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Integration of Biology and Engineering into an Economical and Energy-Efficient 2G Bioethanol Biorefinery

Final Report Summary - PROETHANOL2G (Integration of Biology and Engineering into an Economical and Energy-Efficient 2G Bioethanol Biorefinery)

Executive Summary:
The European Union has set a 10 % mandatory target for the share of renewables (including biofuels) in transportation sector by 2020 in Europe. To achieve this goal sustainable biofuels from lignocellulosic biomass must deploy in Europe very soon since the competition between food vs. energy prevents further (significant) increase on the current first-generation biofuels already in the market. This new generation of biofuels still needs intensive efforts on R&D to fulfill significant GHG reductions in the production chain and to represent an alternative to food-crops derived fuels. Additionally, the use of agricultural residues, industrial or domestic wastes will improve significantly the environmental performance of 2G biofuels.
PROETHANOL2G project focus on the effective integration and development of advanced technologies through the combined use of Biology and Engineering for the production of second generation (2G) bioethanol, from the most representatives European (wheat straw) and Brazilian (sugarcane bagasse and straw) feedstocks.

The research activities are concentrated in the following areas:
i) Feedstock pretreatment
ii) Conversion technologies to second generation (2G) bioethanol
iii) Low-temperature distillation
iv) Conversion technologies (using the bioprocess-derived materials) for electricity and other added-value materials
v) Full process integration and sustainability assessment

The effective combination of pretreatment, enzymatic hydrolysis and fermentation using adapted and robust strains displaying new phenotype features, is a clear objective of this proposal since is the key to the economic lignocellulosic ethanol production.

At the end of the project, ProEthanol2G aims to get a meaningful technological process integration of:
i) For Europe: energy-efficient production of bioethanol and electricity from 100% of wheat straw.
ii) For Brazil: energy-efficient production of bioethanol, sugar and electricity from 100% utilization of sugarcane crop, including bagasse and straw.

Project Context and Objectives:
The ultimate goal of ProEthanol2G was the combination of the most cost and energy-efficient fuel biorefinery technologies providing optimal utilization of land through 100% feedstock utilization and minimal consumption of fossil energy from field to end-products, which means a low carbon based economy.
To reach that, the project focused on the development of advanced technologies (mainly biochemical-based) for the production of second generation bioethanol, from the most representatives European (wheat straw) and Brazilian (sugarcane straw and bagasse) feedstocks. The research activities have been concentrated in the following areas:
i) feedstock pretreatment;
ii) conversion technologies to second generation (2G) bioethanol;
iii) low-temperature distillation
iv) conversion technologies (using the bioprocess-derived materials) for electricity and other added-value materials;
v) full process integration and sustainability assessment.
In this context, a clear ProEthanol2G objective was the effective combination of pretreatment, cost-effective in-situ enzyme production (through the development of novel enzyme-producing strains from biomass pretreatment effluents), enzymatic hydrolysis at high solids and C6 and C5 fermentation (mainly glucose and xylose, respectively) using adapted and robust strains displaying new phenotype features, since it is the key to the economical and sustainable lignocellulosic bioethanol production.
Particular attention was given to the integration of 2G technologies in existing 1G technology in Brazil.

ProEthanol2G bases its conceptual 2G bioethanol biorefinery on INBICON’s demo plant in Kalundborg, Denmark, and on its IBUS technology. The research activities on hydrothermal pretreatment (WP2) have been developed by DTU in the mini-IBUS system (the down-scaled INBICON’s system). The optimization of wheat straw and sugarcane bagasse processing has been performed in batch and assessed through Standard Operational Procedures (SOPs) for enzymatic hydrolysis and fermentation settled and validated in the framework of this project.
In the scope of WP2, innovative pretreatments have been assessed: i) ensiling prior to hydrothermal pretreatment of wheat straw and sugarcane bagasse (DTU); ii) use of ionic liquids in fractionation of wheat straw and sugarcane bagasse (LNEG); iii) use of supercrital CO2 in hydrothermal pretreatment of wheat straw (LNEG). These activities have been, or will be, coordinated with Brazilian partners working on the same topics (INT and FURB).
In parallel, INBICON has been adapting their core business hydrothermal pretreatment, from SSF, to SHF, to generate C6/C5 sugar syrups, and consequent C6/C5 fermentation. The large-scale campaigns in 2012 and 2013 with sugarcane bagasse and wheat straw provided, to ProEthanol2G partners, pretreated material for enzymatic hydrolysis and fermentation studies and, late 2013, C6/C5 sugar syrups for fermentation tests. The flexibility of INBICON demo plant to operate under SSF and SHF, with the production of the C6/C5 sugar solution, was aligned with two major goals of ProEthanol2G: the integration of 2G technology in existing commercial 1G plants in Brazil; the fermentation of the C5 fraction to ethanol to increase process yields.

