Evolution for enhanced extracellular electron transfer in Geobacter sulfurreducens over seventeen years of continuous...

Bioelectrochemical systems that harvest electricity from wastes, soils, or sediments are designed to operate over long periods of time. However, little is known about the long-term adaptation of electroactive microbes to such non-natural environments. Geobacter sulfurreducens strain KN400 produces the highest current densities and columbic efficiencies of described pure culture electroactive microorganisms. To investigate long-term adaptation to current production, strain KN400 was grown continuously for 17 years on graphite anodes poised at -400 mV. Isolates recovered from the 17-year-old anode biofilms (designated LT strains) exhibited markedly enhanced extracellular electron transfer capacity, growing ~1.8-fold faster than the parental KN400 strain on Fe(III)-oxide. Crystal violet assays revealed increased surface-associated biofilm biomass on glass by the adapted strains. Quantitative transcriptomics revealed strong upregulation of pilA, omcZ, and omcB during Fe(III) oxide reduction, consistent with enhanced extracellular electron transfer capability. Comparative genomic analysis demonstrated genome reduction relative to KN400 and identified numerous mutations in genes for c-type cytochromes, signal transduction proteins, and transcriptional regulators, including modifications in c-di-GMP riboswitches and diguanylate cyclases that likely promoted expression of extracellular electron transport genes. Genes dispensable for growth on electrodes, including those associated with motility or hydrogen utilization, accumulated mutations consistent with relaxed selective pressure and genetic drift. These results demonstrate that prolonged electrode cultivation can drive genome reduction and regulatory remodeling that optimize electroactive microbes for efficient, biofilm-based electron transfer and may help stabilize long-term bioelectronic systems.