Thermoanaerobacter Thermohydrosulfuricus Research Articles

Lipase Congeners Designed by Genetic Code Engineering

AbstractClassical enzyme optimization exploits the chemistry confined to the 20 canonical amino acids encoded by the standard genetic code. ‘Genetic code engineering’ allows the global substitution of particular residues with synthetic analogues, endowing proteins with chemical diversity not found in nature. These proteins are congeners of the parent protein because they originate from the same gene sequence, but contain a fraction of noncanonical amino acids. Global substitutions of methionine, proline, phenylalanine, and tyrosine have been carried out with related analogues in Thermoanaerobacter thermohydrosulfuricus lipase. This study represents the first extensive report of an important biocatalyst substituted with a high number of noncanonical amino acids. The generated lipase congeners displayed special features such as enhanced activation, elevated enzyme activity (by up to 25 %) and substrate tolerance (by up to 40 %), and changes in optimal temperature (by up to 20 °C) and pH (by up to 3). These emergent features achieved by genetic code engineering might be important not only for academic research, but also for numerous economical applications in the food, detergent, chemical, pharmaceutical, leather, textile, cosmetic, and paper industries.

Cloning, expression and characterization of xylose isomerase, XylA, from Caldanaerobacter subterraneus subsp. yonseiensis

The xylA gene, coding for xylose isomerase, from the extreme thermophile, Caldanaerobacter subterraneus subsp. yonseiensis was cloned, sequenced, and expressed in Escherichia coli. The nucleotide sequence of the xylA gene encoded a polypeptide of 438 residues with a calculated molecular weight of 50,170 Da. The purified XylA showed high sequence homology (92% identity) with that of Thermoanaerobacter thermohydrosulfuricus. The recombinant enzyme expressed in Escherichia coli was purified by heat treatment and gel chromatography. The purified enzyme was thermostable with optimal activity at 95 degrees C. The enzyme required divalent cations including Zn(2+) for its maximal activity and thermostability.

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 Ayaş, Turkey. The cells were straight to curved rods, 0.4–0.6 μm in diameter and 3.5–10 μm in length. Spores were terminal and round. The temperature range for growth was 40–80°C, with an optimum at 70°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, H2, and CO2. 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 H2 and CO2 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.

Thermostable lipases from the extreme thermophilic anaerobic bacteria Thermoanaerobacter thermohydrosulfuricus SOL1 and Caldanaerobacter subterraneus subsp. tengcongensis

Two novel genes encoding for heat and solvent stable lipases from strictly anaerobic extreme thermophilic bacteria Thermoanaerobacter thermohydrosulfuricus (LipTth) and Caldanaerobacter subterraneus subsp. tengcongensis (LipCst) were successfully cloned and expressed in E. coli. Recombinant proteins were purified to homogeneity by heat precipitation, hydrophobic interaction, and gel filtration chromatography. Unlike the enzymes from mesophile counterparts, enzymatic activity was measured at a broad temperature and pH range, between 40 and 90°C and between pH 6.5 and 10; the half-life of the enzymes at 75°C and pH 8.0 was 48 h. Inhibition was observed with 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride and phenylmethylsulfonylfluorid indicating that serine and thiol groups play a role in the active site of the enzymes. Gene sequence comparisons indicated very low identity to already described lipases from mesophilic and psychrophilic microorganisms. By optimal cultivation of E. coli Tuner (DE3) cells in 2-l bioreactors, a massive production of the recombinant lipases was achieved (53–2200 U/l) Unlike known lipases, the purified robust proteins are resistant against a large number of organic solvents (up to 99%) and detergents, and show activity toward a broad range of substrates, including triacylglycerols, monoacylglycerols, esters of secondary alcohols, and p-nitrophenyl esters. Furthermore, the enzyme from T. thermohydrosulfuricus is suitable for the production of optically pure compounds since it is highly S-stereoselective toward esters of secondary alcohols. The observed E values for but-3-yn-2-ol butyrate and but-3-yn-2-ol acetate of 21 and 16, respectively, make these enzymes ideal candidates for kinetic resolution of synthetically useful compounds.

