A proposal to rename the hyperthermophile Pyrococcus woesei as Pyrococcus furiosus subsp. woesei

Pyrococcus species are hyperthermophilic members of the order Thermococcales, with optimal growth temperatures approaching 100 degrees C. All species grow heterotrophically and produce H2 or, in the presence of elemental sulfur (S(o)), H2S. Pyrococcus woesei and P. furiosus were isolated from marine sediments at the same Vulcano Island beach site and share many morphological and physiological characteristics. We report here that the rDNA operons of these strains have identical sequences, including their intergenic spacer regions and part of the 23S rRNA. Both species grow rapidly and produce H2 in the presence of 0.1% maltose and 10-100 microM sodium tungstate in S(o)-free medium. However, P. woesei shows more extensive autolysis than P. furiosus in the stationary phase. Pyrococcus furiosus and P. woesei share three closely related families of insertion sequences (ISs). A Southern blot performed with IS probes showed extensive colinearity between the genomes of P. woesei and P. furiosus. Cloning and sequencing of ISs that were in different contexts in P. woesei and P. furiosus revealed that the napA gene in P. woesei is disrupted by a type III IS element, whereas in P. furiosus, this gene is intact. A type I IS element, closely linked to the napA gene, was observed in the same context in both P. furiosus and P. woesei genomes. Our results suggest that the IS elements are implicated in genomic rearrangements and reshuffling in these closely related strains. We propose to rename P. woesei a subspecies of P. furiosus based on their identical rDNA operon sequences, many common IS elements that are shared genomic markers, and the observation that all P. woesei nucleotide sequences deposited in GenBank to date are > 99% identical to P. furiosus sequences.     

Metabolic and evolutionary relationships among Pyrococcus Species: genetic exchange within a hydrothermal vent environment

Pyrococcus furiosus and Pyrococcus woesei grow optimally at temperatures near 100 degrees C and were isolated from the same shallow marine volcanic vent system. Hybridization of genomic DNA from P. woesei to a DNA microarray containing all 2,065 open reading frames (ORFs) annotated in the P. furiosus genome, in combination with PCR analysis, indicated that homologs of 105 ORFs present in P. furiosus are absent from the uncharacterized genome of P. woesei. Pulsed-field electrophoresis indicated that the sizes of the two genomes are comparable, and the results were consistent with the hypothesis that P. woesei lacks the 105 ORFs found in P. furiosus. The missing ORFs are present in P. furiosus mainly in clusters. These clusters include one cluster (Mal I, PF1737 to PF1751) involved in maltose metabolism and another cluster (PF0691 to PF0695) whose products are thought to remove toxic reactive nitrogen species. Accordingly, it was found that P. woesei, in contrast to P. furiosus, is unable to utilize maltose as a carbon source for growth, and the growth of P. woesei on starch was inhibited by addition of a nitric oxide generator. In P. furiosus the ORF clusters not present in P. woesei are bracketed by or are in the vicinity of insertion sequences or long clusters of tandem repeats (LCTRs). While the role of LCTRs in lateral gene transfer is not known, the Mal I cluster in P. furiosus is a composite transposon that undergoes replicative transposition. The same locus in P. woesei lacks any evidence of insertion activity, indicating that P. woesei is a sister or even the parent of P. furiosus. P. woesei may have acquired by lateral gene transfer more than 100 ORFs from other organisms living in the same thermophilic environment to produce the type strain of P. furiosus.     

