Enrichment and Kinetic Profiling of

The discovery of comammox bacteria has revolutionized our understanding of nitrification, challenging the conventional paradigm that this process is mediated by two distinct microbial groups. Although comammox bacteria, particularly Candidatus Nitrospira nitrosa, are prevalent with significant activities in wastewater treatment systems, their physiological and biochemical properties, particularly growth substrates-based kinetics, are yet to be fully disclosed. To this end, we first attempted to enrich Ca. N. nitrosa in a laboratory-scale continuous stirred tank reactor fed with mainstream-level ammonium. Following the 40 d operation, a suite of microbial analyses jointly confirmed the unprecedentedly rapid specific enrichment and absolute dominance of Ca. N. nitrosa with the entire gene repertoire needed for completing the full nitrification process in the sludge. Dedicated batch tests then revealed and validated the affinity constants of Ca. N. nitrosa for total ammonium, nitrite and oxygen (0.393 ± 0.058 mg-N/L, 0.365 ± 0.001 mg-N/L and 0.006 ± 0.001 mg-O2/L, respectively), demonstrating high affinities for substrates that could underpin its widely reported competitive presence and functionality in wastewater treatment systems. This work provides the first kinetic characterization of Ca. N. nitrosa, complementing the currently limited understanding of the comammox process while enabling a better evaluation of its important role in the nitrogen cycle across diverse environments.