Biological Reduction of Np(V) and Np(V) Citrate by Metal-Reducing

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Environ. Sci. Technol. 2007, 41, 2764-2769

Biological Reduction of Np(V) and Np(V) Citrate by Metal-Reducing Bacteria GARY A. ICOPINI,† HAKIM BOUKHALFA, AND MARY P. NEU* Chemistry Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545

Oxidized actinide species are often more mobile than reduced forms. Bioremediation strategies have been developed to exploit this chemistry and stabilize actinides in subsurface environments. We investigated the ability of metal-reducing bacteria Geobacter metallireducens and Shewanella oneidensis to enzymatically reduce Np(V) and Np(V) citrate, as well as the toxicity of Np(V) to these organisms. A toxic effect was observed for both bacteria at concentrations of g4.0 mM Np(V) citrate. Below 2.0 mM Np(V) citrate, no toxic effect was observed and both Fe(III) and Np(V) were reduced. Cell suspensions of S. oneidensis were able to enzymatically reduce unchelated Np(V) to insoluble Np(IV)(s), but cell suspensions of G. metallireducens were unable to reduce Np(V). The addition of citrate enhanced the Np(V) reduction rate by S. oneidensis and enabled Np(V) reduction by G. metallireducens. The reduced form of neptunium remained soluble, presumably as a polycitrate complex. Growth was not observed for either organism when Np(V) or Np(V) citrate was provided as the sole terminal electron acceptor. Our results show that bacteria can enzymatically reduce Np(V) and Np(V) citrate, but that the immobilization of Np(IV) may be dependent on the abundance of complexing ligands.

Introduction Neptunium is often considered less important from a regulatory perspective than U or Pu because of its relatively low concentration in spent nuclear fuel (0.03%). However, 237Np concentrations in spent nuclear fuel increase over time from the decay of 241Am (t1/2 ) 432.7 years). It has been estimated that 237Np will be the most hazardous material remaining in fuel waste over about 10000 to 30 million years following disposal (1). Since 237Np has a long half-life (2.14 × 106 years) and is very toxic, it is important to understand Np speciation and mobility under environmentally relevant conditions. The oxidation state of Np controls its solubility, toxicity, bioavailability, complexation reactions, and migration behavior. Although oxidation states from Np(III) through Np(VII) are possible (2), Np(V) and Np(IV) are dominant under environmental conditions (1). Under oxic conditions at nearneutral pH, Np(V) forms free NpO2+, NpO2(OH), and carbonato complexes (1), which have low affinities for mineral * Corresponding author phone: (505) 667-7717; fax: (505) 6679905; e-mail: [email protected]. † Present address: Montana Bureau of Mines and Geology, Butte, MT 59701. 2764

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surfaces (3, 4) and are mobile in subsurface environments. Under reducing conditions, Np(IV) will likely dominate (1) and form aqueous hydoxo and carbonato species such as Np(OH)n and Np(CO3)m (5). However, Np(IV) is sparingly soluble in the absence of complexing ligands (1), which is similar to the behavior of U(IV) and Pu(IV) (5). This chemical behavior has led to the development of remedial strategies that involve the reduction of oxidized actinide species and the in situ stabilization of reduced actinide species (6). Reducing conditions in the subsurface are created naturally by anaerobic bacteria given sufficient organic carbon, water, and nutrients (6), which can reduce metals enzymatically or indirectly by reaction with the reduced species generated by metabolic activity. Dissimilatory metal-reducing bacteria (DMRB), which reduce metals enzymatically, may play an important role in radionuclide speciation in anoxic environments (7). In natural environments, DMRB typically utilize solid Fe or Mn minerals of various reduction potentials (depending on the mineral structure) as terminal electron acceptors (Figure 1). The lower limit of the redox potentials for possible terminal electron acceptors is controlled by the reduction potential of the c-type cytochromes, which range from -0.233 to -0.400 V (normal hydrogen electrode, NHE) (8, 9). Compounds with reduction potentials greater than -0.400 V are theoretically capable of being reduced by DRMB, and environmentally relevant Np species are within this range as shown in Figure 1, along with potentials for environmentally relevant Fe, Mn, and U species for perspective (see also Table S1 in the Supporting Information). Bacterial reduction of Np(V) to Np(IV) has been reported under nongrowth conditions, although the reduction mechanism was not determined (10). Reduction was speculated to have occurred as a result of either reduction by ascorbic acid or via enzymatic reduction by Shewanella putrefaciens, but reduction alone did not remove Np from solution (10). Only with the addition of a phosphate-producing bacterium was Np precipitated. The biological reduction of Np(V) with the subsequent precipitation of Np(IV) has been observed in an anaerobic consortium of sulfate-reducing bacteria (11). While these studies demonstrate that bacteria can reduce Np(V) to Np(IV), it is not clear whether the reduction was a result of enzymatic activity or indirect reduction resulting from reactions with reduced species, such as Fe2+ or S2(12). Here we present data on the interactions of Geobacter metallireducens GS15 and Shewanella oneidensis MR1 with Np(V) in monocultures. The ability of these organisms to reduce both Np(V) and Np(V) citrate under cell suspension and growth conditions was the focus of this study. The toxicity of Np(V) to these organisms is also an important bounding condition and therefore is also presented.

