Environ. Sci. Technol. 2009, 43, 82–87
Effect of NO2- on Stable Isotope Fractionation during Bacterial Sulfate Reduction
reported NO2- concentrations of up to 20 µM in freshwater and marine habitats may not significantly impact the ability to use stable isotope analysis in assessing bacterial sulfate reduction.
FLORIAN EINSIEDL Helmholtz Center Munich-German Research Center for Environmental Health, Institute of Groundwater Ecology, Ingolsta¨dter Landstrasse 1, D-85764 Neuherberg, Germany, and National University of Ireland, Galway, Department of Earth and Ocean Sciences
In marine and freshwater habitats, nitrite is formed as an intermediate during denitrification and nitrification processes (1-4). During denitrification processes the accumulation of NO2- is possible when the reduction of NO3- to NO2and NO2- to N2 is uncoupled. Nitrite can also be enriched in marine and groundwater ecosystems where nitrification processes are oxygen limited. In this case, the nitrite intermediate may not be oxidized to nitrate and may accumulate in the system. However, it was found that at oxic-anoxic interfaces overlapping distributions of ammonia and nitrite lead to the conversion of nitrite and ammonia to N2 by anammox (anaerobic ammonia oxidation) bacteria (5, 6). In other studies Green et al. (7, 8) reported a partial and complete inhibition effect on bacterial sulfate reduction by nitrite. It was concluded that the extent to which nitrite acts as an inhibitor is inversely related to the nitrite reductase activity of sulfate reducing microorganisms. It was also demonstrated that the dissimilatory sulfite reductase (DSR), which represents a key enzyme in the sulfate reduction pathway, has a high affinity for nitrite (8-10). Therefore, a high activity for the reduction of nitrite may be widely distributed in sulfate-reducing bacteria. In field studies it was found that higher numbers of sulfate reducers are present near the oxic-anoxic boundaries of sediments and in water bodies representing hotspots for microbial activity (11). More recent studies have shown that sulfate reducers have the potential to oxidize organic contaminants at the fringe of contaminant plumes and are involved in the bioremediation of toxic metals and radionuclides (e.g., (12)). Since sulfate-reducing bacteria show a high potential in the degradation of contaminants and are often found in zones of high microbial activity it is important to understand the microbial response of sulfate reducers to adverse environmental factors such as nitrate and nitrite common in contaminated environments. The inhibition effect of low NO2- concentrations on stable isotope fractionation of sulfate during bacterial sulfate reduction is largely unknown. Mangalo et al. (13) demonstrated that nitrite concentrations between 500 and 1000 µM affect sulfur isotope fractionation during sulfate reduction and result in a shift to higher sulfur fractionation (more negative sulfur enrichment factors ε). However, in marine and freshwater environments nitrite concentrations are much lower and are up to 20 µM (14). Dalsgaard and Bak (15) and Cypionka (16) demonstrated that a strain of Desulfovibrio desulfuricans can use different electron acceptors and switch from sulfate reduction to denitrification, depending on the redox conditions. These findings agreed with earlier results by Mitchell et al. (17), which focused on nitrite reduction by Desulfovibrio species. Based on these results Dalsgaard and Bak (15) suggested that members of the genus Desulfovibrio have a high nitrite reductase activity within the group of sulfate-reducing bacteria. In addition, Pereira et al. (18) found that Desulfovibrio desulfuricans possesses a nitrite reductase that has a high specific activity in the reduction of the sulfuroxy intermediate sulfite. Therefore, Desulfovibrio desulfuricans was used to study stable isotope fractionation during (dissimilatory) sulfate reduction in the presence of low nitrite concentrations.
Received June 10, 2008. Revised manuscript received October 26, 2008. Accepted November 3, 2008.
