Environ. Sci. Technol. 2001, 35, 1599-1603
CoIIIEDTA- Reduction by Desulfovibrio vulgaris and Propagation of Reactions Involving Dissolved Sulfide and Polysulfides TODD C. BLESSING,† BRUCE W. WIELINGA,‡ MATTHEW J. MORRA,† AND A N D S C O T T F E N D O R F * ,‡ Soil Science Division, University of Idaho, Moscow, Idaho 83844-2339, and Department of Geological and Environmental Sciences, Stanford University, Stanford, California 94305-2115
The migration of 60Co, dominantly via transport of CoEDTA complexes, into surface water and groundwater is a recognized concern at many nuclear production and storage sites. Reduction of CoIIIEDTA- to CoIIEDTA2- should decrease the mobility of 60Co in natural environments by stimulating ligand displacement with Fe(III) or Al(III) or by precipitation of CoSx in sulfidic environments. In this study, we examine direct (enzymatic) and indirect (metabolite) reduction processes of CoIIIEDTA- by the sulfatereducing bacterium Desulfovibrio vulgaris. D. vulgaris reduces CoIIIEDTA- to CoIIEDTA2-, but growth using it as a terminal electron acceptor was not demonstrated. Rather than acting as a competing electron acceptor and limiting cobalt reduction, introducing sulfate with D. vulgaris enhances the reduction of CoIIIEDTA- as a result of sulfide production. Sulfide reduces CoIIIEDTA- in a pathway involving polysulfide formation and leads to a CoS precipitate. Thus, both direct and indirect (i.e., through the production of sulfide) microbial reduction pathways of CoIIIEDTA- may help to retard its migration within soils and waters.
Introduction Radioactive 60Co is a priority pollutant at Department of Energy (DOE) sites. Codisposal of this radioisotope with organic chelates such as EDTA enhance its mobility in surface and subusurface environments (1, 2). EDTA complexes of both di- and trivalent cobalt are present in natural environments; however, CoIIIEDTA- is much more stable and hence mobile than CoIIEDTA2- (1, 3). Cations such as Al(III) and Fe(III) can dissociate the CoIIEDTA2- complex with concomitant formation of an Al/Fe-EDTA complex (2-4). Unfortunately, soil materials such as manganese, and to a lesser extent iron, oxides promote the oxidation of CoIIEDTA2to CoIIIEDTA- (2, 3, 5) leading to enhanced transport of Co. Processes that lead to the reduction of Co(III) to Co(II) thus increase the potential release of 60Co from the soluble EDTA complex and help to resist the transport of this radioisotope. One means of reducing Co(III) to Co(II) is through microbially mediated redox transformations. Microbial re* Corresponding author e-mail:
[email protected]; phone: (650)723-5238; fax: (650)725-2199. † University of Idaho. ‡ Stanford University. 10.1021/es001576r CCC: $20.00 Published on Web 03/02/2001
2001 American Chemical Society
mediation of heavy metal-contaminated sites is being actively investigated, particularly for elements that are stabilized by reductive processes (see refs 6-9). For example, the ironreducing bacterium Shewanella alga strain BrY can use CoIIIEDTA- as a terminal electron acceptor (TEA), reducing it to CoIIEDTA2- (10, 11), and thus diminish the hazard imposed by this radioisotope. Enzymatic reduction of CoIIIEDTA- by sulfate-reducing bacteria (SRB) has not been demonstrated; however, SRB have shown the ability to reduce Mo (12), U(VI) (8, 9), Cr(VI) (6, 7), Fe(III) (13), and Mn (14). SRB may reduce metals by two means: either by direct, enzymatic reduction or through production of dissolved sulfide that secondarily reduces the metal. Recent studies help to define optimal conditions for enzymatic metal reduction by SRB (9, 12). The results of these experiments implicate heavy metal concentrations, electron donors, electron acceptors, and solid phases as influencing reaction rates and products. Limited research has focused on indirect means of reducing heavy metals via the production of sulfide despite the fact that it may predominate in metal-contaminated environments (6). Sulfide is a strong reductant capable of reducing metal complexes such as CoIIIEDTA-. Furthermore, under many conditions, dissolved sulfide leads to the formation of other kinetically facile reactants such as polysulfides. An additional benefit of dissolved sulfide production is the possible formation of cobalt-sulfide solid phases (15-18). Thus, SRB and associated sulfide may exert a strong influence on CoIIIEDTA- reduction and stabilization in natural environments. It seems likely that the fate of Co will be determined by a combination of biotic and abiotic processes, many of which may be coupled. Our objective in this work is to elucidate both direct (enzymatic) and indirect (reaction with the metabolite sulfide) mechanisms of SRB-mediated reduction of CoIIIEDTA-.
