The LignoRef project; - A national research initiative to enhance biorefinery process developments in Norway -



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The LignoRef project; - A national research initiative to enhance biorefinery process developments in Norway - Nasjonalt Seminar Industriell Bioteknologi, Oslo, 06.06.2013 Karin Øyaas, Kai Toven 1, Ingvild A. Johnsen 1, Swarnima Agnihotri 2, Størker Moe 2, Al MacKenzie 3, Vincent Eijsink 3, Nils Dyrset 4, Roman Netzer 4, Bjarte Holmelid 5, Tanja Barth 5, Ingvar Eide 6. 1 (PFI), 2 Norwegian University of Science and Technology (NTNU), 3 Norwegian University of Life Sciences (UMB), 4 SINTEF Materials and Chemistry, 5 University of Bergen (UoB), 6 STATOIL Research Centre

LignoRef project Competence building (KMB) type project Period: 2009 2013 Budget: 22,5 mnok (70% public RCN, 30% industry) Project partners (value-chain set-up): R&D: o Universities: Norwegian University of Science and Technology (NTNU), Norwegian University of Life Sciences (UMB), University of Bergen (UoB) o R&D Institutes: SINTEF Materials and Chemistry, Paper and Fibre Research Institute (PFI,project owner and coordinator) Industry: o Forest owners: Allskog o Biomass converters/biorefineries: Borregaard, Weyland, Xynergo/Norske Skog o Technology providers: Cambi o Energy producers: Statoil, Hafslund

LignoRef - Objectives LignoRef = Lignocellulosics as a basis for second generation biofuels and the future biorefinery Main objective: Develop fundamental knowledge about central processes for cost effective conversion of lignocellulosic materials into 2G biofuels and value-added products. Central processes studied Pretreatment / decrystalisation and separation of biomass Biochemical and thermochemical conversion routes Enzymatic hydrolysis of biomass carbohydrates (cellulose, hemicelluloses) Fermentation of carbohydrates Thermochemical conversion of process by-products (e.g. lignin)

The LignoRef project - Layout Research focus: Biofuels and chemicals from lignocellulose

R&D partners Trondheim: PFI Norwegian Univ. of Science and Technology (NTNU) SINTEF Bergen: University of Bergen Ås: Univ. of Life Sciences

The lignocellulosic raw material Biofuels / Biochemicals / Biomaterials Extractives 2-5% Lignin 15-30% Cellulose 35-50% Fibre products / Biofuels / Biochemicals / Biomaterials Hemicelluloses 25-30%

LignoRef Inital definition of challenges Inhomogeneity of raw material robust/flexible pretreatment processes Low energy density and high moisture content high dry matter concentration Physical inaccessibility; Cell wall barrier; Cellulose crystallinity effective biomass fragmentation to obtain high yields of fermentable sugars and good separation of components Inhibitor formation during pretreatment reduce inhibitor formation Lignin separation efficiency improve enzyme accessibility avoid inhibition High enzyme costs improve enzyme efficiency Ineffective utilization of carbohydrates C5-fermenting organisms Need to improve overall process economy effective utilization of process by-products (e.g. lignin, hemicelluloses); biorefinery mindset

SP1: Pre-treatment PFI / NTNU / UMB Optimizing pretreatment for cost- and energy effective conversion; Pretreatment for biochemical conversion; Maximise yield of fermentable sugars Minimize formation of inhibitors fate of hemicellulose Raw material flexibility Methods studied: Pre-extraction of hemicelluloses; Organosolv pulping; Sulphite based pulping; Steam pretreatment Pretreatment for thermochemical conversion; Energy densification by torrefaction

SP1: Pre-treatment PFI / NTNU / UMB Sulfite pretreatment low temperature sulfonation process effective lignin dissolution (>75%), lignosulfonates as by-product high enzymatic saccharification yields good control of hemicellulose degradation Organosolv (ethanol/water) pretreatment Up to 70% of spruce lignin could be removed; more effective dissolution for bagasse than for spruce at lower temperatures. High enzymatic saccharification yield; up to 100 %. Saccharification of spruce less sensitive to lignin content.

