The thermophilic, anaerobic, propionate-oxidizing bacterial populations present in the methanogenic granular sludge in a thermophilic (55°C) upflow anaerobic sludge blanket reactor were studied by cultivation and in situ hybridization analysis. For isolation of propionate-degrading microbes, primary enrichment was made with propionate as the sole energy source at 55°C. After several attempts to purify the microbes, a thermophilic, syntrophic, propionate-oxidizing bacterium, designated strain SI, was isolated in both pure culture and coculture with Methanobacterium thermoautotrophicum. Under thermophilic (55°C) conditions, strain SI oxidized propionate, 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 known species. To elucidate the abundance and spatial distribution of organisms of the strain SI type within the sludge granules, a 16S rRNA-targeted oligonucleotide probe specific for strain SI was developed and applied to thin sections of the granules. Fluorescence in situ hybridization combined with confocal laser scanning microscopy revealed that a number of rod-shaped cells were present in the middle and inner layers of the thermophilic granule sections and that they formed close associations with hydrogenotrophic methanogens. They accounted for approximately 1.1% of the total cells in the sludge. These results demonstrated that strain SI was one of the significant populations in the granular sludge and that it was responsible for propionate oxidation in the methanogenic granular sludge in the reactor.Anaerobic processes have been widely used over the past decades for the treatment of municipal and industrial wastewaters as well as solid wastes such as sewage sludge (16,32). Over decades, a number of technologies for anaerobic treatment have been created (32), and applications of these processes are now being extended to more complex wastewaters (5,11,18), to low-strength wastewaters (12, 17), and to wastes and wastewaters under extreme temperature conditions (19,26,41). Thermophilic anaerobic processes (normally operated between 50 and 60°C) are those developed in recent decades for the treatment of industrial wastes and wastewaters which are discharged at high temperatures (47, 48). In addition, because of the higher metabolic activities of thermophiles, thermophilic processes are capable of accommodating a very high loading rate at a feasible removal efficiency (43, 48, 49). Consequently, thermophilic processes also offer an attractive alternative for the treatment of middle-and high-strength wastes and wastewaters holding an ambient temperature. In spite of these advantages, the processes are often found to be less stable and more sensitive to environmental changes than mesophilic processes (42,48). In many cases, poorer effluent quality was reported for thermophilic proce...