Long-range electron transport in Geobacter sulfurreducens biofilms is redox gradient-driven

Abstract

Geobacter spp. can acquire energy by coupling intracellular oxidation of organic matter with extracellular electron transfer to an anode (an electrode poised at a metabolically oxidizing potential), forming a biofilm extending many cell lengths away from the anode surface. It has been proposed that long-range electron transport in such biofilms occurs through a network of bound redox cofactors, thought to involve extracellular matrix c-type cytochromes, as occurs for polymers containing discrete redox moieties. Here, we report measurements of electron transport in actively respiring Geobacter sulfurreducens wild type biofilms using interdigitated microelectrode arrays. Measurements when one electrode is used as an anode and the other electrode is used to monitor redox status of the biofilm 15 μm away indicate the presence of an intrabiofilm redox gradient, in which the concentration of electrons residing within the proposed redox cofactor network is higher farther from the anode surface. The magnitude of the redox gradient seems to correlate with current, which is consistent with electron transport from cells in the biofilm to the anode, where electrons effectively diffuse from areas of high to low concentration, hopping between redox cofactors. Comparison with gate measurements, when one electrode is used as an electron source and the other electrode is used as an electron drain, suggests that there are multiple types of redox cofactors in Geobacter biofilms spanning a range in oxidation potential that can engage in electron transport. The majority of these redox cofactors, however, seem to have oxidation potentials too negative to be involved in electron transport when acetate is the electron source.

Fig. 1.

SEM of a fully grown WT G. sulfurreducens biofilm grown on a gold interdigitated microelectrode array (IDA). The edges of the array were masked with photoresist, defining the electroactive area where the biofilm grew, which was removed during preparation for SEM imaging. An unmasked edge of the array is shown at the bottom, where alternate microelectrode bands comprising electrode 1 are electrically connected. (Scale bar: 45 μm.) (Inset) Schematic representation of a portion of the IDA depicting 10 of 100 microelectrode bands (not to scale; dimensions provided in the text).

Fig. 2.

Schematic depiction of a cross-section of biofilm-coated IDA. (A) Anode/open circuit experiment in which electrode 1 is used as an anode that collects electrons coupled to cellular oxidation of acetate throughout the biofilm, whereas electrode 2 is at open circuit and therefore, does not accept electrons; however, it is used to measure oxidation state of the biofilm in the vicinity of electrode 2. White arrows indicate flux of the electrons to microelectrode bands comprising electrode 1 coupled to cellular oxidation of acetate throughout the biofilm continuously supplied by diffusion from adjacent media. A specific case is shown, in which the potential applied to electrode 1 is +0.300 V and the open circuit potential measured at electrode 2 is −0.463 V based on the results depicted in Fig. 3B. In this case, while the biofilm is fully oxidized in vicinity of electrode 1, it is only 44% oxidized in vicinity of electrode 2 (Fig. 3B). (B) Electrochemical gate experiment in which different potentials are applied to electrodes 1 and 2 while maintaining a constant potential offset between the electrodes, resulting in electron transport through the biofilm from the more negative electrode (electron source is electrode 2) to the more positive electrode (electron drain is Electron 1), which is indicated by white arrows. A specific case is shown, in which the potential applied to electrode 1 is −0.475 V and the potential applied to electrode 2 is −0.575 V, resulting in the largest conducted current based on the results depicted in Fig. 5A. Unless otherwise noted, potentials are vs. Ag/AgCl.

Fig. 3.

Anode/open circuit experiment. (A) Catalytic (turnover) CV recorded by scanning potential of electrode 1 at 0.002 V/s from +0.300 V to −0.750 V (cathodic scan) and back to +0.300 V (anodic scan), whereas electrode 2 is at open circuit (asterisk indicates cathodic scan). Catalytic current, , results from the electron transport to microelectrode bands comprising electrode 1 coupled to cellular acetate oxidation throughout the biofilm. The x axis corresponds to the potential applied to electrode 1. Fits are based on Eq. 1. (A, Inset) Nonturnover CV of electrode 1 at 0.002 V/s recorded in absence of acetate, revealing voltammetric peaks attributable to biofilm redox cofactors (same axis scales). (B, right axis) Potential measured for electrode 2 vs. potential applied to electrode 1. (B, left axis) Corresponding biofilm oxidation state in the vicinity of electrode 1, , and electrode 2, , vs. potential applied to electrode 1 calculated using Eq. 2.

Fig. 4.

Electrochemical gate experiment recorded under nonturnover condition (no acetate present). (A) Conducted current, vs. average potential applied to electrodes 1 and 2, . Electrode potentials were scanned at 0.002 V/s while maintaining electrode 2 (electron source) at a fixed −0.100 V offset vs. electrode 1 (electron drain). Fits are based on Eq. 3. (B) The corresponding plot of and vs. was calculated using Eq. 2. Arrows indicate the condition when difference between and is the greatest, resulting in the largest value of .

Fig. 5.

Electrochemical gate experiment recorded under turnover condition (with acetate present). (A) Because each electrode also acts as an anode, the resulting current measured at electrode 1 (drain) is the sum of catalytic current and conducted current out of the biofilm, and at electrode 2 (source) is the sum of catalytic current and conducted current into the biofilm, plotted here vs. potential applied to each electrode. Electrode potentials were scanned at 0.002 V/s while maintaining electrode 2 at a fixed −0.100 V offset vs. electrode 1. (A, Inset) CV recorded for the two electrodes simultaneously without an offset such that for background subtraction of for each of electrode (same axis scales). (B) vs. after background subtracting . Qualitative fits are based on Eq. 3 (same as in Fig. 4A).