Complete Nutrient Removal Coupled to Nitrous Oxide Production as a Bioenergy Source by Denitrifying Polyphosphate-Accumulating Organisms

Coupled aerobic anoxic nitrous decomposition operation (CANDO) is a promising emerging bioprocess for wastewater treatment that enables direct energy recovery from nitrogen (N) in three steps: (1) ammonium oxidation to nitrite; (2) denitrification of nitrite to nitrous oxide (N2O); and (3) N2O conversion to N-2 with energy generation. However, CANDO does not currently target phosphorus (P) removal. Here, we demonstrate that denitrifying polyphosphate accumulating organism (PAO) enrichment cultures are capable of catalyzing simultaneous biological N and P removal coupled to N2O generation in a second generation CANDO process, CANDO+P. Over 7 months (>300 cycles) of operation of a prototype lab-scale CANDO+P sequencing batch reactor treating synthetic municipal wastewater, we observed stable and near-complete N removal accompanied by sustained high-rate,. high-yield N2O production with partial P removal. A substantial increase in abundance of the PAO Candidatus Accumulibacter phosphatis was observed, increasing from 5% of the total bacterial community in the inoculum to over 50% after 4 months. PAO enrichment was accompanied by a strong shift in the dominant Accumulibacter population from clade IIC to clade IIC, based on qPCR monitoring of polyphosphate kinase 1 (ppk1) gene variants. Our work demonstrates the feasibility of combining high-rate, high-yield N2O production for bioenergy production with combined N and P removal from wastewater, and it further suggests a putative denitrifying PAO niche for Accumulibacter Glade IA.