Single-Cell/Molecule Energetic Elucidation of Shuttle-Enhanced Extracellular Electron Transfer: Implications for Iron...

Electron shuttles (ESs) critically enhance microbial extracellular electron transfer (EET), a key biogeochemical process that drives iron cycling and the activation of nutrients like phosphorus. However, existing studies are largely qualitative, focusing on EET pathway identification or current density measurements without quantitatively resolving the underlying energetics. Here, we establish an atomic force microscopy-based single-cell and single-molecule force spectroscopy platform, enabling the first direct quantification of microbial-ES-mineral interfacial energies. We find that riboflavin amplifies the adhesion energy between Shewanella oneidensis MR-1 and ferrihydrite-phosphate complex from 0.81 ± 0.064 fJ to 1.92 ± 0.049 fJ, accelerating microbe-mineral bonding up to 5-fold and boosting electron utilization efficiency from 0.015-0.028 h^-1 to 0.048-0.12 h^-1 across diverse Fe(III) minerals. We further reveal riboflavin binding hotspots on outer membrane c-Cyts, with a binding free energy of -25.6 kJ mol^-1. These thermodynamic findings facilitate EET, which in turn significantly enhances the bioavailability of iron and phosphorus. For the first time, we quantified microbial cell-mineral and ES-cell binding energetics, thereby bridging interfacial thermodynamics across molecular and cellular scales to establish a mechanistic basis for EET and its role in nutrient mobilization. Such insights open avenues for control of ES-mediated functions in nutrient cycling, pollutant transformation, and sustainable bioenergy.