Genetically Boosting Electron Transfer in Electroactive Biofilms for Improved Sensitivity of Microbial Fuel Cell‐Based Biosensing

ABSTRACT Electroactive biofilms (EABs) hold significant potential for applications in bioenergy, biosensing and wastewater treatment; however, their performance is frequently hindered by inefficient extracellular electron transfer (EET). A notable example of this limitation is the significant impact of low EET efficiency on the sensitivity of EAB‐based biosensors. To address this, we engineered Geobacter sulfurreducens anode biofilms by enhancing intracellular levels of cyclic GMP‐AMP (cGAMP) through the overexpression of cGAMP synthase. This approach resulted in the formation of thin yet highly conductive biofilms. When utilized in a microbial fuel cell biosensor, the engineered biofilm demonstrated a 20‐fold improvement in detection limit for Cd(II), achieving 0.03 mg L −1 compared to 0.6 mg L −1 for the wild‐type strain. Furthermore, mechanistic studies that integrated RNA sequencing and quantitative analysis revealed an increased expression of c ‐cytochromes and nanowires while simultaneously reducing exopolysaccharide production, thereby enhancing EET and biosensor sensitivity. Given the widespread role of cGAMP across Geobacter spp., dominant microorganisms in natural EABs, this study presents a broadly applicable strategy for optimizing EABs to advance technologies based on these systems.