Abstract
Despite advancements in urban wastewater treatment, final disinfection remains the primary, yet often inefficient or costly, measure to control fecal bacteria and antibiotic resistance genes (ARGs). A solution to enhance performance while reducing the impact of chemical disinfection could be found in a different management of the often-overlooked tertiary clarifiers integrated into the coagulation–flocculation stage. By analyzing three different wastewater treatment plants (WWTPs) combining amplicon and shotgun metagenomics, flow cytometry, and water chemistry, we demonstrated that tertiary clarifiers shape microbial communities, reducing bacterial abundance while maintaining community richness. Whereas fecal/opportunistic pathogens decline, environmental bacteria either persist or increase, suggesting selective pressures at play. Furthermore, while ARG abundance decreased, their relative prevalence remained constant or even increased, raising concerns about potential horizontal gene transfer. Biofilms within clarifiers act as reservoirs, stabilizing diversity despite environmental fluctuations. These findings challenge the traditional perception of tertiary clarifiers as mere sedimentation tanks and highlight their potential role for microbial control. Through optimized retention times and ecological management, tertiary clarifiers could complement disinfection by reducing environmental footprints and operational costs, lowering the overall intensity required to mitigate bacterial and genetic pollution. This study provides a foundation for an additional use of tertiary clarifiers in WWTP design, integrating eco-engineered solutions to enhance treatment efficacy.
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Acknowledgements
This study was supported by the ECOTREAT Project funded by the National Research Council of Italy (CNR) (call @CNR 2021). This study also was partially funded under the National Recovery and Resilience Plan (NRRP), Mission 4 Component 2 Investment 1.4 – Call for tender No. 3138 of 16 December 2021, rectified by Decree n.3175 of 18 December 2021 of Italian Ministry of University and Research funded by the European Union – NextGenerationEU (code CN_00000033, Concession Decree No. 1034 of 17 June 2022 adopted by the Italian Ministry of University and Research, CUP B83C22002930006, Project title “National Biodiversity Future Center – NBFC”). We would like to thank Acqua Novara. VCO SpA for the collaboration in the sampling activity and generally for the support of the research activity, Simona Rossetti for the valuable suggestions, and Gabriele Tartari (CNR-IRSA Verbania) for the support in the analysis of the chemical parameters.
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Di Cesare, A., Sabatino, R., Sbaffi, T. et al. An additional use of tertiary clarifiers: potential for ecology-based control of bacterial and genetic pollution in wastewater.
npj Clean Water (2026). https://doi.org/10.1038/s41545-026-00582-7
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DOI: https://doi.org/10.1038/s41545-026-00582-7
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