Effect of sulfide on nitrate reduction in mixed methanogenic cultures

The effect of sulfide on nitrate reduction and methanogenesis was investigated in two mixed, mesophilic (35 degrees C) methanogenic cultures: sulfide-free and sulfide-acclimated (67 mg S/L total sulfide). A mixture of dextrin/peptone served as the carbon/electron donor source for the two stock cultures, as well as in all assays reported here. The sulfide-free enriched culture was amended with both nitrate (75-350 mg N/L) and sulfide (10-100 mg S/L). Denitrification was the predominant pathway at all sulfide levels tested and methanogenesis did not recover in any of the sulfide- and nitrate-amended cultures, except in the 10 mg S/L culture. Accumulation of denitrification intermediates such as NO and N(2)O took place, which irreversibly inhibited the methanogens and resulted in the complete cessation of methane production. In contrast, conversion of nitrate to nitrite and then to ammonia via dissimilatory nitrate reduction to ammonia (DNRA) prevented the accumulation of denitrification intermediates and led to the recovery of methanogenesis in the nitrate-amended, sulfide-acclimated, mixed methanogenic culture. The effect of the COD/N value on nitrate reduction was assessed with the sulfide-acclimated, methanogenic culture at COD/N values of 10, 20, and 60. As the COD/N value increased, the fraction of nitrate reduced through DNRA also increased. The results of this study have significant implications relative to the combined anaerobic treatment of carbon-, nitrogen-, and/or sulfur-bearing wastes.     

Glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus woesei: characterization of the enzyme, cloning and sequencing of the gene, and expression in Escherichia coli.

The glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus woesei (optimal growth temperature, 100 to 103 degrees C) was purified to homogeneity. This enzyme was strictly phosphate dependent, utilized either NAD+ or NADP+, and was insensitive to pentalenolactone like the enzyme from the methanogenic archaebacterium Methanothermus fervidus. The enzyme exhibited a considerable thermostability, with a 44-min half-life at 100 degrees C. The amino acid sequence of the glyceraldehyde-3-phosphate dehydrogenase from P. woesei was deduced from the nucleotide sequence of the coding gene. Compared with the enzyme homologs from mesophilic archaebacteria (Methanobacterium bryantii, Methanobacterium formicicum) and an extremely thermophilic archaebacterium (Methanothermus fervidus), the primary structure of the P. woesei enzyme exhibited a strikingly high proportion of aromatic amino acid residues and a low proportion of sulfur-containing residues. The coding gene of P. woesei was expressed at a high level in Escherichia coli, thus providing an ideal basis for detailed structural and functional studies of that enzyme.     

Solution structure and thermal stability of ribosomal protein L30e from hyperthermophilic archaeon Thermococcus celer

To understand the structural basis of thermostability, we have determined the solution structure of a thermophilic ribosomal protein L30e from Thermococcus celer by NMR spectroscopy. The conformational stability of T. celer L30e was measured by guanidine and thermal-induced denaturation, and compared with that obtained for yeast L30e, a mesophilic homolog. The melting temperature of T. celer L30e was 94 degrees C, whereas the yeast protein denatured irreversibly at temperatures >45 degrees C. The two homologous proteins also differ greatly in their stability at 25 degrees C: the free energy of unfolding was 45 kJ/mole for T. celer L30e and 14 kJ/mole for the yeast homolog. The solution structure of T. celer L30e was compared with that of the yeast homolog. Although the two homologous proteins do not differ significantly in their number of hydrogen bonds and the amount of solvent accessible surface area buried with folding, the thermophilic T. celer L30e was found to have more long-range ion pairs, more proline residues in loops, and better helix capping residues in helix-1 and helix-4. A K9A variant of T. celer L30e was created by site-directed mutagenesis to examine the role of electrostatic interactions on protein stability. Although the melting temperatures of the K9A variant is approximately 8 degrees C lower than that of the wild-type L30e, their difference in T(m) is narrowed to approximately 4.2 degrees C at 0.5 M NaCl. This salt-dependency of melting temperatures strongly suggests that electrostatic interactions contribute to the thermostability of T. celer L30e.     

Purification and characterization of an iron-nickel hydrogenase from Thermococcus celer.

