can also be uncoupled from distinct redox boundaries owing to ongoing changes in reduced host minerals (Root et al., 2007). Previous investigations of hydrothermal sediments from Nea Kameni Island at Santorini have identified abundant HFO, and sediment that was depleted in levels of most trace metals typical of hydrothermal systems (e.g. Mn, Cu, Ni, Zn, V), but highly enriched in arsenic (Bostrom and Widenfalk, 1984; Varnavas and Cronan, 1988; Cronan et al., 2000). The sediment underlies shallow (<1 m deep) circum-neutral pH marine water, warmed by rising hydrothermal fluids (maximum reported temperature, 40 °C; Bostrom and Widenfalk, 1984). Analyses of this sediment have revealed that it is biogeochemically stratified, and host to a diverse bacterial community capable of redox cycling of iron, nitrogen, sulfur and arsenic species (Holm, 1987; Handley et al., 2010). In a similar ferruginous environment (Tutum Bay, Papua New Guinea) with high arsenic concentrations, up to 6% (Fe:As  6), arsenic was found to be almost entirely captured within HFO (Pichler and Veizer, 1999). Examination of arsenic partitioning in the surface layer of the Tutum Bay sediment showed that arsenic was predominantly (>98%) associated with an oxalate-extractable poorly-crystalline iron oxide fraction, with comparably minor quantities of arsenic present in carbonates, as easily exchangeable outer-sphere complexes, or in residual phases (Price and Pichler, 2005). The effect of microbial respiration on arsenic partitioning within either of these sediments has not previously been studied, although it is anticipated to have a significant effect on iron mineralogy and hence the nature of arsenic-iron associations. In the present study, laboratory experiments were undertaken in order to investigate the effect of anaerobic microbial respiration on arsenic-cycling in iron-rich hydrothermal sediment from the Nea Kameni site, Santorini, and also the impact of re-oxidation on arsenic speciation. The arsenic- and iron-rich nature of the sediment made it possible to use techniques, such as XANES and EXAFS, to study arsenic speciation and coordination environments, which is not possible with lower arsenic concentrations more typically found in sediments. The fate of arsenic under reducing conditions was determined by stimulating Fe(III) and sulfate respiration in the indigenous microbial

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community. Re-oxidizing conditions were stimulated by the addition of nitrate or air in order to examine the stability or mobility of arsenic associated with reduced host minerals. For comparison, the depth-profile of arsenic species and mineral associations in the natural sediment was also determined. Results contribute to knowledge of arsenic behavior in sediments with low As-to-Fe ratios. 2. MATERIALS AND EXPERIMENTAL METHODS 2.1. Field site description and sample material Sampling was conducted in a shallow marine embayment on the northwest side of Nea Kameni island (see Bostrom and Widenfalk, 1984; Varnavas and Cronan, 2005), situated within the flooded caldera of Santorini, on the Hellenic Volcanic Arc. Active hydrothermal venting occurs in the embayment, depositing iron-rich (44–52%) and arsenic-rich (349–424 mg/kg) sediments that have an As:Fe ratio of approximately 1:1000 wt/wt (Handley et al., 2010). The temperature range is 20–40 °C (Bostrom and Widenfalk, 1984). Detailed descriptions of the sampling, and of the geochemical and microbiological analyses of the Nea Kameni sediment, are reported elsewhere (Handley et al., 2010, and references therein). After collection, sediment cores were kept sealed to prevent oxidation and stored at 4 °C to limit ongoing microbial activity. Sediment for microcosm experiments was derived from a suboxic–anoxic transition zone (5–20 cm depth, Eh 60 to 138 mV, pH 6.1–6.3). 2.2. Microcosm preparation, treatment and sampling A slurry of sediment representing the suboxic–anoxic transition zone from Nea Kameni (5–20 cm depth) was prepared by a 1:3 w/v dilution in artificial seawater (ASW). The ASW comprised (per liter): NaCl (31.64 g), MgCl2
6H2O (9.08 g), MgSO4
7H2O (10.97 g), KCl (0.87 g), NaHCO3 (0.2 g), CaCl2
2H2O (1.80 g), and was autoclaved and then sparged with filter-sterilized N2 prior to combining with the sediment. The slurry was aliquoted (under a stream of N2) into sterile, N2-purged glass serum bottles. Aliquots were sparged with N2 with 3.15% CO2 to maintain the original pH value of the sediment (pH 6.10–6.30), and bottles were sealed with butyl rubber stoppers. All glassware used was first washed in 2 M HNO3 and rinsed with ultra-clean deionized water. Microcosms were initially supplemented with 50 mM acetate as a source of organic carbon and electrons for microbial respiration, and incubated for 111 days. Parallel controls either contained acetate and were heat-sterilized, or contained no added acetate and were not heat-sterilized. After 74 days, a number of the acetate-treated microcosms were incubated for a further 37 days under oxidizing conditions. Oxidation was achieved by either: (1) adding 50 mM KNO3 (± heat-sterilization) or, (2) placing sediment into sterile Erlenmeyer Flasks (10% w/v slurry/flask), sealed with foam plugs permeable to air, and incubating in an orbital shaker at 150 rpm. The effect of evaporation on the concentrations of slurry exposed to air was determined by weight, and geochemical analyses were corrected for

evaporation occurring between each sampling period (approx. 0.6%). All treatments, excluding heat-sterilized controls, were conducted in triplicate. Microcosms were incubated in the dark at 25 °C (the temperature of the original sediment at time of collection). Samples were taken from anaerobic vessels using sterile hypodermic syringes and were replaced by equal volumes of filtered N2–CO2. Manipulations of anaerobic samples were conducted in an anaerobic cabinet. Sampling from aerobic incubations was conducted within a laminar flow cabinet to prevent contamination. Samples for aqueous phase analyses were separated by centrifugation and filtration using MillexÒ GP 0.45 lm MF or sterile 0.22 lm PES Express membrane filters (Millipore, Bedford, MA, USA). Filtrates and uncentrifuged slurry for analysis of anions/organic acids, DNA or solid-phase geochemistry/mineralogy were stored with a N2 headspace at 80 °C. Filtrates for total element analysis were acidified with 2% (v/v) HNO3 (final concentration). Slurry aliquots for the determination of reactive HCl-soluble iron species were analyzed immediately after sampling. 2.3. Analytical methods 2.3.1. Physicochemical and aqueous analyses Intermittent sampling was undertaken for ongoing analysis of the aqueous phases, including Eh and pH measurements. Total element concentrations were determined with a Perkin–Elmer Optima 5300 Dual View Inductively Coupled Plasma Atomic Emission Spectrometer (ICP-AES; MA, USA). Anions and organic acids were quantified using a Dionex DX600 Ion Chromatograph (IC) fitted with a Dionex AG4A-SC (solvent-compatible) guard column, and a Dionex AS4A-SC (250  4 mm) analytical column. A 25-ll sample loop was used for injection. Analytes were eluted isocratically with 2.1 mM Na2CO3/4.9 mM NaHCO3 at 1.7 mL/min (1700 psi). Background conductance was chemically suppressed using sulfuric acid. 2.3.2. Analysis of reactive Fe(II) and Fe(III) iron Reactive Fe(II) was quantified by ferrozine colorimetric assay after digestion of slurry or sediment in 0.5 M HCl following the method described by Lovley and Phillips (1986b). Reactive, easily reducible Fe(III) was determined after reduction by 0.25 M hydroxylamine hydrochloride (NH2OH
HCl) in 0.5 M HCl (Lovley and Phillips, 1987; Anderson and Lovley, 1999). Spectrophotometric measurements were made at 562 nm. 2.3.3. Solid-phase analyses Sample preparation for solid-phase analyses was carried out within an anaerobic cabinet. The crystalline mineral phases present in microcosm treatments were determined using a Philips 1730 PW X-ray diffractometer (XRD) with CuKa radiation (Eindhoven, The Netherlands). Sediment slurries were dried at room temperature, finely ground with 100% ethanol, placed either onto glass slides or a PTFE holder sealed with Kapton tape, and transported anaerobically for immediate analysis by XRD over 10–70° with steps of 0.01° for 5 s (run time: 1 h 20 min).

