Sulfidogenesis from 2-Aminoethanesulfonate (Taurine) Fermentation by a Morphologically Unusual Sulfate-Reducing Bacterium, Desulforhopalus singaporensis sp. nov.

A pure culture of an obligately anaerobic marine bacterium was obtained from an anaerobic enrichment culture in which taurine (2-aminoethanesulfonate) was the sole source of carbon, energy, and nitrogen. Taurine fermentation resulted in acetate, ammonia, and sulfide as end products. Other sulfonates, including 2-hydroxyethanesulfonate (isethionate) and cysteate (alanine-3-sulfonate), were not fermented. When malate was the sole source of carbon and energy, the bacterium reduced sulfate, sulfite, thiosulfate, or nitrate (reduced to ammonia) but did not use fumarate or dimethyl sulfoxide as a terminal electron acceptor for growth. Taurine-grown cells had significantly lower adenylylphosphosulfate reductase activities than sulfate-grown cells had, which was consistent with the notion that sulfate was not released as a result of oxidative C-S bond cleavage and then assimilated. The name Desulforhopalus singaporensis is proposed for this sulfate-reducing bacterium, which is morphologically unusual compared to the previously described sulfate-reducing bacteria by virtue of the spinae present on the rod-shaped, gram-negative, nonmotile cells; endospore formation was not discerned, nor was desulfoviridin detected. Granules of poly-β-hydroxybutyrate were abundant in taurine-grown cells. This organism shares with the other member of the genus Desulforhopalus which has been described a unique 13-base deletion in the 16S ribosomal DNA. It differs in several ways from a recently described endospore-forming anaerobe (K. Denger, H. Laue, and A. M. Cook, Arch. Microbiol. 168:297–301, 1997) that reportedly produces thiosulfate but not sulfide from taurine fermentation. D. singaporensis thus appears to be the first example of an organism which exhibits sulfidogenesis during taurine fermentation. Implications for sulfonate sulfur in the sulfur cycle are discussed.     

A combined pathway of sulfur compound disproportionation in Desulfovibrio desulfuricans

The fates of the two different sulfur atoms of the thiosulfate molecule during anaerobic disproportionation by the sulfate-reducing bacterium Desulfovibrio desulfuricans were followed by isotope mass spectrometry. During disproportionation, ³²S-thiosulfate was preferentially metabolized, and the residual thiosulfate became enriched in ³⁴S. The sulfate formed was isotopically heavier than the inner sulfur of the consumed thiosulfate. Vice versa, the sulfide formed was isotopically lighter than the outer sulfur of the consumed thiosulfate. These results indicate that thiosulfate is cleaved to intermediates that undergo further disproportionation to sulfate and sulfide in a second step. These intermediates are probably elemental sulfur and sulfite. It is concluded that disproportionation of thiosulfate, sulfite and elemental sulfur includes a combined pathway.     

Fermentation of cysteate by a sulfate-reducing bacterium

We isolated a strictly anaerobic bacterium, strain GRZCYSA, from a sludge digestor for its ability to ferment cysteate (2-amino-3-sulfopropionate). The organism also fermented the organosulfonates isethionate (2-hydroxyethanesulfonate) and aminomethanesulfonate, but taurine (2-aminoethanesulfonate) was not a substrate. Strain GRZCYSA, a gram-negative, oxidase-negative and catalase-positive vibrio that could reduce sulfate and contained desulfoviridin, was tentatively identified as Desulfovibrio sp. Utilization of cysteate as a substrate for fermentative growth led to the formation of four products identified as acetate, ammonia, and equimolar amounts of sulfide and sulfate. The fermentation was in balance. Some reactions involved in this novel process were detected in cell-free extracts in which ammonia and acetate were formed from cysteate. Received: 10 March 1997 / Accepted: 14 May 1997     

Oxygen-dependent growth of the sulfate-reducing bacterium Desulfovibrio oxyclinae in coculture with Marinobacter sp. Strain MB in an aerated sulfate-depleted chemostat

