Cooperation between comammox and anammox bacteria for nitrogen removal has been recently reported in laboratory-scale systems, including synthetic community constructs; however, there are no reports of full-scale municipal wastewater treatment systems with such cooperation. Here, we report intrinsic and extant kinetics as well as genome-resolved community characterization of a full-scale integrated fixed film activated sludge (IFAS) system where comammox and anammox bacteria co-occur and appear to drive nitrogen loss. Intrinsic batch kinetic assays indicated that majority of the aerobic ammonia oxidation was driven by comammox bacteria (1.75 ± 0.08 mg-N/g TS-h) in the attached growth phase, with minimal contribution by ammonia-oxidizing bacteria. Interestingly, a portion of total inorganic nitrogen (∼8%) was consistently lost during these aerobic assays. Aerobic nitrite oxidation assays eliminated the possibility of denitrification as a cause of nitrogen loss, while anaerobic ammonia oxidation assays resulted in rates consistent with anammox stoichiometry. Full-scale experiments at different dissolved oxygen (DO = 2 – 6 mg/L) setpoints indicated persistent nitrogen loss that was partly sensitive to DO concentrations. Genome-resolved metagenomics confirmed the high abundance (relative abundance 6.53 ± 0.34%) of two Brocadia-like anammox populations, while comammox bacteria within the Ca. Nitrospira nitrosa cluster were lower in abundance (0.37 ± 0.03%) and Nitrosomonas-like ammonia oxidizers were even lower (0.12 ± 0.02%). Collectively, our study reports for the first time the co-occurrence and cooperation of comammox and anammox bacteria in a full-scale municipal wastewater treatment system.

Partial denitrification-anammox (PdNA) has a significant application potential for retrofitting plants for more intensified and cost-effective nitrogen removal. Demonstrating the reliability and robustness of PdNA in polishing filters will advance...

Cooperation between comammox and anammox bacteria for nitrogen removal has been recently reported in laboratory-scale systems including synthetic community construct; however, there are no reports of full-scale municipal wastewater treatment systems with such cooperation. Here, we report intrinsic and extant kinetics as well as genome-resolved community characterization of a full-scale integrated fixed film activated sludge (IFAS) system where comammox and anammox bacteria co-occur and appear to drive nitrogen loss. Intrinsic batch kinetic assays indicated that majority of the aerobic ammonia oxidation was driven by comammox bacteria (1.75 ± 0.08 mg-N/g TS-h) in the attached growth phase with minimal contribution by ammonia oxidizing bacteria. Interestingly, a portion of total inorganic nitrogen (~8%) was consistently lost during these aerobic assays. Aerobic nitrite oxidation assays eliminated the possibility of denitrification as a cause of nitrogen loss, while anaerobic ammonia oxidation assays resulted in rates consistent with anammox stoichiometry. Full-scale experiments at different dissolved oxygen (DO = 2-6 mg/L) set points indicated persistent nitrogen loss that was partly sensitive to DO concentrations. Genome-resolved metagenomics confirmed high abundance (relative abundance 6.53 ± 0.34%) of two Brocadia-like anammox populations while comammox bacteria within the Ca. Nitrospira nitrosa cluster were lower in abundance (0.37% ± 0.03%) and Nitrosomonas-like ammonia oxidizers even lower (0.12% ± 0.02%). Collectively, our study reports for the first time the co-occurrence and co-operation of comammox and anammox bacteria in a full-scale municipal wastewater treatment system.