DIFFUSION IN BIOFILMS RESPIRING ON ELECTRODES

Abstract

The goal of this study was to measure spatially and temporally resolved effective diffusion coefficients (D(e)) in biofilms respiring on electrodes. Two model electrochemically active biofilms, Geobacter sulfurreducens PCA and Shewanella oneidensis MR-1, were investigated. A novel nuclear magnetic resonance microimaging perfusion probe capable of simultaneous electrochemical and pulsed-field gradient nuclear magnetic resonance (PFG-NMR) techniques was used. PFG-NMR allowed noninvasive, nondestructive, high spatial resolution in situ D(e) measurements in living biofilms respiring on electrodes. The electrodes were polarized so that they would act as the sole terminal electron acceptor for microbial metabolism. We present our results as both two-dimensional D(e) heat maps and surface-averaged relative effective diffusion coefficient (D(rs)) depth profiles. We found that 1) D(rs) decreases with depth in G. sulfurreducens biofilms, following a sigmoid shape; 2) D(rs) at a given location decreases with G. sulfurreducens biofilm age; 3) average D(e) and D(rs) profiles in G. sulfurreducens biofilms are lower than those in S. oneidensis biofilms-the G. sulfurreducens biofilms studied here were on average 10 times denser than the S. oneidensis biofilms; and 4) halting the respiration of a G. sulfurreducens biofilm decreases the D(e) values. Density, reflected by D(e), plays a major role in the extracellular electron transfer strategies of electrochemically active biofilms.

Keywords: Geobacter; Shewanella; biofilm; diffusion; diffusion coefficient; diffusivity; electrochemically active; electron transfer; magnetic resonance; modeling.

Figure 1. Nuclear magnetic resonance microimaging system for studying EABs

An illustration of the experimental arrangement for NMR used to study diffusion in EABs. The diagrams show (from left to right): the vertical bore superconducting magnet with perfusion lines leading to the bottom-loaded NMR probe (medium flowing against gravity); a cutaway view of the custom NMR probe, shown holding the NMR biofilm reactor; an external view of the NMR biofilm reactor; and a cutaway view of the NMR biofilm reactor containing a perfused EAB growing on a gold disc electrode.

Figure 2. G. sulfurreducens biofilm growth

Left) Time series of three normal-plane 2D MRI showing progression of the growth of a G. sulfurreducens biofilm. The white arrows indicate the top of the biofilm. The ages shown are 24, 35, and 52 days. In the initial image the biofilm is 170 μm thick, and in the final image the biofilm is 370 μm thick. Right) A face-plane 2D MRI of the biofilm. The white arrows indicate the edges of the biofilm on top of the electrode. A 1 mm scale bar is provided at the top of each MRI (the normal-plane and face-plane images have the identical scale).

Figure 3. Diffusion mapping of a G. sulfurreducens biofilm over time

The top row shows two-dimensional D_e maps obtained using PFG-NMR, normalized against D_aq, of the middle 2 mm of the biofilm. The dark regions represent low D_e. The bottom row shows D_rs depth profiles derived by averaging the D_e of the middle 2 mm of the biofilm (shown in the maps). The top of the biofilm as determined using magnetic resonance imaging is indicated by the vertical lines. From left to right, the panels show the G. sulfurreducens biofilm at 24, 35, and 52 days old.

Figure 4. S. oneidensis biofilm growth

Left) Time series of two normal-plane 2D MRI showing the progression of the growth of an S. oneidensis biofilm. The white arrows indicate the top of the biofilm. The ages shown are 12 and 16 days. In the initial image the biofilm is 410 μm thick, and in the final image the biofilm is 450 μm thick. Right) A face-plane 2D MRI of the biofilm. The white arrows indicate the edges of the biofilm on top of the electrode. A 1 mm scale bar is provided at the top of each MRI (the normal-plane and face-plane images have the identical scale).

Figure 5. Diffusion mapping of S. oneidensis biofilms over time

The top row shows two-dimensional D_e maps obtained using PFG-NMR, normalized against D_aq, showing the middle 2 mm of the biofilm. Dark regions represent low D_e. The bottom row shows D_rs profiles, derived by averaging the D_e of the middle 2 mm of the biofilm (shown in the maps). The top of the biofilm as determined by magnetic resonance imaging is indicated by the vertical lines. The panel on the lefts shows the S. oneidensis biofilm at 12 days, and the panel on the right shows it at 16 days.

Figure 6. Diffusion mapping of a G. sulfurreducens biofilm with and without a polarized electrode

D_e for a ~41-day-old G. sulfurreducens biofilm. Left) Electrode polarized to +300 mV_Ag/AgCl. Middle) Polarization switched off: open circuit potential. Right) Polarized again to +300 mV_Ag/AgCl.

Figure 7. Diffusion mapping of an S. oneidensis biofilm with and without a polarized electrode

Diffusion mapping of an S. oneidensis biofilm with and without a polarized electrode. Left) Polarization off: open circuit potential. Right) Polarized to +300 mV_Ag/AgCl.