Energy-efficient nitrogen removal in activated sludge (AS) treatment requires process control strategies that limit the oxidation of ammonia at nitrite. Previous reports have revealed the adaptation of nitrite-oxidizing bacteria (NOB) to various out-selection strategies in AS, likely due to their high metabolic versatility and diversity. Thus, it is crucial to illuminate the biokinetic mechanisms driving NOB adaptation to out-selection pressures. Current wastewater treatment process models do not account for functional diversity, which limits their ability to capture the ecophysiology of co-existing NOB populations. A process modeling framework that resolves functional degeneracy is therefore needed. This study combined genome-centric metagenomic abundance data with AS process modelling to capture the biokinetic characteristics of physiologically diverse and co-existing NOB. A lab-scale mainstream AS system was operated with continual treatment of 20% return activated sludge with 200 mg-N/L free ammonia (FA) in a side-stream flow. A genome-centric metagenomic approach was used to recover metagenome-assembled genomes (MAGs), and metabolic reconstruction was performed on the MAGs to assess their genomic potentials. A genome-centric process model was formulated in AQUASIM by integrating highly-paralleled respirometry, in-situ reactor monitoring, and MAG abundance data over a 100-day operational period. Routine exposure of the AS community to FA in a side-stream reactor led to transient nitrite accumulation in the mainstream and a regime change in Nitrospira NOB lineages. Genome annotation indicated that certain Nitrospira MAGs could use formate as an electron donor, and also had the potential for reactive oxygen species degradation and osmolyte production, to aid their survival in the side-stream FA-exposure. The genome-centric process model resolved the biokinetics of two Nitrospira lineages, and better predicted reactor nitrogen dynamics than a conventional AS model.
