Thermophilic Sulfate-reducing Bacterium Research Articles

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 micrometer wide and 1.5 micrometers long. They were motile by means of a single polar flagellum. Growth was observed between 40 degrees and 70 degrees C with optimal growth at 65 degrees C. Cultures remained viable for one year at 27 degrees 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 16 S 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.

Phylogenetic analysis ofDesulfotomaculum thermobenzoicumusing polymerase chain reaction-amplified 16S rRNA-specific DNA

The 16S rRNA gene of the thermophilic sulfate-reducing bacterium Desulfotomaculum thermobenzoicum was amplified by polymerase chain reaction using two eubacterial consensus oligodeoxynucleotide primers flanking the majority of the 16S rRNA gene, cloned, and sequenced. Phylogenetic analysis revealed that D. thermobenzoicum belongs to the Gram-positive (low G + C content) branch and is more related to the thermophilic sulfate-reducing bacterium, D. australicum than the moderate thermophile D. nigrificans, or the mesophiles D. orientis, and D. ruminis. This relationship is further strengthened by the presence of an unusual idiosyncrasy in helix 6 of the 16S rRNA gene of D. thermobenzoicum resembling that of D. australicum but not found in other desulfotomacula species and in any other bacteria sequenced to date.

Phylogenetic analysis of Desulfotomaculum thermobenzoicum using polymerase chain reaction-amplified 16S rRNA-specific DNA

The 16S rRNA gene of the thermophilic sulfate-reducing bacterium Desulfotomaculum thermobenzoicum was amplified by polymerase chain reaction using two eubacterial consensus oligodeoxynucleotide primers flanking the majority of the 16S rRNA gene, cloned, and sequenced. Phylogenetic analysis revealed that D. thermobenzoicum belongs to the Gram-positive (low G + C content) branch and is more related to the thermophilic sulfate-reducing bacterium, D. australicum than the moderate thermophile D. nigrificans, or the mesophiles D. orientis, and D. ruminis. This relationship is further strengthened by the presence of an unusual idiosyncrasy in helix 6 of the 16S rRNA gene of D. thermobenzoicum resembling that of D. australicum but not found in other desulfotomacula species and in any other bacteria sequenced to date.

Effects of hydrogen and formate on the degradation of propionate and butyrate in thermophilic granules from an upflow anaerobic sludge blanket reactor

Degradation of propionate and butyrate in whole and disintegrated granules from a thermophilic (55 degrees C) upflow anaerobic sludge blanket reactor fed with acetate, propionate, and butyrate as substrates was examined. The propionate and butyrate degradation rates in whole granules were 1.16 and 4.0 mumol/min/g of volatile solids, respectively, and the rates decreased 35 and 25%, respectively, after disintegration of the granules. The effect of adding different hydrogen-oxidizing bacteria (both sulfate reducers and methanogens), some of which used formate in addition to hydrogen, to disintegrated granules was tested. Addition of either Methanobacterium thermoautotrophicum delta H, a hydrogen-utilizing methanogen that does not use formate, or Methanobacterium sp. strain CB12, a hydrogen- and formate-utilizing methanogen, to disintegrated granules increased the degradation rate of both propionate and butyrate. Furthermore, addition of a thermophilic sulfate-reducing bacterium (a Desulfotomaculum sp. isolated in our laboratory) to disintegrated granules improved the degradation of both substrates even more than the addition of methanogens. By monitoring the hydrogen partial pressure in the cultures, a correlation between the hydrogen partial pressure and the degradation rate of propionate and butyrate was observed, showing a decrease in the degradation rate with increased hydrogen partial pressure. No significant differences in the stimulation of the degradation rates were observed when the disintegrated granules were supplied with methanogens that utilized hydrogen only or hydrogen and formate. This indicated that interspecies formate transfer was not important for stimulation of propionate and butyrate degradation.

Desulfotomaculum australicum, sp. nov., a Thermophilic Sulfate-Reducing Bacterium Isolated from the Great Artesian Basin of Australia

A new thermophilic species of spore-forming sulfate-reducing bacterium, Desulfotomaculum australicum is described. D. australicum is physiologically different to all other validated species of Desulfotomaculum . It has a high iso-saturated branched (15:0 and 17:0) phospholipid composition (87%), a feature in common with other true thermophiles. Phylogenetically, D. australicum clusters with the mesophilic D. orientis and D. ruminis . However, a set of signature nucleotides that would support the clustering of these three organisms within the radiation of Gram-positive bacteria could not be detected.