A chemostat coculture of the sulfate-reducing bacterium Desulfovibrio oxyclinae and the facultatively aerobic heterotroph Marinobacter sp. strain MB was grown for 1 week under anaerobic conditions at a dilution rate of 0.05 h(-1). It was then exposed to an oxygen flux of 223 micromol min(-1) by gassing the growth vessel with 5% O(2). Sulfate reduction persisted under these conditions, though the amount of sulfate reduced decreased by 45% compared to the amount reduced during the initial anaerobic mode. After 1 week of growth under these conditions, sulfate was excluded from the incoming medium. The sulfate concentration in the growth vessel decreased exponentially from 4.1 mM to 2.5 microM. The coculture consumed oxygen effectively, and no residual oxygen was detected during either growth mode in which oxygen was supplied. The proportion of D. oxyclinae cells in the coculture as determined by in situ hybridization decreased from 86% under anaerobic conditions to 70% in the microaerobic sulfate-reducing mode and 34% in the microaerobic sulfate-depleted mode. As determined by the most-probable-number (MPN) method, the numbers of viable D. oxyclinae cells during the two microaerobic growth modes decreased compared to the numbers during the anaerobic growth mode. However, there was no significant difference between the MPN values for the two modes when oxygen was supplied. The patterns of consumption of electron donors and acceptors suggested that when oxygen was supplied in the absence of sulfate and thiosulfate, D. oxyclinae performed incomplete aerobic oxidation of lactate to acetate. This is the first observation of oxygen-dependent growth of a sulfate-reducing bacterium in the absence of either sulfate or thiosulfate. Cells harvested during the microaerobic sulfate-depleted stage and exposed to sulfate and thiosulfate in a respiration chamber were capable of anaerobic sulfate and thiosulfate reduction.     

Ectothiorhodospira mobilis Pelsh, a Photosynthetic Sulfur Bacterium Depositing Sulfur Outside the Cells

From salt flats on the Galapagos Islands, two strains of a red photosynthetic bacterium were isolated and identified as Ectothiorhodospira mobilis, an organism first described by Pelsh in 1937. The cells are curved in a short spiral, 0.7 to 1.0 mu wide and 2.0 to 4.8 mu long. They are motile by a polar tuft of flagella. Cells contain several large stacks of lamellar membranes, carrying the pigments bacteriochlorophyll a and carotenoids of the spirillo xanthin series. Cell division occurs by binary fission, not budding. The organism is strictly anaerobic and obligately photosynthetic. Its ability to grow well with sulfide, sulfur, thiosulfate, or sulfite as photosynthetic H donors puts it taxonomically in the Thiorhodaceae. During growth with sulfide, elementary sulfur is deposited outside the cells in the medium and disappears during further growth. A limited number of organic carbon compounds can be utilized as hydrogen donors in place of inorganic sulfur compounds. Under these conditions, sulfate can serve as the sulfur source. The enzymes catalase and hydrogenase are present. The newly isolated strains require vitamin B(12). They also require a salinity of 2 to 3% NaCl, but they are not extreme halophiles. The organism is not identical with any of the species listed in Bergey's Manual.     

Nutritional studies with Pseudomonas aeruginosa grown on inorganic sulfur sources.

Pseudomonas aeruginosa was grown on a succinate-basal salts medium supplemented with various inorganic sulfur compounds as its sole source of sulfur. The organism was able to grow on the sodium salts of sulfide, thiosulfate, tetrathionate, dithionite, metabisulfite, sulfite, or sulfate, but not on those of dithionate. Analyses of the culture media after 24 h of growth indicated accumulation of sulfate from each inorganic sulfur source except sulfate. Manometric studies with resting cells obtained by growth on each of these sulfur sources yielded net oxygen uptake for all substrates except sulfite and dithionate. Similar results were obtained with extracts from these cells by spectrophotometric techniques. Thiosulfate oxidase activity appeared to be induced by growth on sulfide, thiosulfate, or tetrathionate, with little or no activity observed when cells were grown on inorganic sulfur sources of higher oxidative states. Metabisulfite oxidase appeared to be associated with growth on all inorganic sulfur compounds. Rhodanese activity appeared to be constitutively present, and its activity, observed only in soluble fraction, seemed independent of the growth medium employed. Thiosulfate and tetrathionate oxidase activities were studied in greater detail than some of the other sulfur oxidases, and both were found to be distributed between particulate and soluble fractions.     

