Bioreduction of Uranium(VI) Complexed with Citric Acid by Clostridia

Oct 22, 2008 - Uranium(VI) complexed with citrate is reduced by anaerobic bacteria, but the reduced form of ... However, the presence of naturally occ...
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Environ. Sci. Technol. 2008, 42, 8277–8282

Bioreduction of Uranium(VI) Complexed with Citric Acid by Clostridia Affects Its Structure and Solubility A. J. FRANCIS* AND C. J. DODGE Environmental Sciences Department, Brookhaven National Laboratory, Upton, New York 11973

Received April 15, 2008. Revised manuscript received August 1, 2008. Accepted August 13, 2008.

Uranium contamination of the environment from mining and milling operations, nuclear-waste disposal, and ammunition use is a widespread global problem. Natural attenuation processes such as bacterial reductive precipitation and immobilization of soluble uranium is gaining much attention. However, the presence of naturally occurring organic ligands can affect the precipitation of uranium. Here, we report that the anaerobic spore-forming bacteria Clostridia, ubiquitous in soils, sediments, and wastes, capable of reduction of Fe(III) to Fe(II), Mn(IV) to Mn(II), U(VI) to U(IV), Pu(IV) to Pu(III), and Tc(VI) to Tc(IV); reduced U(VI) associated with citric acid in a dinuclear 2:2 U(VI): citric acid complex to a biligand mononuclear 1:2 U(IV):citric acid complex, which remained in solution, in contrast to reduction and precipitation of uranium. Our findings show that U(VI) complexed with citric acid is readily accessible as an electron acceptor despite the inability of the bacterium to metabolize the complexed organic ligand. Furthermore, it suggests that the presence of organic ligands at uranium-contaminated sites can affect the mobility of the actinide under both oxic and anoxic conditions by forming such soluble complexes.

Intoduction Mixtures of naturally occurring and synthetic complexing organic ligands coexist with radionuclides and toxic metals at several waste sites (1). These ligands include citric acid, oxalic acid, phthalic acid, ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), and natural organic matter (NOM). Their presence in radioactive wastes and in contaminated soils is a major concern because of their potential for complexing with radionuclides and subsequently migrating from the disposal site. Such movements of radionuclides (e.g., U, Pu, and, Co) were observed even when the concentrations of the organic ligands was relatively low (2-7). For example, citric acid was detected at several sites: at Oak Ridge Bear Creek Burial Grounds and Low-level Waste Trenches and Pits areas, and S-3 Ponds Area 1 (which contains 108 mg/L dissolved organic carbon (DOC) and 21 mg/L citric acid); the Hanford 300 Area at North/South Process Ponds; North Crib, Savannah River Old Testing and Experiment Basins; Idaho National Laboratory Radioactive Management Complex; and, in the TRU waste at the Waste Isolation Pilot Plant (WIPP) (8-11). * Corresponding author phone: (631) 344-4534; fax: (631) 3447303; e-mail: [email protected]. 10.1021/es801045m CCC: $40.75

Published on Web 10/23/2008

 2008 American Chemical Society

Anaerobic conditions are common in subsurface environments and an understanding of the fate of the actinides complexed with organics under such conditions is important in predicting their mobility and bioavailability. Although there are extensive studies on the microbial reduction and precipitation of the uranium(VI) associated with carbonate, nitrate, and acetate as insoluble uraninite under anaerobic conditions, there is a paucity of information on the mechanisms of microbial transformation of uranium complexed with naturally occurring low-molecular-weight soluble organic ligands (12, 13). Understanding the mechanisms of microbially catalyzed transformations of radionuclides complexed with organic and inorganic ligands is critical to the success of in situ stabilization of radionuclides in the subsurface because they may impact the long-term stability of the radionuclides and stewardship of the contaminated sites. We examined the biotransformation of U(VI) complexed with citric acid by Clostridia under anaerobic conditions. We report that the naturally occurring low-molecular-weight organic complexing agents such as citric acid may not only affect the anaerobic microbial reductive precipitation of uranium but also facilitate the transport of uranium.

