Evaluating the potential of indigenous methanogenic consortium for enhanced oil and gas recovery from high temperature depleted oil reservoir.

In past years, lots of research has been focused on the indigenous bacteria and their mechanisms, which help in enhanced oil recovery. Most of the oil wells in Indian subcontinent have temperature higher than 60 °C. Also, the role of methanogenic consortia from high temperature petroleum reservoir for enhanced oil recovery (EOR) has not been explored much. Hence, in the present study methanogens isolated from thermophilic oil wells (70 °C) were evaluated for enhanced oil recovery. Methane gas is produced by methanogens, which helps in oil recovery from depleted oil wells through reservoir re-pressurization and also can be recovered from reservoir along with crude oil as alternative energy source. Therefore, in this study indigenous methanogenic consortium (TERIL146) was enriched from high temperature oil reservoir showing (12 mmol/l) gas production along with other metabolites. Sequencing analysis revealed the presence of Methanothermobacter sp., Thermoanaerobacter sp., Gelria sp. and Thermotoga sp. in the consortium. Furthermore, the developed indigenous consortium TERIL146 showed 8.3% incremental oil recovery in sandpack assay. The present study demonstrates successful recovery of both oil and energy (gas) by the developed indigenous methanogenic consortium TERIL146 for potential application in thermophilic depleted oil wells of Indian subcontinent.

Thermoanaerobacter species differ in their potential to reduce organic acids to their corresponding alcohols.

The reduction of organic acids to their corresponding alcohols has been shown for some bacterial species within the Firmicutes super-phylum and a genetically modified strain of the hyperthermophilic archaeon Pyrococcus furiosus. In the latter strain, an aldehyde:ferredoxin oxidoreductase (AOR) catalyzed the reduction of a variety of organic acids to their corresponding aldehydes, as shown by the deletion of the corresponding aor gene. Here, we found that the genomes of a few thermophilic bacterial species within the genus Thermoanaerobacter which have been described to efficiently ferment sugars to ethanol harbor a copy of aor, while others do not. Specific AOR activity was only found in strains with aor, and the gene was highly expressed in Thermoanaerobacter sp. strain X514. The reduction of a variety of organic acids was observed for several Thermoanaerobacter sp.; however, strains with aor reduced, e.g., isobutyrate at much higher rates of up to 5.1 mM h-1 g-1. Organic acid reduction also led to increased growth rates in Thermoanaerobacter sp. strain X514 and in Thermoanaerobacter pseudethanolicus. Organic acid activation may proceed via acyl-CoA with subsequent NADH-dependent reduction by an aldehyde dehydrogenase (ALDH), or via direct reduction by AOR. Cell-free extracts of Thermoanaerobacter sp. strain X514 exhibited both enzyme activities at comparable rates. Therefore, the biochemistry of organic acid reduction to alcohols in Thermoanaerobacter sp. remains to be elucidated; however, relatively high specific activities and the correlation of AOR specific activities with alcohol production rates suggest a role for AOR.

High-rate, High Temperature Acetotrophic Methanogenesis Governed by a Three Population Consortium in Anaerobic Bioreactors.

A combination of acetate oxidation and acetoclastic methanogenesis has been previously identified to enable high-rate methanogenesis at high temperatures (55 to 65°C), but this capability had not been linked to any key organisms. This study combined RNA-stable isotope probing on 13C-labelled acetate and 16S amplicon sequencing to identify the active micro-organisms involved in high-rate methanogenesis. Active biomass was harvested from three bench-scale thermophilic bioreactors treating waste activated sludge at 55, 60 and 65°C, and fed with 13-C labelled and 12C-unlabelled acetate. Acetate uptake and cumulative methane production were determined and kinetic parameters were estimated using model-based analysis. Pyrosequencing performed on 13C- enriched samples indicated that organisms accumulating labelled carbon were Coprothermobacter (all temperatures between 55 and 65°C), acetoclastic Methanosarcina (55 to 60°C) and hydrogenotrophic Methanothermobacter (60 to 65°C). The increased relative abundance of Coprothermobacter with increased temperature corresponding with a shift to syntrophic acetate oxidation identified this as a potentially key oxidiser. Methanosarcina likely acts as both a hydrogen utilising and acetoclastic methanogen at 55°C, and is replaced by Methanothermobacter as a hydrogen utiliser at higher temperatures.

