was cultured with acetate as the electron donor and fumarate as the electron acceptor as previously described (2
). Cells were collected via centrifugation and resuspended in an equal volume of medium lacking electron donors or acceptors. Cells (5%) were inoculated into 250-ml anaerobic glass chambers which contained only a graphite electrode (62 cm2
) as an electron acceptor. The medium in the chambers was identical to that used for growth, except that fumarate was omitted and 1 mM acetate was provided as the electron donor (2
). The anode in the growth chamber was connected via a 500-Ω resistor to an identical electrode (cathode) in a second chamber, which was flushed with air. The two chambers were linked by a Nafion 117 apparatus, as previously described (2
After a lag (typically 3 to 5 days), current production increased exponentially. When current production declined, due to exhaustion of the donor (verified by high-pressure liquid chromatography) (2
), more acetate (1 mM) was added, and current production resumed. Typically, a second addition of acetate resulted in higher rates of current production (peak values of 0.08 mA), but subsequent additions of acetate typically did not increase current output significantly. In mature cultures, acetate declined as power was produced (Fig. ), and recovery of electrons (integrated current over time versus acetate disappearance) was 94 ± 6% (mean ± standard deviation; n
= 3). Acetate (open symbols; Fig. ) initially disappeared faster than could be accounted for by current recovery (solid line; Fig. ), suggesting storage rather than oxidation of some of the acetate, but as acetate concentrations declined, near-complete electron recovery levels were observed.
FIG. 1. Acetate disappearance (•), electron recovery predicted on the basis of acetate disappearance (○), electron recovery on the basis of integrated amperage (solid line), and current production (inset) for a representative microbial fuel cell (more ...)
Addition of propionate (5 mM), malate (5 mM), lactate (5 mM), or succinate (5 mM) resulted in higher rates of current production than addition of acetate. Propionate and lactate produced the highest peak rates of current flow (0.31 and 0.26 mA, respectively), with malate and succinate producing less (0.11 and 0.15 mA). Propionate and lactate were only partially oxidized to acetate, likely due to the additional substrate-level ATP gain associated with acetate production. Electron recovery data (comparison of integrated current and high-pressure liquid chromatography data) indicated that acetate produced from these compounds was further oxidized in the latter stages of the incubation, when propionate and lactate were depleted. Succinate and malate were completely oxidized.
While ΔE values (calculated from ΔGf
) indicated that all of these organic acids were stronger electron donors than acetate, increases in the rate of current flow did not correlate with their formal potentials. For instance, the ΔE′ of the CO2
-malate half-reaction is −0.35 V, while that for the acetate-propionate half-reaction is −0.28 V. Current production correlated more strongly with the compounds which were partially (and more rapidly) oxidized than with compounds for which only a complete oxidation pathway was available. Peptone (1 g/liter) or yeast extract (1 g/liter) also resulted in short periods of current production for ca. 48 h that peaked at rates lower than those observed with acetate (0.04 and 0.03 mA, respectively), suggesting that some components of each could be oxidized by the organism. Flushing of chambers with hydrogen as a potential electron donor did not result in any current production, an observation consistent with the fact that G. fermentans
cannot use hydrogen to reduce other electron acceptors.
To reduce limitations imposed by the resistive load and oxygen reduction reactions at the cathode and to measure current production by attached G. fermentans
cells under controlled conditions, the potential of the anode was poised with a potentiostat (2
). G. fermentans
could be grown on an anode surface with the anode poised at +200 mV versus an Ag/AgCl reference electrode, and the counter electrode chamber was flushed with 80:20 N2
. Under these conditions, the rate of current production was 0.6 mA at 1 mM acetate, with an electron recovery of 97 ± 4% (n
An average of 3.3 ± 0.7 mg of protein/electrode (n
= 4) was recovered when electrode surfaces from acetate-grown cultures were treated with 0.2 N NaOH and scraped to remove attached biomass. When electrode surfaces were fixed with 1% glutaraldehyde, dehydrated in a series of buffers with increasing ethanol concentrations, subjected to CO2
critical-point drying, and visualized via scanning electron microscopy (SEM) (2
), the cells attached to the electrodes appeared to be coated in a thick matrix (Fig. ). This differed from previous studies with members of the Proteobacteria
family in which individual cells appeared to be attached to the electrode surface without substantial extracellular material (2
SEM image of a graphite anode after growth of G. fermentans in a microbial fuel cell.