The ability of Geobacter species to transfer electrons outside the cell enables them to play an important role in a number of biogeochemical and bioenergy processes. Gene deletion studies have implicated periplasmic and outer-surface c-type cytochromes in this extracellular electron transfer. However, even when as many as five c-type cytochrome genes have been deleted, some capacity for extracellular electron transfer remains. In order to evaluate the role of c-type cytochromes in extracellular electron transfer, Geobacter sulfurreducens was grown in a low-iron medium that included the iron chelator (2,2'-bipyridine) to further sequester iron. Haem-staining revealed that the cytochrome content of cells grown in this manner was 15-fold lower than in cells exposed to a standard iron-containing medium. The low cytochrome abundance was confirmed by in situ nanoparticle-enhanced Raman spectroscopy (NERS). The cytochrome-depleted cells reduced fumarate to succinate as well as the cytochrome-replete cells do, but were unable to reduce Fe(III) citrate or to exchange electrons with a graphite electrode. These results demonstrate that c-type cytochromes are essential for extracellular electron transfer by G. sulfurreducens. The strategy for growing cytochrome-depleted G. sulfurreducens will also greatly aid future physiological studies of Geobacter species and other microorganisms capable of extracellular electron transfer.

SummaryMicrobial electrochemical technologies (METs) rely on the control of interactions between microorganisms and electronic devices, enabling to transform chemical energy into electricity. We report a new approach to construct ready‐to‐use artificial bioelectrodes by immobilizing Geobacter sulfurreducens cells in composite materials associating silica gel and carbon felt fibres. Viability test confirmed that the majority of bacteria (ca. 70 ± 5%) survived the encapsulation process in silica and that cell density did not increase in 96 h. The double entrapment within the silica–carbon composite prevented bacterial release from the electrode but allowed a suitable mass transport (ca. 5 min after electron donor pulse), making the electrochemical characterization of the system possible. The artificial bioelectrodes were evaluated in three‐electrode reactors and the maximum current displayed was ca. 220 and 150 μA cm−3 using acetate and lactate as electron donors respectively. Cyclic voltammetry of acetate‐fed bioelectrodes revealed a sigmoidal catalytic oxidation wave, typical of more advanced‐stage biofilms. The presence of G. sulfurreducens within composites was ascertained by SEM analysis, suggesting that only part of the bacterial population was in direct contact with the carbon fibres. Preliminary analyses of the transcriptomic response of immobilized G. sulfurreducens enlightened that encapsulation mainly induces an osmotic stress to the cells. Therefore, ready‐to‐use artificial bioelectrodes represent a versatile time‐ and cost‐saving strategy for microbial electrochemical systems.

Abstract:Microbial electrochemical technologies (METs) have a number of potential technological applications. In this work, we report the use of screen-printed electrodes (SPEs) as a tool to analyze the microbial electroactivity by using Geobacter sulfurreducens as a model microorganism. We took advantage of the small volume required for the assays (75 µL) and the disposable nature of the manufactured strips to explore short-term responses of microbial extracellular electron transfer to conductive materials under different scenarios. The system proved to be robust for identifying the bioelectrochemical response, while avoiding complex electrochemical setups, not available in standard biotechnology laboratories. We successfully validated the system for characterizing the response of Geobacter sulfurreducens in different physiological states (exponential phase, stationary phase, and steady state under continuous culture conditions) revealing different electron transfer responses. Moreover, a combination of SPE and G. sulfurreducens resulted to be a promising biosensor for quantifying the levels of acetate, as well as for performing studies in real wastewater. In addition, the potential of the technology for identifying electroactive consortia was tested, as an example, with a mixed population with nitrate-reducing capacity. We therefore present SPEs as a novel low-cost platform for assessing microbial electrochemical activity at the microscale level.

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