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The iron speciation of microcosm sediments and of the natural un-treated Nea Kameni sediment was investigated using in a FAST ComTec Mo¨ssbauer spectrometer (Oberhaching, Germany) with a 50 mCi 57Co/Rh c-ray source. Samples were prepared by sealing anaerobically desiccated sediment between sheets of MylarÒ polyethylene terephthalate (PET) (Goodfellow Cambridge Ltd., Huntingdon, UK) with an epoxy resin, to prevent oxidation during measurement. Mo¨ssbauer spectra were recorded at room temperature (RT) and, in some cases, liquid N2 temperature with a FAST ComTec 1024-multichannel analyser system employing a constant acceleration drive. For line fitting, the Lagarec/Rancourt Recoil (v. 1.0) fitting routine was utilized (Intelligent Scientific Applications Inc., Ottawa, ON, Canada). Spectra were fitted using Lorentzian line shape symmetric doublets. Isomer shift data were calibrated with reference to the spectrum of metallic iron foil recorded at room temperature (RT). The absorber thickness was <4 mg iron/cm2. Arsenic speciation and coordination environment were determined at the arsenic K-edge using X-ray absorption near edge structure (XANES) and X-ray absorption fine structure (EXAFS) spectroscopies, respectively, on the ultra-dilute XAS spectroscopy beamline (16.5) at the CLRC Daresbury Synchrotron Radiation Source Laboratory (UK). The Synchrotron Radiation Source (SRS) operated at 2 GeV with an average current of 150 mA. Data were collected using a Si(220) double-crystal monochromator, with harmonic contamination minimized by using a vertically focusing mirror and detuning to 70% of the maximum beam intensity. Samples of slurry or natural Nea Kameni sediment were prepared anaerobically, and placed within aluminum holders that were masked on either side with Kapton tape. Immediately prior to analysis at liquid nitrogen temperature, samples were removed from the anaerobic cabinet and frozen in liquid nitrogen. XANES and EXAFS data for all samples, and the 0.05 M As-glutathione standard solution were collected in fluorescence mode, using a Canberra 30-element solid state detector. The As-glutathione consisted 1:3 of NaAsO2 (GPR, BDH, Poole, UK) and L-glutathione reduced (Sigma, Steinheim, Germany). The sodium As(III) (NaAsO2, GPR; BDH), disodium As(V) heptahydrate (Na2HAsO4
7H2O, Analar; BDH), arsenopyrite (FeAsS; Panasqueira, Portugal, Polya et al., 2000), orpiment (As2S3; British Museum, BM1975,479), and realgar (As2S2; University of Manchester Harwood Collection, 921) standards were analyzed in transmission mode. Treatment of the EXAFS and XANES spectra followed the methods described by Coker et al. (2006). Additionally, in order to determine the proportions of the forms of arsenic present, the background subtracted XANES spectra were modeled as linear combinations of the standard spectra (Salt et al., 1999), using the STFC Daresbury Laboratory program LINCOM, and the relative proportion of each standard was refined so that a least squares residual was minimized.

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2.4. Sequential extractions A six-stage sequential extraction procedure was used that targeted a range of arsenic and iron phases in both the microcosm samples and the different depth samples of the un-treated Nea Kameni sediment, following the methods of Wenzel et al. (2001) and Gault et al. (2003). Extractions were performed in 15 or 50 mL Falcon tubes (Fisherbrand), and reagents were reacted with microcosm samples at 20 °C and shaking at 200 rpm, unless indicated otherwise. Extracts were removed after each stage (1–5) by centrifugation at 3000 rpm for 15 min, and were analyzed by ICP-AES. Aliquots of 1 mL slurry or 1 g sediment (9.09% v/v or v/w) were added to 10 mL of 0.05 M (NH4)2SO4 solution, and reacted for 4 h (20 °C) to remove easily exchangeable arsenic species (1). The residue was subsequently reacted in turn with: (2) 10 mL of 0.05 M NH4H2PO4 for 16 h, for specifically-sorbed surface-bound arsenic; (3) 10 mL of 0.2 M ammonium oxalate buffer (pH 3.0) for poorly-crystalline hydrous oxides of iron (and aluminum), after incubation for 4 h in the dark; (4) 10 mL of 0.2 M ammonium oxalate buffer (pH 3.0) and 0.1 M ascorbic acid, for 30 min (in the light) at 90 °C, targeting the dissolution of well-crystallized hydrous oxides of iron (and aluminum); and (5) 10 mL of 5% (w/v) KClO4/HCl(conc) with intermittent shaking for 45 min, for arsenic associated with sulfides and organics. Sample manipulations for stages 1–4 were performed in an anaerobic cabinet. Final and complete digestion of the recalcitrant residue (e.g. silicates) was achieved in 1.2 mL HF in PFA Savillex vials for 72 h, followed by the addition of 0.4 mL HNO3 and 6 mL H3BO3 (QS to 18 mL). 2.5. Phylogenetic analyses Molecular phylogenetic analyses were carried out as described by Handley et al. (2010). To summarize, genomic DNA was extracted from microcosm slurries and amplified using small subunit 16S rRNA gene primers (8F and 519R) as a general bacterial phylogenetic marker, and dsrAB (dissimilatory sulfite reductase) gene primers (1F and 4R) to target sulfate-reducing bacteria. The dsrAB genes were amplified using the following modification: touchdown PCR run at 95 °C for 5 min; 23 cycles of 94 °C for 30 s, 65 °C (decreasing by 0.5 °C per cycle) for 1 min, and 72 °C for 2 min; 15 cycles of 94 °C for 30 s, 54 °C for 1 min, and 72 °C for 2 min; and extension at 72 °C for 10 min. For each reaction 0.25 ll of HotStar Taq DNA polymerase was used (Qiagen, Crawley, USA). Amplicons were differentiated by cloning (using a StrataClonee PCR Cloning Kit, Agilent Technologies, TX, USA) and Restriction Fragment Length Polymorphism (RFLP) methods, and representative clone inserts were sequenced on an Applied Biosystems 3730 DNA Analyzer. Phylogenetic affiliations of the nucleotide sequences were determined using BLAST analysis (Altschul et al., 1990).