Materials and Methods Cultures and Growth Media. Clostridium sp. (ATCC 53464), a N2-fixing bacterium that ferments glucose but not citrate, was grown in a medium consisting of the following ingredients (per liter): glucose, 5.0 g; NH4Cl, 0.5 g; glycerolphosphate, 0.3 g; MgSO4 · 7H2O, 0.2 g; CaCl2 · 2H2O, 0.5 g; peptone, 0.1 g; yeast extract, 0.1 g; FeSO4 · 7H2O, 2.78 mg. Clostridium sphenoides (ATCC 19403) able to ferment both citric acid and glucose was grown in the following medium (per liter): sodium citrate, 8.2 g; NH4Cl, 0.5 g; glycerolphosphate, 0.3 g; MgSO4 · 7H2O, 0.2 g; CaCl2 · 2H2O, 0.5 g; peptone, 0.1 g; L-cysteine · HCl, 0.03 g. The pH of the media was adjusted to 6.8 and they were prereduced by boiling and purging with nitrogen gas for 15 min. Forty milliliters of the culture medium was dispensed into 60 mL serum bottles; the bottles were sealed with butyl rubber stoppers and aluminum crimp seals in an anaerobic glovebox and autoclaved. They were then inoculated with 2 mL of a 24 h old culture and incubated in the dark at 26 ( 1 °C. Preparation of Uranyl-Citrate Complex. Stock solutions containing 13.0 mM citric acid (Sigma, MO) and UO2(NO3)2 · 6H2O (BDH Chemicals, Analar, Poole, England) were prepared in deionized water. The U(VI)-citrate complex was made by addition of U stock solution to equimolar, 10fold excess or 100-fold excess citric acid as previously described (14). Ionic strength was adjusted to 0.1 M by adding KCl, and the pH was adjusted to 6.1 by using KOH. The solution was purged with N2 gas, transferred to the anaerobic glovebox, filter-sterilized (0.45 µm) into sterile tubes, and stored in the dark to prevent photodegradation of the complex. Biotransformation of Uranyl-Citrate. The extent of uranium reduction by resting cells of C. sphenoides was determined as follows. The bacterial culture was grown in the citrate medium for 36 h, and harvested by centrifugation at 11 000 × g for 15 min. The cell pellet was washed twice with a defined mineral-salts medium containing (mg/L): CaCl2 · 2H2O, 2.75; MgCl2 · 6H2O, 1.1. The ionic strength of the medium was adjusted to 0.1 M with KCl, and the pH adjusted to 6.1 with KOH. We resuspended the cells in the defined mineral salts medium, and adjusted the OD (600 VOL. 42, NO. 22, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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nm) to 0.8; the cells then were incubated with 0.15 mM uranium and citric acid to obtain a 1:1 or 1:10 ratio of U(VI): citric acid in serum bottles under anaerobic conditions at 26 ( 1 °C. Controls without bacterial cells were included (data not shown). Uranium reduction by growing culture of Clostridium sp. was investigated by adding 1:100 U(VI):citric acid complex to 40 mL of an 18 h old log phase culture with an OD 0.60 at 600nm and pH 3.4 grown at 26 ( 1 °C. The pressure was vented from the culture bottles before the addition of citric acid or U-citrate complex after which the pH of the medium increased from 3.4 to 4.6. All treatments were performed in triplicate. Chemical Analyses. Aliquots were removed periodically from the cultures in serum