Article pubs.acs.org/est Stimulation of Fe(II) Oxidation, Biogenic Lepidocrocite Formation, and Arsenic Immobilization by Pseudogulbenkiania Sp. Strain 2002 † ‡ † ‡ ‡ ‡ ‡ † ‡ Wei Xiu, , Huaming Guo,*, , Jiaxing Shen, Shuai Liu, Susu Ding, Weiguo Hou, Jie Ma, † and Hailiang Dong † State Key Laboratory of Biogeology and Environmental Geology, China University of Geosciences, Beijing 100083, P.R. China ‡ School of Water Resources and Environment, China University of Geosciences, Beijing 100083, P.R. China *S Supporting Information ABSTRACT: An anaerobic nitrate-reducing Fe(II)-oxidizing bacterium, Pseudogulbenkiania sp. strain 2002, was used to investigate As immobilization by biogenic Fe oxyhydroxides under different initial molar ratios of Fe/As in solutions. Results showed that Fe(II) was effectively oxidized, mainly forming lepidocrocite, which immobilized more As(III) than As(V) without changing the redox state of As. When the initial Fe/As ratios were kept constant, higher initial Fe(II) concentrations immobilized more As with higher Asimmobilized/Feprecipitated in biogenic lepidocrocite. EXAFS analysis showed that variations of initial Fe(II) concentrations did not change the As−Fe complexes (bidentate binuclear complexes (2C)) with a fixed As(III) or As(V) initial concentration of 13.3 μM. On the other hand, variations in initial As concentrations but fixed Fe(II) initial concentration induced the co-occurrence of bidentate binuclear and bidentate mononuclear complexes (2E) and bidentate binuclear and monodentate mononuclear complexes (1V) for As(III) and As(V)-treated series, respectively. The coexistence of 2C and 2E complexes (or 2C and 1V complexes) could contribute to higher As removal in experimental series with higher initial Fe(II) concentrations at the same initial Fe/As ratio. Simultaneous removal of soluble As and nitrate by anaerobic nitrate- reducing Fe(II)-oxidizing bacteria provides a feasible approach for in situ remediation of As-nitrate cocontaminated groundwater. ■ INTRODUCTION As(V) were effectively removed from aquatic systems by biogenic Fe(III) oxide precipitation under oxic conditions by Arsenic (As), a prevalent contaminant found in drinking 21 21 ff Gallionella ferruginea, Leptothrix ochracea, Pseudomonas sp. groundwater, a ects hundreds of millions of people via 17 consumption of As-contaminated groundwater.1 Arsenic strain GE-1, etc. and under anoxic conditions by Acidovorax sp. strain BoFeN1,16 Rhodobacter ferrooxidans strain SW2,16 and contamination in groundwater is usually a result of both 16 natural processes and anthropogenic activities. Natural strain KS, among others. processes include geothermal sources, weathering of As-bearing Because groundwater As is usually mobilized under anoxic minerals, and release of mineral-adsorbed As due to microbial conditions, anaerobic nitrate-reducing Fe(II) oxidizers may be beneficial for in situ remediation of As-contaminated ground- activity, whereas anthropogenic activities include mining, wood 22 1−8 water. Different Fe(III) (oxyhydr)oxide phases were formed preservation, and As-containing pesticide utilization. The 16,19,25,26 dominant inorganic species of As in aqueous environments in the presence of anaerobic Fe(II) oxidizers. More- (groundwater and surface water) are arsenate [As(V)] and over, relative to synthetic abiogenic Fe(III) minerals, biogenic 1 Fe(III) minerals are mostly characterized by cell−mineral arsenite [As(III)]. Arsenic(III) is more mobile at neutral pH 23,24 − and 25−60 times more toxic (acute poisoning) than As(V).8,9 aggregates and possibly contain cell Fe/As complexes in − 2− the presence of As(III) and As(V), which would contribute to Arsenate oxyanions (e.g., H2AsO4 and HAsO4 ) and arsenite 16 species (e.g., H AsO 0) are usually adsorbed onto the surfaces greater As immobilization. Furthermore, binding mechanisms 3 3 of As would be expected to vary depending on Fe(III) mineral of Fe(III) (oxyhydr)oxide minerals over a wide pH range, 2 forming strong inner-sphere as well as outer-sphere surface phases, including bidentate binuclear complexes ( C), bidentate − 2 complexes.10 12 mononuclear complexes ( E), and monodentate mononuclear Fe(II) oxidation by Fe(II)-oxidizing bacteria produces not only poorly crystalline Fe(III) oxyhydroxide (e.g., ferrihydrite) Received: February 2, 2016 but also crystalline phases (e.g., green rusts, goethite, and Revised: May 24, 2016 − magnetite),13 20 which have the potential to coprecipitate or Accepted: May 25, 2016 adsorb As. Previous studies showed that both As(III) and Published: May 25, 2016 © 2016 American Chemical Society 6449 DOI: 10.1021/acs.est.6b00562 Environ. Sci. Technol. 