BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING Use of Pseudomonas species producing phenazine-based metabolites in the anodes of microbial fuel cells to improve electricity generation The Hai Pham & Nico Boon & Katrien De Maeyer & Monica Höfte & Korneel Rabaey & Willy Verstraete Received: 19 May 2008 / Revised: 10 July 2008 / Accepted: 11 July 2008 / Published online: 8 August 2008 # Springer-Verlag 2008 Abstract The rate of anodic electron transfer is one of the factors limiting the performance of microbial fuel cells (MFCs). It is known that phenazine-based metabolites produced by Pseudomonas species can function as electron shuttles for Pseudomonas themselves and also, in a syntrophic association, for Gram-positive bacteria. In this study, we have investigated whether phenazine-based metabolites and their producers could be used to improve the electricity generation of a MFC operated with a mixed culture. Both anodic supernatants obtained from MFCs operated with a Pseudomonas strain (P-PCA) producing phenazine-1-carboxylic acid (PCA) and those from MFCs operated with a strain (P-PCN) producing phenazine-1- carboxamide (PCN) exerted similarly positive effects on the electricity generation of a mixed culture. Replacing super- natants of MFCs operated with a mixed culture with supernatants of MFCs operated with P-PCN could double the currents generated. Purified PCA and purified PCN had similar effects. If the supernatant of an engineered strain overproducing PCN was used, the effect could be main- tained over longer time courses, resulting in a 1.5-fold increase in the production of charge. Bioaugmentation of the mixed culture MFCs using slow release tubes contain- ing P-PCN not only doubled the currents but also maintained the effect over longer periods. The results demonstrated the electron-shuttling effect of phenazine- based compounds produced by Pseudomonas species and their capacity to improve the performance of MFCs operated with mixed cultures. Keywords Microbial fuel cell . Electrochemically active bacteria . Phenazines . Bacterial interactions . Mixed culture Introduction Microbial fuel cells (MFCs) are known as bioelectrochem- ical systems (BESs) that enable conversion of chemical energy into electrical energy through the catalytic activity of microorganisms (Allen and Bennetto 1993; Rabaey et al. 2007; Rao et al. 1976). Due to their unique characteristics, in addition to their putative application in wastewater treatment for energy recovery, the application field of BESs has been expanded (Clauwaert et al. 2008; Rabaey et al. 2007). To render the use of MFCs in particular, and BESs in general, feasible in practice, several limiting factors that cause losses in these systems need to be overcome (Clauwaert et al. 2008; Kim et al. 2007; Pham et al. 2006; Rabaey and Verstraete 2005). Among these factors, the rate of electron transfer between microbes and the anodic electrode is critical. In order to improve the anodic electron transfer, it is essential that the catalytic activity of the electrode material, Appl Microbiol Biotechnol (2008) 80:985–993 DOI 10.1007/s00253-008-1619-7 Electronic supplementary material The online version of this article (doi:10.1007/s00253-008-1619-7) contains supplementary material, which is available to authorized users. T. H. Pham : N. Boon : W. Verstraete (*) Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Coupure Links 653, 9000 Ghent, Belgium e-mail: Willy.Verstraete@UGent.be K. De Maeyer : M. Höfte Laboratory of Phytopathology, Ghent University, Coupure Links 653, 9000 Ghent, Belgium K. Rabaey Advanced Water Management Centre, University of Queensland, Brisbane, QLD 4072, Australia