Rates of sulfate reduction and thiosulfate consumption in a marine microbial mat

Abstract The sulfur cycle in a microbial mat was studied by determining viable counts of sulfate-reducing bacteria, chemolithoautotrophic sulfur bacteria and anoxygenic phototrophic bacteria. All three functional groups of sulfur bacteria revealed a maximum population density in the uppermost 5 mm of the mat: 1.1 × 10⁸ cells of sulfate reducers cm⁻³ sediment, 2.0 × 10⁹ cells of chemolithoautotrophs cm⁻³ sediment, and 4.0 × 10⁷ cells of anoxygenic phototrophs cm⁻³ sediment. Bacterial dynamics were studied by sulfate reduction rate measurements, both under anoxic conditions (dark incubation) and oxic conditions (incubation in the light), and determination of the vertical distribution of the potential rate of thiosulfate consumption under oxic conditions. Sulfate reduction rates in the top 5 mm of the sediment were 566 nmol cm⁻³ d⁻¹ in the absence of oxygen, and 123 nmol cm⁻³ d⁻¹ in the presence of oxygen. In the latter case, the maximum rate was found in the 5–10-mm depth horizon (361 nmol cm⁻³ d⁻¹). Biological consumption of amended thiosulfate was rapid and decreased with depth, while in the presence of molybdate, thiosulfate consumption decreased to 10–30% of the original rate.     

Sulfur cycling in Catenococcus thiocyclus

Abstract: Since its isolation from marine volcanic areas, Catenococcus thiocyclus has been known to be able to oxidize thiosulfate to tetrathionate, but the benefits gained from the reaction were unknown. The energy to be gained from such a reaction is so small (1 electron per mol of thiosulfate, compared with 8 electrons if the thiosulfate is oxidized to sulfate) that it seemed unlikely to be a useful metabolic reaction. However, continuous culture experiments have now revealed that C. thiocyclus is able to gain metabolically useful energy from this oxidation (biomass yields increased by approximately 20% after the addition of 7.75 mM thiosulfate to medium containing 20 mM acetate) by combining it with the chemical reduction of the tetrathionate by sulfide. The enzymes for thiosulfate oxidation appear to be constitutive. Moreover, with a suitable primary energy source (e.g. glucose), C. thiocyclus can reduce sulfur (S°) to sulfide and Fe³⁺ to Fe²⁺. A chemical reaction then generates FeS. Such reactions may have important implications for the sulfur cycle at oxic:anoxic interfaces in marine and freshwater systems.     

Competition between anoxygenic phototrophic bacteria and colorless sulfur bacteria in a microbial mat

Abstract The populations of chemolithoautotrophic (colorless) sulfur bacteria and anoxygenic phototrophic bacteria were enumerated in a marine microbial mat. The highest population densities were found in the 0–5 mm layer of the mat: 2.0 × 10⁹ cells cm⁻³ sediment, and 4.0 × 10⁷ cells cm⁻³ sediment for the colorless sulfur bacteria and phototrophs, respectively. Kinetic parameters for thiosulfate-limited growth were assessed for Thiobacillus thioparus T5 and Thiocapsa roseopersicina M1, both isolated from microbial mats. For Thiobacillus T5, growing at a constant oxygen concentration of 43 μmol l⁻¹, μ_max was 0.336 h⁻¹ and K _s 0.8 μmol l⁻¹. Phototrophically grown Thiocapsa strain M1 displayed a μ_max of 0.080 h⁻¹ and a K _s of 8 μmol l⁻¹ when anoxically grown under thiosulfate limitation. In a competition experiment with thiosulfate as electron donor, Thiocapsa became dominant during a 10-h oxic/14-h anoxic regimen at continuous illumination, despite the higher affinity for thiosulfate of Thiobacillus .     

