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

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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.

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