Electron donors supporting growth and electroactivity of Geobacter sulfurreducens anode biofilms

Abstract

Geobacter bacteria efficiently oxidize acetate into electricity in bioelectrochemical systems, yet the range of fermentation products that support the growth of anode biofilms and electricity production has not been thoroughly investigated. Here, we show that Geobacter sulfurreducens oxidized formate and lactate with electrodes and Fe(III) as terminal electron acceptors, though with reduced efficiency compared to acetate. The structure of the formate and lactate biofilms increased in roughness, and the substratum coverage decreased, to alleviate the metabolic constraints derived from the assimilation of carbon from the substrates. Low levels of acetate promoted formate carbon assimilation and biofilm growth and increased the system's performance to levels comparable to those with acetate only. Lactate carbon assimilation also limited biofilm growth and led to the partial oxidization of lactate to acetate. However, lactate was fully oxidized in the presence of fumarate, which redirected carbon fluxes into the tricarboxylic acid (TCA) cycle, and by acetate-grown biofilms. These results expand the known ranges of electron donors for Geobacter-driven fuel cells and identify microbial constraints that can be targeted to develop better-performing strains and increase the performance of bioelectrochemical systems.

Fig 1

Current generation (solid line) and electron donor uptake (circles [red, acetate; green, formate; blue, lactate]) (A to D) and CLSM micrographs of anode biofilms (E to H) in fuel cells fed with acetate (A and E), H₂ (B and F), formate (C and G), or lactate (D and H). The inset in panel B shows controls with no electron donor. The biofilms in panels E to H were stained with the BacLight viability dyes (green, live cells; red, dead cells). Top views and the corresponding projections in the x (bottom) and y (right) axes are shown. Scale bar, 20 μm.

Fig 2

Iron reduction (A) and growth (B) of G. sulfurreducens with acetate (circles), formate (triangles), or lactate (squares) as the electron donor and Fe(III) citrate (A) or fumarate (B) as the electron acceptor. (C) Lactate oxidation coupled to fumarate reduction and generation of malate and succinate in lactate-fumarate cultures shown in panel B.

Fig 3

Metabolic routes for the oxidation (ev̄) and carbon assimilation (C) of acetate, formate, and lactate (in bold). Alternative routes predicted to also be operative when fumarate serves as the electron acceptor are shown with dashed lines. Enzyme abbreviations: PFL, pyruvate formate lyase; FDH, formate dehydrogenase; ACK, acetate kinase; PTA, phosphotransacetylase; PDH, pyruvate dehydrogenase; POR, pyruvate oxidoreductase; LDH, lactate dehydrogenase.

Fig 4

(A and B) Effect of acetate supplementation (0.1 mM) on current generation (A) and substrate uptake (B) in fuel cells with acetate (red), formate (black), and lactate (blue). Left axes in panel B, acetate in mM; right axes, formate or lactate in mM; x axes, time in days. (C and D) CLSM micrographs of the anode biofilms from formate (C) and lactate (D) fuel cells supplemented with 0.1 mM acetate. The biofilms were stained with the BacLight viability dyes (green, live cells; red, dead cells). Top views and the corresponding projections in the x (bottom) and y (right) axes are shown. Scale bar, 20 μm.

Fig 5

Lactate oxidation by acetate-grown biofilms in BESs. (A) Current generation coupled to acetate and then lactate use as electron donors. The addition of lactate to the anode chamber is shown with an arrow. (B) CLSM micrograph of lactate-oxidizing anode biofilms previously grown with acetate. The biofilms were stained with the BacLight viability dyes (green, live cells; red, dead cells). Top view and the corresponding projections in the x (bottom) and y (right) axes are shown. Scale bar, 20 μm.