Growth and metabolic profiling of the novel thermophilic bacterium Thermoanaerobacter sp. strain YS13.

A strictly anaerobic, thermophilic bacterium, designated strain YS13, was isolated from a geothermal hot spring. Phylogenetic analysis using the 16S rRNA genes and cpn60 UT genes suggested strain YS13 as a species of Thermoanaerobacter. Using cellobiose or xylose as carbon source, YS13 was able to grow over a wide range of temperatures (45-70 °C), and pHs (pH 5.0-9.0), with optimum growth at 65 °C and pH 7.0. Metabolic profiling on cellobiose, glucose, or xylose in 1191 medium showed that H2, CO2, ethanol, acetate, and lactate were the major metabolites. Lactate was the predominant end product from glucose or cellobiose fermentations, whereas H2 and acetate were the dominant end products from xylose fermentation. The metabolic balance shifted away from ethanol to H2, acetate, and lactate when YS13 was grown on cellobiose as temperatures increased from 45 to 70 °C. When YS13 was grown on xylose, a metabolic shift from lactate to H2, CO2, and acetate was observed in cultures as the temperature of incubation increased from 45 to 65 °C, whereas a shift from ethanol and CO2 to H2, acetate, and lactate was observed in cultures incubated at 70 °C.

Ecology and biotechnological potential of the thermophilic fermentative Coprothermobacter spp.

Thermophilic bacteria have been isolated from several terrestrial, marine and industrial environments. Anaerobic digesters treating organic wastes are often an important source of these microorganisms, which catalyze a wide array of metabolic processes. Moreover, organic wastes are primarily composed of proteins, whose degradation is often incomplete. Coprothermobacter spp. are proteolytic anaerobic thermophilic microbes identified in several studies focused on the analysis of the microbial community structure in anaerobic thermophilic reactors. They are currently classified in the phylum Firmicutes; nevertheless, several authors showed that the Coprothermobacter group is most closely related to the phyla Dictyoglomi and Thermotoga. Since only a few proteolytic anaerobic thermophiles have been characterized so far, this microorganism has attracted the attention of researchers for its potential applications with high-temperature environments. In addition to proteolysis, Coprothermobacter spp. showed several metabolic abilities and may have a biotechnological application either as source of thermostable enzymes or as inoculum in anaerobic processes. Moreover, they can improve protein degradation by establishing a syntrophy with hydrogenotrophic archaea. To gain a better understanding of the phylogenesis, metabolic capabilities and adaptations of these microorganisms, it is of importance to better define the role in thermophilic environments and to disclose properties not yet investigated.

Characterization of enriched aerotolerant cellulose-degrading communities for biofuels production using differing selection pressures and inoculum sources.

Ethanol production from direct cellulose fermentation has mainly been described as a strictly anaerobic process. The use of air-tolerant organisms or consortia for this process would reduce the need for prereduction of the medium and also permit continuous feed of aerobic feedstock. To this end, moderately thermophilic (60 °C) consortia of fermentative, cellulolytic bacteria were enriched from 3 distinct environments (manure, marsh, and rotten wood) from a farm in southeast Saskatchewan, Canada. Community phenotypic and metabolic profiles were characterized. Selection methods included direct plating under an aerobic atmosphere and repeated passaging; the methods were designed to select for robust, stable aerotolerant cellulose-degrading communities. Several of the isolated communities exhibited an increase in total cellulose degradation and total ethanol yield when compared with a monoculture of Clostridium thermocellum DSMZ 1237. Owing to stringent selection conditions, low diversity enrichments were found, and many appeared to be binary cultures via density gradient gel electrophoresis analysis. On the basis of 16S rRNA gene sequencing, aerobic conditions selected for a mix of organisms highly related to C. thermocellum and Geobacillus species, while anaerobic conditions led to the development of consortia containing strains related to C. thermocellum with strains from either the genus Geobacillus or the genus Thermoanaerobacter. The presence of a Geobacillus-like species appeared to be a prerequisite for aerotolerance of the cellulolytic enrichments, a highly desired phenotype in lignocellulosic consolidated bioprocessing.

