Structure and reactivity of zinc sulfide precipitates formed in the presence of sulfate-reducing bacteria

Applied Geochemistry 26 (2011) 1673–1680

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Structure and reactivity of zinc sulfide precipitates formed in the presence of sulfate-reducing bacteria Edward Peltier a,⇑, Pavan Ilipilla a, David Fowle b a b

Department of Civil, Environmental and Architectural Engineering, University of Kansas, 1530 West 15th Street, Lawrence, KS 66045, USA Department of Geology, University of Kansas, 1475 Jayhawk Boulevard, Lawrence, KS 66045, USA

a r t i c l e

i n f o

Article history: Received 19 July 2010 Accepted 24 April 2011 Available online 29 April 2011 Editorial handling by R. Fuge

a b s t r a c t The biologically mediated formation of metal sulfide precipitates in anoxic sediments represents a potentially important mechanism for the sequestration of toxic metals. Current knowledge of the structure and reactivity of these biogenic metal sulfides is scarce, limiting the ability to effectively assess contaminant sequestration in, and remobilization from, these solids. In this study, SO4-reducing bacteria (Desulfovibrio sp.) were grown for 5 days in a high-SO4, minimal metal media amended with Zn at either 30 or 300 micromolar. Zinc speciation in the reactor solids was determined using X-ray absorption spectroscopy, and the results compared to spectra of known metal sulfide mineral phases and freshly formed metal sulfides synthesized through purely chemical processes. Biogenically mediated Zn sulfides showed significantly more short range crystallographic order than the abiotically prepared amorphous precipitates. The presence of dissolved Fe2+ at similar concentrations did not affect the nature of the Zn precipitates formed. The biogenic ZnS solids were also more resistant to re-oxidation than the chemical precipitates but more soluble than sphalerite mineral samples. These results suggest that Zn sulfides formed in anaerobic sediments are likely to be more resistant to re-oxidation than would be expected based on dissolution of Fe sulfides and/or sediment acid volatile sulfides. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction The speciation of metal elements in contaminated sediments is a crucial factor in determining their toxicity and bioavailability. Typically, the formation of inorganic metal precipitates reduces metal toxicity to plants and benthic organisms (Gambrell, 1994; Luoma, 1983). In anaerobic sediments, the formation of metal sulfide phases is a potentially significant pathway for immobilizing trace element pollutants. Recent studies have shown that sediment acid volatile sulfide (AVS) content controls both the mobility of metals within pore waters (Boothman et al., 2001; Fang et al., 2005; Lee et al., 2000b) and metal bio-accumulation (Burton et al., 2005; Lee et al., 2000a; Lee and Lee, 2005). AVS, defined operationally as the fraction of solid sediment phase sulfides that are soluble in cold acid, is generally considered to include most freshly-formed trace metal and Fe monosulfides (DiToro et al., 1992). This approach, however, does not provide direct speciation information on individual elements present in the sediments and does not always correlate well with observed metal behavior (De Jonge et al., 2009). Sulfate reduction in sediments is mediated by various groups of bacteria (e.g., Desulfovibrio sp., Geobacter sulfurreducens, etc.) that ⇑ Corresponding author. Address: 4112 Learned Hall, 1530 West 15th Street, Lawrence, KS 66045, USA. Tel.: +1 785 864 2941; fax: +1 785 864 5379. E-mail address: [email protected] (E. Peltier). 0883-2927/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.apgeochem.2011.04.024

