Micromolar chlorate sensitivity of wastewater-associated comammox Nitrospira: Endogenous toxification mechanism and...

Chlorate (ClO₃^-) is widely employed as a specific inhibitor of nitrite-oxidizing bacteria (NOB) to differentiate the contributions of ammonia-oxidizing microorganisms (AOMs). However, the discovery of completed ammonia oxidizing (comammox) Nitrospira, which performs both ammonia and nitrite oxidation, challenges the assumed specificity of chlorate, raising the question of whether it also inadvertently inhibits the ammonia oxidation activity of comammox Nitrospira, potentially leading to an underestimation of total ammonia oxidation activity in mixed communities. This study investigates the inhibitory kinetics and mechanisms of chlorate on wastewater-associated comammox Nitrospira. Batch assays revealed that comammox ammonia oxidation was hypersensitive to chlorate, with a half-maximal inhibitory concentration (IC₅₀) of 9.11 µM, orders of magnitude lower than canonical NOB inhibition levels. Metabolic assays showed that 10 μM chlorate reduced ammonia oxidation activity to 49.1% during exposure, with recovery requiring multiple cultivation cycles. Integrated metagenomic and meta-transcriptomic analyses uncovered an endogenous toxification mechanism: chlorate is reduced by nitrite oxidoreductase (NXR) to cytotoxic chlorite, which triggers the upregulation of chlorite dismutase (CLD) and reactive chlorine species (RCS) defense genes, ultimately leading to metabolic arrest. This study confirms the inhibitory effect of chlorate on comammox Nitrospira and deciphers its RCS-mediated mechanism, emphasizing the need for caution when using chlorate-based assays to assess ammonia oxidation activity and providing a theoretical basis for selectively suppressing comammox Nitrospira in WWTPs.