The effects of low NO2- concentrations on stable isotope fractionation during dissimilatory sulfate reduction by strain Desulfovibrio desulfuricans were investigated. Nitrite, formed as an intermediate during nitrification and denitrification processes in marine and freshwater habitats, inhibits the reduction of the sulfuroxy intermediate SO32- to H2S even at low concentrations. To gain an understanding of the inhibition effect of the reduction of the sulfuroxy intermediate on stable isotope fractionation in sulfur and oxygen during bacterial sulfate reduction, nitrite was added in the form of short pulses. In the batch experiments that contained 0.02, 0.05, and 0.1 mM nitrite, sulfur enrichment factors ε of -12 ( 1.6, -15 ( 1.1, and -26 ( 1.3‰, respectively were observed. In the control experiment (no addition of nitrite) a sulfur enrichment factor ε of around -11‰ was calculated. In the experiments that contained no 18O enriched water (δ18O: -10‰) and nitrite concentrations of 0.02, 0.05, and 0.1 mM, δ18O values in the remaining sulfate were fairly constant during the experiments (δ18O sulfate: ≈10‰) and were similar to those obtained from the control experiment (no nitrite and no enriched water). However, in the batch experiments that contained 18O enriched water (+700‰) and nitrite concentrations of 0.05 and 0.1 mM increasing δ18O values in the remaining sulfate from around 15‰ to approximately 65 and 85‰, respectively, were found. Our experiments that contained isotopic enriched water and nitrite show clear evidence that the ratio of forward and backward fluxes regulated by adenosine-5′phosphosulfate reductase (APSR) controls the extent of sulfur isotope fractionation during bacterial sulfate reduction in strain Desulfovibrio desulfuricans. Since the metabolic sulfuroxy intermediate SO32- exchanges with water, evidence of 18O enriched water in the remaining sulfate in the experiments that contained nitrite also demonstrates that SO32- recycling to sulfate affects sulfur and oxygen isotope fractionation during bacterial sulfate reduction to some extent. Even though reduction of adenosine-5′-phosphosulfate (APS) to sulfite of -25‰ was not fully expressed, SO32- was recycled to SO42-. On the basis of the results of this study a sulfur isotope fractionation for APSR of up to approximately -30‰ can be assumed. However,
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Introduction
10.1021/es801592t CCC: $40.75
2009 American Chemical Society
Published on Web 12/05/2008
In this study we investigated the inhibition of the reduction of the sulfuroxy intermediate, which is formed during the sulfate reduction pathway, to hydrogen sulfide on stable sulfur isotope fractionation in the presence of low nitrite concentrations. To reach this goal we used strain Desulfovibrio desulfuricans characterized by a low sulfur isotope enrichment factor of around -11‰, δ18O enriched water with a final concentration of +700 ‰, and injected pulses of NO2- with different concentrations to our growth media.
Materials and Methods Enrichment Cultures. A set of batch experiments was used in this study to investigate the effect of nitrite on stable isotope fractionation during bacterial sulfate reduction. Each batch test was inoculated with strain Desulfovibrio desulfuricans. The pure culture Desulfovibrio desulfuricans (DSMZ 642) was purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ). The strain was grown in a bicarbonate-buffered (30 mM) basal freshwater mineral medium under strictly anaerobic conditions with lactate as a carbon source (19). The medium was reduced with 1 mM sulfide. Lactate and sulfate were added to each batch experiment from 1 M sterile stock solutions. The final concentration of each test was 20 mM. The batch experiments focusing on the effect of stable isotope fractionation had a final volume of 120 mL filled with 60 mL of growth media. After a short growth phase of approximately 15 h, during which time H2S increased from around 1 to 2 mM, an anoxic nitrite solution was added to 11 of the batch experiments. The initial nitrite concentration of the batch test was 0.02 (n ) 2), 0.05 (n ) 4), and 0.1 mM (n ) 5). Water enriched in 18O with a final δ18O value of approximately +700 ( 70‰ (18O content: 10%; Hyox; Rotem GmbH) was used in four of the batch experiments (each was performed in duplicate) that contained 0.05 and 0.1 mM nitrite. Approximately every 24 h (Figure 1), nitrite concentration decreased below detection in the batch tests and additional nitrite was added in the form of short pulses to achieve the initial nitrite concentrations described above. A control experiment contained the strain Desulfovibrio desulfuricans without any addition of