Materials and Methods Cell Suspension Preparation. Desulfovibrio vulgaris (Hildenborough) (ATCC 29579) was obtained from the American Type Culture Collection, Rockville, MD. D. vulgaris was routinely cultured at 30 °C on modified Baar’s medium (ATCC Medium No. 1249). The medium contained the following components (in g/L of deionized water): MgSO4, 2.0; sodium citrate, 5.0; CaSO4‚2H2O, 1.0; NH4Cl, 1.0; K2HPO4, 0.5; yeast extract, 1.0; sodium lactate, added to provide a final concentration of 30 mM from a 60% syrup. All components were dissolved in deionized water, and the pH was adjusted to 7.5. The medium was boiled and cooled under a stream of oxygen-free N2 gas, transferred to 125-mL serum vials, and capped with thick butyl rubber stoppers prior to sterilization. Oxygen levels were measured with an O2sensitive electrode. Ferrous ammonium sulfate was omitted from the medium to avoid the precipitation of ferrous sulfide, and the headspace of the vials was replaced with fresh N2 gas 2-3 times per day to prevent the accumulation of sulfide. Cells were harvested by centrifugation (5500g, 15 min, 4 °C) and washed twice with anoxic 30 mM Tris buffer (pH 7.2). Washed cell suspensions were transferred to pressure tubes with an O2-free N2 gas headspace, stored at 4 °C, and used within 15 min. Cell density in the final suspension was determined by direct counts using a Petroff-Hausser counting chamber. Chemical Preparation. CoIIIEDTA- was prepared according to Taylor and Jardine (19) by heating 20 g of cobalt(II) chloride hexahydrate, 25 g of EDTA, and 50 g of potassium acetate in 150 mL of deionized water to near boiling and VOL. 35, NO. 8, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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then adding 32 mL of 7% sodium peroxide. Cobalt was then speciated as described below to ensure complete production of Co(III). The oxidized solution was cooled and then precipitated with ethanol. This process was repeated two additional times to form the precipitate CoIIIKC10H12N2O8. CoIIEDTA2- was made by combining equimolar concentrations of cobalt(II) chloride hexahydrate and EDTA. Sulfide standard solutions were prepared by dissolving reagent-grade Na2S‚6H2O into N2-purged deionized water and used immediately after preparation. All other standards or chemicals used were of reagent-grade quality and were not further purified. A modified protocol from Rosen and Tegman (20) was used to synthesize Na2S5. Elemental sulfur and sodium sulfide were mixed in a test tube at a 4:1 molar ratio. The tube was then placed under a vacuum aspirator and sealed with a propane torch. A cannula was used to purge the bottom of the test tube with nitrogen. This step in the procedure was an effective way to purge oxygen and gaseous hydrogen sulfide from the system. The test tube was then heated to 400 °C in a muffle furnace. The color of the product was brick red, matching the published description (21). Once cooled, the sealed test tube was placed in a glovebox to inhibit product oxidation. The Raman spectrum of the synthesized material was consistent with that of S52(22). Microbial Reactions. CoIIIEDTA- reduction experiments were performed in 125-mL serum vials containing 30 mM Tris buffer (pH 7.2) supplemented with the following (in g/L): NH4Cl, 1.0; KCl, 0.2; CaCl2, 0.2; NaCl, 0.5; MgCl2, 0.4. Lactate (30 mM) or ethanol (20 mM) was provided as the electron donor. When ethanol was used, sodium tungstate (0.1 mM) was added to stimulate ethanol metabolism (23). CoIIIEDTA- was added to give an initial concentration