Thermococcus celer cells contain a single hydrogenase located in the cytoplasm, which has been purified to apparent homogeneity using three chromatographic steps: Q-Sepharose, DEAE-Fast Flow, and Sephacryl S-200. In vitro assays demonstrated that this enzyme was able to catalyze the oxidation as well as the evolution of H2. T. celer hydrogenase had an apparent MW of 155,000+/-30,000 by gel filtration. When analyzed by SDS polyacrylamide gel electrophoresis a single band of 41,000+/-2,000 was detected. Hydrogenase activity was also detected in situ in a SDS polyacrylamide gel followed by an activity staining procedure revealing a single band corresponding to a protein of apparent Mr 84,000+/-3,000. Measurements of iron and acid-labile sulfide in different preparations of T. celer hydrogenase gave values ranging from 24 to 30 g-atoms Fe/mole of protein and 24 to 36 g-atoms of acid-labile sulfide per mole of protein. Nickel is present in 1.9-2.3 atoms per mole of protein. Copper, tungsten, and molybdenum were detected in amounts lower than 0.5 g-atoms per mole of protein. T. celer hydrogenase was inactive at ambient temperature, exhibited a dramatic increase in activity above 70 degrees C, and had an optimal activity above 90 degrees C. This enzyme showed no loss of activity after incubation at 80 degrees C for 28 h, but lost 50% of its initial activity after incubation at 96 degrees C for 20 h. Hydrogenase exhibited a half-life of approximately 25 min in air. However, after treating the air-exposed sample with sodium dithionite, more than 95% of the original activity was recovered. Copper sulfate, magnesium chloride and nitrite were also inactivators of this enzyme.     

A barophilic response by two hyperthermophilic, hydrothermal vent Archaea: An upward shift in the optimal temperature and acceleration of growth rate at supra-optimal temperatures by elevated pressure

Abstract The influence of elevated hydrostatic pressure on the growth rates of two hyperthermophilic Archaea isolated from hydrothermal vent environments (strains ES1 and ES4) was investigated over their entire temperature range for growth. Thermococcus celer , a shallow marine hyperthermophile was included in the study for comparative purposes. For one strain (ES4), the pressure at the site of collection (22 MPa) caused an upward shift in the optimal growth temperature of about 6°C compared to growth at 1 MPa. Although the optimal temperature for ES1 was unaffected by 22 MPa, elevated pressure stimulated the growth rate at supra-optimal temperatures. The temperature range for growth for both organisms was extended upward 2°C at 22 MPa pressure. For both strains 22 MPa had little effect on growth rates at sub-optimal temperatures. Growth was observed at pressures as high as 89 MPa for ES1 and 67 MPa for ES4, but with these higher pressures the temperature range for growth was narrowed, and the optimal temperature was shifted downward. Growth of Thermococcus celer was slightly stimulated by 22 MPa at its reported optimal temperature of 88°C, but was inhibited by higher pressure.     

Effect of carbon and nitrogen sources on growth dynamics and exopolysaccharide production for the hyperthermophilic archaeon Thermococcus litoralis and bacterium Thermotoga maritima

Batch and continuous cultures were used to compare specific physiological features of the hyperthermophilic archaeon, Thermococcus litoralis (T(opt) of 85 degrees to 88 degrees C), to another fermentative hyperthermophile that reduces S degrees facultatively, that is, the bacterium Thermotoga maritima (T(opt) of 80 degrees to 85 degrees C). Under nutritionally optimal conditions, these two hyperthermophiles had similar growth yields on maltose and similar cell formula weights based on elemental analysis: CH(1.7)O(0. 7)N(0.2)S(0.006) for T. litoralis and CH(1.6)O(0.6)N(0.2)S(0.005) for T. maritima. However, they differed with respect to nitrogen source, fermentation product patterns, and propensity to form exopolysaccharides (EPS). T. litoralis could be cultured in the absence or presence of maltose on an amino acid-containing defined medium in which amino acids served as the sole nitrogen source. T. maritima, on the other hand, did not utilize amino acids as carbon, energy, or nitrogen sources, and could be grown in a similar defined medium only when supplemented with maltose and ammonium chloride. Not only was T. litoralis unable to utilize NH(4)Cl as a nitrogen source, its growth was inhibited at certain levels. At 1 g/L ( approximately 20 mM) NH(4)Cl, the maximum growth yield (Y(x/s(max))) for T. litoralis was reduced to 13 g cells dry weight (CDW)/mol glucose from 40 g CDW/mol glucose in media lacking NH(4)Cl. Alanine production increased with increasing NH(4)Cl concentrations and was most pronounced if growth on NH(4)Cl was carried out in an 80% H(2) atmosphere. In T. maritima cultures, which would not grow in an 80% H(2) atmosphere, alanine and EPS were produced at much lower levels, which did not change with NH(4)Cl concentration. EPS production rose sharply at high dilution rates for both organisms, such that maltose utilization plots were biphasic. Wall growth effects were also noted, because cultures failed to wash out at dilution rates significantly above maximum growth rates determined from batch growth experiments. This study illustrates the importance of effective cultivation methods for addressing physiological issues related to the growth of hyperthermophilic heterotrophs.     

