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COMMUNICATIONS Enzymatic Uranium Precipitation Yurl A. Gorby and Derek R. Lovky' Water Re80urCBs Division, 430 National Center, U.S. Geological Survey. Reston. Virginia 22092 Introduction Techniques for the removal of dissolved uranium from waters are needed for a variety of environmental applications. Surface waters or groundwaters may have undesirably high concentrations of dissolved uranium as the result of natural processes or from contamination resulting from uranium mining and processing activities or the release of nuclear materials in the environment (1-4). It was recently demonstrated that two dissimilatory Fe(II1)-reducing microorganisms, GS-15 and Shemanella putrefacierw, can use U(VI) as a terminal electron acceptor (5). Under anaerobic conditions, acetate (GS-15) or hydrogen (S. putrefacierw) is oxidized with the concomitant reduction of U(VI) to U(IV). The organisms obtain energy to support growth from this metabolism (5). Subsequent studies have further indicated that U(V1) reduction is an enzymatic reaction coupled to electron-transport chains within these organisms (6). Although U(V1) is soluble in most surface waters and groundwaters, U(IV) is highly insoluble (7,8). Thus, microbial U(VI) reduction has the potential to convert uranium from a soluble form to an insoluble form (5). However, the ability of U(VI)-reducing microorganisms to precipitate uranium from solution has not been previously investigated. This report represents the first phase in attempts to evaluate the potential for removing uranium from contaminated waters with U(V1)-reducing microorganisms. Materials and Methods Cell Suspensions. The U(VI)- and Fe(II1)-reducing microorganism, GS-15, was cultured under an anaerobic atmosphere of NzXOz (8090) in a defined medium (9) with acetate (10 mM) as the sole electron donor and Fe(III)-citrate (ca.50 mM) as the terminal electron acceptor. GS-15 cannot use citrate as an electron donor for Fe(II1) reduction (9). AU manipulations of the cells were carried out under N2-COZ. The cells were harvested by centrifugation and washed three times in anaerobic bicarbonate buffer (30 mM). Groundwater was taken from an uncontaminated site in a stratified sand and gravel outwash aquifer located in Cape Cod, MA (10). The dilute calcium bicarbonate groundwater which had a specific conductance of 46 pQ-'/cm and a pH of 6.5 was amended with sodium hicarbonate (30 mM) and uranyl acetate (0.4 or 1.0 mM). The bicarbonate was added to ensure that all of the uranium was in the form of a uranyl carbonate complex. This is important because (1) this is the most common form of U(V1) in most natural waters (7,8)and (2) bicarbonate is the extractant most likely to be used in attempts to remove uranium from contaminated sediments and soils. The water was dispensed into large culture vessels (500 mL of medium in a 1-L bottle; Figure l),pressure tubes (10 mL in a 26-mL tube; Figure 2), or serum bottles (40 mL of medium in a 160-mL serum bottle; Figure 3). The Not subject to U.S. Copylght.
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28 Predpbte tamed as a res* of enzymaIh WI)reduction
ngun 1. by GS-15. Uhoa#f~ted !qmmdwnW (A. left) and appearam of water 24 h after addition of GS-15 (A. Wt). Filters (6) following passage of unlmulated (left) and lmu$ted (Wt)groundwater 24 h after hoculatbn. X-ray diffractionpattern of biogenic WIV) preclpltate (C). Lattice spaclng values matched moSe for uranlntte from me Joint Commmee on Powder DiffractionFile data card 5-549(Inset).
water was sparged with NZXOzto remove dissolved oxygen and the bottles were stoppered. The pH was 6.8. Washed cells were anaerobically injected into the water with a syringe and needle and the resultant cell suspension was incubated a t 30 OC. In the serum bottle incubations, subsamples (0.5 mL) of the suspension were withdrawn over t i e with a syringe and needle and processed in a glovebox containing an atmosphere of Nz-COZ-Hz(85510). Suhsamples passed through a 0.2-pm polycarbonate filter as well as untreated subsamples were acidified with 12 N HC1 (0.25 mL) and then diluted 1:loOO with anaerobic deionized water. Diluted samples (1mL) were mixed with an anaerobic complexing reagent (Uraplex, 1.5 mL), sealed in an anaerobic cuvette, and analyzed with a kinetic phosphorescence analyzer (KPA-10, Chemchek Instruments), which uses a pulsed nitrogen dye laser to measure U(VI) in solution. In order to determine U(IV) concentrations, samples were aerated for 60 min prior to acidification and dilution. This treatment oxidized the U(IV) to U(V1). The difference between the U(V1) concentration in the oxidized and unoxidized treatments could be attributed to U(1V) in the original sample. X-ray Diffraction. A black precipitate resulting from microbial U(V1) reduction in a cell suspension of GS-15 and uranyl acetate (1mM) was collected on filter paper (whatman No. 5), dried under a stream of Orfree nitrogen, and ground to a fine powder with a mortar and pestle. The randomly oriented powder was mounted on a glass slide
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uranium was determined by transmission electron microscopy using a Philips EM400T operating at 100 kV. Elemental composition of visible precipitates were determined by energy-dispersive X-ray analysis using a Link EDS system (IO-nm spot size) attached to a Link light element detector.