Disproportionation of inorganic sulfur compounds by the sulfate-reducing bacterium Desulfocapsa thiozymogenes gen. nov., sp. nov.

A new strictly anaerobic, gram-negative bacterium was isolated from the sediment of a freshwater lake after enrichment with thiosulfate as the energy source. The strain, named Bra2 (DSM 7269), is able to grow by disproportionation of thiosulfate or sulfite to sulfate plus sulfide. Elemental sulfur is also disproportionated to sulfate and sulfide, but this only supports growth if free sulfide is chemically removed from the culture, e.g., by precipitation with amorphous ferric hydroxide. Growth is also possible by coupling the reduction of sulfate to sulfide with the oxidation of ethanol, propanol, or butanol to the corresponding fatty acid. The cells are rod-shaped, motile, and have genomic DNA with a mol% G+C content of 50.7. Cytochromes are present, but desulfoviridin is not. The new strain was shown to be related to, but distinct from members of the genus Desulfobulbus on the basis of physiological characteristics and by comparative sequence analysis of its 16S rDNA. Strain Bra2 is described as the type strain of a new taxon, Desulfocapsa thiozymogenes gen. nov., sp. nov. Received: 29 January 1996 / Accepted: 31 May 1996     

Isolation and characterization of an anaerobic benzoate-degrading spore-forming sulfate-reducing bacterium, Desulfotomaculum sapomandens sp. nov.

Abstract Spore-forming sulfate-reducing bacteria (SRB) were enriched selectively from various kinds of aerobic soils with fatty acids as the sole carbon and energy source. A Gram-negative motile rod-shaped bacterium, which produced gas vacuoles during sporulation was isolated. It degraded alcohols, aromatic and n-fatty acids (up to C₁₈) except for propionate, completely to CO₂. Sulfate, sulfite, thiosulfate or elemental sulfur served as electron acceptors. Because of its sensitivity to H₂S, the isolate never produced more than 8 mM dissolved sulfide at pH 7.0. G + C-content of the DNA was 48.0 mol %. The isolated strain Pato is described as a new species Desulfotomaculum sapomandens .     

Microbial Recovery of Sulfur from Thiosulfate-Bearing Wastewater with Phototrophic and Sulfur-Reducing Bacteria

The spent caustic wastewater from the oxidation of sulfide present in offshore natural gas production mainly comprises thiosulfate and sulfate. A biocatalytic process, employing phototrophic green sulfur bacteria in symbiosis with sulfate-reducing bacteria, is described in this paper for the production of sulfur from the spent caustic wastewater, with synthetic wastewater as the model system. The process entails the conversion of thiosulfate to sulfur and sulfate by photosynthetic green sulfur bacteria Chlorobium vibrioforme f. thiosulfatophilum. Sulfate formed in turn is removed by Desulfovibrio desulfuricans to sulfide, which is further converted to sulfur by Chlorobium limicola through photooxidation. Sulfide is also oxidized to sulfur and sulfate via thiosulfate as an intermediate by Chlorobium vibrioforme f. thiosulfatophilum.     

A new ecological adaptation to high sulfide by a Hydrogenobacter sp. growing on sulfur compounds but not on hydrogen

Thermophilic bacteria were isolated from a sulfide-rich, neutral hot spring in Iceland on gelrite minimal medium with 16 mM thiosulfate. The isolates were aerobic, obligate chemolithoautotrophs and used thiosulfate and sulfur as electron donors, producing sulfate from both substrates. No growth was observed with hydrogen as the sole electron donor, and no hydrogenase activity was detected. The cells were gram-negative and usually single, 4-5 microm long and 0.7 microm in diameter and formed sulfur globules after a few days of incubation. By SSU rRNA sequence comparisons, the bacterium was placed in the genus Hydrogenobacter with the closest relative to be Calderobacterium hydrogenophilum with 98.3% sequence similarity. This novel bacterium shows an ecological adaptation to high sulfide springs and is differentiated from its closest known relatives by lack of H2 oxidation, deposition of sulfur and lower growth temperature.     