Comparative performance and microbial diversity of hyperthermophilic and thermophilic co-digestion of kitchen garbage and excess sludge

The objective of this study was to evaluate the performance characteristics of a hyperthermophilic digester system that consists of an acidogenic reactor operated at hyperthermophilic (70°C) conditions in series with a methane reactor operated at mesophilic (35°C), thermophilic (55°C), and hyperthermophilic (65°C) conditions. Lab-scale reactors were operated continuously, and were fed with co-substrates composed of artificial kitchen garbage (TS 9.8%) and excess sludge (TS 0.5%) at the volumetric ratio of 20:80. In the acidification step, COD solubilization was in the range of 22–46% at 70°C, while it was 21–29% at 55°C. The average protein solubilization was 44% at 70°C. The double bond fatty acid removal ratio at 70°C was much higher than at 55°C. These results suggested that the optimal operation conditions for the acidogenic fermenter were about 3.1 days of HRT and 4 days of SRT at 70°C. Methane conversion efficiency and the VS removal percentage in the methanogenic step following acidification was around 65% and 64% on average at 55°C, respectively. The optimal operational conditions for this system are acidogenesis performed at 70°C and methanogenesis at 55°C. The key microbes determined in the hyperthermophilic acidification step were Anaerobic thermophile IC-BH at 6.4 days of HRT and Thermoanaerobacter thermohydrosulfuricus DSM 567 at 2.4 days of HRT. These results indicated that the hyperthermophilic system provides considerable advantages in treating co-substrates containing high concentrations of proteins, lipids, and nonbiodegradable solid matter.

Bioprospecting Thermophilic Microorganisms from Icelandic Hot Springs for Hydrogen and Ethanol Production

Fermentations can be used to produce sustainable energy carriers, such as hydrogen and ethanol (EtOH), from biomass or organic waste materials. The aim of this research was to prospect efficient H2- and EtOH-producing thermophilic microorganisms derived from hot spring environments in Iceland. Hydrogen- and EtOH-producing enrichment cultures were obtained from various hot spring samples over a temperature range of 50–78 °C. The temperature dependencies for the most promising enrichments were determined with a temperature-gradient incubator. One of the enrichments (33HL) produced 2.10 mol of H2/mol of glucose at 59 °C. Another enrichment (9HG), dominated by bacteria closely affiliated with Thermoanaerobacter thermohydrosulfuricus, produced 0.68 mol of H2/mol of glucose, and 1.21 mol of EtOH/mol of glucose at 78 °C. Hydrogen and EtOH production by 9HG was characterized further in a continuous-flow bioreactor at 74 °C. The highest H2 and EtOH yields of 9HG were obtained at pH 6.8 ± 0.3. Lactate production de...

Isolation from canned foods of a novel Thermoanaerobacter species phylogenetically related to Thermoanaerobacter mathranii (Larsen 1997): Emendation of the species description and proposal of Thermoanaerobacter mathranii subsp. Alimentarius subsp. Nov

Several anaerobic, thermophilic, Gram-positive bacteria were isolated from dairy products and canned meats. While some isolates were identified as Thermoanaerobacter thermohydrosulfuricus, comparisons of 16S rDNA genes indicated that others were phylogenetically closely related to Thermoanaerobacter mathranii, and more distantly related to Thermoanaerobacter thermocopriae and Thermoanaerobacter italicus. Biochemical characteristics, phylogenetic analysis, G+C content, and DNA–DNA hybridization experiments demonstrated that the strains AIP 504.99, AIP 505.99 T and AIP 431.03, notwithstanding their high sequence similarities differ from T. mathranii and represent a novel T. mathranii subspecies for which the name T. mathranii subsp. alimentarius is proposed. The type strain is strain AIP 505.99 T=CIP 108280 T=CCUG 49566 T. Emendation of the species description for T. mathranii is proposed to include this subspecies.