can utilize SO2 4 as a terminal electron acceptor and are collectively referred to as SO4-reducing bacteria (SRB). The reduced S species (H2S and HS) produced by this reaction react rapidly with Fe and many trace metals (Zn, Cd, Cu, Pb, Ni, Co and Hg) to produce low-solubility metal sulfide compounds. Zinc appears to be particularly susceptible to inclusion in sulfide phases. Multiple studies using X-ray absorption spectroscopy to directly determine sediment metal speciation have identified ZnS formation in wetlands (Bostick et al., 2001; Peltier et al., 2003, 2005), estuaries (O’Day et al., 2000) and reduced soils (Hesterberg et al., 1997). Weber et al. (2009) have found that, when more reactive metals (primarily Cu) were present in excess of the available reduced S, dissolved Zn concentrations in flooded soil porewaters remained relatively stable, suggesting a lack of other competing reactions for Zn removal. By contrast, other metals, especially Pb, that should similarly precipitate with reduced S are sometimes present as carbonate or oxide phases (Carroll et al., 2002; Hesterberg et al., 1997; O’Day et al., 2000). Under oxic conditions, metal sulfides undergo dissolution reactions that can result in metal re-release into solution. These re-oxidation processes are generally abiotic (Burton et al., 2009), although they may be expedited by biological processes such as O2 leakage from plant roots (Choi et al., 2006; Jacob and Otte, 2004). The rate and extent of metal re-oxidation is strongly dependent on the nature of the metal sulfide phases present. Some anoxic sediments oxidize rapidly and have significant loss of AVS

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after only 2–3 h exposure to oxic waters (DiToro et al., 1996). Caetano et al. (2003) also saw significant release of Cd, Cu and Pb from dredged anoxic sediments after only 4 h, consistent with a rapid oxidation process. Alternatively, model phases of Cd, Pb and Zn sulfides were shown to be resistant to oxidation over a 24-h period in several other experiments (Simpson et al., 1998, 2000). In a longerterm study, de Carvalho et al. (1998) observed mobilization of Zn, Cd, Cr, Ni and Pb from sulfidic sediments only after 7–15 days. These latter results suggest that trace metal sulfides may oxidize much more slowly than the Fe monosulfides that dominate AVS. Clearly, there is highly variable reactivity of different metal contaminants in sulfide-dominated systems, a complexity not captured by the AVS model. Determining speciation and sequestration patterns for trace element pollutants will therefore require information on the actual nature and stability of individual element sulfide phases formed in anaerobic sediments. Here the results are presented of studies on Zn sulfide phases formed in the presence of SO4-reducing bacteria and (in some cases) dissolved Fe2+. Zinc sulfide precipitates formed from solutions initially containing dissolved Zn2+, SO2 and SO4-reducing bacteria were 4 examined by X-ray absorption spectroscopy and compared to both known mineral phases and amorphous, chemically precipitated Zn sulfide. A short-term re-oxidation experiment in O2-saturated water was also completed to assess the stability of these biogenic ZnS phases. The results provide new insights on the formation and reactivity of freshly-formed Zn sulfides in contaminated sediments. 2. Materials and methods 2.1. Medium preparation This experiment used a modified version of the metal toxicity medium developed by Sani et al. (2001) to minimize the potential for trace metal complexation and precipitation in solution. This medium contains 26 mM total S, which was added as a mixture of Na and Mg sulfate. The original medium was modified by the substitution of HEPES buffer (2.83 g of HEPES acid plus 6.06 g sodium HEPES salt/L solution) for PIPES and the absence of chloramphenicol. Following preparation, the solution pH was adjusted to 7.2 using small additions of NaOH. After sterilization through autoclaving, 8.1 mL of a 60% Na lactate solution was added immediately prior to inoculation. The media solution was prepared using analytical or trace metal grade chemicals and ultrapure water (resistivity >18 MX) obtained from a MilliQ water system.

