Thermophilic Sulfate-reducing Bacterium Research Articles

Coexistence of Psychrophilic, Mesophilic, and Thermophilic Sulfate-Reducing Bacteria in a Deep Subsurface Aquifer Associated with Coal-Bed Methane Production.

The microbial community of subsurface environments remains understudied due to limited access to deep strata and aquifers. Coal-bed methane (CBM) production is associated with a large number of wells pumping water out of coal seams. CBM wells provide access to deep biotopes associated with coal-bed water. Temperature is one of the key constraints for the distribution and activity of subsurface microorganisms, including sulfate-reducing prokaryotes (SRP). The 16S rRNA gene amplicon sequencing coupled with in situ sulfate reduction rate (SRR) measurements with a radioactive tracer and cultivation at various temperatures revealed that the SRP community of the coal bed water of the Kuzbass coal basin is characterized by an overlapping mesophilic-psychrophilic boundary. The genus Desulfovibrio comprised a significant share of the SRP community. The D. psychrotolerans strain 1203, which has a growth optimum below 20°C, dominated the cultivated SRP. SRR in coal bed water varied from 0.154 ± 0.07 to 2.04 ± 0.048nmol S cm-3day-1. Despite the ambient water temperature of ~ 10-20°C, an active thermophilic SRP community occurred in the fracture water, which reduced sulfate with the rate of 0.159 ± 0.023 to 0.198 ± 0.007nmol S cm-3day-1 at 55°C. A novel moderately thermophilic "Desulforudis audaxviator"-clade SRP has been isolated in pure culture from the coal-bed water.

Chemosynthesis: a history of innovation

BACKGROUND. Despite the fact that the process of chemosynthesis has been known for more than a hundred years, its significance and importance are still relevant today in the transformation of chemical elements in biogeochemical cycles. Today, the vital processes of nitrifying bacteria, which lead to the oxidation of ammonia to nitric acid, require scientific substantiation and additional research. The ability of bacteria to convert inorganic substances into organic ones suggests that chemosynthetics can accumulate valuable resources for human needs.
 OBJECTIVE. To analyze the history of the discovery of chemosynthesis and identify the main ways of its implementation in innovative technologies.
 MATERIALS AND METHODS. Generalization, analysis and synthesis of thematic scientific publications.
 RESULTS AND DISCUSSION. The article is presents the history of the discovery of the process of chemosynthesis and its author Serhiy Winogradsky. Peculiarities and conditions of transformation of chemical elements in biogeochemical cycles are given. The main directions of scientific research are the analysis of the peculiarities of the process of chemosynthesis, the reasons for their occurrence, as well as the definition of the main possibilities of chemosynthesis in innovative technologies. The relevance of chemosynthesis in life processes and innovative technologies of today is confirmed.
 CONCLUSIONS. Chemosynthetic communities in different environments are important biological systems in terms of their ecology, evolution and biogeography, as well as their potential as indicators of the availability of permanent hydrocarbon- based energy sources. In the process of chemosynthesis, bacteria produce organic matter where photosynthesis is impossible. Isolation of thermophilic sulfate-reducing bacteria Thermodesulfovibrio yellowstonii and other types of chemosynthetics provides prospects for further research. Thus, the importance of chemosynthesis remains relevant for use in innovative technologies, conservation of ecosystems, human life in general. The role of Serhiy Winogradsky in discovering the phenomenon of chemosynthesis is underestimated and needs further research and popularization.

