M. Stöckl, C. Schlegel, A. Sydow, D. Holtmann, R. Ulber, K.-M. Mangold
Understanding the attachment of electro-active bacteria to electrode surfaces and their subsequent biofilm formation is one of the major challenges for the establishment of bacterial bioelectrochemial systems (BES). For a constant observation of biofilm growth, providing information on different stages of biofilm formation, continuous monitoring methods are required. In this paper a combination of two powerful analytical methods, Electrochemical Impedance Spectroscopy (EIS) and Confocal Laser Scanning Microscopy (CLSM), for biofilm monitoring is presented. A custom-built flow cell with a transparent indium tin oxide working electrode (WE) was constructed allowing monitoring of cell attachment to a working electrode simultaneously by EIS and CLSM. Cyclic Voltammetry (CV) and EIS of an iron (II)/iron (III) redox couple indicate that the flow cell is suitable for electrochemical experiments. An engineered Shewanella oneidensis MR-1 (ATCC700550) producing eGFP was used as electro-active model organism to demonstrate the practical application of the flow cell as BES to monitor cell attachment simultaneously with EIS and CLSM. Applying the flow cell as MFC (transparent working electrode poised as anode) produced a typical current curve for such a system. From the equivalent circuit used to interpret EIS data the charge transfer resistance RCT is sensitive to attachment of microorganisms. Fitted RCT was increased initially after cell inoculation and then lowered constantly with progressing experimental time. In parallel taken CLSM images show that bacteria already adhered to the WE 5 min after inoculation. A mono- respectively bilayer of electro-active cells was observed after 17 h on the WE surface. With the presented flow cell interpretation of EIS measurements is significantly improved by simultaneous CLSM imaging, leading to a wide range of attachment information and pointing to the investigation of less studied and less established electro-active bacteria.