The electron transfer (ET) processes of electroactive microbial biofilms have been investigated by combining electrochemistry and time-resolved surface-enhanced resonance Raman (TR-SERR) spectroscopy. This experimental approach provides selective information on the ET process across the biofilm–electrode interface by monitoring the redox-state changes of heme cofactors in outer membrane cytochromes (OMCs) that are in close vicinity (i.e., within 7 nm) to the Ag working electrode. The rate constant for heterogeneous ET of the surface-confined OMCs (sc-OMCs) of a mixed culture derived electroactive microbial biofilm has been determined to be 0.03 s−1. In contrast, according to kinetic simulations the ET between sc-OMCs and their redox partners, embedded within the biofilm, is a much faster process with an estimated rate constant greater than 1.2 s−1. The slow rate of heterogeneous ET and the lack of high-spin species in the SERR spectra rule out the direct attachment of the sc-OMCs to the electrode surface.
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