maximum production of Zn sulfide precipitates for analysis.) During this growth phase, samples were periodically withdrawn and analyzed for dissolved metal and reduced S concentrations, as described below. At the end of this period, the medium was transferred to 250 mL centrifuge bottles and centrifuged at 10,000 rpm for 20 min. The supernatant was then discarded and a rinse and centrifugation step performed with deionized water. The solids remaining after separation of the rinse solution were flash-frozen in liquid N2, freeze-dried under vacuum at 40 °C, and then stored at 20 °C until XAS analysis. In order to determine the effects (if any) of the freeze-drying process on metal speciation in the reactor solids, a portion of the solid material from each reactor was separated from solution by filtration onto a 0.45 lm nylon filter and rinsed with deionized water. The filters were then mounted between two pieces of Kapton tape, flash-frozen in liquid N2, and stored at 20 °C to prevent further reaction prior to XAS analysis. These filters are referred to hereafter as ‘‘wet’’ samples. For all of the activities in this section, up until freeze-drying of the reactor solids, media solutions and centrifuge tubes were only opened in a glovebox containing a reducing atmosphere (5% H2 in N2) in order to minimize exposure of the samples to O2. 2.3. Preparation of amorphous metal sulfide precipitates An amorphous Zn sulfide precipitate phase was prepared using an adapted version of the Fe monosulfide synthesis method described by Michel et al. (2005). Fifty mL of a 0.3 M solution of ZnSO4 was mixed with an equal amount of 0.3 M Na2S in the anaerobic glovebox. After stirring for 24 h, a portion of the sample was filtered through a 0.45 lm PTFE filter and washed with 1–2 mL of deionized water to displace any remaining solution. The filter was mounted between two pieces of X-ray transparent Kapton tape, shock frozen in liquid N2 and then stored at 20 °C until XAS analysis. The remaining solution was centrifuged at 10,000 rpm for 20 min, after which the supernatant solution was removed and discarded. The solids were then rinsed, frozen, freeze-dried, and stored in the same manner as the biogenic precipitates. An amorphous Fe monosulfide precipitate was prepared in a similar manner. Equal volumes of ferrous ammonium sulfate and Na2S solutions, both at 0.3 M, were mixed together in the anaerobic glovebox. The mixture was allowed to stir for 30 min after the initial mixing, after which the precipitated FeS was centrifuged, washed, and freeze-dried in an identical manner to the ZnS precipitates. The short reaction time was designed to produce nanocrystalline mackinawite with a disordered structure (Wolthers et al., 2003).

2.2. SRB growth and metal sulfide formation 2.4. X-ray absorption spectroscopy (XAS) experiments Desulfovibrio sulfuricans (ATCCC 29577, obtained from DMSZ, Brunswick, Germany) were rehydrated from frozen samples in a standard anaerobic growth medium. They were subsequently inoculated into the low-metal medium and allowed to grow for 48–72 h. Following this growth stage, the solution was transferred to 50 mL test tubes and centrifuged at 4000 rpm for 10 min. The supernatant was then discarded and the solid pellet rinsed with fresh medium. After a second centrifugation and separation cycle, the settled solids were transferred to a new medium solution amended with Zn and/or Fe. Five distinct amendments were used in this experiment: Zn at 20 and 2 mg/L, Fe at 20 mg/L, and mixed metal amendments containing either (i) 5 mg/L Zn and 10 mg/L Fe or (ii) 2 mg/L Zn and 2 mg/L Fe. Zinc and Fe were added to the media as ZnSO4 and FeSO4⁄5H2O, respectively. SRB growth was continued in the metal-amended medium for 7 days. (Initial studies had determined that SRB growth in this medium peaked at 3–5 days. The 7-day period was used to ensure

XAS experiments were conducted in April, 2009 on both freezedried and wet biogenic metal sulfide samples on the bending magnet beamline at Sector 20 (PNC/XOR) of the Advanced Photon Source at Argonne National Laboratory. This beamline uses a Si h1 1 1i crystal monochromator, which was detuned by 10% to avoid harmonic interferences. Zinc and Fe K-edge spectra were collected from 200 eV below the absorption edge to 750 eV above the absorption edge, with multiple (P3) scans collected for each sample to improve the signal to noise ratio. Fluorescence data were collected using a 13-element Ge detector, while incident and transmitted signals were monitored using ion chambers. Spectra were also collected from multiple reference compounds, including powdered ZnS and FeS (<100 lm) purchased from Fisher Scientific, and mineral samples of sphalerite (ZnS), pyrite (FeS2) and pyrrhotite (FeS) purchased from Alfa Aesar. XAS samples of the chemically precipitated samples described in Section 2.3 were collected sepa-