Genomic evaluation of Thermoanaerobacter spp. for the construction of designer co-cultures to improve lignocellulosic biofuel production.

The microbial production of ethanol from lignocellulosic biomass is a multi-component process that involves biomass hydrolysis, carbohydrate transport and utilization, and finally, the production of ethanol. Strains of the genus Thermoanaerobacter have been studied for decades due to their innate abilities to produce comparatively high ethanol yields from hemicellulose constituent sugars. However, their inability to hydrolyze cellulose, limits their usefulness in lignocellulosic biofuel production. As such, co-culturing Thermoanaerobacter spp. with cellulolytic organisms is a plausible approach to improving lignocellulose conversion efficiencies and yields of biofuels. To evaluate native lignocellulosic ethanol production capacities relative to competing fermentative end-products, comparative genomic analysis of 11 sequenced Thermoanaerobacter strains, including a de novo genome, Thermoanaerobacter thermohydrosulfuricus WC1, was conducted. Analysis was specifically focused on the genomic potential for each strain to address all aspects of ethanol production mentioned through a consolidated bioprocessing approach. Whole genome functional annotation analysis identified three distinct clades within the genus. The genomes of Clade 1 strains encode the fewest extracellular carbohydrate active enzymes and also show the least diversity in terms of lignocellulose relevant carbohydrate utilization pathways. However, these same strains reportedly are capable of directing a higher proportion of their total carbon flux towards ethanol, rather than non-biofuel end-products, than other Thermoanaerobacter strains. Strains in Clade 2 show the greatest diversity in terms of lignocellulose hydrolysis and utilization, but proportionately produce more non-ethanol end-products than Clade 1 strains. Strains in Clade 3, in which T. thermohydrosulfuricus WC1 is included, show mid-range potential for lignocellulose hydrolysis and utilization, but also exhibit extensive divergence from both Clade 1 and Clade 2 strains in terms of cellular energetics. The potential implications regarding strain selection and suitability for industrial ethanol production through a consolidated bioprocessing co-culturing approach are examined throughout the manuscript.

Thermoanaerobacter pentosaceus sp. nov., an anaerobic, extremely thermophilic, high ethanol-yielding bacterium isolated from household waste.

An extremely thermophilic, xylanolytic, spore-forming and strictly anaerobic bacterium, strain DTU01(T), was isolated from a continuously stirred tank reactor fed with xylose and household waste. Cells stained Gram-negative and were rod-shaped (0.5-2 µm in length). Spores were terminal with a diameter of approximately 0.5 µm. Optimal growth occurred at 70 °C and pH 7, with a maximum growth rate of 0.1 h(-1). DNA G+C content was 34.2 mol%. Strain DTU01(T) could ferment arabinose, cellobiose, fructose, galactose, glucose, lactose, mannitol, mannose, melibiose, pectin, starch, sucrose, xylan, yeast extract and xylose, but not cellulose, Avicel, inositol, inulin, glycerol, rhamnose, acetate, lactate, ethanol, butanol or peptone. Ethanol was the major fermentation product and a maximum yield of 1.39 mol ethanol per mol xylose was achieved when sulfite was added to the cultivation medium. Thiosulfate, but not sulfate, nitrate or nitrite, could be used as electron acceptor. On the basis of 16S rRNA gene sequence similarity, strain DTU01(T) was shown to be closely related to Thermoanaerobacter mathranii A3(T), Thermoanaerobacter italicus Ab9(T) and Thermoanaerobacter thermocopriae JT3-3(T), with 98-99 % similarity. Despite this, the physiological and phylogenetic differences (DNA G+C content, substrate utilization, electron acceptors, phylogenetic distance and isolation site) allow for the proposal of strain DTU01(T) as a representative of a novel species within the genus Thermoanaerobacter, for which the name Thermoanaerobacter pentosaceus sp. nov. is proposed, with the type strain DTU01(T) ( = DSM 25963(T) = KCTC 4529(T) = VKM B-2752(T) = CECT 8142(T)).

