Enrichment and Characterization of a Haloalkaline-tolerant Anammox Bacterium

Metagenomic surveys have substantially expanded the known diversity of anaerobic ammonium-oxidizing (anammox) bacteria. However, the physiological traits of newly proposed anammox genera remain largely hypothetical due to the lack of cultured representatives. Although niche differentiation driven by salinity, organic substrates, and oxygen is well documented in anammox bacteria, adaptations to alkaline environments remain uncharacterized. Moreover, no anammox lineage has been identified as an alkaline specialist. Herein, we report the enrichment (>80% relative abundance) of an anammox bacterium, provisionally named Candidatus Loosdrechtia alkalitolerans, which represents the cultured member of the genus Ca. Loosdrechtia. Ca. L. alkalitolerans exhibits marked haloalkaline tolerance, outcompeting other freshwater anammox genera under long-term saline-alkaline stress conditions (0.5% NaCl; pH~9.13). Comparative genomics revealed that among freshwater anammox lineages, only Ca. L. alkalitolerans encodes a complete multiple resistance and pH adaptation (Mrp) cation/proton antiporter operon. Batch assays with transcriptional profiling demonstrated that sodium addition upregulated mrp expression and recovered anammox activity under alkaline stress, suggesting that Mrp-mediated cation/proton exchange may contribute to pH homeostasis in Ca. L. alkalitolerans. Furthermore, database mining revealed that the habitat of Ca. L. alkalitolerans is not restricted to haloalkaline environments, but extends to diverse freshwater and wastewater ecosystems. These findings provide mechanistic insight into saline-alkaline tolerance in anammox bacteria, expanding understanding of their physiological plasticity and ecological roles.