Experimental Section All solutions were prepared with doubly distilled water. Np(V) stock solutions were prepared by digestion of neptunium carbide in concentrated HClO4. The neptunium(VI) stock solutions were reduced to Np(V) by the addition of sodium sulfite. The purity of the Np(V) stock solution (∼99%) was monitored by UV-vis spectroscopy (described below). The pH of the Np(V) stock solution was raised to above 9, which caused Np(V) to precipitate, presumably as NpO2(OH). The solution was centrifuged at 5000 rpm for 15 min, washed with 0.10 M NaOH, and then redissolved with 1.0 M HCl. The pH was then raised to approximately 3, and aliquots were dispensed into anoxic, doubly distilled water in sealed 10.1021/es0618550 CCC: $37.00

 2007 American Chemical Society Published on Web 03/09/2007

FIGURE 1. Distribution of the redox potential (mV) for Np and U complexes along with the redox potential of representative mineral phases at neutral pH. Redox data for Np species are from Table S1 (in the Supporting Information) and those for uranium from ref 30. serum bottles to create stocks of the appropriate Np(V) concentrations for the individual experiments. Stock cultures of G. metallireducens and S. oneidensis were obtained from the American Type Culture Collection. Cultures were grown anaerobically at pH 7.0 on ferric citrate growth medium containing the following constituents: 3.4 g/L NaOH, 12.25 g/L ferric citrate anhydrous, 0.25 g/L NH4Cl, 1.08 g/L glycerol 2-phosphate, 0.025 g/L KCl, 10.46 g/L MOPS, 10.0 mL/L mineral stock solution, 10 mL/L vitamin stock solution, and 10 mM acetate (Geobacter) or 20 mM lactate (Shewanella). The vitamin stock solution contained the following constituents: 0.02 g/L biotin, 0.02 g/L folic acid, 0.10 g/L pyridoxine hydrochloride, 0.05 g/L thiamine hydrochloride, 0.05 g/L riboflavin, 0.05 g/L nicotinic acid, 0.05 g/L pantotheric acid, 0.05 g/L thiottic acid, 2.0 g/L choline chloride, and 0.01 g/L vitamin B12, (pH 7.0). The mineral stock solution contained the following constituents: 1.5 g/L NTA, 3.0 g/L MgSO4‚H2O, 0.5 g/L MnSO4‚H2O, 1.0 g/L NaCl, 0.1 g/L FeSO4‚7H2O, 0.1 g/L CoCl2‚6H2O, 0.1 g/L CaCl2, 0.1 g/L ZnSO4‚7H2O, 0.1 g/L Al,K(SO4)2‚12H2O, 0.1 g/L H3BO3, and 0.1 g/L Na2MoO4‚2H2O, (pH 6.5). Oxygen was removed from the medium by bubbling high-purity (UHP) Ar gas through the solutions. The solutions were then sealed in serum bottles and autoclaved.

