Environ. Sci. Technol. 1994, 28,636-639
XPS and XANES Studies of Uranium Reduction by Closfridium sp. Arokiasamy J. Francls,"gt Cleveland J. Dodge,? Fulong Lu,? Gary P. Halada,* and Clive R. Clayton* Department of Applied Science, Brookhaven National Laboratory, Upton, New York 11973, and Department of Materials Science and Engineering, SUNY at Stony Brook, Stony Brook, New York 11794
Speciation of uranium in cultures of Clostridium sp. by X-ray absorption near-edge spectroscopy (XANES) at the National Synchrotron Light Source and by X-ray photoelectron spectroscopy (XPS) showed that U(V1) was reduced to U(1V). In addition to U(IV), a lower oxidation state of uranium, most probably U(III), was detected by XANES in the bacterial cultures. Reduction of uranium occurred only in the presence of growing or resting cells, Organic acid metabolites, the extracellular components of the culture medium, and heat-killed cells failed to reduce uranium under anaerobic conditions. The addition of uranyl acetate or uranyl nitrate (>210 pM U) to the culture medium retarded the growth of the bacteria as evidenced by an increase in the lag period before resumption of growth, by decreases in turbidity, and in the total production of gas and organic acid metabolites. These results show that uranium in wastes can be stabilized by the action of anaerobic bacteria. Introduction
The direct implication of microorganisms in the reduction of uranium is a subject of considerable interest because of its potential application in the bioremediation of contaminated sites, in the pretreatment of radioactive wastes, and in processes critical to the performance of nuclear waste repositories (1-3). Although the presence of a wide variety of microorganisms in radioactive wastes and in natural deposits of radioactive minerals has been reported, the extent to which microbes can alter the mobility of radionuclides is not fully known (4-6). The reduction of uranium has been reported in axenic cultures of iron reducing bacteria (3, sulfate-reducingbacteria (8IO),and cell-free extracts of Micrococcus lactilyticus (111, and in uranium wastes by Clostridium sp. (12, 13). Determination of the oxidation states of uranium in natural or modified materials has become increasingly important from the standpoint of its mobility or stability in the environment. Several conventional techniques have been used to speciate uranium, including hydrogen consumption (1I ) , direct measurement of U(V1) in solution (14),conversion of insoluble species such as U(1V) to soluble U(V1) by extraction from aqueous acidification and oxidation (3, phase using solvents such as thenoyltrifluoroacetone (1517), and separation of U(V1) and U(1V) by ion-exchange resin (18). These methods involve extensive preparation of the sample that can result in changes in its oxidation state (19, 20). X-ray diffraction methods are useful in identifyingthe mineral phases but not the oxidation states. X-ray spectroscopic techniques (XRS) involve very little manipulation of the sample and provide more accurate information on the oxidation state of elements than conventional methods. We used XPS and XANES to determine the oxidation states of uranium in microbial cultures. _lll___
t Brookhaven t
National Laboratory. SUNY at Stony Brook.
