Activity and diversity of sulfate-reducing bacteria in a petroleum hydrocarbon-contaminated aquifer

Microbial sulfate reduction is an important metabolic activity in petroleum hydrocarbon (PHC)-contaminated aquifers. We quantified carbon source-enhanced microbial SO(4)(2-) reduction in a PHC-contaminated aquifer by using single-well push-pull tests and related the consumption of sulfate and added carbon sources to the presence of certain genera of sulfate-reducing bacteria (SRB). We also used molecular methods to assess suspended SRB diversity. In four consecutive tests, we injected anoxic test solutions (1,000 liters) containing bromide as a conservative tracer, sulfate, and either propionate, butyrate, lactate, or acetate as reactants into an existing monitoring well. After an initial incubation period, 1,000 liters of test solution-groundwater mixture was extracted from the same well. Average total test duration was 71 h. We measured concentrations of bromide, sulfate, and carbon sources in native groundwater as well as in injection and extraction phase samples and characterized the SRB population by using fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE). Enhanced sulfate reduction concomitant with carbon source degradation was observed in all tests. Computed first-order rate coefficients ranged from 0.19 to 0.32 day(-1) for sulfate reduction and from 0.13 to 0.60 day(-1) for carbon source degradation. Sulfur isotope fractionation in unconsumed sulfate indicated that sulfate reduction was microbially mediated. Enhancement of sulfate reduction due to carbon source additions in all tests and variability of rate coefficients suggested the presence of specific SRB genera and a high diversity of SRB. We confirmed this by using FISH and DGGE. A large fraction of suspended bacteria hybridized with SRB-targeting probes SRB385 plus SRB385-Db (11 to 24% of total cells). FISH results showed that the activity of these bacteria was enhanced by addition of sulfate and carbon sources during push-pull tests. However, DGGE profiles indicated that the bacterial community structure of the dominant species did not change during the tests. Thus, the combination of push-pull tests with molecular methods provided valuable insights into microbial processes, activities, and diversity in the sulfate-reducing zone of a PHC-contaminated aquifer.     

Activity and Diversity of Sulfate-Reducing Bacteria in a Petroleum Hydrocarbon-Contaminated Aquifer

Microbial sulfate reduction is an important metabolic activity in petroleum hydrocarbon (PHC)-contaminated aquifers. We quantified carbon source-enhanced microbial SO₄²⁻ reduction in a PHC-contaminated aquifer by using single-well push-pull tests and related the consumption of sulfate and added carbon sources to the presence of certain genera of sulfate-reducing bacteria (SRB). We also used molecular methods to assess suspended SRB diversity. In four consecutive tests, we injected anoxic test solutions (1,000 liters) containing bromide as a conservative tracer, sulfate, and either propionate, butyrate, lactate, or acetate as reactants into an existing monitoring well. After an initial incubation period, 1,000 liters of test solution-groundwater mixture was extracted from the same well. Average total test duration was 71 h. We measured concentrations of bromide, sulfate, and carbon sources in native groundwater as well as in injection and extraction phase samples and characterized the SRB population by using fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE). Enhanced sulfate reduction concomitant with carbon source degradation was observed in all tests. Computed first-order rate coefficients ranged from 0.19 to 0.32 day⁻¹ for sulfate reduction and from 0.13 to 0.60 day⁻¹ for carbon source degradation. Sulfur isotope fractionation in unconsumed sulfate indicated that sulfate reduction was microbially mediated. Enhancement of sulfate reduction due to carbon source additions in all tests and variability of rate coefficients suggested the presence of specific SRB genera and a high diversity of SRB. We confirmed this by using FISH and DGGE. A large fraction of suspended bacteria hybridized with SRB-targeting probes SRB385 plus SRB385-Db (11 to 24% of total cells). FISH results showed that the activity of these bacteria was enhanced by addition of sulfate and carbon sources during push-pull tests. However, DGGE profiles indicated that the bacterial community structure of the dominant species did not change during the tests. Thus, the combination of push-pull tests with molecular methods provided valuable insights into microbial processes, activities, and diversity in the sulfate-reducing zone of a PHC-contaminated aquifer.     