from 100 to 500 µM depending on the experiment. Sodium sulfate was supplied to specified systems as an alternate electron acceptor at concentrations ranging between 0.5 and 25 mM. Ethanol, tungstate, CoIIIEDTA-, and sodium sulfate were all added from prereduced, filter-sterilized stock solutions. D. vulgaris cell suspension was added to give a final concentration of approximately 1 × 107 cells/mL. Heat-killed cells were prepared by holding the cell suspension at 80 °C for 20 min. Chemical Reactions. All chemical reactions were conducted in 20 mM Tris buffer (pH 7.2) under an N2 atmosphere at 23 °C in 125-mL serum vials. Solutions were deoxygenated by purging with oxygen-free N2 gas for 30 min. Reactions were initiated by adding CoIIIEDTA- and sulfide to the buffer from concentrated stock solutions. Reduction of CoIIIEDTAwas explored using sulfide concentrations ranging from 0.1 to 1.5 mM, with 110 µM CoIIIEDTA-. During the reactions, aliquots were taken from the serum vials and analyzed as discussed below. The potential for polysulfide-mediated CoIIIEDTA- reduction was tested in reactions with conditions similar to those described above. In one set of experiments, elemental sulfur was added to solutions containing 1 mM S2- and 110 µM CoIIIEDTA to promote in situ formation of polysulfides. Since S0 was present in excess, equilibrium concentrations of all polysulfide species were presumed (24-27). In a second set of reactions, pentasulfide (S52-) in concentrations of 550, 750, and 950 µM was added to serum vials containing 350 µM CoIIIEDTA giving Co(III):S52- ratios of 1:1.6, 1:2.1, and 1:2.7. Analytical Methods. CoIIEDTA2-, CoIIIEDTA-, and sulfate concentrations were quantified by ion chromatography (IC) using a method outlined by Taylor and Jardine (19). A Dionex DX500 IC (Dionex Corp., Sunnyvale, CA) equipped with an Ionpac AG5 guard column, GP40 gradient pump, AS40 auto sampler, ARSII ultra supressor (4 mm), ED40 electrochemical detector, and an Ionpac AS11 anion exchange column (4 mm) was used. Typically the IC was run isocratically (2 mL 1600
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min-1) with a bicarbonate (2.8 mM) and carbonate (1.2 mM) eluent. In this method, CoIIIEDTA- elutes at 1 min, CoIIEDTA2elutes at 3.5 min, and sulfate elutes at 10.5 min. In contrast to Taylor and Jardine (19), we did not observe any interference of CoIIIEDTA- with chloride, most likely due to the use of different columns. Peak Net Software 5.01 (Dionex Corp.) was used for peak detection and integration. For reactions involving D. vulgaris and media, we observed an anionic peak interference with CoIIIEDTA-; in this case, CoIIIEDTAwas measured spectrophotometrically at 535 nm. Sulfide concentrations in samples were determined by the methylene blue method (28); polysulfides do not interfere with sulfide detection by this colorimetric procedure when the ratio of hydrogen sulfide species to polysulfides is large (26, 27). Polysulfides were measured by differential pulse polarography (DPP) using a technique similar to that described by Jordan et al. (29). Polarograms were obtained using a Bioanalytical Systems (BAS) 100B electrochemical analyzer equipped with a controlled growth mercury electrode (CGME 530). They were recorded at a scan rate of 10 mV s-1 with the CGME in the static mercury drop electrode (SMDE) mode with a 1-s drop time. High-purity argon gas (Oxarc, 99.999%) was used to purge oxygen from the cell. Polysulfide formation was monitered by a potential dependent adsorption-desorption phenomena giving rise to minima near -1000 mV.