Effect of zinc on ethanol production by two Thermoanaerobacter strains

Ethanol production ratios of Thermoanaerobacter ethanolicus (JW200) and two other thermophilic anaerobic bacteria Thermoanaerobacter (65-2) and Thermoanaerobacter thermohydrosulfuricus (70-1) were determined at different sucroses (15, 20 and 30 g l −1) and molasses (30, 40 and 60 g l −1) concentrations, in batch culture conditions. As a result, bacterial growth and ethanol production ratios were optimum at the same conditions. Also, effect of zinc as ZnSO 4·7H 2O on the ethanol formation of bacteria were investigated and it was found that, addition of 0.17 g l −1 of ZnSO 4·7H 2O to the fermentation medium of T. ethanolicus (JW200) was increased the ethanol ratio by ca. 39% and 23% for Thermoanaerobacter strain 65-2. Addition of 0.22 g l −1 ZnSO 4·7H 2O, increased the ethanol ratio by 80% for T. thermohydrosulfuricus (70-1). The highest ethanol ratio was obtained at the 20 g l −1 sucrose and 40 g l −1 molasses concentrations by all the bacteria studied and ethanol production ratio was not proportional to substrate concentration. T. ethanolicus (JW200) produced 5.04 g l −1 ethanol from sucrose (20 g l −1) and Thermoanaerobacter strain (65-2) produced 6.50 g l −1 ethanol from 40 g l −1 molasses in modified medium 2. T. ethanolicus (JW200) yielded ( Y x/s) up to 0.47 of ethanol at 15 g l −1 sucrose concentration.

Development of affinity microparticles for extracorporeal blood purification based on crystalline bacterial cell surface proteins.

In this article, the development of specific adsorbents for extracorporeal blood purification are described. Affinity microparticles were prepared by linking Protein A to crystalline cell surface layers (S-layers) from Thermoanaerobacter thermohydrosulfuricus 1111-69. S-layers were used in the form of cell wall fragments obtained by breaking whole cells by ultrasonification, resulting in cup-shaped structures (average size 0.5 x 1 microm) completely covered with S-layer protein. Protein A was covalently bound to carboxylic acid groups of the S-layer protein after activation with 1-ethyl-3,3'(dimethylamino)propylcarbodiimide. In batch adsorption experiments with fresh frozen human plasma, the resulting S-layer based affinity microparticles showed a high adsorption capacity for IgG (40 mg IgG were bound per g wet pellet of S-layer based affinity microparticles). Fractions eluted from the microparticles were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. They contained only IgG demonstrating that adsorption was specific. In biocompatibility tests, preparations of the S-layer microparticles showed no low-density lipoprotein-reactivity, no cytotoxicity, and no cytokine inducing activity.

Differentiation of Thermophilic Anaerobic Gram-positive Bacteria by Random Amplified Polymorphic DNA Analysis.

Random amplified polymorphic DNA (RAPD) analysis was applied to the differentiation of 11 thermophilic clostridia species, 11 type strains and 14 reference strains, at the species level. The 6 primers used in amplification generated 2 to 11 bands with 170-3,300 bp that were different depending on each of the strains and the primers used. From a comparison of RAPD fingerprints: sizes, numbers, and intensities of bands, it was demonstrated that (Clostridium) thermocellum, (Clostridium) thermolacticum, Moorella thermoacetica, and Thermoanaerobacterium thermosaccharolyticum were genotypically homogeneous at each species level. Each species showed species-specific and -common bands in RAPD profiles that were most pertinent for the discrimination of strains at the species level. In contrast, it was shown that each species of Thermoanaerobacter thermohydrosulfuricus, Thermoanaerobacter thermocopriae and Thermoanaerobacterium thermosulfurigenes was genotypically heterogeneous because none of the 6 primers produced species-common band patterns. However, each type strain of thermophilic clostridia species gave different RAPD fingerprints with each primer. Results obtained by the RAPD analysis therefore complement and support the current classification of these species and this study also shows that RAPD has the potential to differentiate between these species.

14] Chaperonins from Thermoanaerobacter species

Publisher Summary The chapter presents a study on chaperonins from Thermoanaerobacter species. A notable difference between the Escherichia coli GroE proteins and the Thermus chaperonins is that whereas the former interact only in the presence of ATP, the latter interact enough strongly to be isolated together as a complex (cpn60) 14 (cpnl0) 7 . Thermus species are aerobic bacteria, found free living in many hot water environments, there are many anaerobic thermophilic bacteria occurring in more sheltered unaerated parts of hot springs. Of the most common are strains of Thermoanaerobacter , one of which, Thermoanaerobacter thermohydrosulfuricus , often makes up more than 90% of the microbial biomass in these environments. On the basis of its ability to form spores it was originally called Clostridium thermohydrosulfuricum , which has now been reclassified along with a number of other organisms in the Thermoanaerobacter genus. In addition, the much studied and closely related strain of this species Thermoanaerobium brockii, which has been reclassified as Thermoanaerobacter brockii is the presented in the chapter.

