Growth during electricity production by Shewanella 31 Fig. 1. Current production, lactate consumption and acetate accumula- tion by Shewanella oneidensis MR-1 growing with lactate as an electron donor and an anode as the sole terminal electron acceptor in (a) deﬁned FW medium; (b) FW medium supplemented with 0.05% yeast extract. Fuel cells fed with acetate in deﬁned FW medium (c) or hydrogen in deﬁned FW medium (d) were ﬁrst started with lactate as an electron donor, and medium was exchanged with the appropriate electron donor when current production was steady. First (1), second (2) or third (3) medium exchange in the anode chamber was accompanied by addition of 3 mM lactate (lac), 4 mM acetate (ac), or bubbling with hydrogen (as H2/CO2/N2) (H), as noted. Sampling of medium and electrode at the end of experiment was dubbed ‘ﬁnal’ (ﬁnal). The medium in fuel cell C was only exchanged twice. FEMS Microbiol Lett 278 (2008) 29–35 the cathode, similar studies were also conducted in which the anode was poised with a potentiostat at 1300 mV (vs. Ag/AgCl reference) as described previously (Bond & Lovley, 2003). However, sustained power production in the poised system was no higher than in fuel cells (data not shown). Therefore, all remaining studies were conducted as fuel cells. Growth and electron recovery The protein content from planktonic cells in the anode chambers was quantiﬁed after each exchange of the medium and after termination of the experiments (as indicated in Fig. 1). Protein on the anode was determined only after termination of the experiments (Fig. 1). There were sub- stantial amounts of planktonic protein biomass in the anode chambers with higher quantities of planktonic protein biomass in the yeast-extract amended medium than the deﬁned medium (Fig. 2). Planktonic cells became abundant each time the anode medium was exchanged, as indicated by the return of visible turbidity and measured by high planktonic cell protein after each medium exchange (Fig. 2). Planktonic protein biomass declined with each medium exchange accompanied by lactate amendment. The decrease in planktonic protein biomass over the course of the experiment was not as large in deﬁned medium as in yeast extract amended medium. After multiple medium replace- ments (Fig. 1a, b and d), the anodes were removed from the anode chamber and analyzed for protein. There was less attached protein biomass than planktonic protein biomass at the termination of the experiment (Fig. 2). There was more protein biomass on the anodes from yeast extract medium than deﬁned medium. Growth of MR-1 with the anode serving as the sole electron acceptor was further evaluated by inoculating a fuel cell containing deﬁned FW medium and 10 mM lactate with a small inoculum of cells (0.04 mg protein). After 10 days, current was produced at a level equivalent to that observed with the larger inocula shown in Fig. 1. After 14 days, the total protein in the anode chamber and on the anode surface was 2.29 mg (anode: 0.28 mg). These results demonstrate that S. oneidensis may conserve energy to support growth using the anode in a fuel cell as the sole terminal electron acceptor. Growth on the anodes was also apparent with CLSM and SEM (Fig. 3). Cells were scattered over the surface of the anodes from fuel cells with deﬁned medium (Fig. 3a and b). There was greater coverage of the anodes from fuel cells with yeast extract medium, but there were still substantial por- tions of the anode that were not colonized with cells and in no instance were thick bioﬁlms of multiple stacked cells observed (Fig. 3c and d). CLSM images indicate a higher density of live cells on the anode recovered from the fuel cell c 2007 The Authors Journal compilation c 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd.