Adaptation and Resistance of Nitrifiers to Harsh Engineered Conditions during Partial Nitritation

Selective suppression of nitrite-oxidizing bacteria (NOB) is essential for stable partial nitritation (PN), yet NOB often persist or recover under engineered suppression conditions. This study investigated the kinetic, microbial, and genomic responses of NOB and ammonia-oxidizing bacteria (AOB) in a PN system subjected to low pH and high free nitrous acid (FNA) sidestream treatment. A sequencing batch reactor was operated for 283 days under varying treatment intensities. Long-term operation selectively enriched a Candidatus nitrotogafabula-related MAG (bin.69), characterized by a relatively high apparent growth rate (∼1.3 d-1) and low substrate affinity (Ks = 2.3 ± 0.4 mg NO2--N L-1; Ko = 1.1 ± 0.3 mg O2 L-1). Comparative genomic analyses identified stress-tolerance-associated traits related to proton transport, ion homeostasis, protein stabilization, and cellular stress responses. Proteome isoelectric (pI) point analysis further showed a lower genomic pI profile than other known NOB, potentially consistent with persistence under acidic and FNA stress. These findings provide genome-informed insights into NOB persistence under long-term suppression and implications for improving PN stability.