Dissimilatory arsenate and sulfate reduction in Desulfotomaculum auripigmentum sp. nov.

A newly discovered arsenate-reducing bacterium, strain OREX-4, differed significantly from strains MIT-13 and SES-3, the previously described arsenate-reducing isolates, which grew on nitrate but not on sulfate. In contrast, strain OREX-4 did not respire nitrate but grew on lactate, with either arsenate or sulfate serving as the electron acceptor, and even preferred arsenate. Both arsenate and sulfate reduction were inhibited by molybdate. Strain OREX-4, a gram-positive bacterium with a hexagonal S-layer on its cell wall, metabolized compounds commonly used by sulfate reducers. Scorodite (FeAsO₄2· H₂O) an arsenate-containing mineral, provided micromolar concentrations of arsenate that supported cell growth. Physiologically and phylogenetically, strain OREX-4 was far-removed from strains MIT-13 and SES-3: strain OREX-4 grew on different electron donors and electron acceptors, and fell within the gram-positive group of the Bacteria, whereas MIT-13 and SES-3 fell together in the ɛ-subdivision of the Proteobacteria. Together, these results suggest that organisms spread among diverse bacterial phyla can use arsenate as a terminal electron acceptor, and that dissimilatory arsenate reduction might occur in the sulfidogenic zone at arsenate concentrations of environmental interest. 16S rRNA sequence analysis indicated that strain OREX-4 is a new species of the genus Desulfotomaculum, and accordingly, the name Desulfotomaculum auripigmentum is proposed. Received: 22 October 1997 / Accepted: 16 June 1997     

Influence of oxygen on sulfate reduction and growth of sulfate-reducing bacteria

The ambivalent relations of sulfate-reducing bacteria to molecular O₂ have been studied with ten freshwater and marine strains. Generally, O₂ was reduced prior to sulfur compounds and suppressed the reduction of sulfate, sulfite or thiosulfate to sulfide. Three strains slowly formed sulfide at O₂ concentrations of below 15 M (6% air saturation). In homogeneously aerated cultures, two out of seven strains tested, Desulfovibrio desulfuricans and Desulfobacterium autotrophicum, revealed weak growth with O₂ as electron acceptor (up to one doubling of protein). However, O₂ was concomitantly toxic. Depending on its concentration cell viability and motility decreased with time. In artificial oxygen-sulfide gradients with sulfide-containing agar medium and also in sulfide-free agar medium under an oxygen-containing gas phase, sulfate reducers grew in bands close to the oxic/anoxic interface. The specific O₂ tolerance and respiration capacity of different strains led to characteristically stratified gradients. The maximum O₂ concentration at the surface of a bacterial band (determined by means of microelectrodes) was 9 M. The specific rates of O₂ uptake per cell were in the same order of magnitude as the sulfate reduction rates in pure cultures. The bacteria stabilized the gradients, which were rapidly oxidized in the absence of cells or after killing the cells by formaldehyde. The motile strain Desulfovibrio desulfuricans CSN slowly migrated in the gradients in response to changing O₂ concentrations in the gas phase.     

Sulfate reduction from phosphogypsum using a mixed culture of sulfate-reducing bacteria

Phosphogypsum (CaSO₄), a primary by-product of phosphoric acid production, is accumulated in large stockpiles and occupies vast areas of land. It poses a severe threat to the quality of water and land in countries producing phosphoric acid. In this study, the potential of sulfate-reducing bacteria for biodegradation of this sulfur-rich industrial solid waste was assessed. The effect of phosphogypsum concentration, carbon and nitrogen sources, temperature, pH and stirring on the growth of sulfate-reducing bacteria was investigated. Growth of sulfate-reducing bacteria was monitored by measuring sulfide production. Phosphogypsum was shown to be a good source of sulfate, albeit that the addition of organic carbon was necessary for bacterial growth. Biogenic sulfide production occurred with phosphogypsum up to a concentration of 40 g L⁻¹, above which no growth of sulfate-reducing bacteria was observed. Optimal growth was obtained at 10 g L⁻¹ phosphogypsum. Both the gas mixture H₂/CO₂ and lactate supported high amounts of H₂S formation (19 and 11 mM, respectively). The best source of nitrogen for sulfate-reducing bacteria was yeast extract, followed by ammonium chloride. The presence of nitrate had an inhibitory effect on the process of sulfate reduction. Stirring the culture at 150 rpm slightly stimulated H₂S formation, probably by improving sulfate solubility.     

