Reduction of Iron Oxides Enhanced by a Sulfate-Reducing Bacterium ...

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Savannah River Ecology Laboratory, University of Georgia, Aiken, South Carolina 29803, ... Address correspondence to Chuanlun L. Zhang, Savannah River.

Geomicrobiology Journal, 23:103–117, 2006 c Taylor & Francis Group, LLC Copyright  ISSN: 0149-0451 print / 1521-0529 online DOI: 10.1080/01490450500533965

Reduction of Iron Oxides Enhanced by a Sulfate-Reducing Bacterium and Biogenic H2 S Yi-Liang Li Savannah River Ecology Laboratory, University of Georgia, Aiken, South Carolina 29803, USA

Hojatollah Vali Department of Anatomy & Cell Biology, McGill University, Montreal, Quebec, H3A 2B2, Canada

John Yang Environmental Research Center, Lincoln University, Jefferson City, Missouri 65201, USA

Tommy J. Phelps Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, USA

Chuanlun L. Zhang Savannah River Ecology Laboratory, University of Georgia, Aiken, South Carolina 29803, USA

Interactions between bacteria and minerals at low temperatures often lead to accelerated alteration and transformation of mineral phases through dissolution and precipitation. Here we report the reductive dissolution of ferrihydrite, goethite, hematite, and magnetite by the sulfate-reducing bacterium Desulfovibrio desulfuricans strain G-20. The goal of this study was: (1) to investigate iron reduction by G-20 using iron as the sole electron acceptor and (2) to determine whether iron reduction could be enhanced during bacterial sulfate reduction. In the absence of sulfate, G-20 was capable of enzymatically reducing structural Fe3+ from different ironoxide phases including ferrihydrite (4.6% of total iron reduced), goethite (5.3%), hematite (3.7%), magnetite (8.8%) and ferric citrate (23.0%). Enzymatic reduction of goethite and hematite was comparable to abiotic reduction by N2 S using the same medium. Within 3 weeks, the maximum cells-density increased 13-fold in the magnetite culture and 5-fold in the ferric-citrate culture com-

Received 12 July 2005; accepted 31 October 2005. Dr. Judy Wall is acknowledged for providing the G-20 culture. Comments from two anonymous reviewers, and the Editor enhanced the quality of the manuscript. We thank Ms. Jeannie Mui of the Facility for Electron Microscopy Research for assistance in sample preparation for TEM. Financial support for this research was provided by the Donors of the American Chemical Society Petroleum Research Fund (CLZ), the Department of Energy through Oak Ridge National Laboratory (TJP), the U.S. Department of Energy Financial Assistance Award No. DE-FC09-96SR18546 to the University of Georgia Research Foundation (CLZ), and the Natural Sciences and Engineering Research Council of Canada and the Canadian Institute of Health Science (HV). Address correspondence to Chuanlun L. Zhang, Savannah River Ecology Laboratory, University of Georgia, Drawer E, Aiken, SC 29803, USA. E-mail: [email protected]

pared to cell densities at the beginning. In the presence of sulfate, iron reduction was significantly enhanced in all bacterial cultures. The amount of reduced iron was 64.3% of total iron for hematite, 73.9% for goethite, 97.3% for magnetite, and nearly 100% for ferric citrate and ferrihydrite after incubation for 156 hours. The accelerated dissolution of the iron oxides under sulfate-reducing conditions was due to strong interplay between cell growth and redox-reactions between ferric iron and biogenic sulfides. Analysis by transmission electron microscopy and electron-dispersion spectroscopy indicated extensive alteration of the crystals of goethite, hematite, and magnetite, and revealed changes in stoichiometry of iron sulfides after 1 year’s incubation. Keywords

sulfate-reducing bacteria, iron oxides, enzymatic iron reduction, iron sulfide

INTRODUCTION Iron is one of the few major elements that undertakes extensive redox cycling in surface and near-surface environments and plays a critical role in essential biochemical processes. The iron present in the form of (oxyhydr)oxides represents approximately 2 wt.% of the oxidized sediments transported to lacustrine and marine environments on a global scale (Poulton and Raiswell 2002). Iron (oxyhydr)oxides make up a dominant pool of iron in the crust and significantly influenced the evolution of the biosphere and geological features of Earth’s surface. Though sulfate-reducing bacteria (SRB) have long been recognized to be able to reduce ferric iron enzymatically (Jones et al. 1984; Coleman et al. 1993; Lovley et al. 1993), its impact on geochemical cycling of iron is still unclear. Coleman et al. (1993) indirectly indicated that SRB is capable of ferric iron

