Simple Assay for Accurate Determination of [35S]sulfate Reduction Activity

A method is described for the assay of [³⁵S]sulfate reduction in which filter paper wicks are used to trap [³⁵S]sulfide. The simplicity of the technique enables large numbers of samples to be conveniently processed. Enhanced sensitivity is achieved since all acid-volatile [³⁵S]sulfides produced during the incubation period are counted. Recovery of radioactivity from added Na₂³⁵S is excellent (mean, 100.1%; standard deviation, 1.81; n = 9) and is unaffected by sulfide concentrations of up to 400 μg per sample. Field trial results with anoxic sediment samples are presented.     

Barite in hydrothermal environments as a recorder of subseafloor processes: a multiple‐isotope study from the Loki's Castle vent field

Barite chimneys are known to form in hydrothermal systems where barium‐enriched fluids generated by leaching of the oceanic basement are discharged and react with seawater sulfate. They also form at cold seeps along continental margins, where marine (or pelagic) barite in the sediments is remobilized because of subseafloor microbial sulfate reduction. We test the possibility of using multiple sulfur isotopes (δ³⁴S, Δ³³S, ?³⁶S) of barite to identify microbial sulfate reduction in a hydrothermal system. In addition to multiple sulfur isotopes, we present oxygen (δ¹⁸O) and strontium (⁸⁷Sr/⁸⁶Sr) isotopes for one of numerous barite chimneys in a low‐temperature (~20 °C) venting area of the Loki's Castle black smoker field at the ultraslow‐spreading Arctic Mid‐Ocean Ridge (AMOR). The chemistry of the venting fluids in the barite field identifies a contribution of at least 10% of high‐temperature black smoker fluid, which is corroborated by ⁸⁷Sr/⁸⁶Sr ratios in the barite chimney that are less radiogenic than in seawater. In contrast, oxygen and multiple sulfur isotopes indicate that the fluid from which the barite precipitated contained residual sulfate that was affected by microbial sulfate reduction. A sulfate reduction zone at this site is further supported by the multiple sulfur isotopic composition of framboidal pyrite in the flow channel of the barite chimney and in the hydrothermal sediments in the barite field, as well as by low SO₄ and elevated H₂S concentrations in the venting fluids compared with conservative mixing values. We suggest that the mixing of ascending H₂‐ and CH₄‐rich high‐temperature fluids with percolating seawater fuels microbial sulfate reduction, which is subsequently recorded by barite formed at the seafloor in areas where the flow rate is sufficient. Thus, low‐temperature precipitates in hydrothermal systems are promising sites to explore the interactions between the geosphere and biosphere in order to evaluate the microbial impact on these systems.     

Assay of sulfotransferases.

Two methods are described for the assay of sulfotransferases which are active with sulfate acceptors bearing the hydroxyl functional group. Assays were developed for enzymes which transfer sulfate from 3′-phosphoadenosine–5′-phosphosulfate (PAPS) to sterols, phenols, and simple alcohols thereby forming the corresponding sulfate esters. With a filter binding assay, useful with crude and purified enzyme preparations, a radioactive sterol substrate is used and subsequently separated from labeled product, allowing the determination of between 50 and 400 pmol of product. In a second method, [³⁵S]PAPS is used and the labeled product is separated from PAPS and inorganic sulfate by a thin-layer technique in which product migrates close to the solvent front; the assay is useful with a broad array of substrates and is more sensitive than the filter binding assay.     

A simplified assay for phospholipase C.

A new assay for phospholipase C activity that uses alkaline phosphatase to convert phosphorylcholine to inorganic phosphate is described. The determination of inorganic phosphate is performed in the presence of phosphatidylcholine and protein after the addition of sodium dodecyl sulfate. Phospholipase C activity determined by this coupled enzyme assay agrees well with data obtained by extracting and measuring phosphoryl[¹⁴C]choline produced from phosphatidyl[methyl-¹⁴C]choline. The assay is sensitive to 1 nmol of phosphate, requires no removal of protein or phospholipid, and will work with a variety of phospholipid substrates. The assay is faster and more sensitive than previously published procedures. Stimulation of phospholipase C from Clostridium perfringens by ammonium sulfate is also reported.     

