Continuous-flow outperforms sequencing batch operation: Mechanistic insights into comammox Nitrospira enrichment under low-ammonium oligotrophic regimes

The discovery of complete ammonia oxidizing bacteria (comammox bacteria) has offered a new direction for low-energy and high-efficiency nitrogen removal in wastewater treatment, but their enrichment still remains challenging. The choice of reactor, fundamental to microbial enrichment, plays a crucial role: continuous-flow reactors (CFR) and sequencing batch reactors (SBR) simulate stable and dynamic oligotrophic conditions, respectively. Although both activated sludge and biofilm systems have achieved selective comammox bacteria cultivation, the optimal reactor configuration for enrichment remains undetermined. In this study, SBR and CFR were used to simulate dynamic and stable oligotrophic environments, respectively, targeting comammox bacteria enrichment at low ammonium levels. CFR operation induced remarkable comammox bacteria proliferation, with the relative abundance of Nitrospirota increasing from 0.21% in the inoculated sludge to 10.28%, and comammox bacteria amoA gene copy number reaching 1.39 x 100000 copies/mg-biomass, which were 4.6-fold and 6.0-fold increases over SBR performance. Substrate affinity experiments showed that SBR-enriched microorganisms exhibited higher ammonium affinity and more stable ammonia removal efficiency, whereas CFR upregulated AmoABC and other nitrogen metabolism genes. These results indicate that continuous-flow reactor operation is more effective in increasing the relative abundance of comammox bacteria. These findings establish continuous-flow regimes as an optimal strategy for comammox bacteria cultivation, with direct implications for energy-efficient nitrogen removal in mainstream wastewater treatment.