Extracellular electron transfer (EET) is a key process in microbial electrochemical systems (MES), enabling electron exchange between microbes and solid external materials.^[1] While electroactive bacteria (EAB), such as Shewanella oneidensis MR-1, have been extensively studied for their native EET capability, non-electroactive bacteria (n-EAB), including Escherichia coli, lack intrinsic EET pathways and remain largely underutilized in MES.^[2] Recently, we reported that polyethyleneimine with ferrocene side chains (Fc-LPEI) facilitates EET in both gram-positive and gram-negative n-EAB, yet its underlying mechanism remains to be fully elucidated.^[3]

In this study, we present a comparative investigation of Fc-LPEI-enhanced EET in both EAB and n-EAB. Acting as an artificial molecular conduit, Fc-LPEI forms an efficient charge-transfer interface between bacterial cells and electrodes. Carbon felt electrodes modified with Fc-LPEI exhibited remarkable current enhancements, with E. coli showing a ~200-fold and S. oneidensis MR-1 a ~12-fold increase relative to unmodified electrodes. Scanning electron microscopy confirmed that the polymer layer enhances bacterial adhesion via electrostatic interactions, while electrochemical techniques (CA, CV, EIS) revealed reduced interfacial resistance and increased electrochemically active surface area (EASA), thereby enabling efficient electron transport.

Our detailed analysis revealed distinct EET mechanisms across the two bacterial species. In E. coli, current generation was closely associated with interactions between ferrocene moieties and secreted redox-active species or planktonic cells, indicative of a mediated electron transfer (MET)-like process. By contrast, S. oneidensis MR-1 primarily relied on direct contact-based mechanisms, wherein Fc-LPEI reinforces native electron pathways by promoting robust cell-electrode contact. Cyclic voltammetry demonstrated clear ferrocene redox peaks for E. coli, while such peaks were suppressed in MR-1, suggesting fundamentally different electron access modes. Electrochemical impedance spectroscopy further confirmed the facilitative role of Fc-LPEI: R_bac decreased from 38 kΩ to 6.5 kΩ for E. coli and from 25.3 kΩ to 0.56 kΩ for MR-1.

To investigate the electron transport pathway in MR-1, we employed cytochrome-deletion mutants (Δomc-all and ΔcymA). Our findings indicate that Fc-LPEI partially bypasses the need for outer membrane cytochromes, likely interacting with redox components in the periplasm but not across the inner membrane. A split-recombined MES configuration further demonstrated that EET in E. coli depends strongly on planktonic interactions, whereas in MR-1, direct electrode attachment remains dominant for current generation.

Lastly, we observed that applying higher electrode potentials significantly enhanced E. coli glucose consumption and current output, suggesting that metabolic activity and EET efficiency can be tuned electrochemically. These findings demonstrate that Fc-LPEI can serve as a versatile and tunable interface for dissecting and enhancing bacterial EET, providing valuable mechanistic insight and offering promising strategies for engineering sustainable bioelectronic systems.