Thermophilic sulfate-reducing bacteria Moorela thermoacetica Nadia-3, isolated from “Nadiia” pit spoil heap of Chervonohrad mining region

Introduction. Thermophilic sulfate-reducing bacteria attract attention of scientists as the potential agents of purification of wastewater polluted by sulfur and its compounds, heavy metal ions and organic compounds. These bacteria oxidize different organic substrates using metals with variable valency as electron acceptors and transform them into non-toxic or less toxic forms for living organisms. However, wastewater contains high concentrations of different toxic xenobiotics, particularly, metal ions that have negative influence on living organisms. For this reason, it is important to use resistant strains of microorganisms for the purification of wastewater. The aim of this work was to identify the thermophilic sulfur-reducing bacteria, isolated from “Nadiia” pit spoil heap of Chervonohrad mining region, and to study their properties. Materials and Methods. Thermophilic sulfur-reducing bacteria were isolated from the samples of rock of “Nadiia” pit heap at 50 cm depth. Bacteria were cultivated in TF medium under the anaerobic conditions in anaerostates. Cell biomass was measured turbidimetrically using the photoelectric colorimeter KFK-3 (λ = 340 nm, 3 mm cuvette). Hydrogen sulfide content was measured photoelectrocolorymetrically by the production of methylene blue. Organic acids content was measured by high performance liquid chromatography. Cr(VI), Fe(III), Мn(IV) and NO3– content was measured turbidimetrically. Results. Thermophilic sulfur-reducing bacteria were isolated from the rock of “Nadiia” pit heap of Chervonohrad mining region. They were identified as Moorela thermoacetica based on the morpho-physiological and biochemical properties and on the results of phylogenetic analysis. M. thermoacetica Nadia-3 grow in the synthetic TF medium, have the shape of elongated rods, are gram-positive, endospore-forming. They form light brown colonies. Optimal growth was observed at 50–55 °C, pH 6.5–7. The bacteria utilize glucose, starch, fructose, maltose, lactose, sodium lactate, arabinose, cellulose, maltose, glycerol, fumarate, and ethanol as carbon sources. The highest sulfidogenic activity of M. thermoacetica Nadia-3 was found in media with glycerol, lactose, and glucose. M. thermoacetica Nadia-3 reduce SO42-, S2O32-, Fe(III), NO3–, Cr(VI) compounds besides elemental sulfur. They accumulate biomass at K2Cr2O7 concentrations of 0.1–1 mM. Sulfur reduction is not the main way of energy accumulation. Conclusions. Thermophilic chromium-resistant sulfur-reducing bacteria M. thermoacetica Nadia-3, that produce hydrogen sulfide during the oxidation of different organic compounds, were isolated from the rock of “Nadiia” pit heap. They reduce Fe(III), Cr(VI), NO3–, SO42-, S2O32-, besides elemental sulfur.

Microbial Diversity and Potential Sulfide Producers in the Karazhanbas Oilfield (Kazakhstan)

Biogenic sulfide production in oilfields results in lower quality of oil and gas and in corrosion of oil-producing equipment. Bactericidal agents are not used at the Karazhanbas oilfield (Kazakhstan), since sulfate concentrations in both injected and formation water are low or zero. However, increasing corrosion of steel equipment and sulfide release into formation water were observed in the course of the oilfield development. The goal of the present work was to reveal the potential agents of microbial corrosion and to investigate the possibility to suppress development of sulfidogens using nitrate. Environmental conditions and the composition of microbial communities in production and injection water were studied for the sites with different temperatures. High-throughput sequencing of the V3–V4 region of the 16S rRNA gene revealed predominance of thermophilic sulfate-reducing bacteria (genera Thermodesulfobacterium, Thermodesulfovibrio, Desulfotomaculum, Thermodesulforhabdus, Desulfovirgula, Defluviitoga, and Desulfonauticus) and archaea (genus Archaeoglobus) at the high-temperature horizon, as well as of thiosulfate-reducing (genera Thermoanaerobacter, Anaerobaculum, and Coprothermobacter) and syntrophic bacteria, while abundance of methanogens (genera Methanolinea and Methanothermobacter) was low. Communities from low-temperature horizons contained mesophilic methanogens (genera Methanococcus, Methanobacterium, and Methanothrix), thermophilic and mesophilic sulfate-reducing bacteria (genera Desulfovibrio, Desulfomicrobium, Desulfosarcina, Desulfoglaeba, Desulfotignum, and Desulfocurvus), syntrophic bacteria (genus Smithella), and members of the genera Marinobacter, Paracoccus, Alcaligenes, Arcobacter, and Halomonas. Enrichment cultures of bacteria producing sulfide were obtained from formation water, as well as the cultures reducing nitrate to nitrite and suppressing growth of sulfate- and thiosulfate-reducing bacteria. These findings may indicate the possibility of using nitrate as a competitive inhibitor of sulfidogenesis in this oilfield. Corrosion-active hydrogen-utilizing methanogens and acetogens were also found, which implies the necessity for monitoring the oilfield microbial communities.

