Biofilm-associated microbial risks in a mega water diversion project: distribution of putative pathogen-associated taxa and concrete biocorrosion potential in the Middle Route canal of the South-to-North water diversion project

The ecological assembly of putative pathogen-associated taxa and the biogeochemical potential related to concrete biocorrosion have rarely been examined within an integrated framework in large freshwater diversion systems. We conducted quarterly biofilm sampling at eight stations along the Middle Route canal of the South-to-North Water Diversion Project, and combined 16S rRNA gene amplicon sequencing with shotgun metagenomics to characterize longitudinal and seasonal microbial patterns. Taxonomy-based screening identified 279 putative pathogen-associated ASVs with a mean relative abundance of 3.40%, primarily affiliated with Bacillus and Brevundimonas. Their relative abundance was lowest in the middle reaches, where higher flow velocity and dissolved oxygen may reduce biofilm-associated retention. Total nitrogen accounted for the largest individual contribution among the measured environmental variables (6.13%), whereas normalized stochasticity ratios indicated that stochastic processes predominated in overall community assembly. Metagenomic analysis further revealed spatially structured nitrogen- and sulfur-cycling potential, including biocorrosion-associated taxa such as Thiobacillus and Desulfovibrio, with several related functional pathways showing comparatively higher abundances in upstream biofilms. These findings establish an integrated ecological framework in which stochastic assembly, nutrient-associated selection, and hydrodynamic modulation jointly shape biofilm-associated microbial risks. The study provides critical insights for safeguarding both water-quality monitoring and century-scale infrastructure performance in mega water diversion systems.