Complete genome sequence of Conexibacter woesei type strain (ID131577)

The genus Conexibacter (Monciardini et al. 2003) represents the type genus of the family Conexibacteraceae (Stackebrandt 2005, emend. Zhi et al. 2009) with Conexibacter woesei as the type species of the genus. C. woesei is a representative of a deep evolutionary line of descent within the class Actinobacteria. Strain ID131577(T) was originally isolated from temperate forest soil in Gerenzano (Italy). Cells are small, short rods that are motile by peritrichous flagella. They may form aggregates after a longer period of growth and, then as a typical characteristic, an undulate structure is formed by self-aggregation of flagella with entangled bacterial cells. Here we describe the features of the organism, together with the complete sequence and annotation. The 6,359,369 bp long genome of C. woesei contains 5,950 protein-coding and 48 RNA genes and is part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Effect of hydrogen and carbon dioxide partial pressures on growth and sulfide production of the extremely thermophilic archaebacterium Pyrodictium brockii

The effect of hydrogen and carbon dioxide partial pressure on the growth of the extremely thermophilic archaebacterium Pyrodictium brockii at 98 degrees C was investigated. Previous work with this bacterium has been done using an 80:20 hydrogen-carbon dioxide gas phase with a total pressure of 4 atm; no attempt has been made to determine if this mixture is optimal. It was found in this study that reduced hydrogen partial pressures affected cell yield, growth rate, and sulfide production. The effect of hydrogen partial pressure on cell yield and growth rate was less dramatic when compared to the effect on sulfide production, which was not found to be growth-associated. Carbon dioxide was also found to affect growth but only at very low partial pressures. The relationship between growth rate and substrate concentration could be correlated with a Monod-type expression for either carbon dioxide or hydrogen as the limiting substrate. The results from this study indicate that a balance must be struck between cell yields and sulfide production in choosing an optimal hydrogen partial pressure for the growth of P. brockii.     

一株氨氧化链霉菌的分类鉴定及其氨氧化特性的研究

从硝化反应器中分离获得一株链霉菌。根据其形态特征、培养特征、生理生化特性,（G+C）mol％含量以及16S rDNA序列和DNA杂交结果,将其归入链霉菌属中的比基尼链霉菌（Streptomycesbikiniensis）。该菌株既能在YD培养基上异养生长,也能在无机培养基上自养生长,异养生长速率(Vmax为0.39mg/L\5d)明显高于自养生长速率（Vmax为0.22mg/L.d）。异养生长时,氨氮主要用于合成细胞物质;自养生长时,部分氨氮用于合成细胞物质,部分氨氮转化成亚硝酸盐。在无机培养基上自养生长时,最适氨浓度为118mgN/L。最适生长pH值为9.36,最适氨氧化pH值为9.29。最适生长温度为31℃,最适氨氧化温度为40.6℃。提高溶解氧浓度有利于该菌株生长和氨氧化,菌体生长对溶解氧浓度的敏感性高于氨氧化。     

Isolation and characterization of a marine methanogenic bacterium from the biofilm of a shiphull in Los Angeles harbor

A marine mesophilic, irregular coccoid methanogen, which shows close resemblance toMethanococcus sp., was isolated from the biofilm of shiphulls docked in Los Angeles harbor. Hydrogen plus carbon dioxide or formate served as substrates for methanogenesis in a mineral salt medium. The isolate did not use acetate and methanol as sole source of carbon and energy. The organism had an optimal pH range of 6.8–7.0 and a temperature optimum of 37°C. Elevated levels of sodium chloride were required for optimum growth. Optimum levels of total sulfide and magnesium chloride for growth were 1.0mm and 10mm respectively. The isolate used ammonia as nitrogen source. The concentration of 30mm ammonium chloride supported maximum growth of the isolate.     

