Using planktonic microorganisms to supply the unpurified multi-copper oxidases laccase and copper efflux oxidases at a biofuel cell cathode Sabine Sané a , Katrin Richter b , Stefanie Rubenwolf a , Nina Joan Matschke a , Claude Jolivalt c,f , Catherine Madzak d , Roland Zengerle a,e , Johannes Gescher b , Sven Kerzenmacher a,⇑ a Laboratory for MEMS Applications, IMTEK – Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany b Institute for Applied Biosciences, Department of Applied Biology, Karlsruhe Institute of Technology, Fritz-Haber-Weg 2, 76131 Karlsruhe, Germany c Sorbonne Universités, UPMC Univ Paris 06, UMR 7197 Laboratoire de Réactivité de surface, F-75005 Paris, France d INRA, UMR 1319 Micalis, Domaine de Vilvert, F-78352 Jouy-en-Josas, France e BIOSS – Centre for Biological Signalling Studies, University of Freiburg, 79110 Freiburg, Germany f CNRS, UMR 7197 Laboratoire de Réactivité de surface, F-75005 Paris, France highlights  Supply of unpurified laccase and copper efflux oxidase to a biofuel cell cathode.  Using crude culture supernatant from planktonic microorganisms.  Live cells at a cathode provide copper efflux oxidase.  Easy to operate and cost effective enzymatic biofuel cells. article info Article history: Received 7 January 2014 Received in revised form 7 February 2014 Accepted 10 February 2014 Available online 17 February 2014 Keywords: Laccase Copper efflux oxidase Biofuel cell Cathode Bioelectrochemistry abstract The feasibility to apply crude culture supernatants that contain the multicopper oxidases laccase or cop- per efflux oxidase (CueO) as oxygen reducing catalysts in a biofuel cell cathode is shown. As enzyme- secreting recombinant planktonic microorganisms, the yeast Yarrowia lipolytica and the bacterium Esch- erichia coli were investigated. The cultivation and operation conditions (choice of medium, pH) had dis- tinct effects on the electro-catalytic performance. The highest current density of 119 ± 23 lA cm 2 at 0.400 V vs. NHE was obtained with the crude culture supernatant of E. coli cells overexpressing CueO and tested at pH 5.0. In comparison, at pH 7.4 the electrode potential at 100 lA cm 2 is 0.25 V lower. Lac- case-containing supernatants of Y. lipolytica yielded a maximum current density of 6.7 ± 0.4 lA cm 2 at 0.644 V vs. NHE. These results open future possibilities to circumvent elaborate enzyme purification pro- cedures and realize cost effective and easy-to-operate enzymatic biofuel cells. Ó 2014 Elsevier Ltd. All rights reserved. 1. Introduction Biofuel cells (BFC) have great potential for the eco-friendly di- rect conversion of biochemically stored energy into electricity (Bullen et al., 2006; Osman et al., 2010, 2011). A prominent appli- cation is to combine the treatment of wastewater with the gener- ation of electricity (Logan, 2005), hydrogen, or fine chemicals (Rosenbaum et al., 2011). Furthermore, implantable, glucose powered fuel cells which supply medical implants (Cinquin et al., 2010; Kerzenmacher et al., 2008), or miniature biofuel cells that digest organic matter to power energy-autonomous robots (Ieropoulos et al., 2012) are currently under development. Usually, enzymatic catalysts or the biochemical pathways of complete microorganisms are employed to catalyse the electrode reactions in biofuel cells. In this way, the use of expensive and unsustainable noble metal catalysts is circumvented and renew- able electrode materials such as carbon can be utilized as elec- trodes (Bullen et al., 2006; Lapinsonniére et al., 2012; Osman et al., 2010, 2011). However, to bring biofuel cells from the lab-scale into practical application, in particular the cathode performance needs to be improved (Harnisch and Schröder, 2010; Schaetzle et al., 2009). http://dx.doi.org/10.1016/j.biortech.2014.02.038 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved. ⇑ Corresponding author. Tel.: +49 761 203 73218; fax: +49 761 203 73299. E-mail addresses: sabine.sane@imtek.uni-freiburg.de (S. Sané), katrin.richter@ kit.edu (K. Richter), claude.jolivalt@upmc.fr (C. Jolivalt), Catherine.Madzak@ grignon.inra.fr (C. Madzak), zengerle@imtek.uni-freiburg.de (R. Zengerle), johannes. gescher@kit.edu (J. Gescher), kerzenma@imtek.uni-freiburg.de (S. Kerzenmacher). Bioresource Technology 158 (2014) 231–238 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech