Abstract

Granular anaerobic ammonium oxidation (anammox) sludge enables effective biomass retention and supports the stable operation of anammox reactors. During long-term operation, however, effluent-released sludge (ERS) is continuously washed out with the effluent, exhibiting physicochemical and microbial characteristics that differ markedly from those of retained sludge (RS). The functional role and biosafety implications of ERS remain poorly understood. In this study, RS and ERS from the same granular anammox reactor were systematically compared in terms of nitrogen removal performance, microbial community composition, functional gene profiles, and biosafety-related features. RS maintained high anammox activity, whereas ERS showed reduced anammox performance but was characterized by a pronounced enrichment of comammox Nitrospira , supporting more diverse nitrogen transformation pathways. Metagenomic and 16S rRNA analyses further indicated the co-occurrence of comammox Nitrospira and heterotrophic denitrifiers in ERS, suggesting a potential metabolic linkage involving nitrate production and partial reduction to nitrite that may complement anammox activity. Functional pathway analysis revealed diminished autotrophic carbon fixation in ERS, alongside enhanced heterotrophic metabolism and cobalamin biosynthesis. In parallel, ERS exhibited elevated abundances of antibiotic resistance genes and pathogenic taxa. Collectively, these results demonstrate that ERS represents a functionally distinct biomass fraction with unique microbial and metabolic characteristics, as well as potential biosafety implications, warranting further consideration in the evaluation and management of granular anammox systems.