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3. RESULTS 3.1. Changes in aqueous chemistry and reactive HCl-soluble iron 3.1.1. Acetate-stimulated sediment Analysis of HCl/HCl–NH2OH
HCl slurry digests demonstrated that acetate addition stimulated Fe(III) reduction, and later also, sulfate reduction. Within the first month of incubation, the initial dull orange-brown of the sediment changed to a dark brown color. Reactive HCl-soluble Fe(II) increased 2.4-fold, from 13.5 ± 3.9 g/L in the sediment slurry to 32.6 ± 2.1 g/L in 37 days, and fluctuated between 28.2 ± 2.5 g/L and 38.7 ± 2.1 g/L over the remaining 10 weeks (Fig. 1a). Ferric iron from the HCl–NH2OH
HCl reducible fraction was 11.5 ± 0.2 g/L at timezero, and 1.6 ± 6.0 g/L at day 111, suggesting this fraction was essentially reduced to completion. The quantity of initial HCl–NH2OH
HCl reducible Fe(III) (i.e. total easilyreducible ferric iron), however, accounts for only 62% of the HCl-soluble Fe(II) produced during incubation, suggesting there were contributions from the reduction of less reactive iron minerals not extracted using the dilute acid extraction protocol. These observations are in accordance with data for the Fe(III):Fe(II) ratios in the solid-phase as determined by Mo¨ssbauer spectroscopy (see below). Major sulfate reduction (78% sulfate loss from solution) occurred between days 13 and 50, and was largely concomitant with Fe(III) reduction (Fig. 1b). Thereafter, sulfate reduction persisted at a very slow rate, evidently owing to the lack of acetate, which was consumed completely within 43 days (Fig. 1c). The microcosm pH rapidly increased by one unit (Fig. 1d), concomitant with a decrease in redox values (Eh) from 164.0 ± 2.6 mV to an average of 317.7 ± 43.1 mV within 13 days of acetate addition (Fig. 1e). These respective changes in pH and Eh are consistent with changes in solution physico-chemistry induced by microbial respiration of either Fe(III) or sulfate (e.g. Jørgensen, 1977; Thamdrup et al., 2000; Quantin et al., 2001; Zachara et al., 2002; Lloyd et al., 2004). In heat-sterilized controls, a loss of approximately 22% of the acetate present occurred over 111 days; however, no significant change in Fe(II), sulfate, pH or Eh was evident in heat-sterilized controls or controls without acetate. As discussed below, the lack of change in Fe(II) concentration was also seen from the Mo¨ssbauer data for the solid-phase iron component. 3.1.2. Re-oxidation experiments Microcosm supplementation with 50 mM (3100 mg/L) nitrate or exposure to air was used in order to test the response of pre-reduced (acetate-stimulated) systems to the effects of re-oxidation. During nitrate-treatment, sediment changed from dark brown to black in color, and a decrease of 801 ± 86 mg/L (25%) nitrate occurred; however, there were no detectable changes in reactive HCl-soluble Fe(II), sulfate, pH or Eh relative to controls (Fig. 2; sulfate data not shown). Aerobic incubation resulted in the formation of bright rust-colored sediment, and a decrease in reactive HCl-solu-

Fig. 1. Plots of: (a) reactive HCl-soluble Fe(II) accumulation, (b) sulfate and (c) acetate depletion, and (d) pH and (e) Eh values with acetate stimulation of Nea Kameni hydrothermal sediment. Results depict microcosm sediments with acetate (solid squares), without acetate (unfilled squares), and heat-sterilized with acetate (crosses). Error bars represent replicate microcosms. Where error bars are not evident for the microbially viable acetate treated data, their range is smaller than the solid square data point markers.

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ble Fe(II) of 70% from 32.7 ± 4.3 to 9.8 ± 0.96 g/kg within 13 days of aeration, and a 2.5-fold to 24.5 ± 8.1 g/kg for the remaining 23 days (Fig. 3a). No change in HCl–NH2OH
HCl soluble Fe(III) was evident throughout the incubation period (data not shown). Studies of solid-phase iron

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speciation by Mo¨ssbauer spectroscopy, reported in detail below, are again in line with these observations from solution species. Sulfate was also produced throughout the aerobic incubation period, reaching a concentration (5.3 ± 0.6 g/L) be-

Fig. 2. Plots of: (a) reactive HCl-soluble Fe(II) depletion, (b) nitrate depletion, and (c) pH and (d) Eh values with nitrate re-oxidation. Results are shown for microcosm sediments with nitrate (solid squares), without nitrate (unfilled squares), and heat-sterilized with nitrate (crosses). Error bars represent replicate microcosms. Where error bars are not apparent for the microbially viable nitrate-treated data, their range is smaller than the solid square data point markers.

Fig. 3. Plots of: (a) reactive HCl-soluble Fe(II) depletion, (b) sulfate depletion, and (c) pH and (d) Eh values with re-oxidation by aeration. Results are given for microcosm sediments with air (solid squares), without air (unfilled squares), and heat-sterilized with air (crosses). Error bars represent replicate microcosms. Where error bars are not evident for the microbially viable aerated sediment data, their range is smaller than the solid square data point markers.

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yond initial levels present in solution (Fig. 3b). This may be accounted for by the oxidation of sulfur or reduced sulfur species originally present in the solid-phase (4.25–4.67 g/ kg of sediment; Handley et al., 2010), and also the re-oxidation of sulfate products reduced during acetate-amendment. The potential contribution of each fraction would be approximately 57% from experimentally reduced sulfate, and 43% from pre-existing sulfur species in the sediment (based on a dilution estimate of 1:6 solid:solution). The endpoint of sulfate generation was not evident in the experimental timeframe. Sediment pH remained essentially constant throughout incubation with air (Fig. 3c). Sediment slurries became progressively more oxidizing, although conditions were still within the suboxic range after a month of aeration, evidently due to a strong natural buffering capacity (Fig. 3d). Overall, no changes in Eh, pH, nitrate, sulfate or reactive HCl-soluble Fe(II) were evident in control microcosms (sediment without air or heat-sterilized sediment with air), with the exception of Eh and reactive Fe(II) values in heat-sterilized controls, which increased in a similar manner to those in the unsterilized, aerated sediment, suggesting Fe(II) and Eh were abiotically controlled. In contrast, sulfate was only regenerated in unsterilized sediments that were exposed to air, possibly owing to microbial re-oxidation of sulfides, although the possibility that sulfide was altered into a less oxidizable form during autoclaving cannot be excluded. 3.2. Mineralogy and speciation of solid-phase iron and arsenic 3.2.1. Iron speciation and mineralogy Analysis of sediments by XRD gave some evidence for goethite [a-FeO(OH)] and oligonite [manganese-rich siderite, Fe(Mn,Zn)(CO3)2] in all samples. However, manganese contents of 36–104 mg/kg and 0.02–0.27 mg/L (i.e. 60.01% total; Handley et al., 2010) in the original sediment and pore water, respectively, suggest that manganese substitution in any iron carbonate phases was negligible. No differences were apparent amongst the different microcosm treatments based on XRD analyses. In contrast, Mo¨ssbauer spectroscopy (at room temperature) of the time-zero microcosm sample compared with acetate-amended sediment slurries showed marked differences. The room temperature spectra of all samples consist of overlapping doublets arising from Fe(II) and Fe(III) components from which Fe(III):Fe(II) ratios could be determined. Initially, simple fits comprising two overlapping doublets were applied to the spectra in order to obtain consistent data on the iron redox state (see Fig.6). These fits show Fe(III):Fe(II) in the time-zero sediment slurry with values of 59:41. After 37 days incubation with acetate, the concentration of Fe(III) was substantially (20%) lower (Fe(III):Fe(II) of 42:58). As suggested by the decrease in aqueous Fe(II) accumulation (Fig. 1), Mo¨ssbauer spectroscopy data also indicate Fe(III) reduction had essentially ceased after this period of incubation; after 74 days the corresponding ratio had changed little (38:62). Re-oxidation experiments showed a ratio of 60:40, close to the original

Fig. 4. Normalized As K edge XANES of microcosm treatments and different sediment depths at Nea Kameni. Standards are Na– As(V) and Na–As(III) (thin lines); microcosm samples (1–5b, thick black lines), including heat-sterilized controls (killed, k); Nea Kameni sediment horizons NK1–3, 0–15 cm depth (thick gray lines, 6–8).

