The ability of Geobacter sulfurreducens to utilize electrodes as electron acceptors provides a system for monitoring mechanisms of electron transfer beyond the cell surface. This study examined the physiology of extracellular electron transfer during many stages of growth, and in response to short- and long-term changes in electron acceptor potential. When G. sulfurreducens was grown on planar potentiostat-controlled electrodes, the magnitude of early cell attachment increased with initial cell density. However, the first cells to attach did not demonstrate the same electron transfer rates as cells grown on electrodes. For example, following initial attachment of fumarate-grown cells, the electron transfer rate was 2 mA/mg protein, but increased to nearly 8 mA/mg protein within 6 h of growth. Once attached, all biofilms grew at a constant rate (doubling every 6 h), and sustained a high specific electron transfer rate and growth yield, while current density was below 300 mA/cm2. Beyond this point, the rate of current increase slowed and approached a stable plateau. At all phases, slow scan rate cyclic voltammetry of G. sulfurreducens showed a similar well-defined sigmoidal catalytic wave, indicating the general model of electron transfer to the electrode was not changing. Short-term exposure to reducing potentials (3 h) did not alter these characteristics, but did cause accumulation of electrons which could be discharged at potentials above -0.1 V. Sustained growth at lower potentials (0.16 V) only slightly altered the pattern of detectable redox species at the electrode, but did eliminate this pattern of discharge from the biofilm. Single-turnover voltammetry of colonized electrodes showed at least 3 redox couples at potentials similar to other recent observations, with redox protein coverage of the electrode on the order of ca. 1 nmol/cm2. The consistent electrochemistry, growth rate, and growth yield of the G. sulfurreducens biofilm at all stages suggests an initial phase where cells must optimize attachment or electron transfer to a surface, and that after this point, the rate of electron production by cells (rate electrons are delivered to the external surface) remains rate limiting compared to the rate electrons can be transferred between cells, and to electrodes.
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