Chemical Speciation and Association of Plutonium with Bacteria

Mar 24, 2007 - Japan, The National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8567, Japan, Eco Topia. Science Insti...
0 downloads 0 Views 216KB Size
Download PDF
Environ. Sci. Technol. 2007, 41, 3134-3139

Chemical Speciation and Association of Plutonium with Bacteria, Kaolinite Clay, and Their Mixture T O S H I H I K O O H N U K I , * ,†,‡ T A K A H I R O Y O S H I D A , †,§ T A K U O O Z A K I , † NAOFUMI KOZAI,† FUMINORI SAKAMOTO,† TAKUYA NANKAWA,† Y O S H I N O R I S U Z U K I , †,| A N D AROKIASAMY J. FRANCIS⊥ Advanced Science Research Center, Japan Atomic Energy Agency, Tokai, Ibaraki 319-1195, Japan, Geological Survey Japan, The National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8567, Japan, Eco Topia Science Institute and Department of Materials, Physics, and Energy Engineering, Graduate School of Engineering, Nagoya University, Furocho, Chikusa-ku, Nagoya 464-8603, Japan, and Environmental Sciences Department, Brookhaven National Laboratory, Upton, New York 11973

We investigated the interactions of Pu(VI) with Bacillus subtilis, kaolinite clay, and a mixture of the two to determine and delineate the role of the microbes in regulating the environmental mobility of Pu. The bacteria, the kaolinite, and their mixture were exposed to a 4 × 10-4 M Pu(VI) solution at pH 5.0. The amount of Pu sorbed by B. subtilis increased with time, but had not reached equilibrium in 48 h, whereas equilibrium was attained in kaolinite within 8 h. After 48 h, the oxidation state of Pu in the solutions exposed to B. subtilis and the mixture had changed to Pu(V), whereas the oxidation state of Pu associated with B. subtilis and the mixture was Pu(IV). Exudates released from B. subtilis reduced Pu(VI) to Pu(V). In contrast, there was no change in the oxidation state of Pu in the solution or on kaolinite after exposure to Pu(VI). Scanning electron microscopy-energy dispersive spectrometry analysis indicated that most of the Pu in the mixture was associated with B. subtilis. These results suggest that Pu(IV) is preferably sorbed to bacterial cells in the mixture and that Pu(VI) is reduced to Pu(V) and Pu(IV).

Introduction A major environmental concern is the possibility of the migration of plutonium released from nuclear weapon tests, accidents at nuclear power plants, reprocessing of nuclear fuels, and from the disposal of radioactive waste (1-3). Since the mobility of Pu is determined by its interaction with soils and rocks, many studies have explored its adsorption by soil * Corresponding author phone: (81) 29 282 5361; fax: (81) 29282 5927; e-mail: [email protected]. † Japan Atomic Energy Agency. ‡ Eco Topia Science Institute, Nagoya University. § The National Institute of Advanced Industrial Science and Technology. | Graduate School of Engineering, Nagoya University. ⊥ Brookhaven National Laboratory. 3134

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 9, 2007

minerals (4-7). A sorption database compiled by Yui et al. (8) shows that most studies focused on the interaction of Pu with inorganic components, but few on those with microorganisms. The former revealed that the Kd value, i.e., the ratio of Pu adsorbed on an adsorbent to that dissolved in solution, ranged between 103 and 105 mL g-1, demonstrating the high sorption ability of inorganic substances (9-13). Microorganisms, ubiquitous in the environment, also have a high capacity to sorb and bind actinides on their surfaces (14-18), and may affect the movement of Pu; however, our knowledge of their role in the migration of Pu is limited (18-21). Plutonium can exist in several oxidation states in the environment; as III, IV, V, and VI (22). In aqueous solutions, Pu(III) and Pu(IV) are present as simple cations, while Pu(V) and Pu(VI) exist as dioxo cations, such as PuO2+ and PuO22+, respectively (23). Various physical, chemical, and biological processes alter Pu speciation (24-27). Hence, the presence of multiple Pu species and their complex chemistry make it difficult to predict their migration behavior in the environment. Since the sorption of actinides depends, in principle, on the effective charge, Pu(IV) has a higher propensity for sorption than Pu(III), Pu(V), and Pu(VI). Approximately 7090% of Pu was present as Pu(VI) in chlorinated drinking waters and in neutral and basic brines (28, 29). In neutral waters, Pu(V) predominates (30). These studies suggest that the behavior and movement of the more mobile species of Pu(V) and Pu(VI) should be investigated. In this study, we investigated the association of Pu(VI) with a common soil bacterium, Bacillus subtilis, with clay (kaolinite), and with a mixture of the two. This microorganism and kaolinite are widespread in the terrestrial environment, and their surfaces are well characterized.

