Kirsten S. Habicht

Sulfur isotope fractionation during bacterial sulfate reduction in organic-rich sediments.

Isotope fractionation during sulfate reduction by natural populations of sulfate-reducing bacteria was investigated in the cyanobacterial microbial mats of Solar Lake, Sinai and the sediments of Logten Lagoon sulfuretum, Denmark. Fractionation was measured at different sediment depths, sulfate concentrations, and incubation temperatures. Rates of sulfate reduction varied between 0.1 and 37 micromoles cm-3 d-1, with the highest rates among the highest ever reported from natural sediments. The depletion of 34S during dissimilatory sulfate reduction ranged from 16% to 42%, with the largest 34S-depletions associated with the lowest rates of sulfate reduction and the lowest 34S-depletions with the highest rates. However, at high sulfate reduction rates (>10 micromoles cm-3 d-1) the lowest fractionation was 20% independent of the rates. Overall, there was a similarity between the fractionation obtained by the natural populations of sulfate reducers and previous measurements from pure cultures. This was somewhat surprising given the extremely high rates of sulfate reduction in the experiments. Our results are explained if we conclude that the fractionation was mainly controlled by the specific rate of sulfate reduction (mass cell-1 time-1) and not by the absolute rate (mass volume-1 time-1). Sedimentary sulfides (mainly FeS2) were on average 40% depleted in 34S compared to seawater sulfate. This amount of depletion was more than could be explained by the isotopic fractionations that we measured during bacterial sulfate reduction. Therefore, additional processes contributing to the fractionation of sulfur isotopes in the sediments are indicated. From both Solar Lake and Logten Lagoon we were able to enrich cultures of elemental sulfur-disproportionating bacteria. We suggest that isotope fractionation accompanying elemental sulfur disproportionation contributes to the 34S depletion of sedimentary sulfides at our study sites.

Diversity of Sulfur Isotope Fractionations by Sulfate-Reducing Prokaryotes

ABSTRACT Batch culture experiments were performed with 32 different sulfate-reducing prokaryotes to explore the diversity in sulfur isotope fractionation during dissimilatory sulfate reduction by pure cultures. The selected strains reflect the phylogenetic and physiologic diversity of presently known sulfate reducers and cover a broad range of natural marine and freshwater habitats. Experimental conditions were designed to achieve optimum growth conditions with respect to electron donors, salinity, temperature, and pH. Under these optimized conditions, experimental fractionation factors ranged from 2.0 to 42.0‰. Salinity, incubation temperature, pH, and phylogeny had no systematic effect on the sulfur isotope fractionation. There was no correlation between isotope fractionation and sulfate reduction rate. The type of dissimilatory bisulfite reductase also had no effect on fractionation. Sulfate reducers that oxidized the carbon source completely to CO2 showed greater fractionations than sulfate reducers that released acetate as the final product of carbon oxidation. Different metabolic pathways and variable regulation of sulfate transport across the cell membrane all potentially affect isotope fractionation. Previous models that explained fractionation only in terms of sulfate reduction rates appear to be oversimplified. The species-specific physiology of each sulfate reducer thus needs to be taken into account to understand the regulation of sulfur isotope fractionation during dissimilatory sulfate reduction.

Isotope fractionation by sulfate-reducing natural populations and the isotopic composition of sulfide in marine sediments

Isotope fractionations during sulfate reduction by natural bacterial populations were measured in seven different marine sediments and compared with the isotopic composition of solid-phase sulfides in the same sediments. The measured fractionations during sulfate reduction could explain only between 41% and 85% of the 34S depletion in the sedimentary sulfides. This result directly demonstrates that the depletion of 34S in solid sulfides is an expression of the combined activity of sulfate-reducing organisms with additional fractionations accumulated during the oxidative part of the sulfur cycle. The only known process to significantly augment the fractionations created during sulfate reduction is the microbial disproportionation of the intermediate sulfur compounds: elemental sulfur, thiosulfate, and sulfite. In a simple model, we show how important each of these disproportionation processes could be in generating the isotopic composition of sedimentary sulfides. The sulfate reduction rates in the sediments studied varied by a factor of 1000 and did not correlate with the fractionation during sulfate reduction. By contrast, a correlation was observed between sulfate- reduction rate and the extent of 34S depletion into sedimentary sulfides, the most 34S-depleted sulfides found in sediments supporting the lowest rates of sulfate reduction. Thus, the fractionations imposed during the disproportionating processes are better expressed in sediments with low sulfate-reduction rates. The direct phototrophic oxidation of sulfide to sulfate, with minimal fractionation, may have been important in the sediments that had a high sulfate-reduction rate.