In the framework of biomass conversion technologies for 2G bioethanol (WP3), the interaction with the Brazilian Consortium focused on:
- the screening of xylanolytic microbial strains (LNEG)
- assessment of enzymatic hydrolysis performance of novel cellulolytic and xylanolytic enzyme cocktails (CIEMAT and LNEG)
- the characterization of novel and efficient C5-fermenting non-conventional yeasts (LNEG)
- the construction of novel C5-fermenting S. cerevisiae strains and its detailed characterization (ULUND and LNEG).

Moreover, LNEG tested different configurations, at lab scale, for operating enzymatic hydrolysis and fermentation in order to evaluate process flexibility (cellulolignin fermentation vs clear mash technology) towards integration of 2G bioethanol technology in commercial 1G bioethanol technology operating in Brazil. CIEMAT evaluated SS(C)F processes at high solid loadings (20% w/w or above) for maximum ethanol production.
The distillation process was assessed in WP4, by HOLM, using a novel energy efficient diabatic vacuum distillation system operating at low temperature, which, simultaneously, avoids thermal inactivation of the cellulolytic enzymes. The potential market in Brazil for a novel horizontal vacuum stripper has been evaluated and a special focus has been given on the conceptual design of process configurations for implementation of 2G bioethanol technology and vacuum stripping, with enzyme recovery, in existing 1G bioethanol process in operation in Brazil. The benchmarking performed by HOLM on Brazilian ethanol market has been essencial to address, in the framework of ProEthanol2G, most of the technological challenges for the implementation of 2G technology in Brazil, preferentially through 1G+2G processes.
WP5 aims at contributing for 100% utilization of plant feedstock with the most cost and energy efficient 2G bioethanol biorefinery, minimizing CO2 emissions. Therefore, the activities tackled the development of technologies for residues upgrading – lignin as valuable market product, lignin/solids for electricity and wastewaters for value added products through electrochemical processes.
The solid residue (wet cake) produced by INBICON and CIEMAT was used by GreenValue for lignin recovery and purification and further assessment for end-use applications. The potential of lignin upgrading obtained from ionic liquid fractionation (in LNEG) was also evaluated by GreenValue.
Simultaneously, LNEG evaluated gasification properties of the same residue towards electricity production.
Wastewaters from 2G bioethanol biorefinery was used by UGent for the production of high-value biochemical, such as butyrate and caproate, by means of electrochemical systems.
Process integration and LCA was performed in WP6 by EPFL, while environmental and social impact has been addressed by FRAUNHOFER in WP7. The major goal was to perform a sustainability assessment of EU/Brazilian 2G bioethanol plants using wheat straw/sugarcane bagasse and considering integration of 2G into 1G bioethanol in Brazil. IBUS concept was used as reference case and several alternatives have been considered according to project technological breakthroughs. The interaction with Brazilian partners and other external entities (e.g. CTBE in Brazil) was extremely important to understand the specificities of Brazilian scenario and to address properly the LCA of an integrated 2G technology in existing 1G ethanol(/sugar) plants.

Project Results:
The ProEthanol2G project focused on the development of advanced technologies (mainly biochemical-based) for the production of second generation bioethanol, from the most representatives European (wheat straw) and Brazilian (sugarcane straw and bagasse) feedstocks. With its conceptual 2G bioethanol biorefinery based on INBICON demo plant in Kalundborg, Denmark, and on its IBUS technology, the research activities have been concentrated in the following areas:
i) feedstock pretreatment (WP2)
ii) conversion technologies to second generation (2G) bioethanol (WP3)
iii) low-temperature distillation (WP4)
iv) conversion technologies (using the bioprocess-derived materials) for electricity and other added-value materials (WP5)
v) full process integration and sustainability assessment (WP6/7)