Materials and Methods Microorganism, Kaolinite, and Pu Solution. B. subtilis (IAM 1069), a Gram-positive, rod-shaped heterotrophic bacterium, was obtained from the Institute of Molecular and Cellular Biosciences, The University of Tokyo. The cells were grown for 40-48 h in 500 mL conical flasks at 30 °C in 250 mL of sterilized liquid growth medium containing meat extract (3 g L-1), polypeptone (5 g L-1), and NaCl (5 g L-1). They were harvested at the stationary growth phase by centrifugation at 2580 x g for 10 min, washed twice by 0.1 M NaCl, resuspended in a sterile 0.01 M NaCl solution, and immediately used in the experiments. An aliquot of the cell suspension was centrifuged and dried overnight at 70 °C to determine the dry weight of the cells. We used commercial kaolinite from Nihon Chikagakusha Co. Ltd., Kyoto, Japan; X-ray powder diffraction identified it as the only mineral present. The specific surface area of kaolinite determined by the BET method was 26.4 m2 g-1, and its cation exchange capacity at pH 7 was 3.3 mequiv/100 g. The kaolinite was washed twice with distilled deionized water and suspended as a slurry (100 g L-1). A 239Pu stock solution was purified by ion exchange chromatography using a Dowex AG1-X8 anion exchange resin. Pu in an 8 M HNO3 solution was loaded into the resin column, which then was washed with this solution. The sorbed Pu was eluted with a 0.2 M HNO3 solution. The Pu(VI) oxidation state was obtained by heating it in a concentrated HClO4 solution on a hot plate by fuming after the organic compounds in the solution were destroyed with concentrated nitric acid solution. The stock solution was 10.1021/es061207g CCC: $37.00

 2007 American Chemical Society Published on Web 03/24/2007

diluted to 20 mL containing 0.08 M Pu(VI). The purity of the oxidation state was verified by UV/vis spectroscopy. Accumulation Experiments. Samples consisting of 40 g L-1 kaolinite or 1.5 g L-1 B. subtilis were incubated in a 0.01 M NaCl solution containing 4 × 10-4 M Pu(VI) for up to 48 h at pH 5.0 ( 0.1 maintained by adding 1 M NaOH or HCl at 0, 2, 8, 18, and 24 h. Duplicate samples of Pu(VI) were incubated at room temperature in polypropylene centrifuge tubes previously washed with a 0.1 M HClO4 solution and rinsed with deionized water. Aliquots of 1 mL of the slurry were removed at 2 min and 2, 3, 8, 18, 24, and 48 h for measuring Pu concentrations and at 2 min and 2, 18, and 48 h for determination of Pu oxidation states. The solution and the bacteria or the clay from the samples were separated by centrifugation for 10 min at 2580 x g unless otherwise noted. The oxidation state of the Pu(VI) was verified immediately before each experiment by the spectroscopy described below. To determine the accumulation of Pu(VI) by a mixture of the bacterium and the clay, B. subtilis at 0.75 g L-1 and kaolinite at 20.0 g L-1 were incubated with 4 × 10-4 M Pu(VI) solution for 48 h at pH 5.0 ( 0.1. The concentrations of plutonium were measured at 2, 18, and 48 h, and its oxidation state was measured at 48 h. Association of Pu with B. subtilis, Kaolinite, and the Mixture. The association of Pu in the mixture containing bacteria and kaolinite clay was examined after desorption by a 1 M CH3COOK solution. The B. subtilis or kaolinite sample was incubated for 48 h with Pu, and the mixtures were incubated for 2, 18, and 48 h. Then the products were separated from the Pu solutions by centrifugation and washed repeatedly with deionized water to remove any Pu in solution retained through surface tension. The solid samples were extracted with a 1 M CH3COOK solution at pH 5 for 8 h; then the CH3COOK solutions were separated by centrifugation, and the concentrations of Pu in the CH3COOK solutions were measured by liquid scintillation. The desorbed fraction of Pu was normalized to 100 when all of the sorbed Pu was removed with CH3COOK. Effects of Exudates from B. subtilis on Pu Oxidation States and Pu Accumulation. To examine the reduction of Pu(VI) by exudates released from B. subtilis, the washed bacterial cells were incubated in sterile Pu-free 0.01 M NaCl solution for 48 h at pH 5.0 ( 0.1, and the solution was separated from the cells by centrifugation. This solution hereinafter is called the exudate solution. An aliquot of Pu(VI) was then added to the exudate solution to a concentration of 4.0 × 10-4 M at pH 5.0 ( 0.1. The oxidation state of Pu in the exudate solution was determined by UV/vis spectroscopy. This solution hereafter is termed the Pu-exposed exudate solution. We assessed the sorption of Pu in the Pu-exposed exudate solution on kaolinite of 40 g L-1 for 48 h at pH 5.0 ( 0.1. The sample was centrifuged, and the concentrations and oxidation states of Pu were measured in 1 mL supernatant solutions. We determined the total amount of dissolved organic carbon (DOC) in the Pu-free exudate solution using a Shimadzu TOC 5000 after filteration through a 0.2 µm membrane filter. The molecular weights of the organic compounds present were determined by HPLC (Hitachi Instruments Service Co., Ltd.) using a size exclusion column (GL-W540) that separated the materials by molecular weight and UV detector. This column has an exclusion limit of MW 600000 and a linear fractionation range of MW 1000-600000; it was calibrated for molecular weight using a standard protein (31, 32). The mobile phase in HPLC was a 0.01 M Tris-HCl buffer solution of pH 7.4. Measurement of Pu Concentration and pH. Pu concentrations were measured by a combination of a liquid scintillation analyzer with R/β discrimination (Packard TriCarb 2550TR/AB) and liquid scintillation cocktails (Packard

