Operating strategy drives microbial succession with minimal vertical stratification in a full-scale vertical-flow...

A full-scale vertical-flow air-lifting reactor (VA/RPIR) integrates anoxic, oxic and settling functions within a single compact unit, but its microbial community dynamics and their relationship with nitrogen removal remain insufficiently understood. In this study, activated sludge samples were collected from three depths (0.5, 2.0 and 5.5 m) from the seed sludge and across three operational stages with different aeration strategies. Reactor performance and dissolved oxygen (DO) profiles were analyzed together with 16S rRNA amplicon sequencing to evaluate stage-dependent temporal and vertical spatial variation in microbial communities. The system achieved stable organics matter removal and improved nitrogen removal under low-DO operation, with effluent TN decreasing from Stage I to Stage III. Community composition was dominated by Proteobacteria and Bacteroidota, while key functional taxa exhibited clear stage-dependent succession. Importantly, vertical differences among depths were minor compared with stage-dependent temporal differences, indicating limited vertical differentiation and a well-mixed spatial structure inferred from microbial and bulk water-quality patterns. Spearman correlations analysis (performed using date-level averages to avoid pseudo-replication) indicated that TN removal efficiency was associated with the relative abundances of several key taxa (e.g., Nitrospira and Dechloromonas). Overall, this full-scale study demonstrates that operating strategy, rather than vertical position, predominantly shapes microbial assembly in VA/RPIR, and provides microbiological insights for optimizing compact low-DO nitrogen removal systems.