838 Envlron. Sci. Technol., Vol. 28, No. 4, 1994
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Materials and Methods
Culture. Clostridium sp. (ATCC No. 53464) was grown in the following medium: glucose, 5.0 g; NH4C1, 0.5 g; glycerolphosphate,0.3g;MgSOr.7H20,0.2g; CaC12.2H20, 0.5 g; FeS04 .7H20,2.8 mg; peptone, 0.1 g; yeast extract, 0.1 g; deionized water, 1 L; pH, 6.8. The medium was pre-reduced by boiling for 15 min while purging with filtered ultra-high-purity (UHP) nitrogen gas. It was then cooled and transferred to an anaerobic glovebox, and 40mL aliquots of the medium were dispensed into 60-mL serum bottles. The bottles were sealed with butyl rubber stoppers and autoclaved. Reagents were pre-reduced by boiling and purging with a stream of Nz prior to use. All manipulations of the samples were carried out inside an anaerobic glovebox filled with NP. Effect of Uranium on Bacterial Growth. The influence of the uranyl ion on the growth of the bacteria was determined by adding to the medium 0,42,210, and 420 pM of uranium as uranyl acetate dihydrate or uranyl nitrate hexahydrate (ICN Biomedicals, Costa Mesa, CA). The pH was adjusted to 6.8 by adding pre-reduced, filtersterilized 0.1 M NaQH. The bottles were inoculated with a 24-h-old culture (early log phase growth) of bacteria, and triplicate samples were incubated in the dark at 26 "C. Uninoculated medium containing uranium was used as a control. After 45-h incubation (late log phase growth), total gas production in the head space of the culture sample was measured by a pressure transducer (Model 665-D/030, Wallace and Tiernan). Ten-milliliter aliquots of the culture were removed to determine bacterial growth, changes in pH, and production of organic acid metabolites. The growth of the bacterium was determined by measuring the turbidity a t 600 nm, using a Bausch and Lomb Spectronic 20 spectrophotometer. The culture sample was filtered through a 0.22-pm filter, acidified with 0.05 mL of concentrated HC1 (Ultrex), and analyzed by highpressure liquid chromatography (HPLC) for organic acid metabolites using a UV-vis detector a t 210 nm (Spectra Physics) and for glucose using a refractive index detector (Shimadzu). The culture was centrifuged at 17000g for 20 min, and the cell pellet and the supernatant were separated. The cell pellet was dried and stored in the dark in a desiccator under vacuum for uranium analysis by XPS and XANES. Uranium Analysis in Solution. A spectrophotometric method specific for U(V1) in solution was used to determine the soluble uranium in the supernatant (14). The supernatant was filtered through a 0.22-pm Millex filter, and the U(V1) in solution was determined. To determine total uranium in solution, the supernatant was purged with oxygen for 30 min to oxidizethe reduced forms of uranium to U(VI), which was then analyzed spectrophotometrically. The difference between total uranium and U(V1) gave the amount of reduced forms of uranium in solution. 0013-936X/94/0928-Q636$04.50/0
0 1994 American Chemlcal Society
Table 1. Effect of Uranyl Acetate on Growth of Clostridium S P . ~
uranium (pM)
turbidity (600nm)
gas produced (mL)
PH
acetic acidb (mM)
butyric acid (mM)
0 42 210 420
0.84 f 0.02c 0.80 f 0.00 0.34 f 0.20 0.09 f 0.01
31.6f 0.7 28.2f 2.1 9.6 f 6.1 0.9 f 0.1
3.1 f 0.0 3.2f 0.0 5.0f 0.7 6.4-+ 0.1
8.44 f 0.30 7.57 f 0.05 3.33 f 1.23 0.90 f 0.12
13.7f 0.8 11.1 f 0.4 4.36 f 2.89 0.69 A 0.13
a Growth was determined after 45 h. b Values corrected for the addition of acetate from uranyl acetate. fl standard error of the mean (fl SEM).
