Ammonium and nitrate fluxes drive metabolic functional shifts in tap water biofilms

Nitrogen availability critically influences biofilm development in drinking water distribution systems (DWDSs), yet the distinct impacts of ammonium (NH₄⁺) and nitrate (NO₃⁻) on biofilm and pathogen risks remain unclear. Using bench-scale annular reactors simulating DWDSs, we investigated how ammonium depletion/supplementation (AD/AS) and nitrate depletion/supplementation (ND/NS) regulate biofilm profiles over 28 days. Water chemistry analyses and functional annotation (KEGG, FAPROTAX) revealed that AS and NS increased total nitrogen retention (AS: 2.046-2.261 mg/L; NS: 10.625-11.800 mg/L) and biofilm prokaryotic cell abundance. AS enriched biosynthesis and virulence pathways (e.g., amino acid transport, energy production), while AD activated stress-response mechanisms (e.g., DNA repair, carbohydrate metabolism). NS favored Gram-negative bacteria, stress-tolerant taxa, and pathogenic genera (e.g., Acinetobacter, Mycobacterium), whereas ND upregulated organic nitrogen scavenging. Nitrogen supplementation preferentially enriched genera including WHO-listed critical-risk pathogens, which correlated strongly with inorganic nitrogen levels (Mantel r > 0.5, P < 0.05). In contrast, genera including high/medium-risk pathogens exhibited increased relative abundance under depletion via organic nutrient reliance. These findings demonstrate that NH₄⁺ and NO₃⁻ differentially regulate biofilm pathogenicity: NH₄⁺ drives growth and virulence, while NO₃⁻ enhances structural resilience and redox flexibility. The study advocates targeted nitrogen management in DWDS to monitor and mitigate biofilm-associated health risks and for optimizing microbial stability. KEY POINTS: • Nitrogen species (NH₄⁺ and NO₃⁻) enrichment and availability elevated biofilm cell density and altered functional groups and metabolic processes, revealing strong coupling between nutrient availability and microbial proliferation in DWDSs. • Ammonium enrichment drove virulence- and metabolism-related functions, whereas nitrate promoted oxidative resilience and biofilm matrix stability. • Nitrogen limitation activated stress-adaptive and organic nitrogen scavenging pathways, reshaping community composition toward stress-tolerant, opportunistic taxa.