The main objective of WP2 was to develop pretreatment processes for the production of easily accessible cellulosic fibres for ethanol fermentation on wheat straw and sugar cane bagasse/straw at lab scale investigating: 1) hydrothermal treatment; 2) treatment by ensiling combined with hydrothermal treatment; 3) novel strategies of biomass deconstruction using ionic liquids, 4) supercritical fluids. Demonstration activities through large scale experiments are also involved in this WP to provide pretreated material to ProEthanol2G partners as well as to establish correlation factor between bench and large scale on hydrothermal pretreatments.
DTU performed hydrothermal pretreatment experiments in pilot scale reactor (Mini IBUS), both on wheat straw and sugarcane bagasse, and studied the effect of dry matter loading, reaction time, temperature and addition of catalyst. DTU focused on the combination of hydrothermal treatment with ensiling, where several positive effects of ensiling prior to hydrothermal treatment were revealed: glucan was up-concentrated in the solid fraction and the solubilisation of hemicellulose was significantly increased. Subsequent enzymatic hydrolysis of the solid fraction showed that ensiling significantly improved the effect of wheat straw pretreatment on cellulose digestibility, especially at the lower temperatures (>30% higher yield at 170ºC). In turn, ensiling of sugarcane bagasse coupled to hydrothermal pretreatment promoted a 5-10% improvement of enzymatic hydrolysis yield when comparing to non-ensiled pretreated sugarcane bagasse, whatever the temperature tested. DTU has provided some of the pretreated material to project partners in WP3.
LNEG worked on biomass deconstruction based on the use of ionic liquids and supercritical fluids as media for the biomass pretreatment. The new methodology for wheat straw pretreatment with the ionic liquid (IL) 1-ethyl-3-methylimidazolium acetate ([emim][OAc]) allowed the production of cellulose, hemicellulose and lignin-rich fractions in a rapid and simple three-step fractionation process. The cellulose-rich fraction denoted high enzymatic digestibility. The methodology of wheat straw fractionation with ionic liquids was scaled up to generate an adequate amount of lignin to be tested in WP5, by GreenValue, for potential commercialization. Moreover, the CO2-assisted autohydrolysis was used as wheat straw pretreatment method. The in-situ formed carbonic acid was found to result in a higher dissolution of xylose as well as XOS (xylo-oligosaccharides) in comparison to CO2-free pretreatments under the same conditions.
INBICON is now able to operate their hydrothermal treatment technology in SSF and SHF mode with improved ethanol yields at demonstration scale, making their process suitable for deployment in Brazil. Mixed sugar hydrolysis (C5/C6) and fermentation has been proved in demonstration scale. The process is flexible obtaining positive business cases with several lignocellulosic materials, including wheat straw and sugarcane bagasse. INBICON has provided pretreated wheat straw and sugarcane bagasse to partners, for enzymatic hydrolysis and fermentation processes, as well as the new C6/C5 sugar solution (from SHF) for testing new C5-fermenting strains. INBICON has also provided lignin for LNEG and GreenValue activities in WP5.

