A positive contribution to nitrogen removal by a novel NOB in a full-scale duck wastewater treatment system

Nitrite-oxidizing bacteria (NOB) are undesirable in the anaerobic ammonium oxidation (anammox)-driven nitrogen removal technologies in the modern wastewater treatment plants (WWTPs). Diverse strategies have been developed to suppress NOB based on their physiological properties that we have understood. But our knowledge of the diversity and mechanisms employed by NOB for survival in the modern WWTPs remains limited. Here, Three NOB species (NOB01-03) were recovered from the metagenomic datasets of a full-scale WWTP treating duck breeding wastewater. Among them, NOB01 and NOB02 were classified as newly identified lineage VII, tentatively named Candidatus (Ca.) Nitrospira NOB01 and Ca. Nitrospira NOB02. Analyses of genomes and in situ transcriptomes revealed that these two novel NOB were active and showed a high metabolic versatility. The transcriptional activity of Ca. Nitrospira could be detected in all tanks with quite different dissolved oxygen (DO) (0.01-5.01 mg/L), illustrating Ca. Nitrospira can survive in fluctuating DO conditions. The much lower Ca. Nitrospira abundance on the anammox bacteria-enriched sponge carrier likely originated from the intensification substrate (NO2-) competition from anammox and denitrifying bacteria. In particular, a highlight is that Ca. Nitrospira encoded and treanscribed cyanate hydratase (CynS), amine oxidase, urease (UreC), and coppercontaining nitrite reductase (NirK) related to ammonium and NO production, driving NOB to interact with the co-existed AOB and anammox bacteria. Ca. Nitrospira strains NOB01 and NOB02 showed quite different niche preference in the same aerobic tank, which dominanted the NOB communities in activated sludge and biofilm, respectively. In addition to the common rTCA cycle for CO2 fixation, a reductive glycine pathway (RGP) was encoded and transcribed by NOB02 likely for CO2 fixation purpose. Additionally, a 3b group hydrogenase and respiratory nitrate reductase were uniquely encoded and transcribed by NOB02, which likely confer a survival advantage to this strain in the fluctuant activated sludge niche. The discovery of this new genus significantly broadens our understanding of the ecophysiology of NOB. Furthermore, the impressive metabolic versatility of the novel NOB revealed in this study advances our understanding of the survival strategy of NOB and provides valuable insight for suppressing NOB in the anammox-based WWTP.