FIGURE 1. Time course of the accumulation of Pu on B. subtilis and on kaolinite. Average fractions of Pu obtained in a duplicate set of experiments were plotted; measurement error is shown by the error bar. The initial concentration of Pu was 4 × 10-4 M, initial pH 5.0, ionic strength 0.01 M, cell density 1.5 g L-1, and kaolinite density 40 g L-1. Ultima-Gold AB and FG). A TOA HM-30S pH meter with a combined electrode of TOA GS-5015C measured the solution’s pH. Oxidation States of Pu. The oxidation states of Pu in the solutions were measured by UV/vis spectroscopy (Hitachi, model 323) using 1 mL of the supernatant. The oxidation state in the initial solution of Pu was VI. The Pu associated with B. subtilis, kaolinite, or the mixture was extracted by a 50% H3PO4 solution (prepared by mixing a concn H3PO4 solution with deionized water) for 2 min after the Puaccumulated solids had been removed by centrifugation. The H3PO4 extract solution was filtered through a 0.2 µm membrane filter. Scanning Electron Microscopy (SEM) Analysis. The bacteria and clay in the Pu-sorbed mixture for 48 h were separated by centrifugation, washed with deionized water, and then air-dried overnight in sterile polypropylene centrifuge tubes. Samples coated with carbon were examined by SEM and energy dispersive spectrometry (EDS) using a JEOL JMS-6330 F microscope equipped with an EDS instrument to determine and localize the Pu.

Results and Discussion Accumulation of Pu by B. subtilis and Kaolinite. Figure 1 shows the accumulation of Pu by B. subtilis and kaolinite up to 48 h. The fraction accumulated by the bacterium (solid line) increased to 35% within 8 h and thereafter gradually rose; at 48 h it had not reached equilibrium. In contrast, the accumulation of Pu by kaolinite (broken line) attained equilibrium within 8 h. At 48 h, the bacteria and the clay had accumulated a similar amount of Pu. The UV/vis spectra of Pu in solution after exposure to B. subtilis are shown in Figure 2a, those of Pu extracted from B. subtilis by a 50% H3PO4 solution in Figure 2b, and those of a Pu solution after exposure to kaolinite and Pu extracted from kaolinite by a 50% H3PO4 solution in Figure 2c. In Figure 2c, the UV/vis spectrum of the Pu-exposed exudate solution after 48 h at pH 5.0 is also shown. The UV/vis spectrum of the Pu(VI) stock solution exhibited only one sharp peak at 831 nm (data not shown), verifying that Pu originally is in the VI oxidation state. We note that the increase in the spectrum’s baseline with a decrease of the wavelength from 400 to 800 nm probably is due to organic materials released from B. subtilis. After 2 min of exposure to B. subtilis, the spectrum of Pu in solution revealed one peak around 831 nm, indicating that most of it still was present as Pu(VI). After 2 h of exposure, an additional peak had appeared around 570 nm, of the same intensity as that at 831 nm (Figure 2a). The molar absorption of Pu(V) at 570 nm is 17.1 cm-1 mol-1 (33), and that of Pu(VI) at 831 nm is 58.1 cm-1 mol-1; accordingly, approximately 80% of Pu in solution after 2 h of exposure to B. subtilis was VOL. 41, NO. 9, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

