Identification of active denitrifiers in full-scale nutrient removal wastewater treatment systems

Denitrification is essential to the removal of nitrogen from wastewater during treatment, yet an understanding of the diversity of the active denitrifying bacteria responsible in full-scale wastewater treatment plants (WWTP) is lacking. In this study stable isotope probing (SIP) was applied in combination with microautoradiography (MAR)-fluorescence in situ hybridisation (FISH) to identify the active denitrifiers in a full-scale WWTP with biological N and P removal. Recognising that a range of carbon sources likely drive denitrification, a fully 13 C-labelled complex substrate was used for SIP incubations, under nitrite reducing conditions, in order to better capture the diversity of active denitrifiers present. Members of the Rhodoferax, Dechloromonas and Sulfuritalea were well represented in the 16S rRNA gene clone libraries from DNA enriched in 13 C, with FISH probes designed here for their in situ characterisation. FISH and MAR confirmed that they were core active denitrifiers in the community. The SIP clone library was additionally represented by a phylogenetically diverse group of organisms, with many previously not considered as important denitrifiers. The combined approach of SIP and MAR-FISH represents an excellent approach for identifying and characterising un-described diversity of active denitrifiers in full-scale systems.