Quantifying genome-specific carbon fixation in a 750-meter deep subsurface hydrothermal microbial community.

Dissolved inorganic carbon has been hypothesized to stimulate microbial chemoautotrophic activity as a biological sink in the carbon cycle of deep subsurface environments. Here, we tested this hypothesis using quantitative DNA stable isotope probing of metagenome-assembled genomes (MAGs) at multiple 13C-labeled bicarbonate concentrations in hydrothermal fluids from a 750-m deep subsurface aquifer in the Biga Peninsula (Turkey). The diversity of microbial populations assimilating 13C-labeled bicarbonate was significantly different at higher bicarbonate concentrations, and could be linked to four separate carbon-fixation pathways encoded within 13C-labeled MAGs. Microbial populations encoding the Calvin-Benson-Bassham cycle had the highest contribution to carbon fixation across all bicarbonate concentrations tested, spanning 1-10 mM. However, out of all the active carbon-fixation pathways detected, MAGs affiliated with the phylum Aquificae encoding the reverse tricarboxylic acid (rTCA) pathway were the only microbial populations that exhibited an increased 13C-bicarbonate assimilation under increasing bicarbonate concentrations. Our study provides the first experimental data supporting predictions that increased bicarbonate concentrations may promote chemoautotrophy via the rTCA cycle and its biological sink for deep subsurface inorganic carbon.

High methane ebullition throughout one year in a regulated central European stream.

Ebullition transports large amounts of the potent greenhouse gas methane (CH 4 ) from aquatic sediments to the atmosphere. River beds are a main source of biogenic CH 4 , but emission estimates and the relative contribution of ebullition as a transport pathway are poorly constrained. This study meets a need for more direct measurements with a whole-year data set on CH 4 ebullition from a small stream in southern Germany. Four gas traps were installed in a cross section in a river bend, representing different bed substrates between undercut and slip-off slope. For a comparison, diffusive fluxes were estimated from concentration gradients in the sediment and from measurements of dissolved CH 4 in the surface water. The data revealed highest activity with gas fluxes above 1000 ml m - 2  d - 1 in the center of the stream, sustained ebullition during winter, and a larger contribution of ebullitive compared to diffusive CH 4 fluxes. Increased gas fluxes from the center of the river may be connected to greater exchange with the surface water, thus increased carbon and nutrient supply, and a higher sediment permeability for gas bubbles. By using stable isotope fractionation, we estimated that 12-44% of the CH 4 transported diffusively was oxidized. Predictors like temperature, air pressure drop, discharge, or precipitation could not or only poorly explain temporal variations of ebullitive CH 4 fluxes.

Carbon metabolism and biogeography of candidate phylum "Candidatus Bipolaricaulota" in geothermal environments of Biga Peninsula, Turkey.

Terrestrial hydrothermal springs and aquifers are excellent sites to study microbial biogeography because of their high physicochemical heterogeneity across relatively limited geographic regions. In this study, we performed 16S rRNA gene sequencing and metagenomic analyses of the microbial diversity of 11 different geothermal aquifers and springs across the tectonically active Biga Peninsula (Turkey). Across geothermal settings ranging in temperature from 43 to 79°C, one of the most highly represented groups in both 16S rRNA gene and metagenomic datasets was affiliated with the uncultivated phylum "Candidatus Bipolaricaulota" (former "Ca. Acetothermia" and OP1 division). The highest relative abundance of "Ca. Bipolaricaulota" was observed in a 68°C geothermal brine sediment, where it dominated the microbial community, representing 91% of all detectable 16S rRNA genes. Correlation analysis of "Ca. Bipolaricaulota" operational taxonomic units (OTUs) with physicochemical parameters indicated that salinity was the strongest environmental factor measured associated with the distribution of this novel group in geothermal fluids. Correspondingly, analysis of 23 metagenome-assembled genomes (MAGs) revealed two distinct groups of "Ca. Bipolaricaulota" MAGs based on the differences in carbon metabolism: one group encoding the bacterial Wood-Ljungdahl pathway (WLP) for H2 dependent CO2 fixation is selected for at lower salinities, and a second heterotrophic clade that lacks the WLP that was selected for under hypersaline conditions in the geothermal brine sediment. In conclusion, our results highlight that the biogeography of "Ca. Bipolaricaulota" taxa is strongly correlated with salinity in hydrothermal ecosystems, which coincides with key differences in carbon acquisition strategies. The exceptionally high relative abundance of apparently heterotrophic representatives of this novel candidate Phylum in geothermal brine sediment observed here may help to guide future enrichment experiments to obtain representatives in pure culture.