Kinetics of sulfate respiration by free-living and particle-associated sulfate-reducing bacteria

Abstract Effects on sulfate respiration of association of sulfate-reducing bacteria (SRB) with solid particles (anion exchange resin and FeS-precipitate) were examined using Desulfovibrio desulfuricans . The rates of sulfide production by resin- and FeS-associated cells were 2–3% and 19–56% of that by free-living ones, respectively, under sulfate- and lactate-rich conditions. On the other hand, under sulfate-poor (less than 50 μM) and lactate-rich conditions the rate by FeS-associated cells was higher than that by free-living ones. The values of K _m (μM), half saturation constant of the Michaelis-Menten model, for sulfate were 244 for free-living cells, 8.96 for resin-associated ones and 8.42 for FeS-associated ones. Under lactate-poor and sulfate-rich conditions the rate by FeS-associated cells was similar to that by free-living ones. These results suggest that FeS-associated SRB are more advantageous than free-living ones under sulfate-poor environments such as freshwater sediments.     

Development of a downstream process for the isolation of Staphylococcus aureus arsenate reductase overproduced in Escherichia coli

Arsenate reductase (ArsC) encoded by Staphylococcus aureus arsenic-resistance plasmid pI258 reduces intracellular As(V) (arsenate) to the more toxic As(III) (arsenite). In order to study the structure of ArsC and to unravel biochemical and physical properties of this redox enzyme, wild type enzyme and a number of cysteine mutants were overproduced soluble in Escherichia coli. In this paper we describe a novel purification method to obtain high production levels of highly pure enzyme. A reversed-phase method was developed to separate and analyze the many different forms of ArsC. The oxidation state and the methionine oxidized forms were determined by mass spectroscopy.     

Mineralization of monofluorobenzoate by a diculture under sulfate-reducing conditions

Abstract A mesophilic, dehalogenating, sulfate-reducing diculture was isolated from an anaerobic lake sediment. One strain of the diculture is proposed to be an endospore-forming Desulfotomaculum species, the second strain was a vibrioid, motile and non-sporeforming species which is tentatively assigned to the genus Desulfovibrio . The diculture was able to mineralize 4- and 2-fluorobenzoate both isomers being incompletely oxidized with the release of acetate, which was subsequently used by both sulfate-reducing strains. Other electron donors used for growth included benzoate, 3- and 4-hydroxybenzoate, protocatechuate, catechol, phenol, 2,5-dimethoxyphenol, fatty acids up to C₈, malate and pyruvate. The culture obtained from a freshwater habitat grew optimally at NaCl concentrations of 0.3–0.5 g 1⁻¹, 33–37°C, and pH 7.4. Our experiments showed that certain fluorinated aromatic hydrocarbons could serve as sole sources of carbon and energy for sulfate-reducing bacteria.     

Characterization of polycyclic aromatic hydrocarbons degradation and arsenate reduction by a versatile Pseudomonas isolate

A Pseudomonas isolate, designated PAHAs-1, was found capable of reducing arsenate and degrading polycyclic aromatic hydrocarbons (PAHs) independently and simultaneously. This isolate completely reduced 1.5 mM arsenate within 48 h and removed approximately 100% and 50% of 60 mg l⁻¹ phenanthrene and 20 mg l⁻¹ pyrene within 60 h, respectively. Using PAHs as the sole carbon source, however, this isolate showed a slow arsenate reduction rate (4.62 μM h⁻¹). The presence of arsenic affected cell growth and concurrent PAHs removal, depending on PAH species and arsenic concentration. Adding sodium lactate to the medium greatly enhanced the arsenate reduction and pyrene metabolism. The presence of the alpha subunit of the aromatic ring-hydroxylating dioxygenase (ARHD) gene, arsenate reductase (arsC) and arsenite transporter (ACR3(2)) genes supported the dual function of the isolate. The finding of latter two genes indicated that PAHAs-1 possibly reduced arsenate via the known detoxification mechanism. Preliminary data from hydroponic experiment showed that PAHAs-1 degraded the majority of phenanthrene (>60%) and enhanced arsenic uptake by Pteris vittata L. (from 246.7 to 1187.4 mg kg⁻¹ As in the fronds). The versatile isolate PAHAs-1 may have potentials in improving the bioremediation of PAHs and arsenic co-contamination using the plant-microbe integrated strategy.     