Kinetics of Sulfate Reduction in a Coastal Aquifer Contaminated with Petroleum Hydrocarbons

An integrated field and laboratory study was conducted to quantify the effect of environmental determinants on the activity of sulfate reducers in a freshwater aquifer contaminated with petroleum hydrocarbons (PHC). Within the contaminated zone, PHC-supported in␣situ sulfate reduction rates varied from 11.58±3.12 to 636±53 nmol cm⁻³ d⁻¹ and a linear increase (R ²=0.98) in reduction rate was observed with increasing in situ sulfate concentrations suggesting sulfate limitation. Half-saturation concentration (K _s) for sulfate reduction coupled to PHC mineralization was determined for the first time. At two different sites within the␣aquifer, maximum sulfate reduction rate under␣non-limiting conditions (R _max) was 5,000 nmol cm⁻³ d⁻¹, whereas the retrieved K _s values were 3.5 and 7.5 mM, respectively. The K _s values are the highest ever reported from a natural environment. Furthermore, the K _s values were significantly higher than in situ sulfate concentrations confirming sulfate limited growth. On addition of lactate and formate, sulfate reduction rate increased indicating that reactivity and bioavailability of organic substrate may also have played a role in rate inhibition in certain parts of the aquifer. Experiments with sulfide amendments show statistically minor decrease in sulfate reduction rates on addition of sulfide and analogous increase in sulfide toxicity with increasing sulfide concentrations (0.5–10 mM) was not apparent.     

A spin-label assay for metal ion chelation and complex formation.

The electron spin resonance (ESR) lines of nitroxide spin labels are broadened by electron spin exchange reactions that take place during collisions with paramagnetic ions. The degree of line broadening is greatly reduced when the paramagnetic ion forms a coordination bond with certain functional groups on organic molecules. These observations form the basis for a spin-label assay for metal ion chelation and complex formation. This paper describes the characteristics of such an assay for divalent nickel ions and the spin label TEMPONE (2,2,6,6-tetramethylpiperidone-N-oxyl). The chelation of Ni²⁺ by cysteine and the interaction of Ni²⁺ with sodium dodecyl sulfate micelles and phospholipid vesicles are demonstrated. In addition to monitoring interactions of paramagnetic ions, the assay also allows the detection of interactions of nonparamagnetic ions that compete with the paramagnetic ions for binding sites. A kinetic analysis of competition between Ni²⁺ and Zn²⁺ ions for binding sites on phospholipid vesicles is presented. There are several advantages of the spin-label line-broadening assay compared to other conventional assays for metal chelation and complex formation. The line-broadening assay does not require that the sample be optically clear or chemically defined, it requires only very small quantities of material, it can detect as little as 0.4 to 1 μmol of complexing agent, and it may be utilized in complex biological systems including subcellular organelles and macromolecules.     

Indicators of Microbial Sulfate Reduction in Acidic Sulfide-Rich Mine Tailings

Sulfate-reducing bacteria (SRB) are thought to be actively involved in the cycling of sulfur in acidic mine tailings. However, most studies have used circumstantial evidence to assess microbial sulfate activity in such environments. In order to fully ascertain the role of sulfate-reducing bacteria (SRB) in sulfur cycling in acidic mine tailings, we measured sulfate reduction rates, sulfur isotopic composition of reduced sulfide fractions, porewaters and solid-phase geochemistry and SRB populations in four different Cu-Zn tailings located in Timmins, Ontario, Canada. The tailings were sampled in the summer and in the spring, shortly after snowmelt. The results first indicate that all four sites showed very high sulfate reduction rates in the summer (～100–1000 nmol cm^{? 3}d^?1), which corresponded to the presence of sulfide in the porewaters and to high SRB populations. In some of the sites, zones of microbial sulfate reduction also corresponded to a decline of organic carbon and to an apparent pyrite (with slightly negative δ³⁴S values) enrichment around the same depth. Microbial sulfate reduction was also important in permanently acidic (pH 2–3) mine tailings sites, suggesting that SRB can be active under very acidic conditions. Secondly, the results showed that microbial sulfate reduction was greatly reduced in the spring, suggesting that temperature might be a key factor in the activity of SRB. However, a closer look at the results indicated that temperature was not the sole factor and that acidic conditions and limited substrate availability in the spring appeared to be important as well in limiting microbial sulfate par reduction in sulfidic mine tailings. Finally, the results indicate that sulfur undergoes rapid cycling throughout the year and that microbial sulfate reduction and metal sulfide precipitation do not appear to be a permanent sink for metals.     