Draft Genome Sequence of a Sulfate-Reducing Bacterium, "Desulfofundulus salinum" 435T, Isolated from a High-Temperature Gas Field in Russia.

The draft genome sequence of the thermophilic sulfate-reducing bacterium "Desulfofundulus salinum" strain 435T, isolated from condensate water of the Igrim high-temperature gas field (Western Siberia, Russia), is presented here. The genome is annotated to elucidate the taxonomic position of strain 435T.

Influence of thermophilic sulfate-reducing bacteria and deposited CaCO3 on the corrosion of water injection system

Abstract Herein, a schematic model was established to assess the combined effect of deposited CaCO3 and thermophilic sulfate-reducing bacteria (SRB), at high CO2 and low O2 dissolution levels on the corrosion behavior and growth of corrosion scale on X65 pipeline carbon steel. Field emission scanning electron microscopy, energy-dispersive spectrometry, X-ray diffraction, confocal laser scanning microscopy, open circuit potential, and electrochemical impedance spectroscopy were used to characterize the corrosion pattern of water injection pipe. The results showed that a synergistic effect exists between CaCO3 and thermophilic SRB, both uniform and localized corrosion of CaCO3-coated X65 steel were promoted when inoculated with SRB. The corrosion process was comprised of three stages depending on the metabolism of SRB.

The deep-subsurface sulfate reducer Desulfotomaculum kuznetsovii employs two methanol-degrading pathways

Methanol is generally metabolized through a pathway initiated by a cobalamine-containing methanol methyltransferase by anaerobic methylotrophs (such as methanogens and acetogens), or through oxidation to formaldehyde using a methanol dehydrogenase by aerobes. Methanol is an important substrate in deep-subsurface environments, where thermophilic sulfate-reducing bacteria of the genus Desulfotomaculum have key roles. Here, we study the methanol metabolism of Desulfotomaculum kuznetsovii strain 17T, isolated from a 3000-m deep geothermal water reservoir. We use proteomics to analyze cells grown with methanol and sulfate in the presence and absence of cobalt and vitamin B12. The results indicate the presence of two methanol-degrading pathways in D. kuznetsovii, a cobalt-dependent methanol methyltransferase and a cobalt-independent methanol dehydrogenase, which is further confirmed by stable isotope fractionation. This is the first report of a microorganism utilizing two distinct methanol conversion pathways. We hypothesize that this gives D. kuznetsovii a competitive advantage in its natural environment.

Effect of Thermophilic Nitrate Reduction on Sulfide Production in High Temperature Oil Reservoir Samples.

Oil fields can experience souring, the reduction of sulfate to sulfide by sulfate-reducing microorganisms. At the Terra Nova oil field near Canada’s east coast, with a reservoir temperature of 95°C, souring was indicated by increased hydrogen sulfide in produced waters (PW). Microbial community analysis by 16S rRNA gene sequencing showed the hyperthermophilic sulfate-reducing archaeon Archaeoglobus in Terra Nova PWs. Growth enrichments in sulfate-containing media at 55–70°C with lactate or volatile fatty acids yielded the thermophilic sulfate-reducing bacterium (SRB) Desulfotomaculum. Enrichments at 30–45°C in nitrate-containing media indicated the presence of mesophilic nitrate-reducing bacteria (NRB), which reduce nitrate without accumulation of nitrite, likely to N2. Thermophilic NRB (tNRB) of the genera Marinobacter and Geobacillus were detected and isolated at 30–50°C and 40–65°C, respectively, and only reduced nitrate to nitrite. Added nitrite strongly inhibited the isolated thermophilic SRB (tSRB) and tNRB and SRB could not be maintained in co-culture. Inhibition of tSRB by nitrate in batch and continuous cultures required inoculation with tNRB. The results suggest that nitrate injected into Terra Nova is reduced to N2 at temperatures up to 45°C but to nitrite only in zones from 45 to 65°C. Since the hotter zones of the reservoir (65–80°C) are inhabited by thermophilic and hyperthermophilic sulfate reducers, souring at these temperatures might be prevented by nitrite production if nitrate-reducing zones of the system could be maintained at 45–65°C.