2016, 50, 6449−6458 Environmental Science & Technology Article − complexes (1V).27 29 These different As binding modes As(V) molar ratios (37.5, 75, 375, and 750 with initial As(V) contribute to the differences in As immobilization, with 2E concentration of 13.3 μM) and three initial Fe/As(V) molar and 1V complexes showing greater As immobilization than 2C ratios (37.5, 750, and 1500 with the corresponding initial As(V) complexes.6 Therefore, more investigation is required to reveal concentrations of 266.7, 13.3, and 6.67 μM) were set for Fe/ phases of biogenic Fe(III) (oxyhydr)oxides and binding fixed As(V) series and fixed Fe/As(V) series, respectively. The mechanisms of As in the presence of anaerobic Fe(II) oxidizers. higher toxicity of As(III) compared to As(V) made it difficult to Pseudogulbenkiania sp. strain 2002 has been identified as an work with such high initial concentrations of As(III). anaerobic neutrophilic Fe(II)-oxidizer, which oxidizes Fe(II) Therefore, we obtained the similar Fe/As(III) ratios by using nitrate as the electron acceptor and results in the proportionally decreasing initial concentrations of Fe(II) and formation of biogenic Fe(III) minerals.30,31 However, it is As(III). Four initial Fe/As(III) ratios (37.5, 75, 150, and 375 unclear how As immobilization occurs during the formation of with initial As(III) concentration of 13.3 μM) and three initial biogenic Fe(III) oxides induced by strain 2002. The effects of Fe/As(III) ratios (37.5, 75, and 375 with the corresponding As speciation on the formation of biogenic Fe(III) oxides and initial As(III) concentrations of 133.3, 66.7, and 13.3 μM) were the mechanism of As immobilization during the Fe(II) used for Fe/fixed As(III) and fixed Fe/As(III) series, oxidation also are not well understood. respectively. Because of these knowledge gaps, the objectives of this study For biotic control free of As, we used anoxic PIPES (10 mM, ff ff are to (i) investigate the e ects of As species on biogenic Fe(II) pH 7.0) bu er amended with Fe(II) (5 mM, FeCl2) as the sole oxidation by strain 2002; (ii) determine the effects of molar electron donor and nitrate (10 mM) as electron acceptor. Heat- ratios of initial Fe(II) relative to As(III) or As(V) on As killed controls were prepared by pasteurizing the inoculum in removal by biogenic Fe(III)-oxide minerals; (iii) reveal the an autoclave at 121 °C for 20 min. Assays were carried out mechanisms of As(III) and As(V) removal by biogenic Fe(III)- under nongrowth medium without an organic carbon source in ff oxides. an anoxic (100% N2 atmosphere) PIPES bu er (10 mM, pH 7.0) at 30 °C. Supernatant samples were anoxically taken at ■ MATERIALS AND METHODS different time intervals, filtered with 0.22 μm membrane filter, Bacterial Strain and Cultivation Conditions. Pseudogul- and analyzed for total soluble As, As species, Fe species, nitrate, benkiania sp. strain 2002 (ATCC BAA-1479; DSM 18807), an and nitrite. Iron oxide precipitates were sampled following anaerobic nitrate-dependent Fe-oxidizing bacterium isolated repeated centrifugation (1000 rpm for 10 min) and rinsed with from a freshwater lake in Illinois,32 was retrieved from lab stock deionized water. The precipitates were then dried in the (20% glycerol at −80 °C, Geomicrobiology Lab at the China anaerobic glovebox and preserved in anaerobic amber glass University of Geosciences (Beijing)). Strain 2002 was bottles with a headspace of N2/H2 (92.5/7.5, v/v) and analyzed anaerobically cultured to the early stationary growth phase within 1 week. All experiments were conducted in duplicate, with an initial incubation in 5% (v/v) freshwater basal medium and results were reported using averages. · −1 · −1 · −1 · (0.25 g L NH4Cl, 0.6 g L NaH2PO4, 0.1 g L KCl, 0.42 g Sample Analysis. Details on the analysis of total soluble As, −1 · −1 · −1 L NaNO3, 2.52 g L NaHCO3,10mLL vitamin, and 10 As species, total Fe, nitrate, nitrite, and As K-edge X-ray mL·L−1 trace mineral solution). Contents of various absorption spectroscopy can be found in the Supporting components in vitamin and trace mineral solution were Information. Fe(II) concentration was determined spectro- detailed in the Supporting Information. Nitrate (10 mM) and metrically with a modified ferrozine assay at 562 nm.33 Samples acetate (10 mM) were used as the electron acceptor and donor, were mixed with 40 mM sulfamic acid (pH ∼ 1.8) instead of respectively. The cells were harvested by centrifugation (6000g, HCl because the sulfamic acid could react rapidly with nitrite ° 33 10 min, 30 C), washed twice with anoxic (100% N2 and prevent Fe(II) oxidation by nitrite at acidic pH. atmosphere) PIPES [piperazine-N,N-bis(2-ethanesulfonic Solid samples were examined by scanning electron acid)] buffer (10 mM, pH 7.0), and resuspended to serve as microscopy (SEM) to obtain the morphology information an inoculum for nongrowth Fe(II) oxidation experiments. All using a Zeiss Supra 35VP SEM at an accelerating voltage of 3 to chemicals used in this study were of analytical reagent grade, 10 kV, which was equipped with energy dispersive spectroscopy and all volumetric flasks and vessels were cleaned by soaking in (EDS) for chemical analysis.