All experiments were conducted using sealed serum bottles. Anaerobically grown bacteria were pelleted, washed once with anoxic 100 mM MOPS, and then resuspended in fresh anoxic 100 mM MOPS. The cell density of the stock culture used to inoculate the experimental cultures was quantified by optical density, for which a calibration curve was developed using direct cell counts. Manipulations of cultures containing Np were done within a radiological fume hood. All experimental cultures were incubated at 30 °C. The toxicity of Np(V) to G. metallireducens and S. oneidensis was assessed by adding Np(V) to cultures of these organisms grown on ferric citrate growth medium (described above) and monitoring the decrease in growth relative to the growth of controls without Np(V). The initial cell density for the toxicity experiments was 2 × 107 cells/mL. Cells used in all experiments were harvested in the late log stage of growth. Concentrations used in the toxicity studies were 0.10, 0.50, 2.0, 4.0, 6.0, and 10.0 mM Np(V). Growth was monitored by measuring the optical density of cells that had been pelleted and resuspended in the same volume of doubly distilled water containing 100 mM MOPS. The relative toxicity is defined as the ratio of the cell density after 48 h of growth in the Npcontaining cultures to the cell density in control cultures without Np inoculated at the same time and under the same conditions. All toxicity experiments were conducted in triplicate. For select toxicity experiments, Fe(II) was measured using the ferrozine method (13). The ability of G. metallireducens and S. oneidensis to reduce Np(V) and Np(V) citrate was assessed using cell suspensions of approximately 4 × 108 cells/mL in 100 mM MOPS at pH 7.0 with 20 mM acetate (Geobacter) or lactate (Shewanella) as the electron donor. The effect of Np(V) complexation on Np(V) reduction was examined by adding 50 mM sodium citrate to the cultures. The initial Np(V) concentration for all cell suspension experiments was 0.50 mM Np(V). In addition to the experimental conditions, each cell suspension experiment also had controls consisting of no cells, no electron donor, and heat-killed cells. All control and experimental conditions were conducted in triplicate. Growth of G. metallireducens and S. oneidensis with Np(V) as the sole terminal electron acceptor was examined at pH 7.0 using the growth medium described above with the following exceptions: no NaOH, no iron(III) citrate, 20 mM acetate (Geobacter) or lactate (Shewanella), and 20.92 g/L MOPS (100 mM). The initial cell density for the growth experiments was 2 × 107 cells/mL. The initial concentrations of Np(V) for the growth experiments were 1.0, 2.0, and 4.0 mM. In addition to the experimental condition, each growth experiment also had controls consisting of no cells and no electron donor. Samples were collected using sterile syringes with metal needles that were purged with sterile Ar. Generally, 1.0-1.4 mL of culture suspension was collected for each sample. For the cell suspension experiments, 0.6 mL was filtered through a 0.2 µm PTFE filter (Millex SLLG013SL) into a vessel containing 15 µL of concentrated HCl. Concentrated HCl was used to immediately lower the pH to below 2 in an effort to prevent Np(IV) from reoxidizing to Np(V). The filtered sample was mixed with 2.0 M HClO4 in a 1:1 volume ratio, giving a final concentration of 1.0 M HClO4. The concentrations of Np(V) and Np(IV) were determined spectrophotometrically by measuring the maximum absorbance at 980 and 727 nm corresponding to Np(IV) and Np(V), respectively. Molar absorptivity coefficients of 395 M-1 cm-1 for Np(V) and 127 M-1 cm-1 for Np(IV) were used (14). The unfiltered aliquots were digested with a 1:1 dilution of 2.0 M HClO4 overnight, then filtered through a 0.2 µm filter, and analyzed for Np(V) and Np(IV), as described above. In solutions without citrate added, Np(IV) was insoluble and the concentration was quantified using the acid digestion solution. In solutions VOL. 41, NO. 8, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. Time-dependent variation of Np(V) and Np(IV) concentrations in the presence of cell suspensions of S. oneidensis and G. metallireducens. Conditions: cell density of S. oneidensis and G. metallireducens, 4 × 108 cells/mL; [Np(V)] ) 0.50 mM; pH 7.0; T ) 30 °C. Key: (2) G. metallireducens live cells with acetate Np(V), (9) S. oneidensis live cells with lactate Np(V), (b) S. oneidensis live cells with lactate Np(IV), (O) S. oneidensis lactate-free control Np(V), (]) S. oneidensis cell-free control Np(V), (0) S. oneidensis heat-killed cell control Np(V).

FIGURE 2. Relative toxicity of complexed Np(V) on S. oneidensis (A) and G. metallireducens (B). The relative toxicity is defined here as a decrease in growth relative to the growth of controls with no Np(V) added. with citrate, Np(IV) remained soluble and the concentration was quantified in the filtered aliquot.