Uranium Analysis in Solids and Bacteria. X-ray photoelectron spectroscopy (XPS) was used to determine the oxidation states of uranium on the solid phase using a VG Scientific ESCA 3 MK I1 spectrometer. The cell pellet and the precipitate from uninoculated control medium were dried, pressed firmly onto indium foil, and transferred to the spectrometer under argon to minimize surface modification. Samples were cooled with liquid N2 to prevent heating and decomposition. Spectra were obtained for U 4f5/2 and U 4f7/2 peaks using an A1 Ka1,2 (1486.6 eV) source. The binding energies of reference compounds Au 4f7/2 and Cu 2p3/2 were 83.8 (FWHM 1.4 eV) and 932.5 eV (FWHM 1.7 eV), respectively. Spectra were corrected for charge shifting using the Cls spectrum from adventitious hydrocarbon at 284.6 eV. All spectra were smoothed with modificationto cover truncation errors at their ends (21,22). Curve fitting gave the peak position, height, width, and shape, in addition to the background slope. X-ray absorption near-edge structure (XANES),usually within 40 eV of the absorption edge, provides information on the local structure and oxidation state of the metal. The cell pellet was mounted on Mylar foil using a mixture of acetone and DUCO cement (duPont de Nemours Co.) and placed in a helium flushed sample chamber. Measurements were made at the MV (3545 eV) absorption edge using an electron yield detector and a double crystal Si(111) monochromator on the X-19A beam line at the National Synchrotron Light Source,Brookhaven National Laboratory. Data points in the absorption peak region were collected with a resolution of 0.1 eV and 3 s time average, using a scanning program. Background subtraction, normalization of the data to the edgejump, and curve fitting were done to determine the shifts in peak energy. High-purity uranium metal, cleaned in 6 M concentrated nitric acid, and uranium oxides were used to calibrate the peak energy values for both XPS and XANES analysis. All samples for analysis were prepared in an anaerobic glovebox under nitrogen. Mechanisms of Uranium Reduction. The methods involved in elucidating the mechanisms of uranium reduction were described previously (23). Briefly, indirect reduction of uranium due to metabolic products as well as changes in Eh and pH of the medium, were determined by adding 210 pM uranyl acetate to (i) pre-reduced, autoclaved medium (control); (ii) pre-reduced synthetic medium containing organic acids in the same amounts as found in a late logarithmic growth phase of the bacterial culture; (iii) cell-free spent medium obtained from a 41h-old culture of bacteria (OD6mnm,0.60), grown without uranium and passed through a 0.22-pm Millex filter to remove the cells, and (iv) autoclaved cell-free spent medium to inactivate any enzymes. This last medium was refiltered to remove any denatured proteins as well as other extracellular components. Reduction of uranium by direct enzymatic action was determined by adding 210 pM uranyl acetate to a 41-h-old
Table 2. Analysis of Uranium in Solution
uranium (pM) 42 210 420
treatment
total (pM)
uraniuma U(V1) (*M)
U(1V) (pM)
uninoculated 0.04 f O.OOb 0.04 f 0.00 CO.01 inoculated 0.38 f 0.28 CO.01 0.38f 0.28 uninoculated 2.41 f 0.28 2.43 f 0.31 CO.01 inoculated 1.55 f 0.55 0.04 f 0.02 1.51 f 0.54 uninoculated 16.3f 3.6 16.3 f 3.6 CO.01 inoculated 3.4 f 1.1 3.4 f 1.1 CO.01
Uranium was analyzed after 45 h. fl SEM.
late logarithmicgrowth phase of bacterial culture (OD6wnm, 0.60) containing (i) resting cells and (ii) heat-killed (autoclaved) cells. The cell pellet was separated from the supernatant by centrifugation. Soluble forms of uranium in the supernatant were analyzed spectrophotometrically, and the precipitated form in the cell pellet was analyzed by XPS. Concentration of uranium in the solid phase was determined spectrophotometrically after digestion with "03. All samples were analyzed for uranium after 2and 24-h incubation.
