Nitrate and Pipe Material Jointly Govern Biofilm Ecology in Drinking Water Networks

The combined effects of pipe materials and nitrate availability on the biofilm assembly in drinking water networks (DWNs) remain unclear. This study used bench-scale annular reactors to investigate biofilm dynamics on plastic [polyethylene, unplasticized polyvinyl chloride (UPVC)] and metal (stainless steel, galvanized steel) pipes under nitrate depletion (ND) and nitrate supplementation (NS) over 28 days. NS significantly altered water chemistry, increasing dissolved organic nitrogen and turbidity while reducing total organic carbon compared to ND (P < 0.05). Dominant dissolved organic matter shifted from protein-like in ND to humic-like in NS, mirrored by a transition in extracellular polymeric substances (EPS) from protein- to polysaccharide-dominated. Deterministic processes governed biofilm assembly (R2: ND = 0.38; NS = 0.48), favoring Burkholderia under ND and Sphingomonas under NS. Biofilm growth was most robust on UPVC, with plastics enriching Staphylococcus and Pseudomonas, while metals favored Acinetobacter and Methylobacterium. Network analysis showed >90% positive interspecies associations, potentially linking Pseudomonas to EPS production and Bacillus to EPS degradation. Functionally, ND enriched hydrocarbon-degrading taxa, whereas NS promoted anaerobic chemoheterotrophs and methylotrophs. These findings highlight the role of nitrate and material type in shaping biofilm ecology, informing targeted strategies to control microbial risks in DWNs.