Prolonged Stagnation Reduces Treated Wastewater Biostability by Altering Microbial Community: Insights From Metaproteomics.

Reclaimed wastewater is increasingly reused for irrigation and other non-potable applications; however, inadequately treated effluent has raised concerns regarding environmental and public health impacts. Water quality in reclaimed distribution systems is shaped by multiple factors, particularly hydraulic stagnation in pipes and storage reservoirs. Stagnation can alter microbial community stability and facilitate persistence of pathogenic taxa. To investigate how prolonged stagnation affects microbial community structure and function, we integrated metagenomics and metaproteomics analyses of biofilms under flow and stagnant conditions over 3, 5 and 7 months. Prolonged stagnation caused pronounced compositional shifts, including strong reductions in nitrogen-removing taxa such as Nitrospira and Nitrosomonas. Correspondingly, key nitrification and denitrification proteins were depleted ≥ twofold under stagnation, indicating impaired nitrogen conversion processes. Stagnation also enriched motility- and transport-related functions and promoted Acidovorax persistence, a genus including phytopathogenic species. In contrast, flow conditions sustained nitrogen-cycling activity, contaminant-degrading enzymes, and quorum-quenching proteins, supporting greater biostability. Overall, our findings show that prolonged stagnation disrupts microbial community balance, suppresses essential nitrogen-cycling and detoxification pathways, and reduces the functional robustness of treated wastewater. Maintaining hydraulic flow within reclaimed water systems is therefore critical for preserving microbial functionality and ensuring safe and reliable reuse in irrigation and other non-potable applications.