nitrite and 18O enriched water was performed to calculate the sulfur enrichment factor ε for Desulfovibrio desulfuricans and to compare microbial growth with the amended nitrite and earlier findings (16, 20). A second experiment was conducted to follow the nitrite and sulfide concentration during nitrite and sulfate reduction. To follow this goal two 1 L serum bottles filled with 0.45 L of growth medium were inoculated with Desulfovibrio desulfuricans allowing for frequent sample withdrawal. Similar to the batch experiments described above, sulfate and lactate concentrations were added to the batch experiment from 1 M sterile stock solutions. One batch experiment was designated as a control and did not receive an injection of nitrite. The second batch test contained an initial nitrite concentration of 0.1 mM and no further additions of nitrite were made to the batch test. In all experiments the headspace was completely replaced by a N2/CO2 gas mixture (80:20) [vol/vol]. All experiments were conducted in the dark at 30 °C. Bacterial sulfate reduction was determined by analysis of accumulating H2S as described by Cline (21). Nitrite concentration was determined using a DIONEX DX-100 ion chromatography unit. The minimum detection limit for nitrite for this experiment was 0.0025 mM, whereas all samples of nitrite were measured on a dilution ratio of 1:10. Isotope Analysis. For sulfur and oxygen isotope analyses, microbially produced H2S was precipitated from aliquots of 2 and 10 mL with up to 3 mL of a 20% Zn-acetate solution in order to prevent reoxidation of sulfide to sulfate. After ZnS precipitation, the samples were filtered through 0.45 µm pore size Millipore syringe filters.
All samples were acidified to pH < 4 to remove HCO3- and sulfate was precipitated as BaSO4 with 3 mL of a 10% BaCl2 solution. The precipitate was recovered by centrifuging or filtration and was carefully washed and dried prior to isotope analysis. The isotope analyses were performed by isotope ratio mass spectrometry (IRMS Thermo Electron MAT 253) after complete conversion of BaSO4 to SO2 via high temperature reaction with WO2 and V2O5. For the measurements of δ18O in sulfate, CO was produced through thermal composition of BaSO4 with pure graphite under vacuum at 1450 °C and subsequent isotope analysis was conducted by IRMS. Sulfur and oxygen isotope ratios are reported in parts per thousands (‰) using the conventional delta-notation (δ) (eq 1). δR )
(
)
Rsample - 1 × 1000[‰] Rstandard
(1)
The values Rsample and Rstandard are the 34S/32S or 18O/16O ratios of the sample and the standard, respectively. δ34S values are reported relative to the Canon Diablo Troilite standard (VCDT). The δ18O values of sulfate refer to Vienna Standard Mean Ocean Water (V-SMOW). Laboratory internal standards and NBS 127 were used to ensure accurate measurements. Reproducibilities for δ34S measurements were ( 0.2 ‰ and ( 0.4 ‰ for δ18O of sulfate. The Rayleigh equation was used to assess stable isotope fractionation for sulfur (eq 2). Rt ⁄ R0 ) (Ct ⁄ C0)(R-1)
(2)
Rt and R0 denote the stable isotope ratios of sulfur at times t and zero. Ct and C0 represent the respective concentrations of sulfate during times t and zero. The fractionation factor (R) describes the relationship between the initial isotopic composition (R0) of a substrate relative to the isotopic composition of the substrate (Rt) at any given time point during the microbial degradation. The isotopic enrichment factor ε is commonly used to quantify the apparent isotopic shift during a reaction and was determined as the slope of a linear regression according to the Rayleigh equation: ln
Ct Rt ) ε ⁄ 1000 · ln R0 C0
(3)
where the enrichment factor ε is a measure for the isotopic enrichment as an average over all positions in a molecule, and Rt and R0 are the isotope ratios of heavy versus light isotopes at a given time and at the beginning of the reaction, respectively. Principles of Stable Isotope Fractionation during Bacterial Sulfate Reduction. The model that is commonly used to explain stable isotope fractionation during bacterial sulfate reduction was reported by Harrison and Thode (22) and Rees (23) and was further developed by, e.g., Brunner et al. (24) and Johnston et al. (25). Following earlier results (eq 4) published by Rees (23), the four important steps regulating stable isotope fractionation during sulfate reduction are as follows: the uptake of sulfur into the cell (step 1, ε: +3‰); the activation of sulfate to adenosine-5′-phosphosulfate (step 2, APS) by ATP sulfurylase; the reduction of the sulfate bound in APS to sulfite (step 3, ε: -25 ‰) by the APS reductase (APSR); and the conversion of sulfite to H2S (step 6, ε: - 25‰) by DSR.
