Using Femtosecond Transient Absorption Spectroscopy to Detect Early Redox Shifts in Engineered Bacterial Biofilms

Microbial biofilms exhibit complex redox processes that are critical for bioelectrochemical applications, yet the earliest electron-transfer events occur on femtosecond timescales and remain largely unobserved. Here, we applied femtosecond transient absorption spectroscopy (fs-TAS) to engineered Geobacter sulfurreducens DL-1 biofilms expressing redox-active flavocytochromes to probe ultrafast electron dynamics. Biofilms were grown on quartz substrates and genetically modified to enhance optical accessibility. Fs-TAS measurements, complemented by transient absorption microscopy and time-resolved fluorescence, revealed sub-picosecond excitation and long-lived charge-transfer intermediates that reveal early redox shifts in engineered biofilms compared to wild-type and Δeps controls. Measurement viability assays confirmed that the fs-TAS procedure was non-destructive and that extracellular electron transfer (EET) function was preserved. These results are an indication that targeted genetic modifications can enhance ultrafast electron-transfer pathways and that fs-TAS provides a sensitive, non-invasive method to detect early redox events in living bacterial biofilms. Our findings provide a basis for understanding and improving biofilm-based electrochemical systems. Keywords: Geobacter sulfurreducens, biofilm, femtosecond transient absorption spectroscopy, ultrafast electron transfer, extracellular electron transfer, redox dynamics, genetic engineering