Melike Balk

Thermotoga lettingae sp. nov., a novel thermophilic, methanol-degrading bacterium isolated from a thermophilic anaerobic reactor.

A novel, anaerobic, non-spore-forming, mobile, Gram-negative, thermophilic bacterium, strain TMOT, was isolated from a thermophilic sulfate-reducing bioreactor operated at 65 C with methanol as the sole substrate. The G+C content of the DNA of strain TMOT was 39.2 mol%. The optimum pH, NaCl concentration, and temperature for growth were 7.0, 1.0%, and 65 degrees C, respectively. Strain TMOT was able to degrade methanol to CO2 and H2 in syntrophic culture with Methanothermobacter thermautotrophicus AH or Thermodesulfovibrio yellowstonii. Thiosulfate, elemental sulfur, Fe(III) and anthraquinone-2,6-disulfonate were able to serve as electron acceptors during methanol degradation. In the presence of thiosulfate or elemental sulfur, methanol was converted to CO2 and partly to alanine. In pure culture, strain TMOT was also able to ferment methanol to acetate, CO2 and H2. However, this degradation occurred slower than in syntrophic cultures or in the presence of electron acceptors. Yeast extract was required for growth. Besides growing on methanol, strain TMOT grew by fermentation on a variety of carbohydrates including monomeric and oligomeric sugars, starch and xylan. Acetate, alanine, CO2, H2, and traces of ethanol, lactate and alpha-aminobutyrate were produced during glucose fermentation. Comparison of 16S rDNA genes revealed that strain TMOT is related to Thermotoga subterranea (98%) and Thermotoga elfii (98%). The type strain is TMOT (= DSM 14385T = ATCC BAA-301T). On the basis of the fact that these organisms differ physiologically from strain TMOT, it is proposed that strain TMOT be classified as a new species, within the genus Thermotoga, as Thermotoga lettingae.

Constraints on the biological source(s) of the orphan branched tetraether membrane lipids

A soil profile from the Saxnäs Mosse peat bog, Sweden, has been analysed for glycerol dialkyl glycerol tetraether (GDGT) membrane lipids and 16S rRNA genes in order to constrain the source of the yet ‘orphan,’ but supposedly bacterial, branched GDGTs. Branched GDGT lipids dominate over archaeal membrane lipids. The Acidobacteria comprise the dominant bacterial group, accounting for the majority of total Bacteria, and are generally more abundant than methanogenic archaea. Analysed acidobacterial strains did not contain branched GDGT lipids. Thus, the source organism must likely be searched for in other acidobacterial phyla or in another abundant group within the remaining bacteria.

Desulfotomaculum thermobenzoicum subsp. thermosyntrophicum subsp. nov., a thermophilic, syntrophic, propionate-oxidizing, spore-forming bacterium.

From granular sludge from a laboratory-scale upflow anaerobic sludge bed reactor operated at 55 degrees C with a mixture of volatile fatty acids as feed, a novel anaerobic, moderately thermophilic, syntrophic, spore-forming bacterium, strain TPO, was enriched on propionate in co-culture with Methanobacterium thermoautotrophicum Z245. The axenic culture was obtained by using pyruvate as the sole source of carbon and energy. The cells were straight rods with pointed ends and became lens-shaped when sporulation started. The cells were slightly motile. The optimum growth temperature was 55 degrees C and growth was possible between 45 and 62 degrees C. The pH range for growth of strain TPO was 6-8, with an optimum at pH 7-7.5. Propionate was converted to acetate, CO2 and CH4 by a co-culture of strain TPO with Methanobacterium thermoautotrophicum Z245. In pure culture, strain TPO could grow fermentatively on benzoate, fumarate, H2/CO2, pyruvate and lactate. Sulphate could serve as inorganic electron acceptor when strain TPO was grown on propionate, lactate, pyruvate and H2/CO2. The G+C content was 53.7 mol%. Comparison of 16S rDNA sequences revealed that strain TPO is related to Desulfotomaculum thermobenzoicum (98%) and Desulfotomaculum thermoacetoxidans (98%). DNA-DNA hybridization revealed 88.2% reassociation between strain TPO and D. thermobenzoicum and 83.8% between strain TPO and D. thermoacetoxidans. However, both organisms differ physiologically from strain TPO and are not capable of syntrophic propionate oxidation. It is proposed that strain TPO should be classified as new subspecies of D. thermobenzoicum as D. thermobenzoicum subsp. thermosyntrophicum.

(Per)chlorate Reduction by the Thermophilic Bacterium Moorella perchloratireducens sp. nov., Isolated from Underground Gas Storage

ABSTRACT A thermophilic bacterium, strain An10, was isolated from underground gas storage with methanol as a substrate and perchlorate as an electron acceptor. Cells were gram-positive straight rods, 0.4 to 0.6 μm in diameter and 2 to 8 μm in length, growing as single cells or in pairs. Spores were terminal with a bulged sporangium. The temperature range for growth was 40 to 70°C, with an optimum at 55 to 60°C. The pH optimum was around 7. The salinity range for growth was between 0 and 40 g NaCl liter−1 with an optimum at 10 g liter−1. Strain An10 was able to grow on CO, methanol, pyruvate, glucose, fructose, cellobiose, mannose, xylose, and pectin. The isolate was able to respire with (per)chlorate, nitrate, thiosulfate, neutralized Fe(III) complexes, and anthraquinone-2,6-disulfonate. The G+C content of the DNA was 57.6 mol%. On the basis of 16S rRNA analysis, strain An10 was most closely related to Moorella thermoacetica and Moorella thermoautotrophica. The bacterium reduced perchlorate and chlorate completely to chloride. Key enzymes, perchlorate reductase and chlorite dismutase, were detected in cell extracts. Strain An10 is the first thermophilic and gram-positive bacterium with the ability to use (per)chlorate as a terminal electron acceptor.