Improved Lumped-Parameter and Numerical Modeling of Unsaturated Water Flow and Stable Water Isotopes.

Characterizing unsaturated water flow in the subsurface is a requirement for understanding effects of droughts on agricultural production or impacts of climate change on groundwater recharge. By employing an improved lumped-parameter model (LPM) approach that mimics variable flow we have interpreted stable water isotope data (δ18 O and δ2 H), taken over 3 years at a lysimeter site located in Germany. Lysimeter soil cores were characterized by sandy gravel (Ly1) and clayey sandy silt (Ly2), and both lysimeters were vegetated with maize. Results were compared with numerical simulation of unsaturated flow and stable water isotope transport using HYDRUS-1D. In addition, both approaches were extended by the consideration of preferential flow paths. Application of the extended LPM, and thus varying flow and transport parameters, substantially improved the description of stable water isotope observations in lysimeter seepage water. In general, findings obtained from the extended LPM were in good agreement to numerical modeling results. However, observations were more difficult to describe mathematically for Ly2, where the periodicity of seasonal stable water isotope fluctuation in seepage water was not fully met by numerical modeling. Furthermore, an extra isotopic upshift improved simulations for Ly2, probably controlled by stable water isotope exchange processes between mobile soil water and quasi-immobile water within stagnant zones. Finally, although LPM requires less input data compared with numerical models, both approaches achieve comparable decision-support integrity. The extended LPM approach can thus be a powerful tool for soil and groundwater management approaches.

Strategies to Overcome Intermediate Accumulation During in situ Nitrate Remediation in Groundwater by Hydrogenotrophic Denitrification.

Bioremediation of polluted groundwater is one of the most difficult actions in environmental science. Nonetheless, the clean-up of nitrate polluted groundwater may become increasingly important as nitrate concentrations frequently exceed the EU drinking water limit of 50 mg L-1, largely due to intensification of agriculture and food production. Denitrifiers are natural catalysts that can reduce increasing nitrogen loading of aquatic ecosystems. Porous aquifers with high nitrate loading are largely electron donor limited and additionally, high dissolved oxygen concentrations are known to reduce the efficiency of denitrification. Therefore, denitrification lag times (time prior to commencement of microbial nitrate reduction) up to decades were determined for such groundwater systems. The stimulation of autotrophic denitrifiers by the injection of hydrogen into nitrate polluted regional groundwater systems may represent a promising remediation strategy for such environments. However, besides high costs other drawbacks, such as the transient or lasting accumulation of the cytotoxic intermediate nitrite or the formation of the potent greenhouse gas nitrous oxide, have been described. In this article, we detect causes of incomplete denitrification, which include environmental factors and physiological characteristics of the underlying bacteria and provide possible mitigation approaches.

Dechloromonas and close relatives prevail during hydrogenotrophic denitrification in stimulated microcosms with oxic aquifer material.

Globally occurring nitrate pollution in groundwater is harming the environment and human health. In situ hydrogen addition to stimulate denitrification has been proposed as a remediation strategy. However, observed nitrite accumulation and incomplete denitrification are severe drawbacks that possibly stem from the specific microbial community composition. We set up a microcosm experiment comprising sediment and groundwater from a nitrate polluted oxic oligotrophic aquifer. After the microcosms were sparged with hydrogen gas, samples were taken regularly within 122 h for nitrate and nitrite measurements, community composition analysis via 16S rRNA gene amplicon sequencing and gene and transcript quantification via qPCR of reductase genes essential for complete denitrification. The highest nitrate reduction rates and greatest increase in bacterial abundance coincided with a 15.3-fold increase in relative abundance of Rhodocyclaceae, specifically six ASVs that are closely related to the genus Dechloromonas. The denitrification reductase genes napA, nirS and clade I nosZ also increased significantly over the observation period. We conclude that taxa of the genus Dechloromonas are the prevailing hydrogenotrophic denitrifiers in this nitrate polluted aquifer and the ability of hydrogenotrophic denitrification under the given conditions is species-specific.