Fe(III):Fe(II) ratio after 37 days of exposure to air. However, in agreement with other measurements reported above, attempted re-oxidation by the addition of nitrate produced no measurable effects. The Mo¨ssbauer parameters given by the two doublet fits for the time-zero and acetate-amended sediment slurries were remarkably consistent through the time series. The doublet attributed to Fe(II) had isomer shifts of 1.26– 1.27 mm/s and quadruple splitting values of 1.92– 1.96 mm/s. The doublet attributed to Fe(III) displayed isomer shifts of 0.35–0.37 mm/s and quadruple splittings of 0.66–0.70 mm/s. The consistency of these parameters suggests that the solid-phase iron-containing species vary in their proportions but not their identities. The parameters noted above suggest that the Fe(III) was present in the form of a hydrated oxide or oxyhydroxide (i.e. HFO), such as ferrihydrite (5Fe2O3
9H2O) (e.g. Stolyar et al., 2007), and may also be associated with nano-crystalline clay silicates (Weaver et al., 1967), given the presence of approximately 8.8% w/w Si and 0.5% w/w Al. Parameters for the Fe(II) model fit were not consistent with iron oxide phases, but were analogous to spectra of Fe(II) carbonate (e.g. Greenwood and Gibb, 1971; Ram et al., 1998; Wade et al., 1999). The suggested association of Fe(II) with carbonate phases may be accounted for by 4% (3.3 mol/kg) inorganic carbon in the Nea Kameni sediment (Handley et al., 2010). On this basis, up to 19% (w/w) of Fe(II) could have formed Fe(II)-carbonate (FeCO3). The remaining Fe(II) (at least 8% w/w) may be associated with nano-crystalline clay silicates. The time-zero microcosm sample and the sample following incubation with acetate for 37 days were subjected to

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Fig. 5. As K edge EXAFS (a) and related Fourier transforms (b) of microcosm treatments and different sediment depths at Nea Kameni. Microcosm data are represented by black lines: time-zero (1), un-treated day 111 (2), acetate treated day 111 (3), nitrate day 111 (4), aerobic day 111 (5). Results for sediment NK1–3 (0–15 cm depth) at Nea Kameni is shown in gray (6–8). Sample numbering is as for Fig. 4, and experimental and model data are shown as thick vs. thin lines, respectively.

further fitting of the room temperature spectra and to measurements made at low temperature (84 K). The additional room temperature spectra fits (data not shown) yield spectral parameters consistent with at least two types of Fe(II), one associated with a carbonate (siderite) phase (isomer shift = 1.26 mm/s; quadrupole splitting = 1.73 mm/s; see Ram et al., 1998), and the other associated with Fe(II) in a silicate, almost certainly a clay mineral (isomer shift = 1.11 mm/s; quadrupole splitting = 2.31 mm/s) where the larger quadrupole splitting value is generally associated with Fe(II) on an octahedral site (see Stevens and Zhu, 1986). The low temperature spectra are also characterized by the emergence of at least one sextet of peaks due to magnetic hyperfine interactions (i.e. magnetic ordering) in addition to the presence of overlapping doublets. The overall value of the hyperfine field (magnetic field at the nucleus) is 460 kOe, although this value needs to be treated with caution as the spectra exhibit some of the distortion associated with small particle sizes (i.e. superparamagnetism). These data are consistent with the Fe(III) being present chiefly as an iron oxyhydroxide. Best fit parameters for the room temperature spectra (isomer shift = 0.34 mm/s; quadrupole splitting = 0.70 mm/s) are

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Fig. 6. Mo¨ssbauer spectra obtained at room temperature are shown for (a) microcosm time-zero sediment, and (b) sediment incubated with acetate for 37 days. Actual data is represented by black dots; the lines of overall best fits for data are given by black lines; and the model best fit component spectra are shown by gray lines.

the same as reported for ‘amorphous iron oxide gels’ (see Coey and Readman, 1973). As noted above, this material could (at least in part) be a ferrihydrite, but other iron oxyhydroxides phase cannot be discounted. In particular, goethite (tentatively reported from XRD data) may play a role here. Whatever the precise phases present, it is clear that a poorly-crystalline iron oxyhydroxide phase (or phases) plays a key role in these sediments. One further point related to the Mo¨ssbauer data is the possible role of an iron sulfide phase (such as mackinawite, FeS) in these sediments. Given that the bulk concentration of sulfur detected is only <0.5%, whereas iron bulk concentration is approximately 50%, only <1% of the iron could be present as iron sulfide. This would not be detectable using the Mo¨ssbauer technique, particularly as the signal from iron in iron sulfide has a very small isomer shift and quadrupole splitting (see Vaughan and Ridout, 1971), and would be masked by the signal from Fe(III) in oxidic components. Hence we cannot confirm or eliminate the presence of an iron sulfide-type phase in any of these samples (although, given the evidence for the presence of sulfur derived from sulfate reduction, this has to be a strong possibility). 3.2.2. Arsenic solid-phase speciation and coordination environments The oxidation states and coordination environments of arsenic within the microcosm slurries and an un-treated

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sediment core were determined by XANES and EXAFS, respectively. XANES spectra of arsenic were consistent with the presence of As(III) in anoxic microcosm slurries, that is, those collected at time-zero, and at day 111 after no treatment, acetate treatment, nitrate-treatment or heatsterilization (Fig. 4). LINCOM fits of XANES data demonstrate that the arsenic in these samples was between 88% and 100% As(III) (Table 1). By contrast, arsenic was approximately 71% As(V) in the air-exposed slurries, and 48% As(V) in the aerobic heat-sterilized control. XANES analyses of un-treated sediment at different core depths revealed a transition in arsenic species with depth, from oxidized to reduced. Sediment cores consisted of a top 5 cm of suboxic sediment, a suboxic–anoxic transition zone sediment at 5–20 cm depth, and a lower anoxic zone at >20 cm depth. 66% As(V) was identified in the surface horizon (0–5 cm depth), and 96% As(III) was measured in the lower two zones. Complementary results were obtained from fits of corresponding EXAFS data (Fig. 5, Table 2). In both un-treated sediment and microcosm slurries, arsenic displayed first shell bonding with oxygen atoms. In the time-zero, and day 111 un-treated and acetate-treated microcosm slurries, and the 5 to >20 cm deep fraction of the un-treated sediment core, arsenic was predominantly trigonal pyramidal As(III) (arsenic trioxide, As2O3). Arsenic analyzed in these sediments had three oxygen atoms in the first coordination sphere, and an average As–O interatomic distance of 1.74– ˚ (As(III) = 1.78 A ˚ ). Results indicate that a lesser 1.78 A amount of arsenic trioxide was present in nitrate-treated sediment. In comparison, EXAFS confirm that As(V) was the primary form of arsenic in more oxic sediments. In the aerated microcosm slurry, terahedrally coordinated ˚ As(V) had a measured As–O bond distance of 1.71 A ˚ (As(V) = 1.68 A), and a first coordination sphere of four oxygen atoms. Fits for EXAFS measurements of the untreated sediment, at 0 to 5 cm depth, indicate that the arsenic had a three oxygen atom coordination sphere, but ˚ . This suggests a mixture an average bond distance of 1.72 A of As(III) and As(V), as was also indicated by XANES.

