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BIOREFINE-2G Report Summary

Project ID: 613771
Funded under: FP7-KBBE
Country: Denmark

Periodic Report Summary 2 - BIOREFINE-2G (Development of 2nd Generation Biorefineries – Production of Dicarboxylic Acids and Bio-based Polymers Derived Thereof)

Project Context and Objectives:
The existing 2nd generation biorefineries utilize less than 20% of the biomass feedstock for ethanol production. Major side-streams are produced such as pentose and lignin streams that are respectively used for biogas and energy production. Converting the carbon from these waste streams into added-value products would increase the otherwise low profitability and improve the environmental benefits of the biorefineries. BioREFINE-2G aims at developing commercially attractive processes for efficient conversion of pentose-rich side-streams from biorefineries into dicarboxylic acids, to be used as precursors for bio-based polymers including biodegradable polymers (Figure 1).
The project covers the whole value chain, from characterization of side streams from forest and other non-food feedstocks, development of novel robust industrial yeast cell factories, fermentation and downstream process development, to polymerization methods development for the production of biodegradable polymers applicable as plastics, coatings or adhesives, scale-up and demonstration and to life cycle and economic viability analyses. The consortium involves eight distinguished industrial and academic partners. The strong industry drive is ensured by participation of 4 SMEs and one large enterprise, Borregaard biorefinery, which is directly interested in demonstrating and integrating the new technology into the current and future biorefinery plants. All industry partners will assure demonstration activities and investigate the technical, environmental and commercial feasibility of the new process with regard to scale up to industrial production.
Overall, an innovative process for bio-based chemicals production from bio-waste will represent a paradigm shift with a tremendous impact with regard to commercial viability and environmental issues such as waste reduction, less pollution and less greenhouse gas emissions.
During the first year promising achievements in strain, process, both up and down-stream, as well as in novel polymer development could be made. The development of diacid-producing strains and efficient purification methods will remain a major challenge to meet the required low production production cost. Remarkably, the consortium developed novel tools for rapid engineering of robust industrial polyploid yeast, and the first generation purification methods for separation of diacids from various fermented biomass hydrolysates look very promising. The consortium looks particularly into the development of novel polyester and polyurethanes, and some new bio-based and biodegradable polymers with relevant properties have been designed. The overall BioREFINE-2G project is being monitored by Life Cycle Sustainability Analysis (LCSA) to assess and compare the impacts of the developed products. A methodological framework on environmental, economic and social aspects of Life Cycle Sustainability Analysis (LCSA) to guide all project partners during the whole project has been created and adapted to the project context.

Project Results:
BioREFINE-2G has reached significant progress in the areas of engineering of industrial yeast strains for applications in 2nd generation biorefineries, in development of downstream processing of dicarboxylic acids from complex fermentation broths, on obtaining novel polyesters based on diacids, and on life cycle analysis. The project results have been widely disseminated in the scientific community, among industrial stakeholders and general public.
Strain engineering. BioREFINE-2G has developed an efficient and versatile genetic toolbox for engineering industrial S. cerevisiae strains. The toolbox includes the usage of CRISPR/Cas9 technology and allows creating stable strains without antibiotic selection markers, which is important for large-scale fermentations. Three peer-reviewed articles have been published. BioREFINE-2G has created industrial yeast strains capable of efficient utilization of xylose and tolerant to Borregaard hardwood hydrolysate even at low pH. We also created proof-of-concept industrial strains capable of producing smaller amounts of dicarboxylic acids (fumaric, succinic, and malic) from xylose. In the engineering of alpha-ketogluratic acid production from xylose via the Weimberg pathway, we have identified the bottleneck in xylonic acid conversion, which will be addressed in the following metabolic engineering efforts.
Fermentation&Downstream Process. A 2-stage purification protocol has been developed and refined in order to increase both the yield and the purity of the fumaric acid. It was concluded that recycling the filtrate stream of the second filtration step, saturated in FA, improved the FA recovery yields. The selective precipitation of contaminants was attempted by testing a sequential pH decrease without apparent success. A polishing step based on ion exchange was introduced in order to improve the purity of the final product. Samples of different grades of FA were produced to assess whether less stringent, thus lest costly, purification protocols can be envisaged. The information required to develop a purification process for glutaric acid has been compiled and reported.
Novel biopolymers. Synthetic routes to obtain polyesters using glutaric acid have been identified. Presently, the most promising combination is glutaric acid and 1,12-dodecanediol for production of medium and small polymers that can be polyurethanes precursors. The feasibility to transfer the synthesis to a continuous reactive extrusion process has been tested and mathematically simulated, and found to be scalable to a large scale. In order to determine the molecular weight of new biopolyesters a gel permeation chromatography method and a fast nmr method have been developedas well as and an easy method to determine the polymer purity. The syntheses have been thoroughly optimized, and polymers were conveniently characterized (melting point, hydroxyl number and acid number, FTIR and DSC). The obtained polyesters were converted into commercially interesting products, such as thermoplastic polyurethane polymers,polyurethane dispersions and PLA copolymers. Polyurethane and PLA copolymers have been analysed according to their appearance, transparency, melting point, flexibility and brittleness. From novelty and commercial point of view some good results have been obtained. A market analysis has been conducted showing that it will be important to develop efficient processes for the production of the diacids.
Life Cycle Analysis (LCA). Guidelines for conducting sustainability LCA have been prepared. A report for a comparative preliminary sustainability assessment has been send to all project partners. The preliminary comparison showed significantly higher impacts for the BioREFINE approach than for the conventional production of succinic acid. This is mainly due to unrefined process models for both assessments but also because of the lack of reasonable data available for the newly developed fermentation process. The social assessment showed only little to no impacts for the foreground assessment on country, sector and site specific level.
Dissemination & Exploitation. A project website was designed and is continuously updated (; a project logo and common reporting templates were developed, as well as marketing material (project flyer) and a dissemination and exploitation plan (PUDF). The project results have been presented as 28 poster and oral presentations at national and international conferences, workshops and exhibitions. 6 press releases have been published. Three papers have been published in peer-reviewed journals and several more scientific manuscripts are in preparation. Various technologies developed by AIMPLAS, DTU, Ecolpol Tech and Lund University are checked for patentability.

