Unique characteristics of acid-tolerant comammox bacteria revealed by multi-omics analyses

Complete ammonia oxidation (comammox) is a critical biogeochemical process in the nitrogen cycle. In this study, we utilized comammox Nitrospira to convert urine wastewater into ammonium nitrate by operating a laboratory-scale membrane bioreactor at pH 3 ~ 4. During the process, the acid-tolerant comammox Nitrospira was highly enriched. The metagenomic and metatranscriptomic analyses were applied to reveal its unique characteristics. Comparative genomic analysis among previously reported comammox Nitrospira demonstrated that this species was phylogenetically novel, named Candidatus Nitrospira aciditolerans. Key mechanisms were further identified to enable this species to thrive in acidic environments. These include active proton efflux, regulation of proton consumption, inhibition of proton influx, and cellular strategies for acid stress management and repair. Remarkably, different from other comammox Nitrospira and acid-tolerant ammonia-oxidizing bacteria (AOB), Candidatus Nitrospira aciditolerans possesses highly expressed V-type ATPases that are typically associated with acidophilic ammonia-oxidizing archaea (AOA). This may indicate an ecologically significant role for comammox bacteria and AOA in co-maintaining ammonia oxidation activity in low pH environments. Kinetic characterization revealed an apparent ammonium half-saturation coefficient K _m of 0.50 ± 0.05 μM NH₃ and an apparent ammonium inhibition constant K _i of 241.43 ± 45.64 μM NH₃. The enrichment culture demonstrated optimal ammonia oxidation activity at neutral pH but maintained functionality across a broader pH range between 4 and 8. Like other nitrifying bacteria, this comammox culture was sensitive to temperature and salinity changes. The findings enhance our understanding of the nitrogen cycle under acidic conditions and also present opportunities for engineering applications of acid-tolerant ammonia oxidizers.