Estimating the Abundance of Endospores of Sulfate-Reducing Bacteria in Environmental Samples by Inducing Germination and Exponential Growth

ABSTRACTIt is a challenge to quantitatively distinguish active from dormant microbial populations in environmental samples. Here we present an approach for estimating the abundance of dormant sulfate-reducing bacteria (SRB), present as viable endospores in environmental samples. This is achieved by inducing endospores to germinate and grow exponentially. We demonstrate this approach for thermophilic SRB in temperate sediment from Aarhus Bay, Denmark. The approach is based on measuring bulk sulfate reduction rates (SRRs) in pasteurized sediment and calculating associated cell-specific SRRs based on average values for SRB growth yield and cell size known from exponentially growing pure cultures. The method presented is a faster bioassay than most probable number enumerations and has the potential to distinguish between slow- and fast-growing SRB populations in the same sample. This bioassay is attractive given the challenges posed by endospores with respect to cell permeabilization and lysis, which are prerequisite in quantitative microscopy- and nucleic acid extraction-based strategies. These molecular approaches additionally rely on designing target-appropriate oligonucleotide probes, whereas the method presented here considers the trait of interest more broadly. This strategy thus presents a useful complement to other methods in ecological investigations of microbial biogeography and for specific industrial applications such as reservoir souring and corrosion risk assessments in the oil and gas sector.

Thermophilic archaea activate butane via alkyl-coenzyme M formation

The anaerobic formation and oxidation of methane involve unique enzymatic mechanisms and cofactors, all of which are believed to be specific for C1-compounds. Here we show that an anaerobic thermophilic enrichment culture composed of dense consortia of archaea and bacteria apparently uses partly similar pathways to oxidize the C4 hydrocarbon butane. The archaea, proposed genus 'Candidatus Syntrophoarchaeum', show the characteristic autofluorescence of methanogens, and contain highly expressed genes encoding enzymes similar to methyl-coenzyme M reductase. We detect butyl-coenzyme M, indicating archaeal butane activation analogous to the first step in anaerobic methane oxidation. In addition, Ca. Syntrophoarchaeum expresses the genes encoding β-oxidation enzymes, carbon monoxide dehydrogenase and reversible C1 methanogenesis enzymes. This allows for the complete oxidation of butane. Reducing equivalents are seemingly channelled to HotSeep-1, a thermophilic sulfate-reducing partner bacterium known from the anaerobic oxidation of methane. Genes encoding 16S rRNA and methyl-coenzyme M reductase similar to those identifying Ca. Syntrophoarchaeum were repeatedly retrieved from marine subsurface sediments, suggesting that the presented activation mechanism is naturally widespread in the anaerobic oxidation of short-chain hydrocarbons.

Corrosion characteristics of sulfate-reducing bacteria (SRB) and the role of molecular biology in SRB studies: an overview