Results Np(V) Toxicity to G. metallireducens and S. oneidensis. The toxicity of Np(V) to these organisms is an important bounding condition, because it may influence the ability of DMRB to utilize Np as a terminal electron acceptor. The toxicity was determined by comparing the growth of these organisms in the presence of increasing concentrations of Np(V) to growth in Np-free medium. The growth medium used in these experiments contained ferric citrate, which resulted in the formation of Np(V) citrate complexes (see the Supporting Information). The data in Figure 2 show the relative toxicity of Np(V) citrate complexes on S. oneidensis and G. metallireducens in 50 mM ferric citrate growth medium. The toxicity is defined here as a function of decreased growth relative to the growth of a no-Np(V) control. A decrease of at least 50% in growth is considered a significant toxic effect (15). The data in Figure 2 show that concentrations of e2.0 mM Np(V) did not inhibit the growth of either organism. On the other 2766

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hand, concentrations of g4.0 mM Np(V) did significantly inhibit growth. Analysis of the toxicity cultures grown with 2.0 mM Np(V) showed that both iron(III) citrate and Np(V) citrate were rapidly reduced to their lower oxidation states (Figure S1 in the Supporting Information). Np(V) reduction along with Fe(III) reduction was observed for G. metallireducens and S. oneidensis. Enzymatic Reduction of Np(V) by G. metallireducens and S. oneidensis in Cell Suspensions. The ability of these organisms to utilize Np(V) as a terminal electron acceptor was assessed in cell suspensions with 0.50 mM uncomplexed Np(V) (without ferric citrate). The disappearance of Np(V) from solution and the appearance of Np(IV) in the precipitated phase were observed for cultures with S. oneidensis (Figure 3). The production of Np(IV) was monitored by solubilizing the Np(IV) precipitates with a 1.0 M HClO4 digestion. The unreduced fraction of Np(V) remained soluble throughout the experiment, and concentrations were consistent between the filtered aqueous and acid-digested aliquots for a given sample. The rate of Np(V) reduction was very slow compared with the rate of reduction of other metals (see, e.g., refs 6 and 16). However, both the heat-killed and cell-free controls showed little or no decrease in Np(V) concentration and Np(IV) production. Also, the experimental control with live cells and without lactate showed an approximately 20% overall decrease in Np(V) concentrations. Some metal reduction in the absence of an electron donor (i.e., lactate) has been observed by others and is not unexpected (16). These data indicate that S. oneidensis is capable of direct enzymatic reduction of aqueous Np(V). In addition, at the end of the experiment the cultures containing precipitated Np(IV) were exposed to atmospheric oxygen for a period of one week, and no reoxidation of Np(IV) was observed during that time. Neither a decrease in Np(V) nor production of Np(IV) was observed in G. metallireducens cell suspension experiments with 0.50 mM Np(V) (Figure 3). This experiment was

FIGURE 4. Direct reduction of Np(V) citrate by a cell suspension of S. oneidensis. Conditions: cell density 4 × 108 cells/mL suspended in 100 mM MOPS, [Np(V)] ) 0.50 mM, [citrate] ) 50 mM, T ) 30 °C. Key: (9) S. oneidensis live cells with lactate Np(V), (b) S. oneidensis live cells with electron donor Np(IV), (O) S. oneidensis lactate-free control Np(V), (]) S. oneidensis cell-free control Np(V), (0) S. oneidensis heat-killed cell control Np(V). performed (in triplicate) on three separate occasions with the same result each time. It is possible that 0.50 mM uncomplexed Np(V) is toxic to G. metallireducens, and therefore, it was unable to reduce Np(V). The toxicity data indicated that cultures of G. metallireducens were able to grow normally in up to 2.0 mM Np(V); however, the toxicity data were for Np(V) complexed with citrate. Np(V) Respiration by G. metallireducens and S. oneidensis To Support Growth. The ability of both G. metallireducens and S. oneidensis to utilize uncomplexed Np(V) as a terminal electron acceptor for growth was assessed with growth cultures containing initial concentrations of 1.0 and 2.0 mM unchelated Np(V) for S. oneidensis and 2.0 mM Np(V) for G. metallireducens. These concentrations of Np(V) were in excess of the solubility for NpO2(OH) (17) and resulted in aqueous concentrations of between 0.5 and 1.0 mM Np(V) depending on the time of analysis and initial concentration. The 1.0 M HClO4 digestion was used to quantify Np concentrations in these cultures. Since solid Fe and Mn substrates are normally utilized by these bacteria in nature and since there was a significant amount of Np(V) remaining in solution, the precipitation of Np(V) was not likely to have been a limiting factor for the growth of these organisms. Cultures were monitored for 4-7 days. There were no observed increases in cell density, decreases in Np(V), or production of Np(IV) during any of the growth experiments, indicating that these bacteria could not utilize Np(V) for growth under these conditions. Enzymatic Reduction of Chelated Np(V) by G. metallireducens and S. oneidensis in Cell Suspensions. Np(V) was added to the bacterial cell suspensions containing a large excess of citrate (50 mM sodium citrate) to ensure that Np(V) was present as a citrate complex, NpV(citrate)2-. The results obtained for S. oneidensis cell suspensions indicate that the addition of citrate to the medium caused an increase in the rate of Np(V) reduction (Figure 4) relative to rate in the cultures without citrate (Figure 3). The Np(IV) did not produce an insoluble precipitate in the citrate-amended