Results Effect of Uranium on Bacterial Growth. Uranyl acetate inhibited the growth of the bacterium as evidenced by a decrease in turbidity, a decrease in the production of total gas and the major organic acid metabolites, acetic and butyric acids, and changes in pH (Table 1). The effect was more pronounced at higher concentrations of uranium. In a medium containing 420 pM U, only slight growth was noted. The addition of uranyl nitrate also retarded bacterial growth, but to a much lesser extent (data not shown). In addition to acetic and butyric acids, a significant amount of propionic acid was detected in the culture medium. The concentration varied between 3 % and 21% of total acid production and increased with increasing uranium concentration Uranium Analysis in Solution. Only a small amount of the total uranium added to the medium (0.1-4%) was present in solution in the uninoculated control sample (Table 2). This was due to the formation of a colloidal precipitate as a result of hydrolysis and condensation reactions with the medium components. Analysis of the soluble uranium in the supernatant of the uninoculated control sample showed that it was present as U(VI), whereas in the inoculated sample it was in the reduced form as U(1V). Much less soluble U(V1) was detected in the supernatant of the inoculated samples because of the reduction to the insoluble U(1V) by the bacteria. Uranium Analysis in Solids and Bacteria. The cell pellets recovered from the uranium-containing inoculated samples were dark green, indicating the presence of a reduced form of uranium, whereas the pellet from the uninoculated control was yellow, characteristic of uranyl salts. Environ. Scl. Technol.. Vol. 28, No. 4, 1994
637
I
I
I
Uninoculated E ib
Inoculated
\ Uninoculated [U(Vl)]
400
395
390
385
380
375
1
1
1
I
1
I
I
I
I
l
1
0 ~
Binding Energy (eV) Figure 1. XPS spectra of uranium(V1) in the uninoculated control sample and uranium(1V) in the sample inoculated with ClosfrMiumsp.
XPS analyses of the oxidation states of uranium in the cell pellet and in the precipitate from the control sample were determined by comparing the shifts in binding energy with known standards. Figure 1shows the U 4f XPS data of the uranium samples before and after bacterial treatment. The presence of two peaks in the spectrum (e.g., A,a) is due to multiplet splitting of electrons with unpaired spins in the atomic shells representing the U 4f7p and U 4f5p spin states. In the control sample, the binding energy of the U 4f712 peak (A) is at 382.0 eV, which is identical with the standard spectrum of uranyl acetate (not shown), confirming that the uranium in the uninoculated culture medium is present in the U(V1) oxidation state. The cell pellet obtained from the inoculated sample (42 pM U or 210 pM U) showed a marked shift to lower binding energy at the U 4f7/2 peak (B)to 380.6 eV, which is in the range of the binding energyfor the U(1V)oxidation state. These results confirm the reduction of uranium from U(V1) to U(1V) oxidation state in the bacterial culture sample. Curve fitting of the data, however, indicated the presence of a small amount of U(V1) oxidation state (A) in the inoculated samples. The X-ray source was not monochromatic and produced an additional peak, labeled C. XANES spectra for uranium at the MV absorption edge for the inoculated and uninoculated control samples containing uranium are shown in Figure 2. In the uninoculated control sample, the absorption peak for uranium is at 3551.1eV, which is identical to the absorption peak for uranyl acetate standard (not shown) and confirms that uranium was not reduced by the components of the medium under anaerobic conditions. However, in the inoculated sample, there was a shift in the absorption peak to lower energy at 3550.1 eV, which was lower than the absorption peak position for U(1V) (3550.4 eV) but higher than that of uranium metal (3549.6 eV), indicating the 838 Environ. Scl. Technol., Vol. 28, No. 4, 1994
3540
~~
3550 3560 Energy (eV)
3: '0
Figure 2. XANES spectra of U(VI), U(IV), U metal, and sample inoculated with Clostridlum sp.
Table 3. Mechanisms of Uranium Reduction by Clostridium sp. treatmenta
detected (pM)
uranium reduced ( % )
control organic acids cell-free spent medium cell-free spent medium (autoclaved) resting cella heat-killed cells
NDb ND
ND ND
u4+
2
1
ND
ND
190 94 4 2 210 pM U as uranyl acetate was added. ND = not detected. (Samples were analyzed at 2 and 24 h after the addition of uranium, and the results were identical.)