Denitrification at extremely high pH values by the alkaliphilic, obligately chemolithoautotrophic, sulfur-oxidizing bacterium Thioalkalivibrio denitrificans strain ALJD

Thioalkalivibrio denitrificans is the first example of an alkaliphilic, obligately autotrophic, sulfur-oxidizing bacterium able to grow anaerobically by denitrification. It was isolated from a Kenyan soda lake with thiosulfate as electron donor and N2O as electron acceptor at pH 10. The bacterium can use nitrite and N2O, but not nitrate, as electron acceptors during anaerobic growth on reduced sulfur compounds. Nitrate is only utilized as nitrogen source. In batch culture at pH 10, rapid growth was observed on N2O as electron acceptor and thiosulfate as electron donor. Growth on nitrite was only possible after prolonged adaptation of the culture to increasing nitrite concentrations. In aerobic thiosulfate-limited chemostats, Thioalkalivibrio denitrificans strain ALJD was able to grow between pH values of 7.5 and 10.5 with an optimum at pH 9.0. Growth of the organism in continuous culture on N2O was more stable and faster than in aerobic cultures. The pH limit for growth on N2O was 10.6. In nitrite-limited chemostat culture, growth was possible on thiosulfate at pH 10. Despite the observed inhibition of N2O reduction by sulfide, the bacterium was able to grow in sulfide-limited continuous culture with N2O as electron acceptor at pH 10. The highest anaerobic growth rate with N2O in continuous culture at pH 10 was observed with polysulfide (S8(2-)) as electron donor. Polysulfide was also the best substrate for oxygen-respiring cells. Washed cells at pH 10 oxidized polysulfide to sulfate via elemental sulfur in the presence of N2O or O2. In the absence of the electron acceptors, elemental sulfur was slowly reduced which resulted in regeneration of polysulfide. Cells of strain ALJD grown under anoxic conditions contained a soluble cd1-like cytochrome and a cytochrome-aa3-like component in the membranes.     

Anaerobic Desulfonation of 4-Tolylsulfonate and 2-(4-Sulfophenyl) Butyrate by a Clostridium sp

Alkyl- and arylsulfonates were tested as sole added sources of sulfur for the growth of enrichment cultures under strictly anaerobic denitrifying or fermentative conditions. Cultures that utilized taurine, ethylsulfonate, the dyestuffs orange II and acid red I, tolylsulfonate, 2-(4-sulfophenyl)butyrate (SPB), a dialkyltetralinesulfonate, and 1-(4-sulfophenyl)octane were readily obtained. We chose to work with the simple aromatic compounds and isolated a fermentative bacterium, strain EV4, which utilized SPB as the sole added source of sulfur in glucose-mineral medium. The organism was identified as a Clostridium sp. related to Clostridium beijerinckii. Clostridium sp. strain EV4 utilized seven of seven tested arylsulfonates quantitatively. The growth yield was about 3 kg of protein per mol of sulfur, whether sulfonate or sulfate was utilized. A major product specific to each sulfonate could be observed. Although no product was identified, the existence of anaerobic desulfonation has been established.     

Isotope effects associated with the anaerobic oxidation of sulfite and thiosulfate by the photosynthetic bacterium, Chromatium vinosum

Abstract The purple photosynthetic bacterium Chromatium vinosum , strain D, catalyzes several oxidations of reduced sulfur compounds under anaerobic conditions in the light: e.g., sulfide → sulfur → sulfate, sulfite → sulfate, and thiosulfate → sulfur + sulfate. Here it is shown that no sulfur isotope effect is associated with the last of these processes; isotopic compositions of the sulfur and sulfate produced can differ, however, if the sulfane and sulfonate positions within the thiosulfate have different isotopic compositions. In the second process, an observed change from an inverse to a normal isotope effect during oxidation of sulfite may indicate the operation of 2 enzymatic pathways. In contrast to heterotrophic anaerobic reduction of oxidized sulfur compounds, anaerobic oxidations of inorganic sulfur compounds by photosynthetic bacteria are characterized by relatively small isotope effects.     

