Integration of real-time NH₄⁺-N control and spatial microbial engineering achieves high removals of nitrogen and...

The imbalance among nitrite supply, nitrate accumulation and aeration demand poses significant challenges in single-stage partial nitrification-anammox (PN/A) systems for stably treating high-strength anaerobically digested swine wastewater (ADSW) at low C/N ratio. Here, we propose a novel process that integrates real-time NH₄⁺-N control with a floc-granule partitioned biomass architecture in a sequence anoxic-oxic-anoxic (SAOA) system to dynamically modulate free ammonia (FA) concentration while preventing free nitrous acid (FNA) inhibition. By maintaining an NH₄⁺-N endpoint of 50 mg/L, FA was stabilized at 5.2 mg N/L, and FNA was effectively suppressed. Thus, the SAOA system achieved 94.14% TN removal at a loading rate of 0.24 kg N/(m³·d) and 92.71% COD at low influent COD/TN ratio of 1.71, respectively. Metagenomic and enzymatic profiling revealed a distinct ecological stratification: floccular biomass was enriched with Candidatus Kuenenia, whereas granular microenvironments favored ammonia-oxidizing bacteria (AOB), accompanied by the upregulation of key nitrification and anammox genes. Kinetic analysis of COD and NH₄⁺-N removals revealed a stage-specific metabolic transition from carbon-driven to autotrophic nitrogen-dominated removal. This study provides mechanistically robust and scalable control paradigm for advancing simultaneous, high-efficiency nitrogen and carbon removal from nitrogen-rich and carbon-limited wastewater.