Results and Discussion Biogenic U(IV) Precipitation. As GS-15 reduced the U(VI) in groundwater amended with 1 mM uranyl acetate, the yellow color of the water prior to inoculation (Figure lA, left) disappeared and a hlack precipitate accumulated (Figure lA, right). When water ( 5 mLJ from the bottles was pamed through a glass fiber filter (Gelman type A, E), the black precipitate from the bottle receiving GS-15 accumulated on the filter (Figure lR, right) whereas there was no precipitate from the bottle that had not received GS-15 (Figure lH, left). X-ray diffraction analysis indicated that the precipitate was comprised of the U(IV) mineral, uraninite (UO& (Figure 1cJ. Uraninite is the most common naturally occurring U(IV) mineral and is present in many anoxic sediments and aquifers (7.J l , 12). Cellular Location of U(IV) Precipitation. An electron-dense precipitate formed in U(V1)-reducing cell suspensions of GS-15 (Figure 2A). T h e precipitate was entirely extracellular, as confirmed by viewing the specimen at several angles on a goniometric stage. The precipitate was identified as uranium (Figure 2R). No intracellular uranium was detected. Uranium precipitates were absent from heat-killed (80O C , 20 min) suspensions incubated at 30 "C or live suspensions incubated at 4 ' C since these treatments inhibited U(VU reduction. The formation of the fine-grained, extracellular precip itate of U(IV) during U(VI) reduction by GS-15 is reminiscent of the formation of extracellular magnetite by this organism when is uses Fe(1lI) as the electron acceptor (13, 14).
Figure 2. Electron micrograph (A) of U(Vr)-reducingcell suspension (bar = 1.0 !A).The electrondense precipitate which accumulated in the surrounding medium was identified as uranium (U)by energydispersive X-ray spectroscopy (B). The copper signal (Cu)was from the sample grid.
with amyl acetate and analyzed by X-ray diffraction. Electron Microscopy a n d Energy-Dispersive X-ray Spectroscopy. The cellular location of precipitated
Time Course of U(V1) Reduction e n d Uranium Precipitation. In order to examine the relationship hetween U(V1) reduction and the precipitation of U(IV), UtVI) reduction and the formation of a U(1V) precipitate were examined on a short time scale (Figure 3). Throughout the experiment, the loss of U(V1) in the suspension was accompanied (within ca. 10%) by a corresponding increase in U(W) (Figure 3A). However, in the early stages of U(V1) reduction, the U(IV) that was produced pawed through a 0.2-um pore diameter filter. Only
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Figure 3. Time course
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after more than half of the U(V1) was reduced did U(1V) begin to be retained on the filter. These results indicated that the formation of large (i.e., >0.2-pm diameter) U(1V) precipitates was not instantaneous and was not directly linked to U(V1) reduction. As U(1V) began to be retained on the filters, a portion of the remaining U(V1) was also retained, evidenced as a discrepancy between the concentration of U(V1) in the filtered and unfiltered samples (Figure 3A). This was in contrast to earlier time points in which all of the U(V1) present in the cell suspensions passed through the filters. The retention of U(V1) on the filter coincided with a decrease in the rate of U(VI) reduction. Thus, it seems likely that, after ca. 10 h of incubation, some of the remaining U(V1) had become incorporated into the U(1V) precipitate and was no longer available for microbial reduction. This phenomenon requires further study. In contrast to U(V1)-reducingsuspensions, no U(V1) was reduced and no precipitate formed in cell suspensions incubated a t 4 "C (Figure 3B). As an additional control, no U(V1) was reduced and no precipitate formed in heat-killed cell suspensions incubated a t 30 "C (data not shown). These results demonstrate that U(V1)-reducing microorganisms have the potential to readily precipitate uranium from water. Thus, this metabolism may provide a novel mechanism for the removal of uranium from a variety of contaminated waters. Previously, biosorption had been the bioremediation method of choice for removal of uranium from contaminated waters (15). Microbial U(V1) reduction which results from the direct enzymatic metabolism of U(V1) is a fundamentally different process from biosorption, which typically relies on a passive interaction between U(V1) and the biosorbant. The relative effectiveness of microbial U(V1) reduction versus biosorption strategies in treating a variety of typical uranium-contaminated waters is currently under investigation. Acknowledgments
We thank Dr. Terry Beveridge and Robert Harris for electron microscopy, Daniel Webster for X-ray diffraction,
Warren Wood for providing the groundwater, and Ed Landa and Harvey Bolton for helpful suggestions on the manuscript. Registry No. U, 7440-61-1;UOz, 1344-57-6. Literature Cited (1) Waite, D. T.; Joshi, S. R.; Sommerstad, H. Arch. Environ.