FIGURE 5. Direct reduction of Np(V) citrate by a cell suspension of G. metallireducens. Conditions: cell density 4 × 108 cells/mL suspended in 100 mM MOPS, [Np(V)] ) 0.50 mM, [citrate] ) 50 mM, T ) 30 °C. Key: (9) G. metallireducens live cells with acetate Np(V), (b) G. metallireducens live cells with acetate Np(IV), (O) G. metallireducens acetate-free control Np(V), (2) G. metallireducens acetate-free control Np(IV), (]) G. metallireducens cell-free control Np(V), (0) G. metallireducens heat-killed cell control Np(V). cultures and remained soluble throughout the experiment. In citrate-amended cultures containing S. oneidensis all of the Np(V) was reduced to Np(IV) within 24 h (∼85% within 8 h), whereas in cultures without citrate only approximately 12% of the Np(V) was reduced within 24 h. A more pronounced change is observed in G. metallireducens cell suspensions with citrate added (Figure 5). Approximately 90% of the Np(V) added to the citrateamended G. metallireducens cultures was reduced to Np(IV) within 24 h, whereas no reduction was observed in cultures without citrate added. The experimental control cultures with live cells and no electron donor (acetate) showed the same Np(V) reduction profile as the cultures with live cells and acetate, which was also reflected in the Np(IV) production rate. Again, this is not wholly unexpected and has been observed by others in similar systems (18). As with the S. oneidensis citrate-amended cultures, the Np(IV) remained soluble throughout the experiment. The controls with heatkilled cells and no cells showed a slight decrease in Np(V) concentration for both bacteria, but no evidence of Np(IV) production for either bacteria. The concentration of Np(V) in these experiments was near the solubility limit for NpO2(OH) (17), and the slow decrease in concentration is likely the result of precipitation of NpO2(OH). Np(V) Citrate Respiration by G. metallireducens and S. oneidensis To Support Growth. The ability of both G. metallireducens and S. oneidensis to utilize uncomplexed Np(V) citrate as a terminal electron acceptor for growth was assessed with growth cultures containing an initial concentration of 2.0 mM Np(V) with 50.0 mM citrate. The cultures were monitored for 7 days (data not shown), and no decreases in Np(V), appearance of Np(IV), or increases in cell densities were observed. Np(V) remained soluble throughout the experiment.