A kinetic model of growth and product formation of the anaerobic microorganism Thermoanaerobacter thermohydrosulfuricus

A model is developed to describe the growth, product formation and enzyme synthesis of the microorganism Thermoanaerobacter thermohydrosulfuricus. It differentiates between extracellular enzymatic starch degradation and the microorganism's constant specific growth rate, combined with the ingestion of maltose and maltotriose (the products of starch cleavage). The formation of growth dependent products like ethanol, acetate and the synthesis of the cell-associated enzyme, amylopullulanase, are formulated with constant yield coefficients. In contrast, both the non-growth dependent product lactate, and extracellular enzyme activity are linked to an intracellular substrate concentration. Most of the parameters used in the model are evaluated from batch culture or literature. The calculated results are compared with experimental data. Keywords: Continuous culture; Thermoanaerobacter thermohydrosulfuricus; Starch; Model

Immunoreactivity of allergen (Bet v 1) conjugated to crystalline bacterial cell surface layers (S-layers)

Background: Crystalline cell surface layers (S-layers) from Gram-positive eubacteria had been demonstrated as carrier/adjuvants for chemically synthesized tumor-associated oligosaccharide haptens and capsular polysaccharide antigens of Streptococcus pneumoniae strains. Objectives: The applicability of S-layers as vaccine carrier for treatment of Type I allergy was investigated. Study design: Native or cross-linked S-layer self-assembly products and cell wall preparations from Bacillus sphaericus CCM 2177 and Thermoanaerobacter thermohydrosulfuricus L111-69 and L110-69 were used for immobilization of recombinant major birch pollen allergen Bet v 1. Results and conclusions: Depending on the carrier used, amounts of approximately 20–40 μg allergen per mg conjugate could be immobilized. By application of l-glutamic acid dimethyl ester as a spacer, this value could be increased approximately 10-fold. The functionality of the rBet v 1-conjugates was assessed in immunological systems. (i) The presence of intact B-cell epitopes was demonstrated in inhibition experiments using human Bet v 1-specific IgE. (ii) The rBet v 1 -S-layer conjugates were immunogenic in mice. (iii) The proliferation of rBet v 1-specific T-cell clones suggested that the peptides created by processing of immobilized Bet v 1 were similar to those derived from natural allergen. (iv) Stimulation of human allergen-specific TH2 lymphocytes with S-layer-conjugated Bet v 1 led to a modulation of the cytokine production pattern from TH2 to TH0TH1. This study indicates that S-layers may be suitable carriers for new immunotherapeutical vaccines for Type 1 hypersensitivity.

Two-dimensional paracrystalline glycoprotein S-layers as a novel matrix for the immobilization of human IgG and their use as microparticles in immunoassays

In the present study, cup-shaped 1–3 μm large cell wall fragments from Thermoanaerobacter thermohydrosulfuricus L111-69 covered with a hexagonal S-layer lattice composed of glycoprotein subunits were shown to act as a matrix for the immobilization of human IgG. After cross-linking the S-layer glycoprotein lattice with glutaraldehyde (S-layer microparticles), IgG was either bound to carbodiimide activated carboxyl groups from acidic amino acids from the protein moiety or to the carbohydrate chains activated with cyanogen bromide or oxidized with periodate. After determining the binding capacity of the S-layer lattice for human IgG, the orientation of the immobilized antibody molecules was investigated using anti-human IgG peroxidase conjugates with different specificity. Attachment of S-layer microparticles with covalently bound human IgG to microplates precoated with anti-human IgG of different specificity led to clear correlations between the amount of applied human IgG and the absorption values in the immunoassays. The steepest absorption curves were obtained when human IgG was bound to the carbohydrate chains exposed on the surface of the S-layer lattice. This confirmed that the location and the accessibility of the immobilized antibodies on S-layer microparticles is of major importance for the response in immunoassays. In addition to the high reproducibility of the amount of IgG which could be bound to the S-layer lattice and the high reproducibility of the absorption curves in the immunoassays, one major advantage of using cup-shaped S-layer microparticles can be seen in the considerable increase of the actual surface available for binding processes and immunological reactions.

