Ambar SUSILORUKMI Diversity and Physiology of Syntrophic Substrate-Oxidizing Anaerobes in Methanogenic Ecosystems. Akiyoshi OHASHI, Hideki HARADA Syntrophic bacteria are among the most important microbial populations in the degradation of complex organic compounds in methanogenic ecosystems. Despite their significant metabolic functions in methanogenic ecosystem, information on the physiological characteristics and phylogenetic diversity of such organisms was limited, especially due to the fastidious characteristics of the syntrophic microbes, such as slow growth, low growth yield and syntrophic association with hydrogenotrophic microbes. In this study, the diversity and physiology of syntrophic substrate-oxidizing anaerobes were studied by applying conventional cultivation techniques combined with rRNA-based molecular approaches. For cultivation syntrophic bacteria, various anaerobic samples were used as inoculum in primary enrichments with ethanol, benzoate and propionate as the substrate. The almost all enrichment cultures comprised of F420 autofluorescent methanogen-like cells and bacterial-type cells, producing methane along with substrate depletion. To identify the syntrophic bacteria that might occur in the enrichments, 16S rDNA-based cloning analysis was cunducted for all enrichment cultures. Among the predominant clones recovered from each enrichment culture, some clones showed close relation with known bacteria to date as syntroph, such as Desulfovibrio sp. Nonetheless, several clones seemed to indicate novel bacterial lineages that have never cultivated and isolated so far, such as clones related with the genus Geobacter in two mesophilic ethanol enrichments, clones representing a deeply branched lineage of the phylum Firmicutes in a thermophilic ethanol enrichment culture, and clones related with the genus Desulfobulbus in a mesophilic propionate degrading anaerobes. To determine whether the dominant clones were derived from the dominant microbes in enrichment cultures, specific DNA probes were designed and applied for the cultures in fluorescence in situ hybridization analyses. This resulted in the detection of a number of DNA probe-reacted cells in all the cultures, suggesting the probe-positive cells were the dominant microbes in the cultures. To isolate such identified bacteria, a molecular-directed isolation strategy was applied. Through this approach, two novel syntrophic, ethanol-oxidizing bacteria and a novel benzoate-degrading bacterium were successfully isolated. This study strongly suggested that the strategy employing conventional techniques combined with 16S rRNA-based approaches is advantageous to determine the diversity of recalcitrant microbes like syntrophic microorganisms and to attempt at subsequent isolation of targeted cells.