Results and Discussion CoIIIEDTA- Reduction by D. vulgaris. To ascertain whether D. vulgaris could directly reduce CoIIIEDTA-, washed cell suspensions were inoculated into media containing 500 µM CoIIIEDTA- with ethanol as the electron donor. Increasing concentrations of sulfate were also added to assess its roles as a competing electron acceptor and to examine the possibility that sulfide production may stimulate the reduction of CoIIIEDTA-. When ethanol is present without sulfate, there is a loss of CoIIIEDTA- with time and concomitant production of CoIIEDTA2- (Figure 1). In contrast, addition of heat-killed cells results in minimal to no loss of CoIIIEDTA-. In separate experiments, we noted that D. vulgaris reduced CoIIIEDTA- when lactate was provided as an electron donor but not in its absence (data not shown). Thus, both viable cells and the availability of an electron donor are required for the reduction of CoIIIEDTA- by D. vulgaris, suggesting an enzymatic reductive process. When sulfate is eliminated from Modified Baar’s medium and replaced with CoIIIEDTA- as the sole electron acceptor, no increase in D. vulgaris cell density is observed, despite repeated spiking with fresh CoIIIEDTA- and the consequent reduction to CoIIEDTA2-. While not conclusive, this suggests that D. vulgaris is unable to conserve energy for growth via the reduction of Co(III). The inability of SRB to grow using metal ions as the electron acceptor has also been noted for Mo (12) and U (30). An increase in the amount of sulfate added promotes an increase in CoIIIEDTA- reduction in the presence of D. vulgaris (Figure 1). One might expect decreased reduction of CoIIIEDTA- with increasing amounts of sulfate since the latter would serve as an alternate terminal electron acceptor in place of CoIIIEDTA-. Alternatively, we propose that the increase in CoIIIEDTA- reduction with increasing amounts of sulfate is caused by the production of sulfide, which then acts as a reductant of CoIIIEDTA-. CoIIIEDTA- is rapidly reduced in all samples by D. vulgaris during the first 10 h. However, after this initial period the rate of CoIIIEDTA2- reduction is enhanced in the presence of sulfate (Figure 1). At early reaction times, the concentration of sulfide is minimal in all samples, and thus dissimilatory reductive processes should dominate. As sulfide concentrations increase, CoIIIEDTA- reduction is promoted. The increase in CoIIEDTA2- production as compared to samples
FIGURE 1. Reduction of CoIIIEDTA- by D. vulgaris (a) and the resulting production of CoIIEDTA2- (b) with varying concentrations of dissolved sulfate. The nonviable control was achieved with heat-killed cells; error bars represent the standard deviation among triplicate samples.
