Sulfide-Dependent Methane Production and Growth of a Thermophilic Methanogenic Bacterium

Two strains of a thermophilic methanogenic bacterium, designated MT1 a and b, were isolated from a coastal surface sediment in the northern part of the Swedish West Coast. The two strains were identical in appearance and nutritional requirements and resembled Methanobacterium thermoautotrophicum. MT1 had an extraordinary requirement for sulfide. At sulfide levels below 0.1 mM, growth was poor and the methane production rate decreased. Other sources of sulfur, such as cysteine, sulfate, or thiosulfate, could not replace sulfide. The results indicate that a fast turnover rate of reduced sulfur compounds is involved in the energy metabolism of this organism.     

Sulfur requirement of Gonium (Volvocaceae)

The sulfur requirement of six strains of three species of Goniumhas been investigated. These strains can grow well with sulfide,sulfite, bisulfite, thiosulfate or sulfate in light and darkness.They are the first algae shown to utilize sulfide as a sulfursource. However, organic sulfur sources (methionine, cystine,cysteine, homocysteine, homocystine and taurine) were ineffectivefor growth of Gonium. (Received December 6, 1975; )     

Rhodopseudomonas sulfidophila,nov. spec., a new species of the purple nonsulfur bacteria

Summary From marine mud flats a new type of photosynthetic purple bacterium was isolated. This type is described as a new species of the Rhodospirillaceae and is named Rhodopseudomonas sulfidophila. The cells are rod-shaped, 0.6 to 0.9 wide and 0.9 to 2.0 long, and motile by means of polar flagella. Cell division occurs by binary fission. The photosynthetic membrane system is of the vesicular type. The pigments consist of bacteriochlorophyll a and of carotenoids, most probably of the spheroidene group. A wide range of organic compounds can be utilized anaerobically in the light. Growth on organic compounds aerobically in the dark is also possible. Niacin, thiamin, biotin and p-aminobenzoic acid are required as growth factors. The new species needs 2.5% (w/v) sodium chloride for optimal growth. All strains show excellent photolithotrophic growth on hydrogen, hydrogen sulfide, and thiosulfate. They show a remarkably high sulfide tolerance. Sulfide and thiosulfate are oxidized to sulfate without an intermediate accumulation of elemental sulfur. The new species seems to be one of the most versatile types of photosynthetic bacteria isolated thus far.     

Ecophysiology of lithotrophic sulfur-oxidizing <Emphasis Type="Italic">Sphaerotilus</Emphasis> species from sulfide springs in the Northern Caucasus

Six strains of sulfur-oxidizing bacteria of the known organotrophic species Sphaerotilus natans were isolated from two North Caucasian sulfide springs. Similar to known colorless sulfur bacteria, all the strains accumulated elemental sulfur when grown in media with sulfide. Unlike previously isolated S. natans strains, new isolates had higher temperature growth optimum (33–37°C) and variable metabolism. All the strains were capable of organotrophic, lithoheterotrophic, and mixotrophic growth with sulfur compounds as electron donors for energy metabolism. Variable metabolism of new Sphaerotilus isolates is a highly important adaptation mechanism which facilitates extension of their geographic range and supports their mass development in new habitats, e.g. sulfide springs. Within the cluster of new isolates, the physiological heterogeneity was shown to result from the inducible nature of the enzymes of oxidative sulfur metabolism and from their resistance to aerobic cultivation.     

Oxidation of Inorganic Sulfur Compounds by Obligately Organotrophic Bacteria

New data obtained by the author and other researchers on two different groups of obligately heterotrophic bacteria capable of inorganic sulfur oxidation are reviewed. Among culturable marine and (halo)alkaliphilic heterotrophs oxidizing sulfur compounds (thiosulfate and, much less actively, elemental sulfur and sulfide) incompletely to tetrathionate, representatives of the gammaproteobacteria, especially from the Halomonas group, dominate. Some denitrifying species from this group are able to carry out anaerobic oxidation of thiosulfate and sulfide using nitrogen oxides as electron acceptors. Despite the low energy output of the reaction of thiosulfate oxidation to tetrathionate, it can be utilized for ATP synthesis by some tetrathionate-producing heterotrophs; however, this potential is not always realized during their growth. Another group of marine and (halo)alkaliphilic heterotrophic bacteria capable of complete oxidation of sulfur compounds to sulfate mostly includes representatives of the alphaproteobacteria which are most closely related to nonsulfur purple bacteria. They can oxidize sulfide (polysulfide), thiosulfate, and elemental sulfur via sulfite to sulfate but neither produce nor oxidize tetrathionate. All of the investigated sulfate-forming heterotrophic bacteria belong to lithoheterotrophs, being able to gain additional energy from the oxidation of sulfur compounds during heterotrophic growth on organic substrates. Some doubtful cases of heterotrophic sulfur oxidation described in the literature are also discussed.     