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sumed (26, 27). Recent work suggested that the fractionation capability of microbial sulfate reduction may exceed the upper 47‰ when the trithionate pathway (28) (step 4) or an internal H2S reservoir (step 5) is assumed and sulfur enrichment factors of up to -75‰ were hypothesized (24). Wortmann et al. (29) reported, for hypersulfidic sediments, large isotopic difference of up to 72‰ between the coexisting dissolved sulfide and sulfate. Furthermore, Mangalo (20) showed some evidence that the ratio of forward and backward reactions of the intermediate control the stable isotope fractionation of sulfur and oxygen of sulfate for different sulfate reducers during dissimilatory sulfate reduction. In a second study performed by Mangalo et al. (13) it was demonstrated that the dissimilatory sulfite reductase (DSR) regulates the lifte-time of the intermediate sulfite and affects the isotope fractionation of sulfur and oxygen during bacterial sulfate reduction.
Results and Discussion Growth in Batch Experiments. To determine at what concentration nitrite affects the extent of sulfur isotope fractionation, Desulfovibrio desulfuricans was grown with sulfate in the presence of different nitrite concentrations. When Desulfovibrio desulfuricans was grown in the absence of nitrite (control experiment) approximately 42% of the sulfate was consumed and a final H2S concentration of about 8.5 mM was reached within approximately 40 h (Figure 1). Thereafter, it is assumed that H2S concentrations of around 9 mM are toxic to Desulfovibrio desulfuricans and microbial growth will cease (30). In contrast, H2S production reached a value of approximately 5.4 mM after 72 h in the batch experiments that contained a NO2- concentration of 0.1 mM. This corresponds to an average sulfate consumption of 27% of the initially supplied sulfate. The batch experiments that contained initial nitrite concentrations of 0.05 and 0.02 mM also had lower H2S production compared to the control experiment. Hydrogen sulfide concentrations increased up to 7.5 mM within 70 h and approximately 38% of the sulfate was consumed. It is suggested that in the batch experiments that contained nitrite, the reduction of the sulfuroxy intermediate to hydrogen sulfide may be partially inhibited. In addition, these results demonstrate that sulfate reduction did not go to completion within 72 h in all of the batch experiments that contained nitrite. However, after the final injection of nitrite (65 h) sulfate reduction ceases within 20 h in the batch experiments that contained 0.1 mM NO2-. At this point (≈ 90 h) final hydrogen sulfide concentration of approximately 9 mM was reached which is similar to hydrogen sulfide concentrations observed for the control experiment. Our experimental results suggest that the observed delay in H2S production in experiments containing Desulfovibrio desulfuricans and nitrite concentrations between 0.1 and 0.02 mM is caused by an inhibition effect of sulfate reduction on strain Desulfovibrio desulfuricans. This interpretation is consistent with the results obtained by Dalsgaard and Bak (15) and Green et al. (8) showing that Desulfovibrio desulfuricans can use different electron acceptors such as nitrate, nitrite, and sulfate. The results from this study show that sulfate-reducing microorganisms can be inhibited by low nitrite concentrations and sulfate reduction slowed down about up to 40% compared to the control experiments. However, this study also demonstrates that environmental nitrite concentrations of up to 20 µM in marine and freshwater habitats may not affect microbial growth of Desulfovibrio desulfuricans to a strong extent. Nitrite Reduction during Dissimilatory Sulfate Reduction. Figure 2 shows the H2S and nitrite profiles in the second set of batch experiments which contained one batch test with no added nitrite (control) and a second batch test with an initial nitrite concentration of 0.1 mM. In the control 84
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FIGURE 1. Sulfide production by Desulfovibrio desulfuricans in the batch experiments that contained no 18O enriched water and no nitrite (2n ) 1), a nitrite concentration of 0.1 mM (), 4, 0; n ) 3), nitrite concentrations of 0.05 (+, ×; n ) 2) and a nitrite concentration of 0.02 mM (°, -; n ) 2).