AbstractSulfate-reducing bacteria (SRB), an anaerobic bacterial group, are found in many environments like freshwater, marine sediments, agricultural soil, and oil wells where sulfate is present. SRB derives energy from electron donors such as sulfate, elemental sulfur or metals, and fermenting nitrate. It is the major bacterial group involved in the microbiologically influenced corrosion (MIC), souring, and biofouling problems in oil-gas-producing facilities as well as transporting and storage facilities. SRB utilizes sulfate ions as an electron acceptor and produce H2S, which is an agent of corrosion, causing severe economic damages. Various theories have been proposed on the direct involvement of H2S and iron sulfides in corrosion; H2S directly attacks and causes corrosion of metals and alloys. Many reviews have been presented on the aforementioned aspects. This review specifically focused on SRB corrosion and the role of molecular biology tools in SRB corrosion studies viz. cathodic and anodic depolarization theories, corrosion characteristics of thermophilic SRB and influence of hydrogenase, temperature, and pressure in thermophilic SRB corrosion, SRB taxonomy, molecular approaches adopted in SRB taxonomical studies, sulfate and citrate metabolism analyses in completed SRB genomes, and comparative studies on SRB’s dissimilatory sulfite reductase structures.

Desulfotomaculum tongense sp. nov., a moderately thermophilic sulfate-reducing bacterium isolated from a hydrothermal vent sediment collected from the Tofua Arc in the Tonga Trench

A novel, strictly anaerobic, moderately thermophilic, endospore-forming, sulfate-reducing bacterium, designated TGB60-1T, was isolated from a hydrothermal sediment vent collected from the Tofua Arc in the Tonga Trench. The strain was characterized phenotypically and phylogenetically. The isolated strain was observed to be Gram-positive, with slightly curved rod-shaped cells and a polar flagellum. Strain TGB60-1T was found to grow anaerobically at 37–60 °C (optimum, 50 °C), at pH 6.0–8.5 (optimum, pH 7.0) and with 1.0–4.0 % (w/v) NaCl (optimum, 3.0 %). The electron acceptors utilised were determined to be sulfate, sulfite, and thiosulfate. Strain TGB60-1T was found to utilise pyruvate and H2 as electron donors. Strain TGB60-1T was determined to be related to representatives of the genus Desulfotomaculum and the closest relatives within this genus were identified as Desulfotomaculum halophilum SEBR 3139T, Desulfotomaculum alkaliphilum S1T and Desulfotomaculum peckii LINDBHT1T (92.7, 92.1, and 91.8 % 16S rRNA gene sequence similarity, respectively). The major fatty acids (>20 %) were identified as C16:0 and C18:1 ω7c. The G+C content of the genomic DNA of this novel bacterium was determined to be 53.9 mol%. Based on this polyphasic taxonomic study, strain TGB60-1T is considered to represent a novel species in the genus Desulfotomaculum, for which the name Desulfotomaculum tongense sp. nov. is proposed. The type strain of D. tongense is strain TGB60-1T (= KTCT 4534T = JCM 18733T).

Desulfotomaculum peckii sp. nov., a moderately thermophilic member of the genus Desulfotomaculum , isolated from an upflow anaerobic filter treating abattoir wastewaters

A novel anaerobic thermophilic sulfate-reducing bacterium designated strain LINDBHT1(T) was isolated from an anaerobic digester treating abattoir wastewaters in Tunisia. Strain LINDBHT1(T) grew at temperatures between 50 and 65 °C (optimum 55-60 °C), and at pH between 5.9 and 9.2 (optimum pH 6.0-6.8). Strain LINDBHT1(T) required salt for growth (1-40 g NaCl l(-1)), with an optimum of 20-30 g l(-1). In the presence of sulfate as terminal electron acceptor, strain LINDBHT1(T) used H2/CO2, propanol, butanol and ethanol as carbon and energy sources but fumarate, formate, lactate and pyruvate were not utilized. Butanol was converted to butyrate, while propanol and ethanol were oxidized to propionate and acetate, respectively. Sulfate, sulfite and thiosulfate were utilized as terminal electron acceptors but elemental sulfur, iron (III), fumarate, nitrate and nitrite were not used. The G+C content of the genomic DNA was 44.4 mol%. Phylogenetic analysis of the small-subunit rRNA gene sequence indicated that strain LINDBHT1(T) was affiliated to the genus Desulfotomaculum with the type strains of Desulfotomaculum halophilum and Desulfotomaculum alkaliphilum as its closest phylogenetic relatives (about 89% similarity). This strain represents a novel species of the genus Desulfotomaculum, Desulfotomaculum peckii sp. nov.; the type strain is LINDBHT1(T) (=DSM 23769(T)=JCM 17209(T)).

