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Arsenic(V) incorporation in vivianite during microbial reduction of arsenic(V)-bearing biogenic Fe(III) (oxyhydr)oxides E. Marie Muehe, Guillaume Morin, Lukas Scheer, Pierre Le Pape, Imène Esteve, Birgit Daus, and Andreas Kappler Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b04625 • Publication Date (Web): 01 Feb 2016 Downloaded from http://pubs.acs.org on February 2, 2016
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Arsenic(V) incorporation in vivianite during microbial reduction
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of arsenic(V)-bearing biogenic Fe(III) (oxyhydr)oxides
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E. Marie Muehe1,2*, Guillaume Morin3, Lukas Scheer1, Pierre Le Pape3,
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Imène Esteve3, Birgit Daus4, Andreas Kappler1
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1
Geomicrobiology, Center for Applied Geosciences, University of Tuebingen, Germany
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2
Now: Environmental Soil Biogeochemistry, Earth System Science, Stanford University, USA
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3
Environmental Mineralogy, Institut de Minéralogie, de Physique des Matériaux et de
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Cosmochimie (IMPMC), UMR7590 - CNRS - UPMC, France
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Germany
UFZ – Helmholtz Centre for Environmental Research, Department Analyt. Chem., Leipzig,
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For re-submission to Environmental Science and Technology
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*To whom correspondence should be sent:
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E. Marie Muehe, Environmental Soil Biogeochemistry, Earth System Science, Stanford
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University, 473 Via Ortega, Stanford, CA 94305, USA
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Phone: +1-650-291-9715, email:
[email protected] 20 21
Running title: As-incorporation in microbially produced vivianite
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Keywords: Arsenate, arsenite, biomineralization, biogenic iron minerals, Fe(III)-reducing
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bacteria
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ABSTRACT
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The dissolution of arsenic-bearing iron(III) (oxyhydr)oxides during combined microbial
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iron(III) and arsenate(V) reduction is thought to be the main mechanism responsible for arsenic
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mobilization in reducing environments. Besides its mobilization during bio-reduction, arsenic
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is often re-sequestered by newly forming secondary iron(II)-bearing mineral phases. In
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phosphate-bearing environments, iron(II) inputs generally lead to vivianite precipitation. In
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fact, in a previous study we observed that during bio-reduction of arsenate(V)-bearing biogenic
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iron(III) (oxyhydr)oxides in phosphate-containing growth media, arsenate(V) was immobilized
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by the newly-forming secondary iron(II) and iron(II)/iron(III)mineral phases, including
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vivianite. In the present study, changes in arsenic redox state and binding environment in these
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experiments were analyzed. We found that arsenate(V) partly replaced phosphate in vivianite,
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thus forming a vivianite-symplesite solid solution identified as Fe3(PO4)1.7(AsO4)0.3•8H2O. Our
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data suggests that in order to predict the fate of arsenic during the bio-reduction of abiogenic
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and biogenic iron(III) (oxyhydr)oxides in arsenic-contaminated environments, the formation of
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symplesite-vivianite minerals needs to be considered. Indeed, such mineral phases could
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contribute to a delayed and slow release of arsenic in phosphate-bearing surface and
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groundwater environments.
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TOC art:
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INTRODUCTION
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Arsenic (As) is one of the most widely studied metalloids since it has been recognized as a
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major threat to human health, especially when present at concentrations above the
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recommended WHO levels (10 µg L-1) in groundwater aquifers.1,
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concentrations as found in the aquifers of Bengal and Mekong deltas are caused by a
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combination of various geochemical and microbial processes.1-3 The transformation of As-
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bearing minerals during their riverine transport from Himalaya leads to the binding of As to
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Fe(III) (oxyhydr)oxide minerals within the deltaic sediments.4-6 Arsenic may then be released
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from the surface of these Fe(III) minerals by sorption competitors, such as phosphate,7
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bicarbonate,8 silicic acid,9 natural organic matter (NOM, e.g. humic substances)10, 11 as well as
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by the formation of mobile humic-Fe-As complexes and colloidal aggregates.12, 13 Additionally,
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microbial reduction of the mineral-bound As(V) to the more mobile As(III) form was suggested
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to lead to As mobilization.14-17 The most important factor responsible for the high As
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concentrations in the groundwater, however, is thought to be related to reductive dissolution of
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the As-bearing Fe(III) (oxyhydr)oxide minerals by Fe(III)-reducing bacteria in anoxic As-
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contaminated aquifers.16, 18 By using allochthonous and autochthonous organic matter these
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bacteria reductively dissolve Fe(III) (oxyhydr)oxides, leading to the release of the bound As.2-
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4, 18, 19
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which As release was accompanied by the progressive export of aqueous Fe(II) from the
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columns.20, 21
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Fe(III) (oxyhydr)oxides in the environment are formed either via chemical or microbial
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oxidation of Fe(II).22,
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environments24-30 and were typically associated with cell-derived organic matter.31-35 Organic
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carbon and ion impurities hinder homogenous crystal growth,22 leading to smaller and less
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crystalline biogenic minerals compared to abiogenic Fe(III) (oxyhydr)oxides.36-39 Phosphate is
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one such ion impurity and is thus frequently encountered as a major constituent of Fe(III)
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Elevated aqueous As
The feasibility of this process was also simulated with long-time column experiments, in
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Biogenic Fe(III) (oxyhydr)oxides have been found in various
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(oxyhydr)oxides found in many different environmental systems.40-45 Bacteriogenic Fe(III)
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(oxyhydr)oxides were shown to be highly susceptible to microbial reduction compared to
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synthetic, poorly crystalline Fe phase analogues, though the rate of bio-reduction varied
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according to the mineralogy of the biogenic Fe(III) (oxyhydr)oxide.46, 47 The resulting biogenic
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Fe(II) mineral products able to capture As include siderite (FeCO3),48 magnetite (FeIIFeIII2O4),20,
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48-50
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column experiments. Especially vivianite is of interest as secondarily formed Fe(II) mineral
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phase in culture media,47,48 aquifers,52 and in fertilized and non-fertilized paddy soils.53, 54 More
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specifically, Islam et al.48 showed that vivianite sorbs As(V) after bio-reduction of soluble
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Fe(III) but did not observe direct evidence for As(V) incorporation in the vivianite structure.