Ectothiorhodospira vacuolata sp. nov., a new phototrophic bacterium from soda lakes

A new phototrophic bacterium was isolated from Jordanian and Kenyan alkaline salt lakes. Cells are rod shaped, 1.5 m wide and 2–4 m long, and motile by polar flagella. They divide by binary fission, and possess photosynthetic membranes as lamellar stacks similar to those in the other species of the genus Ectothiorhodospira and the brown colored Rhodospirillum species. The presence of bacteriochlorophyll a and carotenoids of the normal spirilloxanthin series is indicated by the absorption spectra of living cells. Under certain growth conditions the cells form gas vacuoles, may become immotile and float to the top of the culture medium. Sulfide and thiosulfate are used as photosynthetic electron donors. During the oxidation of sulfide to sulfate, elemental sulfur is formed, which is accumulated outside the cells. The organisms are strictly anaerobic, do not require vitamins, are moderately halophilic and need alkaline pH-values for growth. The new species Ectothiorhodospira vacuolata is proposed.     

Sulfide utilization by the photosynthetic bacterium Chlorobium phaeobacteroides

Sulfide and sulfur are used by the photosynthetic bacterium Chlorobium phaeobacteroides as electron donors. Sulfide and sulfur consumption was found to be affected by sulfide concentration in the medium. Raising the sulfide concentration from 0.28 mM to 5.05 mM caused an increase in the amount of S⁼ utilized per growth unit from 0.58 mM to 2.32 mM. This increase in sulfide utilization was not reflected in a higher photosynthetic activity. Sulfide and sulfur consumption was also influenced by light intensity, with higher light intensity sulfide consumption was increased. In Lake Kinneret, Chlorobium phaeobacteroides did not bloom in the thermocline layer until sulfide concentrations reached 0.03–0.06 mM.     

Sulfur Production by Obligately Chemolithoautotrophic Thiobacillus Species

Transient-state experiments with the obligately autotrophic Thiobacillus sp. strain W5 revealed that sulfide oxidation proceeds in two physiological phases, (i) the sulfate-producing phase and (ii) the sulfur- and sulfate-producing phase, after which sulfide toxicity occurs. Specific sulfur-producing characteristics were independent of the growth rate. Sulfur formation was shown to occur when the maximum oxidative capacity of the culture was approached. In order to be able to oxidize increasing amounts of sulfide, the organism has to convert part of the sulfide to sulfur (HS(sup-)(symbl)S(sup0) + H(sup+) + 2e(sup-)) instead of sulfate (HS(sup-) + 4H(inf2)O(symbl)SO(inf4)(sup2-) + 9 H(sup+) + 8e(sup-)), thereby keeping the electron flux constant. Measurements of the in vivo degree of reduction of the cytochrome pool as a function of increasing sulfide supply suggested a redox-related down-regulation of the sulfur oxidation rate. Comparison of the sulfur-producing properties of Thiobacillus sp. strain W5 and Thiobacillus neapolitanus showed that the former has twice the maximum specific sulfide-oxidizing capacity of the latter (3.6 versus 1.9 (mu)mol/mg of protein/min). Their maximum specific oxygen uptake rates were very similar. Significant mechanistic differences in sulfur production between the high-sulfur-producing Thiobacillus sp. strain W5 and the moderate-sulfur-producing species T. neapolitanus were not observed. The limited sulfide-oxidizing capacity of T. neapolitanus appears to be the reason that it can convert only 50% of the incoming sulfide to elemental sulfur.     

