Towards smarter viral risk management: Unraveling viral RNA loss dynamics via virus-in-sludge mass balance in activated sludge treatment process.

Conventional activated sludge (CAS) and membrane bioreactor (MBR) systems are effective for virus removal in wastewater treatment, resulting in low virus concentrations in treated effluent. Virus removal performance is commonly evaluated using log reduction values (LRVs); however, LRVs reflect the combined effects of multiple controllable and uncontrollable factors, including virus decay within activated sludge. Accurate estimation of viral infectivity decay and the RNA loss rates depend on reliable quantification of viruses in the solid phase, where viruses are predominantly distributed, yet no consensus method for such quantification has been established. In this study, we investigated virus recovery and distribution and developed a sludge-based mass balance model to estimate apparent viral RNA loss. A highly sensitive RNA quantification approach targeting the solid phase was applied to improve accuracy. Viruses were predominantly associated with the solid fraction, with concentrations exceeding influent levels by over one order of magnitude, indicating accumulation during sludge retention. Using the proposed mass balance model, apparent viral RNA loss rates (day⁻¹) were estimated as 3.23 × 10² (CAS) and 2.25 × 10¹ (MBR) for PMMoV, 1.72 × 106 (CAS) and 7.16 × 104 (MBR) for norovirus GII, and 3.62 × 10¹ (CAS) and 2.18 × 100 (MBR) for Aichi virus. These findings highlight the critical role of the solid phase in governing virus fate and apparent viral RNA loss in activated sludge processes.