Table 1 LINCOM fits of background subtracted XANES data from microcosm treatments and original Nea Kameni sediment (NK1– 3 = 0–15 cm depth), depicting arsenic identities. In each case, values represent percentages of total arsenic present. Heat-sterilized controls (killed, k). Sample

As(V)

As(III)

Fit index

Time-zero Un-treated day 111 Acetate day 111 Acetate (k) day 111 Nitrate day 111 Nitrate (k) day 111 Aerobic day 111 Aerobic (k) day 111 NK1 NK2 NK3

0 14 0 12 5 0 71 48 66 5 4

100 86 100 88 95 100 29 52 34 96 96

5.63 7.78 10.34 2.6 1.52 4.27 2.17 1.67 1.22 5.39 5.91

Table 2 Model fit parameters for the As K edge EXAFS of different microcosm treatments and Nea Kameni sediment horizons (NK1– 3 = 0–15 cm depth). Coordination number (N, ±25%); average ˚ for the first shell, ±0.05 A ˚ for the interatomic distance (r, ±0.02 A outer shells); Debye–Waller factor (2d2, ±25%); least squares residual (R). Outer shell scatterers are shown as As or Fe according to best fit; however, in all cases fits for As and Fe atoms were similar, and results do not conclusively distinguish between either atom. ˚ ) 2d2 (A ˚ 2) R Sample Atom type N R (A Time-zero

O As

3 2

1.76 3.73

0.003 0.003

37.3

Un-treated day 111

O Fe

3 2

1.76 3.41

0.002 0.019

46.2

Acetate day 111

O As

3 1

1.76 3.34

0.006 0.006

26.6

Nitrate day 111

O Fe

3 2

1.74 2.91

0.007 0.032

29.2

Aerobic day 111

O

4

1.71

0.009

27.8

NK1

O Fe

3 2

1.72 2.89

0.004 0.027

22.9

NK2

O Fe

3 2

1.74 3.41

0.008 0.015

38.9

NK3

O Fe

3 2

1.78 3.43

0.007 0.016

29.4

Additional modeling of EXAFS data produced best fits for iron outer shell scatterers in most cases (Table 2), although overall similar fits were obtained for arsenic or iron outer shell scatterers, such that either of these atoms could represent the scatterers in each sample. It is probable that iron represents the universal outer shell scatterers, particularly given the inconsistency otherwise arising between similar sediments with regard to their outer shell scatterers (i.e. time-zero un-treated and day 111 un-treated slurries) – indeed, although we have not rigorously calculated the impact, this does highlight the value of analyzing a series of related samples in increasing the confidence in best fit models of EXAFS data, where there are several models with similar best fit parameters. Only in the aerated slurry treatments were no outer shell scatterers determined. EXAFS showed no evidence of any As–S species. 3.3. Solid-phase partitioning of arsenic Partitioning of arsenic in the microcosm sediments was elucidated by sequential extractions targeting sorbed or iron-bound arsenic, or arsenic associated with either sulfide or organic phases. Results demonstrate a significant shift in the solid-phase associations of arsenic in microcosm experiments after acetate treatment and re-oxidation (Fig. 7a). In microcosms where acetate was added to stimulate Fe(III) reduction, within 37 days the proportions of arsenic that were specifically-sorbed increased from 15 ± 2% (s.d.) to 30%, arsenic associated with well-crystallized iron fractions increased from 3 ± 1% to 22%, while that associated with poorly-crystalline iron fractions decreased from 69 ± 5%

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Fig. 7. Solid-phase partitioning of arsenic in microcosm experiments (a) and the Nea Kameni sediment column (0–35 cm depth in 5 cm increments, NK1–7). Heat-sterilized controls (killed, k). Partitions comprise: (1) (NH4)2SO4 for easily exchangeable arsenic; (2) NH4H2PO4 for specifically-sorbed arsenic; (3) NH4-oxalate (dark) for arsenic associated with amorphous and poorly-crystalline hydrous iron oxides; (4) NH4-oxalate/ascorbic acid (light, 90 °C) for arsenic associated with well-crystallized hydrous iron oxides; and (5) KClO4/HCl for arsenic associated with sulfides and organics. Error bars represent extractions from replicate microcosms. Error bars are shown for samples for which between two to five extraction replicates were performed.

to 32% (Table EA-1-1 gives raw values for each experimental replicate). The proportions attained at day 37 were stable over the remaining two-month incubation period, and no change in these fractions was observed in heat-sterilized or no-acetate controls at days 37, 74 or 111. The small quantities of arsenic associated with the sulfide/organic fraction appeared to differ little amongst treatments, and tended to be only very slightly higher in the acetateamended sediments at days 0–111 (13 ± 4% and 10 ± 1%, respectively) compared with the anaerobic no-acetate control (5 ± 1%). Loosely sorbed arsenic was negligible in both time-zero and subsequent acetate-amended anaerobic treatments, and was 60.5% for all analyses of incubation experiments. Addition of nitrate and air at day 74 resulted in a partial reversal of arsenic partitioning, most notably with respect to the arsenic associated with poorly-crystallized iron oxides. This latter fraction increased in re-oxidation experiments. In the presence of nitrate, the poorly-crystallized iron-associated fraction increased from 30 ± 1% to 46 ± 1%. No change between day 74 and 111 was evident in heat-sterilized, nitrate-amended sediment. Exposure to air resulted in a more dramatic reversal, causing an increase

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of the poorly-crystallized fraction from 30% to 59 ± 4%. Also, in the presence of air the specifically-sorbed fraction decreased from 34 ± 2% to 18 ± 1%, and the sulfide/organic fraction decreased from 16 ± 1% to 3 ± 2%. Changes in arsenic fractions in the air-exposed and microbially active sediment were essentially identical to those observed in the corresponding heat-sterilized control sediment. No arsenic was detected following the final hydrofluoric acid extraction step (for digestion of all residual material, e.g. silicates) owing to extremely low quantities (3.0– 6.5 mg) of residue material remaining after the KCIO4/ HCl extraction step. In all cases (i.e. microcosm and sediment column) the loosely sorbed arsenic fraction was negligible. Arsenic concentrations in the aqueous phase sampled throughout the course of the incubation experiments were not detectable with ICP-AES, and were determined with IC–ICP-MS to be at most tens of lg/L As(III) (data not shown). Total concentrations of arsenic sequentially extracted from microcosm sediment slurries were 44 ± 6 mg/ L. A total extraction procedure was not conducted to determine the recovery efficiency; however, given the estimated solid-solution ratio (1:6) the total sequentially extracted arsenic equated to approximately 65% recovery based on initial arsenic concentrations of the Nea Kameni transition zone sediment (405 mg/kg average arsenic concentration; Handley et al., 2010). Arsenic partitioning at different depths in an un-treated sediment core displays similar trends to microcosm treatments, whereby increased sediment depth and anoxia in the reduced-iron zone (5–30 cm depth) equated with a greater proportion of arsenic bound within well-crystallized phases (Fig. 7b; Table EA-1-2). In the surface sediment horizon arsenic was predominantly (57%) associated with the poorly-crystalline iron fraction, and only 29% was associated with the well-crystallized fraction. In the deeper (5– 30 cm), more reduced sediment, this fraction hosted only 18–37% of the arsenic, whereas the well-crystallized fraction hosted 49–60% of arsenic. In the deepest sediment (30– 35 cm depth), arsenic within the well-crystallized fraction was only slightly elevated (38%) compared to the surface; however, arsenic associated with the sulfide/organics fraction was an additional 26–35% above concentrations in all shallower sediment (0–30 cm depth). Differences in the arsenic proportionation between the slurry used in timezero microcosms, and the depths (5–20 cm) representing the same sediment in the un-treated core (Fig. 7a and b), most likely reflect changes that occurred during storage in the sediment used for microcosm slurries. Specifically, a greater proportion of arsenic occurs in the specificallysorbed and, in particular, the poorly-crystalline iron fractions in the time-zero and no-acetate control microcosm slurries. 3.4. Bacterial phylogenies and sulfate reducers 3.4.1. Bacterial community dynamics Analysis of the sediment bacterial communities using PCR-amplified 16S rRNA gene clone libraries (57–60 clones/library) depicted significant differences between microcosm treatments with contrasting incubation periods

