Cultivation and in situ detection of a thermophilic bacterium capable of oxidizing propionate in syntrophic association with hydrogenotrophic methanogens in a thermophilic methanogenic granular sludge

The thermophilic, anaerobic, propionate-oxidizing bacterial populations pre sent in the methanogenic granular sludge in a thermophilic (55 degrees C) u pflow anaerobic sludge blanket reactor were studied by cultivation and in s itu hybridization analysis. For isolation of propionate-degrading microbes, primary enrichment was made with propionate as the sole energy source at 5 5 degrees C. After several attempts to purify the microbes, a thermophilic, syntrophic, propionate-oxidizing bacterium, designated strain SI, was isol ated in both pure culture and coculture with Methanobacterium thermoautotro phicum. Under thermophilic (55 degrees C) conditions, strain SI oxidized pr opionate, ethanol, and lactate in coculture with M. thermoautotrophicum. In pure culture, the isolate was found to ferment pyruvate. 16S ribosomal DNA sequence analysis revealed that the strain was relatively close to members of the genus Desulfotomaculum, but it was only distantly related to any kn own species. To elucidate the abundance and spatial distribution of organis ms of the strain SI type within the sludge granules, a 16S rRNA-targeted ol igonucleotide probe specific for strain SI was developed and applied to thi n sections of the granules. Fluorescence in situ hybridization combined wit h confocal laser scanning microscopy revealed that a number of rod-shaped c ells were present in the middle and inner layers of the thermophilic granul e sections and that they formed close associations with hydrogenotrophic me thanogens, They accounted for approximately 1.1% of the total cells in the sludge. These results demonstrated that strain SI was one of the significan t populations in the granular sludge and that it was responsible for propio nate oxidation in the methanogenic granular sludge in the reactor.