Molecular identification of heterotrophic nitrification and aerobic denitrification bacteria: From methods development to application demonstration

Although heterotrophic nitrification and aerobic denitrification (HN-AD) bacteria, a novel functional group involved in nitrogen conversion, have been isolated and characterized, the lack of specific molecular markers for identification severely limits the study of their role in geochemical cycling and the contribution in ecosystems. Here, a set of molecular markers was developed for the rapid identification of HN-AD bacteria, via delving into the genomics and transcriptomics of a HN-AD isolate (Pseudomonas aeruginosa SNDPR-01). Among the nine candidate genes that were significantly expressed during heterotrophic nitrification, three were involved in the conversion of hydroxylamine to nitrite, a characteristic process of HN-AD. The universality and stability of the identification methods based on the gene primer set were validated using pure HN-AD strains, mixed cultures of pure HN-AD strains, and activated sludge from laboratory-scale and real wastewater treatment plants. In all cases, the amplification outcome was positively correlated with the function and population of HN-AD bacteria, demonstrating its validity as a molecular marker. This study supports the paradigm of heterotrophic nitrification from hydroxylamine to nitrite. As an effective tool for the identification of classic HN-AD bacteria, this study lays the groundwork for research on environmental ecology and biotechnological application of HN-AD bacteria.