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More recently, Muehe et al.46 found that microbial reduction of As(V)-bearing biogenic Fe(III)
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(oxyhydr)oxides in freshwater medium containing 1 mM phosphate resulted mainly in the
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formation of vivianite. They discussed that As(V) is very likely to be associated with vivianite
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but so far, the mechanism of As(V) immobilization during this process is unknown.
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The goal of the present study was to investigate the fate of As after microbial reduction of
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As(V)-bearing biogenic Fe(III) (oxyhydr)oxides. More specifically, the objectives were (I) to
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identify the localization and binding mode of As(V) and As(III) in the newly formed Fe mineral
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phases using electron microscopy and synchrotron-based X-ray absorption spectroscopy and
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(II) to identify arsenic speciation changes in the solid and liquid phase to better understand the
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mobility of As in the system. The presentation of the data will conclude to the discussion of the
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mechanisms of As scavenging and release processes under the reducing conditions investigated.
, green rust (FeII4FeIII2(OH)12CO3),51 and vivianite (Fe3(PO4)2)48, 49 in batch and short-time
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MATERIAL AND METHODS
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Source of microorganism, microbial growth conditions, and biogenic Fe(III)
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(oxyhydr)oxide synthesis. We used the Fe(III)-reducing γ-Proteobacterium Shewanella
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oneidensis strain MR-1, which utilizes a wide range of organic substrates and uses Fe(III) as an ACS Paragon Plus Environment
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electron acceptor in the absence of O2.55 Iron-free pre-cultures (20 mM lactate, 40 mM
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fumarate) and experimental cultures (20 mM lactate, 5-7 mM Fe(III) biogenic minerals) of
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strain MR-1 were grown in freshwater medium (0.14 g L-1 KH2PO4, 0.2 g L-1 NaCl, 0.3 g L-1
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NH4Cl, 0.5 g L-1 MgSO4·7H2O, 0.1 g L-1 CaCl2·2H2O, 22 mM bicarbonate buffer, pH 7, 1 mL
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L-1 vitamin solution, 1 mL L-1 trace element solution, 1 mL L-1 selenate-tungstate solution) and
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prepared under an N2/CO2 (80/20 v/v) atmosphere according to Muehe et al.46 The biogenic
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material used in the experiments here was produced independently of the Muehe et al.46 study,
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as large quantities of minerals were needed for analysis. However, these experiments were run
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at the same time when the Muehe et al.46 study was performed, using the same chemicals,
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apparatuses, and incubators, ensuring maximum compliance between both studies. As the
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geochemical, XRD, and microscopic data of this study and Muehe et al.46 are in accordance,
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we assume that the mineralogy of the here-used biogenic material is the same as in Muehe et
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al.46.
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The As(V)-bearing biogenic Fe(III) (oxyhydr)oxides were synthesized using Acidovorax sp.
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strain BoFeN1 according to Hohmann et al.56 and Muehe et al.46, and harvested and processed
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according to Muehe et al.46. Biogenic Fe(III) minerals with two initial Fe(III) to As(V) ratios
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of 250 to 1 and of 50 to 1 [w/w] were investigated, which approximates to 335 to 1 and 67 to 1
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[Fe mol/ As mol] or 0.003 to 1 and 0.015 to 1 [As mol/ Fe mol], respectively.
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For Fe(III) reduction experiments with strain MR-1, 50 mL of freshwater medium were set-up
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in 100 mL bottles and amended with 5 to 7 mM of As-bearing biogenic Fe(III) minerals, 20 mM
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of Na-lactate, and 100 µM of 9,10-anthraquinone-2,6-disulfonate (AQDS). 2×105 cells mL-1 of
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strain MR-1 were added to the set-ups from an iron-free pre-culture grown on 20 mM lactate
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as electron donor and 40 mM fumarate as electron acceptor. The synthesis of the biogenic
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Fe(III) (oxyhydr)oxide and their reduction with MR-1 were performed in the dark at 28°C.