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rately at Sector 12 of the Advanced Photon Source in February, 2010, following the same procedure. XAS data extraction was performed using the ATHENA software program, followed by theoretical fitting of the Fourier-transformed data using ARTEMIS (Ravel and Newville, 2005). Background subtraction and edge normalization were carried out using a first order polynomial fit to the pre-edge region from 150 to 50 eV below the absorption edge, and a second order polynomial fit from 150 to 700 eV above the edge, respectively. Extraction of the extended X-ray absorption fine structure (EXAFS) v function was performed by fitting a spline from 0.5 to 13.5 Å1, with the result weighted by k3 to compensate for dampening of the spectrum at higher k. Data were Fourier transformed from 3 to 12 Å1 using a Kaiser-Bessel window. Theoretical fittings of the Fourier-transformed data were performed with the amplitude reduction factor (S20 ) fixed at 0.85. For the Zn samples, fits were carried out on the Fourier-transformed data between 1.3 and 4.65 Å, corresponding to the first two major peaks observed in the sample spectra. Ab-initio amplitude and phase functions were generated using FEFF7 (Zabinsky et al., 1995). The input file was based on a sphalerite structure, with four symmetrical S atoms at 2.342 Å, 12 Zn atoms at 3.825 Å and 12 S atoms at 4.485 Å. For the Fe samples, fits were carried out between 1.0 and 3.0 Å. The input file was based on a mackinawite structure, with 4 S atoms at 2.256 Å and 4 Fe atoms at 2.598 Å. 2.5. Reoxidation The solubility of biogenic ZnS precipitates under re-oxidizing conditions was tested in a second set of experiments. Biogenic Zn sulfides were prepared as described above, with Zn amendments of 20 and 2 mg/L. At the end of the 7 day growth period, the wet solids from each reactor were divided in half. One half was washed and freeze-dried as described above, and then acid digested to determine total metal content. Acid digestion was carried out using inverse aqua regia (75% concentrated HNO3, 25% concentrated HCl). The other half of the wet precipitate was transferred to 100-mL glass bottles which were filled with O2-saturated water at 25 °C and then capped. The bottles initially contained minimal headspace, which increased as samples were removed. The bottles were continuously stirred for 6 days, with samples taken at periodic intervals throughout the experiment and analyzed for dissolved metal content. In separate experiments, the chemically precipitated ZnS precipitates and ground sphalerite powder were reacted under identical conditions, except a greater volume (1000 mL) of solution was used. Zinc loadings in these experiments ranged from 4 to 30 mg Zn/L. 2.6. Analytical methods For all experiments, dissolved concentrations were determined after filtration through 0.45 lm filters. Samples for metal analysis were preserved by addition of concentrated HNO3 immediately after collection. Aqueous metal concentrations were determined using atomic absorption spectroscopy in either flame or graphite furnace mode, as appropriate. Analysis for free sulfide phases during the Desulfovibrio growth experiments were conducted immediately after sample collection using the methylene blue method (American Public Health Association, 2001). 3. Results and discussion 3.1. Zinc sulfide formation Measured concentrations of reduced sulfide (Fig. 1a) show that microbial activity begins immediately after transfer of Desulfovib-