Isolation and properties of acetate kinase- and phosphotransacetylase-negative mutants of Thermoanaerobacter thermohydrosulfuricus

Summary: Thermoanaerobacter thermohydrosulfuricus (Clostridium thermohydrosulfuricum) ferments carbohydrate substrates to acetate, ethanol and L-lactate. To block acetate production, mutants defective in acetate kinase and/or phosphotransacetylase were isolated after mutagenesis with N-methyl-N'-nitro-N-nitroguanidine by selection for fluoroacetate resistance and subsequent counterselection for growth on pyruvate. Upon growth on glucose, all mutants formed only minor amounts of acetate and markedly less ethanol than the wild-type. The mutants produced L-lactate as the main fermentation product, yielding up to 1-8 mol lactate (mol glucose)−1 as compared to 0-7 mol lactate (mol glucose)−1 obtained with the parent strain. Expression of the genes encoding acetate kinase and phosphotransacetylase was found to be negatively controlled by acetate.

Nutritional interdependence betweenThermoanaerobacter thermohydrosulfuricus andClostridium thermocellum

Thermoanaerobacter thermohydrosulfuricus strain YM3 andClostridium thermocellum strain YM4, obtained originally as a stable coculture, required yeast extract to grow separately. Cell-free broths ofT. thermohydrosulfuricus strain YM3 andC. thermocellum strain YM4 monocultures replaced yeast extract in supporting the growth of strains YM4 and YM3, respectively.T. thermohydrosulfuricus strain YM3 produced vitamin B6, B12 analog(s),p-aminobenzoic acid and folic acid, which were required byC. thermocellum strain, YM4. Likewise, strain YM4 produced niacin-active compound(s), thiamine, and methionine required by strain YM3.

Hyperbolic growth of Thermoanaerobacter thermohydrosulfuricus (Clostridium thermohydrosulfuricum) increases ethanol production in pH-controlled batch culture

Thermoanaerobacter thermohydrosulfuricus Rt8.B1 exhibited hyperbolic growth (i.e. a continuous rate of growth, without diauxie, during growth and utilization of two carbon sources) on mixed carbohydrate substrates when grown in pH-controlled batch culture. Hyperbolic growth was observed with xylose in combination with either glucose or cellobiose. Diauxic growth ways observed when T. thermohydrosulfuricus Rt8.B1 was grown on a glucose plus cellobiose substrate mix. The major fermentation end-products under all substrate conditions were ethanol and acetate. Ethanol production varied depending on the substrate supplied and was always greatest on mixtures that included xylose (i.e. hyperbolic growth). High ethanol-to-acetate ratios could not be explained on the basis of a greater substrate uptake and thus more ethanol production under these conditions, or by variations in the levels of acetate kinase and NADP-linked alcohol dehydrogenase synthesis. The high ethanol-to-acetate ratio could not be increased by growing T.thermohydrosulfuricus Rt8.B1 under a partial pressure of hydrogen (1 atm) or by growth at different pH. Growth under these conditions decreased the ethanol-to-acetate ratio.

Dual mechanisms of xylose uptake in the thermophilic bacteriumThermoanaerobacter thermohydrosulfuricus

Thermoanaerobacter thermohydrosulfuricus Rt8.B1 catabolized xylose by the pentose phosphate pathway, and xylose isomerase and xylulokinase were inducible. The uptake of xylose was by two low-affinity, inducible systems. Both systems were resistant to the protonophore, tetrachlorosalicylanilide, the F1F0-ATPase inhibitor, N,N-dicyclohexylcarboiimide, and the sodium/proton antiporter, monensin. The high capacity system (100 nmol min−1 (mg protein)−1) was only expressed when the bacterium was grown with a high concentration of xylose (50 mM). It took more than 60 mM xylose to saturate the high capacity system. When T. thermohydrosulfuricus was grown with a low concentration of xylose (5 mM), xylose uptake was saturated by as little as 10 mM xylose (18 nmol min−1 (mg protein)−1). Cells grown with 50 mM xylose could not transport glucose, and high capacity xylose transport was not inhibited by glucose or non-metabolizable glucose analogues. Cells grown with 5 mM xylose transported glucose at a rapid rate (30 nmol min−1 (mg protein)−1), and low capacity xylose uptake was competitively inhibited by either glucose or 2-deoxy-glucose. Because the glucose uptake of cells grown on 5 mM xylose was competitively inhibited by xylose, it appeared that the low capacity xylose uptake system was a glucose/xylose carrier.

