Environ. Sci. Technol. 2000, 34, 2190-2195
Inhibition of Bacterially Promoted Uranium Reduction: Ferric (Hydr)oxides as Competitive Electron Acceptors B R U C E W I E L I N G A , * ,† BENJAMIN BOSTICK,† COLLEEN M. HANSEL,† R. FRANK ROSENZWEIG,‡ AND SCOTT FENDORF† Department of Geological and Environmental Sciences, Stanford University, Stanford, California 94305-2115, and Department of Biological Sciences, University of Idaho, Moscow, Idaho 83844
The reduction of uranyl (U(VI)) to the relatively insoluble tetravalent form (U(IV)) by Shewanella alga (BrY), a dissimilatory metal-reducing bacteria, was studied in the presence of environmentally relevant iron hydrous oxides. Because this process is dependent on U(VI) being used as the terminal electron acceptor (TEA) during anaerobic respiration, it is important to understand how other potential TEAs might affect this process. When cell suspensions of BrY were added to uranyl acetate (400 µM), uranyl was removed from solution within 10 h. Similarly, uranyl in the presence on goethite (11.1 µmol of U/m2 of solid) underwent dramatic reduction (>90%) with active BrY cells. In contrast, when ferrihydrite was available (0.67 µmol of U/m2 of solid) only 48% of the initial U(VI) was removed after 10 h. When varying ratios of goethite and ferrihydrite were incorporated into cell suspensions, the extent of uranyl reduction was inversely related to the fraction of ferrihydrite present. Increasing uranyl concentrations retarded the inhibition, but the effects were transient. Using Raman spectroscopy, we observed that the initial solid product was UO2.17, but with continued exposure to a reducing environment a relatively pure uraninite phase resulted.
Introduction A consequence of the nuclear age has been the release of radionuclides, toxic heavy metals, and organic co-contaminants into the environment (1, 2). Due in part to historical waste disposal methods, large quantities of these materials now contaminate soils and have migrated to surface and groundwater systems. Because uranium contamination of soils is widespread and extensive, in situ remedial approaches are desirable (2). It has therefore become important to identify processes that can stabilize such contaminants, thus limiting their environmental risk. Biotranformation of wastes containing uranium, other radionuclides, and heavy metals is an attractive alternative to traditional methodologies due to their potential costeffectiveness (3, 4). Several organisms common to soil and * Corresponding author phone: (650)724-3220; fax: (650)725-0979; e-mail:
[email protected]. † Stanford University. ‡ University of Idaho. 2190
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 34, NO. 11, 2000
subsurface environments have now been identified that can enzymatically reduce U(VI) to U(IV) under anoxic conditions. These include the dissimilatory metal-reducing bacteria (DMRB) Geobacter metallireducens (5), Shewanella putrefaciens (6), and Shewanella alga strain BrY (7) as well as the sulfate-reducing bacteria (SRB) Desulfovibrio desulfuricans (8), Desulfovibrio vulgaris (9), and Desuflotomaculum reducens sp. nov. strain MI-1 (10). In its most oxidized state, uranium (U(VI)) forms very stable aqueous complexes (e.g., carbonate, hydroxide), which greatly increase its solubility and mobility in natural water systems (6, 11). In contrast, the reduced state (U(IV)) generally forms phases of limited solubility such as the mineral uraninite (UO2) (4, 6, 11). Microbial reduction of uranyl therefore has the potential to restrict the risk and the transport of uranium in aqueous environments by producing solids of limited solubility. Organisms that can utilize U(VI) as a terminal electron acceptor (TEA) in respiration also commonly have the ability to use a variety of oxidized metals as a TEA. In any given environment there often are several possible electron acceptors and co-contaminants present simultaneously. In attempting to evaluate the potential for in situ metal stabilization, it will be important to understand how the presence of multiple TEAs and contaminants affect this process. In anoxic environments, microorganisms preferentially use different TEAs (NO3-, Mn(IV), Fe(III), SO42-) with the sequence of use principally based on availability and redox potential of the acceptor (12, 13). When metal (hydr)oxides act as the TEA, the mineralogical form of the (hydr)oxide determines its redox potential (12). For example, the reduction potential for Fe(III) in hematite (Fe2O3) will differ appreciably from that in a hydrous ferric oxide (Fe(OH)3‚ nH2O) (E°HFO ) -70 mV, E°Fe2O3 ) -260 mV; 14). In addition, it is well-established that the relative surface area of a particular iron(III) oxide can affect the rate and extent to which the oxide is reduced (12, 15, 16). Truex et al. (17) have recently reported that S. alga strain BrY reduced iron at a rate that was 3 times greater than the rate at which it reduced U(VI) under equivalent conditions. This observation suggests that, in environmental settings, ferric iron may inhibit the reduction of U(VI). In the aforementioned experiments (17), iron was provided as a soluble ferric-citrate complex, whereas in most natural settings iron (hydr)oxides generally predominate (12, 18). In studies on the reduction of U(VI) by D. desulfuricans, Lovley et al. (8) demonstrated that the presence of sulfate had no effect on the rate of U(VI) reduction when both were available. To our knowledge, the reduction of U(VI) by DMRB in the presence of Fe(III) minerals has not been investigated. Shewanella alga strain BrY (hereafter referred to as BrY) is a facultatively anaerobic DMRB that can couple the oxidation of organic acids or H2 to the reduction of iron, manganese, and uranium (7). Here we examine the reduction of uranyl by BrY in the presence of environmentally relevant iron hydrous oxides that have reduction potentials that vary over a range of ca. 200 mV.
