Electroactive biofilms from activated sludge: Mechanistic insights into electron transport on nanostructured electrodes for the development of biosensors and microbial fuel cell devices

Understanding the mechanisms of extracellular electron transport (EET) in biofilms is critical for advancing bioelectrochemical systems (BES). In this study, four bacterial strains - Pseudarthrobacter scleromae , Arthrobacter halodurans , Brevundimonas vesicularis and Rhodococcus fascians - were isolated from activated sludge and shown to form electroactive biofilms (EAB) on graphite-paste electrodes, including those modified with single-walled carbon nanotubes (SWCNTs). Microscopy and spectroscopy confirmed biofilm formation and matrix structure, as well as biofilm viability under SWCNTs cultivation conditions. High-performance liquid chromatography–mass spectrometry (HPLC-MS/MS) identified pyocyanin and 2-hydroxyphenazine as endogenous redox mediators. Electrochemical analyses revealed that SWCNTs significantly enhanced redox conductivity and reduced charge transfer resistance by integrating into the polysaccharide matrix and facilitating direct electron flow from phenazines to the electrode. The resulting biofilm-based biosensors exhibited 5-min BOD5 detection with lower limits down to 0.12 mg O2/dm3. Microbial fuel cells using these systems generated power densities up to 6 mW/m2. The ability of aerobic microorganisms to form functional EAB and independently generate effective redox mediators presents a promising approach for creating sustainable, affordable, and scalable BES. Additionally, this research fosters the advancement of next-generation biosensors and eco-friendly energy devices by combining biological activity with nanomaterial-enhanced interfaces. © 2025 Elsevier B.V.