Stable carbon isotope fractionation by sulfate-reducing bacteria

Biogeochemical transformations occurring in the anoxic zones of stratified sedimentary microbial communities can profoundly influence the isotopic and organic signatures preserved in the fossil record. Accordingly, we have determined carbon isotope discrimination that is associated with both heterotrophic and lithotrophic growth of pure cultures of sulfate-reducing bacteria (SRB). For heterotrophic-growth experiments, substrate consumption was monitored to completion. Sealed vessels containing SRB cultures were harvested at different time intervals, and delta(13)C values were determined for gaseous CO(2), organic substrates, and products such as biomass. For three of the four SRB, carbon isotope effects between the substrates, acetate or lactate and CO(2), and the cell biomass were small, ranging from 0 to 2 per thousand. However, for Desulfotomaculum acetoxidans, the carbon incorporated into biomass was isotopically heavier than the available substrates by 8 to 9 per thousand. SRB grown lithoautotrophically consumed less than 3% of the available CO(2) and exhibited substantial discrimination (calculated as isotope fractionation factors [alpha]), as follows: for Desulfobacterium autotrophicum, alpha values ranged from 1.0100 to 1.0123; for Desulfobacter hydrogenophilus, the alpha value was 0.0138, and for Desulfotomaculum acetoxidans, the alpha value was 1.0310. Mixotrophic growth of Desulfovibrio desulfuricans on acetate and CO(2) resulted in biomass with a delta(13)C composition intermediate to that of the substrates. The extent of fractionation depended on which enzymatic pathways were used, the direction in which the pathways operated, and the growth rate, but fractionation was not dependent on the growth phase. To the extent that environmental conditions affect the availability of organic substrates (e.g., acetate) and reducing power (e.g., H(2)), ecological forces can also influence carbon isotope discrimination by SRB.     

Culture-dependent comparison of microbial diversity in deep granitic groundwater from two sites considered for a Swedish final repository of spent nuclear fuel

Site selection for a spent nuclear fuel (SNF) repository required analysis of microbial abundance and diversity at two Swedish sites, Forsmark and Laxemar-Simpevarp. Information about sulphate-reducing bacteria (SRB) was required, as sulphide could corrode copper SNF canisters. Total number of cells (TNC) and ATP were analysed, and plate counts and most probable number (MPN) analyses were conducted using eight media based on different electron donors and acceptors for specific microorganism physiological groups. Groundwater chemical composition and E(h) were analysed; sampling depths were 112-978 m below sea level. TNC was 5.5 × 10(3) to 4.7 × 10(5) cells mL(-1), correlating with ATP concentrations. Culturability in TNC percentage was 0.01-35.9, averaging 5.12. Culturable numbers varied greatly between sample positions and uncorrelated with depth. SRB were found in 29 samples and were below detection in three; the MPN of SRB correlated negatively with E(h), as did the MPN of acetogens. Data indicated that microbial sulphate reduction was ongoing in many sampled aquifers; published stable isotope data and modelling results supported this observation. The sites did not differ significantly, but the large data range suggested that analysis of more samples would enable detailed evaluation of microbial processes and their relationship with geochemical information.     

Characterization of Specific Membrane Fatty Acids as Chemotaxonomic Markers for Sulfate-Reducing Bacteria Involved in Anaerobic Oxidation of Methane

