Nitrogen removal mechanisms in improved hybrid activated sludge-membrane aerated biofilm reactor: Insights from metagenomics and metaproteomics.

Abstract

Simultaneous nitrification-denitrification (SND) is a promising process for low C/N wastewater treatment. A hybrid activated sludge-membrane aerated biofilm reactor (H-MABR) capable of achieving two‑stage SND was developed and exhibited satisfactory nitrogen removal performance; however, the underlying mechanisms remain unclear. To address this, an improved H-MABR was developed and integrated metagenomic and metaproteomic analyses were performed to systematically elucidate the spatial distribution of microbial communities and their functions in the reactor. The results showed that the improved H-MABR achieved removal efficiencies of 94.4 ± 0.4% for chemical oxygen demand, 92.9 ± 0.7% for total nitrogen, and 97.8 ± 0.8% for ammonia nitrogen. Multi‑omics analysis identified seven dominant microbial genera and three highly expressed nitrogen metabolic pathways. The outer ring was the primary nitrogen removal zone, where the aerobic zone mainly mediated nitrification and the anoxic zone exhibited significantly upregulated activities of NosZ, NirK, NirS, and NorC (ORA vs. ORO), strongly supporting the denitrification function in this zone. The inner ring served as an important auxiliary nitrogen removal unit, showing particularly strong ammonia nitrogen removal performance, which was attributed to the highly expressed hydroxylamine dehydrogenase. This study provides a foundation for advancing the understanding and application of SND systems.