In situ hybridization of 16S rRNA-targeted oligonucleotide probes was used to elucidate the spacial distribution of several microorganisms within two types of granular sludge, mesophilic (35°C) and thermophilic (55°C), in upflow anaerobicsludge blanket (UASB) reactors which have been adapted to the identical composition of artificial wastewater. To visualize the microbial distributions, sectionsof both granules were hybridized with fluorescent oligonucleotide probes, and viewed by confocal laser scanning microscopy (CLSM). Hybridization of oligonucleotide probes for Archaea and Bacteria with microbes in the granule sections clearly showed the layered structure of both granules, indicating that the outer layerof the granules harbors mainly bacterial cells, and the inward consists of mainly archaeal cells. Methanosaeta, Methanobacterium, Methanospirillum, and Methanosarcina-like cells were detected and localized by the probes for several phylogenetic groups of methanogens, in which the most dominant methanogens in both granules were related with the genus Methanosaeta. For the detection of some unknown bacteria which were suggested to be present in the granules by 16S rDNA-based cloning analysis, we designed probes specific for clonal 16S rDNAs relative to thegreen non-sulfur bacteria, and clonal rDNAs which are relatives of the Syntrophobacter species. Hybridization of the probes for both granule sections showed particular locations of the targeted cells. The cells related with the green non-sulfur bacteria were detected as filamentous, and located in the most outer layer of the thermophilic granule sections. On the other hand, the cells related with Syntrophobacter species were coccoides, and located in the inward of only the mesophilic granules. These observations demonstrated a part of the layered structure, and revealed the overall microbial topography of both granules.
