Spatiotemporal niche differentiation enables stable autotrophic nitrogen removal in a continuous-flow system coupling...

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.