Molecular characterization of microbial community in nitrate-removing activated sludge.

Abstract The microbial community composition and dominant denitrifying populations in high-nitrate-removing (CR-I) and low-nitrate-removing (CR-II) activated sludge from continuous bioreactors were investigated with most probable number (MPN) enumeration, fluorescence in situ hybridization (FISH) and 16S rDNA characterization. MPNs of nitrate-reducing bacteria of sludge CR-I and sludge CR-II were 2.82x10(7) and 2.69x10(4) colony-forming units ml(-1), respectively. Eight denitrifying bacteria and two nitrate-reducing bacteria were isolated from sludge CR-I, and four denitrifying bacteria and three nitrate-reducing bacteria from sludge CR-II. Small subunit rDNA characterization of the isolates showed that the majority belonged to the genus Pseudomonas. By using FISH up to 76% (CR-I) and 52% (CR-II) of total 4,6-diamidino-2-phenylindole cell counts hybridized to the bacterial probe EUB338. Members of beta-Proteobacteria were the most abundant proteobacterial group in both sludges, accounting for up to 41.6% and 37.1% of those detected by EUB338, respectively, whereas a higher number of Cytophaga-Flexibacter cluster members were observed in CR-I sludge compared to CR-II sludge. In contrast with culture-based results, the numbers of rRNA group I Pseudomonads accounted for less than 0.01% of those detected by EUB338 in both sludges. Ribosomal DNA clone library analysis showed that the beta-Proteobacteria were also dominant in both sludges. In CR-I sludge, they were related to Zooglorea ramigera, Alcaligenes defragrans, denitrifying Fe-oxidizing bacteria and Dechlorimonas sp., whereas in CR-II sludge, they were related to Nitrosomonas sp. and Dechlorimonas agitatus. When this reactor was operated under anaerobic and anoxic conditions, nitrifying bacteria could adapt to the anoxic environment. We inferred that anaerobic ammonium oxidation and nitrite oxidation may occur in low-nitrate-removing sludge CR-II and inhibit denitrification.