Dispersal of thermophilic Desulfotomaculum endospores into Baltic Sea sediments over thousands of years.

Patterns of microbial biogeography result from a combination of dispersal, speciation and extinction, yet individual contributions exerted by each of these mechanisms are difficult to isolate and distinguish. The influx of endospores of thermophilic microorganisms to cold marine sediments offers a natural model for investigating passive dispersal in the ocean. We investigated the activity, diversity and abundance of thermophilic endospore-forming sulfate-reducing bacteria (SRB) in Aarhus Bay by incubating pasteurized sediment between 28 and 85 °C, and by subsequent molecular diversity analyses of 16S rRNA and of the dissimilatory (bi)sulfite reductase (dsrAB) genes within the endospore-forming SRB genus Desulfotomaculum. The thermophilic Desulfotomaculum community in Aarhus Bay sediments consisted of at least 23 species-level 16S rRNA sequence phylotypes. In two cases, pairs of identical 16S rRNA and dsrAB sequences in Arctic surface sediment 3000 km away showed that the same phylotypes are present in both locations. Radiotracer-enhanced most probable number analysis revealed that the abundance of endospores of thermophilic SRB in Aarhus Bay sediment was ca. 10(4) per cm(3) at the surface and decreased exponentially to 10(0) per cm(3) at 6.5 m depth, corresponding to 4500 years of sediment age. Thus, a half-life of ca. 300 years was estimated for the thermophilic SRB endospores deposited in Aarhus Bay sediments. These endospores were similarly detected in the overlying water column, indicative of passive dispersal in water masses preceding sedimentation. The sources of these thermophiles remain enigmatic, but at least one source may be common to both Aarhus Bay and Arctic sediments.

Desulfosoma profundi sp. nov., a thermophilic sulfate-reducing bacterium isolated from a deep terrestrial geothermal spring in France

A novel strictly anaerobic bacterium designated SPDX02-08(T) was isolated from a deep terrestrial geothermal spring located in southwest France. Cells (1-2×2-6μm) were non-motile, non sporulating and stained Gram negative. Strain SPDX02-08(T) grew at a temperature between 40 and 60°C (optimum 55°C), pH between 6.3 and 7.3 (optimum 7.2) and a NaCl concentration between 0 and 5g/l (optimum 2g/l). Sulfate, thiosulfate and sulfite were used as terminal electron acceptors, but not elemental sulfur, nitrate, nitrite, Fe (III) or fumarate. In the presence of sulfate, strain SPDX02-08(T) completely oxidized pyruvate, propionate, butyrate, isobutyrate, valerate, isovalerate and hexadecanoate. Stoichiometric measurements revealed a complete oxidation of part of lactate (0.125mol of acetate produced per mole lactate oxidized). Strain SPDX02-08(T) required yeast extract to oxidize formate and H(2) but did not grow autotrophically on H(2). Among the substrates tested, only pyruvate was fermented. The G+C content of the genomic DNA was 57.6mol%. Major cellular fatty acids of strain SPDX02-08(T) were iso-C(15:0), C(15:0), and C(16:0). Phylogenetic analysis of the 16S small-subunit (SSU) ribosomal RNA gene sequence indicated that strain SPDX02-08(T) belongs to the genus Desulfosoma, family Syntrophobacteraceae, having Desulfosoma caldarium as its closest phylogenetic relative (97.6% similarity). The mean DNA/DNA reassociation value between strain SPDX02-08(T) and Desulfosoma caldarium was 16.9±2.7%. Based on the polyphasic differences, strain SPDX02-08(T) is proposed to be assigned as a new species of the genus Desulfosoma, Desulfosoma profundi sp. nov. (DSM 22937(T)=JCM 16410(T)). GenBank accession number for the 16S rRNA gene sequence of strain SPDX02-08(T) is HM056226.