Monte Carlo Simulations as a Decision Support to Interpret δ15 N Values of Nitrate in Groundwater.

Intense farming is often associated with the excessive use of manure or fertilizers and the subsequent deterioration of the groundwater quality in many aquifers worldwide. Stable isotopes of dissolved nitrate (δ15 N and δ18 O) are widely used to determine sources of nitrate contamination and denitrification processes in groundwater but are often difficult to interpret. Thus, Monte Carlo simulations were carried out for a site in lower Bavaria, Germany, in order to explain δ15 N observations in a porous groundwater system with two aquifers, the main aquifer (MA) and several smaller perched aquifers (PA). For evaluating potential contributions, frequency distributions of δ15 N were simulated deriving from (I) the mixing of different nitrate sources, related to land use, as input to groundwater, combined with (II) transport of nitrate in groundwater and (III) microbial denitrification. Simulation results indicate a source-driven isotopic shift to heavier δ15 N values of nitrate in groundwater, which may be explained by land use changes toward a more intensified agriculture releasing high amounts of manure. Microbial denitrification may play a role in the PA, with simulated δ15 N distributions close to the observations. Denitrification processes are however unlikely for the MA, as reasonable simulation curve fits for such a scenario were obtained predominantly for unrealistic portions of nitrate sources and related land use. The applied approach can be used to qualitatively and quantitatively evaluate the influence of different potential contributions, which might mask each other due to overlapping δ15 N ranges, and it can support the estimation of nitrate input related to land use.

Characterizing Water Flow in Vegetated Lysimeters with Stable Water Isotopes and Modeling.

We have used stable water isotopes (δ18 O, δ2 H) in combination with lumped-parameter modeling for characterizing unsaturated flow in two lysimeters vegetated with maize. The lysimeters contained undisturbed soil cores dominated by sandy gravel (Ly1) and clayey sandy silt (Ly2). Stable water isotopes were analyzed in precipitation and lysimeter outflow water over about 3 years. The mean transit time of water T and dispersion parameter PD , obtained from modeling, were higher for the silt soil in Ly2 than for the gravel soil in Ly1 (T of 362 vs. 129 d, PD of 0.7 vs. 0.12). The consideration of preferential flow (PF) paths could substantially improve the model curve fits, with 13 and 11% contribution of PF for Ly1 and Ly2 as best estimates. Different assumptions were compared to estimate the input function, that is, stable water isotope content in the recharging water. Using the isotopic composition of precipitation as input (no modification) resulted in reasonable model estimations. Best model fits for the entire observation were obtained by weighting the recharging isotopes according to average precipitation within periods of 3 and 6 months, in correspondence to changing vegetation phases and seasonal influences. Input functions that consider actual evapotranspiration could significantly improve modeling at some periods, however, this led to deviations between modeled and observed δ18 O at other periods. This may indicate the influence of variable flow, so that dividing the whole observation period into hydraulically characteristic sub-periods for lumped-parameter modeling (which implements steady-state flow) is recommended for possible further improvement.

Evaluation and source attribution of freshwater contributions to Kinvarra Bay, Ireland, using (222)Rn, EC and stable isotopes as natural indicators.

Freshwater discharge into the coastal sea is of general interest for two reasons: (i) It acts as vehicle for the transport of contaminants or nutrients into the ocean, and (ii) it indicates the loss of significant volumes of freshwater that might be needed for irrigation or drinking water supply. Due to the large-scale and long-term nature of the related hydrological processes, locating and quantitatively assessing freshwater discharge into the sea require naturally occurring tracers that allow fast, inexpensive and straightforward detection. In several studies, the standard water parameters electrical conductivity (EC) and pH have proven their suitability in this regard. However, while distribution patterns of EC and pH in the coastal sea indicate freshwater discharge in general, a separation between discharging surface water and submarine groundwater discharge (SGD) is not possible with these alone. The naturally occurring radionuclide radon-222 has been shown to be useful in the quantification of SGD and its distinction from surface runoff. This study aimed to evaluate and compare the informative value of the three parameters-EC, pH and radon concentration-in detecting and quantifying SGD by carrying out a case study in a bay located in western Ireland. The results reveal that radon activity is the most sensitive parameter for detecting SGD. However, only the combined evaluation of radon, EC and pH allows a quantitative allocation of groundwater and surface water contributions to the overall freshwater discharge into the sea. This conclusion is independently supported by stable isotope data measured on selected samples.