Putative ammonia-oxidizing Crenarchaeota in suboxic waters of the Black Sea: a basin-wide ecological study using 16S ribosomal and functional genes and membrane lipids

Within the upper 400 m at western, central and eastern stations in the world's largest stratified basin, the Black Sea, we studied the qualitative and quantitative distribution of putative nitrifying Archaea based on their genetic markers (16S rDNA, amoA encoding for the alpha-subunit of archaeal ammonia monooxygenase), and crenarchaeol, the specific glycerol diphytanyl glycerol tetraether of pelagic Crenarchaeota within the Group I.1a. Marine Crenarchaeota were the most abundant Archaea (up to 98% of the total archaeal 16S rDNA copies) in the suboxic layers with oxygen levels as low as 1 microM including layers where previously anammox bacteria were described. Different marine crenarchaeotal phylotypes (both 16S rDNA and amoA) were found at the upper part of the suboxic zone as compared with the base of the suboxic zone and the upper 15-30 m of the anoxic waters with prevailing sulfide concentrations of up to 30 microM. Crenarchaeol concentrations were higher in the sulfidic chemocline as compared with the suboxic zone. These results indicate an abundance of putative nitrifying Archaea at very low oxygen levels within the Black Sea and might form an important source of nitrite for the anammox reaction.     

Influence of temperature and pH on xylitol production from xylose by Debaryomyces hansenii

The production of xylitol from concentrated synthetic xylose solutions (S(o) = 130-135 g/L) by Debaryomyces hansenii was investigated at different pH and temperature values. At optimum starting pH (pH(o) = 5.5), T = 24 degrees C, and relatively low starting biomass levels (0.5-0.6 g(x)/L), 88% of xylose was utilized for xylitol production, the rest being preferentially fermented to ethanol (10%). Under these conditions, nearly 70% of initial carbon was recovered as xylitol, corresponding to final xylitol concentration of 91.9 g(P)/L, product yield on substrate of 0.81 g(P)/g(S), and maximum volumetric and specific productivities of 1.86 g(P)/L x h and 1.43 g(P)/g(x) x h, respectively. At higher and lower pH(o) values, respiration also became important, consuming up to 32% of xylose, while negligible amounts were utilized for cell growth (0.8-1.8%). The same approach extended to the effect of temperature on the metabolism of this yeast at pH(o) = 5.5 and higher biomass levels (1.4-3.0 g(x)/L) revealed that, at temperatures ranging from 32-37 degrees C, xylose was nearly completely consumed to produce xylitol, reaching a maximum volumetric productivity of 4.67 g(P)/L x h at 35 degrees C. Similarly, both respiration and ethanol fermentation became significant either at higher or at lower temperatures. Finally, to elucidate the kinetic mechanisms of both xylitol production and thermal inactivation of the system, the related thermodynamic parameters were estimated from the experimental data with the Arrhenius model: activation enthalpy and entropy were 57.7 kJ/mol and -0.152 kJ/mol x K for xylitol production and 187.3 kJ/mol and 0.054 kJ/mol x K for thermal inactivation, respectively.     

Characterization of a fourth tungsten-containing enzyme from the hyperthermophilic archaeon Pyrococcus furiosus

Pyrococcus furiosus grows optimally near 100 degrees C using peptides and carbohydrates as carbon sources, and it reduces elemental sulfur (S(0)), if present, to H(2)S. Tungsten (W), an element rarely used in biology, is required for optimal growth, and three different tungsten-containing enzymes have been previously purified from this organism. They all oxidize aldehydes of various types and are thought to play primary roles in the catabolism of sugars or amino acids. Here, the purification of a fourth tungsten-containing enzyme, termed WOR 4, from cell extracts of P. furiosus grown with S(0) is described. This was achieved by monitoring through multiple chromatography steps the W that is not associated with the three characterized tungstoenzymes. The N-terminal sequence of WOR 4 and the approximate molecular weight of its subunit determined electrophoretically (69,000) correspond to the product of an ORF (PF1961, wor4) present in the complete genome sequence of P. furiosus. WOR 4 is a homodimer and contains approximately one W, three Fe, three or four acid-labile sulfide, and one Ca atom per subunit. The visible and electron paramagnetic resonance spectra of the oxidized and reduced enzyme indicate the presence of an unusual iron-sulfur chromophore. WOR 4 does not oxidize aliphatic or aromatic aldehydes or hydroxy acids, nor does it reduce keto acids. Consistent with prior microarray data, the protein could not be purified from P. furiosus cells grown in the absence of S(0), suggesting that it may have a role in S(0) metabolism.     