Membrane fatty acids were extracted from a sediment core above marine gas hydrates at Hydrate Ridge, NE Pacific. Anaerobic sediments from this environment are characterized by high sulfate reduction rates driven by the anaerobic oxidation of methane (AOM). The assimilation of methane carbon into bacterial biomass is indicated by carbon isotope values of specific fatty acids as low as m 103. Specific fatty acids released from bacterial membranes include C 16:1 y 5c , C 17:1 y 6c , and cyC 17:0 y 5,6 , all of which have been fully characterized by mass spectrometry. These unusual fatty acids continuously display the lowest i 13 C values in all sediment horizons and two of them are detected in high abundance (i.e., C 16:1 y 5c and cyC 17:0 y 5,6 ). Combined with microscopic examination by fluorescence in situ hybridization specifically targeting sulfate-reducing bacteria (SRB) of the Desulfosarcina/Desulfococcus group, which are present in the aggregates of AOM consortia in extremely high numbers, these specific fatty acids appear to provide a phenotypic fingerprint indicative for SRB of this group. Correlating depth profiles of specific fatty acid content and aggregate number in combination with pore water sulfate data provide further evidence of this finding. Using mass balance calculations we present a cell-specific fatty acid pattern most likely displaying a very close resemblance to the still uncultured Desulfosarcina/Desulfococcus species involved in AOM.     

Seasonal oscillation of microbial iron and sulfate reduction in saltmarsh sediments (Sapelo Island, GA, USA)

Seasonal variations in anaerobic respiration pathways were investigated at three saltmarsh sites using chemical data, sulfate reduction rate measurements, enumerations of culturable populations of anaerobic iron-reducing bacteria (FeRB), and quantification of in situ 16S rRNA hybridization signals targeted for sulfate-reducing bacteria (SRB). Bacterial sulfate reduction in the sediments followed seasonal changes in temperature and primary production of the saltmarsh, with activity levels lowest in winter and highest in summer. In contrast, a dramatic decrease in the FeRB population size was observed during summer at all sites. The collapse of FeRB populations during summer was ascribed to high rates of sulfide production by SRB, resulting in abiotic reduction of bioavailable Fe(III) (hydr)oxides. To test this hypothesis, sediment slurry incubations at 10, 20 and 30 °C were carried out. Increases in temperature and labile organic carbon availability (acetate or lactate additions) increased rates of sulfate reduction while decreasing the abundance of culturable anaerobic FeRB. These trends were not reversed by the addition of amorphous Fe(III) (hydr)oxides to the slurries. However, when sulfate reduction was inhibited by molybdate, no decline in FeRB growth was observed with increasing temperature. Addition of dissolved sulfide adversely impacted propagation of FeRB whether molybdate was added or not. Both field and laboratory data therefore support a sulfide-mediated limitation of microbial iron respiration by SRB. When total sediment respiration rates reach their highest levels during summer, SRB force a decline in the FeRB populations. As sulfate reduction activity slows down after the summer, the FeRB are able to recover.     

Sulfate Reduction Dynamics and Enumeration of Sulfate-Reducing Bacteria in Hypersaline Sediments of the Great Salt Lake (Utah, USA)

Bacterial sulfate reduction activity (SRA) was measured in surface sediments and slurries from three sites in the Great Salt Lake (Utah, USA) using radiolabeled 35S-sulfate. High rates of sulfate reduction (363 ± 103 and 6,131 ± 835 nmol cm-3 d-1) were measured at two sites in the moderately hypersaline southern arm of the lake, whereas significantly lower rates (32 ± 9 nmol cm-3 d-1) were measured in the extremely hypersaline northern arm. Bacterial sulfate reduction was strongly affected by salinity and showed an optimum around 5-6% NaCl in the southern arm and an optimum of around 12% NaCl in the more hypersaline northern arm of the lake. High densities of sulfate-reducing bacteria (SRB) ranging from 2.2 × 107 to 6.7 × 108 cells cm-3 were determined by a newly developed tracer MPN-technique (T-MPN) employing sediment media and 35S-sulfate. Calculation of specific sulfate reduction rates yielded values comparable to those obtained in pure cultures of SRB. However, when using a conventional MPN technique with synthetic media containing high amounts of Fe(II), the numbers of SRB were underestimated by 1-4 orders of magnitude as compared to the T-MPN method. Our results suggest that high densities of slightly to moderately halophilic and extremely halotolerant SRB are responsible for the high rates of sulfate reduction measured in Great Salt Lake sediments.     