The main objectives of WP3 “Biomass conversion technologies for 2G bioethanol” were (i) the reduction of enzyme costs through the coordinated research carried out with the Brazilian Consortium, (ii) the generation of robust microbial factories for efficient hexose and pentose fermentation improved on hydrolysates obtained from feedstock pre-treatment and (iii) the integration of simultaneous saccharification and co-fermentation of pentose and hexose sugars by the newly developed strains, preferentially with pretreated wheat straw and sugarcane bagasse at high solids.
ProEthanol2G progressed towards WP3 objectives, with respect to characterization of novel xylanases and cellulolytic and xylanolytic cocktails, identification of novel genes for pentose (D-xylose) utilization, metabolic engineering of S. cerevisiae and subsequent characterization of strains and key metabolic enzymes, and on process intensification through studies on different process configuration for enzymatic hydrolysis and fermentation and SSF at high solids in bench and large scale bioreactors.
Novel enzyme cocktails, obtained from collaborative work with Brazilian partners, have been tested in the hydrolysis of wheat straw and/or sugarcane bagasse, and their cellulolytic and xylanolytic, performance evaluated. As revealed by collaborative work between CIEMAT and UFRJ, cellulolytic enzyme cocktails from UFRJ/BIOMM (Brazilian Consortium) present advantages in the liquefaction process (first 6 h of enzymatic hydrolysis, although those enzymes are more affected by product inhibition than Cellic CTec2 (Novozymes). Xylanolytic cocktails obtained from collaborative work between LNEG and UFMG (Brazilian Consortium) perform, in some cases, better than the hemicellulose Cellic HTec2 (Novozymes), especially at temperatures required for SSF processes (35ºC). Special attention has been given to β-xylosidases from yeasts, their identification and heterologous expression in S. cerevisiae for xylo-oligosaccharide fermentation, also in collaboration with the Brazilian Consortium (both UFMG and UFSC).
ULUND screened industrial Saccharomyces cerevisiae strains towards robust fermentation capacity in hydrolysates produced from hydrothermal pretreatments. The best performing strains (Ethanol Red, Proethanol2G reference strain, and SAF Instant) were identified as suitable hosts for heterologous expression of xylose assimilation pathways.
In a collaborative work between LNEG and UFMG (Brazilian consortium) a novel C5-fermenting non-conventional yeast, Spathaspora passalidarum UFMG-HMD-1.1 revealed superior xylose fermentation capacity and a xylose reductase (XR) activity with preference for NADH. The gene XYL1.2 and the corresponding gene in Spathaspora passalidarum CBS 10155 were expressed in appropriate recombinant S. cerevisiae lab strain and characterized at ULUND. The generated strains displayed superior C5-fermentation properties, with both increased yield and productivity, and no significant difference using the XYL1.2 genes from one or the other Sp. passalidarum strain. The S. cerevisiae strain carrying Sp. passalidarum UFMG XR (called TMB3504) was further tested at CIEMAT for fermentation of hydrolysates under different configurations. The novel C5 yeast strain revealed high xylose fermentation capacity and robustness towards the pretreated materials and respective hydrolysates generated in ProEthanol2G. In mixtures with glucose, xylose fermentation was delayed and incomplete, which suggests that this novel trait should be combined with other properties to promote efficient glucose/xylose mixtures.
CIEMAT contributed to the integration of enzymatic hydrolysis and fermentation by means of simultaneous saccharification and fermentation (SSF) approaches. The main objective is to work at high solids (20% w/w and above) and with minimum enzyme and yeast loading. Under those conditions, CIEMAT reaches ethanol concentrations higher than 60 g/L in 72h from pretreated sugarcane bagasse, which correspond to slightly higher ethanol titres than those obtained from wheat straw. At large scale, and 30% (w/w) solid content, CIEMAT was able to reach 92 g/L ethanol from an SSF process (24-h pre-hydrolysis) with pretreated sugarcane bagasse.
LNEG tested different configurations for operating enzymatic hydrolysis and fermentation in order to evaluate process flexibility (cellulolignin fermentation vs clear mash technology) against final ethanol yield and potential of enzyme recovery at different process steps. Comparable ethanol yields were obtained (>0.43 and >0.40 g/g glucan) from 10% and 20% (w/w) wheat straw, respectively, suggesting that the hydrothermal pretreated feedstock from INBICON and enzymes and yeasts used can be implemented both in Europe and Brazil.

The main objectives of WP 4 were the development and implementation in Brazil of a novel energy efficient diabatic distillation which avoids thermal inactivation of the cellulolytic enzymes of the 2G bioethanol process and reduces the energy consumption for distillation more than 50% for both 1G and 2G bioethanol.
Based on intensive research and discussion with Brazilian stakeholders, HOLM developed two versions of a conceptual design for Brazilian ethanol plants (1G) comprising 2G bioethanol production. Apart from using bagasse as raw material for 2G bioethanol, both versions also integrates utilisation of green tops and leaves and brown leaves in the sugarcane campaign and sweet sorghum outside the campaign. Version A provides maximum recycling of enzymes, and relies on ultrafiltration (UF) for separation of enzymes and sugars and on reverse osmosis for concentration of the sugar solution to 14-20% fermentable sugars. Version B provides less enzyme recycling, but is more robust, since problems may occur with fouling and clogging of the UF equipment, due to residual lignin particles in the hydrolysate after delignification. Version B keeps the enzymes in the mash, and relies on evaporation for concentration of the mash after delignification. Both versions rely on a horizontal vacuum stripper to increase already existing distillation capacity, and to maintain enzyme activity through the stripper.