Isolation of a new Thermoanaerobacterium thermosaccharolyticum strain (FH1) producing a thermostable dextranase

A Gram-positive spore-forming thermophilic strict anaerobic bacterium, designated FH1, was isolated from enrichments at 65 degrees C with dextran as sole carbon and energy source. A sequence analysis of the 16S rRNA gene revealed 99.2% identity of FH1 to Thermoanaerobacterium thermosaccharolyticum. Furthermore, the substrate spectra of both organisms were similar. It was therefore concluded that FH1 represents a new strain within the species T. thermosaccharolyticum. The optimal growth temperature of strain FH1 was 68 degrees C. The isolated organism produced a thermostable and thermoactive dextranase with a native molecular mass of approximately 200,000 Da. The enzyme was concentrated from the cell-free culture supernatant by ammonium sulfate precipitation. The resulting crude dextranase exhibited optimal activity from 65 to 70 degrees C and a pH optimum of 5.5.     

A simplified method for the cultivation of extreme anaerobic Archaea based on the use of sodium sulfite as reducing agent

The extreme sensitivity of many Archaea to oxygen is a major obstacle for their cultivation in the laboratory and the development of archaeal genetic exchange systems. The technique of Balch and Wolfe (1976) is suitable for the cultivation of anaerobic Archaea but involves time-consuming procedures such as the use of air locks and glove boxes. We describe here a procedure for the cultivation of anaerobic Archaea that is more convenient and faster and allows the preparation of liquid media without the use of an anaerobic chamber. When the reducing agent sodium sulfide (Na₂S) was replaced by sodium sulfite (Na₂SO₃), anaerobic media could be prepared without protection from oxygen outside an anaerobic chamber. Exchange of the headspace of serum bottles by appropriate gases was sufficient to maintain anaerobic conditions in the culture media. Organisms that were unable to utilize sulfite as a source for cellular sulfur were supplemented with hydrogen sulfide. H₂S was simply added to the headspace of serum bottles by a syringe. The use of H₂S as a source for sulfur minimized the precipitation of cations by sulfide. Representatives of 12 genera of anaerobic Archaea studied here were able to grow in media prepared by this procedure. For the extremely oxygen-sensitive organism Methanococcus thermolithotrophicus, we show that plates could be prepared outside an anaerobic chamber when sulfite was used as reducing agent. The application of this method may faciliate the cultivation and handling of extreme anaerobic Archaea considerably. Received: January 4, 2000 / Accepted: April 5, 2000     

Lithotrophic growth ofSulfurospirillum deleyianum with sulfide as electron donor coupled to respiratory reduction of nitrate to ammonia

Sulfurospirillum deleyianum grew in batch culture under anoxic conditions with sulfide (up to 5 mM) as electron donor, nitrate as electron acceptor, and acetate as carbon source. Nitrate was reduced to ammonia via nitrite, a quantitatively liberated intermediate. Four moles of sulfide were oxidized to elemental sulfur per mole nitrate converted to ammonia. The molar growth yield per mole sulfide consumed, Y_m, was 1.5 ± 0.2 g mol^–1 for the reduction of nitrate to ammonia. By this type of metabolism, S. deleyianum connected the biogeochemical cycles of sulfur and nitrogen. The sulfur reductase activity in S. deleyianum was inducible, as the activity depended on the presence of sulfide or elemental sulfur during cultivation with nitrate or fumarate as electron acceptor. Hydrogenase activity was always high, indicating that the enzyme is constitutively expressed. The ammonia-forming nitrite reductase was an inducible enzyme, expressed when cells were cultivated with nitrate, nitrite, or elemental sulfur, but repressed after cultivation with fumarate. Received: 13 March 1995 / Accepted: 29 May 1995     

Taurine reduction in anaerobic respiration of Bilophila wadsworthia RZATAU.

