Denitrification is of crucial importance for nitrogen removal in wastewater treatment. However, due to the polyphyletic taxonomy of denitrifiers, little is known about their community composition and ecophysiology and the available knowledge derives mainly from culture-dependent studies or enriched reactor studies. To obtain better identification of active denitrifying communities in full-scale wastewater treatment plants (WWTPs) we applied DNA-SIP with 13C-labelled substrates, and RT-PCR of expressed denitrification genes (nirS, nirK and nosZ) upon various substrate-inductions. To come around the bias of horizontal gene transfer, the identities were verified by microautoradiography (MAR) combined with fluorescence in situ hybridization (FISH) with incorporation of radio-labelled substrates under denitrifying conditions. The in situ abundances of the identified denitrifiers in different WWTPs were determined with quantitative FISH, while their active metabolic pathways were investigated directly in activated sludge with a tag-based metatranscriptomic approach under acetate-utilizing and denitrifying conditions. The different methods revealed a majority of denitrifiers in all WWTPs belonging to Betaproteobacteria (Acidovorax, Azoarcus, Curvibacter and Thauera) and to a lesser extent Alphaproteobacteria (Paracoccus), while few denitrifying Gammaproteobacteria and Firmicutes were identified. A taxonomic discrepancy of the denitrifying communities was highly correlated to the configuration of the WWTPs and when external carbon sources were supplemented to the activated sludge the composition of the denitrifying communities was significantly affected. Transcriptome profiling provided detailed insight in the metabolic pathways in several of the active denitrifiers in activated sludge. In conclusion, this study has provided novel leads for the identity and distribution of denitrifiers in WWTPs as well as their physiological and metabolic capabilities in activated sludge.
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13th International Symposium on Microbial Ecology, 2010