Contam. Toxic01 1989,18, 881-887. (2) Bradford, G. R.; Bakhtar, D.; Westcot, D. J. Enuiron. Qual.
1990,19, 105-108. (3) Osiensky, J. L.; Williams, R. E. Ground Water Montit. Rev. 1990, 10, 107-112. (4) Strandberg, G. W.; Shumate, S. E., 11; Parrott, J. R., Jr. Appl. Enuiron. Microbiol. 1981, 41, 237-245. (5) Lovley, D. R.; Phillips, E. J. P.; Gorby, Y. A,; Landa, E. R. Nature 1991, 350, 413-416. (6) Gorby, Y. A.; Lovley, D. R. Appl. Environ. Microbiol., in preparation. (7) Langmuir, D. Geochim. Cosmochim. Acta 1978,42,547-569. (8) Taylor, G. H. In Biogeochemical Cycling of MineralForming Elements; Trudinger, P. A., Swaine, D. J., Eds.; Elsevier: New York, 1979; Chapter 8. (9) Lovley, D. R.; Phillips, E. J. P. Appl. Environ. Microbiol. 1988,54, 1472-1480. (10) LeBlanc, D. R. Sewage plume in a sand and gravel aquifer, Cape Cod, Massachusetts. U. S. Geol. Surv. Water-Supply Pap. 1984, No. 2218. (11) Hostetler, P. B.; Garrels, R. M. Econ. Geol. 1962, 57, 137-167. (12) Durrance, E. M. Radioactivity in Geology; John Wiley & Sons: New York, 1986. (13) Lovley, D. R.; Stolz, J. F.; Nord, G. L.; Phillips, E. J. P. Nature 1987, 330, 252-254. (14) Lovley, D. R. In Iron Biominerals; Frankel, R. B., Blakemore, R. P. Eds.; Plenum Press: New York, 1990; pp 151-166. (15) Tsezos, M. In Microbial Mineral Recovery; Ehrlich, H. L., Brierley, C. L., Eds.; McGraw-Hill: New York, 1990; Chapter 14. Received for review August 7,1991. Revised manuscript received September 9,1991. Accepted September 26,1991. This study was supported by the U. S. Geological Survey Toxic Waste and Nuclear Waste Hydrology Programs.
Measurement of the Absorption Constants for Nitrate in Water between 270 and 335 nm Jeffrey S. Gaffney," Nancy A. Marley, and Mary M. Cunningham
Environmental Research Division, Building 203, Argonne National Laboratory, Argonne, Illinois 60439 Introduction
Acidic species in rain have received substantial attention in recent years. Most of this attention has been placed upon the acidity (Le., H+) of the solutions, with little attention being placed upon the corresponding inorganic and organic ions ( I ) . Sulfate and nitrate are known to be the dominant inorganic species in acidic precipitation (2). However, substantial numbers and amounts of organic species can also be present, as pointed out in previous articles and books ( I , 2). A number of questions remain regarding the formation of organic acids and diacids (i.e., oxalic acid) and their subsequent aqueous deposition. The production of these acids in the gas phase is difficult, since the formation of peracids or peroxyacetyl nitrates is the dominant reactive pathway for organic oxidations ( 3 ) . Therefore, the potential for aqueous oxidation in aerosols and clouds has 0013-936X/92/0926-0207$03.00/0
been given recent attention. Zellner and co-workers have examined the potential photolysis of nitrate, nitrite, and dissolved hydrogen peroxide at 308 and 351 nm by using single-line lasers ( 4 ) . They observed that the photolysis of nitrate in acidic water yielded OH in solution and determined that when compared to aqueous nitrite and hydrogen peroxide photolysis under relevant conditions this photochemical reaction was likely to be the dominant source of this oxidant in the aqueous phase. Thus, this reaction may be an important source of organic oxidation in clouds and aerosols (5). It is now well documented that the release of chlorofluorocarbons into the troposphere affects the levels of ozone in the stratosphere (6). The reduction of stratospheric ozone will increase the levels of ultraviolet radiation in the troposphere because ozone is a key ultraviolet radiation absorber in the stratosphere (2). Thus, increases
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