30 Experimental Growth conditions and microbial fuel cells Shewanella oneidensis strain MR-1 (ATCC 7005500), was grown in anaerobic, freshwater, lactate (20 mM)-Fe(III) citrate (50 mM) medium (FW medium) as described pre- viously (Lovley et al., 1989) with the exception that the three amino acids typically added to the medium (L-arginine, L-glutamine, and DL-serine) were deleted and the medium was amended with 0.05% (w/v) yeast extract. The cells from a 100 mL culture near the end of log phase were collected by centrifugation and resuspended in FW medium (5 mL) without yeast extract or Fe(III) citrate. These cells (3.7 mg of total protein) were inoculated into the anode chamber of previously described (Bond & Lovley, 2003) fuel cells in which graphite sticks (surface area 61.2 cm2) serve as the anode and cathode. The electrical connection between the anode and cathode included a resistor of 560 O (Bond & Lovley, 2003). Current was measured hourly with a Keithley Model 2700 Digital Multimeter (Keithley Instruments, Cleveland, OH). The anode chamber contained 225 mL of FW medium described above or ‘defined FW medium’ in which the yeast extract was omitted and substituted with L-arginine (22 mg L 1), L-glutamine (22 mg L 1), and DL-serine (44 mg L 1). The initial lactate concentration was 10 mM. The cathode chamber contained 225 mL of FW medium without yeast extract, amino acids, or Fe(III). The anode chamber was continuously bubbled with N2/CO2 (80 : 20) and the cathode chamber was continuously bubbled with air. All incubations were at 30 1C. Protein quantification Total protein content from planktonic cells in the anode chambers was quantiﬁed after each exchange of the medium and after termination of the experiments. Protein concen- tration was determined on centrifuged cell pellets and on bioﬁlm scraped from electrodes with sterile razorblades by the bicinchoninic acid method with bovine serum albumin as a standard (Smith et al., 1985). Microscopy Cells associated with the anodes were stained with the BacLight Live/Dead viability kit (Invitrogen–Molecular Probes Inc., Eugene, OR), as described previously (Korber et al., 1996) before examination with confocal laser scanning microscopy (CLSM). Samples were examined on a Zeiss Axiovert LSM 510 Meta Confocal System equipped with a 63 Zeiss plan-apochromat oil immersion objective (numerical aperture of 1.4) and a Meta detector (Carl Zeiss MicroImaging Inc., Thornwood, NY). The confocal microscope was equipped with an argon laser (lines at 458, c 2007 The Authors Journal compilation c 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. M. Lanthier et al. 477, 488 and 514 nm; 25 mW total), and two HeNe lasers (543 nm, 1 mW; 633 nm, 5 mW), krypton–argon dual laser (488 and 568 nm) and a diode laser (638 nm). Representa- tive three-dimensional scans of bioﬁlm sections on the electrodes were taken, and displayed as ortho view. Images were averaged by Kalman ﬁltration with eight running scans per image (Manz et al., 1999). The acquisition software was LSM 510 META, version 3.2 SP2. The ratio of live/dead cells on the anodes was determined by quantifying the ratio of dead cells (red) and live cells (green) from confocal images as described previously (Lanthier et al., 2005). The IMAGEJ software v1.31 (US National Institutes of Health, Bethesda, MD, http://rsb.info.nih.gov/ij/) was used for image analysis. Anodes sacrificed for SEM were prepared as described previously (Bond & Lovley, 2003) except that the whole electrode was chemically dehydrated with hexamethyldisilazane. Results and discussion Current production With either the yeast extract-amended or deﬁned medium, current production increased over time to a maximum of 0.2–0.3 mA (Fig. 1a and b). Maximum current was typically reached earlier in the medium containing yeast extract than the deﬁned medium, but there was no consistent difference in the maximum current between the two medium types (Fig. 1). The anode chambers were turbid with planktonic cells and higher turbidity was observed with yeast extract- amended medium (see biomass quantiﬁcation by protein assay in the next section). The medium in the anode chambers was exchanged by removing the medium and planktonic cells (but retaining anode-associated cells) and replacing with new FW medium (Bond & Lovley, 2003). After addition of 3 mM lactate, current production resumed at levels comparable (0.2–0.3 mA) to those before exchanging the medium within 12 h in deﬁned medium and within 3 h in medium containing yeast extract. This observation was con- sistent after each exchange of the medium (Fig. 1a and b). Addition of yeast extract did not increase current produc- tion, but increased protein biomass both in suspension and on the anode (Fig. 2). Current was not observed in the absence of cells (data not shown), or in the presence of 4 mM acetate (Fig. 1c), an electron donor not utilized by S. oneidensis under anaerobic conditions (Lovley et al., 1992). When the acetate medium was exchanged with medium containing lactate, power production resumed, but at a somewhat lower initial rate (Fig. 1c). This is presumably due to the inability of cells to maintain as active a population during the incubation with acetate. In order to ensure that the current production in the presence of cells and lactate was not limited by reactions at FEMS Microbiol Lett 278 (2008) 29–35