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rio to the Zn-containing solution. While preliminary growth experiments had shown no inhibitory effect of Zn on Desulfovibrio growth within the range of concentrations used in this experiment, aqueous reduced S concentrations increased much more rapidly in the 2 mg/L Zn solution (e.g. at 8 h, total reduced S concentrations were 170 lmol/L in the 2 mg/L Zn solution, compared to 56 lmol/L in the 20 mg/L solution). In conjunction with this increase in reduced S species, aqueous metal concentrations declined substantially (Fig. 1b). At 20 mg/L initial Zn addition, 50% of added Zn was removed from solution during the first 48 h, while the 2 mg/L Zn reactor saw 80% removal over the same time period. 3.2. Solid-phase zinc speciation Fig. 2 shows the v-extracted EXAFS data and Fourier-transformed spectra from each of the Zn-amended SRB reactors, along with those from chemically precipitated ZnS and relevant reference phases. Inspection of the EXAFS data of the biologically prepared samples shows strong similarities to the reference sphalerite phase, including the presence of small structural features at 5 and 6.5 Å1. For each of the biogenic samples, the first shell distance in the Fourier transform is consistent with S as the nearest neighboring atom. The second shell is consistent with Zn–Zn scattering from second-nearest neighboring atoms and is generally diagnostic for inorganic precipitate phases. By comparison, the spectrum for zinc diethyldithiocarbamate (Zn[(C2H5)2NCS2]2), an organic Zn–S complex, has no significant second shell component (Fig. 2, spectrum vii). This is because the C atoms present as second-nearest neighbors to the S complexed Zn atoms scatter X-rays poorly at the range of incident energies used in this experiment. Taken together, these spectra indicate both that the Zn in this sample is primarily associated with S as the nearest neighboring atom and that this association is likely part of an inorganic precipitate phase with at least some longerrange crystalline structure. Using a single-scattering model, the first shell fits were consistent with a sphalerite-type ZnS, but second and third shell coordination numbers were unrealistically high (20–40 atoms per shell). This discrepancy is due to the contribution of multiple-scattering paths at approximately the same effective radius as the combined second and third shell single-scattering paths. The final model, therefore, also included the two multiple-scattering paths present at 4.255 Å and the three multiple-scattering paths present at 4.685 Å. Debye–Waller factors for these paths were obtained by averaging the values for the relevant single-scattering paths, while coordination numbers and path distances were constrained such that the ratio of the value of the multiple-scattering path to the single-scattering paths was consistent with that in the sphalerite structure. This approach provided good fits to the experimental data for both the reference standard phases and the biotic and abiotic ZnS precipitates (Fig. 3). The full results of the fitting model for all experimental samples and ZnS reference phases are presented in Table 1. Based on the error estimates calculated by the fitting software, the uncertainty in the first shell coordination numbers is approximately 10%, while second (Zn) and third (S) shell uncertainties are 40% and 50%, respectively. Bond distances are accurate to within 0.005, 0.01 and 0.023 Å for the first, second and third shells, respectively. The model provided good fits to the two standard ZnS phases, with negligible changes in the Zn–S and Zn–Zn bond distance. Second shell coordination numbers for the sphalerite sample are higher than expected based on the known structure, while the third shell Zn–S bond distances for both samples are longer than the theoretical predictions (4.52 vs 4.48 Å). Both of these factors may be affected by differences in scattering intensity between the Zn and S atoms present in the overlapping second and third shells.

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Fig. 1. Aqueous concentrations of reduced S (A) and Zn (B) in the growth media.

Fig. 2. Zn EXAFS data for prepared samples and reference standards: (A) v-extracted EXAFS spectra for (i) biogenic ZnS from 20 mg/L Zn solution, (ii) biogenic ZnS from 2 mg/ L Zn solution, (iii) biogenic ZnS from 5 mg/L Zn + 10 mg/L Fe solution, (iv) biogenic ZnS from 2 mg/L Zn + 2 mg/L Fe solution, (v) chemically precipitated ZnS, (vi) sphalerite mineral sample, (vii) organic Zn–S complex (zinc diethyldithiocarbamate) and (B) Fourier-transformed spectra of the same samples.

The theoretical fitting results indicate that the local environment of Zn in the biotic samples is very similar to that of ZnS mineral phases. The distance of both the first shell S and second shell Zn atoms are essentially indistinguishable from those in the sphalerite structure, and the third shell bond distances, though less definite, show evidence of some longer-range ZnS ordering. Zinc coordination numbers, however, are somewhat lower than the 12 second-nearest neighbors expected for a fully crystalline phase, suggesting that these precipitates are still somewhat amorphous. The model also indicates that the ratio of second shell Zn to third shell S atoms in these samples is 0.8–0.9, less than the 1:1 ratio observed in ZnS minerals. This may be a compensation effect of the

model stemming from the lower scattering intensity of the low molecular weight S atoms at these incident energies. Based on these results, the Zn solids formed in the Desulfovibrio medium are likely ZnS particles with small long-range order. The abiotically prepared ZnS precipitate, by contrast, shows substantially more deviation from the sphalerite model. The wet precipitate has a less well-defined EXAFS spectrum and substantially lower first and second shell coordination numbers than the biotic samples, with approximately one-half the expected number of S and Zn atoms. For this sample, there is no significant contribution to the fit from the third shell S atoms, as the best fit produces a coordination number <0.2 and a negative Dr2 value. These results