3135

FIGURE 2. (a) UV/vis spectra of the Pu(VI) solution 2 min and 2, 18, and 48 h after exposure to B. subtilis. (b) UV/vis spectra of the extracted 50% H3PO4 solution from B. subtilis and kaolinite on which Pu accumulated for 2 min and 2, 18, and 48 h. (c) UV/vis spectra of the Pu solution 48 h after exposure to kaolinite, that of the extracted 50% H3PO4 solution from kaolinite on which Pu was accumulated for 48 h, and that of the Pu-exposed exudate solution after 48 h at pH 5.0. The absorption peak around 831 nm represents the original valence of Pu(VI). The absorption peak around 570 nm denotes Pu(V), and those around 645 and 670 nm reflect the presence of Pu(IV). A Pu-free 0.01 M NaCl solution was used as the reference for the supernatant of the Pu solutions; a Pu-free 50% H3PO4 solution similarly served for the 50% H3PO4 extract. The UV/vis spectrum of distilled water determined the baseline of the spectrum. Scans were at a rate of 200 nm min-1. in the V oxidation state. A distinct peak at 570 nm also was apparent after 18 and 48 h of exposure. Thus, 18 h after exposure to the bacterium, more than 90% of the Pu(VI) in the solution had been converted to Pu(V). Comparison of these findings with the spectra of the 50% H3PO4 extract from the Pu-accumulated B. subtilis showed differences with time; after 2 min, a sharp peak at 831 nm indicated that most of the accumulated Pu was present as Pu(VI) (Figure 2b). Two hours later, additional peaks were apparent at 642 and 667 nm (Pu(IV)), although the intensity of the peaks at 642 and 667 nm was much less than that at 831 nm. The molar absorptivity of Pu(IV) around 642 nm in the 50% H3PO4 solution was lower by 5-fold than that at 831 nm of Pu(VI), showing that both Pu(IV) and Pu(VI) were present in the extract. The oxidation state of Pu(V) in acid solution is known to change to IV and VI in a disproportionation reaction expressed by the following equation:

2PuVO2+ + 4H+ a PuVIO22+ + Pu4+ + 2H2O The peak of Pu(V) disappeared in the 50% H3PO4 solution (see Supporting Information Figure S1). These results strongly suggested that the oxidation state of the same fraction of Pu accumulated to B. subtilis was V at 2 h after exposure; residual fractions may be Pu(VI) and/or Pu(IV). The intensity of the peak at 831 nm in the spectra of the extracted 50% H3PO4 solution declined with prolonged exposure, indicating that the concentration of both Pu(V) and Pu(VI) fell in B. subtilis. On the other hand, the intensity of the Pu(IV) absorption peak rose with exposure time, implying that, in the presence of the bacteria, the oxidation state of Pu changed from VI to V and then to IV. Two sharp peaks at 831 and 850 nm apparent in the Pu solution after a 48 h exposure to kaolinite (Figure 2c) reflected the presence of plutonyl ion and plutonyl hydroxides, respectively. Only a sharp peak at 831 nm was seen in the spectrum of the extracted 50% H3PO4 solution from the Puaccumulated kaolinite sample (Figure 2c). Hence, there is no change in the oxidation state of Pu(VI) in the solution and of Pu associated with kaolinite. 3136

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 9, 2007

FIGURE 3. Percent fractions of Pu adsorbed by kaolinite, the mixtures containing kaolinite and B. subtilis, and B. subtilis and desorbed with a 1 M CH3COOK solution of pH 5.0 from these samples. The solid lines and circles show the fractions adsorbed and broken, and triangles represent the fractions desorbed. The desorbed fraction of Pu was normalized to 100 when all of the sorbed Pu was removed with the CH3COOK. Error bars show the measurement errors in the sorption and desorption experiments. The times at the bottom of the columns denote the time of exposure of the Pu solution to these samples. Extraction of Pu from B. subtilis by a CH3COOK solution (Figure 3, B. subtilis at 48 h) showed desorption of approximately 40% of its accumulated Pu, compared to approximately 85% from kaolinite (Figure 3, kaolinite at 48 h). Seemingly, Pu is associated more strongly with B. subtilis than with kaolinite. About a 90% fraction of the adsorbed Pu from B. subtilis and kaolinite was extracted by the 50% H3PO4 solution, as denoted by the peak intensities in the UV/ vis spectra and molar absorptivities. The presence of Pu(IV) on B. subtilis caused the desorption of a lower fraction of Pu by a 1 M CH3COOK solution compared with that from kaolinite. There was 14 mg L-1 DOC in the exudate solution after a 48 h incubation of the cells in Pu-free solution, with molecular weights between 1000 and 500000 (Figure 4). Exposure of Pu(VI) to the exudates for 48 h, and analysis by UV/vis spectroscopy (Figure 2c) revealed the presence of Pu(V) with absorption peaks at 570 and 780 nm. Note that

