Lack of electricity production by Pelobacter carbinolicus indicates that the capacity for Fe(III) oxide reduction does not necessarily confer electron transfer ability to fuel cell anodes

Abstract

The ability of Pelobacter carbinolicus to oxidize electron donors with electron transfer to the anodes of microbial fuel cells was evaluated because microorganisms closely related to Pelobacter species are generally abundant on the anodes of microbial fuel cells harvesting electricity from aquatic sediments. P. carbinolicus could not produce current in a microbial fuel cell with electron donors which support Fe(III) oxide reduction by this organism. Current was produced using a coculture of P. carbinolicus and Geobacter sulfurreducens with ethanol as the fuel. Ethanol consumption was associated with the transitory accumulation of acetate and hydrogen. G. sulfurreducens alone could not metabolize ethanol, suggesting that P. carbinolicus grew in the fuel cell by converting ethanol to hydrogen and acetate, which G. sulfurreducens oxidized with electron transfer to the anode. Up to 83% of the electrons available in ethanol were recovered as electricity and in the metabolic intermediate acetate. Hydrogen consumption by G. sulfurreducens was important for ethanol metabolism by P. carbinolicus. Confocal microscopy and analysis of 16S rRNA genes revealed that half of the cells growing on the anode surface were P. carbinolicus, but there was a nearly equal number of planktonic cells of P. carbinolicus. In contrast, G. sulfurreducens was primarily attached to the anode. P. carbinolicus represents the first Fe(III) oxide-reducing microorganism found to be unable to produce current in a microbial fuel cell, providing the first suggestion that the mechanisms for extracellular electron transfer to Fe(III) oxides and fuel cell anodes may be different.

FIG. 1.

Fuel cells with 5 mM ethanol, inoculated with P. carbinolicus (A) or a coculture of G. sulfurreducens and P. carbinolicus (B). In the coculture, the anode chamber medium was replaced several times and supplemented with 5 mM ethanol (1) or ethanol was added without medium replacement (2). OD₆₀₀, optical density at 600 nm.

FIG. 2.

Comparison of current generation, ethanol consumption, and acetate production in fuel cells with sterile control plus ethanol (A), P. carbinolicus plus ethanol (B), G. sulfurreducens grown with acetate until day 16 and then fed with ethanol (EtOH) (C), and a coculture of P. carbinolicus and G. sulfurreducens fed with ethanol (D). In panels C and D, G. sulfurreducens was grown with acetate in the fuel cell until current production was stable and then media were exchanged (acetate was omitted, and ethanol or ethanol and P. carbinolicus were added).

FIG. 3.

Current generation and metabolite concentrations in cocultures of P. carbinolicus and a G. sulfurreducens strain with the uptake hydrogenase gene deleted (A) or wild-type G. sulfurreducens (B). The anode was poised at +300 mV with a potentiostat. Cultures were initiated with vigorous sparging in order to remove hydrogen produced as outlined in the text. Time courses are shown after the sparging was stopped on day 1.

FIG. 4.

Current and concentrations of ethanol and acetate in a coculture fuel cell in an anaerobic glove bag. First the fuel cell was set up to sparge the cathode (air) and anode (N₂-CO₂). It was then inoculated with G. sulfurreducens plus 10 mM acetate (arrow 1). After initial growth, the anode medium was exchanged (P. carbinolicus and 5 mM ethanol were added) (arrow 2). Medium plus ethanol was replaced two more times until stable current was achieved (arrows 3 and 4). Ethanol, acetate, and current were measured in “sparging mode” until ethanol was completely consumed (arrows 4 and 5). Then oxygen was sparged out of the cathode chamber with N₂-CO₂, while the cathode medium was supplemented with K₃Fe(III)(CN)₆. Sparging of both chambers was stopped, and the fuel cell was put into an anaerobic glove bag (arrow 5). Electron recovery was then determined without evaporative loss of metabolites (arrows 5 and 6).

FIG. 5.

Top views (large squares) and orthogonal side views (top and side rectangles) of P. carbinolicus and G. sulfurreducens biofilm growing on an anode and hybridized with probes targeting Deltaproteobacteria (probe DELTA495, red) (A), Desulfuromonas cluster (probe DMONAS, red) (B), G. sulfurreducens (probe GEO2, red) (C), and P. carbinolicus (probe PCARB1, red) and G. sulfurreducens (GEO2, blue) (D). All biofilm sections are shown with YOYO-1 counterstain (green), except in panel D. Helper probes HGEO2-1 and HGEO2-2 were used in panels C and D. Bar = 10 μm.