Effect of different carbon substrates on nitrate stable isotope fractionation during microbial denitrification.

In batch experiments, we studied the isotope fractionation in N and O of dissolved nitrate during dentrification. Denitrifying strains Thauera aromatica and "Aromatoleum aromaticum strain EbN1" were grown under strictly anaerobic conditions with acetate, benzoate, and toluene as carbon sources. (18)O-labeled water and (18)O-labeled nitrite were added to the microcosm experiments to study the effect of putative backward reactions of nitrite to nitrate on the stable isotope fractionation. We found no evidence for a reverse reaction. Significant variations of the stable isotope enrichment factor ε were observed depending on the type of carbon source used. For toluene (ε(15)N, -18.1 ± 0.6‰ to -7.3 ± 1.4‰; ε(18)O, -16.5 ± 0.6‰ to -16.1 ± 1.5‰) and benzoate (ε(15)N, -18.9 ± 1.3‰; ε(18)O, -15.9 ± 1.1‰) less negative isotope enrichment factors were calculated compared to those derived from acetate (ε(15)N, -23.5 ± 1.9‰ to -22.1 ± 0.8‰; ε(18)O, -23.7 ± 1.8‰ to -19.9 ± 0.8‰). The observed isotope effects did not depend on the growth kinetics which were similar for the three types of electron donors. We suggest that different carbon sources change the observed isotope enrichment factors by changing the relative kinetics of nitrate transport across the cell wall compared to the kinetics of the intracellular nitrate reduction step of microbial denitrification.

The reversibility of dissimilatory sulphate reduction and the cell-internal multi-step reduction of sulphite to sulphide: insights from the oxygen isotope composition of sulphate.

Dissimilatory sulphate reduction (DSR) leads to an overprint of the oxygen isotope composition of sulphate by the oxygen isotope composition of water. This overprint is assumed to occur via cell-internally formed sulphuroxy intermediates in the sulphate reduction pathway. Unlike sulphate, the sulphuroxy intermediates can readily exchange oxygen isotopes with water. Subsequent to the oxygen isotope exchange, these intermediates, e.g. sulphite, are re-oxidised by reversible enzymatic reactions to sulphate, thereby incorporating the oxygen used for the re-oxidation of the sulphur intermediates. Consequently, the rate and expression of DSR-mediated oxygen isotope exchange between sulphate and water depend not only on the oxygen isotope exchange between sulphuroxy intermediates and water, but also on cell-internal forward and backward reactions. The latter are the very same processes that control the extent of sulphur isotope fractionation expressed by DSR. Recently, the measurement of multiple sulphur isotope fractionation has successfully been applied to obtain information on the reversibility of individual enzymatically catalysed steps in DSR. Similarly, the oxygen isotope signature of sulphate has the potential to reveal complementary information on the reversibility of DSR. The aim of this work is to assess this potential. We derived a mathematical model that links sulphur and oxygen isotope effects by DSR, assuming that oxygen isotope effects observed in the oxygen isotopic composition of ambient sulphate are controlled by the oxygen isotope exchange between sulphite and water and the successive cell-internal oxidation of sulphite back to sulphate. Our model predicts rapid DSR-mediated oxygen isotope exchange for cases where the sulphur isotope fractionation is large and slow exchange for cases where the sulphur isotope fractionation is small. Our model also demonstrates that different DSR-mediated oxygen isotope equilibrium values are observed, depending on the importance of oxygen isotope exchange between sulphite and water relative to the re-oxidation of sulphite. Comparison of model results to experimental data further leads to the conclusion that sulphur isotope fractionation in the reduction of sulphite to sulphide is not a single-step process.