Discussion Np(V) Toxicity to S. oneidensis and G. metallireducens. The toxicity of radionucides to microorganisms is a critical factor VOL. 41, NO. 8, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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in assessing the viability of approaches to bioremediation. Radionuclide toxicity to microorgansims differs from other metal toxicities by having a potential radiological toxicity, as well as a chemical toxicity (15, 19, 20). Neptunium’s decay mode is mainly R, and its radiological toxicity should be lower than that of plutonium (62.1 mCi/g for 239Pu and 0.70 mCi/g for 237Np). However, its chemical toxicity appears to be greater than that of Pu. The concentrations of Pu(IV), U(VI), and Np(V) found to have a toxic effect on D. radiodurans were determined to be 5, 2.5, and 1.6 mM, respectively (15). Similary, the toxic effect of 2-4 mM Np(V) citrate on S. oneidensis observed in this study is greater than that of Pu(VI) (6-7 mM) on S. putrefaciens (15). An apparent growth stimulation at low concentrations was observed (Figure 1), which is similar to observed metal toxicity trends with aerobic bacteria (15) and is not likely a result of Np(V) respiration. The medium used in the toxicity experiments contained excess citrate, and Np was present mainly as a Np(V) citrate complex (see the Supporting Information). Therefore, the toxicity observed here is more likely due to Np(V) citrate complexes rather than unchelated Np(V). Np(V) complexation has been observed to reduce its toxicity to other bacteria (21), and it is likely that uncomplexed Np(V) is more toxic to G. metallireducens and S. oneidensis than Np(V) citrate. The reduction in Np(V) toxicity through complexation by citrate might be due to the reduction of Np(V) bioavailability, because the smaller uncomplexed molecule can more easily enter the cell. Since intracellular transport is not required for Np reduction, the increased toxicity with increased bioavailability is not contradicted by the increased reduction rates observed for complexed Np(V). Np(V) Speciation and Accessibility of the Species Formed to Bacterial Reduction. The accessibility of oxidized neptunium species to direct and indirect bacterial reduction depends on the species formed and their redox potentials. Under oxic conditions, the neptunyl ion (NpO2+), NpO2(OH), and NpO2CO3- will be the dominant aqueous species at nearneutral pH, with the dominance of any given species depending on the pH and pCO2 (1) (see also the Supporting Information). Under our experimental conditions NpO2+ is the important species in solution in the absence of citrate. The addition of organic ligands with hard oxygen donor groups such as citrate, NTA, EDTA, and oxalate causes a redistribution of species with the formation of complexes such as NpO2-L (where L is the ligand). The formation of these complexes is likely to favor the reduction of Np(V) to Np(IV), because the affinity of these ligands toward Np(IV) binding is higher than that of Np(V). The complexation of Np(V) by citrate leads to the formation of 1:1 NpO2+-citrate, whose stability constant is small (log β110 ) 2-4) (22). There are no data available for the complexation of Np(IV) by citrate, but data for Pu(IV) (log K ) 18.8) and U(IV) (log K ) 9.72) suggest that citrate will form a very strong complex with Np(IV) (22). This differential binding affinity will create a thermodynamic driving force for the formation of neptunium(IV) citrate. However, this thermodynamic argument cannot explain why the reduction rate is faster for Np(V) citrate relative to Np(V) without detailed kinetic experiments. Reduction of Uncomplexed Np by S. oneidensis and G. metallireducens. The ability of these organisms to reduce U(VI) to U(IV), as well as other metals, has been demonstrated under cell suspension (nongrowth) (23) and growth conditions (6), and these observations were the basis for selecting these organisms for this study. However, as previously mentioned uncomplexed Np(V) is very toxic, which may interfere with the ability of these bacteria to effectively reduce Np(V). The toxicity of Np(V) was the likely cause for the observed lack of uncomplexed Np(V) reduction in cell suspensions of G. metallireducens and the apparent inability of either organism to grow with Np(V) as the sole electron 2768