Characterization of Desulfomicrobium salsuginis sp. nov. and Desulfomicrobium aestuarii sp. nov., two new sulfate-reducing bacteria isolated from the Adour estuary (French Atlantic coast) with specific mercury methylation potentials

Three strains of sulfate-reducing bacteria (ADR21, ADR26 and ADR28) were isolated from Adour estuary sediments (French South Atlantic coast). Cells of these isolates were rod-shaped, motile and stained Gram-negative. The 16S rRNA and dsrAB genes sequence analyses indicated that these three strains belonged to the genus Desulfomicrobium within the delta Proteobacteria, with Desulfomicrobium escambiense strain DSM10707^T as their closest relative. According to phenotypic characteristics, strains ADR21 and ADR28 could be considered as members of the same species. The relatedness values, based on DNA–DNA hybridization studies, between strains ADR21/DSM10707^T, ADR26/DSM10707^T and ADR21/ADR26 ranged between 30.6–40.8%, 45.2–43.0% and 19.0–26.4%, respectively. Strains ADR21 and ADR28 grew well on lactate, fumarate, malate, formate, ethanol and H₂/acetate in the presence of sulfate as an electron acceptor. Thiosulfate, nitrate, fumarate and DMSO were alternative electron acceptors. Malate was well fermented but pyruvate and fumarate only poorly. Strain ADR26 could not grow on ethanol or fumarate and was unable to use DMSO or fumarate as electron acceptors. The three new strains exhibited differences compared to the type strain of D. escambiense, such as temperature optima, substrate utilization and mercury methylation capacities. On the basis of both genetic and phenotypic evidences, strain ADR21 is proposed as the type strain of the species Desulfomicrobium salsuginis sp. nov., and strain ADR26 as the type strain of the species Desulfomicrobium aestuarii sp. nov.     

Growth, incidence and activities of dissimilatory sulfate-reducing bacteria in the human oral cavity

Abstract Viable counts and activities of sulfate-reducing bacteria were determined in the oral cavities of 12 healthy volunteers. Of these, 10 harboured viable sulfate-reducing bacteria populations. Six separate sites were sampled: the posterior tongue, anterior tongue, mid buccal mucosa, vestibular mucosa, supragingival plaque and subgingival plaque. Sulfate-reducing bacteria occurred in all areas, with the highest incidence in supragingival plaque. Viable counts and sulfate-reducing activities in each of the regions varied from 0 to 10⁸ cfu (g wet weight)⁻¹ and from 0 to 50 nmol (g wet weight) ⁻¹ h⁻¹, respectively. As sulfate-reducing bacteria can be detected in the oral cavity, they may potentially be involved in terminal oxidative processes carried out by the microflora of the mouth.     