FIGURE 2. Enhanced production of CoIIEDTA2- by D. vulgaris resulting from the addition of dissolved sulfate (2.5 mM) and its reduction to sulfide (denoted as SO42- consumed). ∆-CoIIEDTA2- is defined as the difference between the dissolved concentration with sulfate present versus that produced in the absence of sulfate. without sulfate (denoted as ∆-CoIIEDTA2-) correlates well with SO42- consumed during the reaction up to times of 24 h, after which both SO42- and CoIIEDTA2- production decrease asymptotically (Figure 2). CoIIIEDTA- reduction is not inhibited by SO42- but is instead enhanced during sulfide formation by D. vulgaris. There is, however, a point at which higher SO42- concentrations have no effect on the reduction rate. For example, there is minimal change in the rate of CoIIIEDTA- reduction between sulfate additions of 2.5 and 25 mM, most likely resulting from enzyme saturation in sulfate reduction (Figure 1). To determine the potential for
FIGURE 3. (a) CoIIIEDTA- reduction by dissolved sulfide with the concomitant production of CoIIEDTA2-; (b) the resulting oxidative degradation of sulfide. dissolved sulfide to reduce CoIIIEDTA-, we performed abiotic experiments with dissolved sulfide and CoIIIEDTA- described below. Reduction of CoIIIEDTA- by Sulfide. Complete reduction of CoIIIEDTA- (110 µM) to CoIIEDTA2- in the presence of sulfide (1 mM) occurred in less than 160 min (Figure 3). As the reaction proceeds, the pink color of CoIIIEDTA- disappears quickly until the solution is clear with subsequent formation of a light yellow precipitate, suggesting the formation of elemental sulfur. As the reactions aged over several weeks, a black precipitate formed and was identified as CoS (see Supporting Information). Sulfate was below the limits of our detection throughout the period investigated. Interestingly, the rate of the reaction (Figure 3) did not decrease as the reaction progressed but rather increases with time in most cases. This phenomenon suggests the formation of at least one reaction intermediate that increases the reaction rate. In contrast to the accelerated reduction of Co(III), the oxidation rate of sulfide decreased as the reaction progresses (Figure 3b); the concentration of sulfide is relatively constant when CoIIIEDTA- is not present in solution. Comparisons of CoIIIEDTA- reduction and sulfide oxidation rates through the first 90 min show that the loss of sulfide from solution cannot be explained purely from its oxidation by CoIIIEDTA(Figure 3). These results imply that there is at least one additional oxidant in the system or a secondary pathway consuming sulfide. We measured small concentrations of O2 (roughly 20 µM) in our degassed solution that could serve as the initial oxidant for sulfide, but oxidation of aqueous sulfide by oxygen is slow (24, 31) and unlikely to play an appreciable role. It is more plausible that the initial oxidation of sulfide by CoIIIEDTA- catalyzes a secondary reaction involving sulfide. Sulfide oxidized to elemental sulfur may lead to the VOL. 35, NO. 8, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 5. Rate of CoIIIEDTA- reduction at varying concentrations of initial sulfide. Enhanced rates of reduction when elemental sulfur is added are also shown. FIGURE 4. Differntial-pulse polarogram of sulfur species formed during the introduction of 110 µM CoIIIEDTA- to 1000 µM sulfide solution illustrating the minimum at -1000 mV indicative of pentasulfides. An S52- standard is also depicted for comparison. formation of polysulfides (specific formation pathways are noted in reaction 1) and thus consume additional quantities of the former reactant. Since the rate of CoIIIEDTA- reduction increases as the concentration of sulfide decreases (Figure 3), we sought to investigate whether a more reactive reductant formed during sulfide oxidation. The formation of a reactive sulfide oxidation product accounts for both the rapid initial decline in sulfide concentration and the increased rate of CoIIIEDTA- reduction. Using differential pulse polarography, we detected the formation of polysulfides by a current minimum at -1000 mV (Figure 4), which increases with reaction time. This minimum is specific to polysulfides and is proportional to polysulfide concentration (32, 33). The formation of polysulfides thus represents potential reductants for CoIIIEDTA-. To determine whether polysulfides actually increase the rate of CoIIIEDTA- reduction, we ran reactions under the same conditions as before (110 µM CoIIIEDTA-, 1000 µM sulfide) and added elemental sulfur to simulate their formation according to the following equilibrium:
HS- +
(n - 1) S8(ort) T Sn2- + H+ 8
(1)