Isolates of Thermoanaerobacter thermohydrosulfuricus from decaying wood compost display genetic and phenotypic microdiversity.

In this study, 12 strains of Thermoanaerobacter were isolated from a single decaying wood compost sample and subjected to genetic and phenotypic profiling. The 16S rRNA encoding gene sequences suggested that the isolates were most similar to strains of either Thermoanaerobacter pseudethanolicus or Thermoanaerobacter thermohydrosulfuricus. Examination of the lesser conserved chaperonin-60 (cpn60) universal target showed that some isolates shared the highest sequence identity with T. thermohydrosulfuricus; however, others to Thermoanaerobacter wiegelii and Thermoanaerobacter sp. Rt8.G4 (formerly Thermoanaerobacter brockii Rt8.G4). BOX-PCR fingerprinting profiles identified differences in the banding patterns not only between the isolates and the reference strains, but also among the isolates themselves. To evaluate the extent these genetic differences were manifested phenotypically, the utilization patterns of 30 carbon substrates were examined and the niche overlap indices (NOI) calculated. Despite showing a high NOI (> 0.9), significant differences existed in the substrate utilization capabilities of the isolates suggesting that either a high degree of niche specialization or mechanisms allowing for non-competitive co-existence, were present within this ecological context. Growth studies showed that the isolates were physiologically distinct in both growth rate and the fermentation product ratios. Our data indicate that phenotypic diversity exists within genetically microdiverse Thermoanaerobacter isolates from a common environment.

Predicting relatedness of bacterial genomes using the chaperonin-60 universal target (cpn60 UT): application to Thermoanaerobacter species.

D.R. Zeigler determined that the sequence identity of bacterial genomes can be predicted accurately using the sequence identities of a corresponding set of genes that meet certain criteria [32]. This three-gene model for comparing bacterial genome pairs requires the determination of the sequence identities for recN, thdF, and rpoA. This involves the generation of approximately 4.2kb of genomic DNA sequence from each organism to be compared, and also normally requires that oligonucleotide primers be designed for amplification and sequencing based on the sequences of closely related organisms. However, we have developed an analogous mathematical model for predicting the sequence identity of whole genomes based on the sequence identity of the 542-567 base pair chaperonin-60 universal target (cpn60 UT). The cpn60 UT is accessible in nearly all bacterial genomes with a single set of universal primers, and its length is such that it can be completely sequenced in one pair of overlapping sequencing reads via di-deoxy sequencing. These mathematical models were applied to a set of Thermoanaerobacter isolates from a wood chip compost pile and it was shown that both the one-gene cpn60 UT-based model and the three-gene model based on recN, rpoA, and thdF predicted that these isolates could be classified as Thermoanaerobacter thermohydrosulfuricus. Furthermore, it was found that the genomic prediction model using cpn60 UT gave similar results to whole-genome sequence alignments over a broad range of taxa, suggesting that this method may have general utility for screening isolates and predicting their taxonomic affiliations.

Mathematical modeling of hydrolysate diffusion and utilization in cellulolytic biofilms of the extreme thermophile Caldicellulosiruptor obsidiansis.