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Aqueous and solid-phase samples were taken at the end of the microbial exponential growth
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phase after 5 days of reduction to ensure the analysis of freshly formed and non-aged secondary
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Fe(II) minerals according to Muehe et al.46.
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Sampling of batch experiments. All experiments were conducted in triplicate. Samples were
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taken anoxically in a N2-filled glove box before and after inoculation with strain MR-1 as well
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as after 5 days. For each time point, three parallel individual bottles were sampled for
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determining the solution chemistry followed by harvesting the minerals for mineralogical
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analyses. Aqueous samples were taken with anoxic syringes and analyzed for total and
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dissolved Fe(II) and Fe(III), total dissolved As and As speciation according to Muehe et al.46.
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Samples for quantification of total dissolved As were frozen at -20°C until measurement. The
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minerals were harvested in the glove box by decanting the supernatant followed by
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centrifugation (13,200 g for 2 min) and vacuum drying of the mineral pellet. Once dry, the
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minerals of three parallel bottles were pooled in equal amounts for mineral analysis.
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Solution chemistry analysis. Total and dissolved Fe(II) and Fe(III) were quantified spectro-
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photometrically using the ferrozine assay according to Stookey, 197057 and following the
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protocol in Muehe et al.46. While Fe(II) was directly quantified with the ferrozine assay, Fe(III)
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was first reduced by 10% hydroxylamine hydrochloride before determining total Fe by
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complexation with ferrozine. Total dissolved As was quantified by diluting thawed samples in
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2% HNO3 and measuring with an inductively-coupled-plasma mass-spectrometer (7700 series
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ICP-MS, Agilent Technologies). Using a high-pressure-liquid chromatography-coupled ICP-
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MS (7700 series ICP-MS, Agilent Technologies), the As redox speciation present before and
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after incubation were quantified in a 10 mM phosphoric acid matrix, which preserves the redox
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state of As.58
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Mineralogical and spectroscopic analyses. All samples for mineral and Fe-As bonding
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environment analysis were prepared under anoxic conditions in glove boxes or bags, dried
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under vacuum, and stored and transported anoxically in the dark. Samples were only exposed
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to ambient air when taken out of the anoxic transporting container followed by direct insertion
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into the analytical device. The mineralogy and the binding environment, location, and
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speciation of Fe and As in the minerals present before (0 day) and during microbial Fe(III)
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reduction (day 5) were investigated using powder X-ray diffraction (XRD), Scanning Electron
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Microscopy (SEM) combined with Energy Dispersive X-ray Spectrometry (EDXS), as well as
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with X-ray Absorption Near-Edge Structure (XANES) and Extended X-ray Absorption Fine
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Structure (EXAFS) spectroscopy at the Fe and As K-edge.
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XRD patterns were acquired using an X’Pert pro Panalytical Diffractometer equipped with an
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X’celerator detector. Samples were deposited on a zero-background Si sample holder and
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loaded into an anoxic sample chamber equipped with a curved Kapton window (constructed by
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the IMPMC project group). Data were collected over the 5 to 80° two-theta range with a 0.017°
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two-theta step, counting 4 hours per sample. Spectra were interpreted using the ICSD database
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for goethite (# 15-9970), vivianite (# 20-0703), and siderite (# 68298).
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SEM analyses of carbon-coated samples were performed with a Zeiss ultraTM 55 electron
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microscope operating at 10 kV with a 60 µm diaphragm in high current mode. Secondary
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electron images were obtained with an in-lens detector and EDXS analyses were collected using
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a Bruker Si-drift detector. The As/Fe and P/Fe molar ratio for specific mineral phases were
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determined by averaging 10 consecutive spot measurements per mineral phase. Semi-
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quantitative analyses and elemental mapping was performed with the program Esprit 1.8(3)
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using a spectra database for standard materials. Raw EDX elemental maps were built using the
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integrated intensities of the Fe L, P K and As L emission lines. EDXS spectra were plotted
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for selected areas by averaging the spectra recorded for the corresponding pixels. Background
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subtracted EDX elemental maps were calculated via semi-quantitative elemental analysis using ACS Paragon Plus Environment
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the background subtracted intensities of the Fe L, P K and As K emission lines in individual
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EDX spectra obtained by binning the mapping data over four pixels.
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X-ray Absorption Spectroscopy (XAS) data at the Fe K-edge were collected in transmission
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detection mode below 20K using a He cryostat on the SAMBA beamline at SOLEIL, Orsay,
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France. Arsenic K-edge data were collected in fluorescence detection mode using a 30 element
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Ge detector on the FAME-BM30B beamline at ESRF, Grenoble, France. Data for the
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As:vivianite model compound were collected in fluorescence detection mode using a 38
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element Ge array detector on the SAMBA beamline. In order to minimize the photo-reduction
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of As under the X-ray beam, all As K-edge data were recorded below 20K using a He cryostat
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and the focused beam (
