Electrical stimulation-induced π-π stacking drives sludge humification to enhance direct interspecies electron...

Electrical stimulation has been applied to enhance anaerobic digestion (AD), but its underlying mechanisms remain unclear. This study reveals that electrical stimulation accelerates sludge humification during AD by driving π-π stacking of humic precursors, thereby enhancing electroactivity and methanogenesis. Specifically, methanogenic potential and HA content increased by 42 % and 75.1 %, respectively, compared to the control (without electrical stimulation). X-ray photoelectron spectroscopy (XPS) revealed intensified π-π stacking interactions in the electrical stimulation group. Density Functional Theory (DFT) calculations confirmed that the electric field induces strong polarization of humic precursors (dipole moment ∼21 Debye). This polarization significantly strengthens intermolecular noncovalent interactions (lowering binding energy by 25.63 kcal/mol), serving as the thermodynamic driving force for π-π stacking. Consequently, this supramolecular assembly forms an extensive delocalized electron system, reducing charge transfer resistance by 56 % and facilitating Direct Interspecies Electron Transfer (DIET). Furthermore, for the first time, ¹³C isotope tracing confirmed a strong correlation (R² > 0.88) between conductivity and DIET contribution. The findings of this study provide a novel perspective for understanding and optimizing bio-electrochemical systems.