SULFUR ISOTOPE FRACTIONATION DURING BACTERIAL REDUCTION AND DISPROPORTIONATION OF THIOSULFATE AND SULFITE

In bacterial cultures we measured sulfur isotope fractionation during transformations of thiosulfate (S2O32−) and sulfite (SO32−), pathways which may be of considerable importance in the cycling of sulfur in marine sediments and euxinic waters. We documented isotope fractionations during the reduction and disproportionation of S2O32− and SO32− by bacterial enrichments and pure bacterial cultures from marine and freshwater environments. We also measured the isotope fractionation associated with the anoxygenic phototrophic oxidation of H2S to S2O32− by cyanobacteria. Except for SO32− reduction, isotope fractionations for these processes have not been previously reported. During the dissimilatory reduction of SO32−, H2S was depleted in 34S by 6‰, and during the reduction of S2O32− to H2S, depletions were between 7‰ and 11‰. The largest observed isotope fractionation was associated with the bacterial disproportionation of SO32− which caused a 34S depletion in H2S of 20–37‰ and a 34S enrichment in sulfate of 7–12‰. During the bacterial disproportionation of S2O32−, isotope fractionations between the outer sulfane sulfur and H2S and between the inner sulfonate sulfur and SO42− were <4‰. We observed isotope exchange between the two sulfur atoms of S2O32− leading to a depletion of34S in H2S by up to 12‰ with a comparable enrichment of 34S in SO42−. No isotope fractionation was associated with the anoxygenic phototrophic oxidation of H2S to S2O32−. The depletion of 34S into H2S during the bacterial reduction and disproportionation of S2O32− and SO32− may, in addition to sulfate reduction and the bacterial disproportionation of elemental sulfur, contribute to the generation of 34S-depleted sedimentary sulfides.

Effect of Low Sulfate Concentrations on Lactate Oxidation and Isotope Fractionation during Sulfate Reduction by Archaeoglobus fulgidus Strain Z

ABSTRACT The effect of low substrate concentrations on the metabolic pathway and sulfur isotope fractionation during sulfate reduction was investigated for Archaeoglobus fulgidus strain Z. This archaeon was grown in a chemostat with sulfate concentrations between 0.3 mM and 14 mM at 80°C and with lactate as the limiting substrate. During sulfate reduction, lactate was oxidized to acetate, formate, and CO2. This is the first time that the production of formate has been reported for A. fulgidus. The stoichiometry of the catabolic reaction was strongly dependent on the sulfate concentration. At concentrations of more than 300 μM, 1 mol of sulfate was reduced during the consumption of 1 mol of lactate, whereas only 0.6 mol of sulfate was consumed per mol of lactate oxidized at a sulfate concentration of 300 μM. Furthermore, at low sulfate concentrations acetate was the main carbon product, in contrast to the CO2 produced at high concentrations. We suggest different pathways for lactate oxidation by A. fulgidus at high and low sulfate concentrations. At about 300 μM sulfate both the growth yield and the isotope fractionation were limited by sulfate, whereas the sulfate reduction rate was not limited by sulfate. We suggest that the cell channels more energy for sulfate uptake at sulfate concentrations below 300 to 400 μM than it does at higher concentrations. This could explain the shift in the metabolic pathway and the reduced growth yield and isotope fractionation at low sulfate levels.

Sulphur isotopes and the search for life: strategies for identifying sulphur metabolisms in the rock record and beyond.

The search for life can only be as successful as our understanding of the tools we use to search for it. Here we present new sulphur isotope data (32S, 33S, 34S, 36S) from a variety of modern marine environments and use these observations, along with previously published work, to contribute to this search. Specifically, we use these new data to gain a sense of lifes influences on the sulphur isotope record and to distinguish these biologically influenced signatures from their non-biological counterparts. This treatment extends sulphur isotope analyses beyond traditional (34S/32S) measures and employs trace isotope relationships (33S/32S, 36S/32S), as the inclusion of these isotopes provides unique information about biology and its role in the sulphur cycle through time. In the current study we compare and contrast isotope effects produced by sulphur-utilizing microorganisms (experimental), modern and ancient sedimentary records (observational) and non-biological reactions (theoretical). With our collective search for life now extending to neighbouring planets, we present this study as a first step towards more fully understanding the capability of the sulphur isotope system as a viable tool for life detection, both on Earth and beyond.