WP5 aimed at 100% utilization of plant feedstock within the most cost and energy efficient 2G bioethanol biorefinery, with minimal CO2 emissions. Therefore, the activities tackled the development of technologies for residues upgrading – lignin as valuable market product, lignin/solids for electricity and wastewaters for added-value products through electrochemical processes.
Greenvalue worked on the evaluation of lignin technical properties for partial replacement of phenol in plywood, foundry or laminate resins and in the preparation of dispersants for concrete. The upgraded wheat straw lignin provided by INBICON demoplant performed well in several applications, while that from sugarcane bagasse (solids provided by CIEMAT after SSF) is also consider to have the purity required for industrial applications. As an alternative, lignin provided by INBICON was used in gasification studies at bench and pilot scale to be applied in electricity production. No significant changes were required in existing LNEG’s gasification installation. Lignin gasification did not give rise to any operational problems, regarding both the feeding system and gasification operation. Lignin gasification produced a gas suitable for energy production with HHV values between 12 and 16 kJ/NL, depending on gasification conditions. Since side streams and effluents in a biorefinery are rich in unconverted biomass sugars and fermentation products, UGent was focused on wastewater upgrading through an integrated fermentation and electrochemical recovery and upgrading process. Valuable charged fermentation products, such as caproate, are extracted across an ion exchange membrane and reacted or processed into higher value products.

The main objective of WP6 was to develop an integrated assessment methodology and to apply it to case studies.
Firstly two reference cases for stand-alone 2G bioethanol technology were analyzed. Inspired from IBUS concept of INBICON, both processes are an integration of a second generation into a preliminary first generation. At this stage of the project, the second generation was analyzed independently. The scale of the 2G processes was 50 tons per hour of biomass (dry matter basis). The feedstock was wheat straw for the first reference case and sugarcane bagasse for the second one. The process includes five steps: 1) Feed handling; 2) Liquid hot water pretreatment; 3) Cellulose hydrolysis and fermentation; 4) Distillation, dehydration and molasses feed production and, 5) Lignin fuel and power generation. For each reference case, the process design was performed using Aspen Plus® version 7.3 and the results were transferred to an Excel platform (previously developed by the EPFL) for establishing the mass and energy balances. Finally, based on these results, an LCA was performed. Then integrated schemes of 2G with 1G and sugar mill were assessed and an LCA was performed for selected scenarios. The scenarios also included Heat and Power Generation and study of alternative uses of lignin and C5 value chains.

WP7 targeted the sustainability assessment of the production of bioethanol, by- or co-products and energy with the corresponding material flows looking preferentially to the sugarcane crop. The focus was concentrated on the whole process chain (cultivation, fertilization, harvesting, transport, transformation) and on the evaluation of their impact on sustainable development, with an integrative assessment, including social, economic and ecological aspects. WP7 had a strong link and cooperation with WP6, which focused on the technical design and material flows.
A framework was developed that defines relevant criteria and indicators to assess the sustainability of the bioethanol production from straw/bagasse. WP7 concluded the life cycle assessment of 1G, 2G, and an integrated (1G+2G) production of ethanol in Brazil using ProEthanol2G data which was delivered by all partners and validated by the coordinator. The data collection has been challenging due to process interdependencies and changing process design. Due to the novelty of some processes data on industrial scale were hardly accessible and some data had to be completed by an extensive literature research.
Special effort has been set on data assessment for sugarcane production, consideration of local conditions and balancing of by-products. A strong collaboration with the Brazilian and European partners has been built up for improving the life cycle model. The results obtained are the basis for joint publication between European and Brazilian partners to be submitted to a scientific peer-reviewed journal.