Potentially Active Iron,Sulfur, and Sulfate Reducing Bacteria in Skagerrak and Bothnian Bay Sediments

In many marine surface sediments, the reduction of manganese (Mn) and iron (Fe) oxides is obscured by sulfate reduction, which is regarded as the predominant anaerobic microbial respiration process. However, many dissimilatory sulfate and sulfur reducing microorganisms are known to utilize alternative electron acceptors such as metal oxides. In this study, we tested whether sulfate and sulfur reducing bacteria are linked to metal oxide reduction based on biogeochemical modeling of porewater concentration profiles of Mn²⁺ and Fe²⁺ in Bothnian Bay (BB) and Skagerrak (SK) sediments. Steady-state modeling of Fe²⁺ and Mn²⁺ porewater profiles revealed zones of net Fe (0–9 cm BB; ～10 and 20 cm SK) and Mn (0–5 cm BB; 2–8 cm SK) species transformations. 16S rRNA pyrosequencing analysis of the in-situ community showed that Desulfobacteraceae, Desulfuromonadaceae and Desulfobulbaceae were present in the zone of Fe-reduction of both sediments. Rhodobacteraceae were also detected at high relative abundance in both sediments. BB sediments appeared to harbor a greater diversity of potential Fe-reducers compared to SK. Additionally, when the upper 10 cm of sediment from the SK was incubated with lepidocrocite and acetate, Desulfuromonas was the dominant bacteria. Real-time quantitative polymerase chain reaction (qPCR) results showed decreasing dsrA gene copy numbers with depth coincided with decreased Fe-reduction activity. Our results support the idea that sulfur and sulfate reducing bacteria contribute to Fe-reduction in the upper centimeters of both sediments.     

Flowthrough Reactor Flasks for Study of Microbial Metabolism in Sediments

Flowthrough reactor flasks are described that allow continuous low-level nutrient input to mixed anoxic sediments without dilution of the sediment. The flasks were tested by simulating sulfate inputs into sediments collected from a freshwater eutrophic lake. After an initial 2-day adaptation within the reactor system, rates of methane production and sulfate consumption were constant for the duration of a 12-day incubation. A sulfate input rate of 0.15 mmol liter of sediment⁻¹ day⁻¹ resulted in an equivalent rate of sulfate removal, which was unaffected by inputs of acetate (1.0 mmol liter of sediment⁻¹ day⁻¹). The rate of methane production in control reactors, 0.18 mmol liter of sediment⁻¹ day⁻¹, was doubled by the addition of acetate, whereas sulfate consumption was only stimulated by additions of high concentrations of sulfate plus acetate (1.5 and 1.0 mmol liter of sediment⁻¹ day⁻¹, respectively). The reactor system appears to be effective in maintaining the balance between sulfate reduction and methane production in freshwater sediments and is potentially useful for study of the response of sediment populations to varying inputs of naturally occurring substrates, selected inhibitors, or xenobiotic compounds.     

A microassay for analysis of serum sulfate

A simple assay for analysis of sulfate in a 100-μl volume of serum is described. Selective precipitation of ¹³³Ba after removal of protein with trichloroacetic acid is reproducible and accurate. In particular, no major interference from calcium or phosphate within the physiological range of concentration is found.     

Changes in Organic Matter Biodegradability Influencing Sulfate Reduction in an Aquifer Contaminated by Landfill Leachate

In situ experiments were conducted to measure sulfate reduction rates and identify rate-limiting factors in a shallow, alluvial aquifer contaminated with municipal landfill leachate. Single-well, push–pull tests conducted in a well adjacent to the landfill with >8 mM dissolved organic carbon (DOC) exhibited a sulfate reduction rate of 3.2 μmol SO₄ ⁻² (L sediment)⁻¹ day⁻¹, a value in close agreement with laboratory-derived estimates. Identical tests conducted in wells located 90 m downgradient where DOC levels remained high (>3 mM) showed no detectable sulfate consumption, and laboratory assays confirmed this observation. However, the rates of sulfate reduction in sediment samples obtained from this site were three times larger when they were amended with filter-sterilized groundwater from the upgradient location. The effect of various amendments on sulfate reduction rates was further examined in laboratory incubations using sediment collected from the downgradient site amended with ³⁵S sulfate. Unamended sediments showed only weak conversion of the tracer to ³⁵S sulfide (5 to 7 cpm/cm²), whereas the addition of Desulfovibrio cells increased ³⁵S sulfide production to 44 cpm/cm². However, the application of heat-killed Desulfovibrio had a similar stimulatory effect, as did a lactate amendment. Collectively, these findings indicate that the lack of measurable sulfate reduction at the downgradient site was not due to the absence of the necessary metabolic potential, the presence of lower sulfate concentration, or the quantity of electron donor, but by its biodegradability. The findings also indicate that field bioaugmentation attempts should be interpreted with caution.     

