Development of an Azonexus- and Competibacter-enriched phosphate-accumulating community in the anaerobic/anoxic...

Denitrifying polyphosphate-accumulating organisms (DPAOs) enable simultaneous N and P removal, however, reliable strategies for enriching stable DPAO communities and their metabolic interactions remain insufficiently understood. In this study, DPAO-enriched cultures were developed in a sequencing batch reactor operated under anaerobic/anoxic conditions with acetate as C source. For three independent experiments, activated sludge, collected at different times, was used as the inoculum. Within 0.5-2 months, all experiments exhibited definitive DPAO phenotype dynamics. After 100-200 days of operation, the microbial community was consistently co-dominated by two genera: Azonexus (19-35 %), representing DPAOs, and Competibacter (23-31 %), representing denitrifying glycogen-accumulating organisms (DGAOs). Metagenomic reconstruction revealed that neither Azonexus nor Competibacter harbored the full complement of denitrification genes. The Azonexus metagenome-assembled genome encoded napAB (nitrate reductase), nirS (nitrite reductase), and nosZ (nitrous oxide reductase), while the Competibacter MAG possessed only norBC (nitric oxide reductase) genes. This genomic complementarity provides evidence that complete denitrification in this system could be achieved through cooperation between DPAOs and DGAOs. Consequently, the observed lower phosphorus removal efficiency, compared to anaerobic/aerobic systems, is attributed to the reduced biomass yield of DPAOs and the high essential abundance of DGAOs. These results clarify the ecological role of Azonexus as a DPAO dependent on partnership with DGAOs. Furthermore, the selective conditions favoring Azonexus development in enhanced nutrient removal systems, are evaluated. This work reveals a possible mechanism of syntrophic cooperation between DPAO and DGAO, which has direct implications for the development of resource-saving biological processes for nutrient removal.