Periodic Reporting for period 3 - ENGICOIN (Engineered microbial factories for CO2 exploitation in an integrated waste treatment platform) Reporting period: 2020-10-01 to 2022-12-31 Summary of the context and overall objectives of the project Engicoin transforms carbon dioxide from a bulky waste to a raw feedstock in special biotechnological processes. Carbon dioxide is turned into several building blocks, valuable in green chemistry applications. The proof of concept takes place at the ACEA site, which provides the surrounding area with services related to water treatment, sludge/digestate, thermal and electrical energy recovery. In particular, at the ACEA facility, the biogas resulting from different treatments is upgraded into biomethane which in turn can be injected into the grid or used as transport biofuel. Engicoin envisages the full integration of the biotechnological processes with the ACEA site. Indeed, Engicoin relies on the concentrated carbon dioxide stream derived from the biogas-to-biomethane upgrading and the Flue gas coming from the cogenerative system to feed the three Microbial Factories, engineered for optimal production of chemical intermediates. When necessary, the MFs retrieve the energy source from water electrolysis using efficiently the electrical energy available at the ACEA premises. • Microbial Factory 1 is reliant on the photosynthetic microorganism Synechocystis which uses sunlight and carbon dioxide to produce lactic acid • Microbial Factory 2 is reliant on a facultative chemo-litho-autotrophic bacterium, Cupriavidus Necator, which converts carbon dioxide and hydrogen produced by water electrolysis into polyhydroxyalkanoates, which are biodegradable and biocompatible thermoplastics• Microbial Factory 3 is reliant on the acetogen of the genus Clostridium, Acetobacterium woodii, to produce Acetone, which converts carbon dioxide and hydrogen into acetone, which is mainly used as a solvent in products manufacturing. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far The ENGICOIN project started in January 2018 and will last 60 months. The core R&D activities are divided in two progressive phases with increasing TRL levels, from TRL3 to TRL5.Currently, the project is on the conclusion of the last phase of R&D development which is mainly focused on TRL5 pilot plant testing campaign. The first phase, mainly focused on microbial chassis design and improvement, was closed almost at the end of the second RP. In-fact, to make the production competitive on a market scale ENGICOIN partners worked on microorganism modification to increase CO2 capture and conversion ability. Synthetic and systems biology studies was performed to increase the CO2 uptake as substrate as well as its fast and selective transformation into the desired products or to facilitate product extraction. At the same time, a new Alkaline electrolyser for H2 production and integration with MF2 and MF3 was developed. All these R&D activities was performed in accordance with the original plan. Moreover, in view of the final in-field validation (phase 3), the basic site preparation activities have been carried out including the design of the validation site and experimental campaign as well as the feedstocks and services distribution lines deployment and the development of protocols to extract organic substrates from the AD digestate which will be utilized as complementary carbon substrates for the Microbial Factories.The objectives related to the last phase of the project regards the TRL 5 validation tests, data monitoring, and to products, microorganisms, materials & plants durability assessment. During the last period, the partners provided all facilities on the platform to run the prototype. Electric power, water, CO2 inlet streams, connection to waste water system and data network are present on the platform, following specification required by the project. Experimental trials on TRL5 were conducted in the prescribed real environment: results on MF1 production showed that Synechocystis can grow and produce LA by using as carbon source both concentrated CO2 (coming from the biogas upgrading system) and waste gas streams (from cogeneration units). An experimental trial was performed also on MF2, showing that the C. Necator is able to grow and produce PHB by using as carbon source both concentrated CO2 and flue gas. Also the PHB extraction Bio-process performed by B.Bacteriovorus was tested at TRL5 level, showing good results in terms of PHB extraction and feasibility of the continuous process. 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 work performed so far has greatly increased the knowledge of the microbial chassis utilized in the three microbial factories, thus amplifying their effective application in other context with significant production of CO2 raw flue gases.Moreover, coupling ENGICOIN technologies and processes for CO2 capture and conversion into value-added chemicals with waste treatment platform defines an ecologically and economically sustainable strategy. The project is developing a win-win condition perfectly matching with Circular Economy principles and aiming at the demonstration of a “near to zero waste biorefinery”. The present image shows the final configuration of the TRL5 ENGICOIN pilot plant The present image show the main features and compounds of ENGICOIN Project and its processes.