FIGURE 2. Effect of NO2- on H2S production by Desulfovibrio desulfuricans. The concentration of sulfide with (0) and without amended NO2- (4) is shown as a function of time. Nitrite concentrations (+) are also shown as a function of time. The open circles (O) represent measurements in the control, whereas the dashed line represents the detection limit for NO2-. experiment (i.e., no added nitrite) the microbial growth curve shows an exponential growth phase. This is in contrast with the linear growth observed in the experiment containing an initial nitrite concentration of 0.1 mM. At the end of the experiment nitrite concentrations were below detection and sulfide concentrations increased to a value similar to the control experiment. It is likely that Desulfovibrio desulfuricans switches from nitrite reduction to sulfate reduction once nitrite levels are below detection. However, H2S production does not cease during the experiments and it is assumed that Desulfovibrio desulfuricans reduces sulfate and nitrite, simultaneously with lactate as the electron donor. Since Green et al. (8) and Wolfe et al. (9) found that DSR has a strong affinity for nitrite and Pereira et al. (18) reported that Desulfovibrio desulfuricans has a nitrite reductase with a high specific activity in the reduction of sulfite, we assume that sulfur isotope fractionation is regulated on complex cell internal enzymatic regulations. At the end of the experiment after initially added nitrite was completely reduced, the reduction of sulfite to sulfide was fully expressed. To support the interpretation that nitrite and sulfate are reduced simultaneously under experimental conditions and to clarify if the reduction of low nitrite concentrations affects sulfur and oxygen isotope fractionation we used the stable isotope technique. We hypothesize that when sulfate reduction occurs in the presence of nitrite reduction and the reduction of sulfite to hydrogen sulfide is the rate limiting step a larger sulfur isotope fractionation (more negative sulfur enrichment factors) will be observed.
FIGURE 3. Variation of the sulfur isotope enrichment factors (εs) ((standard error) in batch experiments that contain no added nitrite (εs1 ) -11, n ) 1), and in batch experiments which contain nitrite concentration of 0.02 (εs2 ) -12‰; n ) 2), 0.05 (εs3 ) -15‰, n ) 2), and 0.1 mM (εs4 ) -26‰, n ) 3) addition of nitrite. For clarity purposes the data points from duplicate batch experiments are not shown.
FIGURE 4. δ18O in sulfate versus δ34S in batch experiments that contained nitrite concentrations of 0.05 (open symbols) and 0.1 mM (closed symbols) and 18O enriched water of +700 ‰. The experiments (+) that contained nitrite (0.02 mM, 0.05 mM, 0.1 mM) and no isotopic enriched water. In addition, the control experiment (•) that contained no nitrite and no 18O enriched water is shown. Influence of Low Nitrite Concentrations on Stable Isotope Fractionation of Sulfur and Oxygen during Bacterial Sulfate Reduction. Batch Experiments without 18O Enriched Water. The calculated sulfur enrichment factors and isotope data are shown in Figure 3 and Figure 4, respectively. In the control experiment (i.e., absence of nitrite), a sulfur isotope enrichment factor ε of -11‰ was calculated (Figure 3). This value is consistent with previous results obtained by authors that reported sulfur isotope enrichment factors between ε -6 and -13 ‰ for strain Desulfovibrio desulfuricans (13). However, with increasing NO2- concentrations in the batch experiments an increase in the sulfur isotope enrichment factors was observed. In the experiments that contained final nitrite concentrations of 0.02 and 0.05 mM, the sulfur isotope enrichment factors were -12 ( 1.6 and -15 ( 1.6‰, respectively (Figure 3). In the batch experiments that contained nitrite concentrations of 0.1 mM, the sulfur isotope enrichment factor increased to values up to -26 ( 1.3 ‰ (Figure 3), quite similar to those observed by Mangalo et al. (13)for high nitrite concentrations between 500 and 1000 µM. This indicates that NO2- concentrations of about 0.05 mM affect sulfur isotopic fractionation in Desulfovibrio desulfuricans during bacterial sulfate reduction. In the experiments which did not contain any addition of enriched water and had a δ18O water value of ∼ -10 ‰, the δ18O values of residual sulfate were fairly constant during the growth experiment and increased from around 9.5 up to 11‰ (Figure 4). Similar δ18O values in sulfate were also