Occurrence and transport of pharmaceuticals in a karst groundwater system affected by domestic wastewater treatment plants.

The occurrence of two pharmaceuticals, ibuprofen and diclofenac, in a vulnerable karst groundwater system was investigated. The hydrogeology of the karst system was identified by collecting (3)H samples in groundwater over 27years and by performing tracer tests. The isotopes and tracer data were interpreted by mathematical modeling to estimate the mean transit time of water and to characterize the hydrogeological flow paths in the groundwater system. By this approach, a mean (3)H transit time of 4.6 years for the fissured-porous karst aquifer was determined, whereas the fast flowing water in the conduit system showed a mean transit time of days. Both pharmaceuticals which infiltrated along sinkholes and small streams into the karst system were detected in concentrations of up to approximately 1 microg/L in effluent water of the wastewater treatment plants. Diclofenac was present in most samples collected from four springs discharging the karst groundwater to the rivers Altmühl and Anlauter in concentrations between 3.6 and 15.4 ng/L. In contrast, ibuprofen was rarely detected in groundwater. The results of this study suggest that both pharmaceuticals move into the fractured system of the karst system and go into storage. Thus dilution processes are the dominant control on the concentrations of both pharmaceuticals in the fractured system, whereas biodegradation is likely less important.

Biogeochemical and isotopic gradients in a BTEX/PAH contaminant plume: model-based interpretation of a high-resolution field data set.

A high spatial resolution data set documenting carbon and sulfur isotope fractionation at a tar oil-contaminated, sulfate-reducing field site was analyzed with a reactive transport model. Within a comprehensive numerical model, the study links the distinctive observed isotope depth profiles with the degradation of various monoaromatic and polycyclic aromatic hydrocarbon compounds (BTEX/PAHs) under sulfate-reducing conditions. In the numerical model, microbial dynamics were simulated explicitly and isotope fractionation was directly linked to the differential microbial uptake of lighter and heavier carbon isotopes during microbial growth. Measured depth profiles from a multilevel sampling well with high spatial resolution served as key constraints for the parametrization of the model simulations. The results of the numerical simulations illustrate particularly well the evolution of the isotope signature of toluene, which is the most rapidly degrading compound and the most important reductant at the site. The resulting depth profiles at the observation well show distinct differences between the small isotopic enrichment in the contaminant plume core and the much stronger enrichment of up to 3.3 per thousand at the plume fringes.

Effect of NO2(-) on stable isotope fractionation during bacterial sulfate reduction.

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 SO3(2-) 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 epsilon of -12 +/- 1.6, -15 +/- 1.1, and -26 +/- 1.3 per thousand, respectively were observed. In the control experiment (no addition of nitrite) a sulfur enrichment factor epsilon of around -11 per thousand was calculated. In the experiments that contained no 18O enriched water (delta18O: -10 per thousand) and nitrite concentrations of 0.02, 0.05, and 0.1 mM, delta18O values in the remaining sulfate were fairly constant during the experiments (delta18O sulfate: approximately equal to 10 per thousand) 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 per thousand) and nitrite concentrations of 0.05 and 0.1 mM increasing delta18O values in the remaining sulfate from around 15 per thousand to approximately 65 and 85 per thousand, 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 SO3(2-) exchanges with water, evidence of 18O enriched water in the remaining sulfate in the experiments that contained nitrite also demonstrates that SO3(2-) 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 per thousand was not fully expressed, SO3(2-) was recycled to SO4(2-). On the basis of the results of this study a sulfur isotope fractionation for APSR of upto approximately -30 per thousand can be assumed. However, reported NO2(-) concentrations of up to 20 microM in freshwater and marine habitats may not significantly impact the ability to use stable isotope analysis in assessing bacterial sulfate reduction.

Evidence for incorporation of H2S in groundwater fulvic acids from stable isotope ratios and sulfur K-edge X-ray absorption near edge structure spectroscopy.