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reduction in sediment environments; however, studies indicated that only water-soluble Fe3+ , for example, Fe3+ -NTA (Lovley et al. 1993), ferric citrate (Tebo and Obraztsova 1998) or other water-soluble Fe3+ (e.g., Robertson et al. 2001) could be enzymatically reduced by dissimilatory SRB. King and Garey (1999) also studied reduction of ferric oxyhydroxides by dissilimatory SRB. Acid-volatile sulfides are an important reductant of iron oxides and may be the major pathway for the reductive dissolution of iron oxides in sulfidic sediments and euxinic basins (Canfield 1989; Canfield et al. 1992; Krom et al. 2002). The subsequent reduction of iron (oxyhydr)oxides by sulfides constitutes an important mechanism in coupled-cycling of sulfur and iron in the sedimentary environment (e.g., Canfield 1989; Canfield et al. 1992). Abiotic thermal reduction of sulfate by organic matter is limited to temperatures ≥150◦ C (Trudinger et al. 1985; Machel et al. 1995; Machel and Foght 2000); on the other hand, SRB are found to thrive under a wide range of temperatures from extremely cold habitats (e.g., Sagemann et al. 1998) to active hydrothermal vents (Jørgensen et al. 1992; Roychoudhury 2004). Thus, microbial sulfate reduction may be the most significant primary source of sulfide in sediments at temperatures below 100◦ C (e.g., Trudinger 1979; Canfield and Thamdrup 1996). The kinetics and mechanisms of reactions between iron (oxyhydr)oxides and sulfides have been well established. Richard (1974) and Pyzik and Sommer (1981) examined the rates of formation of iron monosulfide during the sulfidation of goethite. Dos Santos Afonso and Stumm (1992) investigated the reductive dissolution of hematite by hydrogen sulfide. Canfield and Berner (1987) estimated rates of in situ reduction of magnetite by dissolved sulfide in anoxic marine sediment. Recently, Poulton (2003) and Poulton et al. (2004) examined the kinetics of dissolved-sulfide removal by ferrihydrite under flow-through conditions. Reductive dissolution of (oxyhydr)oxides by dissolved sulfide has been reported for abiotic systems (e.g., dos Santos Afonso and Stumm 1992; Poulton et al. 2004). The sulfur isotopic effects associated with biological sulfate reduction have also been reported (e.g., Canfield 2001). However, reports on interplays among microbial growth, accumulation of biogenic H2 S, and iron reduction are few. The redox reactions between biogenically reduced sulfur and (oxyhydr)oxides maybe the same as those in the abiotic system (Thamdrup et al. 1993); however, the possible biological enhancement of iron reduction has not been fully evaluated (Neal et al. 2001). In a previous paper (Li et al. 2004), we described iron reduction and alteration of the clay mineral nontronite (NAu-2) by the sulfate-reducing bacterium Desulfovibrio desulfuricans strain G-11 with or without the presence of sulfate. This paper examined the reduction of Fe3+ in a variety of iron oxides (ferrihydrite, magnetite, goethite, and hematite) by D. desulfuricans strain G-20 with or without the presence of sul-

