Spatiotemporal niche differentiation enables stable autotrophic nitrogen removal in a continuous-flow system coupling microalgae, ammonia oxidizers and anammox bacteria.

Natural aquatic aggregates use diel fluctuations in dissolved oxygen and microscale redox gradients to support aerobic and anaerobic processes, providing an ecological blueprint for continuous-flow microalgal-bacterial systems; however, this coupling remains difficult because photosynthetic oxygen supports ammonia oxidation but inhibits oxygen-sensitive anaerobic ammonium-oxidizing bacteria (AnAOB). In this study, a continuous-flow light-driven microalgal-bacterial system was developed. Long-term reactor operation, pathway contribution tests, extracellular polymeric substance (EPS) characterization, granule and FISH-CLSM analysis, and metagenomics were conducted to elucidate the stabilization mechanism of autotrophic nitrogen removal without external organic carbon. During 530 days of operation, the 8 h light/16 h dark regime maintained the dissolved oxygen concentration at 0.2-0.4 mg/L, while TNRE reached 86.2% before external nitrite supplementation and remained approximately 90% during the nitrite-assisted final phase. Pathway contribution tests indicated that the estimated microalgal-associated assimilation contribution was 36.48% during illumination, whereas denitrification and anammox dominated under dark conditions, contributing 40.53% and 39.67%, respectively. During reactor maturation, the average granule size peaked at 450 μm on day 400, the protein/polysaccharide (PN/PS) ratio increased from approximately 3.0 to 5.0, and the amount of tightly bound EPS protein (TB-EPS-PN) increased to approximately 35 mg/g VSS, indicating enhanced granule cohesion and resistance to disturbance. Metagenomic analysis revealed enrichment of Candidatus Kuenenia to 11.62% and coordinated increases in key nitrogen transformation genes. During reactor maturation, granule development, EPS accumulation, and the algal-bacterial association observed by FISH-CLSM were associated with increasing structural organization, while stage-dependent changes in stress- and adaptation-related genes supported a gene-EPS-structure adaptation framework. Collectively, these findings support a spatiotemporal niche‑differentiation framework in which light/dark cycling may temporally partitions nitrogen pathways, while EPS‑associated granule development and structural heterogeneity may enhance diffusion limitation and provide spatial buffering favorable for oxygen‑sensitive anaerobic functions.