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Fig. 3. Theoretical fits to Zn EXAFS spectra, showing both the magnitude (—) and real portion () of the Fourier transform.

Table 1 Theoretical fitting parameters for the biotic and abiotic ZnS precipitates. Sample

DE0 (eV)

Zn–S (1st shell)

Zn–Zn

Zn–S (3rd shell)

N

R (Å)

Dr2 (Å2)  103

N

R (Å)

Dr2 (Å2)  103

N

R (Å)

Dr2 (Å2)  103

Biogenic 20 mg/L Zn (wet) 20 mg/L Zn 2 mg/L Zn 5 mg/L Zn + 10 mg/L Fe 2 mg/L Zn + 2 mg/L Fe

6.5 6.5 6.5 6.3 6.7

4.6 4.2 4.7 4.5 4.5

2.334 2.336 2.337 2.336 2.338

6.15 6.25 6.03 6.70 6.29

9.4 8.9 9.8 12.4 9.5

3.825 3.826 3.826 3.821 3.821

16.5 16.0 15.8 19.5 16.5

11.0 9.5 12.4 11.1 9.7

4.510 4.514 4.509 4.502 4.517

20.4 18.7 22.0 23.9 19.9

Chemical ZnS (wet) ZnS

7.0 6.7

2.3 4.0

2.330 2.326

6.43 7.61

4.9 6.2

3.824 3.812

18.7 19.8

0.1 7.4

4.581 4.510

7.6 25.6

Standards Sphalerite Powder

7.7 7.4

3.8 4.6

2.340 2.340

5.30 6.05

16.4 13.5

3.844 3.836

17.0 15.5

14.4 12.8

4.513 4.519

18.3 16.5

indicate that the chemically precipitated ZnS is a much less ordered phase than that produced by the Desulfovibrio growth experiments. While freeze drying of the sample has the potential to induce changes in the precipitate structure, it also substantially improves the ease of data collection and sample handling. This second consideration can be particularly important when dealing with air sensitive samples, as the wet samples are likely to be more reactive during even brief exposure to oxic environments during sample preparation and collection of XAS data. Comparison of the wet and dried samples for the 20 mg/L biotic sample showed no significant effects of freeze drying on the local Zn coordination. For the abiotic sample, however, freeze drying increased the apparent number of neighboring S and Zn atoms in the precipitate structure. It was also possible to fit the third shell S atoms in the dried sample, although the coordination number in this shell is still lower

than for the biotic samples. These changes indicate that sample drying may have affected the structure of the abiotic precipitate. 3.3. Mixed Zn and Fe samples Iron addition to the Desulfovibrio media had minimal effect on solid-phase Zn speciation. Theoretical fitting results for Zn precipitates in the two mixed Zn + Fe biogenic samples indicate formation of the same sphalerite-type ZnS phases observed in the Zn-only experiments. Iron addition, either alone (at 20 mg/L) or mixed with Zn (2 mg/L of each metal), resulted in the formation of a Fe monosulfide phase (Fig. 4) similar to mackinawite, with XAS spectra almost identical to mackinawite spectra reported elsewhere (Jeong et al., 2008; Spadini et al., 2003). Theoretical fitting results for the biogenic Fe sulfide samples, as well as the chemically precipitated Fe monosulfide, are presented

