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Shewanella oneidensis is able to carry out extracellular electron transfer (EET), although its EET efficiency is largely limited by low flavin concentrations, poor biofilm forming‐ability, and weak biofilm conductivity. After identifying an important role for riboflavin (RF) in EET via in vitro experiments, the synthesis of RF is directed to 837.74 ± 11.42 µm in S. oneidensis. Molecular dynamics simulation reveals RF as a cofactor that binds strongly to the outer membrane cytochrome MtrC, which is correspondingly further overexpressed to enhance EET. Then the cell division inhibitor sulA, which dramatically enhanced the thickness and biomass of biofilm increased by 155% and 77%, respectively, is overexpressed. To reduce reaction overpotential due to biofilm thickness, a spider‐web‐like hybrid biofilm comprising RF, multiwalled carbon nanotubes (MWCNTs), and graphene oxide (GO) with adsorption‐optimized elongated S. oneidensis, achieve a 77.83‐fold increase in power (3736 mW m−2) relative to MR‐1 and dramatically reduce the charge‐transfer resistance and boosted biofilm electroactivity. This work provides an elegant paradigm to boost EET based on a synthetic biology strategy and materials science strategy, opens up further opportunities for other electrogenic bacteria. [ABSTRACT FROM AUTHOR]

Shewanella oneidensis is able to carry out extracellular electron transfer (EET), although its EET efficiency is largely limited by low flavin concentrations, poor biofilm forming-ability, and weak biofilm conductivity. After identifying an important role for riboflavin (RF) in EET via in vitro experiments, the synthesis of RF is directed to 837.74 ± 11.42 µm in S. oneidensis. Molecular dynamics simulation reveals RF as a cofactor that binds strongly to the outer membrane cytochrome MtrC, which is correspondingly further overexpressed to enhance EET. Then the cell division inhibitor sulA, which dramatically enhanced the thickness and biomass of biofilm increased by 155% and 77%, respectively, is overexpressed. To reduce reaction overpotential due to biofilm thickness, a spider-web-like hybrid biofilm comprising RF, multiwalled carbon nanotubes (MWCNTs), and graphene oxide (GO) with adsorption-optimized elongated S. oneidensis, achieve a 77.83-fold increase in power (3736 mW m -2 ) relative to MR-1 and dramatically reduce the charge-transfer resistance and boosted biofilm electroactivity. This work provides an elegant paradigm to boost EET based on a synthetic biology strategy and materials science strategy, opens up further opportunities for other electrogenic bacteria., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)