bottles with a needle and syringe, and analyzed for pH in the unfiltered sample and total U, citric acid, glucose, and organic-acid metabolites after filtration through a 0.45 µm filter. Both the oxidized and reduced forms of uranium were determined in a 0.45 µm-filtered aliquot by the colorimetric method and UV-vis spectrophotometry. U(VI) was determined by the bromo-PADAP method (15). Reduced U(IV) was indirectly assessed using the o-phenanthroline method after its reduction by adding ferrous ion (16), and total uranium was determined by ICP-AES. UV-vis spectra of U(VI)-citrate and U(IV)-citrate in culture samples before and after microbial activity were denoted by the presence of peaks at 438 nm for U(VI), and 560 and 662 nm for U(IV) (17). The pH was measured using a Mettler-Toledo DL 53 titrator with a DG 111 SC electrode. Glucose was analyzed by high performance liquid chromatography (HPLC) using an Aminex HPX-87H column and a refractive index detector (Shimadzu). Citric acid and organic-acid metabolites were determined using a UV-vis detector at 210 nm. Molecular Characterization of U(VI)-Citrate and U(IV)Citrate Complexes. We determined the oxidation state of the uranium complexed with citric acid by X-ray absorption near-edge structure (XANES) spectroscopy. Uranium standards uranyl acetate dihydrate, and U(IV) dioxide (Atomergic Chemicals, NY) were ground in an agate mortar and mixed with boron nitride (10% w/v). The standard U(VI)-citrate and aqueous sample of U(IV)-citrate after bacterial action were placed in heat-sealed polyethylene bags (0.2 mil) and analyzed at beamline X11A at the NSLS in the fluorescence mode at the U LIII absorption edge (17.166 keV) (18). To prevent oxidation of the reduced uranium, we prepared the U(IV)-citrate complex sample in an anoxic glovebox and brought to the beamline in a nitrogen-flushed anaerobic jar. The sample chamber of the detector was continually flushed with nitrogen during the analysis. We collected multiple scans (3-10) for each sample and averaged them to minimize the signal-to-noise ratio. The spectra were background-subtracted and normalized to the edge jump. The inflection point for uranium dioxide’s absorption edge was set to 17.166 keV. The oxidation state of uranium in the samples was determined by comparing the energy position at the inflection point with that of the two standards. Extended X-ray Absorption Fine Structure (EXAFS). The coordination environment of uranium after reduction by Clostridium sp. in the culture sample containing U(IV)-citrate (pH 4.3 ( 0.1) was analyzed by EXAFS after filtration through a 0.45 µm filter and compared with uranyl-citrate standard (pH 6.1). Fourier-transformed EXAFS data, a pseudoradial distribution function (PRDF) representing the radial coordination shells of the near-neighbor atoms surrounding the uranium, were obtained using the UW EXAFS analysis software package. Data processing included background subtraction and normalization to the height of the edge jump, followed by Fourier transformation of the k3-weighted spectra (19). Individual coordination shells were signaled out by a 8278