Organosulfonates are important natural and man-made compounds, but until recently (T. J. Lie, T. Pitta, E. R. Leadbetter, W. Godchaux III, and J. R. Leadbetter. Arch. Microbiol. 166:204-210, 1996), they were not believed to be dissimilated under anoxic conditions. We also chose to test whether alkane- and arenesulfonates could serve as electron sinks in respiratory metabolism. We generated 60 anoxic enrichment cultures in mineral salts medium which included several potential electron donors and a single organic sulfonate as an electron sink, and we used material from anaerobic digestors in communal sewage works as inocula. None of the four aromatic sulfonates, the three unsubstituted alkanesulfonates, or the N-sulfonate tested gave positive enrichment cultures requiring both the electron donor and electron sink for growth. Nine cultures utilizing the natural products taurine, cysteate, or isethionate were considered positive for growth, and all formed sulfide. Two clearly different pure cultures were examined. Putative Desulfovibrio sp. strain RZACYSA, with lactate as the electron donor, utilized sulfate, aminomethanesulfonate, taurine, isethionate, and cysteate, converting the latter to ammonia, acetate, and sulfide. Strain RZATAU was identified by 16S rDNA analysis as Bilophila wadsworthia. In the presence of, e.g., formate as the electron donor, it utilized, e.g., cysteate and isethionate and converted taurine quantitatively to cell material and products identified as ammonia, acetate, and sulfide. Sulfite and thiosulfate, but not sulfate, were utilized as electron sinks, as was nitrate, when lactate was provided as the electron donor and carbon source. A growth requirement for 1,4-naphthoquinone indicates a menaquinone electron carrier, and the presence of cytochrome c supports the presence of an electron transport chain. Pyruvate-dependent disappearance of taurine from cell extracts, as well as formation of alanine and release of ammonia and acetate, was detected. We suspected that sulfite is an intermediate, and we detected desulfoviridin (sulfite reductase). We thus believe that sulfonate reduction is one aspect of a respiratory system transferring electrons from, e.g., formate to sulfite reductase via an electron transport system which presumably generates a proton gradient across the cell membrane.     

The structure and biogeochemical activity of the phototrophic communities from the Bol'sherechenskii alkaline hot spring

Microbial communities growing in the bed of the alkaline, sulfide hot spring Bol'sherechenskii (the Baikal rift area) were studied over many years (1986-2001). The effluent water temperature ranged from 72 to 74 degrees C, pH was from 9.25 to 9.8, and sulfide content was from 12 to 13.4 mg/ml. Simultaneous effects of several extreme factors restrict the spread of phototrophic microorganisms. Visible microbial fouling appears with a decrease in the temperature to 62 degrees C and in the sulfide content to 5.9 mg/l. Cyanobacteria predominated in all biological zones of the microbial mat. The filamentous cyanobacteria of the genus Phormidium are the major mat-forming organisms, whereas unicellular cyanobacteria and the filamentous green bacterium Chloroflexus aurantiacus are minor components of the phototrophic communities. No cyanobacteria of the species Mastigocladus laminosus, typical of neutral and subacid springs, were identified. Seventeen species of both anoxygenic phototrophic bacteria and cyanobacteria were isolated from the microbial mats, most of which exhibited optimum growth at 20 to 45 degrees C. The anoxygenic phototrophs were neutrophiles with pH optimum at about 7. The cyanobacteria were the most adapted to the alkaline conditions in the spring. Their optimum growth was observed at pH 8.5-9.0. As determined by the in situ radioisotope method, the optimal growth and decomposition rates were observed at 40-32 degrees C, which is 10 to 15 degrees C lower than the same parameter in the sulfide-deficient Octopus Spring (Yellowstone, United States). The maximum chlorophyll a concentration was 555 mg/m2 at 40 degrees C. Total rate of photosynthesis in the mats reached 1.3 g C/m2 per day. The maximum rate of dark fixation of carbon dioxide in the microbial mats was 0.806 g C/m2 per day. The maximum rate of sulfate reduction comprised 0.367 g S/m2 per day at 40 degrees C. The rate of methanogenesis did not exceed 1.188 micrograms C/m2 per day. The role of methanogenesis in the terminal decomposition of the organic matter was insignificant. Methane formation consumed 100 times less organic matter than sulfate reduction.