In this study, a hydrolysate diffusion and utilization model was developed to examine factors influencing cellulolytic biofilm morphology. Model simulations using Caldicellulosiruptor obsidiansis revealed that the cellulolytic biofilm needs to generate more hydrolysate than it consumes to establish a higher than bulk solution intra-biofilm substrate concentration to support its growth. This produces a hydrolysate surplus that diffuses through the thin biofilm structure into the bulk solution, which gives rise to a uniform growth rate and hence the homogeneous morphology of the cellulolytic biofilm. Model predictions were tested against experimental data from a cellulose-fermenting bioreactor and the results were consistent with the model prediction and indicated that only a small fraction (10-12%) of the soluble hydrolysis products are utilized by the biofilm. The factors determining the rate-limiting step of cellulolytic biofilm growth are also analyzed and discussed.

[Enhanced role of the co-culture of thermophilic anaerobic bacteria on cellulosic ethanol].

Fermentation of the type of cellulosic materials to ethanol was evaluated in batch system of mono-cultures of cellulolytic ethanol producing strains (Clostridium thermocellum strain LQRI), and co-cultures of LQRI in combination with one of the non-cellulolytic ethanol producing strains (Thermoanaerobacter pseudoethanolicus strains X514 or Thermoanaerobacter ethanolicus 39E). Results showed that ethanol yields and cellulose degradation abilities were significantly improved by the establishment of co-cultures consisting of LQRI and Thermoanaerobacter ethanolicus partner. A factorial experimental comparison revealed that the co-culture of LQRI + X514 provided the higher ethanol yield than the co-culture of LQRI + 39E, but no significant difference on cellulose degradation by LQRI was found in these co-cultures. In the absence of yeast extract, the highest ethanol concentrations in the co-cultures of LQRI + X514 and LQRI + 39E were about 71 mmol/L and 36.5 mmol/L, which were approximately 5-11 and 3-5 times higher than that of the mono-culture LQRI under the same concentration substrate, respectively. In the presence of 0.6% yeast extract, the highest ethanol concentrations in the co-cultures of LQRI + X514 and LQRI + 39E were rapidly improved and reached 263.5 mmol/L and 143.5 mmol/L, which were approximately 8-22 and 8-12 times higher than that of the mono-culture LQRI under the same concentrations substrate, respectively. The maximum ethanol concentration reached about 263.5 mmol/L (1.2%) in the co-culture of LQRI + X514 grown on 5% Solka Floc in the presence of 0.6% yeast extract, while the maximum ethanol concentration reached 143.5 mmol/L (1.2%) in the co-culture of LQRI + 39E grown on 2% Solka Floc in the presence of 0.6% yeast extract.

[Isolation and identification of a thermophilic anaerobic bacterium].

OBJECTIVE: To find new microbial resources from a high-temperature oil reservoir. METHODS: Strain HL-3 was isolated by Hungate Anaerobic Technique from oil reservoir water sampled from Dagang oilfield, China. Through physiological, biochemical and phylogenetic analysis, the strain HL-3 was classified. RESULTS: Cells were Gram-positive. The temperature range for growth was 40 degrees C-75 degrees C (optimum at 60 degrees C) and the pH range was 5.0-8.0 (optimum at 6.5). The isolate could grow in the presence of 0%-3.2% NaCl (optimum at 0.25%). Glucose, ribose, mannose, xylose and cellobiose could be metabolized. Metabolites of glucose were ethanol, acetate, CO2 and trace amount of propionate and butanol. The G + C content of DNA was 33.9 mol%. Based on 16S rRNA studies,strain HL-3 was most close to T. uzonensis DSM 18761T (EF530067) with 98.8% similarity and to T. sulfurigignens DSM 17917T (AF234164) with the 98.1% similarity. Strain HL-3 tolerated to high sulfite (0. 1mol/L) ions and extremely high concentration of thiosulfate (0.8 mol/L). When the concentration of thiosulfate was higher than 0.075 mol/L, the cell would generate S element granular. The presence of H2S gas was detected inside of space at the top of serum bottle. Strain HL-3 together with T. uzonensis DSM 18761T differed greatly in toleration of thiosulfate and sulfite. The toleration of strain HL-3 to thiosulfate and sulfite was most close to T. sulfurigignens DSM 17917T (AF234164). In addition, strain HL-3 to metabolite thiosulfate and sulfite was also similar with T. sulfurigignens DSM 17917T (AF234164). However, it differs largely from both of them to metabolize glucose. CONCLUSION: Therefore, strain HL-3 may be a new spieces of the Thermoanaerobacter, and the definitive classification positioning is still awaiting for further verified with the method of determination of whole-genome DNA-DNA similarity

