In situ spectroelectrochemical investigation of electrocatalytic microbial biofilms by surface-enhanced resonance Raman spectroscopy.

Metal-reducing bacteria not only play a key role in geochemical redox cycles, but also attract increasing attention in view of their relevance for microbial bioelectrochemical systems, a seminal sustainable technology. This growing research interest is triggered by the bacteria s capability to oxidize substrates such as acetate and to transfer the released electrons to an insoluble terminal electron acceptor, for example, iron-containing minerals in nature or a fuel cell anode in bioelectrochemical applications. The underlying electron-transfer (ET) mechanisms between the bacteria and the terminal electron acceptor may occur by different mechanisms, including direct and mediated electron transfer denoted as DET and MET, respectively. In the case of DET, the electrons are transferred from the respiratory chain in the cell to extracellular inorganic material via a complex architecture involving several outer membrane cytochromes (OMCs). These cytochromes are multiheme proteins whose function and number of heme groups may vary largely within the same family. Although several studies investigated the behavior of these proteins embedded in microbial biofilms of wild-type and mutant Geobacter sulfurreducens, the archetype bacteria family employing DET, the role of these cytochromes in the heterogeneous ET across the biofilm/electrode interface is far from clearly understood. This is particularly true since structural data are currently only known for two OMCs, namely, OmcF and OmcZ. 11] In this respect, spectroscopic techniques that can be applied to biofilms in situ may provide important structural information about the OMCs involved in the DET. To date, only two spectroscopic studies were devoted to the investigation of OMCs embedded in the cellular membrane. 13] The spectroscopic measurements of these works were carried out with washed and re-suspended cells, but did not refer to intact biofilms grown on an electrode. Herein, we present for the first time in situ spectroscopic characterization of OMCs in a catalytically active microbial biofilm. By measuring the electrochemical and spectroscopic properties of microbial cells embedded in their natural biofilm habitat, a more realistic picture on the natural electron transfer will be provided. Therefore, we have employed surface-enhanced resonance Raman (SERR) spectroscopy in combination with cyclic voltammetry (CV). SERR spectroscopy exploits the combination of the molecular resonance Raman (RR) and the surface-enhanced Raman (SER) effect to probe selectively the heme groups solely of the proteins in proximity of the electrode surface. 14] This powerful technique, in our case performed under strict electrochemical control, reveals the redox, coordination and spin states of the heme iron as well as the nature of its axial ligand, thereby providing important structural information that may complement the interpretation of electrochemical data obtained by CV. 16] The biofilms were grown at a constant potential on roughened (i.e. SER-active) silver electrodes using 10 mm acetate as substrate (see the Supporting Information for experimental details). These biofilms produced a maximum chronoamperometric current density of 600 mAcm 2 (Figure SI2 in the Supporting Information), which is in good agreement with previous studies using graphite anodes. The voltammetric behavior of the biofilms was monitored under turnover (Figure SI3) and nonturnover conditions [that is, with and without the substrate (e.g. acetate), respectively]. Figure 1 shows the CV behavior of such a biofilm for nonturnover conditions. The two redox couples that are proposed to be involved in the DET, Ef,1 and Ef,2, are centered at formal potentials of 282 mV and 363 mV, respectively (all potentials are reported versus the Ag/AgCl (3.0m KCl) reference electrode). The main overall shape and peak positions of the cyclic voltammogram shown in Figure 1 are very similar to those obtained on graphite electrodes in parallel experiments and in previous studies, showing that biofilm formation is not affected by the nature of the electrode material. The similarity between these CV traces and those obtained solely from biofilms of Geobacter sulfurreducens indicates that the biofilm is highly dominated [*] Dr. D. Millo, H. K. Ly, Prof. Dr. P. Hildebrandt Institut f r Chemie, Sekr. PC14, Technische Universit t Berlin Strasse des 17. Juni 135, 10623 Berlin (Germany) Fax: (+ 49)30-3142-1122 E-mail: diego.millo@tu-berlin.de