The preferred electron acceptor of Desulfovibrio desulfuricans CSN

Abstract: The sulfate-reducing bacterium Desulfovibrio desulfuricans strain CSN (DSM 104) oxidized H₂ with thiosulfate, sulfate, sulfite, nitrite, nitrate and oxygen with rates increasing (in the order listed) from 20 to 525 nmol H₂ min⁻¹ mg⁻¹ protein. Nitrate reduction was induced by nitrate or limiting concentrations of sulfate during growth, while all other activities were constitutive. Oxygen prevented reduction of all other electron acceptors, while nitrate and nitrite blocked the reduction of the sulfur compounds. In the presence of H₂ and reduced sulfur compounds, H₂ was the preferred electron donor. The cells oxidized thiosulfate or sulfite coupled to the reduction of nitrate to ammonia. This represents a novel type of metabolism connecting the sulfur and nitrogen cycles. It is concluded that oxygen is the preferred electron acceptor of D. desulfuricans . Sulfate reduction in oxic environments must be due to different organisms or mechanisms.     

Methanogenesis in the hypersaline Solar Lake (Sinai)

Abstract Enrichment studies on microbial mat sediments (potential stromatolites) from the hypersaline Solar Lake (Sinai) indicated high numbers of methanogenic bacteria (up to 10⁵ ml⁻¹ sediment) in spite of the high sulfate reduction rate, sulfate concentration and salinity. Among H₂/CO₂, acetate and monomethylamine, the methylated amine was the preferred substrate. The predominant species enriched was a Methanosarcina sp. The findings indicate that methanogenic bacteria play an important role in hypersaline sulfate-enriched anoxic sediments and stromatolithic microbial mats.     

Microbial consumption of dimethyl sulfide and methanethiol in coastal marine sediments

Abstract: Samples were taken from oxic and anoxic zones of three ecosystems: a cyanobacterial mat, a diatom film and a carbonate sediment. Dimethylsulfide (DMS) concentrations were determined by headspace analysis of sediment slurries; maximal amounts were in the upper 5–10 mm of the sediments of 20 μM (cyanobacterial mat), 8 μM (diatom film) and < 1 μM in the carbonate sediment. Dissolved DMS in the cyanobacterial mat, determined by centrifugation and cryogenic trapping, was about two orders of magnitude lower than from slurry estimations but its variation with depth was similar. CH₃SH concentrations in slurried samples, determined after treatment with tributylphosphine, ranged from 2 to 7 μM in the diatom mat and was below the limit of detection (< 0.1 μM) in the carbonate sediment. MPN counts of bacteria that grew on DMS under oxic and anoxic (nitrate added) conditions were determined at all three sites. Aerobic DMS utilizers peaked in the surface and decreased with depth, while the population of anaerobic DMS utilizers was relatively constant in the top 20 mm. Populations of DMS utilizers were highest in the cyanobacterial mat and lowest in the carbonate sediment. MPN's of thiosulfate utilizers, aerobic and anaerobic (nitrate added) were determined in the cyanobacterial mat. Populations of aerobic and anaerobic S₂O₃²⁻ utilizers were similar throughout the top 20 mm and comparable to those of DMS utilizers in the top 5 mm, but higher by about 100-fold below that zone. DMS and CH₃SH consumption rates were measured in slurries of sediments and aerobic rates were similar or only slightly higher than anaerobic rates; the latter were stimulated by nitrate.     

Dimethylsulfoxide reduction by marine sulfate-reducing bacteria

Abstract Dimethylsulfoxide (DMSO) reduction occurred in five out of nine strains of sulfate-reducing bacteria from marine or saline environments, but not in three freshwater isolates. DMSO reduction supported growth in all positive strains. In Desulfovibrio desulfuricans strain PA2805, DMSO reduction occurred simultaneously with sulfate reduction and was not effectively inhibited by molybdate, a specific inhibitor of sulfate reduction. The growth yield per mol lactate was 26% higher with DMSO than with sulfate as electron acceptor. In extracts of cells of strain PA2805 grown on sulfate, a low level of DMSO-reducing activity was present (0.013 μmol (mg protein)⁻ min⁻); higher levels were found in cells grown on DMSO (0.56 μmol (mg protein)⁻ min⁻). In anoxic marine environments DMSO reduction by sulfate-reducing bacteria may lead to enhanced dimethylsulfide emission rates.     