Mercury methylation in Sphagnum moss mats and its association with sulfate-reducing bacteria in an acidic Adirondack forest lake wetland

Processes leading to the bioaccumulation of methylmercury (MeHg) in northern wetlands are largely unknown. We have studied various ecological niches within a remote, acidic forested lake ecosystem in the southwestern Adirondacks, NY, to discover that mats comprised of Sphagnum moss were a hot spot for mercury (Hg) and MeHg accumulation (190.5 and 18.6 ng g?1 dw, respectively). Furthermore, significantly higher potential methylation rates were measured in Sphagnum mats as compared with other sites within Sunday Lake's ecosystem. Although MPN estimates showed a low biomass of sulfate-reducing bacteria (SRB), 2.8 × 10? cells mL?1 in mat samples, evidence consisting of (1) a twofold stimulation of potential methylation by the addition of sulfate, (2) a significant decrease in Hg methylation in the presence of the sulfate reduction inhibitor molybdate, and (3) presence of dsrAB-like genes in mat DNA extracts, suggested that SRB were involved in Hg methylation. Sequencing of dsrB genes indicated that novel SRB, incomplete oxidizers including Desulfobulbus spp. and Desulfovibrio spp., and syntrophs dominated the sulfate-reducing guild in the Sphagnum moss mat. Sphagnum, a bryophyte dominating boreal peatlands, and its associated microbial communities appear to play an important role in the production and accumulation of MeHg in high-latitude ecosystems.     

In vitro and in vivo sulfate reduction in the gut contents of the termite Mastotermes darwiniensis and the rose-chafer Pachnoda marginata

Sulfate-reducing bacteria (SRB) from termites have been assigned to the genus Desulfovibrio. Desulfovibrio intestinalis lives in the gut of the Australian termite Mastotermes darwiniensis. For the first time we were able to enrich and identify a sulfate-reducing bacterium from the gut of the rose-chafer Pachnoda marginata, which showed the highest 16S rDNA sequence identity (93%) to Desulfovibrio intestinalis and Desulfovibrio strain STL1. Compared to Mastotermes darwiniensis (1x10(7) cells of SRB per ml gut contents), sulfate-reducing bacteria occurred in higher numbers in the gut contents of Pachnoda marginata reaching cell titers of up to 2x10(8) cells per ml gut contents. In vitro sulfate reduction rates were determined with SRB from the gut contents of the termite Mastotermes darwiniensis and the beetle Pachnoda marginata. Due to the higher cell titer, the sulfate reduction rate of Pachnoda marginata was 10(4) nmolxh-1xml-1 and therefore, 21 times higher than that of Mastotermes darwiniensis. In addition, we detected in vivo sulfate reduction in Mastotermes darwiniensis, which indicates that sulfate reducers play an active role in the sulfur metabolism in the termite gut.     

Diversity and vertical distribution of cultured and uncultured Deltaproteobacteria in an intertidal mud flat of the Wadden Sea

The diversity and distribution of Deltaproteobacteria in an intertidal mud flat of the German Wadden Sea was characterized by molecular biological techniques and cultivation. A 16S rRNA gene library generated with general primers (303 clones) suggested that sulfate-reducing bacteria (SRB) related to Desulfobulbaceae and Desulfosarcina were abundant. Fluorescence in situ hybridization (FISH) with probes targeting these groups was used to characterize their vertical distribution. The combination of FISH with catalysed reporter deposition (CARD-FISH) significantly enhanced the detection of selected subgroups of Deltaproteobacteria, particularly in deeper sediment layers. Up to 11% of all cells were assigned to SRB. Organisms related to Desulfosarcina and Desulfobulbaceae were the dominant SRB in the surface sediments. Two abundant subpopulations of Desulfosarcina-related bacteria were identified by FISH. The SRB community differed between the sampling site and a sandy intertidal flat chosen as a reference. Enrichments and MPN cultures inoculated with surface sediment were monitored by FISH. Nine strains of Deltaproteobacteria were isolated. Four strains were related to Desulfobulbaceae, such as Desulfobacterium catecholicum and Desulfocapsa spp. A subgroup including clone sequences and strains related to D. catecholicum could be detected in situ by a specific FISH probe. The first physiological experiments suggested specific functional roles for the isolates. Two strains utilized environmentally relevant compounds in coastal areas such as catechol and nitrate. One strain related to Desulfocapsa spp. disproportionated thiosulfate and might thus contribute to the sulfur isotope fractionation at the study site. A Fe(III)-reducing strain was obtained that affiliated with the Pelobacter-Desulphuromonas group. This group accounted for up to 6% of total cell numbers and even exceeded SRB numbers in upper sediment layers. These bacteria might substantially contribute to carbon mineralization via dissimilatory reduction of, e.g. Fe(III).     