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back-Fourier transform from real to k space. These partial EXAFS functions were then least-squares-fitted by a theoretical function to determine the sample’s structural and chemical parameters. With the theoretical EXAFS modeling code FEFF6 we calculated the backscattering phases and amplitudes of the individual neighboring atoms for curvefitting the raw data (19) Information on phase shift and amplitude was obtained from the crystallographic parameters using uranyl-acetate dehydrate (20).

Results Clostridium sphenoides (ATCC 19403) capable of fermenting both glucose and citrate reduced U(VI)-citrate to U(IV) in the presence of glucose or excess citrate (21, 22). However, the bacterium did not metabolize the citrate complexed to the uranium; hence, the reduced uranium remained in solution as a U(IV)-citrate complex. Figure 1A illustrates the metabolism of noncomplexed citric acid, and 1:1 U:citrate, and 1:10 U:citrate complexes by resting cells of C. sphenoides. The bacterium completely metabolized the uncomplexed citric acid in less than 5 h; concurrently, the pH increased from 6.8 ( 0.1 to 8.7 ( 0.2 because of degradation of the acid. The bacterium did not metabolize the 1:1 U:citric acid complex with a slight change from the initial pH of 6.2 ( 0.1 to 6.4 ( 0 at the end of the experiment. In contrast, the excess citric acid in the 1:10 U: citric acid complex was degraded from 0.87 ( 0.01 mM to 0.16 ( 0.01 mM, until it reached a ratio of 1:1 U:citric acid, and the uranium remained in solution. The pH of the medium increased from 6.5 to 7.1 ( 0.1. This concentration of citric acid is similar to the amount of uranium present for the equimolar studies. The oxidation state of uranium after bacterial activity was determined in these samples by XANES spectroscopic analysis. XANES spectra acquired at the U Llll edge of the equimolar U(VI)-citrate sample after incubation with C. sphenoides showed no shift in absorption edge energy compared to the equimolar U(VI)-citrate standard indicating the oxidation state of uranium was U(VI) (Figure 1B). However, in 1:10 U(VI):citrate samples wherein the bacterium metabolized the excess citric acid, the uranium absorption edge energy shifted from 17168 to 17166 eV, similar to the U(IV)-oxide sample indicating the reduction of uranium (Figure 1C). We also investigated Clostridium sp. (ATCC 53464), which ferments glucose but not citrate to reduce U(VI) complexed withcitrate.ThebacteriumreducedU(VI)-citratetoU(IV)-citrate when supplied with the electron-donor glucose. Adding 0.15 mM U(VI) as 1:100 U:citric acid to an 18 h old culture of Clostridium sp. growing in the presence of glucose resulted in a 93% reduction of the added U(VI) to U(IV) (Figure 2A). The bacterium did not metabolize citric acid, and the U remained in solution. The pH of the medium was 4.3 ( 0.1. UV-vis spectra revealed that the control sample (without bacteria) exhibited an absorption peak maximum at 438 nm for hexavalent uranium, while the inoculated sample showed two peaks at 560 and 662 nm, which are characteristic of U(IV) species (Figure 2B). The absorbance of the reduced uranium peaks increased with time indicating the reduction was essentially complete at 12 h. In addition, XANES analysis at the U Llll edge of the equimolar U(VI)-citrate sample after incubation with Clostridium sp. showed that the absorption edge shifted to lower energy (17166 eV) compared to the equimolar U(VI)-citrate without bacterial action (uninoculated control, 17168 keV), indicating the uranium was present in the reduced form as U(IV) (Figure 2C). Extended X-ray absorption fine structure (EXAFS) analysis of the samples before bacterial action demonstrated the presence of a binuclear 2:2 U(VI):citric acid complex; after reduction by Clostridium sp., we found a mononuclear 1:2

FIGURE 1. Biotransformation of uranyl citrate by Clostridium sphenoides. (A) Resting cells of C. sphenoides metabolized free citric acid, but not citric acid complexed with uranium. In the presence of 1:10 U:citric acid, the excess citric acid was metabolized by the bacterium until a ratio of 1:1 U:citric acid was reached. (B) XANES analysis at U LIII edge showed that uranium was not reduced in the 1:1 U:citric acid complex. (C) In the presence of excess citric acid, the uranium absorption edge energy shifted from 17168 to 17166 eV, similar to the U(IV)-oxide sample indicating the reduction of uranium.

FIGURE 2. (A) Reduction of uranium complexed with citric acid by an 18 h old growing culture of Clostridium sp. in medium containing 1:100 U(VI):citric acid and glucose as carbon source; error bars are ( 1 SEM. (B) Changes in UV-vis spectra due to the reduction of U(VI) to U(IV) in the culture medium. (C) XANES spectra at the U Llll edge showing change in the uranium absorption edge from 17168 to 17166 eV, indicating the reduction of uranium in the inoculated sample.

U(IV): citric acid complex. EXAFS analysis provides information on the local three-dimensional environment surrounding a central atom that is obtained using a multistep analysis procedure to identify nearest-neighbor atoms, determine atomic distances, and also the coordination number of the central atom. Figure 3 and Table 1 presents the raw

k3-weighted (3.7-12.5 Å-1) and Fourier-transformed EXAFS spectra at the U LIII edge for U(VI)-acetate, U(IV)-dioxide, and U(VI):citrate standards and U(IV)-citrate after bacterial action. Uranium acetate dihydrate has 2.2 ( 0.1 axial O atoms at 1.76 ( 0.01 Å and 6.0 ( 1.0 oxygens in the equatorial plane. VOL. 42, NO. 22, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. EXAFS spectra at the U LIII edge showing (A) raw k3-weighted (3.7-12.5 Å-1) and (B) Fourier transform for uranyl acetate dihydrate, aqueous U(VI):citric acid, and uranium dioxide standards and reduced U(IV):citric acid after bacterial action by Clostridium sp. Experimental data (-) and fitted data (s).

