Spatiotemporal mapping of bacterial membrane potential responses to extracellular electron transfer

Abstract

Extracellular electron transfer (EET) allows microorganisms to gain energy by linking intracellular reactions to external surfaces ranging from natural minerals to the electrodes of bioelectrochemical renewable energy technologies. In the past two decades, electrochemical techniques have been used to investigate EET in a wide range of microbes, with emphasis on dissimilatory metal-reducing bacteria, such as Shewanella oneidensis MR-1, as model organisms. However, due to the typically bulk nature of these techniques, they are unable to reveal the subpopulation variation in EET or link the observed electrochemical currents to energy gain by individual cells, thus overlooking the potentially complex spatial patterns of activity in bioelectrochemical systems. Here, to address these limitations, we use the cell membrane potential as a bioenergetic indicator of EET by S. oneidensis MR-1 cells. Using a fluorescent membrane potential indicator during in vivo single-cell-level fluorescence microscopy in a bioelectrochemical reactor, we demonstrate that membrane potential strongly correlates with EET. Increasing electrode potential and associated EET current leads to more negative membrane potential. This EET-induced membrane hyperpolarization is spatially limited to cells in contact with the electrode and within a near-electrode zone (<30 μm) where the hyperpolarization decays with increasing cell-electrode distance. The high spatial and temporal resolution of the reported technique can be used to study the single-cell-level dynamics of EET not only on electrode surfaces, but also during respiration of other solid-phase electron acceptors.

High current production ofShewanella oneidensiswith electrospun carbon nanofiber anodes is directly linked to biofilm formation

“…Flavins: red, ox Figure 6: Illustration of microscopic processes in the biofilm and their dependence on pore size, surface area, and the number of cells inside the anode. A) Metabolic activity as a function of the distance to the closest anode surface (free adaption of Pirbadian et al [16]. B) Acidification in the anode as a function of distance from the bulk medium and cell density.…”

“…The biofilm coverage on electrodes is not limited to single layers and can be stimulated by the presence of oxygen [Kitayama et al, 2017], possibly due to increased motility or motility elements that promote the development of three-dimensional biofilm structures [Thormann et al, 2004]. In static multilayer biofilms, electron transport from cells that are not directly attached to the electrode is facilitated by the sequential redox cycling of outer membrane c-type cytochromes and/or MET [Pirbadian et al, 2020]. In dynamic biofilms, motile cells could increase the apparent cell surface coverage [Okamoto et al, 2012], enabling more cells to participate in EET.…”

In SituOptical Quantification of Extracellular Electron Transfer using Plasmonic Metal Oxide Nanocrystals

“…Previous studies found current generation from S. oneidensis can vary, from ∼2 fA • cell −1 in cell suspensions to soluble riboflavin, to ∼100-200 fA • cell −1 in single cells to electrodes (18, 23, 49). Methods measuring larger reduction rates have varied in experimental conditions, including extrapolating single cell EET from biofilms on electrodes or hematite nanoparticles (14, 18–19, 49–50). One primary difference in our study compared to these is the lack of cell immobilization on a surface.…”

“…One primary difference in our study compared to these is the lack of cell immobilization on a surface. Cell motility may inhibit certain EET mechanisms such as flavin shuttling, which occurs primarily in attached cytochromes (19, 51). Therefore, it is possible that both physiological and experimental differences account for increased EET measurement in these platforms compared to ours.…”

“…EET is typically quantified from biofilms formed on electrodes in microbial fuel cells (MFCs) or bioelectrochemical systems (BESs) (1, 14–16). EET from single cells has also been measured in such systems, providing sensitive quantitative measurements and highlighting variability in EET capabilities between individual cells (17–19). However, these measurements often require long incubation times and electrode colonization (∼days), and the contribution of suspended ( i .…”