Complete assimilation of cysteine by a newly isolated non-sulfur purple bacterium resembling Rhodovulum sulfidophilum (Rhodobacter sulfidophilus)

A rod-shaped, motile, phototrophic bacterium, strain SiCys, was enriched and isolated from a marine microbial mat, with cysteine as sole substrate. During phototrophic anaerobic growth with cysteine, sulfide was produced as an intermediate, which was subsequently oxidized to sulfate. The molar growth yield with cysteine was 103 g mol^–1, in accordance with complete assimilation of electrons from the carbon and the sulfur moiety into cell material. Growth yields with alanine and serine were proportionally lower. Thiosulfate, sulfide, hydrogen, and several organic compounds were used as electron donors in the light, whereas cystine, sulfite, or elemental sulfur did not support phototrophic anaerobic growth. Aerobic growth in the dark was possible with fructose as substrate. Cultures of strain SiCys were yellowish-brown in color and contained bacteriochlorophyll a, spheroidene, spheroidenone, and OH-spheroidene as major photosynthetic pigments. Taking the morphology, photosynthetic pigments, aerobic growth in the dark, and utilization of sulfide for phototrophic growth into account, strain SiCys was assigned to the genus Rhodovulum (formerly Rhodobacter) and tentatively classified as a strain of R. sulfidophilum. In cell-free extracts in the presence of pyridoxal phosphate, cysteine was converted to pyruvate and sulfide, which is characteristic for cysteine desulfhydrase activity (l-cystathionine γ-lyase, EC 4.4.1.1). Received: 15 December 1995 / Accepted: 1 April 1996     

Isolation and characterization of strains CVO and FWKO B, two novel nitrate-reducing, sulfide-oxidizing bacteria isolated from oil field brine

Bacterial strains CVO and FWKO B were isolated from produced brine at the Coleville oil field in Saskatchewan, Canada. Both strains are obligate chemolithotrophs, with hydrogen, formate, and sulfide serving as the only known energy sources for FWKO B, whereas sulfide and elemental sulfur are the only known electron donors for CVO. Neither strain uses thiosulfate as an energy source. Both strains are microaerophiles (1% O(2)). In addition, CVO grows by denitrification of nitrate or nitrite whereas FWKO B reduces nitrate only to nitrite. Elemental sulfur is the sole product of sulfide oxidation by FWKO B, while CVO produces either elemental sulfur or sulfate, depending on the initial concentration of sulfide. Both strains are capable of growth under strictly autotrophic conditions, but CVO uses acetate as well as CO(2) as its sole carbon source. Neither strain reduces sulfate; however, FWKO B reduces sulfur and displays chemolithoautotrophic growth in the presence of elemental sulfur, hydrogen, and CO(2). Both strains grow at temperatures between 5 and 40 degrees C. CVO is capable of growth at NaCl concentrations as high as 7%. The present 16s rRNA analysis suggests that both strains are members of the epsilon subdivision of the division Proteobacteria, with CVO most closely related to Thiomicrospira denitrifcans and FWKO B most closely related to members of the genus Arcobacter. The isolation of these two novel chemolithotrophic sulfur bacteria from oil field brine suggests the presence of a subterranean sulfur cycle driven entirely by hydrogen, carbon dioxide, and nitrate.     

Occurrence of Purple Sulfur Bacteria in a Sewage Treatment Lagoon

The ecology of purple sulfur bacteria in a sewage oxidation lagoon was investigated. Chemical changes in the lagoon were investigated by monitoring biochemical oxygen demand (BOD(5)), sulfide, sulfate, phosphate, total carbohydrates, volatile acids, alkalinity, and pH. Lagoon water temperatures were observed daily. Microbial ecological relationships were deduced by enumerating coliforms, total bacteria other than anaerobes [Tryptone Glucose Extract (TGE) agar], methane formers such as Methanobacterium formicicum, sulfate reducers, purple sulfur bacteria, and algae. Finally, two strains of purple sulfur bacteria were characterized. Two populations, purple sulfur bacteria and total bacteria (TGE agar), reached maximal concentrations in the warmest part of the 1967 summer. Purple sulfur bacteria reached maximal numbers as concentrations of sulfide and volatile acids were depleted, whereas carbohydrates and alkalinity remained unchanged. Low sulfate levels, which were not limiting for sulfate reducers, may be attributable to storage of sulfur within purple sulfur bacteria. No biological, chemical, or physical agent was linked to the removal of coliforms. The increase of algae in the late summer of 1967 may have been related to the low organic content of the lagoon during this period. Although lagoon pH (7.7 to 8.2) was favorable for purple sulfur bacterial growth, temperatures and sulfides were not optimal in the lagoon for these organisms. Chromatium vinosum and Thiocapsa floridana (the predominant lagoon purple sulfur organism in 1967 and 1968) utilized certain carbohydrates, amino acids, volatile acids, and Krebs cycle intermediates. Also purple sulfur bacteria lowered BOD levels as demonstrated by the growth of T. floridana in sterilized sewage.     