FIGURE 4. Time course of UV absorption passed through the size exclusion column. Arrows with numbers show the molecular weights determined by reference proteins. The exclusion limit of the column is MW 600000, and it has a linear fractionation range of MW 1000-600000. A 0.01 M Tris-HCl buffer solution of pH 7.4 was used for the mobile phase in HPLC. no peak at 642 and 667 nm for Pu(IV) was distinguished. These findings suggest that the exudates from B. subtilis reduced Pu(VI) to Pu(V) but not to Pu(IV) in the solution of the present experimental conditions. Incubating the Pu(V)exposed exudates with kaolinite resulted in ∼3% Pu accumulation, namely, 10-fold less than that of the accumulated fraction of Pu(VI) by kaolinite. Choppin (30) pointed out that sorption of Pu(V) is weaker than that of Pu(VI), which is in agreement with our findings on the sorption of Pu(V) and Pu(VI) on kaolinite. Organic materials are abundant in the soil’s water. Although the interaction between Pu and humic substances has been studied, the behavior of Pu organic compounds in the natural environment still is poorly understood. Reed et al. (34) reported that organic acids reduced Pu(VI) to Pu(V). The exudates from B. subtilis, containing organic compounds in the MW range of 1000-500000, also reduced Pu(VI) to Pu(V), suggesting that bacterial metabolic products may play a significant role in the reduction of Pu(VI) in the natural environment. Accumulation of Pu by the Mixture. The mixture of B. subtilis and kaolinite accumulated 20% Pu after 2 h of exposure to a Pu(VI) solution (Figure 3, mixture at 2 h), which rose to about 40% at 18 and 48 h (solid line, circles); the amount accumulated by the mixture was nearly the same as that by B. subtilis or kaolinite alone. Approximately 80% of the accumulated Pu was desorbed by CH3COOK when the mixture was exposed to the Pu solution for 2 h, but this value decreased when the samples were exposed for longer to the Pu(VI) solution (broken line, triangles). Thus, with time, Pu became more tightly associated with the mixture. The UV/vis spectrum of the Pu solution in contact with the mixture of B. subtilis and kaolinite for 48 h (Figure 5, solution) had a distinct absorption peak only for Pu(V), while only a distinct peak for Pu(IV) was observed in the spectrum of the extracted 50% H3PO4 solution from the Pu-accumulated mixture (Figure 5, extracted). Thus, the oxidation state of Pu changed from VI to V in the solution and from VI to IV in the solids coexisting with B. subtilis and kaolinite. The SEM image of Pu(VI) in the 48 h exposed mixture (Figure 6a) showed that rod-shaped B. subtilis and laminarshaped kaolinite were homogenously mixed in the mixture. In the EDS spectrum of the regions of kaolinite (Figure 6b), the Pu MR peak was below the detection limit. However, it was apparent in the spectrum of the B. subtilis region in the mixture (Figure 6b, B. subtilis, 48 h), indicating that most of the accumulated Pu was associated with the bacteria. Role of B. subtilis and Kaolinite on Pu Association in the Mixture. Assuming instantaneous equilibrium for the sorption of Pu on a solid, sorption depends only on the final chemical states, but not on an initial one (35). Pu(IV)

FIGURE 5. UV/vis spectra of the Pu solution and the extracted 50% H3PO4 solution from the mixture of B. subtilis and kaolinite on which Pu accumulated for 48 h. The reference and blank solutions and the scan rate for measuring the UV/vis spectrum were the same as those in Figure 2a,b. The scan rate was 200 nm min -1.

FIGURE 6. (a) SEM image of the mixture of B. subtilis and kaolinite exposed to a Pu solution for 48 h. The rod-shaped material is B. subtilis, and the lamella-shaped material is kaolinite. The white particles shown by the black arrowheads were dust that contained no Pu. The operating voltage was 20 kV. (b) EDS spectra of the regions of B. subtilis (48 h) and kaolinite (48 h) in the mixture exposed to a Pu solution for 48 h. undergoes strong hydrolysis in neutral pH solution, causing its extensive sorption by minerals and organic-coated surfaces (36), suggesting a high affinity of reduced Pu(IV) for both kaolinite and B. subtilis. However, our results pointed to its preferential accumulation by B. subtilis in the mixture of the microorganisms and kaolinite (Figures 3 and 6), even though VOL. 41, NO. 9, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