observed for the control experiment that contained no nitrite and no 18O enriched water (Figure 4). Batch Experiments with 18O Enriched Water. The calculated sulfur isotope enrichment factors for the experiments that contained 18O enriched water (+700 ‰) and NO2- concentrations of 0.05 and 0.1 mM were within the range observed in the experiments that contained no 18O enriched water and nitrite concentrations of 0.05 and 0.1 mM. In the experiments that contained 18O enriched water of +700 ‰ and NO2- concentrations of 0.05 and 0.1 mM, an increase in δ18O values in remaining sulfate of around 15‰ up to 65 and 85‰, respectively, were observed (Figure 4). An observed isotopic shift for δ18O values up to 85‰ in the remaining sulfate with nitrite concentrations of up to 0.1 mM may be indicative that the reoxidation of sulfuroxy-intermediates also affects sulfur stable isotope fractionation during sulfate reduction. This interpretation is supported by earlier results by Cypionka (31) who suggested the formation of the sulfuroxy intermediate sulfite in Desulfovibrio desulfuricans during the reduction of thiosulfate. In addition, it has been previously shown that the metabolic sulfuroxy intermediate SO32- exchanges with water oxygen and reoxidizes to the remaining sulfate (20, 32, 33). Furthermore, it was demonstrated that the proportion of the sulfuroxy intermediate recycled, probably by way of APS to sulfate, depends on the lifetime of the sulfuroxy intermediate in the cell (20). Mangalo et al. (20) demonstrated by using four different sulfatereducing bacteria and enriched water (+700‰) that sulfate reducers characterized by high sulfur fractionation (more negative sulfur enrichment factors) reoxidize a high amount of the sulfuroxy intermediate to the remaining sulfate while only a minute fraction of the sulfur intermediate is reoxidized in sulfate reducers characterized by low sulfur fractionation (less negative sulfur enrichment factors). Therefore, it was concluded that the reoxidation process also regulates the extent of sulfur fractionation, where high amounts of sulfur cycling were associated with a low stable sulfur isotope enrichment factors of up to -39‰ (20). In agreement with the findings of Mangalo et al. (13) we also observed a low amount of backward fluxes of the intermediate SO32- to the remaining sulfate during the experiment, demonstrated by the incorporation of a low amount of 18O enriched water in the remaining sulfate (δ18O values of ca. 65 and 85 ‰). Furthermore, we could not promote sulfur isotope fractionation to the maximum value of approximately -47‰. Therefore, we suggest that the decrease of the sulfur isotope enrichment factor from -11 to -26‰ is mainly regulated by the APS reductase (APSR) in the experiments that contained nitrite. It is suggested that the reduction of sulfite to hydrogen sulfide is rate limiting by inhibition of the reduction of the sulfuroxy intermediate to hydrogen sulfide. Therefore, sulfate is always in isotope equilibrium with the sulfate in the medium and the stable isotope enrichment factor is fully measurable in the remaining sulfate. In this study we estimated a sulfur isotope fractionation for the APSR of up to approximately -30‰ experimentally assuming a value of +3‰ for the uptake of sulfate into the cell (23) and a measured sulfur enrichment factor of -26 ( 1.3‰. So far, Kaplan and Rittenberg (34) estimated a sulfur isotope fractionation of -25‰ for the APSR by subtracting an isotope fractionation of 25‰ for the DSR from a total isotope fractionation of 47‰, with the latter measured experimentally, whereas Farquhar (35) calculated a theoretical equilibrium sulfur isotope fractionation between sulfate and sulfite of approximately -24‰. Since we could not extend sulfur isotope fractionation up to -47‰ and Pereira et al. (18) found that the nitrite reductase from Desulfovibrio desulfuricans has a high activity in the reduction of sulfite we speculate that rather the nitrite reducatse, probably characterized by a low sulfur fractionation, than DSR is VOL. 43, NO. 1, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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involved in the reduction of sulfite in Desulfovibrio desulfuricans in the presence of nitrite. In that case nitrite may inhibit DSR for