Overlooked pathways of endogenous simultaneous nitrification and denitrification in anaerobic/aerobic/anoxic sequencing batch reactors with organic supplementation

The anaerobic/aerobic/anoxic (A/O/A) process is a promising biotechnology to intensify denitrification in low carbon/nitrogen (C/N) wastewater treatment, but the neglected typical rate-limiting step-nitrification-would hinder its wider application. Heterotrophic nitrification driven by intracellular carbon (PHAs) could enhance nitrification and achieve endogenous simultaneous nitrification and denitrification (ESND) in the A/O/A process, but its feasibility remains unexamined. Here we established four A/O/A-SBRs at different C/N ratios (3, 7.5, 12, and 16.5) to address the above-mentioned knowledge gaps. The results showed that organic supplementation promoted both nitrification and denitrification (performance and relevant enzymatic activities) until organic overdose (C/N = 16.5) exacerbated niche competitions from other non-functional heterotrophs. qPCR and batch tests indicated that high C/N ratios inhibited autotrophic nitrifiers, and heterotrophic nitrifiers (HNB) dominated in the enhanced nitrification. Given the high HNB contribution (43.7%) and low COD variation (< 10 mg L-1) in the SND (76.4%) of CN12, we proposed a potential SND pathway based on heterotrophic nitrification and denitrification driven by PHAs and verified it with batch tests. Microbial and functional analyses suggested that CN12 favored the intracellular carbon transformation and harbored the minimum autotrophic nitrifiers, supporting the dominance of ESND in the enhanced SND. Our findings expand the understanding of the relationships between intracellular carbon transformation and SND and provide a novel nitrogen removal pathway for the practical application of the A/O/A process.