Periodic Reporting for period 1 - EMES (Enhanced Microbial Electrosynthesis and Visualization of Microbial Metabolism) Reporting period: 2018-01-01 to 2019-12-31 Summary of the context and overall objectives of the project Addressing the global challenge of sustainability calls for cost-effective and eco-friendly pathways to go beyond the existing energy-intense synthetic routes. Biohybrid electrochemical systems can synergistically combine the strengths of biocatalysts and synthetic electrodes to leverage the power of the intercellular metabolism for energy conversion and chemical synthesis using (photo)electrochemistry. Of particular interest are electroactive bacteria with the naturally evolved ability to electrically interact with insoluble metal oxides for anaerobic respiration, which promises broad applications in microbial fuel cells, microbial electrosynthesis and bioremediation. Nevertheless, the development of microbial hybrid systems is perennially plagued by the low power output and volumetric productivity, arising from the imperfect integration of bacteria with solid-state materials. The nexus of breakthroughs, therefore, lies in the electrode architecture and biological interfaces.Electroactive bacteria are present in a whole host of environments and have recently garnered attention for both, fundamental studies, and emerging applications such as microbial fuel cells. G. sulfurreducens is amongst the most common and promising of these and devices featuring these species have attained some of the highest current densities to date. While the mechanistic details of the EET mechanism of G. sulfurreducens – loaded electrodes in anodic mode are beginning to emerge, the recently established cathodic mode remains rather ambiguous. To shed light on the mechanism of G. sulfurreducens EET, we carried out an extensive study on their biofilms as they grow on electrodes in both anodic and cathodic reaction modes, utilizing electrochemistry, Raman spectroscopy, quartz crystal microbalance measurements, and electron microscopy, with a focus on the role of cytochromes under these two conditions.Overall objectives of the project:-Development of a high-performing electrode for microbial electrogenesis and microbial electrosynthesis-Development of photoanodes for microbial electrosynthesis -Investigation of bacterial extracellular electron uptake (cathodic) mechanism Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far We employed an inverse opal-indium tin oxide (IO-ITO) electrode as a platform for microbial electrogenesis and electrosynthesis using G. sulfurreducens. ITO is hydrophilic and the porous electrode architecture provides easy access for bacteria penetration and colonization. When positive potentials are applied, planktonic G. sulfurreducens from the medium solution attaches on the electrode surface. The sessile bacteria metabolize acetate to support its growth through the tricarboxylic acid (TCA) cycle while discharging excess electrons to the electrode via OMCs, which is registered as a continuous anodic current. Transcriptome analysis by RNA sequencing revealed that G. sulfurreducens regulated gene expression in order to respire on electrodes. Furthermore, Shewanella loihica PV-4 (S. loihica) was introduced together with G. sulfurreducens on the IO-ITO electrode to achieve syntrophic electrogenesis by linking their metabolic pathways, which will grant the system additional flexibility in using different electron donors. Electrosynthesis was carried out by poising negative potentials on the resulting IO-ITO|G. sulfurreducens electrode. Under such conditions, G. sulfurreducens accepts electrons from the electrode to sustain its metabolism and disposes of respiratory electrons by reducing soluble fumarate or heterogeneous graphene oxide (GO). Lastly, to outsource the electron supply to a renewable source, the biohybrid electrode was coupled with a photoanode to achieve photoelectrosynthesis without applying an external electrochemical voltage.We carried out a multifaced study on the growth and electrogenesis of G. sulfurreducens in systematically switching between anodic and cathodic modes on inverse-opal indium-doped tin oxide (IO-ITO) electrodes. In addition to the conventional electrochemical experiments, we performed complementary studies using in situ resonance Raman spectroscopy, UV-Vis absorbance spectroscopy, quartz-crystal microbalance with dissipation (QCM-D), and ex-situ scanning and transmission electron microscopy (SEM and TEM) to piece together clues behind the mechanism of their anodic and cathodic electron transfer. Using this comprehensive set of measurements, we found that anodic mode function is mainly linked to the biofilm’s cytochrome expression, but the cathodic mode likely operates through an alternate channel. We propose that a Fe-containing soluble species that can either come from Fe ions in the medium or alternatively be scavenged from cytochromes is contributing to cathodic mode charge transfer under our set of reaction conditions. The presented findings add insight to G. sulfurreducens' function in its natural environments and in emerging biotechnologies, as well as press for a closer look at the multitude of EET pathways present in biological systems. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Societal benefits of the project in the EU: The project matched with two EU thematic strategies- air pollution and renewable energy. After the Paris Climate Meet in December 2015, the EU has decided to continue to support for reducing greenhouse gases (a 40% cut by 2030) through the European Climate Change Program. The project contributed a high performing electrode material to produce electricity and fuels from waste-carbon by employing electric bacteria which provided an improved and sustainable biosynthetic route to value-added products from the waste-carbon.In addition, the project provided a better clue for the mechanism involved in bacterial extracellular electron uptake from a cathode. The insights obtained from the project is anticipated to greatly bolster the community’s understanding of bacterial electron uptake processes and applicability to other reactions. Bacterial respiration on a inverse opal-indium tin oxide (IO-ITO) electrode