Potential Impact:
In WP 8, effective dissemination of the foreground knowledge generated during the time course of this project was pursued (both EU and Brazil have been directly targeted). In addition, the public has been receiving indirect information on the potential of an intercontinental collaboration, merging S&T expertise from EU and Brazil. The following actions have been taken:
• disseminate the project 2G technologies in Brazil, which were mainly achieve through the Workshop and tour in Brazil in July 2013 (M33).
• to deliver scientific and technical information to the public, mainly achieve through the website, newsletters, scientific presentations in congresses and seminars and scientific publications in international peer-reviewed journals.
• to increase public awareness on the technological challenges and the achievements in the field of 2G biofuels, mainly through website, newsletters and seminars;
• to carry out advance training of PhD´s students and to set up a student mobility programme between both EU-Brazil consortia; several students have been exchanged and thesis have been completed and published with results obtained in the framework of ProEthanol2G.
The webpage www.proethanol2g.org is common to EU and Brazilian ProEthanol2G Consortia and has been a tool for communication between partners and to disseminate R&D results to the public.
Moreover, the exploitation of results has been considered namely by INBICON and HOLM regarding the implementation of their technologies in Brazil (IBUS and novel diabatic vacuum distillation system, respectively), but also by other partners like LNEG on the development of novel C5 strains, GV regarding the use of residual solids from INBICON to recover and purify lignin to be used as a valuable marketable product and UGENT in the scaling up of the electrochemical system for wastewater valorization.

DISSEMINATION ACTIVITIES

The website
A continuous dissemination of the project R&D results have been carried out via the World Wide Web with a dedicated project homepage (www.proethanol2g.org) along with an interactive mailing list for communication inside the consortium and a project e-mail address for the public. LNEG, as Project Coordinator was responsible to create and maintain a domain for the website and to build the website with an open source content and a management system. The website has a user-friendly navigation and architecture and contains a public area as well as a restricted area for the consortium. The public area presents: the European and the Brazilian Consortia; information about the project (summary and workpackages); contacts of the coordinators and scientific project officers; a library section with publishable summaries, public project reports and publications; and a section for news, including announcements of workshops, meetings and relevant information on 2G bioethanol and newsletters of the project. The website has been updated by LNEG over the entire project period. The restricted area was structured so that access is only allowed to project partners.
The website domain was recently renewed and will be active at least up till January 27th 2016 in order to maintain dissemination activities beyond the project timeframe.

Participation in conferences
The ProEthanol2G partners participated in several international conferences with 18 oral communications, from which it can be highlighted the participation as speaker in the 3rd and 4th edition of the International Conference of Lignocellulosic Ethanol (ICLE), 22nd European Biomass Conference and Exhibition, Alternative Energy in Developing Countries and Emerging Economies 2013, 6th International Conference on Green and Sustainable Chemistry, DTU international Energy Conference 2013 and ENZITEC 2014 (in Brazil). Moreover, 9 posters were presented in international conferences, as in the 35th and 36th Symposium on Biotechnology for Fuels and Chemicals, 22nd European Biomass Conference and Exhibition, 13th International Congress on Yeasts, 4th International IUPAC Conference on Green Chemistry, 4th International Conference on Engineering for Waste and Biomass Valorisation. Several of these communications have co-authors from both European and Brazilian Consortia. Other communications disseminating ProEthanol2G R&D results are planned and are expected to occur during 2015. The full list of oral and poster communications are available in the Final Report (D1.222) and in the Final Report of Dissemination (D8.800).

Publications in Scientific Journals, Books and Thesis
The European ProEthanol2G Partners published 9 articles in scientific international journals and 1 book chapter. From those, the publications in Science, Biotechnology for Biofuels, Green Chemistry, Bioresource Technology and Fuel can be highlighted. Several of these publications have co-authors from both European and Brazilian Consortia. The book chapter was a joint publication between European and Brazilian Consortia in the Book entitled Biofuels in Brazil – Fundamental aspects, recent developments and future perspectives, Springer-Verlag. Other scientific publications with ProEthanol2G R&D results are foreseen. A total of 6 Thesis were concluded in the framework of ProEthanol2G, 3 PhD and 3 MSc. From those, 3 (2 PhDs and 1 MSc) are from Brazilian students co-supervised by Brazilian and European Professors/Researchers.