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Fig. 4. Fe EXAFS data for prepared samples and reference standards: (A) v-extracted EXAFS spectra for (i) biogenic FeS from 20 mg/L Fe solution, (ii) biogenic FeS from 2 mg/L Fe + 2 mg/L Zn solution, (iii) disordered mackinawite precipitate, (iv) biogenic FeS from10 mg/L Fe + 5 mg/L Zn solution; (B) Fourier-transformed spectra of the same samples.

in Table 2. These fittings were obtained using a single-shell fitting model for the initial Fe–S and Fe–Fe shells, as shown in Fig. 5. Based on the error estimates calculated by the fitting software, the uncertainties in the first (S) and second (Fe) shell coordination numbers are approximately 10% and 30%, respectively. Bond distances are accurate to within ±0.015 Å for both shells. The coordination numbers in both shells are similar to those in the chemical precipitate, while the bond distances are slightly longer than those in both the chemical precipitate and the theoretical mackinawite structure. While no fitting was attempted beyond the first two nearest neighboring atoms, inspection of the Fourier-transformed data between 4 and 6 Å suggests that the biogenic precipitates are less organized than even the nanocrystalline mackinawite precipitates at longer distances. The formation of Fe monosulfides with little long-range order follows the pattern for Fe monosulfides established in the existing literature, where FeS forms rapidly as a nanocrystalline mackinawite structure (Rickard et al., 2006; Wolthers et al., 2003) that transforms over time to a more crystalline mackinawite phase and eventually to pyrite (FeS2) (Rickard and Luther, 1997). While dissolved Zn atoms can easily be associated with mackinawite through both co-precipitation and adsorption reactions (Morse and Arakaki, 1993), the speciation data for Zn indicates that this has not happened in these reactors. This may be due to the presence of excess reduced S phases in the solution phase over the course of the biogenic synthesis reaction. The Fe EXAFS data for the other mixed Fe–Zn experiment (10 mg/ L Fe + 5 mg/L Zn) showed signs of significant oxidation either before or during analysis, particularly in the smeared first shell peak in the Fourier-transformed data. As the Zn EXAFS data from this sample

Fig. 5. Theoretical fits for biogenic and abiotically prepared Fe monosulfide precipitates.

indicated that Zn was present in sulfide phases, it is likely that this oxidation occurred primarily during data collection. This sample also showed visible signs of oxidation at the end of XAS data collec-

Table 2 Theoretical fitting parameters for iron sulfide phases. Fe–S

Biogenic 20 mg/L Fe 2 mg/L Fe + 2 mg/L Zn Chemical FeS (amorphous)

Fe–Fe

N

R (Å)

Dr2 (Å2)  103

N

R (Å)

Dr2 (Å2)  103

1.7 1.7

3.1 3.9

2.275 2.278

3.27 4.52

3.4 1.8

2.719 2.730

13.4 7.94

1.6

3.2

2.232

4.25

3.7

2.595

9.21

DE0 (eV)

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tion, as portions of the sample had turned from a dark brown/black to a light-brown color. Similar difficulties were encountered in collecting XAS spectra from the chemically precipitated FeS samples, particularly those analyzed in the wet (non-freeze-dried) state. While re-oxidation experiments were not performed on the Fe precipitates in this study, this difference in sample behavior during analysis indicates that, as expected, the biogenic FeS phases are significantly more reactive than the corresponding ZnS precipitates. 3.4. Re-oxidation experiments Differences in short-range structure between the biogenic and chemically precipitated ZnS phases were reflected in the data from the re-oxidation study. At the end of the study, aqueous Zn concentrations in bottles containing the biogenic Zn sulfides were between 20 and 100 lg/L. By contrast, the amorphous ZnS precipitate released 1600 lg/L Zn into solution. The difference in Zn solubility is also significant when the amount of Zn released was normalized for the total added Zn concentration (Fig. 6). Dissolution from the biogenic ZnS was 1–1.5% of the total Zn present, while the chemical precipitate released 5% of the total added Zn (the uncertainty for these values was ±0.5%). Over the entire experiment, the abiotic precipitates were more reactive than the biogenic materials, which were in turn more reactive than the sphalerite. These differences correspond to the observed differences in crystallinity of the sulfide precipitates. 3.5. Biotic vs. abiotic precipitates The Zn sulfide precipitates formed in the presence of the Desulfovibrio cells were similar to sphalerite mineral phases, with substantial short-range order. These results are consistent with a previous study by Gramp et al. (2007) examining biogenic ZnS precipitate formation on a macro-scale through X-ray diffraction and scanning electron microscopy. There are several possible reasons why the biogenic process would result in more ordered ZnS phases than rapid chemical precipitation. Under some conditions, biosorption of dissolved metal to the cellular biomass could result in a direct bacterial role in ZnS precipitation (Chen et al., 2000). In this case, however, the rapid growth in dissolved reduced S compared to the

