Integrating methane conversion with multi-pathway nitrogen removal for wastewater in membrane-enabled stratified biofilm: System development and microbial dynamics

Global demands for energy-neutral wastewater treatment drive innovation in sustainable nitrogen removal. A single-biofilm membrane biofilm reactor (MBfR) was constructed for efficient aerobic methane oxidation coupled with simultaneous ammonia oxidation and denitrification (AME-AOD). Through meticulous refinement in aspects such as membrane materials and gas-to-feed ratios, the best-performing biofilm achieved a high total nitrogen (TN) removal efficiency of 97 % ± 2 %. The system ultimately reduced TN from 51.6 ± 0.7 mg l⁻¹ to approximately 5 mg l⁻¹ within 16 h with a methane conversion efficiency of 30.0 ± 0.9 mg-CH₄/mg-N. From startup, the biofilm supported stable coexistence of aerobic and anoxic processes, with gene abundances related to nitrification and denitrification increasing by 1.6-fold and 1.2-fold, respectively. After long-term operation, ecological niche differentiation enabled coexistence and synergistic interaction of methanotrophs, anaerobic ammonium oxidation (anammox), denitrifiers, and nitrifiers within each layer of the biofilm. Overall, this study offers a new strategy to advance sustainable mainstream nitrogen removal in wastewater.