Sulfide-induced dissimilatory nitrate reduction to ammonia in anaerobic freshwater sediments

Abstract: Different reduced sulfur compounds (H₂S, FeS, S₂O₃²⁻) were tested as electron donors for dissimilatory nitrate reduction in nitrate-amended sediment slurries. Only in the free sulfide-enriched slurries was nitrate appreciably reduced to ammonia (     ), with concomitant oxidation of sulfide to S⁰ (     ). The initial concentration of free sulfide appears as a factor determining the type of nitrate reduction. At extremely low concentrations of free S²⁻ (metal sulfides) nitrate was reduced via denitrification whereas at higher S²⁻ concentrations, dissimilatory nitrate reduction to ammonia (DNRA) and incomplete denitrification to gaseous nitrogen oxides took place. Sulfide inhibition of NO- and N₂O- reductases is proposed as being responsible for the driving part of the electron flow from S²⁻ to NH₄⁺.     

Inhibition of respiratory oxidation of elemental sulfur (S0) and thiosulfate in Thiobacillus versutus and another sulfur-oxidizing bacterium

Abstract The rates of thiosulfate, elemental sulfur (S⁰) and sulfite oxidation were measured respirometrically with an oxygen electrode using young cells of Thiobacillus versutus growing chemolithoautotrophically on thiosulfate under normal air pressure. Myxothiazol, an inhibitor of the cytochrome b−c₁ segment, and HQNO (2-N-heptyl-4-hydroxyquiniline N-oxide), acting in the quinone-cytochrome b region, both significantly inhibited the thiosulfate oxidation rate. The effect on the oxidation rate of S⁰ was even stronger. The oxidation of sulfite or ascorbate + TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) (substrates releasing electrons at the level of cytochrome c) was not inhibited by myxothiazol and HQNO. Thiosulfate, S⁰, sulfite and ascorbate + TMPD oxidations were strongly inhibited by KCN. These respiratory activities were almost completely eliminated by cell breakage. The reduction of b-type cytochrome was observed in thiosulfate-reduced minus sulfite-reduced difference spectra. This study confirms that S⁰ is an important intermediate of thiosulfate oxidation in Thiobacillus versutus , and that electrons released by S⁰ oxidation enter the respiratory chain in the quinone-cytochrome b region. This would allow an increased gain of energy, while less energy would probably be required for pyridine-nucleotide reduction.     

H2 oxidation in the presence of thiosulfate, by a Thermoanaerobacter strain isolated from an oil-producing well

Abstract A thermophilic rod (strain SEBR 5268), isolated from an oil-producing well, was identified as a Thermoanaerobacter strain that was phenotypically related to T. finnii . Both SEBR 5268 and T. finnii oxidized H₂ by reducing thiosulfate to sulfide using yeast extract as growth substrate. H₂ oxidation in the presence of thiosulfate was significant at the end of the exponential growth of SEBR 5268 and was maintained during the lysis phase. In the absence of thiosulfate, H₂ was inhibitory for both strains. The role of H₂ consumption by these bacteria is discussed with regard to their metabolism on organic compounds.     

Competition for sulfide among colorless and purple sulfur bacteria in cyanobacterial mats

Abstract The vertical zonation of light, O₂, H₂S, pH, and sulfur bacteria was studied in two benthic cyanobacterial mats from hypersaline ponds at Guerrero Negro, baja California, Mexico. The physical-chemical gradients were analyzed in the upper few mm at ≥ 100 μm spatial resolution by microelectrodes and by a fiber optic microprobe. In mats, where oxygen produced by photosynthesis diffused far below the depth of the photic zone, colorless sulfur bacteria ( Beggiatoa sp.) were the dominant sulfide oxidizing organisms. In a mat, where the O₂–H₂S interface was close to the photic zone, but yet received no significant visible light, purple sulfur bacteria ( Chromatium sp.) were the dominant sulfide oxidizers. Analysis of the spectral light distribution heare showed that the penetration of only 1% of the incident near-IR light (800–900 nm) into the sulfide zone was sufficient for the development of Chromatium in a narrow band of 300 μm thickness. The balance betweem O₂ and light penetration down into the sulfide zone thus deterined in mcro-scale which type of sulfur bacteria becamed dominant.     