The ProEthanol2G Workshop/Tour in Brazil
The European ProEthanol2G Partners participated in the ProEthanol2G Workshop/Tour in Brazil, 1-5 July 2013. This event was organized by the Coordinators of both Consortia and included: a Workshop (July 1) with oral communications of both European and Brazilian Partners, which generated a book of abstracts; scientific visits to Universities and Research Institutes, UFRJ and INT (July 2) in Rio de Janeiro, and USP (Ribeirão Preto), in São Paulo; a technical visit to Usina Batatais (July 3), a 1st generation sugar/ethanol mill; and scientific and technical visits to the Technological Center for Advanced bioethanol (CTBE) in Campinas, São Paulo (July 4), and to the Cane Technological Center (CTC) in Piracicaba, São Paulo (July 5), the two most relevant Research Institutions on sugarcane ethanol in Brazil. During the Workshop/Tour in Brazil, the ProEthanol2G European Consortium accounted for 19 oral communications and fruitful discussions with Research Scientists and Industrials. The abstracts of the oral communications in the Workshop/Tour in Brazil were collected in a book of abstracts (D8.610) and are listed in the Final Report (D1.222) and in the Final Report of Dissemination (D8.800). The interest of the Brazilian scientific community, media and general public for the collaborative action between EU and Brazil was assessed by several press releases (D8.410).

Networking and other targeted dissemination actions
During the timeframe of ProEthanol2G, the following interactions with other EU project and programmes were identified:
1) EERA – European Energy Research Alliance, Joint Research Program on Bioenergy – Sub-programme Sugar Platform
An interlaboratory comparison on methodologies for analysis of (bio)chemical composition of feedstock and pretreatment products was performed in the scope of EERA-Bioenergy – SP2 – WP1 Biomass Deconstruction. The ProEthanol2G Partners DTU, LNEG and CIEMAT are also involved in EERA-Bioenergy with very active participation in this interlaboratory study. Also, in ProEthanol2G WP3, a SOP were developed for enzymatic hydrolysis and fermentation processes, again with the involvement of DTU, LNEG, CIEMAT. These SOPs will be disseminated among EERA partners in order to have common methodology for interlaboratory comparisons of enzymes, cell factories and processes on biochemical conversion of biomass, within EERA Bioenergy – SP2 – WP2 Cell Factories & Enzymes. Moreover within EERA Bioenergy-SP2 the major biochemical routes for advanced biofuels are being identified and the ProEthanol2G reference case is included. This work is being developed in the framework of EERA Bioenergy – SP2 – WP3 Integrated design, development and evaluation of bioprocesses.
2) HYPE project (FP7-213139. “High efficiency consolidated bioprocess technology for lignocellulose ethanol”)
HOLM, in the framework of WP4, collaborated with the University of Copenhagen, under the HYPE project, to determine the activities of recovered enzymes after stripping.
3) SAHYOG project (Strengthening Networking on Biomass Research and Biowaste Conversion – Biotechnology for Europe India Integration)
A ProEthanol2G research grantee from LNEG was selected to integrate the Short Term Exchange Programme EU-India, 18-27 November 2013, to visit several R&D institutions in India and disseminate ProEthanol2G (4 presentations in Indian R&D institutions).
4) BABETHANOL project (FP7-227498 New feedstock and innovative transformation process for a more sustainable development and production of lignocellulosic ethanol)
Contacts between ProEthanol2G and BABETHANOL coordinators have been promoted by the EU Project Officer (Mr. José Ruiz-Espi) in order to find complementary activities beyond each project, namely on fermentation of lignocellulosics. This contact promoted the joint participation in H2020 calls.
5) ERA-IB project (EIB.10.013. “Products from lignocellulose”)
One of the R&D results of the work carried out in the framework of ProEthanol2G WP2, on delignification of wheat straw, was also one of the main objectives of ERA-IB project, in which LNEG is involved. Exchange of knowledge has been beneficial for both projects.

The visibility and credibility brought by a funded Energy FP7 Project allowed ProEthanol2G partners to strengthen and expand their network (R&D Institutions, Industries and policy makers). Directly or indirectly, this large network promoted the participation in several consortia with applications to National (e.g. in partnership with companies) or European (e.g. H2020 calls LCE and SME instrument) funding to further develop and exploit the foreground knowledge of ProEthanol2G.

EXPLOITATION OF RESULTS

The R&D results with exploitation potential at the end of ProEthanol2G are briefly described below. For detailed technical description and R&D results see the Final Report (attached).