Isolation and characterization of a new CO-utilizing strain, Thermoanaerobacter thermohydrosulfuricus subsp. carboxydovorans, isolated from a geothermal spring in Turkey.

A novel anaerobic, thermophilic, Gram-positive, spore-forming, and sugar-fermenting bacterium (strain TLO) was isolated from a geothermal spring in Ayas, Turkey. The cells were straight to curved rods, 0.4-0.6 microm in diameter and 3.5-10 microm in length. Spores were terminal and round. The temperature range for growth was 40-80 degrees C, with an optimum at 70 degrees C. The pH optimum was between 6.3 and 6.8. Strain TLO has the capability to ferment a wide variety of mono-, di-, and polysaccharides and proteinaceous substrates, producing mainly lactate, next to acetate, ethanol, alanine, H(2), and CO(2). Remarkably, the bacterium was able to grow in an atmosphere of up to 25% of CO as sole electron donor. CO oxidation was coupled to H(2) and CO(2) formation. The G + C content of the genomic DNA was 35.1 mol%. Based on 16S rRNA gene sequence analysis and the DNA-DNA hybridization data, this bacterium is most closely related to Thermoanaerobacter thermohydrosulfuricus and Thermoanaerobacter siderophilus (99% similarity for both). However, strain TLO differs from Thermoanaerobacter thermohydrosulfuricus in important aspects, such as CO-utilization and lipid composition. These differences led us to propose that strain TLO represents a subspecies of Thermoanaerobacter thermohydrosulfuricus, and we therefore name it Thermoanaerobacter thermohydrosulfuricus subsp. carboxydovorans.

[Keratinase of an anaerobic thermophilic bacterium Thermoanaerobacter sp. strain 1004-09 isolated from a hot spring in the Baikal Rift zone].

A thermophilic anaerobic bacterial strain 1004-09 belonging to the genus Thermoanaerobacter and capable of growth on protein substrates such as albumin, gelatin, casein, and alpha and beta-keratins was isolated from the Urinskii hot spring (Barguzin river valley, Republic of Buryatia, Russia). A 150-kDa serine proteinase was revealed in the strain supernatant; it exhibited optimal activity at 60 degrees C and pH 9.3 and was capable of keratin hydrolysis. A number of characteristics for the strain 1004-09 keratinase were established including activation by SDS and NaCl and residual activity (15% to the activity of the intact protein) in the presence of 10% ethanol and acetone.

Thermoanaerobacter uzonensis sp. nov., an anaerobic thermophilic bacterium isolated from a hot spring within the Uzon Caldera, Kamchatka, Far East Russia.