Characterization of a new thermophilic sulfate-reducing bacterium

A thermophilic sulfate-reducing vibrio isolated from thermal vent water in Yellowstone Lake, Wyoming, USA is described. The gram-negative, curved rod-shaped cells averaged 0.3 m wide and 1.5 m long. They were motile by means of a single polar flagellum. Growth was observed between 40° and 70 °C with optimal growth at 65 °C. Cultures remained viable for one year at 27 °C although spore-formation was not observed. Sulfate, thiosulfate and sulfite were used as electron acceptors. Sulfur, fumarate and nitrate were not reduced. In the presence of sulfate, growth was observed only with lactate, pyruvate, hydrogen plus acetate, or formate plus acetate. Pyruvate was the only compound observed to support fermentative growth. Pyruvate and lactate were oxidized to acetate. Desulfofuscidin and c-type cytochromes were present. The G+C content was 29.5 mol%. The divergence in the 16S ribosomal RNA sequences between the new isolate and Thermodesulfobacterium commune suggests that these two thermophilic sulfate-reducing bacteria represent different genera. These two bacteria depict a lineage that branches deeply within the Bacteria domain and which is clearly distinct from previously defined phylogenetic lines of sulfate-reducing bacteria. Strain YP87 is described as the type strain of the new genus and species Thermodesulfovibrio yellowstonii. Yellowstone Lake (Wyoming, USA) is located within one of the most tectonically active regions in the world (Klump et al. 1988; Remsen et al. 1990). Hydrothermal springs, hot gas fumaroles and elevated substrata temperatures have been observed within the lake itself (e.g., Remsen et al. 1990). Hydrothermal vent waters were reported to be anoxic, high in dissolved nutrients relative to the lake water and to have temperatures in excess of 80 °C (Klump et al. 1988; Remsen et al. 1990). Sulfate concentrations averaged 380 M in vent waters and 80 M in bulk lake water (Klump et al. 1988; Remsen et al. 1990). On the basis of on these physical and chemical characteristics, and the observation (e.g., Zeikus et al. 1983) that microbial sulfate reduction is prevalent in the thermal aquatic environments of Yellowstone National Park, we hypothesized that hydrothermal vent waters in Yellowstone Lake could support the growth of thermophilic sulfate reducers.Here we describe the general characteristics of a new thermophilic sulfate reducing bacterium, Thermodesulfovibrio yellowstonii, which was isolated from hydrothermal vent water in Sedge Bay of Yellowstone Lake, Wyoming, USA. In addition, we report on the phylogenetic relationship of this new isolate with other thermophilic and mesophilic sulfate-reducing bacteria.Dedicated to the memory of Friedhelm Bak     

Isolation and characterization of Desulfovibrio senezii sp. nov., a halotolerant sulfate reducer from a solar saltern and phylogenetic confirmation of Desulfovibrio fructosovorans as a new species

A new halotolerant Desulfovibrio, strain CVL^T (T = type strain), was isolated from a solar saltern in California. The curved, gram-negative, nonsporeforming cells (0.3 × 1.0–1.3 μm) occurred singly, in pairs, or in chains, were motile by a single polar flagellum and tolerated up to 12.5% NaCl. Strain CVL^T had a generation time of 60 min when grown in lactate-yeast extract medium under optimal conditions (37°C, pH 7.6, 2.5% NaCl). It used lactate, pyruvate, cysteine, or H₂/CO₂ + acetate as electron donors, and sulfate, sulfite, thiosulfate, or fumarate as electron acceptors. Elemental sulfur, nitrate, or oxygen were not used. Sulfite and thiosulfate were disproportionated to sulfate and sulfide. The G+C content of the DNA was 62 mol%. Phylogenetic analysis revealed that Desulfovibrio fructosovorans was the nearest relative. Strain CVL^T is clearly different from other Desulfovibrio species, and is designated Desulfovibrio senezii sp. nov. (DSM 8436). Received: 27 February 1998 / Accepted: 15 June 1998     

Oxidation of H2, organic compounds and inorganic sulfur compounds coupled to reduction of O2 or nitrate by sulfate-reducing bacteria