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slow loss of dissolved Zn from solution (Fig. 1) argues against such a role for the Desulfovibrio cells. A more likely possibility is that the slow production of reduced S by bacterial SO4 reduction results in a solution that is much less oversaturated with respect to ZnS than the solution used for the chemical precipitation process. Multiple studies of ZnS formation have indicated that less supersaturated solutions result in the formation of larger, more crystalline ZnS precipitates than those where the reagents (particularly reduced S) are present in greater excess (Bijmans et al., 2009; Mokone et al., 2010; Sampaio et al., 2010). The chemical precipitation process, by contrast, results in a solution with a very high amount of supersaturation, which should result in small, amorphous precipitates. This is consistent with the findings in this paper, although particle size was not explicitly measured. The difference in short-range order between the chemical and biogenic precipitates may also be partially due to the experimental conditions used in this study. The biogenic precipitates were collected and analyzed after 7 days reaction time, while the chemical precipitates were collected after only 24 h in solution. Initially amorphous precipitates can develop increased structure and crystallinity over time through processes such as Ostwald ripening. The chemically precipitated ZnS may, therefore, have gained increased short-range order if allowed to remain in solution for the same length of time as the biogenic solids. However, the existing evidence suggests that amorphous ZnS precipitates formed under similar conditions remain amorphous at room temperatures over this time scale. Gramp et al. (2007) prepared abiotic ZnS precipitates by a similar method at 22, 45 and 60 °C and retained those solids for 2 weeks before analysis. While the two heated samples were more crystalline than their biogenic counterparts, the abiotic precipitate at 22 °C had no defined peaks in the XRD spectrum. This suggests that, at room temperature, the crystallinity of the chemical precipitates would not increase substantially even if aged for 7 days, and that the biogenic formation process produces more stable precipitates with respect to short-term re-oxidation. 4. Conclusions The results presented here show that ZnS phases formed in the presence of SO4-reducing bacteria can have significant short-range order. Zinc sulfide formation in these systems occurred over a period of several days and appeared to proceed directly through aqueous phase reactions between dissolved Zn and reduced S species. Under the conditions of these experiments, which included an excess of available reduced sulfides, the presence of dissolved Fe at similar concentrations had no effect on the nature of the ZnS precipitates formed. The short-range order present in the biogenic Zn sulfides increases their stability under re-oxidizing conditions when compared to amorphous chemically prepared precipitates. This increased order may be related to differences in the rates of precipitate formation, as the chemically prepared ZnS phases were precipitated from solution in a matter of minutes. Overall, these results suggest that ZnS formed in anaerobic sediments are likely to be more resistant to re-oxidation than would be expected based on dissolution of Fe sulfides and/or sediment AVS. Predictions for individual elemental behavior should, therefore, be based on a mixture of estimates from AVS and from thermodynamic models for the specific metal sulfide phases. Acknowledgements

Fig. 6. Zn dissolution in oxygenated aqueous solution from sphalerite (j), biotic ZnS prepared with 2 mg/L Zn (d), and 20 mg/L Zn (s), and chemically precipitated ZnS (), normalized to the amount of Zn added with each sample.

We thank Arne Sturm, of the University of Kansas Department of Geology, for his assistance in growing Desulfovibrio cultures. We also thank Dale Brewe and Michael Pape at Sector 20 and Nadia

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