Control of Oxidative Sulfur Metabolism of Chlorobium limicola forma thiosulfatophilum

A metered blend of anaerobic-grade N₂, CO₂, and H₂S gases was introduced into an illuminated, 800-ml liquid volume, continuously stirred tank reactor. The system, described as an anaerobic gas-to-liquid phase fed-batch reactor, was used to investigate the effects of H₂S flow rate and light energy on the accumulation of oxidized sulfur compounds formed by the photoautotroph Chlorobium limicola forma thiosulfatophilum during growth. Elemental sulfur was formed and accumulated in stoichiometric quantities when light energy and H₂S molar flow rate levels were optimally adjusted in the presence of nonlimiting CO₂. Deviation from the optimal H₂S and light energy levels resulted in either oxidation of sulfur or complete inhibition of sulfide oxidation. Based on these observations, a model of sulfide and sulfur oxidases electrochemically coupled to the photosynthetic reaction center of Chlorobium spp. is presented. The dynamic deregulation of oxidative pathways may be a mechanism for supplying the photosynthetic reaction center with a continuous source of electrons during periods of varying light and substrate availability, as in pond ecosystems where Chlorobium spp. are found. Possible applications for a sulfide gas removal process are discussed.     

A new purple sulfur bacterium isolated from a littoral microbial mat,<Emphasis Type="Italic"> Thiorhodococcus drewsii</Emphasis> sp. nov.

A new strain of purple sulfur bacterium was isolated from a marine microbial mat sampled in Great Sippewissett Salt Marsh at the Atlantic coast (Woods Hole, Mass., USA). Single cells of strain AZ1 were coccus-shaped, highly motile by means of a single flagellum, and did not contain gas vesicles. Intracellular membranes were of the vesicular type. However, additional concentric membrane structures were present. The photosynthetic pigments were bacteriochlorophyll a and carotenoids of the normal spirilloxanthin series, with rhodopin as the dominant carotenoid. Hydrogen sulfide (up to 11 mM), sulfur, thiosulfate, and molecular hydrogen were used as electron donors during anaerobic phototrophic growth. During growth on sulfide, elemental sulfur globules were transiently stored inside the cells. Strain AZ1 is much more versatile than most other Chromatiaceae with respect to electron donor and organic substrates. In the presence of CO(2), it is capable of assimilating C(1)-C(5) fatty acids, alcohols, and intermediates of the tricarboxylic acid cycle. Strain AZ1 could also grow photoorganotrophically with acetate as the sole photosynthetic electron donor. Chemotrophic growth in the dark under microoxic conditions was not detected. Optimum growth occurred at pH 6.5-6.7, 30-35 degrees C, > or =50 micro mol quanta m(-2) s(-1), and 2.4-2.6% NaCl. The DNA base composition was 64.5 mol% G+C. Comparative sequence analysis of the 16S rRNA gene confirmed that the isolate is a member of the family Chromatiaceae. Sequence similarity to the most closely related species, Thiorhodococcus minor DSMZ 11518(T), was 97.8%; however, the value for DNA-DNA hybridization between both strains was only 20%. Because of the low genetic similarity and since strain AZ1 physiologically differs considerably from all other members of the Chromatiaceae, including Trc. minor, the new isolate is described as a new species of the genus Thiorhodococcus, Thiorhodococcus drewsii sp. nov.     

Sulfur metabolism enzymes in thermoacidophilus bacteria Sulfobacillus sibiricus

Sulfur oxygenase, sulfite oxidase, adenylyl sulfate reductase, rhodanase, sulfur:Fe(III) oxidoreductase, and sulfite:Fe(III) oxidoreductase were found in cells of aerobic thermoacidophilic bacteria Sulfobacillus sibiricus strains N1 and SSO. Enzyme activity was revealed in cells grown on the medium with elemental sulfur or in the presence of various sulfide elements and concentrates of sulfide ores. The activity of sulfur-metabolizing enzymes depended little on the degree of aeration during bacterial growth.     

Role of Polysulfides in Reduction of Elemental Sulfur by the Hyperthermophilic Archaebacterium Pyrococcus furiosus

Polysulfides formed through the breakdown of elemental sulfur or other sulfur compounds were found to be reduced to H₂S by the hyperthermophilic archaebacterium Pyrococcus furiosus during growth. Metabolism of polysulfides by the organism was dissimilatory, as no incorporation of ³⁵S-labeled elemental sulfur was detected. However, [³⁵S]cysteine and [³⁵S]methionine were incorporated into cellular protein. Contact between the organism and elemental sulfur is not necessary for metabolism. The sulfide generated from metabolic reduction of polysulfides dissociates to a strong nucleophile, HS⁻, which in turn opens up the S₈ elemental sulfur ring. In addition to H₂S, P. furiosus cultures produced methyl mercaptan in a growth-associated fashion.     

