pH, redox potential and local biofilm potential microenvironments within Geobacter sulfurreducens biofilms and their roles in electron transfer

Abstract

The limitation of pH inside electrode-respiring biofilms is a well-known concept. However, little is known about how pH and redox potential are affected by increasing current inside biofilms respiring on electrodes. Quantifying the variations in pH and redox potential with increasing current is needed to determine how electron transfer is tied to proton transfer within the biofilm. In this research, we quantified pH and redox potential variations in electrode-respiring Geobacter sulfurreducens biofilms as a function of respiration rates, measured as current. We also characterized pH and redox potential at the counter electrode. We concluded that (1) pH continued to decrease in the biofilm through different growth phases, showing that the pH is not always a limiting factor in a biofilm and (2) decreasing pH and increasing redox potential at the biofilm electrode were associated only with the biofilm, demonstrating that G. sulfurreducens biofilms respire in a unique internal environment. Redox potential inside the biofilm was also compared to the local biofilm potential measured by a graphite microelectrode, where the tip of the microelectrode was allowed to acclimatize inside the biofilm.

Figure 1

Diagram of redox and biofilm potentials. A biofilm is grown on top of the electrode. The potential of this electrode against a reference electrode is called the biofilm electrode potential. A microelectrode with a Pt tip is inserted into the biofilm. The Pt tip equilibrates with the redox-active compounds within the biofilm, and the potential under this condition is called the redox potential. A microelectrode with a carbon fiber tip is inserted into the biofilm, and after the carbon fiber tip is polarized, the tip connects electronically to the biofilm. The potential under no-current conditions after the biofilm is electronically connected to the microelectrode tip is called the local biofilm potential (LBP).

Figure 2

A three-electrode bioreactor. The working, counter, and reference electrodes were housed in the same compartment. The inset shows a perspective view.

Figure 3

A: Redox microelectrode, (B) pH microelectrode, and (C) graphite microelectrode. RE represents the Ag/AgCl reference electrode.

Figure 4

A: Current generation of the G. sulfurreducens biofilm over time. The red arrows show the times when the pH and redox potential profiles were measured inside the biofilm. The images show the biofilm during measurement. B: Steady state polarization curve superimposed on slow-scan CV (1 mV/s). At polarization potentials above 0.2 V_Ag/AgCl, a maximum current is achieved.

Figure 5

A: The pH profiles at the biofilm electrode. B: The redox potential profiles at the biofilm electrode. The inset shows the profiles over a smaller y-axis range for clarity. The biofilm electrode was polarized to 0.45 V_Ag/AgCl during measurements. The filled in-circles represent profiles measured at 1.05 mA. The open circles represent profiles measured at a current density of 1.85 mA.

Figure 6

Measurements of pH and redox potential above an exposed biofilm electrode surface. The inset shows a microelectrode measuring a profile above the exposed electrode surface. The redox potential remained constant until it touched the electrode surface.

Figure 7

CV of a G. sulfurreducens biofilm at a large glassy carbon electrode (black trace) and at a graphite microelectrode tip inserted into the same biofilm (red trace). The inset shows the graphite microelectrode tip inserted into the G. sulfurreducens biofilm.

Figure 8

A: pH profiles at the counter electrode. B: Redox potential profiles at the counter electrode. The inset shows the profiles over a smaller y-axis range for clarity. The biofilm electrode was polarized at +450 mV_Ag/AgCl during measurements. The filled-in circles represent profiles measured at 1.05 mA, and the open circles represent profiles measured at a current density of 1.85 mA.

Figure 9

Comparison of all pH and redox potential profiles with the theoretical redox potential and pH relationship for the H₂/H⁺ redox couple using Equation (2). Redox potential is abbreviated as E_h for clarity.