FIGURE 4. Proposed mechanism of uranyl-citrate biotransformation by anaerobic bacteria. EXAFS analysis of the U(IV)-citrate complex shows no U-U interaction at 3-4 Å indicating the presence of a mononuclear complex structure. The crystallographic data indicate that several types of oxygen bond to U, including water, R-hydroxyl, and monocarboxyl coordination at the lower distance (2.30-2.35 Å), with bidentate carboxylate coordination at the greater distance (2.40-2.50 Å). This bonding is reflected in the large degree of disorder (0.008 ( 0.003) of the shell. There are 2.0 ( 1.0 carbons at 2.91 ( 0.02 Å. The presence of carbon at this distance in the complex was determined by comparison with the structure of sodium triacetatodioxouranium(VI) Na[UO2(CH3COO)3]. The results confirmed the distance for bidentate carboxyl-carbon coordination for the uranium salt (23). The k3-weighted and Fourier transform spectra of the U(VI):citric acid complex at the U LIII edge prepared at pH 6.0 reveal 2.2 ( 0.2 axial O atoms at 1.78 ( 0.01 Å. There are two distinct groups of oxygens in the equatorial plane; the first group of 4.6 ( 1.0 oxygens are apparent at 2.33 ( 0.02 Å, with the second group of 0.7 ( 0.3 O atoms at 2.47 ( 0.02 Å. There are 2.0 ( 1.0 C at 2.94 ( 0.02 Å. At 3.83 ( 0.02 Å, we detected scattering from 0.7 ( 0.3 U atoms. The spectra for uranium dioxide show the absence of axial O atoms, as expected for the reduced form of uranium. Its coordination consists of 9.0 ( 1.5 O atoms at a distance of 2.39 ( 0.02 Å. The high-amplitude shell, at approximately 3.5 Å, reflects the presence of 7.9 ( 1.3 U atoms at 3.90 ( 0.01 Å. The U(IV)-citrate complex has a split oxygen shell consisting of 4.5 ( 0.8 O atoms at 2.28 ( 0.02 Å, and 3.8 ( 1.2 O atoms at 2.50 ( 0.01 Å. This dichotomy is caused by the presence of two different types of O atoms associated with the inner sphere of U(IV), giving an overall coordination 8280

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number of 8.3 for uranium. There are 3.5 ( 1.0 U-C interactions at 2.84 ( 0.01 Å pointing to carboxylate bonding with citric acid. We saw no evidence of U-U interactions. This structural configuration has also been reported for the Pu(IV)-citrate complex (24). The best structural fit is that of a mononuclear biligand U(IV)-cit2 complex. Figure 4 depicts the molecular structures for both the 2:2 U(VI):citric acid and the 1:2 U(IV):citric acid complexes.

Discussion The metal complexing capabilities of citrate are attributed to its three carboxylates (-COOH) and a hydroxyl (-OH) group. Complexation between the oxidized form of U(VI) (as UO22+ ions) and citrate has been studied extensively (25-32). The biotransformation of soluble radionuclide-organic complexes may result in precipitation of the uncomplexed radionuclides and retard their migration. However, although the aerobic biotransformations of metal-citrate complexes have been investigated (33-36), we know little of their anaerobic biotransformation (21, 22, 37-39). The presence of chelating agents, such as acetate, malonate, oxalate, or citrate, decreased the extent of U(IV) precipitation from solution during the bioreduction of U(VI) by resting cells of the sulfate-reducing bacteria Desulfovibrio desulfuricans, and by the facultative iron-reducing bacteria Shewanella alga under anaerobic conditions; further, it decreased by ∼80% in the presence of multidentate ligands (37). Similarly, Gu et al. (39) reported that in the presence of humic materials the bioreduction of U(VI) did not cause its precipitation; rather, the uranium remained in the solution phase as U(IV)-humic complex.

