Analysis of Microbial Ecological Structure of Anaerobic Granular Sludge Consortia by In-situ Hybridization of 16S rRNA targeted oligonucleotides

Whole cell in-situ hybridization with fluorescent oligonucleotides was used to study the microbial community structure of anaerobic granular sludges developed in upflow anaerobic sludge blanket (UASB) reactors receiving various types of wastewaters. The molecular probes used were complementary to conserved region of the 16S rRNA of the domain Archaea, genus Methanosaeta (Methanothrix) , and Methanobacter group which includes all species of Methanobacterium, Methanobrevibacter, Methanoshaera. The result indicated that Archaea accounted for approximately 40 to 60% of DAPI (6-diamidino-2phenylindole) stained cells (corresponding to the total cell number) in the respective granular sludges and Methanosaeta species constituted 15 to 35% of DAPI stained cells. A strong positive correlation was observed between the presence ratio of genus Methanosaeta determined by FISH and acetoclastic methanogenic activity of the respective sludges. Topology of microbial structure of two types of granules, mesophilically-grown and thermophilicallygrown granules, was investigated by dividing the individual whole-granules into the outer-layer and the innerlayer portions. The inner-layer portion contained methanogens more significantly than the outer-layer portion. Thermophilic granules had a tendency to contain higher percentage of Methanobacter group, which utilize hydrogen or formate for methanogenesis, compared with mesophilic granule. This supports our previous finding that syntrophic acetate oxidizing methanogenesis through hydrogen (or formate) formation became more prominent under thermophilic conditions.