Stable carbon isotope ratios of lipid biomarkers of sulfate-reducing bacteria

We examined the potential use of natural-abundance stable carbon isotope ratios of lipids for determining substrate usage by sulfate-reducing bacteria (SRB). Four SRB were grown under autotrophic, mixotrophic, or heterotrophic growth conditions, and the delta13C values of their individual fatty acids (FA) were determined. The FA were usually 13C depleted in relation to biomass, with Deltadelta13C(FA - biomass) of -4 to -17 per thousand; the greatest depletion occurred during heterotrophic growth. The exception was Desulfotomaculum acetoxidans, for which substrate limitation resulted in biomass and FA becoming isotopically heavier than the acetate substrate. The delta13C values of FA in Desulfotomaculum acetoxidans varied with the position of the double bond in the monounsaturated C16 and C18 FA, with FA becoming progressively more 13C depleted as the double bond approached the methyl end. Mixotrophic growth of Desulfovibrio desulfuricans resulted in little depletion of the i17:1 biomarker relative to biomass or acetate, whereas growth with lactate resulted in a higher proportion of i17:1 with a greater depletion in 13C. The relative abundances of 10Me16:0 in Desulfobacter hydrogenophilus and Desulfobacterium autotrophicum were not affected by growth conditions, yet the Deltadelta13C(FA - substrate) values of 10Me16:0 were considerably greater during autotrophic growth. These experiments indicate that FA delta13C values can be useful for interpreting carbon utilization by SRB in natural environments.     

Sulfur Cycling in the Terrestrial Subsurface: Commensal Interactions, Spatial Scales, and Microbial Heterogeneity

Abstract Microbiological, geochemical, and isotopic analyses of sediment and water samples from the unconsolidated Yegua formation in east-central Texas were used to assess microbial processes in the terrestrial subsurface. Previous geochemical studies suggested that sulfide oxidation at shallow depths may provide sulfate for sulfate-reducing bacteria (SRB) in deeper aquifer formations. The present study further examines this possibility, and provides a more detailed evaluation of the relationship between microbial activity, lithology, and the geochemical environment on meter-to-millimeter scales. Sediment of varied lithology (sands, silts, clays, lignite) was collected from two boreholes, to depths of 30 m. Our findings suggest that pyrite oxidation strongly influences the geochemical environment in shallow sediments (∼5 m), and produces acidic waters (pH 3.8) that are rich in sulfate (28 mM) and ferrous iron (0.3 mM). Sulfur and iron-oxidizing bacteria are readily detected in shallow sediments; they likely play an indirect role in pyrite oxidation. In consistent fashion, there is a relative paucity of pyrite in shallow sediments and a low ³⁴S/³²S-sulfate ratio (0.2‰) (reflecting contributions from ³⁴S-depleted sulfides) in shallow regions. Pyrite oxidation likely provides a sulfate source for both oxic and anoxic aquifers in the region. A variety of assays and direct-imaging techniques of ³⁵S-sulfide production in sediment cores indicates that sulfate reduction occurs in both the oxidizing and reducing portions of the sediment profile, with a high degree of spatial variability. Narrow zones of activity were detected in sands that were juxtaposed to clay or lignite-rich sediments. The fermentation of organic matter in the lignite-rich laminae provides small molecular weight organic acids to support sulfate reduction in neighboring sands. Consequently, sulfur cycling in shallow sediments, and sulfate transport represent important mechanisms for commensal interaction among subsurface microorganisms by providing electron donors for chemoautotrophic bacteria and electron acceptors for SRB. The activity of SRB is linked to the availability of suitable electron donors from spatially distinct zones. Received: 10 November 1997; Accepted: 10 February 1998     