All of fourteen sulfate-reducing bacteria tested were able to carry out aerobic respiration with at least one of the following electron donors: H₂, lactate, pyruvate, formate, acetate, butyrate, ethanol, sulfide, thiosulfate, sulfite. Generally, we did not obtain growth with O₂ as electron acceptor. The bacteria were microaerophilic, since the respiration rates increased with decreasing O₂ concentrations or ceased after repeated O₂ additions. The amounts of O₂ consumed indicated that the organic substrates were oxidized incompletely to acetate; only Desulfobacter postgatei oxidized acetate with O₂ completely to CO₂. Many of the strains oxidized sulfite (completely to sulfate) or sulfide (incompletely, except Desulfobulbus propionicus); thiosulfate was oxidized only by strains of Desulfovibrio desulfuricans; trithionate and tetrathionate were not oxidized by any of the strains. With Desulfovibrio desulfuricans CSN and Desulfobulbus propionicus the oxidation of inorganic sulfur compounds was characterized in detail. D. desulfuricans formed sulfate during oxidation of sulfite, thiosulfate or elemental sulfur prepared from polysulfide. D. propionicus oxidized sulfite and sulfide to sulfate, and elemental sulfur mainly to thiosulfate. A novel pathway that couples the sulfur and nitrogen cycles was detected: D. desulfuricans and (only with nitrite) D. propionicus were able to completely oxidize sulfide coupled to the reduction of nitrate or nitrite to ammonia. Cell-free extracts of both strains did not oxidize sulfide or thiosulfate, but formed ATP during oxidation of sulfite (37 nmol per 100 nmol sulfite). This, and the effects of AMP, pyrophosphate and molybdate on sulfite oxidation, suggested that sulfate is formed via the (reversed) sulfate activation pathway (involving APS reductase and ATP sulfurylase). Thiosulfate oxidation with O₂ probably required a reductive first step, since it was obtained only with energized intact cells.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - APS adenosine phosphosulfate or adenylyl sulfate     

Anaerobic degradation of 1,3-propanediol by sulfate-reducing and by fermenting bacteria

Three strains of strictly anaerobic Gram-negative, non-sporeforming, motile bacteria were enriched and isolated from freshwater sediments with 1,3-propanediol as sole energy and carbon source. Strain OttPdl was a sulfate-reducing bacterium which grew also with lactate, ethanol, propanol, butanol, 1,4-butanediol, formate or hydrogen plus CO₂, the latter only in the presence of acetate. In the absence of sulfate, most of these substrates were fermented to the respective fatty acids in syntrophic cooperation with Methanospirillum hungatei. Sulfur, thiosulfate, or sulfite were reduced, nitrate not. The other two isolates degraded propanediol only in coculture with Methanospirillum hungatei. Strain OttGlycl grew in pure culture with acetoin and with glycerol in the presence of acetate. Strain WoAcl grew in pure culture only with acetoin. Both strains did not grow with other substrates, and did not reduce nitrate, sulfate, sulfur, thiosulfate or sulfite. The isolates were affiliated with the genera Desulfovibrio and Pelobacter. The pathways of propanediol degradation and the ecological importance of this process are discussed.     

The role of polyglucose in oxygen-dependent respiration by a new strain of Desulfovibrio salexigens

Abstract: Desulfovibrio salexigens strain Mastl was isolated from the oxic/anoxic interface of a marine sediment. Growth under sulfate-reducing conditions was accompanied by polyglucose accumulation in the cell with every substrate tested. Highest polyglucose storage was found with glucose (0.8–1.0 g polyglucose (g protein)⁻¹), but the growth rate with this substrate was very low (0.015 h⁻¹). Anaerobically grown cells of strain Mastl exhibited immediate oxygen-dependent respiration. The endogenous oxygen reduction rate was proportional to the polyglucose content. The rate of aerobic respiration of pyruvate was also directly related to the polyglucose content indicating that this organism was only able to respire with oxygen as long as polyglucose was present. Maximum oxygen reduction rates were found at air saturating concentrations and were relatively low (3–50 nmol O₂ min⁻¹ (mg protein)⁻¹). Catalase was constitutively present in anaerobically grown cells. When batch cultures were exposed to oxygen, growth ceased immediately and polyglucose was oxidized to acetate within 40–50 h. Like the oxygen reduction activity, the nitro blue tetrazolium (NBT)-reduction activity in these cells was proportional to the polyglucose content. Under anaerobic starvation conditions there was no correlation between the NBT-reduction activity and polyglucose concentration and polyglucose was degraded slowly within 240 h. The ecological significance of aerobic polyglucose consumption is discussed.     