fate. The results demonstrated that reduction of Fe3+ in these iron oxides depended on the bio-susceptibility as well as the crystalline states of the minerals. Furthermore, when sulfate was amended, the reductive dissolution of the iron oxides was greatly enhanced by biogenic H2 S in the presence of enhanced biomass. MATERIALS AND METHODS Preparation of Iron Oxides Ferrihydrite, goethite, and magnetite were prepared in the laboratory according to Schwertmann and Cornell (1991). The synthesized two-line ferrihydrite has a surface area of 200–300 m2 /g. Goethite was synthesized by precipitating reddish brown ferrihydrite, following by aging at 70◦ C for 60 hours. The surface area of the goethite was ∼52 m2 /g (Brunauer et al. 1938) with a grain size of about 1.64 µm along the long axis. Hematite was obtained from Sigma Chemical Co. (St. Louis, MO). Although the surface area was not provided by the vendor, commercial hematite is usually produced by calcination, which results in surface areas of less than 5 m2 /g (Cornell and Schwertmann 1996). Magnetite was prepared by heating oxygen-free Fe(NH4 )2 (SO4 )2 ·6H2 O solution to 90◦ C with N2 as the flushing gas, followed by the addition of a NaNO3 -plus-NaOH solution (oxygen-free) of known volume and concentration. The average size of magnetite was 0.4 µm and the specific surface area was ∼4 m2 /g. To remove the soluble phases and free ions, the synthesized materials were washed several times with deionized water using centrifugation. The purity of synthesized ferrihydrite, goethite, and magnetite was confirmed by X-ray diffraction (XRD) and transmission electron microscopy (TEM). Soluble ferric citrate was obtained from Sigma Chemical Co. (St. Louis, MO). Stock solutions of ferric citrate and slurries of iron oxides were prepared in an anaerobic chamber and stored under nitrogen headspace at 4◦ C. Source and Growth Conditions of the Sulfate-Reducing Bacterium Desulfovibrio desulfuricans belongs to δ-proteobacteria (Postgate 1984; Coleman et al. 1993; Lovley et al. 1993) and is able to use sulfate and other electron acceptors for growth (e.g., Postgate 1984). Strain G-20, derived from D. desulfuricans G100A (Wall et al. 1993), is capable of enzymatic reduction of radionuclides (Payne et al. 2002). The bacterium normally uses lactate as an energy and carbon source for sulfate reduction. The medium for growing G-20 was modified from Caccavo et al. (1992), which contained the following ingredients in 1 L deinoized and distilled H2 O: 2.5 g NaHCO3 , 0.1 g KCl, 0.05 g CaCl2 ·H2 O, 1.5 g NH4 Cl, 0.1 g NaCl, 0.1 g MgCl2 ·6H2 O, 5 g yeast extract, 1 mL vitamin solution and 10 mL minerals solution (Phelps et al. 1989), and 1 mL resazurin (1 mg/L, for indication of redox change). The medium was boiled while being

IRON OXIDE REDUCTION BY A SULFATE-REDUCING BACTERIUM

degassed with N2 , and then dispensed into 160-mL bottles with each containing 40 mL of the medium. The bottles were sealed with butyl-rubber stoppers and aluminum crimp-caps and autoclaved. A sterile stock solution of NaH2 PO4 was added to each bottle at 5 mM final concentration before inoculation, which would serve as the source of phosphate for bacterial growth. Final pH of the medium was 7.2. N2 was used as headspace gas for all incubations. A culture of G-20 was grown to late exponential-phase using SO2− 4 as the electron acceptor and lactate as the electron donor. Cells were collected by centrifugation (in air) and washed four times with distilled water to remove residual sulfur species. Previous studies showed that dissimilatory SRB could survive the oxic procedure and became metabolically active under anaerobic conditions (Canfield and Des Marais 1991; Hao et al. 1996). The washed cell-pellet was dispensed into the same amount of medium (40 mL) in an oxygen-free chamber for all experiments. Lactate (50 mM) was used as the electron donor and ferric citrate or iron-oxide slurries (∼50 mM Fe3+ , final concentration) was used as the electron acceptor, either in the presence or absence of SO2− 4 . The average density of bacteria inoculated at the starting point (0 hours) was 2 × 107 cells/mL. Experimental Design This study was performed using a series of experiments including: (a) abiotic iron reduction by water-soluble sulfide (Control 1), (b) incubation of G-20 without electron acceptors (Control 2), (c) reduction of Fe3+ by yeast extract (Control 3), (d) growing G-20 with iron oxides as the sole electron acceptors, and (e) growing G-20 with iron oxides and SO2− 4 serving as the electron acceptors. All culturing experiments were carried out in the dark at 30◦ C. All reported data were averages of two parallel bottles. In order to avoid any discrepancy caused by physiological changes of the culture, all experiments (except G20 + magnetite + sulfate set) were inoculated from the same bottle of inoculum. All experiments started and were sampled at the same time. Iron reduction in selective experiments was reexamined after incubation for 48 days; mineralogy and mineral chemistry were reexamined by TEM and EDS after 1 year’s incubation. Bacterial Cell Counts One mL of a culture was removed from each bottle at selected sampling times and fixed by adding 0.2 mL of 2% glutaraldehyde. Numbers of cells were determined by acridine-orange direct counts (Zhang et al. 1996). Fixed samples were shaken for 1 minute by a Touch Mixer for homogenization and then diluted 10- to 1000-fold using a filter-sterilized phosphate buffer (pH = 7.2). The dilution factor is determined in such a way that the cell number is controlled at about 50–100 cell per field-view under the microscope. One mL of the diluted sample was mixed with 0.5 mL filter-sterilized and particle-free solution of acridineorange (0.1 g acridine orange in 1 L phosphate buffer at pH 7.2). After 2 minutes the solution was filtered onto a black Nuclepore