Field-scale bioremediation of arsenic-contaminated groundwater using sulfate-reducing bacteria and biogenic pyrite

This research demonstrates that biogenic pyrite formed by stimulation of indigenous sulfate-reducing bacteria (SRB) in a natural aquifer can remove dissolved arsenic from contaminated groundwater under strongly reducing conditions. SRB metabolism led to the precipitation of biogenic pyrite nanoparticles capable of sorbing and co-precipitating arsenic. The field site is an industrial site where shallow groundwater in an unconfined sandy aquifer is contaminated by arsenic. Therefore, biodegradable organic carbon, ferrous iron, sulfate, and fertilizer were injected into groundwater and SRB metabolism began about 1 week later. Microscopic, X-ray diffraction, X-ray fluorescence, and electron microprobe analyses confirm the bio-mineralization of pyrite and over time, pyrite nanoparticles grew to form well-formed crystals (1–10?µm in diameter) or spherical aggregates that contain 0.05–0.4?wt. % arsenic, indicative of their capacity to sequester arsenic. Consequently, dissolved arsenic decreased from its initial concentration of 0.3–0.5?mg/L to below the regulatory clean-up standard for the site of 0.05?mg/L in three downgradient wells in a matter of weeks after injection. The main sequestration stage, with total arsenic removal rates greater than 90%, lasted for at least 6 months until the arrival and mixing of untreated groundwater from upgradient. Treated groundwater with most active bacterial sulfate reduction became enriched in heavy ³⁴S (range from 2.02 to 4.00 ‰) compared to unaffected well water (0.40–0.61 ‰). One to three orders of magnitude increases in SRB cells were observed in treated wells for at least 2?months after injection. For a full-scale remediation, the injection of solution should start at positions hydrologically upgradient from the major plume and proceed downgradient. If needed, aquifers may be repeatedly amended with biodegradable organic carbon to reestablish the reducing conditions that favor arsenic sequestration.     

Amino acids as main substrates for sulfate-reducing bacteria in surface sediment of a eutrophic bay

The inner part of Tokyo Bay, Japan, is highly eutrophicated as shown by the frequent occurrence of red tide. The bottom water is anoxic during warm seasons especially at artificially dredged sites. In the sediment slurries prepared from surface sediment samples collected from the dredged sites, substrate addition stimulated the consumption of sulfate during anaerobic incubation. Of the substrates added, the seston composed mainly of diatom stimulated consumption more than lactate and acetate. Its effect was nearly equal to that of casamino acids. Casamino acids and some amino acids also accelerated the rate of sulfate reduction measured by the tracer method in sediment samples more than lactate or acetate. Anaerobic incubation of the sediment slurry amended with casamino acids showed that the consumption of amino acids was retarded by the addition of molybdate (final concentration; 20 mM). In the slurry amended with only molybdate, glutamate was accumulated distinctively and linearly with time. Its accumulation rate in molar base was comparable to the rate of sulfate reduction. These results suggested that amino acids were the main substrates for sulfate-reducing bacteria (SRB) in the sediment. The MPN values of SRB in these sediment samples were often higher with the enumeration medium containing casamino acids instead of lactate. Furthermore, during a week incubation of sediment slurries amended with substrates, casamino acids and seston more greatly stimulated the growth of SRB enumerated by both media than lactate.     