Potential Impact:
The overall aim of BioREFINE-2G is to upgrade C5-rich waste sugar streams in 2nd generation biorefineries by production of higher value added chemicals, such as diacids and polymers. The expected final results and the potential impact remain unchanged compared to the start of the project. The consortium will develop novel robust production organism for diacid production from C5 and mixtures of C5/C6 sugars. Industrial Saccharomyces cerevisiae strains have been selected as they are expected to be more robust and perform better in comparison to other potential production organism in 2nd generation biorefinery processes.
During the project, the selected strains will be engineered and adapted to efficiently grow on complex C5 and C5/C6 side streams, and novel processes and fermentation conditions will be developed. Purification methods will be designed that enable the purification of the diacids from the side-streams to sufficiently high purity, and diacids will be polymerized to bio-based polymers such as polyesters and polyurethanes, which can subsequently be used in the development of plastics, coatings, adhesives, etc. Particularly, the fermentation will be demonstrated at 3,000 L scale at industrial partner Borregaard, who has run biorefinery processes for almost 80 years. The integrated process for fermentation, downstream processing and polymerization will be demonstrated at 20 L scale at pilot plant facility of another industrial partner Biotrend. This represents a deviation from the original plan, where we wanted to demonstrate the whole integrated process at 3,000 L scale. This contingency plan was adopted to compensate for the delay with strain development. Data derived from the demonstration activities will be used to carry out Life Cycle Sustainability Analysis (LCSA) considering regionalization aspects and an economic analysis to validate the commercial viability of the process and whether the process will be more environmentally friendly.
BioREFINE-2G aims to address a major environmental challenge associated with the conversion of biorefinery wastes into sustainable and cost-efficient bulk biochemicals. By integrating chemical production into second generation biorefineries, the consortium develops innovative diacid and bio-based polymer production from waste and side-streams, optimizing the sustainability scheme of biomass treatment towards the full use of biomass, as well as reducing the commercial risk in running a second generation biorefinery.
Results from BioREFINE-2G will be game changers within the biorefinery industry and are aligned with the European Environmental 2020 strategy for sustainable growth. Activities are covered ranging from developing a product that reduces waste and pollution to the development of improved production technologies resulting in positive socio-economic impact, not only within Europe. Also, by supporting sustainability of primary production and favouring new and diversified markets in bio-based products, BioREFINE-2G addresses major objectives of Europe Strategy for 2020 and boosts a recycling bio-economy. Additionally, the integration of transnational and multidisciplinary companies in this process favours the cooperation of market actors along the value chain boosting the bio-based product sector economy and greatly contributing for Europe 2020 flagship initiatives like "Innovation Union", "A Resource Efficient Europe" and the Bio-Economy Action Plans for Europe.

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