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filter (0.2 µm pore-diameter). The filter was then mounted onto a glass slide and viewed under an epifluorescence microscope for cell counts. At least 10 fields of view were examined and the counts of cells were averaged for final report. Measurements of Fe2+ , Fe3+ , and Dissolved Sulfide Acid-extraction can recover near 100% of Fe2+ of iron sulfides by the ferrozine method (data not shown) under anaerobic condition. Thus, “acid volatile sulfides” were not distinguished from the “total reduced sulfides” in this study. The ferrozine method (Stookey 1970; Lovley and Phillips 1987) was employed routinely for rapid analysis of total Fe2+ to avoid variation resulting from aging or oxidization. Water-soluble Fe2+ was determined by the ferrozine method by filtering samples through a 0.2 µm filter. Acid-extractable Fe2+ was determined by treating the sample with an oxygenfree 0.5 M HCl solution for 1 hour, followed by filtration and measurement. Water-soluble Fe3+ in the control experiments was measured according to To et al. (1999). Dissolved sulfide (DS) was measured on filtered (0.2 µm) subsamples by the methylene-blue method (Cline 1969). Concentrations of Fe2+ , Fe3+ and DS were measured on a SUMITSU UV-photometer. TEM, EDS and XRD Morphological and compositional changes of both the starting material and the experimental run-products were investigated by TEM, energy dispersion spectroscopy (EDS), and Xray diffraction (XRD). For TEM analysis, samples were washed with a 1:1 solution of 95% ethyl alcohol and deionized water, dehydrated with 95% ethyl alcohol, and embedded in a lowviscosity Epon epoxy resin. Ultrathin sections (70–100 nm) were cut from the resin blocks with Reichert Ultracut AV ultramicrotome equipped with a Diatome diamond knife. The ultrathin sections were transferred onto 300-mesh formvar-coated Cu grads and imaged in bright-field illumination under conditions of Scherzer defocus with a JEOL JEM-2000FX TEM at an accelerating voltage of 80 kV. The TEM was equipped with a PGT Prism electron dispersive spectroscopy. Samples for XRD were prepared by filtering the run-products onto 0.45 µm glassfiber filters under oxygen-free conditions and rinsing the filters with N2 -bubbled water several times to remove soluble salts. RESULTS Iron Reduction and Speciation without Bacterial Inoculation Control 1: Reduction of iron oxides by inorganic sulfide (Na2 S). Experiments were conducted to determine the extent of iron reduction by dissolved sulfide (Control 1, Table 1) in the same medium as used in cultural experiments described below. In bottles containing ferrihydrite and magnetite, black precipitates occurred immediately upon addition of Na2 S (50 mM); the concentration of dissolved sulfide decreased to

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