Distinct virucidal mechanisms of anaerobic digestion intermediates revealed by multi-scale insights.

Anaerobic digestion (AD) is widely applied for organic waste stabilization, yet the molecular mechanisms governing enteric virus inactivation by key anaerobic intermediates remain poorly understood. Using bacteriophage MS2 as a conservative surrogate for non-enveloped enteric viruses, we investigated virucidal kinetics and mechanistic pathways of acetate, ammonia, and sulfide under defined AD-relevant chemical scenarios. Virus inactivation ranked as acetate > ammonia > sulfide, with acetate showing the highest first-order inactivation rate constant (k = 1.21 ± 0.12 d-1 at pH 5.5). Stage-specific assays revealed that acetate selectively blocked genome injection, whereas ammonia impaired both adsorption and genome injection. The activity of ammonia was governed by the equilibrium between NH4+ and un-ionized NH3 under alkaline conditions (pH> 8.0). Molecular dynamics simulations revealed that these pathways were driven by distinct physicochemical microenvironments: the acetate anion (Ac⁻) exhibited preferential association with positively charged regions of the A-protein (ΔG = -15.55 kcal/mol, moderate affinity), whereas NH3 exhibited pH-dependent interaction behavior with the capsid structure. In complex anaerobic matrices, humic substances accelerated MS2 inactivation, reducing the time required to achieve 99% inactivation by 35.8%, potentially associated with enhanced hydrophobic interactions, whereas inorganic suspended solids showed negligible interference under the simplified conditions tested. These findings provide mechanistic insights into viral attenuation under defined AD-relevant conditions, while further validation using additional viral models will be required to evaluate their broader applicability to clinically relevant enteric viruses.