微生物烟气脱硫中硫酸盐还原阶段的限制性因素及其影响

【目的】利用硫酸盐还原菌(SRB)厌氧活性污泥进行烟气脱硫,探索硫酸盐生物还原的最适条件及重金属离子对硫酸盐生物还原的影响,以提高硫酸盐还原阶段的效率。【方法】对取自污水处理厂的SRB厌氧活性污泥进行高浓度硫酸盐胁迫驯化。分析生物脱硫过程中SRB厌氧污泥还原硫酸盐的限制性因素及影响。【结果】在最适生长条件下(pH 6.5,32°C),经驯化获得的SRB厌氧活性污泥有较强的硫酸盐还原能力。Fe2+的适量添加对硫酸盐还原有一定促进作用。SRB厌氧污泥还原硫酸盐的ThCOD/SO42-最适值为3.00,ThCOD=3.33为最适理论化学需氧量,硫酸盐还原率可达72.15%。SRB厌氧污泥还原硫酸盐反应体系中抑制SRB活性的硫化物浓度为300 mg/L。Pb2+和Ni2+在较低的浓度下(1.0 mg/L和2.0 mg/L)对硫酸盐的还原产生较强的抑制作用,而Cu2+在稍高的浓度下(8.0 mg/L)显示出明显的抑制作用。【结论】经驯化,SRB厌氧活性污泥显示出较强的硫酸盐还原能力,具有应用于工业烟气生物脱硫的潜力。去除重金属离子Pb2+、Ni2+和Cu2+可有效解除对硫酸盐生物还原作用的抑制。     

Biotransformation of phosphogypsum in media containing different forms of nitrogen

Studies on the biotransformation of phosphogypsum (a waste product formed in the course of the production of phosphorous fertilizers) with the use of sulfate reducing bacteria (SRB) demonstrated that it is a good source of sulfates and biogenic elements for these bacteria, though the addition of organic carbon and nitrogen is necessary. The aim of this study was to investigate the form of nitrogen and C:N ratio in the medium on the growth of SRB community in cultures containing phosphogypsum. Batch community cultures of sulfate reducing bacteria were maintained in medium with phosphogypsum (5.0 g/l), different concentrations of sodium lactate (1.6 - 9.4 g/l) and different forms (NH4CI, CO(NH2)2, KNO3) and concentrations (0 - 250 mg/l) of nitrogen. The growth of SRB was studied in the C:N ratio of from 2:1 to 300:1. It was found that: 1 - the best source of nitrogen for SRB is urea, followed by ammonium, the worst were nitrates; 2 - the bacteria were also able to grow in medium without nitrogen but their activity was then by approximately 15% lower than in optimal growth conditions; 3 - in medium with KNO3 inhibition of sulfate reduction by approx. 50% was observed; 4 - the highest reduction of nitrates (removal of nitrate) in media with phosphogypsum and nitrates was at limiting concentrations of sodium lactate. This is probably caused by the selection under these conditions (low concentration of hydrogen sulfide) of denitrifying bacteria or sulfate reducing bacteria capable of using nitrates as an electron acceptor.     

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.     

Metabolic associations with archaea drive shifts in hydrogen isotope fractionation in sulfate‐reducing bacterial lipids in cocultures and methane seeps

Correlation between hydrogen isotope fractionation in fatty acids and carbon metabolism in pure cultures of bacteria indicates the potential of biomarker D/H analysis as a tool for diagnosing carbon substrate usage in environmental samples. However, most environments, in particular anaerobic habitats, are built from metabolic networks of micro‐organisms rather than a single organism. The effect of these networks on D/H of lipids has not been explored and may complicate the interpretation of these analyses. Syntrophy represents an extreme example of metabolic interdependence. Here, we analyzed the effect of metabolic interactions on the D/H biosignatures of sulfate‐reducing bacteria (SRB) using both laboratory maintained cocultures of the methanogen Methanosarcina acetivorans and the SRB Desulfococcus multivorans in addition to environmental samples harboring uncultured syntrophic consortia of anaerobic methane‐oxidizing archaea (ANME) and sulfate‐reducing Deltaproteobacteria (SRB) recovered from deep‐sea methane seeps. Consistent with previously reported trends, we observed a ~80‰ range in hydrogen isotope fractionation (ε_{lipid–water}) for D. multivorans grown under different carbon assimilation conditions, with more D‐enriched values associated with heterotrophic growth. In contrast, for cocultures of D. multivorans with M. acetivorans, we observed a reduced range of ε_{lipid–water} values (~36‰) across substrates with shifts of up to 61‰ compared to monocultures. Sediment cores from methane seep settings in Hydrate Ridge (offshore Oregon, USA) showed similar D‐enrichment in diagnostic SRB fatty acids coinciding with peaks in ANME/SRB consortia concentration suggesting that metabolic associations are connected to the observed shifts in ε_{lipid–water} values.     