Groundwater samples collected in a shallow oxic and reduced deep groundwater system revealed the influence of dissolved sulfide on the chemical and isotopic composition of fulvic acid associated sulfur. Stable isotope compositions of groundwater sulfate and fulvic acid sulfur and sulfur K-edge X-ray absorption near edge structure (XANES) spectroscopy data were used to determine the sources and processes affecting fulvic acid sulfur in the aquifer. A delta34S value of 2.2 per thousand for the shallow groundwater sulfate and a delta34S value of fulvic acids of 4.9 per thousand accompanied by a contribution of up to 49% of the most oxidized sulfur species (S+6) documented that fulvic acid sulfur is mainly derived from soil S compounds such as ester sulfates, with delta34S values similar to those of atmospheric sulfate deposition. In contrast, in the deep groundwater system with elevated delta34S values in groundwater sulfate of up to 20per thousand due to bacterial sulfate reduction, delta34S values in fulvic acid sulfur were negative and were up to 22per thousand lower compared to those of groundwater sulfate. Furthermore, reduced sulfur compounds constituted a significantly higher proportion of total fulvic acid sulfur in the deep groundwater compared to fulvic acids in shallow groundwater, supporting the hypothesis that fulvic acids act as a sink for dissolved hydrogen sulfide in the deep aquifer. Our results suggest that the combination of sulfur K edge XANES spectroscopy and stable isotope analysis on fulvic acids represents a powerful tool to elucidate the role of fulvic acids in the sulfur cycle in groundwater.

Hydrodynamic and microbial processes controlling nitrate in a fissured-porous karst aquifer of the Franconian Alb, southern Germany.

Concentrations and stable isotope compositions of nitrate from 11 karst springs in the Franconian Alb (southern Germany) were determined during low flow and high flow conditions to assess sources and processes affecting groundwater nitrate. During low flow, nitrate concentrations in groundwater were around 0.10 mM in springs draining forested catchments, whereas in agricultural areas nitrate concentrations were typically higher reaching up to 0.93 mM. The isotopic composition of groundwater nitrate during low flow (delta15N values of -3.1 to 6.7% per hundred, delta180 values of +2.1 to 4.0% per hundred) in concert with concentration data suggests that nitrate is formed by nitrification in forest and agricultural soils. In addition, synthetic fertilizer N that has undergone immobilization and subsequent remineralization likely constitutes an additional nitrate source in agriculturally used catchments. During recharge conditions, concentrations and delta15N values of groundwater nitrate changed little, but delta18O values were significantly elevated (up to 24.5%o per hundred suggesting that around 25% of the nitrate was directly derived from atmospheric deposition. Groundwater dating revealed that low nitrate concentrations in groundwater (_> or =0 years) are consistent with a mixture of old low nitrate-containing and young water, the latter being affected by anthropogenic N inputs predominantly in the agriculturally used catchment areas during the last few decades. Thermodynamic and hydrogeological evidence also suggests that denitrification may have occurred in the porous rock matrix of the karst aquifer. This study demonstrates that a combination of hydrodynamic, chemical, and isotopic approaches provides unique insights into the sources and the biogeochemical history of nitrate in karst aquifers, and therefore constitutes a valuable tool for assessing the vulnerability of karst aquifers to nitrate pollution in dependence on land use and assessing their self-purification capacity.

Sources and processes affecting sulfate in a karstic groundwater system of the Franconian Alb, southern Germany.

Chemical and isotope analyses on groundwater sulfate and 3H measurements on groundwaterwere used to determine the sulfate sources and sulfur transformation processes in a heterogeneous karst aquifer of the Franconian Alb, southern Germany. Sulfate was found to be derived from atmospheric deposition. Young groundwater was characterized by high sulfate concentrations and delta34S values similar to those of recent atmospheric sulfate deposition. However, the delta18O values of groundwater SO4(2-) were depleted by several per mil with respect to those of atmospheric deposition. This isotopic shift is indicative of mineralization of carbon-bonded S in the vadose zone of the karst system. In groundwater with mean residence times of more than 60 years, a trend of increasing delta34S values and delta18O values with decreasing sulfate concentrations was observed. This trend could not be solely explained by preindustrial atmospheric sulfate deposition with higher delta34S values, and hence, we conclude that bacterial (dissimilatory) sulfate reduction in the porous matrix of the karst aquifer must have occurred. This process has the potential to contribute to long-term biodegradation of contaminants in the porous rock matrix representing the dominantwater reservoir of the fissured porous karst aquifer.