Decoding anammox granulation: Microbial interactions promote granule formation and indirectly shape antibiotic resistance gene dissemination.

The formation of anaerobic ammonium oxidation (anammox) granules plays a crucial role in biomass retention. However, the microbial interaction, metabolic regulation, and risks associated with the dissemination of antibiotic resistance gene (ARG) during granulation remain insufficiently understood. In this study, an anammox granulation reactor was established and analyzed through integrated physicochemical characterization and multi-omics approaches to investigate changes in sludge properties, microbial communities, metabolic gene expression, and ARG profiles throughout granulation process. The results showed that granule size was closely associated with sludge surface free energy and extracellular polymeric substance (EPS) content, particularly the protein content of tightly bound EPS, which exhibited a significantly positive correlation with granule mechanical strength in the stable phase. Members of Proteobacteria exhibited the most significant shifts during granulation and occupied multiple core nodes in the microbial interaction network, indicating their essential roles in sustaining system stability and functional coordination. Metabolic functional analysis revealed selective regulation of carbon metabolism gene expression, which preferentially provides precursors for amino acid and cofactor biosynthesis. Different microbial taxa displayed significant metabolic complementarity in the synthesis of amino acids and cofactors. ARG analyses revealed that granulation was accompanied by an enhanced potential for mobile genetic element mediated horizontal gene transfer of ARG, with antibiotic target replacement and antibiotic efflux as primary resistance mechanisms. These findings deepen the ecological understanding of anammox granulation and offer theoretical support for managing ARG propagation in engineered systems.