Periodic Reporting for period 3 - eForFuel (Fuels from electricity: de novo metabolic conversion of electrochemically produced formate into hydrocarbons) Reporting period: 2020-09-01 to 2022-04-30 Summary of the context and overall objectives of the project Greenhouse gases (especially CO2) emitted from the burning of fossil fuels are significant drivers of climate change and a global threat to society and the environment. Hence, it is very important to replace fossil fuels with alternative, sustainable sources. Early generation biofuels compete with land resources, limit agricultural production of food, and, therefore, cannot completely replace fossil fuels without severely undermining food security and decreasing biodiversity. In order to overcome this problem, eForFuel provides a novel solution to utilize resources that are (almost) unlimited and that are independent of land use: CO2, electricity and water. What makes eForFuel unique is the sustainable production chain that converts CO2 emissions and renewable electricity into easy to handle formic acid, which is then fed to engineered microbes for the production of renewable hydrocarbon fuels and other biomaterials. The goal of eForFuel was to establish and demonstrate a unique integrated electro-bioreactor, which automatically integrates different steps: 1) CO2 (carbon dioxide) electro-reduction, 2) formate production and 3) formate bioconversion to hydrocarbons, such as fuels that can be used in existing engines. The goal was to first reduce CO2 by applying electricity, then add H+ protons from electrolyzed water to create formate, which is a simple molecule similar to formic acid. The formate is then supplied to genetically engineered bacteria that have been designed to feed on formate and to produce biofuels in so-called fermenters. This integrated system was intended to serve as a stepping stone towards transforming the way we produce fuels and carbon-based chemicals.During the course of the 4 year research project, the research team charted new scientific territories and a number of achievments were made, such as improving the efficiency of producing formate from CO2, water and electricity, or managing to engineer bacteria to accept formate as a food source. At the time of completing the research project in 2022, however, the projected costs for producing formate (and thus fuel) was not yet low enough to compete with fossile fuels. The outcome of eForFuel can still be as a success, as it contributes to a portfolio of techniques and processes that will - hopefully- one day replace fossil fuels and contribute to a circular bioeconomy. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far For biorefineries to displace fossil fuels, a sustainable feedstock for microbial growth must be identified. As agricultural production of sugars has a limited capacity, it cannot completely replace fossil fuels without severely undermining food security and decreasing biodiversity. In order to fully displace fossil fuels we need to utilize resources that are (almost) unlimited and that are independent of agricultural or forestry land use.eForFuel developed an industrial biotechnology solution that uses electricity, water and microorganisms to convert CO2 into hydrocarbon fuels, thus providing a sustainable replacement of energy stored in fossil carbons. We used the advantages of different disciplines to establish an efficient process: carbon dioxide activation via reduction to formic acid is performed via electrochemical means while production of hydrocarbons is carried out in genetically engineered formatotrophic microbes.eForFuel addresseed multiple challenges previously limiting the success of novel fuel technologies. We decoupled production from agricultural resources, instead relying on widely available resources, such as water, (renewable) electricity, and concentrated waste CO2 originating for example from the gas outflow of the steel industry. As a mediator between the electrochemical apparatus and microbial growth we use formic acid, which, unlike hydrogen and carbon monoxide, is fully soluble, easily stored and safe to handle. We focused on the production of two products, gaseous propane and isobutene. Both can be easily separated from the microbial culture, reducing production cost and increasing energy efficiency. Furthermore, the products can be easily integrated into existing fuel facilities: propane as component of Liquefied Petroleum Gas (LPG), and isobutene for production of the superb fuel substitute isooctane.eForFuel was a truly interdisciplinary consortium, bringing together experts from a wide spectrum of fields, including electrochemistry, material science, enzymology, biochemistry, microbiology, chemical engineering, industrial biotechnology, environmental science, and sociology. By focusing on integrated sustainability, eForFuel set the stage for a future environmentally, economically, and societally sustainable value chain to produce renewable chemicals and fuels.The main results achieved in eForFuel were:• optimized structure and composition for both cathode and anode in the CO2 electrolyser;• optimized electrolysis with regard to Energetic and Faraday Efficiencies, Current Densities and Electrode Lifetime;• optimise growth of E. coli on formate via one of the formate assimilation pathways;• proof of concept of production of isobutene in a formatotrophic E. coli strain;• construction of a prototype of the electrobioreactor (EBR), with detailed architecture of hardware control and software; • an integrated Life Cycle Assessment (LCA);• a public perception survey in several European countries, indicating a positive attitude towards eForFuel telated goals and technologies;• a series of video clips for the general public.For more details on the results, publications etc. please visit: www.eforfuel.eu 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) The technical steps (CO2 electro-reduction, formate production and formate bioconversion to hydrocarbons) have been shown to work in some measure in the laboratory, but they have not been shown to work all together, and therefore not on an industrial scale. eForFuel went beyond the state of the art by improving capability, maturity and performance in order to bring the process closer to the market. As long as completley renewable sources cannot be used for everything we need in our lives, the technology developed in eForfuel could help to significantly reduce the impact fossil fuels have on the planet.We have initiated a devising methodology for Life Cycle Assessment (LCA), techno-economical assessment and socio-economical and policy assessment, with follow-up of on-going standardisation work in the field of LCA.A number of public communication activities were carried out and a series of citizen engagement events took place in Portugal, Denmark, the Netherlands, Estonia, Spain and Italy. The feedback from European citizens helped us to understand how the public perceives our project goals and objectives. It was found that the production of efuels from CO2, electricity and water was seen as a positive contribution to reduce CO2 emissions. Infographic eForFuel