Dynamics of Bacterial Sulfate Reduction in a Eutrophic Lake

Bacterial sulfate reduction in the surface sediment and the water column of Lake Mendota, Madison, Wis., was studied by using radioactive sulfate (³⁵SO₄²⁻). High rates of sulfate reduction were observed at the sediment surface, where the sulfate pool (0.2 mM SO₄²⁻) had a turnover time of 10 to 24 h. Daily sulfate reduction rates in Lake Mendota sediment varied from 50 to 600 nmol of SO₄²⁻ cm⁻³, depending on temperature and sampling date. Rates of sulfate reduction in the water column were 10³ times lower than that for the surface sediment and, on an areal basis, accounted for less than 18% of the total sulfate reduction in the hypolimnion during summer stratification. Rates of bacterial sulfate reduction in the sediment were not sulfate limited at sulfate concentrations greater than 0.1 mM in short-term experiments. Although sulfate reduction seemed to be sulfate limited below 0.1 mM, Michaelis-Menten kinetics were not observed. The optimum temperature (36 to 37°C) for sulfate reduction in the sediment was considerably higher than in situ temperatures (1 to 13°C). The response of sulfate reduction to the addition of various electron donors metabolized by sulfate-reducing bacteria in pure culture was investigated. The degree of stimulation was in this order: H₂ > n-butanol > n-propanol > ethanol > glucose. Acetate and lactate caused no stimulation.     

Microbial processes at the Lost City vent field,Mid-Atlantic Ridge

Microbiological and biogeochemical measurements showed that the intensities of CO₂ assimilation, methane oxidation, and sulfate reduction at the Lost City vent field (3° N) reach 3.8 µg C/(1 day), 0.06 µg C/(1 day), and 117 µg S/(1 day), respectively. On the surface of the carbonate structures occurring at this field, two varieties of bacterial mats were found. The first variety, which is specific to the Lost City alkaline vent field, represents jellylike bacterial mats dominated by slime-producing bacteria of several morphotypes. This mat variety also contains chemolithotrophic and heterotrophic microorganisms, either microaerobic or anaerobic. The intensities of CO₂ assimilation, methane oxidation, and sulfate reduction in this variety reach 747 µg C/(dm³ day), 0.02 µg C/(dm³ day), and 28000 µg S/(dm³ day), respectively. Bacterial mats of the second variety are formed by nonpigmented filamentous sulfur bacteria, which are close morphologically to Thiothrix. The intensities of CO₂ assimilation, methane oxidation, and sulfate reduction in the second mat variety reach 8.2 µg C/(dm³ day), 5.8 µg C/(dm³ day), and 17000 µg S/(dm³ day), respectively. These data suggest the existence of subsurface microflora at the Lost City vent field.Translated from Mikrobiologiya, Vol. 74, No. 1, 2005, pp. 111–118.Original Russian Text Copyright © 2005 by Dulov, Lein, Dubinina, Pimenov.     

Multiple sulfur isotope constraints on microbial sulfate reduction below an Archean seafloor hydrothermal system

Microbial sulfate reduction is among the most ubiquitous metabolic processes on earth. The oldest evidence of microbial sulfate reduction appears in the ca. 3.5 Ga Dresser Formation in the North Pole area of Pilbara Craton in Western Australia. That evidence was found through analysis of quadruple sulfur isotopes of sulfate and sulfide minerals deposited on the seafloor. However, the activity of microbial sulfate reduction below the Archean seafloor remains poorly understood. Here, we report the quadruple sulfur isotopic compositions of sulfide minerals within hydrothermally altered seafloor basalt and less altered basaltic komatiite collected from the North Pole Dome area. The Δ³³S values of the sulfide minerals were nonzero negative, suggesting that sulfate reduction occurred below the Archean seafloor. To constrain the substrate sulfate sources and sulfate reduction processes, we constructed a numerical model. Comparing the modeled and observed sulfur isotopes, we show that the substrate sulfate comprises seawater sulfate with a negative Δ³³S anomaly and ³⁴S‐enriched sulfate with no anomalous Δ³³S. The latter component probably represents sulfate produced by local hydrothermal processes. The maximum sulfur isotopic fractionation between the putative substrate sulfate and the observed sulfide minerals within the altered basalt and basaltic komatiite is 35‰, which is consistent with a microbial origin. Alternatively, thermochemical sulfate reduction may also produce sulfide. However, considering the hydrothermal temperature inferred from the metamorphic grade of the altered basalt, the sulfur isotopic fractionation produced by inorganic sulfate reduction is probably below 20‰. Collectively, larger fractionations imply the involvement of biological sulfate reduction processes, both in the hydrothermal system below the seafloor and in less altered subsurface settings.     