Materials and Methods Preparation of Cell Suspensions. Shewanella alga (BrY) (ATCC No. 51181) was maintained on Tryptic Soy Agar (TSA, DIFCO, Detroit, MI) at 25 °C. Cell suspensions were prepared by growing BrY aerobically in Tryptic Soy Broth at 32 °C to late log phase. Cells were harvested by centrifugation (6000g, 15 min), washed twice in 50 mL of anaerobic bicarbonate buffer (2.5 g of NaHCO3/L, pH 7.0), and resuspended in bicarbonate buffer. Cell suspensions were transferred to 10.1021/es991189l CCC: $19.00
2000 American Chemical Society Published on Web 04/21/2000
sterile anaerobic pressure tubes having a headspace of N2 gas, capped with a thick butyl rubber stopper, and stored on ice for less than 15 min before being used to inoculate batch cultures. Heat-killed cells were prepared by holding the cell suspension at 80 °C for 20 min. Uranium reduction studies with mixed iron (hydr)oxides were conducted in batch cultures under nongrowth conditions in 60-mL serum vials containing 50 mL of modified bicarbonate medium (19) with iron (as a hydroxide or oxyhydroxide) at ca. 10 mM final concentration. The medium contained the following components (in g/L, final pH 7.0): NaHCO3, 2.5; NH4Cl, 1.5; KCl, 0.1; CaCl2, 0.05; and 10 mL of each vitamin and mineral solution (20). Phosphate was eliminated from the medium due to its interference with the colorimetric assay used to quantify solution phase U(VI). Serum vials were purged with N2:CO2 at a ratio of 80:20, sealed with thick butyl rubber stoppers, and autoclaved (121 °C, 15 psi, 15 min). After sterilization, uranyl acetate and lactate were added from sterile anaerobic stock solutions to give final concentrations of 400 µM and 10 mM, respectively. Media were inoculated with 1-4 × 107 CFU/mL (on TSA) and incubated at 25 ( 2 °C in the dark without agitation. Studies in which the iron (hydr)oxides were added to provide equivalent surface area (10 m2) were performed in 125-mL serum vials containing 75 mL of bicarbonate buffered medium, and initial uranyl acetate concentration was increased to 1 mM. Competition studies between uranyl and ferrihydrite were conducted under similar conditions with the following changes. Assays were done in 125-mL serum vials containing 100 mL of bicarbonate-buffered medium. Ferrihydrite concentration was reduced and held constant at 5 mM Fe while the uranyl concentration was varied from approximately 0.4 to 1.7 mM. In addition, to avoid the possible ambiguities introduced from sorption reactions, uranyl and lactate were added 24 h prior to the addition of BrY so that systems were at sorption equilibrium. Hematite (Fe2O3; 8.6 m2/g) and goethite (R-FeOOH; 15 m2/g) were purchased from Strem Chemicals (Newburyport, MA). Ferrihydrite (5Fe2O3‚9H2O; 300 m2/g) was synthesized by the titration of Fe(NO3)3‚9H2O (0.4 M) with 1 M NaOH to pH 7 as described by Ryden et al. (21). The resulting ferrihydrite was washed with ultrapure water until the electrical conductivity was