3137

most of the accumulated Pu was present as Pu(IV) at 48 h after exposure of the mixture to Pu(VI) solution. In solution, the reduction of Pu(VI) to Pu(V) proceeded with time. The exudates reduced Pu(VI) to Pu(V) but not to Pu(IV) in the solution (Figure 2c). These results suggest that reduction of Pu(VI) to Pu(IV) by the exudates was not predominant in generating Pu(IV) in the present experimental conditions. On the contrary, Pu(VI) was not reduced when the solution was exposed to kaolinite for 48 h. The time course of sorption by B. subtilis and kaolinite exhibited an abrupt increase within 2 h. The Pu sorbed by B. subtilis at 2 min and by kaolinite at 48 h was present as Pu(VI), suggesting that Pu initially sorbed by both of them in the mixture is present as Pu(VI). The Pu(VI) accumulated by kaolinite retained its oxidation state up to 48 h after exposure. The extensive desorption of Pu(VI) from kaolinite by the CH3COOK solution indicates that the sorbed material is not tightly bound. Exudates in the solution reduced the dissolved Pu(VI) to Pu(V), which exhibited less sorption than Pu(VI). Thus, the accumulated fraction of Pu on kaolinite decreases with increasing exposure. Two hours after exposure to B. subtilis alone, the oxidation states of the sorbed Pu were IV, V, and VI. Sorbed Pu(VI) and probably Pu(V) were reduced to Pu(IV) on the bacterial surfaces, with the accumulated fraction increasing with time. Pu(IV) is hydrolyzed to Pu(OH)4, the solubility of which is very low (30). The cellular surfaces of B. subtilis consist of negatively charged hydrophilic anionic functional groups of phosphoryl, carboxyl, and hydroxyl moieties. Therefore, insoluble plutonium hydroxides are precipitated on the bacterial surfaces, and/or Pu4+ is sorbed by the functional groups thereon. Kudo et al. (37) reported that the distribution coefficient (Kd) of Pu for a mixture of bacteria and bentonite, a backfill material for isolating radioactive wastes, was nearly the same as that of bacteria alone, whether the bacteria were sterilized (killed) or not. However, they did not measure the chemical states of Pu throughout the sorption experiments. Uranium(VI) preferentially associated with microorganisms in a mixture of B. subtilis and kaolinite (38), although throughout the oxidation state of U remained at VI. Comparison with our findings suggests that the mechanism of Pu(VI) accumulation differs from that of U(VI) in a mixture of microorganisms and kaolinite. Panak and Nitsche (17) reported that, after exposure of a Pu(VI) solution of 4.2 × 10-4 mol L-1 to Bacillus sphaericus, it was reduced to Pu(IV). They suggested that Pu(V) was reduced by disproportionation. Since B. subtilis and B. sphaericus are Bacillus species, the reduction of Pu(V) on B. subtilis and in the solution also may be caused by disproportionation. Wildung and Garland (39) considered that Pu initially is immobilized by microbial activities in soil; however, they offered no direct evidence. Microbial siderophores enhance the dissolution of Pu(IV) in solution (40, 41). If B. subtilis releases siderophores, then the accumulated fraction of Pu should fall with exposure time (42). Since such accumulation increased with time instead (Figure 1), the influence of the siderophores was very low under our experimental conditions. Our results indicate that the two-step reduction of Pu(VI) to Pu(IV) through Pu(V) is an important process for the preferential accumulation of Pu by B. subtilis in the mixture, although the sorption of Pu(IV) by the bacteria is the predominant immobilization process. Therefore, bacteria apparently may play an important role in Pu(VI) immobilization in the mixture of bacteria and clay minerals present in the natural environment. 3138

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 9, 2007

Acknowledgments This research was supported in part by a Grant-in-Aid for Scientific Research B from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and in part by the Environmental Remediation Sciences Division, Office of Biological and Environmental Research, Office of Science, U.S. Department of Energy, under Contract No. DE-AC0298CH10886.

Supporting Information Available Details of the solution pH in accumulation experiments to avoid the precipitation of Pu and methodology and results of measurements of UV/vis spectra and oxidation states of Pu standard solutions. This material is available free of charge via the Internet at http://pubs.acs.org.