Gelria glutamica gen. nov., sp. nov., a thermophilic, obligately syntrophic, glutamate-degrading anaerobe

A novel anaerobic, gram-positive, thermophilic, spore-forming, obligately syntrophic, glutamate-degrading bacterium, strain TGO(T), was isolated from a propionate-oxidizing methanogenic enrichment culture. The axenic culture was obtained by growing the bacterium on pyruvate. Cells were rod-shaped and non-motile. The optimal temperature for growth was 50-55 degrees C and growth occurred between 37 and 60 degrees C. The pH range for growth was 5.5-8 with optimum growth at pH 7. In pure culture, strain TGO(T) could grow on pyruvate, lactate, glycerol and several sugars. In co-culture with the hydrogenotrophic methanogen Methanobacterium thermautotrophicum strain Z-245, strain TGO(T) could grow on glutamate, proline and Casamino acids. Glutamate was converted to H2, CO2, propionate and traces of succinate. Strain TGO(T) was not able to utilize sulphate, sulphite, thiosulphate, nitrate or fumarate as electron acceptors. The G+C content was 33.8 mol%. Sequence analysis of the 16S rDNA revealed that strain TGO(T) belongs to the thermophilic, endospore-forming anaerobes, though no close relations were found. Its closest relations were Moorella glycerini (92%) and Moorella thermoacetica (90%). Strain TGOT had an unusually long 16S rDNA of more than 1700 bp. The additional base pairs were found as long loops in the V1, V7 and V9 regions of the 16S rDNA. However, the loops were not found in the 16S rRNA. The name Gelria glutamica gen. nov., sp. nov. is proposed for strain TGO(T).

Desulfatirhabdium butyrativorans gen. nov., sp. nov., a butyrate-oxidizing, sulfate-reducing bacterium isolated from an anaerobic bioreactor.

A novel sulfate-reducing bacterium, strain HB1(T), was isolated from an upflow anaerobic sludge blanket (UASB) reactor treating paper-mill wastewater operated at 37 degrees C. Cells of strain HB1(T) were oval to rod-shaped, 1-1.3 microm wide and 2.6-3.5 microm long and Gram-negative. The optimum temperature for growth was 28-30 degrees C. In the presence of sulfate, the isolate was able to grow on H(2)/acetate, formate, ethanol, propionate, fumarate, succinate, butyrate, crotonate, catechol, benzoate, 4-hydroxybenzoate, palmitate and stearate. The isolate only grew on H(2) when acetate was added as a carbon source; when grown on formate, acetate was not required. Growth was also possible on pyruvate and crotonate without an electron acceptor. The isolate showed very poor growth on acetate. Thiosulfate and sulfate were used as electron acceptors. Phylogenetic analysis of 16S rRNA gene sequences revealed that strain HB1(T) represents a novel lineage within the Deltaproteobacteria; sequence similarities between strain HB1(T) and members of other related genera were less than 91%. Strain HB1(T) was also distinguished from members of related genera based on differences in several phenotypic characteristics. It is a member of the family Desulfobacteraceae. The major cellular fatty acids of strain HB1(T) were C(16:0), iso-C(15:0), anteiso-C(15:0) and C(14:0). beta-Hydroxy fatty acids were also present in the range of C(14:0) to C(18:0), of which C(16:0) was the most abundant. The G+C content of the DNA was 55.1 mol%. Based on physiological, biochemical and chemotaxonomic traits together with results of comparative 16S rRNA gene sequence analysis, strain HB1(T) is considered to represent a novel species in a new genus, for which the name Desulfatirhabdium butyrativorans gen. nov., sp. nov. is proposed. The type strain of Desulfatirhabdium butyrativorans is HB1(T) (=DSM 18734(T) =JCM 14470(T)).

Elucidation of the pathways of catabolic glutamate conversion in three thermophilic anaerobic bacteria.

Abstract. The glutamate catabolism of three thermophilic syntrophic anaerobes was compared based on the combined use of [13C] glutamate NMR measurements and enzyme activity determinations. In some cases the uptake of intermediates from different pathways was studied. The three organisms, Caloramator coolhaasii, Thermanaerovibrio acidaminovorans and strain TGO, had a different stoichiometry of glutamate conversion and were dependent on the presence of a hydrogen scavenger (Methanobacterium thermoautotrophicum Z245) to a different degree for their growth. C. coolhaasii formed acetate, CO2, NH4+ and H2 from glutamate. Acetate was found to be formed through the β-methylaspartate pathway in pure culture as well as in coculture. T. acidaminovorans converted glutamate to acetate, propionate, CO2, NH4+ and H2. Most likely, this organism uses the β-methylaspartate pathway for acetate formation. Propionate formation occurred through a direct oxidation of glutamate via succinyl-CoA and methylmalonyl-CoA. The metabolism of T. acidaminovorans shifted in favour of propionate formation when grown in coculture with the methanogen, but this did not lead to the use of a different glutamate degradation pathway. Strain TGO, an obligate syntrophic glutamate-degrading organism, formed propionate, traces of succinate, CO2, NH4+ and H2. Glutamate was converted to propionate oxidatively via the intermediates succinyl-CoA and methylmalonyl-CoA. A minor part of the succinyl-CoA was converted to succinate and excreted.