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acceptor. Cell suspensions of S. oneidensis were able to reduce Np(V), but the rate of Np(V) reduction was much slower than reduction rates reported for U(VI) and other metals by cell suspensions of S. oneidensis under similar conditions (23). Although the reduction rate is slow, the fact that no reduction was observed in the control solutions indicates that enzymatic reduction of Np(V) was occurring. Similarly slow Np(V) reduction rates have been observed during growth of an anaerobic bacterial consortium (11). A possible explanation of the slow Np(V) reduction rate is related to Np(V) toxicity. Shewanella species are capable of producing electron-shuttling compounds that transfer electrons from the cell to metals and thereby utilize metals for respiration indirectly (24, 25). S. oneidensis might not be able to reduce Np(V) directly due the toxicity of Np(V), but indirectly by way of an electron shuttle. Reduction of Np(V) Citrate by S. oneidensis and G. metallireducens. The addition of citrate as a complexing ligand to metal-reducing cultures has been shown to have very different effects depending on the bacteria involved. The addition of citrate to Shewanella alga increased the reduction rate of U(VI), whereas the addition of citrate to Desulfovibrio desulfuricans decreased the rate of U(VI) reduction (26). The addition of citrate has also been observed to cause the complete cessation of U(VI) reduction by Anaeromyxobacter dehalogens (27). In the experiments reported here, the addition of citrate significantly enhanced the reduction of Np(V). Cell suspensions of both S. oneidensis and G. metallireducens were able to reduce Np(V) citrate to neptunium(IV) citrate, which remained in solution. Np(IV) was not detected in the cell-free, heat-killed, or lactate-free controls, which indicated that these bacteria were enzymatically reducing Np(V) citrate. The apparent rate of Np(V) citrate reduction in S. oneidensis cell suspensions was much faster than the apparent reduction rate of uncomplexed Np(V) under the same conditions, but the rate was still much slower than reduction rates for other metals. The addition of citrate enabled the reduction of Np(V) by G. metallireducens. This phenomenon is presumably due to the lower toxicity of Np(V) citrate compared to NpO2+, which G. metallireducens was unable to reduce. Cell suspensions of G. metallireducens reduced approximately 90% of Np(V) citrate within 50 h of initiating the experiment. Again, this reduction rate is much slower than reduction rates reported for U(VI) and other metals by cell suspensions of G. metallireducens under similar conditions (23). However, it is within the range of observed reduction rates for other metalreducing bacteria (18). The reduction of Np(V) citrate in the acetate-free control was essentially identical to the experimental condition with acetate added. This phenomenon has been observed before (18, 28) and has been attributed to endogenous electron donor reserves where no electron donor is provided. The pronounced effect of citrate on the reduction of Np(V) shows the important effect of organic chelators on bioreduction of metals. Citrate enhanced the rate of Np(V) reduction by S. oneidensis cell suspensions and enabled the reduction of Np(V) by G. metallireducens in part because citrate reduced the toxicity of Np(V) to these microorganisms. The presence of organic chelators capable of strongly complexing the reduced metal species can undermine the precipitation of the reduced form of the metal. The reduction of Np(V) citrate did not produce any precipitation, and the reduced form, neptunium(IV) citrate, remained soluble. Growth was not observed for either bacterium when 2 mM Np(V) citrate was provided as the terminal electron acceptor. The toxicity of Np species may limit the ability of these organisms to utilize Np species for anaerobic respiration. The concentration of Np at contaminated sites is often very low and unlikely to be sufficient to support bacterial

growth at any rate. However, the bioaugmentation of metalreducing bacteria is likely to cause the reduction of Np(V) to Np(IV) either enzymatically or indirectly by reaction with biogenic species (e.g., Fe2+ and sulfide) (12, 29). However, the immobilization of Np(IV) will depend on the abundance of complexing ligands. Bacterial Np(V) Reduction and in Situ Immobilization of Np. The reduction of the oxidized neptunium Np(V) to Np(IV) results in the precipitation of Np(OH)4 and/or NpO2. These solid phases form and are stable in the absence of carbonate or any organic ligands capable of solubilizing Np(IV). The thermodynamic data describing Np(IV) complexation by natural organic ligands are very limited, which makes it difficult to predict the behavior and long-term stability of Np(IV) species in the environment. The reduction of Np(V) species to their tetravalent oxidation state may be a viable technique for the in situ immobilization of Np(V) in the absence of complexing ligands. However, a better understanding of the complexation chemistry and potential reoxidation of Np(IV) species is needed to fully evaluate this remedial approach.

Acknowledgments Preparation of this paper and the research reported herein were supported by the Natural and Accelerated Bioremediation Research Program (NABIR) within the Environmental Remediation and Sciences Division, Office of Biological and Environmental Research of the U.S. Department of Energy. Los Alamos National Laboratory is operated by the University of California for the U.S. Department of Energy.

Supporting Information Available Figure S1 showing the reduction of Fe(III) and Np(V) by S. oneidensis, Figures S2 and S3 showing the speciation diagrams for Np(V) in the presence of carbonate and citrate, Table S1 showing the calculated reduction potentials of various Np(V) species, and Table S2 containing relevant thermodynamic values for neptunium complexes used in this study. This material is available free of charge via the Internet at http:// pubs.acs.org.

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Received for review August 2, 2006. Revised manuscript received December 13, 2006. Accepted January 17, 2007. ES0618550 VOL. 41, NO. 8, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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