Pathways and Microbiology of Thiosulfate Transformations and Sulfate Reduction in a Marine Sediment (Kattegat, Denmark)

Reductive and oxidative pathways of the sulfur cycle were studied in a marine sediment by parallel radiotracer experiments with ³⁵SO₄^2-, H₂³⁵S, and ³⁵S₂O₃^2- injected into undisturbed sediment cores. The distributions of viable populations of sulfate- and thiosulfate-reducing bacteria and of thiosulfate-disproportionating bacteria were concurrently determined. Sulfate reduction occurred both in the reducing sediment layers and in oxidized and even oxic surface layers. The population density of sulfate-reducing bacteria was >10⁶ cm^-3 in the oxic layer, high enough that it could possibly account for the measured rates of sulfate reduction. The bacterial numbers counted in the reducing sediment layers were 100-fold lower. The dominant sulfate reducers growing on acetate or H₂ were gas-vacuolated motile rods which were previously undescribed. The products of sulfide oxidation, which took place in both oxidized and reduced sediment layers, were 65 to 85% S₂O₃^2- and 35 to 15% SO₄^2-. Thiosulfate was concurrently oxidized to sulfate, reduced to sulfide, and disproportionated to sulfate and sulfide. There was a gradual shift from predominance of oxidation toward predominance of reduction with depth in the sediment. Disproportionation was the most important pathway overall. Thiosulfate disproportionation occurred only as cometabolism in the marine acetate-utilizing sulfate-reducing bacteria, which could not conserve energy for growth from this process alone. Oxidative and reductive cycling of sulfur thus occurred in all sediment layers with an intermediate “thiosulfate shunt” as an important mechanism regulating the electron flow.     

Growth of thermophilic obligately autotrophic hydrogen-oxidizing bacteria on thiosulfate

Thermophilic obligately autotrophic H₂-oxidizing bacteria from Icelandic hot springs were tested for growth on thiosulfate. Ten strains were tested and all grew on thiosulfate but not on sulfite or sulfur. The product of thiosulfate oxidation was sulfate. The growth rate on thiosulfate was slower (μ=0.12 h^-1) than on H₂ (μ=0.34 h^-1). Washed cells which had been grown on thiosulfate could oxidize thiosulfate rapidly but H₂-grown cells oxidized thiosulfate much more slowly and with about a 3 h lag time. The bacteria would not grow on agar medium under H₂ but grew on agar medium containing thiosulfate.     

Biogeochemistry of an Iron-Rich Hypersaline Microbial Mat (Camargue, France)

In situ microsensor measurements were combined with biogeochemical methods to determine oxygen, sulfur, and carbon cycling in microbial mats growing in a solar saltern (Salin-de-Giraud, France). Sulfate reduction rates closely followed the daily temperature changes and were highest during the day at 25°C and lowest during the night at 11°C, most probably fueled by direct substrate interactions between cyanobacteria and sulfate-reducing bacteria. Sulfate reduction was the major mineralization process during the night and the contribution of aerobic respiration to nighttime DIC production decreased. This decrease of aerobic respiration led to an increasing contribution of sulfide (and iron) oxidation to nighttime O₂ consumption. A peak of elemental sulfur in a layer of high sulfate reduction at low sulfide concentration underneath the oxic zone indicated anoxygenic photosynthesis and/or sulfide oxidation by iron, which strongly contributed to sulfide consumption. We found a significant internal carbon cycling in the mat, and sulfate reduction directly supplied DIC for photosynthesis. The mats were characterized by a high iron content of 56 mol Fe cm^–3, and iron cycling strongly controlled the sulfur cycle in the mat. This included sulfide precipitation resulting in high FeS contents with depth, and reactions of iron oxides with sulfide, especially after sunset, leading to a pronounced gap between oxygen and sulfide gradients and an unusual persistence of a pH peak in the uppermost mat layer until midnight.     