The relationship between the in situ reduction level of the cytochrome c pool of Azorhizobium caulinodans growing in a chemostat with NH4+ or N2 as the N source and the total activity of cytochrome c oxidases

Abstract The in situ method for determination of reduction levels of cytochromes b and c pools during steady-state growth (Pronk et al., Anal. Biochem. 214, 149–155, 1993) was applied to chemostat cultures of the wild-type, a cytochrome aa₃ single mutant and a cytochrome aa₃/d double mutant of Azorhizobium caulinodans . For growth with NH₄⁺ as the N source, the results indicate that (i) the aa₃ mutant strains growing at a dissolved O₂ tension of 0.5% possess an active alternative cytochrome c oxidase, which is hardly present during fully aerobic growth, and assuming that (i) also pertains to the wild-type, (ii) the wild-type uses cytochrome aa₃ under fully aerobic conditions. For growth with N₂ as the N source, it was found that the aa₃ mutant strains growing at dissolved O₂ tensions ranging from 0.5 to 3.0% also contain an active alternative cytochrome c oxidase.     

Influence of clay particles (Illite) on substrate utilization by sulfate-reducing bacteria

The presence of calcium-or iron-saturated illite had a positive effect on the conversion of ethanol and acetate by non-starved cultures of Desulfobacter postgatei D.A41, but had no effect on non-starved cultures of Desulfobulbus propionicus Lindhorst and Sesulfovibrio baculatus H.L21. Starvation of these cultures at room temperature induced adhesion of cells of D. baculatus H.L21 to the surface of the clay particles. No adhesion of cells of D. propionicus Lindhorst and D. postgatei D.A41 was ever observed. However, for the three strains studied, the presence of clay particles had a positive effect on conservation of the oxidative capacity of the cultures during starvation.     

不同pH值条件下硫酸盐还原菌组成及硫酸盐还原机制分析

【目的】从海洋沉积物中富集获得硫酸盐还原菌群,改变pH值进行培养,分析pH值对硫酸盐还原性质的影响,明确菌群组成和进行硫酸盐还原功能基因预测,探究硫酸盐还原机制。【方法】分析硫酸盐还原菌群在不同pH值条件下的硫酸盐还原率,在此基础上,利用高通量测序技术和PICRUSt软件分析硫酸盐还原菌群优势菌组成及硫酸盐还原相关基因相对丰度。【结果】硫酸盐还原菌群在不同pH值培养条件下的生长和硫酸盐还原率出现显著变化(P<0.01),在pH 5.0时达到峰值,分别为0.34±0.01和96.52%±0.44%。高通量测序数据显示,pH 5.0时菌群丰富度和多样性最高,优势菌属为假单胞菌(Pseudomonas)和芽孢杆菌(Bacillus),相对丰度较高的基因为同化性硫酸盐还原相关基因。【结论】硫酸盐还原菌富集生长的最适pH 5.0,在此条件下的高硫酸盐还原率由同化性硫酸盐还原途径主导,为揭示硫酸盐还原机制提供了实验支持,并拓宽了硫酸盐还原菌实践应用方面的种质资源。     

Na+-dependent accumulation of sulfate and thiosulfate in marine sulfate-reducing bacteria

Uptake of ³⁵S-labelled sulfate and thiosulfate was studied in twenty sulfate-reducing bacteria. Micromolar additions of these substrates were highly accumulated by washed cells of freshwater and marine strains. In marine strains accumulation required Na⁺. Generally, the uptake capacity was increased after sulfate limitation during growth. With two marine species, Desulfovibrio salexigens and Desulfobacterium autotrophicum, the effects of various ionophores and inhibitors affecting the transmembrane pH or Na⁺ gradient or the membrane potential were studied. In both strains transport was reversible. There was no discrimination between sulfate and thiosulfate. With increasing additions the amount taken up increased, while the accumulation factor (C_in/C_out) decreased. Uptake was not directly correlated with the ATP level inside the cells. From these results and the action patterns of the inhibitors tested it is concluded that marine sulfate-reducing bacteria accumulate sulfate and thiosulfate electrogenically in symport with Na⁺ ions, while in freshwater strains protons are symported. The high-accumulating systems are induced only at low sulfate concentration, while low-accumulating systems are active at sulfate-sufficient conditions.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DCCD dicyclohexylcarbodiimide - ETH 157 N, N-dibenzyl-N,N-diphenyl-1,2-diphenylendioxydiacetamide - TCS 3,3,4,5-tetrachlorosalicylanilide