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(Fig. 8; Table EA-1-3). The largest proportions of the bacteria in the anaerobic starting sediment (time-zero) were most closely related to members of the Fe(III)-, sulfurand nitrate-reducing order Desulfuromonadales; however, they were only distantly related to the nearest cultured representative Geothermobacter ehrlichii (90% sequence similarity; 20% of clones) (Kashefi et al., 2003). Other starting sediment clones were members of the phylum Deferribacteres (nearest cultured representative Caldithrix abyssi at 86% similarity; 18% of clones), and the phylum Chlorobi (89% similarity to an uncultured clone; 13% of clones). The Deferribacteres contain genera capable of denitrification, Fe(III) and S0 reduction, and respiration of other inorganic compounds (e.g. Caccavo et al., 1996; Greene et al., 1997; Myhr and Torsvik, 2000; Janssen et al., 2002; Miroshnichenko et al., 2003a,b; Takai et al., 2003); whereas the Chlorobi comprise a phylum of anaerobic, sulfide-oxidizing phototrophs (Pfennig, 1978). The relatively low, 86–90%, similarities to known organisms recorded for these gene sequences suggests a significant degree of novelty for the corresponding organisms detected. According to geochemical analyses, the initial 37 days of incubation corresponded to the period of Fe(III) reduction in acetate-amended sediments. The bacterial community, after this period of acetate treatment, was dominated by Deltaproteobacteria clones (74% community abundance), in particular those most closely related to the families Pelobacteraceae (50% of clones, 90–95% similarity to cul-

tured representatives) and Geobacteraceae (12% of clones, only 85% similarity to an uncultured representative) in the order Desulfuromonadales. Species within these Desulfuromonadales families are known to reduce Fe(III) and S0, but not sulfate (Lovley et al., 1995, 2004; Lonergan et al., 1996; Holmes et al., 2004; Vandieken et al., 2006). Following the remaining two months of incubation, up until day 111, acetate-amended sediment was dominated by both Desulfuromonadales (31% abundance, again most closely related to the Pelobacteraceae and Geobacteraceae, 85 to 92% similarity) and Desulfobacterales (47% abundance). Desulfobacterales are typified by sulfate respiration (Kuever et al., 2005a), and were almost equally divided between members of the families Desulfobulbaceae (89% similarity to Desulfocapsa sp.; 24% community abundance) and Desulfobacteraceae (93% similarity to Desulfotignum toluolica; 18% community abundance). Desulfotignum spp. reduce sulfate (Kuever et al., 2001; Schnik et al., 2002; Ommedal and Torsvik, 2007), while bacteria in the Desulfocapsa genus are capable of growth by reducing sulfur species or sulfate (Janssen et al., 1996; Finster et al., 1998). Although phylogeny cannot be used to conclusively determine function, the greater presence of Desulfobacterales bacteria implies shift in community composition from taxa affiliated with Fe(III)-reduction to those affiliated with sulfate reduction. This is consistent with geochemical data, which show that low-levels of sulfate reduction were ongoing, even after the period of rapid sulfate reduction, be-

Fig. 8. Bacterial community composition determined by 16S rRNA gene clone library analyses of microcosm sediments (a–e).

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tween 43 and 50 days, ceased (Fig. 1b), but that no further reactive HCl-soluble Fe(II) accumulated after day 37. Our results suggest no significant difference between sediment communities treated with nitrate vs. acetate at a broad taxonomic level. After one month of incubation (at day 111), the nitrate-supplemented sediment community was still equally dominated by Desulfuromonadales (43%) and Desulfobacterales (39%), although with different proportions of constituent genera (Table EA-1-3). In contrast, exposure of sediments to air resulted in a substantially altered community characterized by aerobic bacteria. Notably, 32% of the bacteria were Alphaproteobacteria most closely related to Roseobacter sp. (95% sequence similarity), which belongs to a genus characterized by anaerobic denitrification and aerobic photosynthesis (Shiba, 1991; Shiba and Imhoff, 2005). Thirty-eight percent of bacteria were related to Bacteroidetes, predominantly Muricauda sp. (95% similarity), which belong to a genus of facultatively anaerobic or obligately aerobic heterotrophs (Bruns et al., 2001; Yoon et al., 2005). A further 12% were also closely related to the facultatively anaerobic heterotroph, Marinobacter flavimaris (97% similarity; gammaproteobacterium) (Yoon et al., 2004). Marinobacter species are also known to oxidize Fe(II) (Edwards et al., 2003; Handley et al., 2009). 3.4.2. Sulfate-reducing bacterial communities Sulfate-reducing bacteria present in microcosm treatments were verified by sequence analysis of dsr gene clone libraries, which is conserved among all known sulfatereducing bacteria (Wagner et al., 1998). Between 51 and 58 clones were sampled per library (Fig. 9; Table EA-1-4). Based on partial dsrAB gene sequence comparisons, the starting (time-zero) sediment sulfate-reducing community were largely members of either the Desulfobacterales (35% community abundance) or the Desulfovibrionales (36% abundance). Desulfobacterales were dominated by a Desulfobulbus-like bacterium (79% sequence similarity), with less abundant bacteria present related to Desulfosalina, Desulfobacterium, Desulfofustis and Desulforhopalus (74–78% similarity). The majority of Desulfovibrionales were related to Desulfohalobium retbaense (79% similarity). By day 37, acetate stimulation had increased the Desulfobacterales fraction to 76% of the sulfate-reducing community, a large proportion of which (51% overall community abundance) were most closely related to the Desulfosarcina variabilis (82% sequence similarity). Lesser Desulfobacterales members were related to Desulfobacterium cetonicum, Desulfobulbus elongates, and Desulfosalina propionicus (71– 77% similarity). Further analyses showed the sulfate-reducing community composition remained relatively stable over the final two-month incubation period (from day 37 to 111). A comparable fraction of the community, 75%, was most closely related to members of the Desulfobacterales, with 38% related to D. variabilis (83% similarity) and 21% related to D. cetonicum (83% similarity). These results clearly demonstrate that, although bacteria related to known Fe(III)-reducing taxa dominated the community as a whole after one month of incubation with acetate, the sulfate-reducing community had nevertheless shifted within the same period to become dominated by

Fig. 9. Sulfate-reducing bacterial community compositions determined by dsr gene clone library analyses of microcosm sediments (a–c). Abbreviation: uncultured, u.

Desulfobacterales. Consistent with geochemical data, microbial sulfate and Fe(III) reduction evidently occurred as concomitant processes within this period (37 days). Sulfate reduction resulted from the respiratory activities of bacteria sharing closest dsr gene sequence similarity with Desulfosarcina species at day 37 and at day 111. The overall increase in Desulfobacterales bacteria in the general 16S rRNA clones libraries, seen at day 111, is consistent with this. Moreover, additional analyses of the day 74 acetatetreated sediment revealed 17% of the bacterial community was related to D. variabilis (78–97% 16S rRNA gene similarity; Table EA-1-3). Ferric iron reduction, on the other hand, is attributable to the Malonomonas rubra-like Desulfuromonadales bacterium. 4. DISCUSSION 4.1. Mechanisms, nature and products of Fe(III) and sulfate reduction 4.1.1. Microbial respiration of Fe(III) and sulfate Microbial respiration of Fe(III) plays a significant role in changing both the iron mineralogy in the sediment and

