In Situ Detection and Quantification of Uncultured Members of the Phylum Nitrospirae Abundant in Methanogenic Wastewater Treatment Systems

In upflow anaerobic sludge blanket (UASB) reactors treating high-strength organic wastewaters, uncultured phylotypes belonging to the phylum Nitrospirae were assumed to play a key role in the treatment processes because of their high abundance as determined by 16S rRNA gene-based clone library analyses (Narihiro et al. (27)). A comparative nucleotide sequence analysis of the phylotypes, commonly found in anaerobic mesophilic sludges, revealed that these phylotypes formed a coherent clade at the family to genus levels with no cultured representatives. To determine the abundance and distribution in methanogenic waste/wastewater treatment sludges, 16S rRNA gene-based quantitative PCR measurements and 16S rRNA-targeted fluorescence in situ hybridization (FISH) were carried out. A specific gene primer/probe for the uncultured clade was developed and used for quantitative PCR and in situ detection of the targeted cells within the sludge granules. In all the six UASB sludges treating food-processing organic wastewaters, the phylotypes were detected at a range of 0.1 to 10.9% of total 16S rRNA genes. FISH revealed that the gene probe reacted with coccoid or irregular coccoid-like cells in the middle and inner layers of sludge granules of a UASB reactor, in which the Nitrospirae phylotypes were found in abundance by real-time PCR analyses.