Final Report Summary - EA-BIOFILMS (Electroactive Biofilms for Microbial Fuel Cells and Biosensors) Summary of progress towards objectives and details for each taskElectroactive biofilms (EABs) are micro structured communities composed of microorganisms that thrive at the solid/liquid interface. The biofilm is composed by the viable microorganisms and a matrix material (sometimes known as “slime”) that confers mechanical stability and increase biofilm resistance to chemical and physical stresses. EABs are capable of respiring solid materials, like metals and electrodes, exchanging electrons with these solid electron donor/acceptors. Because of their unique characteristics, EABs might be applied to energy recovery from waste, wastewater and drinking water treatment, biosensors, and bioelectrosynthesis. For example, EAB can be grown in fuel cells, where the oxidation of a reduced organic carbon source is coupled to the reduction of oxygen or nitrates. This application has been suggested for energy recovery from wastewater. In other applications, EAB might be used to sense the surrounding environment, including the presence of toxic chemical pollutant, thus generating a signal proportional to the concentration of the pollutant. However, the development of reliable EAB-based technologies requires a thorough understanding of charge transfer mechanism at the interface biofilm/electrode. Furthermore, the scale-up of EAB-based devices for industrial applications is usually accompanied by dramatic loss of power output, and this problem have prevented so far a large scale implementation of EAB-based technology.In this project, we have focused on the fundamental of charge transfer between the EAB and electrode in potentiostat-controlled electrochemical cells. The experimental design uses multichannel potentiostat and miniature electrochemical cells to reduce the costs and increase the experimental throughput. Following initial, unsuccessful attempt to use mixed electroactive consortia, we have worked primarily with model metal-reducing microorganisms such as Shewanella sp. and Geobacter sp. Geobacter uses prevalently direct electron transfer through their outer membrane cytochromes, iron-containing proteins that can connect directly the bacterial cells with the electrode. Shewanella possess outer membrane cytochromes and is capable of direct electron transfer. However, it also produces redox compounds, namely flavins (similar to vitamin B12) and quinones (similar to the organic matter present in the soil) that get reduced at the bacterial surface, and then in turn reduce the electrode, thereby producing a net electron transfer.The relative importance of direct vs. mediated electron transfer in EABs depends on numerous factors, including the biofilm age, the electrode material, the potential at which electron transfer occurs, and so on. In this project, we discover that young Shewanella biofilms transfer electrons prevalently via mediated electron transfer. However, the electron transfer becomes mostly direct as the biofilm ages. In general, the mediated electron transfer is very rapid in thin biofilms, as the diffusion of microbially produced redox agent is not impeded by thick and dense biofilm. However, old biofilms such as those envisioned in practical technological applications will have a predominant direct electron transfer mode, thus producing less current than young biofilms. This finding is relevant to the design of efficient EAB bioreactors, where the biofilm thickness should be kept at a minimum.EAB research requires the development of techniques capable of interrogating several aspect of EAB activity and structure. We developed the first spectroelectrochemical method for the in vivo characterization of EAB. We applied this method to this Geobacter biofilms and were able to determine the redox state of outer membrane cytochromes exposed to increasing electrode potential. The simple setup developed in this project enables rapid and informative characterization of EAB with minimal pretreatment and in non-destructive manner. Overall, this project contributed to the knowledge of EAB and their application to environmental monitoring and wastewater treatment. The results generated in this project have been used to design innovative fuel cells for energy recovery from wastewater and sensitive bioelectrochemical sensors for monitoring of drainage water. Both projects were funded by industrial partner and significant results, in term of technology development, are expected in the next 2-3 years.