Role of Thiosulfate in Bisulfite Reduction as Catalyzed by Desulfovibrio vulgaris

Studies with (35)S-labeled substrates were conducted to investigate the pathway involved in the reduction of sulfite to sulfide by cell-free extracts of the sulfate-reducing organism Desulfovibrio vulgaris. The results showed that accumulation of thiosulfate occurred when crude extracts were incubated under appropriate conditions with sulfite as substrate. With labeled sulfite as substrate, thiosulfate with equal distribution of radioactivity in both sulfur atoms was formed. When the rates of formation of (35)S(2-) from inner- and outer-labeled thiosulfate were compared, the rate of formation from outer-labeled thiosulfate was greater. Time studies with S-(35)SO(3) (2-) showed an increase of (35)S(2-) with time and an increasing ratio of doubly labeled to inner labeled thiosulfate remaining in the reaction mixture. From these studies it is concluded that thiosulfate is a stable intermediate formed from sulfite during the reduction of sulfate by D. vulgaris. Both sulfur atoms are derived from sulfite; during the utilization of thiosulfate, the outer sulfur is reduced to sulfide and the inner sulfur recycles through a sulfite pool.     

Oxygen-Dependent Growth of the Sulfate-Reducing Bacterium Desulfovibrio oxyclinae in Coculture with Marinobacter sp. Strain MB in an Aerated Sulfate-Depleted Chemostat

A chemostat coculture of the sulfate-reducing bacterium Desulfovibrio oxyclinae and the facultatively aerobic heterotroph Marinobacter sp. strain MB was grown for 1 week under anaerobic conditions at a dilution rate of 0.05 h⁻¹. It was then exposed to an oxygen flux of 223 μmol min⁻¹ by gassing the growth vessel with 5% O₂. Sulfate reduction persisted under these conditions, though the amount of sulfate reduced decreased by 45% compared to the amount reduced during the initial anaerobic mode. After 1 week of growth under these conditions, sulfate was excluded from the incoming medium. The sulfate concentration in the growth vessel decreased exponentially from 4.1 mM to 2.5 μM. The coculture consumed oxygen effectively, and no residual oxygen was detected during either growth mode in which oxygen was supplied. The proportion of D. oxyclinae cells in the coculture as determined by in situ hybridization decreased from 86% under anaerobic conditions to 70% in the microaerobic sulfate-reducing mode and 34% in the microaerobic sulfate-depleted mode. As determined by the most-probable-number (MPN) method, the numbers of viable D. oxyclinae cells during the two microaerobic growth modes decreased compared to the numbers during the anaerobic growth mode. However, there was no significant difference between the MPN values for the two modes when oxygen was supplied. The patterns of consumption of electron donors and acceptors suggested that when oxygen was supplied in the absence of sulfate and thiosulfate, D. oxyclinae performed incomplete aerobic oxidation of lactate to acetate. This is the first observation of oxygen-dependent growth of a sulfate-reducing bacterium in the absence of either sulfate or thiosulfate. Cells harvested during the microaerobic sulfate-depleted stage and exposed to sulfate and thiosulfate in a respiration chamber were capable of anaerobic sulfate and thiosulfate reduction.     