Literature Cited (1) Harley, J. H. Plutonium in the Environment-A review. Jpn. J. Radiat. Res. 1980, 23, 83-104. (2) Kulakov, V. M.; Dobrynin, I. L.; Kosyakov, V. M.; Lisin, S. K.; Rodionov, Y. F.; Shvetov, I. K. Plutonium release to the environment during Chernobyl accident. Seminar on Comparative Assessment of the Environmental Impact of the Radionuclides Released during Three Major Nuclear Accidents: Kyshtym, Windscale, Chernobyl; EUR-13574; Commission of the European Communities: Brussels, Belgium, 1991; Vol. 1, pp 437-448. (3) AECL. Environmental Impact Statement on the Concept for Disposal of Canada’s Nuclear Fuel Waste; AECL 10711; Atomic Energy Canada Ltd.: Mississauga, Ontario, Canada, 1994. (4) Ashida, T.; Yui, M.; Kohara, Y. Migration behavior of Pu released from Pu-doped glass in compacted bentonite. Radiochim. Acta 1994, 66/67, 359-362. (5) Yamaguchi, T.; Nakayama, S. Diffusivity of U, Pu, and Am carbonate complexes in a granite from Inada, Ibaraki, Japan studied by through diffusion. J. Contam. Hydrol. 1998, 35, 5565. (6) Rameback, H.; Skalberg, M.; Eklund, U. B.; Kjellberg, L.; Werme, L. Mobility of U, Np, Pu, Am and Cm from spent nuclear fuel into bentonite clay. Radiochim. Acta 1998, 82, 167-171. (7) Muramatsu, Y.; Yoshida, S.; Tagami, K.; Uchida, S.; Ruehm, W. ICP-MS analysis of environmental plutonium. In Plutonium in the environment; Kudo, A., Ed.; Elsevier Science, Ltd.: Amsterdam, 2001; pp 33-40. (8) Yui, M.; Shibutani, T.; Shibutani, S.; Rai, D.; Ochs, M. A plutonium geochemical database for performance analysis of high-level radioactive waste repositories. In Plutonium in the environment; Kudo, A., Ed.; Elsevier Science, Ltd.: Amsterdam, 2001; pp 159174. (9) Runde, W.; Conradson, S. D.; Wes, E. D.; Lu, N.; Vanpelt, C. E.; Tait, C. D. Solubility and sorption of redox-sensitive radionuclides (Np, Pu) in J-13 water from the Yucca Mountain site: comparison between experiment and theory. Appl. Geochem. 2002, 17, 837-853. (10) Mahara, Y.; Matsuzuru, H. Mobile and immobile plutonium in a groundwater environment. Water Res. 1989, 23, 43-50. (11) Russel-Debet, S. Experimental values for 241Am and 239+240Pu Kd’s in French agricultural soils. J. Environ. Radioact. 2005, 79, 171-85. (12) Shibutani, T.; Yui, M.; Yoshikawa, H. Sorption mechanism of Pu, Am and Se on sodium-bentonite. Mater. Res. Soc. Symp. Proc. 1994, 333, 725-730. (13) Ticknor, K. V. Actinide sorption by fracture-infilling minerals. Radiochim. Acta 1993, 60, 33-42. (14) Fowle, D. A.; Fein, J. B.; Martin, A. M. Experimental study of uranyl adsorption onto Bacillus subtilis. Environ. Sci. Technol. 2000, 34, 3737-3741. (15) Haas, J. H.; Dichristina, T. J.; Wade, R., Jr. Thermodynamics of U(VI) sorption onto Shewanella putrifaciens. Chem. Geol. 2001, 180, 33-54. (16) Francis, A. J.; Gillow, J. B.; Dodge, C. J.; Harris, R.; Beveridge, T. J.; Papenguth, H. W. Uranium association with halophilic and non-halophilic bacteria and archaea. Radiochim. Acta 2004, 92, 481-488. (17) Panak, P. J.; Nitsche, H. Interaction of aerobic soil bacteria with plutonium(VI). Radiochim. Acta 2001, 89, 499-504.

(18) John, S. G.; Ruggiero, C. E.; Hersman, L. E.; Tung, C. S.; Neu, M. P. Siderophore mediated plutonium accumulation by Microbacterium flavescens (JG-9). Environ. Sci. Technol. 2001, 35, 2942-1948. (19) Ohnuki, T.; Aoyagi, H.; Kitatsuji, Y.; Samadfam, M.; Kimura, Y.; Purvis, O. W. Plutonium(VI) accumulation and reduction by lichen biomass: Correlation with U(VI). J. Environ. Radioact. 2004, 77, 339-353. (20) Francis, A. J. Microbial transformation of Pu and implications for its mobility. In Plutonium in the environment; Kudo, A., Ed.; Elsevier Science, Ltd.: Amsterdam, 2001; pp 201-219. (21) Neu, M. P.; Ruggiero, C. E.; Francis, A. J. Bioinorganic chemistry of plutonium and interactions of plutonium with microorganisms and plants. In Advances in plutonium chemistry 19672000; Hoffman, D. C., Ed.; The American Nuclear Society: La Grange Park, IL, 2002; pp 169-211. (22) Cleveland, J. M. The chemistry of plutonium; American Chemical Society: Washington, DC, 1979. (23) Silva, R. J.; Nitsche, H. Actinide Environmental Chemistry. Radiochim. Acta 1995, 70/71, 377-396. (24) Rusin, P. A.; Quintana, L.; Brainard, J. R.; Stietelmeier, B. A.; Tait, C. D.; Ekberg, S. A.; Palmer, P. D.; Newton, T. W.; Clark, D. L. Solubilization of plutonium hydrous oxide by iron-reducing bacteria. Environ. Sci. Technol. 1994, 49, 2297-2307. (25) Mahara, Y.; Kudo, A. Plutonium released by the Nagasaki A-bomb: Mobility in the environment. Appl. Radiat. Isot. 1995, 46, 1191-1201. (26) Sanchez, A. L.; Murray, J. W.; Sibley, T. H. The adsorption of plutonium IV and V on goethite. Geochim. Cosmochim. Acta 1985, 49, 2297-2307. (27) McLean, R. J. C.; Fortin, D.; Brown, D. A. Microbial metal-binding mechanisms and their relation to nuclear disposal. Can. J. Microbiol. 1996, 42, 392-400. (28) Larsen, R. P.; Oldham, R. D. Plutonium in drinking water: Effects of chlorination on its maximum permissible concentration. Science 1978, 201, 1008-1009. (29) Nitsche, H.; Roberts, K.; Xi, R.; Prussin, T.; Becraft, K.; Al Mahamid, I.; Silber, H. B.; Carpenter, S. A.; Gatti, R. C.; Novak, C. F. Long term plutonium solubility and speciation studies in a synthetic brine. Radiochim. Acta 1994, 66/67, 3-8. (30) Choppin, G. R.; Morgenstern, A. Distribution and movement of environmental plutonium. In Radioactivity in the environment, Vol 1- Plutonium in the environment; Kudo, A., Ed.; Elsevier: Amsterdam, 2001; pp 91-106. (31) Inagaki, K.; Umemura, T.; Matsuura, H.; Haraguchi, H. Speciation of Trace Elements, Binding and Non-binding with Proteins in Human Blood Serum, by Surfactant-Mediated HPLC with Element-Selective Detection by ICP-MS. Anal. Sci. 2000, 16, 787-788.

