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    EveryCarbon
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    Synthetic engineering of conductive biofilm development in the y-proteobacterium Shewanella oneidensis
    The aim of this proposal is to understand the boundary conditions of conductive biofilm development by Shewanella oneidensis on electrode surfaces and the synthetic development of an advanced ability of the organism to endogenously build these current producing structures. S. oneidensis is the best understood model organism regarding extracellular electron transfer and the ability to thrive with insoluble electron acceptors. Still, the achievable electron transfer rates with these electron acceptors are several fold lower compared to the other model organism for extracellular electron transfer – Geobacter sulfurreducens. Our research builds upon the hypothesis that the reduction of electron acceptors like hematite or the ability to produce a current in a bioelectrochemical system is correlated to the ability to produce conductive biofilms. This is corroborated by the ability of G. sulfurreducens cells to integrate conductive structures in its extracellular polymeric substance. In the proposed work we will fundamentally establish how biofilm production on anode surfaces by S. oneidensis can be steered by the addition of abiotic or biotic conductive structures. To this end we will use a recently establish microfluidic platform that was advanced for use with bioelectrochemical systems. We want to understand how the organism reacts to the integration of this material and what the maximum current densities are that can be established using these exogenous current collectors. This maximum current density will be the benchmark that we want to reach when we engineer S. oneidensis to endogenously produce conductive structures within its extracellular polymeric material. We will export outer membrane cytochromes into the matrix and will synthetically connect them via isopeptide bonds to other proteins on the surface of the cell. To reach this goal we will use the recently established SpyTag/SpyCatcher technology that allows the posttranslational connection of proteins.
      44
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    Structure function relationship of electroactive biofilms in microbial fuel and electrolysis cells
    This proposal aims at identifying generic process and design rules that allow for the development and application of optimized electroactive biofilms in anode reactions. Hence, we will study the correlation between surface chemistry, biofilm formation and architecture as well as current production in bioelectrochemical systems. Moreover, the effect of electron shuttles on the development and activity of anode biofilms as well as their long-term stability will be studied. The ability of microbes in these electroactive biofilms to use electrodes as the terminal electron acceptor of their respiratory chain can be used in wastewater treatment plants for the sustainable removal of organic carbon or in anode-assisted fermentations. The effectiveness of both systems depends on the achievable current density. However, there is still a lack of fundamental knowledge regarding the effect of electrode-materials and their surface chemistry on electroactive biofilm formation. Moreover, we do not know the architecture of an efficient electroactive biofilm and whether the integration of sustainable electron shuttles in these biofilms can lead to higher current densities over longer periods of time. We suggest that it is possible to identify, study and define general design rules for efficient anode-based processes that are congruent to a wide range of applications. Therefore, we will strive to understand the limitations of anode-based processes and formulate design rules for the evolution of anode biofilms.Professor Stom and his research group (Russia) will study the effects of surface chemistry, surfactants, the degree of hydrophilicity and hydrophobicity, polarization of electrodes and electric field on the kinetics, stability and physiology of biofilm formation on the anode electrodes. The group of Professor Gescher (Germany) will develop methods for integrating mediator molecules into a biofilm matrix in order to obtain higher current densities through thicker active biofilms. Moreover, the group will study the long-term activity of anodic biofilm-catalysts and reasons for process heterogeneities. Both tasks will be evaluated in terms of current production, as well as in by assessing biological activity in various areas of the biofilm. The results will be combined in both groups and will be evaluated by application in wastewater treatment systems (Professor Stom's group), as well as in anode fermentation processes (Professor Gescher's group). Compared to control biofilms, long-term stability and functional activity will be evaluated using anoxic fluorescent markers, transcriptomic and metagenomic analysis, as well as, by the rate of elimination of carbon and the formation of metabolites products. Finally, based on the results achieved during the first 28 months, both research groups will jointly design a scalable reactor to study the applicability of the developed design rules under real(istic) conditions.
      38
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    SPP 2240: Elucidating factors necessary to transplant extracellular electron transfer pathways
    It is so far not possible to reconstruct extended respiratory chains from native exoelectrogens in other organisms like Escherichia or Pseudomonas strains without tremendous losses regarding electron transfer rates. Hence, an understanding of fundamental parts of the electron transport chains or factors necessary to gain full activity of electron transfer chain components is missing. We hypothesize that this is mostly due to the fact that research regarding necessary components for extended electron transfer builds upon loss-of-function studies in the native exoelectrogenic organism and the subsequent transfer of only factors identified within these loss-of-function screens into the designated host organisms. We could already establish using a gain-of-function approach with a Shewanella oneidensis fosmid library expressed in an E. coli strain, that so far overseen factors seem to have massive influence on electron transfer kinetics. Moreover, we established that typical biotechnological chassis organisms have to be systematically adapted for an obligat respiratory anaerobic physiology. It is the aim of this research project to systematically identify factors necessary for the efficient reconstruction of extended electron transfer chains in typical biotechnological chassis strains. We will establish this by specifically designed gain of function screens and the identification of genomic alterations after specific selection experiments. In the end we aim for an E. coli strain containing the necessary elements for extended electron transfer to an electrode surface that conducts anode-assisted fermentations with competitive space-time-yields compared to established oxic production routines.We teamed up with engineering groups, that also submitted proposals within this priority programme, to establish not only optimized biocatalysts for electrode assisted fermentations but moreover identify limitations, best possible process conditions and reactor geometries for the realization of this sustainable way of biotechnological production.
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