Synergistic operational optimizations and microbial responses stabilize filamentous-dominated continuous-flow partial...

The operational stability of continuous-flow partial denitrification-anammox (PD/A) systems is frequently constrained by insufficient nitrite supply and temperature sensitivity of anammox bacteria, particularly under low-temperature stress. In such conditions, filamentous bacteria often proliferate excessively, and their overgrowth has long been associated with reactor instability and performance deterioration. Here, we demonstrate stable nitrogen removal in a filamentous-dominated continuous-flow PD/A reactor at an average temperature of 16.7 °C through operational optimizations and microbial responses. The reactor achieved 89.8 % total nitrogen removal, with ammonium and nitrate removal efficiencies of 97.2 % and 91.5 %, respectively, with anammox contributing up to 98.3 % of nitrogen removal. Metagenomic analyses revealed that the filamentous genus Sphaerotilus dominated the microbial community (29.3-41.5 %) but sustained the genomic potential for nitrite availability to support anammox. Genome-centric reconstruction confirmed that a Sphaerotilus-affiliated MAG5 possessed adaptive features under low temperature. Additional heterotrophs, including Leptothrix, Rubrivivax, and Thauera, harbored genomic potential for auxiliary nitrate-to-nitrite conversion. Crucially, the synergy between this genomic potential for nitrite provision and engineered biomass retention (specifically mesh filtration and regular sludge return) facilitated the enrichment of Ca. Brocadia, increasing its relative abundance from 2.4 % to 5.4 %. Concurrently, Ca. Brocadia reinforced low-temperature adaptability by expanding the genetic potential of energy-generating carbon metabolic pathways and increasing its contribution to the cold shock protein gene cspA from 6.6 % to 21.0 %. Collectively, this study reveals that integrating strategic biomass retention with microbial responses provides a viable pathway to sustain stable nitrogen removal in filamentous-dominated continuous-flow PD/A systems.