New insights into how sludge wasting timing steers microbial community assembly and performance in hybrid PAO biofilm reactors.

A limited understanding of how suspended sludge wasting timing (SWT) steers microbial community assembly and biofilm performance has hampered the establishment of ecological connections between operational parameters and system function in phosphate-accumulating organism (PAO) enriched biofilm reactors. Four sequencing batch biofilm reactors (SBBRs) were operated with distinct wasting timings (20, 30, 50, and 60 days). The reactor with a 30-day wasting timing achieved the earliest growth inflection point (day 27), the fastest nitrification recovery (80% within 7-10 days post-discharge), and the highest PAO abundance (8.97% by day 73). Early discharge (20 days) reduced initial attached biomass and prolonged subsequent development, while discharging too late (≥50 days) delayed the inflection point by 6-11 days and extended nitrification recovery to 20-26 days. Stochastic processes (ecological drift) dominated community assembly under extended SWT, whereas deterministic processes (heterogeneous selection) progressively dominated under the optimal 30-day regime. This deterministic shift fostered a larger, more modular co-occurrence network and more complex interspecies interactions. Keystone functional guilds, including PAOs (Thiothrix, Azospira, Dechloromonas, total abundance 8.97%), AOB (Ellin6067, norank_f_NS9_marine_group), NOB (Nitrospira), and GAO (Candidatus Competibacter), were enriched to higher levels. The synergistic cooperation among these functional bacteria facilitated simultaneous nitrogen removal and phosphorus enrichment. These results demonstrate that SWT acts as a deterministic filter, which is strongly associated with the transition of community assembly from stochastic colonization to function-oriented selection, providing insights for startup and microbial management of hybrid biofilm reactors.