The addition of elemental sulfur increases the rate of CoIIIEDTA- reduction, demonstrating that polysulfides are more effective reductants of CoIIIEDTA- than sulfide (Figure 5). Recent evidence documents the rapid reduction rates resulting from polysulfides as compared to sulfide (27, 34), the former of which are over 600 times more reactive toward alkyl polyhalides (34). They are better nucleophiles than sulfide (27) and as such may interact with the CoIIIEDTAcomplex more effectively. We further examined the role of polysulfides by reacting CoIIIEDTA- with S52-. When S52- is added to CoIIIEDTAsolutions maintained under an N2 atmosphere, the color immediately changes from pink to yellowish-black, implying that CoIIIEDTA- is reduced to CoIIEDTA2- and cobalt-sulfur precipitates form (CoS and elemental sulfur). The reaction is very rapidsCoIIEDTA2- was below our limits of detection by the first sampling time at all concentrations of polysulfides investigated. Additionally, 20% of the Co is removed from 1602
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solution at all three S52- concentrations examined (i.e., 80% CoIIEDTA2- remains in solution), likely through the formation of CoS. Given the probable formation and reactivity of polysulfides, we hypothesize a reaction series for the reduction of CoIIIEDTA- by sulfide. Under the conditions we investigated (pH 7.1-7.2), HS- is the dominant sulfide species in solution and therefore is the only species included in the reaction scheme. Initially, CoIIIEDTA- induces the production of elemental sulfur:
HS- + 2CoIIIEDTA- T 2CoIIEDTA2- + H+ + S0 (2) With the formation of elemental sulfur, the following equilibrium applies:
HS- + nS T Sn+12- + H+
(3)
where the pK values are 9.50 for n ) 3, 9.41 for n ) 4, and 9.62 for n ) 6. Under the experimental conditions investigated, S4, S5, and S6 species predominate and thus S2 and S3 are not included (25, 26). With the formation of polysulfides, the following reaction may occur and thus accelerate the reduction of CoIIIEDTA-:
Sn2- + 2CoIIIEDTA- T 2CoIIEDTA2- + nS0
(4)
For the hypothesized pathways of CoIIIEDTA- reduction (reactions 2 and 4), elemental sulfur is a product. If HS- is in excess, the concentration of polysulfides will increase as more elemental sulfur accumulates (reaction 3), thus accelerating CoIIIEDTA- reduction. In support of this scenario, initial reaction rates are independent of sulfide concentration but progressively diverge with increase reaction time due to the formation of S0 and subsequently polysulfides (Figure 5). Environmental Relevance. D. vulgaris uses CoIIIEDTAas its terminal electron acceptor leading to the formation of CoIIEDTA-. However, D. vulgaris does not appear to grow with CoIIIEDTA- as its sole terminal electron acceptor under the conditions we investigated. This work thus expands the list of metals and radionuclides that can be reduced directly by SRB. Additionally, sulfide is an effective reductant of CoIIIEDTA-, with polysulfide intermediates enhancing the reduction rate. The presence of sulfate with D. vulgaris therefore stimulates the reduction process through the generation of sulfide; additionally, when excess sulfide is present, CoS can form. The production of sulfide in anaerobic environments is largely due to SRB activity; thus, defining
whether a direct or indirect process is responsible for CoIIIEDTA- reduction in situ may be difficult. In our experimental system, direct reduction appears to be a more appreciable pathway than indirect reduction by sulfides although the later pathways does contribute to the reduction of CoIIIEDTA-. Regardless of which dominates, both processes provide a benefit for 60CoIIIEDTA- remediation since the less mobile and stable CoIIEDTA2- species is produced. The formation of CoS via the concomitant production of sulfide creates the additional benefit of scavenging 60Co in a solid phase, hence further inhibiting its migration.
Acknowledgments We thank Karl Umiker and Dr. Stephen Kariuki (University of Idaho) for their help in the polarographic analysis and Benjamin Bostick (Stanford University) for his assistance in the XAS experiments and analysis. Helpful suggestions provided by four anonymous reviewers are also greatly appreciated. This work was supported by the Department of Energy’s NABIR program (Grant DE-FG03-97ER62481).
Supporting Information Available Additional experimental procedures and one figure (3 pages). This material is available free of charge via the Internet at http://pubs.acs.org.
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Received for review August 10, 2000. Revised manuscript received January 3, 2001. Accepted January 16, 2001. ES001576R
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