Continuous flow outperforms sequencing batch operation: Mechanistic insights into the enrichment of complete ammonia oxidizing bacteria under low-ammonium, oligotrophic conditions

The discovery of complete ammonia oxidation (comammox) bacteria has introduced a promising approach for low-energy, high-efficiency nitrogen removal in wastewater treatment. While various reactors have been developed under oligotrophic conditions to enrich comammox bacteria, the comparative effects of dynamic versus stable oligotrophic environments on the enrichment process remain insufficiently investigated. In this study, sequencing batch biofilm reactors (SBBRs) and continuous flow biofilm reactors (CFBRs) were employed to simulate dynamic and stable oligotrophic environments, respectively, aiming to enrich comammox bacteria at low ammonium concentrations. CFBR operation significantly enhanced comammox bacteria proliferation, as indicated by an increase in the relative abundance of Nitrospirota from 0.21 % in the inoculated sludge to 10.28 %, and a rise in the amoA gene copy number to 1.39 × 10⁵ copies/mg biomass, representing 3.4-fold and 6.0-fold increases over SBBR performance. Moreover, phylogenetic analysis revealed a dominance of Candidatus Nitrospira nitrosa-like comammox bacteria under CFBR operation. Substrate affinity assays revealed that microorganisms enriched in the CFBR exhibited a higher ammonium affinity (K_NH4⁺_-N = 4.967 ± 1.771 mg-N/L) and more stable ammonia removal efficiency. In contrast, the CFBR environment promoted the upregulation of amoABC and other nitrogen metabolism genes. Microbial network analysis revealed that communities in the dynamic SBBR conditions displayed a denser and more modular topology. Notably, Nitrospirota adopted distinct ecological strategies, by selectively interacting with organic carbon-metabolizing taxa in the stable CFBR environment, thereby enhancing its adaptive capacity. These findings demonstrate that CFBR operation more effectively increases the relative abundance of comammox bacteria.