Transition from anaerobic to aerobic growth conditions for the sulfate-reducing bacterium Desulfovibrio oxyclinae results in flocculation

A chemostat culture of the sulfate-reducing bacterium Desulfovibrio oxyclinae isolated from the oxic layer of a hypersaline cyanobacterial mat was grown anaerobically and then subjected to gassing with 1% oxygen, both at a dilution rate of 0.05 h(-1). The sulfate reduction rate under anaerobic conditions was 370 nmol of SO(4)(2-) mg of protein(-1) min(-1). At the onset of aerobic gassing, sulfate reduction decreased by 40%, although viable cell numbers did not decrease. After 42 h, the sulfate reduction rate returned to the level observed in the anaerobic culture. At this stage the growth yield increased by 180% compared to the anaerobic culture to 4.4 g of protein per mol of sulfate reduced. Protein content per cell increased at the same time by 40%. The oxygen consumption rate per milligram of protein measured in washed cell suspensions increased by 80%, and the thiosulfate reduction rate of the same samples increased by 29% with lactate as the electron donor. These findings indicated possible oxygen-dependent enhancement of growth. After 140 h of growth under oxygen flux, formation of cell aggregates 0.1 to 3 mm in diameter was observed. Micrometer-sized aggregates were found to form earlier, during the first hours of exposure to oxygen. The respiration rate of D. oxyclinae was sufficient to create anoxia inside clumps larger than 3 microm, while the levels of dissolved oxygen in the growth vessel were 0.7 +/- 0.5 microM. Aggregation of sulfate-reducing bacteria was observed within a Microcoleus chthonoplastes-dominated layer of a cyanobacterial mat under daily exposure to oxygen concentrations of up to 900 microM. Desulfonema-like sulfate-reducing bacteria were also common in this environment along with other nonaggregated sulfate-reducing bacteria. Two-dimensional mapping of sulfate reduction showed heterogeneity of sulfate reduction activity in this oxic zone.     

Sulfide oxidation under oxygen limitation by a thiobacillus thioparus isolated from a marine microbial mat

Abstract The colorless sulfur bacterium Thiobacillus thioparus T5, isolated from a marine microbial mat, was grown in continuous culture under conditions ranging from sulfide limitation to oxygen limitation. Under sulfide-limiting conditions, sulfide was virtually completely oxidized to sulfate. Under oxygen-limiting conditions, sulfide was partially oxidized to zerovalent sulfur (75%) and thiosulfate (17%). In addition, low concentrations of tetrathionate and polysulfide were detected. The finding of in vivo thiosulfate formation supports the discredited observations of thiosulfate formation in cell free extracts in the early sixties. In a microbial mat most sulfide oxidation was shown to take place under oxygen-limiting conditions. It is suggested that zerovalent sulfur formation by thiobacilli is a major process resulting in polysulfide accumulation. Implications for the competition between colorless sulfur bacteria and purple sulfur bacteria are discussed.     

Sulfate-Reducing Bacteria and Their Activities in Cyanobacterial Mats of Solar Lake (Sinai, Egypt)

The sulfate-reducing bacteria within the surface layer of the hypersaline cyanobacterial mat of Solar Lake (Sinai, Egypt) were investigated with combined microbiological, molecular, and biogeochemical approaches. The diurnally oxic surface layer contained between 10⁶ and 10⁷ cultivable sulfate-reducing bacteria ml⁻¹ and showed sulfate reduction rates between 1,000 and 2,200 nmol ml⁻¹ day⁻¹, both in the same range as and sometimes higher than those in anaerobic deeper mat layers. In the oxic surface layer and in the mat layers below, filamentous sulfate-reducing Desulfonema bacteria were found in variable densities of 10⁴ to 10⁶ cells ml⁻¹. A Desulfonema-related, diurnally migrating bacterium was detected with PCR and denaturing gradient gel electrophoresis within and below the oxic surface layer. Facultative aerobic respiration, filamentous morphology, motility, diurnal migration, and aggregate formation were the most conspicuous adaptations of Solar Lake sulfate-reducing bacteria to the mat matrix and to diurnal oxygen stress. A comparison of sulfate reduction rates within the mat and previously published photosynthesis rates showed that CO₂ from sulfate reduction in the upper 5 mm accounted for 7 to 8% of the total photosynthetic CO₂ demand of the mat.     