Partial purification and characterization of the first hydrogenase isolated from a thermophilic sulfate-reducing bacterium

A soluble [NiFe] hydrogenase has been partially purified from the obligate thermophilic sulfate-reducing bacterium Thermodesulfobacterium mobile. A 17% purification yield was obtained after four chromatographic steps and the hydrogenase presents a purity index (A398 nm/A277 nm) equal to 0.21. This protein appears to be 75% pure on SDS-gel electrophoresis showing two major bands of molecular mass around 55 and 15 kDa. This hydrogenase contains 0.6-0.7 nickel atom and 7-8 iron atoms per mole of enzyme and has a specific activity of 783 in the hydrogen uptake reaction, of 231 in the hydrogen production assay and of 84 in the deuterium-proton exchange reaction. The H2/HD ratio is lower than one in the D2-H+ exchange reaction. The enzyme is very sensitive to NO, relatively little inhibited by CO but unaffected by NO2-. The EPR spectrum of the native hydrogenase shows the presence of a [3Fe-4S] oxidized cluster and of a Ni(III) species.

Immunomagnetically Captured Thermophilic Sulfate-Reducing Bacteria from North Sea Oil Field Waters

Immunomagnetic beads (IMB) were used to recover thermophilic sulfate-reducing bacteria from oil field waters from oil production platforms in the Norwegian sector of the North Sea. IMB coated with polyclonal antibodies against whole-cell antigens of the thermophilic Thermodesulfobacterium mobile captured strains GFA1, GFA2, and GFA3. GFA1 was serologically and morphologically identical to T. mobile. GFA2 and GFA3 were spore forming and similar to the Desulfotomaculum strains T90A and T93B previously isolated from North Sea oil field waters by a classical enrichment procedure. Western blots (immunoblots) of whole cells showed that GFA2, GFA3, T90A, and T93B are different serotypes of the same Desulfotomaculum species. Monoclonal antibodies (MAb) against T. mobile type strain cells were produced and used as capture agents on IMB. These MAb, named A4F4, were immunoglobulin M; they were specific to T. mobile and directed against lipopolysaccharides. The prevailing cells immunocaptured with MAb A4F4 were morphologically and serologically similar to T. mobile type strain cells. T. mobile was not detected in these oil field waters by classical enrichment procedures. Furthermore, extraction with antibody-coated IMB allowed pure strains to be isolated directly from primary enrichment cultures without prior time-consuming subculturing and consecutive transfers to selective media.

Spore-Forming Thermophilic Sulfate-Reducing Bacteria Isolated from North Sea Oil Field Waters

Thermophilic sulfate-reducing bacteria were isolated from oil field waters from oil production platforms in the Norwegian sector of the North Sea. Spore-forming rods dominated in the enrichments when lactate, propionate, butyrate, or a mixture of aliphatic fatty acids (C(4) through C(6)) was added as a carbon source and electron donor. Representative strains were isolated and characterized. The isolates grew autotrophically on H(2)-CO(2) and heterotrophically on fatty acids such as formate, propionate, butyrate, caproate, valerate, pyruvate, and lactate and on alcohols such as methanol, ethanol, and propanol. Sulfate, sulfite, and thiosulfate but not nitrate could be used as an electron acceptor. The temperature range for growth was 43 to 78 degrees C; the spores were extremely heat resistant and survived 131 degrees C for 20 min. The optimum pH was 7.0. The isolates grew well in salt concentrations ranging from 0 to 800 mmol of NaCl per liter. Sulfite reductase P582 was present, but cytochrome c and desulfoviridin were not found. Electron micrographs revealed a gram-positive cell organization. The isolates were classified as a Desulfotomaculum sp. on the basis of spore formation, general physiological characteristics, and submicroscopic organization. To detect thermophilic spore-forming sulfate-reducing bacteria in oil field water, polyvalent antisera raised against antigens from two isolates were used. These bacteria were shown to be widespread in oil field water from different platforms. The origin of thermophilic sulfate-reducing bacteria in the pore water of oil reservoirs is discussed.

Isolation and characterization of a thermophilic benzoate-degrading, sulfate-reducing bacterium, Desulfotomaculum thermobenzoicum sp. nov.

