Efficient nitrogen removal in nitrite-dependent anaerobic methane oxidation biofilms and associated nitrogen and sulfur transformation pathways

Nitrate/nitrite-dependent anaerobic methane oxidation (n-DAMO) links methane oxidation with nitrogen cycling and provides potential for sustainable wastewater treatment. However, its practical application remains constrained by the slow growth of n-DAMO microorganisms. Besides, the coupled nitrogen and sulfur transformation pathways are still insufficiently understood. Here, a membrane aerated moving bed biofilm reactor (MAMBBR) was operated for 725 days to treat nitrite-rich wastewater. A nitrite removal rate of 580 mg N L⁻¹ d⁻¹ was attained, corresponding to a nitrogen removal efficiency of 99.6 %. 16S rRNA sequencing revealed that Candidatus Methylomirabilis consistently dominated the microbial community throughout the operation, maintaining a relative abundance above 47.5 %. Metagenomic and metatranscriptomic analyses during stable operation also confirmed its dominance, accounting for 72.8 % of metagenomic reads and 89.7 % of metatranscriptomic reads. Functional gene expression patterns suggested that Candidatus Methylomirabilis played a major role in coupling nitrite reduction to N₂ and assimilatory sulfate metabolism. Denitrifying heterotrophs (Hyphomicrobium_A) may have complemented this process by participating in partial nitrite reduction and assimilatory sulfate utilization. Sulfate-reducing bacteria (Pseudorhodoplanes) represented only 0.15 % of metatranscriptomic reads and showed low gene expression, suggesting a minor role in sulfate transformation. These findings demonstrate the high efficiency of MAMBBR-based n-DAMO for nitrite-rich wastewater treatment and provide new insights into nitrogen and sulfur cycling, supporting its future application in sustainable wastewater management.