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the speciation of arsenic associated with reduced and oxidized iron phases. The microbial reduction of Fe(III) in microcosm sediments simulates the biogeochemical cycling of iron at, and below, the suboxic–anoxic transition within the Nea Kameni sediments. Considering the phylogeny of bacteria dominating the microbial community during the early Fe(III)-reduction stage (affiliated with Desulfuromonadales), it is likely that the microbially induced reduction of Fe(III) to Fe(II) was a direct, respiratory (energy-conserving) process, coupled with acetate oxidation. Microbial (Desulfobacterales-driven) sulfate reduction was clearly a dissimilatory, energy-conserving process, dependent on acetate availability. It is possible that microbial sulfate reduction also indirectly contributed to the production of Fe(II), via abiotic Fe(III) reduction by reduced sulfur species (e.g. Pyzik and Sommer, 1981; Miller et al., 1995; Nevin and Lovley, 2000). The two processes (Fe(III) and sulfate reduction) were largely, but not entirely, decoupled within acetate incubations, owing to the lower energy yield of sulfate reduction (Thauer et al., 1977). These reactions are generally separated by depth in marine sediments, with principal sulfate reduction taking place deeper, in zones where Fe(III) has been largely depleted (Burdige, 2006; Jørgensen, 2006). 4.1.2. Speciation, mineralogy and bioavailability Experiments here have shown that microbial activity induces the reduction of both reactive, HCl-soluble and more crystalline Fe(III) fractions. Although the initial reactive HCl–NH2OH
HCl soluble Fe(III) was almost completely reduced within 37 days of incubation, the increase in HClsoluble Fe(II) was a factor of two higher. Mo¨ssbauer spectroscopy indicated that a much larger fraction of solid iron, equating to approximately 10% of the sediment, was transformed to Fe(II) during the same period. This suggests that Fe(III) was reduced from both reactive and more recalcitrant minerals, and also a large proportion of the Fe(II) produced formed recalcitrant Fe(II)-bearing minerals. Sediment digests using dilute HCl and HCl–NH2OH
HCl are primarily selective for poorly-crystalline iron mineral fractions. The method was originally determined based upon the amount of Fe(III) reduced in >1 month by enrichment cultures of a tidal river sediment (Lovley and Phillips, 1986a,b; Lovley and Phillips, 1987; Anderson and Lovley, 1999). Nevertheless, more crystalline Fe(III) minerals (e.g. goethite, hematite) may also be respired by Fe(III)-reducing bacteria, although with more difficulty, and at a slower rate (e.g. Lovley and Phillips, 1987; Roden and Zachara, 1996; Luu and Ramsay, 2003; Roden, 2006). X-ray diffraction measurements were of limited use in elucidating the sediment mineralogy, suggesting that the minerals were chiefly X-ray amorphous and nanocrystalline in character. In view of the potential for microbial Fe(III) and sulfate reduction (this study), and also Fe(II) oxidation (Hanert, 2002; Handley et al., 2009, 2010) in this sediment, a largely nanocrystalline mineral assemblage may be expected. Nano-sized minerals are common products of microbial biomineralization (Lloyd et al., 2008). Moreover, Mo¨ssbauer data were consistent with a HFO mineral, such as poorly-crystalline ferrihydrite, and also (nanocrystalline)

clay. Ferrous iron carbonates, for example siderite (FeCO3), putatively present in the Nea Kameni sediment, tend to be microcrystalline (Dong et al., 2000; Fredrickson et al., 1998; Zachara et al., 2002; Islam et al., 2005b), and have been identified in trace amounts in previous XRD analyses of the Nea Kameni sediment (Cronan et al., 2000). Siderite, as opposed to magnetite (Fe3O4), forms as a result of microbial Fe(III) respiration where an excess of bicarbonate is present (e.g. Dong et al., 2000; Zachara et al., 2002). If present, siderite was most likely removed during the crystalline iron oxide (oxalate/ascorbic acid) extraction step in this study. Other studies have comparably identified strong evidence for siderite removal with (16– 20 h) oxalate extractions (Lovley and Phillips, 1986a; Jakobsen and Postma, 1999; Burton et al., 2007). Aqueous sulfate and solid (reduced) sulfur species were, respectively, one and two orders of magnitude lower in concentration than aqueous and sediment-associated iron concentrations, such that it was not possible to determine the presence of potential amorphous iron sulfide phases and their arsenic content. Sulfate reduced during the initial acetate-supplemented incubation period may have formed a solid precipitate within the sediment, such as poorly-crystalline iron sulfide (Jørgensen, 1977; Neal et al., 2001) and/or partially volatile free sulfide at a ratio of 1:10 H2S(g):HS (aq) at pH 8 (Boyd, 2000). Both iron sulfide and hydrogen sulfide are common products of microbial sulfate reduction found in marine sediments (Jørgensen, 2006). Any localized accumulation of iron sulfide in the microcosm sediments would not have been evident, however, owing to homogenization of slurries prior to frequent sampling events. However, small patches of black precipitate, which could be iron sulfide, were visible in cores of the original Nea Kameni sediment below the zone of Fe(III)-reduction. This signifies the potential for iron sulfide to also form in the microcosm sediments as a result of sulfate respiration and sulfide production. 4.2. Arsenic partitioning and speciation under reductive and oxidative conditions 4.2.1. Arsenic solid-phase cycling in response to Fe(III) and sulfate reduction Examination of arsenic partitioning in the microcosm sediments reveals that arsenic in the initial anoxic (Eh = 164 mV), partially reduced sediment slurry was located predominantly in the poorly-crystalline ferrihydritelike fraction. Ferric iron reduction resulted in a shift of approximately 40% of arsenic bound within the poorlycrystalline iron fraction, so that approximately equal proportions also were incorporated into well-crystallized iron minerals or were specifically-sorbed to mineral surfaces. The incorporation of arsenic into well-crystallized Fe(II)bearing minerals owing to microbial Fe(III) reduction differs from many studies that have observed the release of arsenic into solution. Microbial Fe(III) reductive dissolution of Fe(III) has frequently been found to cause the release of As(V) or As(III) (following microbial As(V) reduction) into the aqueous phase (e.g. Cummings et al., 1999; Islam et al., 2004; Campbell et al., 2006; Pedersen et al., 2006;

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Crowe et al., 2007). However, it is increasingly apparent that Fe(III) reduction can have the reverse effect. Other laboratory-based studies have also demonstrated the capture and immobilisation of trivalent/pentavalent arsenic, owing to the bioreduction of Fe(III) to the Fe(II) and Fe(II)/ Fe(III) minerals, siderite, vivianite and magnetite (Islam et al., 2005a,b). In addition, analyses of high-iron sediments in a drinking-water reservoir found that arsenic remained bound to the sediment as As(III), accompanied by the formation of reduced mixed-valency green rust (Root et al., 2007). Ferric iron respiration and biogenic siderite formation have also been shown to result in the immobilisation of arsenic in a shallow Bengal aquifer, through sorption and entrapment within successive concretionary layers of the host siderite (Sengupta et al., 2004). Further experiments have demonstrated that natural siderite is an efficient mineral for arsenic retention in sediments by both direct sorption of arsenic onto siderite and indirect sorption onto siderite-sorbed Fe(III) oxides/hydroxides (Guo et al., 2007). It has been demonstrated elsewhere that microbial sulfate reduction can result in the capture and precipitation of arsenic (Rittle et al., 1995; Newman et al., 1997; Kirk et al., 2004; Wilkin and Ford, 2006), and that sulfides can act as important hosts for arsenic (Moore et al., 1988; Kneebone and Hering, 2000; Farquhar et al., 2002; Smedley and Kinniburgh, 2002; Bostick and Fendorf, 2003; Bostick et al., 2004; Wolthers et al., 2005), although studies have shown contradictory results for the capacity of certain sulfide minerals to sequester arsenic (Farquhar et al., 2002; Bostick and Fendorf, 2003; O’Day et al., 2004; Wolthers et al., 2005; Wilkin and Ford, 2006). Sulfate reduction in our batch incubation experiments, however, produced no appreciable effect on arsenic partitioning in sediment slurry sulfide fractions. 4.2.2. Effect of oxidation on arsenic partitioning in sediment Arsenic re-partitioned into the well-crystallized iron fraction during Fe(III)-reduction was unstable in the presence of oxidants. The addition of air or nitrate largely reversed this partitioning, inducing a large portion of arsenic to re-associate with the poorly-crystalline iron fraction. The increased amount of specifically-sorbed arsenic achieved during Fe(III) reduction was also susceptible to decline in the presence of air, but not nitrate. In aerobic experiments, results also suggest partial loss of the small portion of arsenic associated with the sulfide/organics (or KCIO4/HCl) fraction. While experimental error cannot be excluded, this apparent depletion may be attributed to the oxidation and release of sulfide from the sediment, indicated by sulfate re-generation beyond initial levels present (Fig 3d; cf. Moore et al., 1988; Kneebone and Hering, 2000; Nickson et al., 2000). We may further speculate that this depleted arsenic would have been associated with previously-formed sulfide, as the arsenic-associated sulfide/ organics fractions did not increase during acetate-amendment experiments, and hence during sulfate reduction. In nitrate addition experiments, although nitrate had no apparent effect on reactive HCl-soluble Fe(II) concentrations or on the total ratio of Fe(II)-to-Fe(III) in anaerobic microcosm slurries, it did impact arsenic solid-phase cy-