Effect of hydrogen sulfide on growth of sulfate reducing bacteria

A culture of sulfate reducing bacteria (SRB) growing on lactate and sulfate was incubated at different pH values in the range of 5.8-7.0. The effect of pH on growth rate was determined in this pH range; the highest growth rate was observed at pH 6.7. Hydrogen sulfide produced from sulfate reduction was found to have a direct and reversible toxicity effect on the SRB. A hydrogen sulfide Concentration of 547 mg/L (16.1 mM) completely inhibited the culture growth. Comparison between acetic acid and hydrogen sulfide inhibition is presented and the concomitant inhibition kinetics are mathematically described. (c) 1992 John Wiley & Sons, Inc.     

Biogeochemistry and biodiversity of methane cycling in subsurface marine sediments (Skagerrak, Denmark)

This biogeochemical, molecular genetic and lipid biomarker study of sediments ( approximately 4 m cores) from the Skagerrak (Denmark) investigated methane cycling in a sediment with a clear sulfate-methane-transition zone (SMTZ) and where CH(4) supply was by diffusion, rather than by advection, as in more commonly studied seep sites. Sulfate reduction removed sulfate by 0.7 m and CH(4) accumulated below. (14)C-radiotracer measurements demonstrated active H(2)/CO(2) and acetate methanogenesis and anaerobic oxidation of CH(4) (AOM). Maximum AOM rates occurred near the SMTZ ( approximately 3 nmol cm(-3) day(-1) at 0.75 m) but also continued deeper, overall, at much lower rates. Maximum rates of H(2)/CO(2) and acetate methanogenesis occurred below the SMTZ but H(2)/CO(2) methanogenesis rates were x 10 those of acetate methanogenesis, and this was consistent with initial values of (13)C-depleted CH(4) (delta(13)C c.-80 per thousand). Areal AOM and methanogenic rates were similar ( approximately 1.7 mmol m(-2) day(-1)), hence, CH(4) flux is finely balanced. A 16S rRNA gene library from 1.39 m combined with methanogen (T-RFLP), bacterial (16S rRNA DGGE) and lipid biomarker depth profiles showed the presence of populations similar to some seep sites: ANME-2a (dominant), ANME-3, Methanomicrobiales, Methanosaeta Archaea, with abundance changes with depth corresponding to changes in activities and sulfate-reducing bacteria (SRB). Below the SMTZ to approximately 1.7 m CH(4) became progressively more (13)C depleted (delta(13)C -82 per thousand) indicating a zone of CH(4) recycling which was consistent with the presence of (13)C-depleted archaeol (delta(13)C -55 per thousand). Pore water acetate concentrations decreased in this zone (to approximately 5 microM), suggesting that H(2), not acetate, was an important CH(4) cycling intermediate. The potential biomarkers for AOM-associated SRB, non-isoprenoidal ether lipids, increased below the SMTZ but this distribution reflected 16S rRNA gene sequences for JS1 and OP8 bacteria rather than those of SRB. At this site peak rates of methane production and consumption are spatially separated and seem to be conducted by different archaeal groups. Also AOM is predominantly coupled to sulfate reduction, unlike recent reports from some seep and gassy sediment sites.