Several strains of heterotrophic, anaerobic thermophilic bacteria were isolated from hot springs of the Uzon Caldera, Kamchatka, Far East Russia. Strain JW/IW010(T) was isolated from a hot spring within the West sector of the Eastern Thermal field, near Pulsating Spring in the Winding Creek area. Cells of strain JW/IW010(T) were straight to slightly curved rods, 0.5 mum in width and variable in length from 2 to 5 mum and occasionally up to 15 mum, and formed oval subterminal spores. Cells stained Gram-negative, but were Gram-type positive. Growth was observed between 32.5 and 69 degrees C with an optimum around 61 degrees C (no growth occurred at or below 30 degrees C, or at or above 72 degrees C). The pH(60 degrees C) range for growth was 4.2-8.9 with an optimum at 7.1 (no growth occurred at or below pH(60 degrees C) 3.9, or at 9.2 or above). The shortest observed doubling-time at pH(60 degrees C) 6.9 and 61 degrees C was 30 min. Strain JW/IW010(T) was chemo-organotrophic; yeast extract, peptone, Casamino acids and tryptone supported growth. Yeast extract was necessary for the utilization of non-proteinaceous substrates, and growth was observed with inulin, cellobiose, maltose, sucrose, glucose, fructose, galactose, mannose, xylose, trehalose, mannitol, pyruvate and crotonate. The G+C content of the genomic DNA of strain JW/IW010(T) was 33.6 mol% (HPLC method). The major phospholipid fatty acids were iso-15 : 0 (53.5 %), 15 : 0 (11.8 %), 16 : 0 (7.3 %), 10-methyl 16 : 0 (7.3 %) and anteiso-15 : 0 (5.3 %). 16S rRNA gene sequence analysis placed strain JW/IW010(T) in the genus Thermoanaerobacter of the family 'Thermoanaerobacteriaceae' (Firmicutes), with Thermoanaerobacter sulfurigignens JW/SL-NZ826(T) (97 % 16S rRNA gene sequence similarity) and Thermoanaerobacter kivui DSM 2030(T) (94.5 %) as the closest phylogenetic relatives with validly published names. The level of DNA-DNA relatedness between strain JW/IW010(T) and Thermoanaerobacter sulfurigignens JW/SL-NZ826(T) was 64 %. Based on the physiological, phylogenetic and genotypic data, strain JW/IW010(T) represents a novel taxon, for which the name Thermoanaerobacter uzonensis sp. nov. is proposed. The type strain is JW/IW010(T) (=ATCC BAA-1464(T)=DSM 18761(T)). The effectively published strain, 1501/60, of 'Clostridium uzonii' [Krivenko, V. V., Vadachloriya, R. M., Chermykh, N. A., Mityushina, L. L. & Krasilnikova, E. N. (1990). Microbiology (English translation of Mikrobiologiia) 59, 741-748] had approximately 88.0 % DNA-DNA relatedness with strain JW/IW010(T) and was included in the novel taxon.

[Isolation and characterization of Thermoanaerobacter mathranii SC-2 from oil-field water].

OBJECTIVE: We studied physiological, biochemical properties and metabolites of Thermoanaerobacter mathranii SC-2 from oil-field water in Shengli oilfield. METHODS: Strain SC-2 was isolated by Hungate anaerobic technique. Through physiological, biochemical and phylogenetic analysis, the strain was identified. Metabolites were analyzed by gas chromatogram. RESULTS: The cells were Gram-negative, rod-shaped, spore-forming. Growth was observed in the temperature range from 40 to 75degrees C (optimum 70 degrees C) and pH range from 5.5 to 9.5 (optimum 6.5). The isolate grew in the presence of 0%-5% NaCl with an optimum without NaCl at pH 7.0 and 65 degrees C. Strain SC-2 used many carbohydrates as carbon sources, including glucose and xylose. Metabolites of glucose were ethanol, acetate, propionate, lactate, CO2 and H2. Based on 16S rDNA studies, strain SC-2 was most close to T. mathranii subsp. mathranii11246T with 99.85% similarity. More ethanol and acetate were produced at initial pH 8.0 than yields at other pH. Yeast extract could significantly increase ethanol and acetate yields. In addition, ethanol (4%) added in the medium obviously inhibited its growth. CONCLUSION: Strain SC-2 was extremely thermophilic, halotolerant anaerobe.

The backbone structure of the thermophilic Thermoanaerobacter tengcongensis ribose binding protein is essentially identical to its mesophilic E. coli homolog.