presence of a highly reduced form of uranium, most probably U(II1). Mechanisms of Uranium Reduction. Uranium was reduced only in the presence of live bacteria. The extracellular components did not reduce uranium to any significant extent (Table 3). Although 95% of the added uranium in the uninoculated control sample was precipitated, analysis of the precipitate and the supernatant showed no reduction of U(VI), similar to the results reported in Table 2. The organic acid metabolites did not reduce U(VI), and all the added uranium was present in solution as U(V1). The cell-free spent medium containing the metabolic acids and other extracellular products showed little (1% ) reduction of U(V1) and, similar to the organic acid metabolites, almost all the added uranium was present as U(V1) in solution. Also, the autoclaved cell-free spent medium did not reduce uranium, and it was present in solution. After the addition of U(V1) to the resting culture, only 7.4 pM (4 % ) of uranium remained in solution, of which 82% was in the reduced form. Analysis of the cell pellet showed that almost all of the
uranium (95%) was reduced to U(1V) and possibly to U(II1). In the autoclaved culture sample, 84 pM uranium was present in solution as U(VI), and the remaining uranium recovered with the cell pellet was present predominantly as U(V1). Uranium was reduced by the growing and resting cells only and not by heat-killed cells nor by the cell-free spent medium. XPS analysis of the cell pellet from active cultures, but not from autoclaved cultures, showed the reduction of U(V1) to U(1V). These results suggest that reduction is due to the enzymatic action of the bacteria. Discussion
Higher concentrations of U(V1) inhibited the growth of Clostridium sp. A similar effect on bacterial growth has been reported (24). Retardation of growth initiation is a
fairly common feature of sublethal concentrations of toxic metals on bacteria (25,26). It was postulated that during this lag period the cells develop some mechanisms of molecular accommodation. The reduction of uranium was not due to the ingredients present in bacterial growth medium, the low molecular weight organic acid metabolites, nor to components in the spent medium. Uranium was reduced from the higher oxidation state to lower oxidation states only by growing cells or by resting cultures. The lack of reduction of uranium by the autoclaved spent medium or by autoclaved resting cultures suggests that the reduction is due to enzymatic action. XPS is a surface analytical technique; therefore, the detection of U(1V) in the cell pellet indicates the presence of this species at the surface. XANES is complementary to XPS, and its higher energy allows probing at greater depth. The detection, for the first time, of a lower oxidation state of uranium other than U(1V) in the bacterial culture indicates that the uranium is located beneath the surface within the biomass, although its exact association is unknown. The absence of U(II1) on the surface of the samples is probably due to the fact that U(II1) is unstable and rapidly oxidized to U(1V) in aqueous medium (27). The Clostridium sp. used in this study has been shown to solubilize ferric iron in hematite, goethite, and ferrites and to solubilizemanganese(1V)in pyrolusite by enzymatic reduction and the oxides of cadmium, copper, lead, and zinc due to the production of organic acid metabolites (23, 28, 29). The change in free energy for the reduction of manganese and iron is -83.4 and -27.2 kcal/mol CH20, respectively (30). The change in free energy for the reduction of hexavalent uranium to the tetravalent state is -63.3 kcal/mol and to the trivalent state -52.2 kcal/mol. Uranium reduction should occur in the sequence Mn(1V) > U(V1) > Fe(II1) (18, 31). Clostridia are ubiquitous in soils, sediments, and wastes and could be very useful in the pretreatment and stabilization of uranium in radioactive wastes. For example, treatment of uranium wastes by Clostridium sp. has resulted in the concentration and stabilization of uranium, and at the same time the volume and mass were reduced (13). Uranyl ion present in the water soluble, exchangeable, and carbonate fractions was reduced to U(1V) by the action of anaerobic bacteria. Acknowledgments
We thank F. J. Wobber, program manager, for continued support and J. B. Gillow and S. Kagwade for assistance
in the preparation of cultures and data collection. This research was performed under the auspices of the Environmental Sciences Division’s Subsurface Science Program, Office of Health and Environmental Research, Office of Energy Research, U. S. Department of Energy, under Contract DE-AC02-76CH00016.
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Received for review June 8,1993. Revised manuscript received December 22, 1993. Accepted January 3, 1994.” @
Abstradpublishedin Advance ACSAbstracts,February 1,1994. Environ. Sci. Technol., Voi. 28, No. 4, 1994 639
