The long-range electron transport of exoelectrogenic bacterium G. sulfurreducens was recently discovered to be facilitated by a network of conductive protein wires. In chapter 1 we provide an overview of our current understanding of these protein wires. This newly discovered feature of the model organism for microbial fuel cells may provide new avenues for optimization of biological power generation. In particular a characterization of the wires’ effect on biofilm formation could pro- vide approaches for increasing biofilm thickness and current density. Peripheral cells may depend more heavily on inner cells’ properties than previously thought, since they are required to utilize the inner cells’ conductive wires for electron transport to the elec- tron acceptor. Such an effect is expected to be most visible in the early stages of biofilm formation where independent single cells grow to interdependent cells in microcolonies. Studies of early G. sulfurreducens microcolony growth are complicated by the toxic effect of oxygen on growth. In chapter 2 we describe a method for observing G. sulfurreducens early microcolony growth under agar pads. We show the method is able to spatially and temporally describe growth of single G. sulfurreducens cells into microcolonies and is able to discern differ- ences between strains with or without wires. However, the significant variation in micro- colony surface area between pads showed the method’s lack in reproducibility. Future work should focus on maintaining equally anaerobic conditions between pads. In addition to the potential for improving biological power generation, the poorly understood electron transport mechanism potentially defines a hitherto unknown class of electron transport proteins. Particularly the long range mechanism for efficient bio- logical electron transport remains poorly understood. Some studies support the known mechanism of hopping along cytochromes, yet other studies in cytochrome-denaturing conditions show conductivity is maintained. Complicating full understanding further is the fact that most of these studies use conventional techniques for measuring biological electron transport, which happen to measure in bulk. To model an unknown mecha- nism of electron transport depends on well-defined systems, such as a single nanowire rather than an entire biofilm. In chapter 3 we describe a novel method for making electrical contact with such single nanowires. Using a stochastic deposition method, passive voltage imaging and atomic force microscopy we visually confirm and make electrical contact with single nanowires. We describe optimization of the sample preparation and chip design show- ing that chemically untreated samples performed best, while an interdigitated chip de- sign improved the chances of making contact. Ultimately we demonstrate the abil- ity to manipulate temperature, providing valuable characterization of the temperature- dependence of nanowires conductivity. In chapter 4 we apply this method to characterize the conductive properties of single nanowires. Current-voltage curves showed conductances in orders of magnitudes from 10−13 S up to 10−6 S. Measurements at varying temperatures identified activation ener- gies from 0.36 eV to 0.41 eV. Arrhenius plots displayed features corresponding to a simple model where electron transport rate was limited by injection barriers as well as a model where electron transport rate is limited by intramolecular hopping. Future modeling is required to fully describe the electron transport. In chapter 5 we demonstrate the flexibility of the wire deposition method by mea- suring another type of conductive biological wire. Cable bacteria are able to facilitate electron transport along their cell membranes across several millimeters. We image suc- cessful deposition of cable bacteria bundles and measure their conductance. Current- voltage curves showed conductance in the orders of magnitude from 10^−10 S to 10^−7 S. These initial proof-of-principle measurements can be followed up by further character- ization of this poorly understood method of long-range biological electron transport. @en

Biological electron transport is classically thought to occur over nanometre distances, yet recent studies suggest that electrical currents can run along centimetre-long cable bacteria. The phenomenon remains elusive, however, as currents have not been directly measured, nor have the conductive structures been identified. Here we demonstrate that cable bacteria conduct electrons over centimetre distances via highly conductive fibres embedded in the cell envelope. Direct electrode measurements reveal nanoampere currents in intact filaments up to 10.1 mm long (>2000 adjacent cells). A network of parallel periplasmic fibres displays a high conductivity (up to 79 S cm-1), explaining currents measured through intact filaments. Conductance rapidly declines upon exposure to air, but remains stable under vacuum, demonstrating that charge transfer is electronic rather than ionic. Our finding of a biological structure that efficiently guides electrical currents over long distances greatly expands the paradigm of biological charge transport and could enable new bio-electronic applications.

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