A sensitive and precise isotopic assay of ATPase activity

A manual ATPase assay which measures the release of ³²P_i from [γ-³²P]ATP is described. Sodium dodecyl sulfate is used to terminate the enzyme reaction and extraction of the phophomolybdate complex into xylene: isobutanol is used to separate ³²P_i from [γ-³²P]ATP for quantitation by scintillation counting. The three-step assay is rapid (75–90 samples/h) and minimizes hydrolysis of ATP due to exposure to acidie conditions. The extraction procedure separates 10⁻¹⁵ to 10⁻⁷ mol of ³²P_i from aqueous solution with an efficiency of 100,7 ± 0.62%. Less than 1% of unhydrolyzed [γ-³²P]ATP is extracted. Extraction efficiency is not affected by protein or salts commonly present in enzyme incubation mixtures. Results obtained with this assay are precise, with an intraassay coefficient of variation of 0.6% and an interassay coefficient of variation of 1.8%. The results are comparable to results obtained with a spectrophotometric assay, with a correlation coefficient of 0,996, though assay performance and sensitivity are greatly improved with the isotopic assay.     

Substrates for Sulfate Reduction and Methane Production in Intertidal Sediments

The activity of and potential substrates for methane-producing bacteria and sulfate-reducing bacteria were examined in marsh, estuary, and beach intertidal sediments. Slow rates of methane production were detected in all sediments, although rates of sulfate reduction were 100- to 1,000-fold higher. After sulfate was depleted in sediments, the rates of methane production sharply increased. The addition of methylamine stimulated methanogenesis in the presence of sulfate, and [¹⁴C]methylamine was rapidly converted to ¹⁴CH₄ and ¹⁴CO₂ in freshly collected marsh sediment. Acetate, hydrogen, or methionine additions did not stimulate methanogenesis. [methyl-¹⁴C]methionine and [2-¹⁴C]acetate were converted to ¹⁴CO₂ and not to ¹⁴CH₄ in fresh sediment. No reduction of ¹⁴CO₂ to ¹⁴CH₄ occurred in fresh sediment. Molybdate, an inhibitor of sulfate reduction, inhibited [2-¹⁴C]acetate metabolism by 98.5%. Fluoracetate, an inhibitor of acetate metabolism, inhibited sulfate reduction by 61%. These results suggest that acetate is a major electron donor for sulfate reduction in marine sediments. In the presence of high concentrations of sulfate, methane may be derived from novel substrates such as methylamine.     

Kinetics of nitrate and sulfate removal using a mixed microbial culture with or without limited-oxygen fed

The biological degradation of nitrate and sulfate was investigated using a mixed microbial culture and lactate as the carbon source, with or without limited-oxygen fed. It was found that sulfate reduction was slightly inhibited by nitrate, since after nitrate depletion the sulfate reduction rate increased from 0.37 mg SO₄ ^2?/mg VSS d to 0.71 mg SO₄ ^2?/mg VSS d, and the maximum rate of sulfate reduction in the presence of nitrate corresponded to 56 % of the non-inhibited sulfate reduction rate determined after nitrate depleted. However, simultaneous but not sequential reduction of both oxy-anions was observed in this study, unlike some literature reports in which sulfate reduction starts only after depletion of nitrate, and this case might be due to the fact that lactate was always kept above the limiting conditions. At limited oxygen, the inhibited effect on sulfate reduction by nitrate was relieved, and the sulfate reduction rate seemed relatively higher than that obtained without limited-oxygen fed, whereas kept almost constant (0.86–0.89 mg SO₄ ^2?/mg VSS d) cross the six R_OS states. In contrast, nitrate reduction rates decreased substantially with the increase in the initial limited-oxygen fed, showing an inhibited effect on nitrate reduction by oxygen. Kinetic parameters determined for the mixed microbial culture showed that the maximum specific sulfate utilization rate obtained (0.098?±?0.022 mg SO₄ ^2?/(mg VSS h)) was similar to the reported typical value (0.1 mg SO₄ ^2?/(mg VSS h)), also indicating a moderate inhibited effect by nitrate.     