Demethylation and cleavage of dimethylsulfoniopropionate in marine intertidal sediments

Abstract Demethylation and cleavage of dimethylsulfoniopropionate (DMSP) was measured in three different types of intertidal marine sediments: a cyanobacterial mat, a diatom-covered tidal flat and a carbonate sediment. Consumption rates of added DMSP were highest in cyanobacterial mat slurries (59 μmol DMSP 1⁻¹) and lower in slurries from a diatom mat and a carbonate tidal sediment (24 and 9 μmol DMSP 1⁻¹ h⁻¹, respectively). Dimethyl sulfide (DMS) and 3-mercaptopropionate (MPA) were produced simultaneously during DMSP consumption, indicating that cleavage and demethylation occurred at the same time. Viable counts of DMSP-utilizing bacteria revealed a population of 2 × 10⁷ cells cm⁻³ sediment (90% of these cleaved DMSP to DMS, 10% demethylated DMSP to MPA) in the cyanobacterial mat, 7 × 10⁵ cells cm⁻³ in the diatom mat (23% cleavers, 77% demethylators), and 9 × 10⁴ cells cm⁻³ (20% cleavers and 80% demethylators) in the carbonate sediment. In slurries of the diatom mat, the rate of MPA production from added 3-methiolpropionate (MMPA) was 50% of the rate of MPA formation from DMSP. The presence of a large population of demethylating bacteria and the production of MPA from DMSP suggest that the demethylation pathway, in addition to cleavage, contributes significantly to DMSP consumption in coastal sediments.     

Bacteriological studies on the sulfur cycle in the anaerobic part of the hypolimnion and in the surface sediments of rotsee in Switzerland scientific report of the advanced course of microbial ecology, sponsored by FEMS and the Swiss society for Microbiology, held at Kastanienbaum, Switzerland, 12 September–9 October 1982

Abstract The microbial ecology of the sulfur cycle in the anaerobic part of Rotsee (Switzerland) was studied. Almost all the sulfate reduction took place at the sediment surface at a rate of 2 mmol SO²⁻₄ reduced m⁻² day⁻¹. Approx. 10⁴ sulfate reducers per ml were present in the surface sediments. The sulfide produced was phototrophically consumed mainly by Thiopedia rosea, Lamprocystis roseopersicina and ' Pelochromatium roseum ' consortia. Thiopedia rosea migrated diurnally about one meter. Bacterial photosynthesis was limited by light and sulfide rather than by temperature.     

A rapid and sensitive ion chromatographic technique for the determination of sulfate and sulfate reduction rates in freshwater lake sediments

Abstract Newly developed low capacity columns were used in suppressed ion chromatography for rapid and highly reproducible determination of SO₄²⁻ in porewater samples from freshwater sediments without preconcentration of samples. With a 50 μl injection the detection limit for SO₄²⁻ was ca. 50 pmol (= 1 μ M) with a precision of 1–3% at the 10–200 μM level and <1% at concentrations above 200 μM. SO₄²⁻ could be measured in 4–5 min with the routinely used eluent (3.0 mM NaHCO₃/0.8 mM Na₂CO₃). When the strength of the eluent was increased to 3.0 mM NaHCO₃/2.0 mM Na₂CO₃, sulfate analysis was possible in less than 3 min, provided that samples were nitrate-free. Under these conditions S₂O₃²⁻ could also be sensitively determined in about 6 min. Examples of application of the method are given for measurements of sulfate reduction rates in freshwater sediment samples from Lake Constance.