32 Fig. 2. Protein content of the medium in the anode chamber and on the anode of fuel cells inoculated with Shewanella oneidensis MR-1. Protein from planktonic cells was determined after each exchange of the medium. Protein from both planktonic and anode associated cells was determined after termination of the experiment. All fuel cells contained FW medium supplemented with lactate, except the fuel cell with hydrogen, which was supplemented with lactate and no hydrogen until the ﬁrst medium exchange, and then bubbled with hydrogen without lactate. No third medium exchanged was performed on the fuel cell with deﬁned medium and hydrogen. amended with yeast extract (97.0 3.5%; n = 7) than in deﬁned medium (75.2 7.2%; n = 7), further suggesting that the yeast extract-amended medium was a superior medium for growth in fuel cells. These anaerobic anode bioﬁlms differ signiﬁcantly from the much more extensive bioﬁlms that S. oneidensis MR-1 forms when grown in aerobic ﬂow-through cells with rich medium (Thormann et al., 2004). Analysis of the stoichiometry of lactate metabolism and electricity production demonstrated that lactate was con- sumed over time with an approximate (1 : 1) stoichiometric accumulation of acetate (Fig. 1a and b). The medium was exchanged after every second addition of lactate in the microbial fuel cells. For the ﬁrst additions of lactate after the medium was exchanged, the accumulation of acetate from lactate was 86.8 1.6% (mean SD; n = 3) in the deﬁned medium, whereas in the yeast extract-amended medium the recovery was 94.9 5.4%. These results show that lactate was partially oxidized by MR-1 according to the reaction Lactate þ 2H2O ! Acetate þ HCO3 þ 5Hþ þ 4e which is consistent with previous studies (Lovley et al., 1989, 1992). The coulombic yield based on partial lactate oxidation (4 e mol 1 lactate) was 55.9 12.9% (n = 3) in deﬁned medium and 56.2 14.8% for the medium amended with yeast extract. In this case, a coulombic yield less than 100% c 2007 The Authors Journal compilation c 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. M. Lanthier et al. reﬂects the fraction of electrons obtained from partial lactate oxidation which were diverted to cell synthesis, rather than electricity production, during growth of MR-1 on the anode. Similar electron recovery was observed with MR-1 during growth on nitrate (Middleton et al., 2003) and are typical of other bacteria which exhibit anaerobic nonfermentative respiration and growth (Lo¨fﬂer et al., 1999). In order to evaluate the potential for electricity produc- tion with hydrogen as the electron donor, cells were initially inoculated into an anode chamber containing deﬁned lactate medium and once current production was estab- lished, the medium was replaced with lactate-free medium and continuously bubbled with a mixture of H2/CO2/N2 (7 : 10 : 83). Current production was comparable to that with lactate as the electron donor (Fig. 1d). As in lactate cultures, there was visible turbidity in the anode chamber, corresponding with the recovery of cell protein in the medium (Fig. 2). Turbidity decreased after the media was exchanged with lactate-free medium, but remained stable each time the medium was exchanged afterward (Fig. 2). There was less protein biomass attached to the anode than planktonic protein biomass, consistent with the results with lactate as the fuel (Fig. 2). Conclusion These results further demonstrate the diversity of strategies that microorganisms may employ for growth with electrodes serving as the electron acceptor. For example, in previous studies with other pure cultures (Bond & Lovley, 2003, 2005; Chaudhuri & Lovley, 2003; Holmes et al., 2004a, b), the electricity-producing microorganisms were predominately attached to the anode surface. In contrast, most of the cellular protein in the S. oneidensis fuel cells presented in this study were planktonic. Shewanella species are capable of producing electron shuttles, which permit them to reduce insoluble Fe(III) oxides without direct cell-electron acceptor contact (Newman & Kolter, 2000; Nevin & Lovley, 2002a, b; Lies et al., 2005). Although these electron shuttles are generally considered to be small, soluble molecules, they might also be comprised of the membrane vesicles contain- ing redox proteins that are known to be released from gram- negative microorganisms (Krause et al., 1996; Schooling & Beveridge, 2006). It seems likely that electron shuttle pro- duction permits S. oneidensis to oxidize lactate or hydrogen without being attached to the anode surface as electroche- mical analyses suggest electron-shuttling vs. direct electron transfer in Shewanella-based microbial fuel cells (Marsili et al., 2007). For some organisms, such as Geobacter species, which do not produce electron shuttles (Nevin & Lovley, 2000; Bond & Lovley, 2003) close association of the cells with the anode is likely to be essential for electron transfer (Bond & Lovley, 2003; Reguera et al., 2006). However, even FEMS Microbiol Lett 278 (2008) 29–35