0.10 0.003 (2) 7.9 ( 1.3

N: number of atoms in each coordination shell. c σ2: the degree of disorder (Debye-Waller factor) b

3.90 ( 0.01 uranium dioxide

R: the distance between the two atoms of each atomic pair. (k3χexp)]2/(k3χtheor)2.

0.003 (2) 0.008 (3) 2.0 ( 1.0 3.5 ( 1.0 2.94 ( 0.02 2.84 ( 0.01 1:2 U(IV):citric acid

a

3.83 ( 0.02 0.003 (2) 2.0 ( 1.0 2.91 ( 0.02

0.008 (3) 0.004 (1) 0.002 (1) 0.007 (3) 0.006(1) 0.009 (2) 6.0 (1.0 4.6 ( 1.0 0.7 ( 0.3 4.5 ( 0.8 3.8 ( 1.2 9.0 ( 1.5 2.35 ( 0.02 2.33 ( 0.02 2.47 (0.02 2.28 ( 0.02 2.50 ( 0.01 2.39 ( 0.02 0.003 (2) 0.003 (1) 1.76 ( 0.01 1.78 ( 0.01 U(VI) acetate 2:2 U(VI):citric acid

2.2 ( 0.1 2.2 ( 0.2

CN R (Å) CN R (Å) R (Å) sample

CN

0.7 ( 0.3

d

R ) Σ[(k3χtheor) -

0.05

0.01 0.29 0.003 (2)

Rd (σ2) CN R (Å)

U,U shell

(σ2) U,C shell

(σ2) U-Oeq shell

(σ2c) U-Oax shell

b a

Edge for U(VI)-Citrate and U(IV)-Citrate LIII

TABLE 1. Structural Parameters at the U

Our results show that the U(VI)-citrate complex at pH 6.0 consists of a bridged binuclear complex with tridentate bonding of citric acid through a (di-µ-OH) core to each of the uranium atoms. In the presence of an electron donor, the hexavalent uranyl ion bound to citric acid was reduced to the tetravalent form without the concomitant dissociation of the complex and the bacterial metabolism of the organic ligand. The reduced uranium remained in solution complexed to citric acid as a biligand mononuclear U(IV)-citrate complex. Oxygen atoms in the inner-sphere of the 8-coordinate uranous ion not related to citrate most probably originate from the hydrolytic dissociation of water or addition of chemically bound water. The EXAFS results indicate that the coordinating oxygen atoms of the citric acid to uranium does not change as uranium is reduced. However, as a consequence of the bacterially mediated electron-transfer reaction to uranium, the axial oxygen atoms are converted to single bonds in the uranous ion. We found that Clostridium sp. and C. sphenoides reduced U(VI) complexed to citrate only when supplied with an electron donor, respectively, glucose and citrate. Although C. sphenoides can metabolize citrate, it did not metabolize citrate complexed to uranium, in agreement with previous findings with Pseudomonas fluorescens that also did not metabolize this complex under aerobic conditions (35, 36). These results confirm that the bioavailability of metal-citrate complexes is influenced by their structure. Thus, bidentate complexes are readily biodegraded, whereas tridentate binuclear complexes, such as U:citrate, are not, thereby denoting the structure-function relationships (35, 36). We have shown that, under anaerobic conditions, uranium complexed with organic ligands, such as citric acid, is readily accessible as an electron acceptor for anaerobic bacteria despite the inability of the bacteria to metabolize the organic ligand complexed to the actinide. Furthermore, the reduced uranium complexed with the organic ligand remains in solution; this finding is contrary to the common belief that reduced uranium will precipitate from solution. Our results also suggest that reduced uranium complexed with chelating agents may persist in the subsurface environments and enhance its solubility thereby becoming a major concern because of the potential for increasing the transport of radionuclides.

Acknowledgments We thank A. D. Woodhead for the editorial help and comments. This research was funded by the Environmental Remediation Sciences Program (ERSP), Environmental Remediation Sciences Division, Office of Biological and Environmental Research (OBER) Office of Science, U.S. Department of Energy, under Contract DE-AC02-98CH10886.

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