Deciphering thiosulfate-driven denitrification and anammox (TDDA) Granulation: Inoculation-Driven Community Assembly and Metabolic Reconfiguration Revealed by Surface Properties and Metagenomics

To achieve rapid startup and stable operation of the thiosulfate-driven denitrification and anammox (TDDA) process, this study systematically evaluated the feasibility and mechanisms of various sludge inoculation strategies for TDDA enrichment and granulation. Five inoculation modes were examined in sequencing batch reactors (R1-R5), and their performance was tracked for 126 days. The reactor inoculated with anammox sludge (R1) achieved the fastest startup, the highest total nitrogen removal efficiency, and the strongest granulation (maximum particle size 859 um). Metagenomic analysis revealed a highly synergistic consortium dominated by Candidatus Kuenenia and a novel sulfur-oxidizing bacterium (bin.R21B.4). A key finding was a shift in metabolic potential: metagenomes showed higher abundances of genes for hydrophobic amino acid biosynthesis (branched-chain and aromatic) and lower abundances of genes for hydrophilic amino acids, consistent with increased EPS hydrophobicity. XDLVO-based thermodynamic analysis confirmed that intensified hydrophobic interactions promoted cell aggregation and granule growth. Meanwhile, sulfur-reducing bacteria converted sulfate to S2-, stimulating the secretion of carboxyl-rich polysaccharides. These polysaccharides formed cation-bridged HAP/FeS cores with Ca2+ and Fe2+, enhancing particle cohesion and settling. Collectively, these results suggest that inoculation strategies are associated with shifts in microbial functional potential, coinciding with changes in EPS composition and surface hydrophobicity that favor TDDA granulation. This work provides new mechanistic insight and an operational basis for the targeted construction and efficient start-up of TDDA systems.