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Thermodesulfovibrio aggregans sp. nov. and Thermodesulfovibrio thiophilus sp. nov., anaerobic, thermophilic, sulfate-reducing bacteria isolated from thermophilic methanogenic sludge, and emended description of the genus Thermodesulfovibrio

¹ Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan
² Subground Animalcule Retrieval (SUGAR) Program, Extremobiosphere Research Center, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka, Kanagawa 237-0061, Japan
³ Department of Social and Environmental Systems Engineering, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8527, Japan
⁴ Department of Civil Engineering, Tohoku University, Sendai, Miyagi 980-8579, Japan
⁵ Research Institute of Genome-Based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo, Hokkaido 062-8517, Japan

Correspondence
Yuji Sekiguchi
y.sekiguchi{at}aist.go.jp

Four obligately anaerobic, thermophilic, sulfate-reducing bacterial strains, designated TGE-P1^T, TDV^T, TGL-LS1 and TSL-P1, were isolated from thermophilic (operated at 55 °C) methanogenic sludges from waste and wastewater treatment. The optimum temperature for growth of all the strains was in the range 55–60 °C. The four strains grew by reduction of sulfate with a limited range of electron donors, such as hydrogen, formate, pyruvate and lactate. In co-culture with the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus H^T, strains TGE-P1^T, TGL-LS1 and TSL-P1 were able to utilize lactate syntrophically for growth. The DNA G+C contents of all the strains were in the range 34–35 mol%. The major cellular fatty acids of the strains were iso-C_{17 : 0}, iso-C_{16 : 0}, C_{16 : 0} and anteiso-C_{15 : 0}. Phylogenetic analyses based on 16S rRNA gene sequences revealed that the strains belong to the Thermodesulfovibrio clade of the phylum ‘Nitrospirae’. On the basis of their physiological, chemotaxonomic and genetic properties, strains TGL-LS1 (=JCM 13214) and TSL-P1 (=JCM 13215) were classified as strains of Thermodesulfovibrio islandicus. Two novel species of the genus Thermodesulfovibrio are proposed to accommodate the other two isolates: Thermodesulfovibrio aggregans sp. nov. (type strain TGE-P1^T =JCM 13213^T =DSM 17283^T) and Thermodesulfovibrio thiophilus sp. nov. (type strain TDV^T =JCM 13216^T =DSM 17215^T). To examine the ecological aspects of Thermodesulfovibrio-type cells in the sludge from which the strains were originally isolated, an oligonucleotide probe targeting 16S rRNA of all Thermodesulfovibrio species was designed and applied to thin sections of thermophilic sludge granules. Fluorescence in situ hybridization using the probe revealed rod- or vibrio-shaped cells as a significant population within the sludge, indicating their important role in the original ecosystem.

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains TGE-P1^T, TDV^T, TGL-LS1 and TSL-P1 and Thermodesulfovibrio yellowstonii DSM 11347^T are AB021302, AB231857, AB021303, AB021304 and AB231858, respectively.

Photomicrographs of the strains isolated in this study, graphs showing lactate degradation by strain TGE-P1^T in the presence and absence of Methanothermobacter thermautotrophicus, in situ hybridization of sections from thermophilic granules viewed by CLSM and probe/target sequences of TDV1015 are available as supplementary material with the online version of this paper.