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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.