Who is actively denitrifying in activated sludge?

Denitrification is of crucial importance in nitrogen removal from wastewater. However, due to the polyphyletic taxonomy of denitrifiers, little is known about the composition and ecophysiology of the actively denitrifying community in activated sludge. To identify the active denitrifiers in a full-scale wastewater treatment plant the transcripts (mRNA) of the nirS, nirK and nosZ denitrification genes expressed under acetate or amino acid consumption were amplified, sequenced and identified. This revealed that the majority of the denitrifiers belonged to Alpha- and Betaproteobacteria, while only few transcripts came from Gammaproteobacteria. A clear taxonomic discrepancy was observed between the nirS and nirK expressing bacteria, which primarily belonged to Beta- and Alphaproteobacteria, respectively. The nosZ gene was expressed by both taxonomic groups and it was therefore surprising that the highest genetic diversity was observed from the nirS transcripts and not the nosZ transcripts. Likewise, denitrifying cultures obtained from the activated sludge affiliated with the same Alpha- and Betaproteobacteria as detected with the denitrification genes, except one culture, which affiliated with Bacteroidetes. Furthermore, potential denitrifying genera of Alpha- and Betaproteobacteria were quantified in the activated sludge with 16S rRNA gene probes for fluorescence in situ hybridization (FISH). This revealed that Aquaspirillum-related bacteria were dominant followed by bacteria related to Azoarcus, Thauera and Paracoccus, respectively. Few bacteria were related to Acidovorax, Zoogloea and Rhodobacter. Except from Aquaspirillum, these genera were also identified with the denitrification genes. The substrate preferences of the bacteria affiliated with these genera were further evaluated by microautoradiography combined with FISH.