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cling, and 25% nitrate was reduced. Nitrate has a high redox potential and, in the absence of oxygen, it is readily utilized as an electron acceptor in anaerobic microbial respiration in the surface layer of marine sediments (Koike and Sørensen, 1988; Herbert, 1999). This process may be directly coupled with the microbial oxidation of, for example, Fe(II), sulfide or As(III) (Oremland et al., 2002; Cardoso et al., 2006; Weber et al., 2006), or result in the abiotic oxidation of reduced geochemical species, such as Fe(II) by nitrate reduction products such as nitrite (Senko et al., 2005). In this study, nitrate reduction occurred only in microbially active sediment slurries, suggesting that it was respired by bacteria, which phylogenetic analyses suggest may be abundant in the nitrate-treated sediments. Nitrate-dependent, Fe(II)-oxidizing bacteria have been identified in, and isolated from, the same sediment at Nea Kameni (Handley et al., 2009, 2010); however, as no re-oxidation of Fe(II) was observed in nitrate-treatments, it seems unlikely that these bacteria contributed to nitrate loss in these experiments. Incomplete reduction of nitrate could be due to the limited availability of organic electron donors for heterotrophic denitrification, or organic carbon required by some mixotrophic bacteria for nitrate-dependent Fe(II) oxidation (Straub et al., 1996; Straub and Buchholz-Cleven, 1998; Kappler et al., 2005; Handley et al., 2009). 4.2.3. Character of arsenic sorption, complexes and speciation Sequential extraction results indicate that very little arsenic formed easily exchangeable (i.e. loosely sorbed) outer-sphere complexes, but significant concentrations were specifically-sorbed forming inner-sphere complexes (cf. Wenzel et al., 2001). During the acetate-amended, Fe(III)reducing experiment, the source of increasing arsenic in the well-crystallized iron and specifically-sorbed arsenic fractions was evidently derived from the reduction and transformation of poorly-crystalline ferrihydrite-like minerals. However, the location of specifically-sorbed arsenic (i.e. whether on poorly or well-crystallized iron or sulfide minerals) after Fe(III) reduction is not clear. Compared with nitrate-treatment, exposure to air led to greater reestablishment of the arsenic fractions associated with the poorly-crystallized iron fraction, and included a greater degree of re-integration of specifically-sorbed arsenic. In terms of bulk analyses, EXAFS data for outer shells suggests arsenic trioxide in the Nea Kameni sediment column formed inner-sphere complexes with iron at all depths, and also with iron in un-treated, acetate- and nitrate-treated microcosm sediments. For comparison, Manning et al. (1998) determined that arsenic trioxide with two iron outer shell scatterers, and similar As–Fe distances (i.e. ˚ ) to this study, were consistent with a bidentate, binu3.4 A clear bridging complex, in models of As(III)-goethite sorption. Similar results were obtained in a XAS study of As(III) binding to green rust (Root et al., 2007). The lower ˚ ) and the As–Fe bond distances in nitrate-treated (2.91 A ˚ ) corNea Kameni surface sediments (0–5 cm depth, 2.89 A respond with shorter As–O distances, and possibly reflect influence from As(V). Although no reasonable fits were obtained from the EXAFS Fourier transform of As(V) outer

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shell scatterers in the aerobic sediment, sequential extractions confirmed the presence of inner-sphere complexed arsenic. In microcosm experiments, As(V) was detected only in sediment incubated aerobically. 4.2.4. Correlation between incubation experiments and natural sediments The presence of 71% As(V) in the air-exposed microcosm sediment slurries compares with the oxidized surface sediment layer at Nea Kameni, in which 66% of the arsenic was As(V). These sediments were also similar in terms of Eh (suboxic at 0 mV), and for being Fe(III)-rich. Results suggest that the oxidation of As(III) to As(V) in air-exposed sediments was at least partially abiotic, as 48% oxidation also occurred in the heat-sterilized control. The activity of As(III)-oxidizing bacteria may have contributed to the greater formation of As(V) in the unsterilized sediment. An aerobic, As(III)-oxidizing bacterium was previously isolated from the same Nea Kameni sediment (Handley et al., 2009), although it was not obviously enriched for in these experiments. Arsenic speciation and trends in Fe-As solidphase associations were also similar between reduced acetate-amended microcosm slurries and deeper Nea Kameni sediment zones. Specifically, both contain arsenic that is almost or entirely As(III), and both have higher ratios of arsenic in the well-crystallized vs. poorly-crystallized iron fraction compared with that found in oxidized slurries and sediments. 5. CONCLUSIONS Here we employed laboratory-based incubation experiments to investigate the impact of acetate-stimulated microbial respiration and abiotic/biotic re-oxidation on arseniccycling in an iron-rich marine sediment. Experiments demonstrated that significant and partially reversible changes in solid-phase arsenic binding patterns occur under reducing and oxidizing conditions, respectively. Arsenic in the initial, partially-oxidized sediment was largely structurally-bound within poorly-crystalline HFO. The biogenic transformation of both crystalline and reactive poorly-crystalline HFO occurs under reducing conditions, and corresponds with the release and co-precipitation of approximately 19% of trivalent arsenic in crystalline Fe(II)-bearing phases (possibly siderite), and an increase in specifically-sorbed arsenic of 15%. The effect can be partially reversed when sediments are exposed to incomplete nitrate reduction, and almost entirely reversed under exposure to air, accompanied by some apparent loss of arsenic from sulfur/sulfide minerals (owing to oxidative dissolution) and subsequent capture by HFO. Arsenic partitioning is extensively controlled by iron redox cycling owing to the high-iron content of the sediment relative to arsenic concentrations. These findings are consistent with principal trends in arsenic solid-phase associations that occur with depth, and increasingly reducing conditions in the un-treated Nea Kameni sediment column. Results corroborate observations elsewhere that identify the association of arsenic with Fe(II)bearing minerals in reducing environments.

ACKNOWLEDGMENTS This study was funded by the following grants: BIOTRACS, EST Marie Curie Fellowship (European Union); and Overseas Research Students Award Scheme (ORSAS) from the Higher Education Funding Council of England (HEFCE). Alastair Bewsher, Paul Lythgoe, Cath Davies, Richard Cutting and John Waters provided assistance with geochemical analyses. We also thank Andrew Gault for valuable advice. XAS analysis was supported by a CLRC Daresbury SRS beamtime allocation award (50/194) to David Polya, Jonathan Lloyd, Aimee Hegan, Marina Hery and Enoma Omoregie.

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