A new thermophilic sulfate-reducing bacterium, strain TSB, that was spore-forming, rod-shaped, slightly motile and gram-positive, was isolated from a butyrate-containing enrichment culture inoculated with sludge of a thermophilic methane fermentation reactor. This isolate could oxidize benzoate completely. Strain TSB also oxidized some fatty acids and alcohols. SOinf4sup2-, SOinf3sup2-, S2Oinf3sup2-and NOinf3sup-were utilized as electron acceptors. With pyruvate or lactate the isolate grew without an external electron acceptor and produced acetate. The optimum temperature for growth was 62°C. The G+C content of DNA was 52.8 mol%. This isolate is described as a new species, Desulfotomaculum thermobenzoicum.

The participation of a ferredoxin of [formula omitted][formula omitted] in sulfite reduction

Clostridium nigrificans , a thermophilic sulfate-reducing bacterium ( Campbell et al ., 1957 ), reduces inorganic sulfate to hydrogen sulfide by a dissimilatory pathway involving adenosine-5'-phosphosulfate as the active intermediate compound ( Peck, 1959; Ishimoto and Fujimoto, 1959). The electron transport system operating in this reductive process was not established for C. nigrificans . Previous attempts, in this laboratory, to isolate a ferredoxin from this organism were unsuccessful ( Akagi, 1964). The failure to detect ferredoxin-activity was not due to the absence of the carrier but rather, was primarily due to the method of assay employed. In this communication evidence for the presence of a ferredoxin in C. nigrificans and a brief description of some of its properties are presented.

STUDIES ON THERMOPHILIC SULFATE-REDUCING BACTERIA III

Akagi, J. M. (University of Kansas, Lawrence) and L. Leon Campbell. Studies on thermophilic sulfate-reducing bacteria. III. Adenosine triphosphate-sulfurylase of Clostridium nigrificans and Desulfovibrio desulfuricans. J. Bacteriol. 84:1194-1201. 1962.-Adenosine triphosphate (ATP)-sulfurylase, which catalyzes the formation of adenosine-5'-phosphosulfate (APS) from ATP and SO(4) (=), has been purified from crude extracts of Clostridium nigrificans and Desulfovibrio desulfuricans by (NH(4))(2)SO(4) fractionation and triethylaminoethyl column chromatography. The enzyme from both sources operates over a broad pH range from 6.0 to 9.5. Below pH 6.0, activity decreases sharply, with no detectable activity at pH 5.0. Of the nucleotides tested (ATP and the triphosphates of deoxyadenosine, uridine, inosine, and guanosine), only ATP was acted upon by the enzyme from either source. The enzyme requires Mg(++) for activity. Incubation of the enzyme from both organisms with ATP and S(35)O(4) (=) in the presence of helium resulted in the formation of an S(35)-labeled nucleotide whose electrophoretic mobility was identical to that of chemically prepared APS. When incubated with ATP and the group VI anions (CrO(4), MoO(4), WO(4)), the enzyme from both organisms formed an unstable intermediate, resulting in the accumulation of pyrophosphate. Thermal stability studies revealed that the ATP-sulfurylase of C. nigrificans was stable at higher temperatures than the enzyme obtained from D. desulfuricans. Exposure of the enzyme from C. nigrificans to 65 C for 2 hr gave virtually no decrease in activity. In contrast, the enzyme from D. desulfuricans was completely inactivated after 30 min at 55 C, after 3 min at 60 C, or after 1 min at 65 C.

Studies on thermophilic sulfate-reducing bacteria. II. Hydrogenase activity of Clostridium nigrificans.

Akagi, J. M. (Western Reserve University, Cleveland, Ohio) and L. Leon Campbell. Studies on thermophilic sulfate-reducing bacteria. II. Hydrogenase activity of Clostridium nigrificans. J. Bacteriol. 82:927-932. 1961.-The hydrogenase of Clostridium nigrificans has been found to be associated with the cell-free particulate fraction which can be sedimented at 105,000 x g in 1 hr. The specific activity of this fraction was increased 2 to 3 fold over that of the crude extract. It was not found possible to liberate the enzyme from the particulate fraction by methods of enzymatic digestion, chemical extraction, or physical disruption. The optimum temperature for H(2) utilization using benzyl viologen as an electron acceptor was found to be 55 C, and the optimum pH range was 7 to 8. Employing metal complexing agents it was found that the enzyme required Fe(++) ions for H(2) utilization. In contrast, Fe(++) ions were not required to catalyze the evolution of H(2) from reduced methyl viologen. The role of Fe(++) ions in the hydrogenase activity of this organism is discussed.