Mechanistic drivers of high-rate nitrification and N₂O emission in thin membrane-aerated biofilms: Insights from...

Membrane-aerated biofilm reactors (MABRs) are promising energy-saving technologies with intrinsically low N₂O emissions. However, even in nitrifying MABRs, the simplest configuration, the biofilm thickness maximizing nitrification performance and the factors driving N₂O emissions remain unclear. Furthermore, achieving ideal nitrification requires retaining ammonia-oxidizing bacteria (AOB) in the aerobic zone adjacent to the membrane by enhancing robust biofilm formation. To address these research gaps, we applied an integrated microscale approach-combining non-invasive optical coherence tomography (OCT) imaging with a newly developed image analysis workflow, microsensor profiling, fluorescence in situ hybridization (FISH), and 16S rRNA gene amplicon sequencing-to a nitrifying MABR equipped with a gear-shaped membrane. A near-maximal nitrification rate (4.45 g-N/m²/day; comparable to the operational average of 4.35 ± 0.49 g-N/m²/day) was achieved at a biofilm thickness of 68 µm. This performance was underpinned by dense AOB colonization retained within the concave regions of the gear membrane, with high transcriptional activity (abundance in transcribed complementary DNA ≈ 79%). A significantly positive correlation between N₂O emission rate and bulk nitrite concentration (Spearman's ρ = 0.51, p = 0.0011) and N₂O/O₂ microprofiles implied N₂O production via denitrification in anoxic regions and its back-diffusion into the membrane lumen. Additionally, biofilm sloughing events were quantitatively detected by the novel image analysis workflow; notably, the basal layer critical for nitrification was firmly retained by the membrane's gear structure, thereby preventing severe performance deterioration. Collectively, this study demonstrates that the gear structure effectively retains a thin, highly active nitrifying biofilm, while identifying nitrite denitrification as the primary driver of N₂O emissions.