Oxidation of Elemental Sulfur by Sulfolobus acidocaldarius

Oxidation of elemental sulfur by Sulfolobus acidocaldarius, an autotroph which grows at high temperatures and low pH, was examined by use of (35)S-labeled elemental sulfur. When cultured at pH 3.2 and 70 C, S. acidocaldarius oxidized elemental sulfur essentially quantitatively to sulfuric acid. Oxidation rate paralleled growth rate and decrease in pH of the culture medium. Elemental sulfur was not oxidized under these conditions if the culture was poisoned with formaldehyde. During the growth phase, the proportion of cells attached to the sulfur crystals increased progressively, and in the later phases of growth over 10 times more cells were attached to sulfur than were free. Doubling times for eight strains growing on elemental sulfur varied from 37 to 55 h. The organism grows much more rapidly on yeast extract than on sulfur. In a medium containing both sulfur and yeast extract, sulfur oxidation was partially inhibited, although growth was excellent.     

Isolation and Characterization of Strains CVO and FWKO B, Two Novel Nitrate-Reducing, Sulfide-Oxidizing Bacteria Isolated from Oil Field Brine

Bacterial strains CVO and FWKO B were isolated from produced brine at the Coleville oil field in Saskatchewan, Canada. Both strains are obligate chemolithotrophs, with hydrogen, formate, and sulfide serving as the only known energy sources for FWKO B, whereas sulfide and elemental sulfur are the only known electron donors for CVO. Neither strain uses thiosulfate as an energy source. Both strains are microaerophiles (1% O₂). In addition, CVO grows by denitrification of nitrate or nitrite whereas FWKO B reduces nitrate only to nitrite. Elemental sulfur is the sole product of sulfide oxidation by FWKO B, while CVO produces either elemental sulfur or sulfate, depending on the initial concentration of sulfide. Both strains are capable of growth under strictly autotrophic conditions, but CVO uses acetate as well as CO₂ as its sole carbon source. Neither strain reduces sulfate; however, FWKO B reduces sulfur and displays chemolithoautotrophic growth in the presence of elemental sulfur, hydrogen, and CO₂. Both strains grow at temperatures between 5 and 40°C. CVO is capable of growth at NaCl concentrations as high as 7%. The present 16s rRNA analysis suggests that both strains are members of the epsilon subdivision of the division Proteobacteria, with CVO most closely related to Thiomicrospira denitrifcans and FWKO B most closely related to members of the genus Arcobacter. The isolation of these two novel chemolithotrophic sulfur bacteria from oil field brine suggests the presence of a subterranean sulfur cycle driven entirely by hydrogen, carbon dioxide, and nitrate.     

Characterization of a new platelet-forming purple sulfur bacterium,Amoebobacter pedioformis sp. nov.

The dominant purple sulfur bacterium of a reddish-colored waste water pond near Taichung, Taiwan, was isolated in pure culture, strain CML2. Individual cells were nearly spherical, nonmotile, and contained in their peripheral parts was vacuoles that appeared like elongated, curved tubes. Four to sixteen cells formed platelet-like aggregates reminiscent of Thiopedia rosea. The intracellular photosynthetic membrane system of the cells was of vesicular type; the photosynthetic pigments consisted of bacteriochlorophyll a and spirilloxanthin as the major carotenoid. The color of cell suspensions was pink to rosered. Under anaerobic conditions photolithoautotrophic growth occurred with sulfide, elemental sulfur or thiosulfate; sulfur globules were stored as an intermediary oxidation product. In the presence of sulfide, acetate, lactate and pyruvate were photoassimilated; strain CML2 lacked assimilatory sulfate reduction. Fastest photoautotrophic growth (11 h doubling time) was obtained at pH 7.5, 35°C and a light intensity of about 1000 lux (tungsten lamp). Chemolithoautotrophic growth in the dark was possible under reduced oxygen partial pressure with reduced sulfur compounds as respiratory substrates. The DNA base composition of strain CML2 was 65.5 mol% G+C. Strain CML2 is described as type strain of a new species, Amoebobacter pedioformis sp. nov., in the family Chromatiaceae.