Engineering extracellular electron transfer for enhanced energy harvesting in microbial electrochemical devices

Microorganisms known as exoelectrogens evolved the ability of transporting electrons across insulating biological membranes. In a physiological context, this enables respiration on extracellular electron acceptors and electron transfer to other organisms. In a broader context, this extracellular electron transfer (EET) can be seen as both an exchange of energy and information between a living organism and its environment, which can be tapped into using an electrode. The resulting connection between cellular metabolic processes, and an external electrical circuit enables microbial electrochemical technologies (METs) for a broad range of applications aiming to address societal challenges, such as the availability of fresh water and energy. The most fundamental process in a MET is the electron exchange between a microorganism and an electrode. Therefore, exoelectrogenic bacteria equipped with EET pathways for respiration on extracellular electron acceptors, such as the model exoelectrogen Shewanella oneidensis MR-1, appear suitable for use in METs. Besides EET, metabolic versatility and adaptability are crucial properties needed to cater for product and substrate requirements while covering the vast range of potential MET applications. This work aims to combine the broad metabolism and genetic amenability of Escherichia coli with the EET capabilities of S. oneidensis MR-1, to obtain E. coli strains which can serve as a platform for the development of METs. To this end, we firstly explored cytochrome-based direct EET in E. coli, by expressing both E. coli native and S. oneidensis MR-1 cytochromes in the inner membrane, periplasmic space and outer membrane. The resulting strains revealed the importance of periplasmic electron shuttles in EET pathway design, with the best strain yielding a 3-fold increased current to graphite felt electrodes under tested conditions. Besides direct EET, S. oneidensis MR-1 relies on flavins as soluble redox mediators for reduction of extracellular electron acceptors. Taking inspiration from this synergistic EET mechanism, we further modified our engineered strain for biosynthesis and secretion of riboflavin and flavinmononucleotide. We showed that this mediated EET pathway can be functional alongside the cytochrome pathway resulting in an additional 50 % improvement in currents, as well as increase EET when expressed on its own. In the context of a MET, the electron transfer at the biotic-abiotic interface is not only governed by the microbe's ability for direct and mediated EET, but also the properties of the device. An important consideration is the electrode design for optimal interaction with the microorganisms. Close proximity of the microbes to the electrode surface may facilitate direct EET, and lead to higher local mediator concentrations at the electrode. In that regard, the last work in this thesis focused on the modification of graphite felt electrodes with a conducting polymer.Overall, this work consolidates previous efforts at engineering E. coli for EET from the literature by offering a systematic comparison of different strains, and providing an optimal EET pathway for future applications.