BACKGROUND: Comparison of experimentally determined mesophilic and thermophilic homologous protein structures is an important tool for understanding the mechanisms that contribute to thermal stability. Of particular interest are pairs of homologous structures that are structurally very similar, but differ significantly in thermal stability. RESULTS: We report the X-ray crystal structure of a Thermoanaerobacter tengcongensis ribose binding protein (tteRBP) determined to 1.9 A resolution. We find that tteRBP is significantly more stable (appTm value approximately 102 degrees C) than the mesophilic Escherichia coli ribose binding protein (ecRBP) (appTm value ~56 degrees C). The tteRBP has essentially the identical backbone conformation (0.41 A RMSD of 235/271 Calpha positions and 0.65 A RMSD of 270/271 Calpha positions) as ecRBP. Classification of the amino acid substitutions as a function of structure therefore allows the identification of amino acids which potentially contribute to the observed thermal stability of tteRBP in the absence of large structural heterogeneities. CONCLUSION: The near identity of backbone structures of this pair of proteins entails that the significant differences in their thermal stabilities are encoded exclusively by the identity of the amino acid side-chains. Furthermore, the degree of sequence divergence is strongly correlated with structure; with a high degree of conservation in the core progressing to increased diversity in the boundary and surface regions. Different factors that may possibly contribute to thermal stability appear to be differentially encoded in each of these regions of the protein. The tteRBP/ecRBP pair therefore offers an opportunity to dissect contributions to thermal stability by side-chains alone in the absence of large structural differences.

Thermoanaerobacter pseudethanolicus sp. nov., a thermophilic heterotrophic anaerobe from Yellowstone National Park.

Strain 39E(T), originally characterized as Clostridium thermohydrosulfuricum strain 39E and later renamed as Thermoanaerobacter ethanolicus strain 39E, shows less than 97 % 16S rRNA gene sequence similarity with the type strain of the type species of the genus Thermoanaerobacter, T. ethanolicus strain JW 200(T). On the basis of a polyphasic analysis that included DNA-DNA hybridization studies with the subspecies of Thermoanaerobacter brockii, its closest phylogenetic relatives, strain 39E(T) represents a novel species of the genus Thermoanaerobacter, for which the name Thermoanaerobacter pseudethanolicus sp. nov. is proposed. The type strain is 39E(T) (=DSM 2355(T)=ATCC 33223(T)).

Thermoanaerobacter sulfurigignens sp. nov., an anaerobic thermophilic bacterium that reduces 1 M thiosulfate to elemental sulfur and tolerates 90 mM sulfite.

Two anaerobic thermophilic bacteria, designated strains JW/SL824 and JW/SL-NZ826(T), were isolated from an acidic volcanic steam outlet on White Island, New Zealand. Cells were rod-shaped, spore-forming, motile and Gram-stain negative, but contained Gram-type positive cell wall. Strain JW/SL-NZ826(T) utilized various carbohydrates including xylose and glucose. The fermentation end products produced from glucose in the absence of thiosulfate were lactate, ethanol, acetate, CO(2) and H(2). The temperature range for growth was 34-72 degrees C, with an optimum at 63-67 degrees C. The pH(60 degrees C) range for growth was 4.0-8.0, with an optimum at 5.0-6.5. The doubling time of strain JW/SL-NZ826(T) under optimal growth conditions was 2.4 h. The DNA G+C content was 34-35 mol% (HPLC). The two strains reduced up to 1 M thiosulfate to elemental sulfur without sulfide formation, which is a trend typically observed among species belonging to the genus Thermoanaerobacterium. Sulfur globules containing short and long sulfur chains but no S(8)-ring sulfur were produced inside and outside the cells. Up to 90 mM sulfite was tolerated. This tolerance is assumed to be an adaptation to the geochemistry of the environment of White Island. The 16S rRNA gene sequence analysis, however, indicated that the two strains belonged to the genus Thermoanaerobacter, with similarities in the range 95.6-92.7 %. Therefore, strains JW/SL-NZ824 and JW/SL-NZ826(T) represent a novel taxon, for which the name Thermoanaerobacter sulfurigignens sp. nov. is proposed, with strain JW/SL-NZ826(T) (=ATCC 700320(T)=DSM 17917(T)) as the type strain. Based on this and previous studies, an emended description of the genus Thermoanaerobacter is given.