Influences of Thermophilic Sulfate-Reducing Bacteria on the Corrosion of Carbon Steel at Different Temperature

The growth and the corrosion behaviors of thermophilic sulfate reducing bacteria (SRB), desulfotomaulum thermocisternum strain ST90, were studied in this work. It was found that the growth curves and the main reduction products were influenced greatly by the growth temperatures. The time of the exponential phase and the stable phase decreased with the increase of growth temperatures. The reduction product of this kind of SRB was mainly H2S when it grew at 60°C. With the decrease of growth temperatures, thiosulfate came up as an internal reduction product. The thiosulfate was re-reduced by SRB at the growth temperatures of 40 and 50°C. When the growth temperature came to 30°C, no H2S was generated. The regulation of corrosion rate changed was coincident with the changes of the main reduction products of the SRB. This kind of SRB could accelerate the corrosion rate of carbon steel when grew at higher temperature. But the corrosion rate of carbon steel could be inhibited when it grew at 30°C.

Desulfotomaculum varum sp. nov., a moderately thermophilic sulfate-reducing bacterium isolated from a microbial mat colonizing a Great Artesian Basin bore well runoff channel

A strictly anaerobic moderately thermophilic bacterium, designated strain RH04-3T (T = type strain), was isolated from a red colored microbial mat that colonizes a Great Artesian Basin (GAB) bore well (Registered Number 17263) runoff channel at 66 °C. The cells of strain RH04-3T were straight to slightly curved, sporulating, Gram-positive rods (2.0–5.0 × 1.0 μm) that grew optimally at 50 °C (temperature growth range between 37 and 55 °C) and at pH 7 (pH growth range of 5.0 and 8.5). Growth was inhibited by NaCl concentrations ≥1.5% (w/v), and by chloramphenicol, streptomycin, tetracycline, penicillin and ampicillin. The strain utilized fructose, mannose, glycerol, lactate, pyruvate and H2 in the presence of sulfate, and fermented pyruvate in the absence of sulfate. Strain RH04-3T reduced sulfate, sulfite, thiosulfate and elemental sulfur, but not nitrate, nitrite, iron(III), arsenate(V), vanadium(V) or cobalt(III) as terminal electron acceptors. The G + C content of DNA was 52.4 ± 0.8 mol % as determined by the thermal denaturation (Tm) method. 16S rRNA sequence analysis indicated that strain RH04-3T was a member of the genus Desulfotomaculum and was most closely related to Desulfotomaculum putei (similarity value of 95.2%) and Desulfotomaculum hydrothermale (similarity value of 93.6%). On the basis of phylogenetic and phenotypic characteristics, strain RH04-3T is considered to represent a novel species of the genus Desulfotomaculum, for which the name Desulfotomaculum varum sp. nov. is proposed. The type strain RH04-3T = JCM 16158T = KCTC 5794T.

Sulfur Isotope Enrichment during Maintenance Metabolism in the Thermophilic Sulfate-Reducing Bacterium Desulfotomaculum putei

Values of Delta(34)S (=delta(34)S(HS)-delta(34)S(SO(4)), where delta(34)S(HS) and delta(34)S(SO(4)) indicate the differences in the isotopic compositions of the HS(-) and SO(4)(2-) in the eluent, respectively) for many modern marine sediments are in the range of -55 to -75 per thousand, much greater than the -2 to -46 per thousand epsilon(34)S (kinetic isotope enrichment) values commonly observed for microbial sulfate reduction in laboratory batch culture and chemostat experiments. It has been proposed that at extremely low sulfate reduction rates under hypersulfidic conditions with a nonlimited supply of sulfate, isotopic enrichment in laboratory culture experiments should increase to the levels recorded in nature. We examined the effect of extremely low sulfate reduction rates and electron donor limitation on S isotope fractionation by culturing a thermophilic, sulfate-reducing bacterium, Desulfotomaculum putei, in a biomass-recycling culture vessel, or "retentostat." The cell-specific rate of sulfate reduction and the specific growth rate decreased progressively from the exponential phase to the maintenance phase, yielding average maintenance coefficients of 10(-16) to 10(-18) mol of SO(4) cell(-1) h(-1) toward the end of the experiments. Overall S mass and isotopic balance were conserved during the experiment. The differences in the delta(34)S values of the sulfate and sulfide eluting from the retentostat were significantly larger, attaining a maximum Delta(34)S of -20.9 per thousand, than the -9.7 per thousand observed during the batch culture experiment, but differences did not attain the values observed in marine sediments.