Diabatic Distillation with Vapor Recompression (HOLM) - WO2014012554 (PCT/DK2013/050238)
HOLM is planning the construction of a prototype for demonstration tests of the diabatic distillation with vapor recompression, which can contribute with more than 50% energy savings than conventional distillation. The potential market in Brazil for a novel horizontal vacuum stripper has been evaluated in the framework of ProEthanol2G. Now, HOLM is pursuing the construction of the prototype through different funding schemes, like SME instrument.

Ensiling treatment used in combination with hydrothermal pretreatment (DTU)
Ensiling significantly improved the effect of wheat straw pretreatment on cellulose digestibility, especially at the lower temperatures (>30% higher yield at 170ºC). In turn, ensiling of sugarcane bagasse coupled to hydrothermal pretreatment promoted a 5-10% improvement of enzymatic hydrolysis yield when comparing to non-ensiled pretreated sugarcane bagasse, whatever the temperature tested.
A publication has been generated in 2013, since a patent application on the topic is registered. However, the results have shown that the process is biomass specific and this can follow different exploitation actions, for example with wheat straw in Europe and with sugarcane bagasse in Brazil. Tests on larger scale should be performed prior to commercialization of the technology.

Technological assessment of novel integrated biomass pretreatments based on ionic liquids (LNEG)
Ionic liquids have shown to be promising for biomass processing through biochemical conversion pathways, but significant R&D is still necessary to progress beyond the proof-of-concept, namely on the scaling up and on ionic liquid recycling.

Novel C5-fermenting yeast strains (LNEG, ULUND and UFMG-Brazil)
A novel C5-fermenting Saccharomyces cerevisiae strain was generated, which displays a novel xylose reductase (XR) with preference for NADH. This strain revealed to be far superior to the benchmarking, i.e. TMB 3422, which was the best C5-fermenting strain available in ULUND (background knowledge) displaying the XR-XDH pathway for xylose fermentation. The novel C5 yeast strain revealed high xylose fermentation capacity and robustness towards the pretreated materials and respective hydrolysates generated in ProEthanol2G. In mixtures with glucose, xylose fermentation was delayed and incomplete, which suggests that this novel trait should be combined with other properties to promote efficient glucose/xylose mixtures. This work is progressing and will continue beyond the end of ProEthanol2G. The submission of a patent application is waiting for authorization from Brazilian authorities since this work involves a Brazilian partner.

Novel oligosaccharide-fermenting yeast strains (LNEG and UFSC/UFMG-Brazil)
The proof-of-concept of novel oligosaccharide-fermenting yeast is being developed and the stage of this invention required further R&D activities, which are in-place. Also, as the previous invention, it requires combination with other properties to promote efficient oligosaccharide fermentation. A patent application may be submitted by the end of 2015, after obtaining several examples supporting the proof-of-concept.

Improved system for liquid/vapor contact and contact between liquid and heat transmission wall (HOLM)
HOLM improved the Jensen modules with the objective to design universal module with optimal evaporation capacity within the container frame and with the ability to work as:
1) Stripper with-or-without vacuum, with MVR of steam for energy supply;
2) Evaporator, with-or-without vacuum, with MVR or steam for energy supply.
The development work has resulted in an improved contact system for the liquid/vapour contact, which is important for the capacity of the stripper and evaporator.
A patent application will be submitted and the exploitation will be performed together with that of Diabatic Distillation with Vapor Recompression (WO2014012554).

Novel lignin applications (GV)
The lignin pellets from INBICON have been tested by GV with potential exploitable results. However, the demonstration nature of INBICON facility, incapable of steady lignin supply, was not attractive for further discussions towards commercialization by GV of lignin products from INBICON’s pellets. If a commercial INBICON facility is commissioned, GV may benefit from lignin pellets to generate marketable lignin products.

Operation of an electrochemical system for separation of fermentation products (UGENT)
The electrochemical system developed by UGENT in the framework of ProEthanol2G was design to produce high-value biochemicals, such as butyrate and caproate, from wastewaters generated in 2G bioethanol biorefineries. This system is expected to be tested at pilot scale after the construction of a prototype during 2015.

List of Websites:

ProEthanol2G website address: www.proethanol2g.org

PROJECT COORDINATOR
Francisco Gírio
francisco.girio@lneg.pt
+351 210924721

PROJECT MANAGER
César Fonseca
cesar.fonseca@lneg.pt
+351 210924717