the reduction of the sulfuroxy intermediate to hydrogen sulfide. In this study, based on the δ18O values in sulfate and the stable isotope fractionation in sulfur we found evidence that the activity of the APSR affects stable isotope fractionation during bacterial sulfate reduction in Desulfovibrio desulfuricans and, to a lesser extent, the reoxidation of the sulfuroxy intermediate sulfite to sulfate. However, the results of this study show strong evidence that sulfite is reoxidized to the remaining sulfate when sulfur isotope fractionation factors do not exceed -25 ‰. This is contradicted to the assumption of Rees (23) that reoxidation of intermediates is zero when the sulfur enrichment factor does not reach -25 ‰. However, the new findings are in line with the modeling results published by, e.g., Brunner et al. (36) showing that reoxidation of sulfite is also possible when sulfur isotopic fractionation does not exceed -25 ‰. A linear slope of increasing δ18O values in experiments with 18O enriched water (+700 ‰) with increasing δ34S values (Figure 4) during bacterial sulfate reduction leads to the assumption that kinetic fractionation dominates over oxygen isotope exchange during bacterial sulfate reduction. However, as shown by Brunner et al. (36) it is likely that based on our data it is not possible to separate equilibrium fractionation from kinetic fractionation which is only observed at extended enrichments in sulfur. Inhibition of sulfate reduction was already reported by Lovley and Phillips (37) in the zone of ferric iron reduction affected by concentrations of hydrogen and acetate. In this study, it was demonstrated that nitrite inhibits bacterial sulfate reduction and regulates sulfur isotope fractionation during bacterial sulfate reduction. However, under environmental conditions with nitrite concentrations of up to 20 µM, stable isotope fractionation during bacterial sulfate reduction may not be significantly affected and represents a powerful tool to quantify sulfate reduction. However, the high affinity of DSR for nitrite may affect sulfate reduction at oxic-anoxic interfaces, e.g., in contaminated environments. Therefore, we hypothesize that the formation of nitrite is a limiting factor for bioremediation in nitrate-containing aquatic environments. As previously discussed, sulfate reducers such as Desulfovibrio desulfuricans reduce nitrate or nitrite to ammonia (15). Earlier results showed that anammox is an important process in marine environments where ammonia acts as electron donor to reduce nitrite to N2 (5). Based on our novel results the question arises whether sulfate reducers with a high nitrite reductase activity can limit the anaerobic oxidation of ammonia in marine environments. Our novel results also provide improved knowledge of the interplay between increasing sulfur fractionation (more negative sulfur enrichment factors) and the activity of the APSR and DSR affecting stable sulfur isotope fractionation, and will consequently lead to a better description of the isotope fractionation model for sulfate-reducing bacteria. This study also supports earlier results of recycling of sulfuroxy intermediates which are formed during the sulfate reduction pathway. This recycling process affects the δ18O value in the remaining sulfate by incorporation of water oxygen. However, mechanisms such as abiotic reactions of H2S with Fe-minerals or the interaction of sulfide with organic matter also lead to the formation of sulfur intermediates such as thiosulfate, sulfite, and elemental sulfur (e.g., (38)). These intermediates can be oxidized, reduced, or disproportionate, and oxygen molecules from water are incorporated in the newly formed sulfate (e.g., (39)) affecting the overall δ18O values of the remaining sulfate. Therefore this study provides a first step in the efforts to quantitatively 86
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separate the biotic and abiotic processes that affect δ18O in the remaining sulfate and to use this parameter as an additional tool in the interpretation of the complex sulfur cycle in aquatic and contaminated systems (e.g., (40)).
Acknowledgments I thank M. Blank for her support during the laboratory work. I am also grateful to three anonymous reviewers whose thoughtful comments contributed significantly to improve this manuscript.
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