Linking spectral and electrochemical analysis to monitor c-type cytochrome redox status in living Geobacter sulfurreducens biofilms.

When transferring electrons to insoluble Fe oxyhydroxide particles, the bacterium Geobacter sulfurreducens must utilize a chain of redox proteins to relay electrons from cytoplasmic electron carriers to external electron acceptors. In the presence of an electrode poised at an oxidizing potential, not only can cells in contact with the electrode respire directly to the surface, but 10 to 20 cell layers can stack upon each other, and each layer is also electrically connected to the electrode. Thus, G. sulfurrueducens naturally self-assembles a three-dimensional network of proteins capable of oxidizing complex fuels, relaying electrons out of the cytoplasm and across their membranes, and through a biofilm as thick as 40 mm, to sustain current densities on the order of 1 mA cm . 5] Such rates of simultaneous enzymatic oxidation and long-range current transfer rival those achieved by pure enzymes embedded in advanced redox hydrogels. The adaptation of electrochemical techniques to study and control this complex microbial electron-transfer process has opened a window into the physiology of these bacteria. 2, 5, 7–9] However, challenges remain in correlating voltammetry data with specific proteins, and understanding the molecular mechanism of long-distance electron relay between living cells. Mutant analyses, immunogold labeling, and proteomic studies 14–18] have suggested roles for many different multiheme ctype cytochromes, as well as pili and multicopper proteins, 14, 16, 18–24] but less is known about their kinetics or activity in vivo. The use of spectroscopic methods during potentiometric analysis of redox enzymes offers a tool to directly measure the redox status of multiple cofactors. When proteins are immobilized on transparent conductive electrodes, potential-dependent changes can be linked to specific redox centers. 26] Two recent reports extended spectral and electrochemical techniques to the study of whole cells capable of electron transfer to electrodes. Busalmen et al. detected signatures characterisic of c-type cytochromes at a Geobacter cell–electrode interface using surface-enhanced infrared absorption, while Nakamura et al. were able to detect redox-dependent changes in the Soret-band characteristic of c-type cytochromes in Shewanella suspensions, using evanscent wave spectroscopy. These previous studies primarily examined concentrated cell suspensions for short periods of time. However, it is well known that biofilm growth requires disctrete attachment, biosynthesis, and secretion events to construct the external network of delicate proteins that transports electrons from cells. 29–33] Thus, the goal of this work was was to design a system able to support growth of metal-reducing bacteria on transparent conductive electrodes to link potential-dependent changes with real-time measurements of spectral signatures in an undamaged biofilm network (Figure 1). Extensive physiological, proteomic, mRNA expression, and imaging data already exists describing Geobacter biofilm growth on graphitic carbon and gold electrodes. To investigate whether data from this well-understood model could be com-