Anaerobic degradation of dimethylsulfoniopropionate to 3-S-methylmercaptopropionate by a marine Desulfobacterium strain

Dimethylsulfoniopropionate, an osmolyte of marine algae, is thought to be the major precursor of dimethyl sulfide, which plays a dominant role in biogenic sulfur emission. The marine sulfate-reducing bacterium Desulfobacterium strain PM4 was found to degrade dimethylsulfoniopropionate to 3-S-methylmercaptopropionate. The oxidation of one of the methyl groups of dimethylsulfoniopropionate was coupled to the reduction of sulfate; this process is similar to the degradation betaine to dimethylglycine which was described earlier for the same strain. Desulfobacterium PM4 is the first example of an anaerobic marine bacterium that is able to demethylate dimethylsulfoniopropionate.Abbreviations DMSP dimethylsulfoniopropionate - DMS dimethyl sulfide - MMPA 3-S-methylmercaptopropionate     

Sulfate-Reducing Bacterium with Unusual Morphology and Pigment Content

A dissimilatory sulfate-reducing bacterium was isolated which differed in morphology and pigment content from previously described species. The organism was mesophilic, obligately anaerobic, gram-negative, nonsporulating, long, and slender with one polar flagellum. Whole cells fluoresced red at neutral pH when excited with light at 365 nm owing to the presence of a pink pigment. Desulfoviridin was present. Reduced minus oxidized spectra of whole cells showed peaks in the position of a c-type cytochrome characteristic of Desulfovibrio species and peaks at about 629 and 603 nm. CO difference spectra showed the presence of a CO-binding pigment with a peak at 593 nm. Lactate and pyruvate supported growth in the presence of sulfate but not in its absence. Sulfate, sulfite, and thiosulfate served as electron acceptors for growth. Hydrogenase was present. The deoxyribonucleic acid had a buoyant density of 1.722 g/cm(3) and a guanosine plus cystosine molar percentage of total bases calculated by two different methods of 61.2 or 63.2.     

Desulfomonile tiedjei gen. nov. and sp. nov., a novel anaerobic,dehalogenating, sulfate-reducing bacterium

An anaerobic, dehalogenating, sulfate-reducing bacterium, strain DCB-1, is described and nutritionally characterized. The bacterium is a Gram-negative, nonmotile, non-sporeforming large rod with an unusual morphological feature which resembles a collar. The microorganism reductively dehalogenates meta substituted halobenzoates and also reduces sulfate, sulfite and thiosulfate as electron acceptors. The bacterium requires nicotinamide, 1,4-naphthoquinone and thiamine for optimal growth in a defined medium. The microorganism can grow autotrophically on H₂:CO₂ with sulfate or thiosulfate as terminal electron acceptors. It can also grow heterotrophically with pyruvate, several methoxybenzoates, formate plus sulfate or benzoate plus sulfate. It ferments pyruvate to acetate and lactate in the absence of other electron acceptors. The bacterium is inhibited by MoO _inf4 ^sup2- or SeO _inf4 ^sup2- as well as tetracycline, chloramphenicol, kanamycin or streptomycin. Cytochrome c₃ and desulfoviridin have been purified from cells grown in defined medium. 16S rRNA sequence analysis indicates the organism is a new genus of sulfate-reducing bacteria in the delta subdivision of the class Proteobacteria. We propose that the strain be named Desulfomonile tiedjei.Non-standard abbreviations PIPES piperazine-N,N-bis[2-ethanesulfonic acid] - MES 2-[N-morpholino]ethanesulfonic acid - TES N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid - HQNO 2-N-heptyl-4-hydroxy-quinoline-N-oxide - CCCP carbonyl-cyanide-m-chlorophenylhydrazine - CM carboxymethyl     

Isolation and characterization of a furfural degrading sulfate-reducing bacterium from an anaerobic digester

A sulfate-reducing bacterium (SRB) was isolated from a continuous anaerobic digester, which converted the furfural-containing wastewater to methane and CO₂. This SRB isolate could use furfural, furfuryl alcohol, and 2-furoic acid as sole source of carbon and energy in a defined mineral sulfate medium. Acetic acid was the major end product of furfural degradation. This organism also used wide varieties of other carbon sources, including ethanol, pyruvate, lactate, succinate, propanol, formate, and malate. The SRB isolate contained the electron carrier desulfoviridin. It used SO₄, NO₃, and thiosulfate as electron acceptors. This isolate used ammonium chloride, nitrate and glutamate as nitrogen source. The characteristics of the SRB isolate were closely similar toDesulfovibrio sp.     

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.