Zinc transport by respiratory epithelial cells and interaction with iron homeostasis

Despite recurrent exposure to zinc through inhalation of ambient air pollution particles, relatively little information is known about the homeostasis of this metal in respiratory epithelial cells. We describe zinc uptake and release by respiratory epithelial cells and test the postulate that Zn²⁺ transport interacts with iron homeostasis in these same cells. Zn²⁺ uptake after 4 and 8 h of exposure to zinc sulfate was concentration- and time-dependent. A majority of Zn²⁺ release occurred in the 4 h immediately following cell exposure to ZnSO₄. Regarding metal importers, mRNA for Zip1 and Zip2 showed no change after respiratory epithelial cell exposure to zinc while mRNA for divalent metal transporter (DMT)1 increased. Western blot assay for DMT1 protein supported an elevated expression of this transport protein following zinc exposure. RT-PCR confirmed mRNA for the metal exporters ZnT1 and ZnT4 with the former increasing after ZnSO₄. Cell concentrations of ferritin increased with zinc exposure while oxidative stress, measured as lipid peroxides, was decreased supporting an anti-oxidant function for Zn²⁺. Increased DMT1 expression, following pre-incubations of respiratory epithelial cells with TNF-α, IFN-γ, and endotoxin, was associated with significantly decreased intracellular zinc transport. Finally, incubations of respiratory epithelial cells with both zinc sulfate and ferric ammonium citrate resulted in elevated intracellular concentrations of both metals. We conclude that exposure to zinc increases iron uptake by respiratory epithelial cells. Elevations in cell iron can possibly affect an increased expression of DMT1 and ferritin which function to diminish oxidative stress. Comparable to other metal exposures, changes in iron homeostasis may contribute to the biological effects of zinc in specific cells and tissues.     

Biofilm Dynamics and Kinetics during High-Rate Sulfate Reduction under Anaerobic Conditions

The sulfate kinetics in an anaerobic, sulfate-reducing biofilm were investigated with an annular biofilm reactor. Biofilm growth, sulfide production, and kinetic constants (K_m and V_max) for the bacterial sulfate uptake within the biofilm were determined. These parameters were used to model the biofilm kinetics, and the experimental results were in good agreement with the model predictions. Typical zero-order volume rate constants for sulfate reduction in a biofilm without substrate limitation ranged from 56 to 93 μmol of SO²₄-cm⁻³ h⁻¹ at 20°C. The temperature dependence (Q₁₀) of sulfate reduction was equivalent to 3.4 at between 9 and 20°C. The measured rates of sulfate reduction could explain the relatively high sulfide levels found in sewers and wastewater treatment systems. Furthermore, it has been shown that sulfate reduction in biofilms just a few hundred micrometers thick is limited by sulfate diffusion into biofilm at concentrations below 0.5 mM. This observation might, in some cases, be an explanation for the relatively poor capacity of the sulfate-reducing bacteria to compete with the methanogenic bacteria in anaerobic wastewater treatment in submerged filters.     

Effects of sulfate concentration in the overlying water on sulfate reduction and sulfur storage in lake sediments

We investigated the effects of sulfate concentration on sulfate reduction and net S storage in lake sediments using³⁴S as a tracer. The water overlying intact sediment cores from the hypolimnion of Mares Pond, MA, was replaced with two Na₂ ³⁴SO₄ solutions at either ambient (70 M) or elevated (260 M) sulfate concentrations. The ³⁴S of the added sulfate was 4974 . Over two months, the net sulfate reduction rate in the ambient sulfate treatment was zero, while the net rate for the high sulfate treatment was 140 moles/m²/d. The water overlying the cores was kept under oxic conditions and the sediment received no fresh carbon inputs, thus the net rate reported may underestimate the in situ rate. Gross sulfate reduction rates calculated by isotope dilution were approximately 350 moles/m²/d for both treatments. While the calculation of gross sulfate reduction rates in intact sediment cores can be complicated by differential diffusion of³⁴S and³²S, isotopic fractionation, and the possible formation of ester sulfates, we believe these effects to be small. The results suggest that sulfate reduction is not strongly sulfate-limited in Mares Pond. The difference in net sulfate reduction rates between treatments resulted from a decrease in sulfide oxidation and suggests the importance of reoxidation in controlling net S storage in lake sediments. In both treatments the CRS and organic S fractions were measurably labelled in³⁴S. Below the sediment surface, the CRS fraction was the more heavily labelled storage product for reduced sulfides.