High Rates of Sulfate Reduction in a Low-Sulfate Hot Spring Microbial Mat Are Driven by a Low Level of Diversity of Sulfate-Respiring Microorganisms

ABSTRACT The importance of sulfate respiration in the microbial mat found in the low-sulfate thermal outflow of Mushroom Spring in Yellowstone National Park was evaluated using a combination of molecular, microelectrode, and radiotracer studies. Despite very low sulfate concentrations, this mat community was shown to sustain a highly active sulfur cycle. The highest rates of sulfate respiration were measured close to the surface of the mat late in the day when photosynthetic oxygen production ceased and were associated with a Thermodesulfovibrio-like population. Reduced activity at greater depths was correlated with novel populations of sulfate-reducing microorganisms, unrelated to characterized species, and most likely due to both sulfate and carbon limitation.

Dominance of a clonal green sulfur bacterial population in a stratified lake.

For many years, the chemocline of the meromictic Lake Cadagno, Switzerland, was dominated by purple sulfur bacteria. However, following a major community shift in recent years, green sulfur bacteria (GSB) have come to dominate. We investigated this community by performing microbial diversity surveys using FISH cell counting and population multilocus sequence typing [clone library sequence analysis of the small subunit (SSU) rRNA locus and two loci involved in photosynthesis in GSB: fmoA and csmCA]. All bacterial populations clearly stratified according to water column chemistry. The GSB population peaked in the chemocline (c. 8 x 10(6) GSB cells mL(-1)) and constituted about 50% of all cells in the anoxic zones of the water column. At least 99.5% of these GSB cells had SSU rRNA, fmoA, and csmCA sequences essentially identical to that of the previously isolated and genome-sequenced GSB Chlorobium clathratiforme strain BU-1 (DSM 5477). This ribotype was not detected in Lake Cadagno before the bloom of GSB. These observations suggest that the C. clathratiforme population that has stabilized in Lake Cadagno is clonal. We speculate that such a clonal bloom could be caused by environmental disturbance, mutational adaptation, or invasion.

Comparative proteomics and activity of a green sulfur bacterium through the water column of Lake Cadagno, Switzerland

Primary production in the meromictic Lake Cadagno, Switzerland, is dominated by anoxygenic photosynthesis. The green sulfur bacterium Chlorobium clathratiforme is the dominant phototrophic organism in the lake, comprising more than half of the bacterial population, and its biomass increases 3.8-fold over the summer. Cells from four positions in the water column were used for comparative analysis of the Chl. clathratiforme proteome in order to investigate changes in protein composition in response to the chemical and physical gradient in their environment, with special focus on how the bacteria survive in the dark. Although metagenomic data are not available for Lake Cadagno, proteome analysis was possible based on the completely sequenced genome of an isolated strain of Chl. clathratiforme. Using LC-MS/MS we identified 1321 Chl. clathratiforme proteins in Lake Cadagno and quantitatively compared 621 of these in the four samples. Our results showed that compared with cells obtained from the photic zone, cells collected from the dark part of the water column had the same expression level of key enzymes involved in carbon metabolism and photosynthetic light harvesting. However, most proteins participating in nitrogen and sulfur metabolism were twofold less abundant in the dark. From the proteome analysis we were able to show that Chl. clathratiforme in the photic zone contains enzymes for fixation of N(2) and the complete oxidation of sulfide to sulfate while these processes are probably not active in the dark. Instead we propose that Chl. clathratiforme cells in the dark part of the water column obtain energy for maintenance from the fermentation of polyglucose. Based on the observed protein compositions we have constructed possible pathways for C, N and S metabolism in Chl. clathratiforme.

Temperature effect on the sulfur isotope fractionation during sulfate reduction by two strains of the hyperthermophilic Archaeoglobus fulgidus

Sulfur isotope fractionation during dissimilatory sulfate reduction by two strains of the thermophilic archaeon Archaeoglobus fulgidus (strains VC-16 and Z) was explored over the entire temperature range of growth. The optimal cell-specific sulfate reduction rate (14 fmol cell(-1) h(-1)) was found at 82-84 degrees C but growth was measured as low as 54 degrees C. The fractionation ranged between 0.52 per thousand and 27 per thousand, with largest fractionations were found at intermediate temperatures and the smallest fractionations at the lowest and highest temperatures. There was an inverse relationship between the cell-specific sulfate reduction rate and fractionation, and the cell-specific rate was a good indicator of the expected fractionations regardless of whether temperature or substrate concentrations controlled the rate. Comparison of the fractionation trend found in this study with similar measurements for seven other sulfate-reducers showed that sulfate-reducing organisms respond to temperature in three different ways and this correlated with their maximum fractionation value, but not with the cell-specific sulfate reduction rate. A sulfur isotope model was used to reproduce the observed variation of fractionation with temperature. This approach predicted the rate of internal sulfur transformations as having the major influence on the observed fractionations in the intermediate temperature range, whereas the exchange of sulfate across the cell membrane controls fractionation at low and high temperatures.