(32) Umemura, T.; Kitaguchi, R.; Inagaki, K.; Haraguchi, H. Direct injection determination of theophylline and caffeine in blood serum by high-performance liquid chromatography using an ODS column coated with a zwitterionic bile acid derivative. Analyst 1998, 123, 1767. (33) Katz, J. J.; Seaborg, G. T. The chemistry of the actinide elements; John Wiley and Sons: New York, 1957; pp 300. (34) Reed, D. T.; Wygmans, D. G.; Aase, S. B.; Banaszak, J. E. The Reduction of Np(VI) and Pu(VI) by Organic Chelating Agents. Radiochim. Acta 1998, 82, 109-114. (35) Parks, G. A. Surface energy and adsorption at mineral-water interfaces: an introduction. In Mineral-water interface geochemistry; Hochella, M. F., White, A. F., Eds.; Mineralogical Society of America: Washington, DC, 1990; pp 133-0175. (36) Morse, J. W.; Choppin, G. R. The chemistry of transuranic elements in natural waters. Rev. Aquat. Sci. 1991, 4, 1-22. (37) Kudo, A.; Zheng, J.; Cayer, I.; Fujikawa, Y.; Asano, H.; Arai, K.; Yoshikawa, H.; Ito, M. Behavior of plutonium interacting with bentonite and sulfate-reducing anaerobic bacteria. Mater. Res. Soc. Symp. Proc. 1997, 465, 879-884. (38) Ohnuki, T.; Yoshida, T.; Ozaki, T.; Samadofam, M.; Kozai, N.; Yabuta, K.; Mitsugashira, T.; Kasama, T.; Francis, A. J. Interactions of uranium with bacteria and kaolinate clay. Chem. Geol. 2005, 220, 237-243. (39) Wildung, R. E.; Garland, T. R. The relationship of microbial processes to the fate and behavior of transuranic elements in soil, plants, and animals; PNL-2416/UC-11; Battelle Pacific Northwest Laboratory: Richland, WA, 1977. (40) Raymond, K. N.; Mohs, T. R.; Romanovski, V.; Veek, A. C. Plutonium sequestering agents: their chemistry and biological evaluation. The transition from actinide to acinide extraction agents. A biomimetic approach to the development of new actinide(IV) extraction technologies. 213th ACS National Meeting; American Chemical Society: Washington, DC, 1997; Conf. 970443, pp 1136-1137. (41) Neu, M. P.; Boukhalfa, H.; Ruggiero, C. E.; Lack, J. G.; Hersman, L. E., Reily, S. D. Microbial siderophore influence on plutonium biogeochemistry. J. Inorg. Biochem. 2003, 96, 69. (42) Yoshida, T.; Ozaki, T.; Ohnuki, T.; Francis, A. J. Adsorption of Th(IV) and Pu(IV) on the surface of Pseudomonas fluorescens and Bacillus subtilis in the presence of desferrioxamine siderophore. J. Nucl. Radiochem. Sci. 2005, 6, 77-80.

Received for review May 18, 2006. Revised manuscript received December 26, 2006. Accepted January 22, 2007. ES061207G

VOL. 41, NO. 9, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

3139