Desulfonauticus autotrophicus sp. nov., a novel thermophilic sulfate-reducing bacterium isolated from oil-production water and emended description of the genus Desulfonauticus

A novel moderately thermophilic and halophilic sulfate-reducing bacterium, strain TeSt(T), was isolated from production water of an oil field in Northern Germany near Hamburg. The cells were Gram-negative, straight to slightly curved rods and motile by a single polar flagellum. Only hydrogen and formate served as electron donors, whereas a wide variety of organic substrates and CO(2) could be used as carbon sources. Sulfate, sulfite, thiosulfate and sulfur were used as electron acceptors, but not nitrate or ferric iron. The novel isolate was negative for oxidase, catalase and desulfoviridin enzyme activity. Cytochromes were present and predominantly of the c-type. Whole-cells fatty acid patterns were dominated by the branched-chain fatty acids anteiso-C(15:0), iso-C(15:0), iso-C(17:0) and anteiso-C(17:0). As major respiratory lipoquinones partially saturated derivates of menaquinone 6 [MK-6(H(2)) and probably MK-6(H(4))] were identified. The G + C content of the genomic DNA was 41.3 mol% (HPLC method). An analysis of the 16S rRNA gene sequence indicated that strain TeSt(T) belongs to the family Desulfohalobiaceae within the class Deltaproteobacteria. The most closely related species with a sequence similarity of 95.0% was Desulfonauticus submarinus suggesting an affiliation of TeSt(T) to the genus Desulfonauticus. The novel isolate could be clearly distinguished from Desulfonauticus submarinus by its ability to grow chemolithoautotrophically and hence should be assigned to a novel species for which the name Desulfonauticus autotrophicus sp. nov. is proposed. The type strain is TeSt(T) (=DSM 4206(T)=JCM 13028(T)).

Desulfotomaculum hydrothermale sp. nov., a thermophilic sulfate-reducing bacterium isolated from a terrestrial Tunisian hot spring

A novel strictly anaerobic, moderately thermophilic, sulfate-reducing bacterium, designated strain Lam5(T), was isolated from a hot spring in north-east Tunisia and was characterized phenotypically and phylogenetically. The isolate stained Gram-negative but had a Gram-positive-type cell wall. The strain comprised endospore-forming, slightly curved rod-shaped cells with peritrichous flagella. It did not possess desulfoviridin. Strain Lam5(T) grew anaerobically at 40-60 degrees C (optimally at 55 degrees C) and at pH 5.8-8.2 (optimally at pH 7.1); it did not require NaCl but tolerated concentrations up to 1.5 % (w/v). It utilized lactate, pyruvate, formate, ethanol, butanol, glycerol, propanol and H(2) (plus acetate) as electron donors. Lactate was oxidized and pyruvate was fermented to acetate. Sulfate, sulfite, thiosulfate, As(V) and Fe(III) (but not elemental sulfur, fumarate, nitrate or nitrite) were used as electron acceptors. The G+C content of the genomic DNA was 46.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequencing showed that strain Lam5(T) was a member of the genus Desulfotomaculum, with Desulfotomaculum putei as its closest relative (96 % similarity to the type strain). On the basis of genotypic, phenotypic and phylogenetic data, strain Lam5(T) represents a novel species of the genus Desulfotomaculum, for which the name Desulfotomaculum hydrothermale sp. nov. is proposed. The type strain is Lam5(T) (=DSM 18033(T) =JCM 13992(T)).