Fluorescence in situ hybridization using 16S rRNA-targeted oligonucleotides reveals localization of methanogens and selected uncultured bacteria in mesophilic and thermophilic sludge granules

16S rRNA-targeted in situ hybridization combined with confocal laser scanni ng microscopy was used to elucidate the spatial distribution of microbes wi thin two types of methanogenic granular sludge, mesophilic (35 degrees C) a nd thermophilic (55 degrees C), in upflow anaerobic sludge blanket reactors fed with sucrose-, acetate-, and propionate-based artificial wastewater. T he spatial organization of the microbes was visualized in thin sections of the granules by using fluorescent oligonucleotide probes specific to severa l phylogenetic groups of microbes. In situ hybridization with archaeal- and bacterial-domain probes within granule sections clearly showed that both m esophilic and thermophilic granules had layered structures and that the out er layer harbored mainly bacterial cells while the inner layer consisted ma inly of archaeal cells, Methanosaeta-, Methanobacterium-, Methanospirillum- , and Methanosarcina-like cells were detected with oligonucleotide probes s pecific for the different groups of methanogens, and they were found to be localized inside the granules, in both types of which dominant methanogens were members of the genus Methanosaeta, For specific detection of bacteria which were previously detected by whole-microbial-community 16S ribosomal D NA (rDNA)-cloning analysis (Y. Sekiguchi, Y. Kamagata, K. Syutsubo, A. Ohas hi, H. Harada, and K. Nakamura, Microbiology 144:2655-2665, 1998) we design ed probes specific for clonal 16S rDNAs related to unidentified green nonsu lfur bacteria and clones related to Syntrophobacter species. The probe desi gned for the cluster closely related to Syntrophobacter species hybridized with coccoid cells in the inner layer of the mesophilic granule sections. T he probe for the unidentified bacteria which were clustered with the green nonsulfur bacteria detected filamentous cells in the outermost layer of the thermophilic sludge granule sections. These results revealed the spatial o rganizations of methanogens and uncultivated bacteria and their in situ mor phologies and metabolic functions in both mesophilic and thermophilic granu lar sludges.