Periodic Reporting for period 3 - BioRECO2VER (Biological routes for CO2 conversion into chemical building blocks) Reporting period: 2021-01-01 to 2021-12-31 Summary of the context and overall objectives of the project The high-level goal of BioRECO2VER was to demonstrate the technical feasibility of more energy efficient and sustainable non-photosynthetic anaerobic and micro-aerobic biotechnological processes for the capture and conversion of CO2 from industrial point sources into 2 valuable platform chemicals, i.e. isobutene and lactate. To overcome several of the existing technical and economic barriers for CO2 conversion by industrial biotechnology, the project focused on minimizing gas pretreatment costs, maximizing gas transfer in bioreactors, preventing product inhibition, minimizing product recovery costs, reducing footprint and improving scalability. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far LTU developed a hybrid CO2 capture approach combining a tertiary-amine based solvent with the enzyme carbonic anhydrase (CA). Several enhanced enzyme mutants were generated that showed 50% increased resistance to selected flue gas inhibitors compared to the original CA. Moreover, a blend of an amino acid ionic liquid, MDEA (methyldiethanol amine) and CA was developed that displayed a good compromise between enzyme compatibility, absorption rate, capacity and desorption potential. When scaling up the CO2 capture process in a pilot plant, reduction in operation times and energy consumption compared to the reference system could be demonstrated, yielding a pre-treated concentrated gas stream of at least 92% CO2. The pilot test rig with sorption and desorption column is available to assess chemo/enzymatic capture processes with real off-gases.Substantial progress was made on the 3 microbial platforms under investigation. (1) GBE identified an optimal strain for the development of the Clostridial platform, implemented an isobutene pathway in this strain and demonstrated isobutene production on CO2/H2. Through a combination of implementing the best enzyme variants into the pathway and modifications in the cultivation medium, isobutene production levels could be increased by a factor of 10. (2) EnobraQ performed metabolic engineering of autotrophic Cupriavidus necator to produce lactate from CO2 and H2. Overexpression of lactate dehydrogenase and deletion of competitive pathways resulted in the improvement of the production of lactate. Further work resulted in significant improvements in the lactate dehydrogenase activity and in biomass production in a bioreactor. (3) CNR developed methods to address an efficient transformation of Thermotoga neapolitana DMS33003 and generated a recombinant strain producing CO2-derived lactate at a 1.7 fold higher level than wild type. For this platform, the bottleneck remains to overtake the production of CO2 from glycolysis. All three microbial platforms were tested with real off gases: 2 of them did not show a negative impact of off-gas impurities and demonstrated growth without gas pretreatment, a third one is sensitive to specific impurities. The three microbial platforms were tested for enhanced bioconversion processes in fermenters and/or bio-electrochemical systems (BES). Biocathodes coated with immobilized proteobacteria were tested for sustained and increased in situ H2 production in BES. For Cupriavidus, the same levels of lactate were produced in BES as in fermentations. Clostridium strain performance was evaluated in a 10-L fermenter at elevated pressures up to 8 bar, and a first proof-of-concept was achieved in BES. Finally, the effect of salinity level, buffering agent, carbon and sulfur sources was investigated on H2 and lactate synthesis in Thermotoga neapolitana and new adapted strains generated. Unique BES and pressurized fermenter set-ups were designed and commissioned. The 1st fully automated bio-electrochemical platform is available at UdG and paves the ground for the intensification of the process (in terms production rates and selectively) by controlling key operational parameters (pCO2, pH, pH2). VITO has a flexible and multifunctional high-pressure fermenter, customized with advanced online sensors, monitoring and control, and a membrane filtration unit to achieve high concentrations of the microbial biocatalysts. The set-up can also be used for other poorly soluble gases, such as methane, oxygen, or synthesis gas. The (partially) calibrated BioRECO2VER process model, optimization tool and metabolic models were used to optimize the various unit processes. Moreover, a conceptual design and basic engineering of the BioRECO2VER process is available.A techno-economic analysis and process replication study were completed. The results are based on the BioRECO2VER virtual plant integrating results of developed metabolic models and the obtained experimental results. Monte-Carlo Simulations for lactic acid production indicated that in the present case, it would be necessary to have a high sale price (with significant premiums) for CO2 based lactic acid to have an attractive case for investors. The project activities generated interesting new insights concerning gas fermentation and CO2 conversions with suggestions for future development routes, including potential new target products.A LCA hotspot assessment was performed for the upscaled production processes for best-case scenarios. If renewable electricity can be used, the developed production processes can outperform the conventional production process depending on the method of solving multifunctionality. Several methodologies for solving multifunctionality arising from CO2 utilisation have been evaluated and compared to the conventional production processes to give insights into the implications of each methodological choice. The project website and the Zenodo Community provide open access to the project publications. Two final stakeholder events were organized which each attracted >200 participants. Presentations are accessible on the project website http://bioreco2ver.eu/webinar/. 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) Significant progress has been made on the knowledge of enzyme and microbial platforms, operational conditions and procedures for the proposed technologies. Experimental work has provided insightful data and information on the physiology and metabolism of the strains to use for the microbial platforms, which could be further exploited biotechnologically. Unique equipment was acquired for biotechnological capture and conversion of CO2 and will be used beyond the project duration to support further research and development activities in the broad area of CO2 capture and conversion. To the best of the authors’ knowledge, it is the first time that process models with this level of detail were developed and implemented for an integrated Carbon Capture and Utilization (CCU) system involving biological processes for CO2 conversion.Because a positive public perception is important for a successful market launch, we studied consumer perceptions of captured CO2 as a feedstock for chemicals and polymers used in daily lives. Many CCU experts had the opinion that consumers would perceive the technology as environmentally friendly if it was explained to them properly. However, only very few producers currently do this. A CO2 label indicating the amount of CO2 captured in a product could help guiding consumers in their purchasing decisions. Overall, little to no knowledge about captured carbon is available among consumers, but if explained in an easy way the responses of the participants are usually positive. We should thus make clear that there is no danger to human health when products are produced from CO2. Visualization of project concept