Primary structure of the O-glycosidically linked glycan chain of the crystalline surface layer glycoprotein of Thermoanaerobacter thermohydrosulfuricus L111-69. Galactosyl tyrosine as a novel linkage unit.

The products of Pronase digestion of the crystalline surface layer (S-layer) glycoproteins of Thermoanaerobacter thermohydrosulfuricus strains L111-69 and L110-69 were isolated by gel permeation chromatography, cation exchange chromatography, chromatofocusing, and reversed phase high performance liquid chromatography. Four compounds were obtained which were analyzed by monosaccharide analysis, one- and two-dimensional 500 and 600 MHz 1H and 13C NMR spectroscopy, methylation analysis, gas-liquid chromatography/mass spectrometry, and matrix-assisted laser desorption ionization mass spectrometry. For all glycopeptides we propose the following glycan structure with galactose as the linkage sugar. [formula: see text] The isolated glycopeptides resulted from Pronase cleavage at the glycosylated tyrosine residues. Tyrosine was found as the linkage amino acid in all fractions but the remaining amino acid sequences varied, indicating the presence of different glycosylation sites in the intact S-layer glycoprotein.

Taxonomic Distinction of Saccharolytic Thermophilic Anaerobes: Description of Thermoanaerobacterium xylanolyticum gen. nov., sp. nov., and Thermoanaerobacterium saccharolyticum gen. nov., sp. nov.; Reclassification of Thermoanaerobium brockii, Clostridium thermosulfurogenes, and Clostridium thermohydrosulfuricum E100-69 as Thermoanaerobacter brockii comb. nov., Thermoanaerobacterium thermosulfurigenes comb. nov., and Thermoanaerobacter thermohydrosulfuricus comb. nov., Respectively; and Transfer of Clostridium thermohydrosulfuricum 39E to Thermoanaerobacter ethanolicus

Two thermophilic, anaerobic, xylan-degrading bacteria, strains B6A-RIT (T = type strain) and LX-11T, were isolated from Frying Pan Springs in Yellowstone National Park. These organisms grew chemoorganotrophically by utilizing xylan and starch but not cellulose, as well as a number of di- and monosaccharides, including glucose and xylose. Both organisms had the same optimum temperature and pH for growth (60°C and pH 6.0). The fermentation products included acetate, ethanol, lactate, CO2, and H2. Both organisms were rod shaped and deposited sulfur on their cells. The major difference between the two isolates was in spore formation; strain LX-11T sporulated, whereas strain B6A-RIT were compared with other thermophilic, anaerobic, xylan-degrading bacteria by performing DNA-DNA hybridizations and total protein analyses in order to determine the relationships of these organisms. Three different groups were identified, and new taxonomic assignments are proposed. Clostridium thermocellum LQRI was least closely related to the other seven strains studied and is placed in group I, retaining its original taxonomic assignment. Clostridium thermosulfurogenes 4BT and new isolates B6A-RIT are closely related and fall into group II, for which the new genus Thermoanaerobacterium is proposed. Isolate LX-11T is designated Thermoanaerobacterium xylanolyticum sp. nov., and isolate B6A-RIT is designated Thermoanaerobacterium saccharolyticum sp. nov. Thermoanaerobacterium thermosulfurigenes 4BT (originally C. thermosulfurogenes) is the type strain of the type species of the genus. Group III strains are placed in the genus Thermoanaerobacter; this group includes Thermoanaerobacter ethanolicus JW200T, Clostridium thermohydrosulfuricum 39E and E100-69T, and Thermoanaerobium brockii HTD4T. Thermoanaerobium brockii HTD4T is renamed Thermoanaerobacter thermohydrosulfuricus comb. nov. C. thermohydrosulfuricum 39E is nearly identical to Thermoanaerobacter ethanolicus JW200T, and these organisms are considered members of the same species. Therefore, C. thermohydrosulfuricum 39E is renamed Thermoanaerobacter ethanolicus 39E; strain JW200 is the type strain of Thermoanaerobacter ethanolicus.