SP1: Pre-treatment PFI / NTNU / UMB Steam pretreatment of wheat straw: high glucose yields, but inhibitor formation remains a challenge Hemicellulose pre-extraction: Hot water extraction suitable. Temp/time conditions aiming at high DP determined for bagasse and spruce Torrefaction of spruce Significant increase in energy density. Suited for energy dense pellets and feed in entrained flow gasifiers (after milling)

SP2: New enzymatic processes UMB (Norw. Univ. of Life Sciences) Optimize enzyme mixtures and process conditions for maximized hydrolysis yield Enzyme development: Accessory proteins (CBP21 and GH61-like) Contribution of hemicellulases and acetyl xylan esterases Optimization of enzyme mixtures/process conditions

SP2: New enzymatic processes UMB (Norw. Univ. of Life Sciences) Use of helper proteins (CBM33s, GH61s) to better degrade biomass Strengthen the effect of more traditional enzyme mixtures used in cellulose hydrolysis. Search for novel biomass degrading enzymes focusing on hemicellulose degradation E.g. genes involved in biomass degradation in the gut of Svalbard reindeer identified and characterized Studies aimed at developing effective enzymatic conversion protocols undertaken

SP3: Strain development and fermentation SINTEF Develop new yeast strains capable of effective C5 and C6 sugar fermentation; New strains for use in bioethanol production; Maximise ethanol yield, production rate, and tolerance to ethanol and inhibitors Microbial products other than ethanol

SP3: Strain development and fermentation SINTEF Strain developments based on S. cerevisiae This strain has excellent glucose fermentation capability, high ethanol tolerance and resistant to inhibitors Cannot utilize xylose Development of S. cerevisiae mutants able to utilize xylose: Genes for xylose utilization from the yeast Pichia stipitis introduced into the chromosome of industrial S. cerevisiae strain Resulting mutants can convert xylose efficiently to cell biomass and recombinant proteins Potential host for further genetic engineering.

SP4: Thermo chemical processing Univ. of Bergen Conversion of lignin to fuel components and value-added products; Mechanistic studies to identify major reaction pathways and ratelimiting steps Catalyst testing to reduce reaction time and/or pressure Optimisation of reaction conditions and product composition Evaluation of optimal product composition

SP4: Thermo chemical processing Univ. of Bergen Solvolytic approach; Lignin-to-liquid (LtL) process further refined (raw material demands, reaction conditions) Analysis of fragmentation patterns has shown: Different lignins behave rather similar during solvolysis, i.e the LtL process is robust. Detailed composition of products may vary somewhat. Mechanistic studies basis for the further optimizations Model compound studies: Central reaction pathways identified. Solvent system studies (e.g. formic acid, paraformaldehyde, water): Water identified as an alternative "green" solvent. Catalyst screenings effective lignin depolymerisation, oxygen removal and hydrogen incorporation at low temp. (< 360 C)

SP5: Evaluation of bio-fuel quality Statoil Analyses/characterisation of bio-fuels/bio-oils Chemical composition Fuel properties needs for upgrading Health and environmental effects

SP5: Bio-oil quality Statoil Comparisons of bio-oils to petro oils. Methods established: Standardized methods for analysing conventional fuels New methods for analysing components not normally found in petrobased products Needs for upgrading of bio-oils to transport fuel quality determined using Mass spectrometry based fingerprinting techniques and chemo metrics central in this work HSE studies of bio-oils ongoing

Other project activities Initial standardisation/calibration of analytical methods among partners Literature surveys describing the possible co-production of chemical/biotechnological products and their value-adding effect Continuous exchange of samples between partners Implementation of joint demo trial in the latter phase of the project involving: Sulphite pretreatment (PFI) Enzymatic hydrolysis (UMB) Fermentation (SINTEF) Solvolysis of by-products (UoB) Researcher education 3 postdocs and 1 PhD Disseminations

Conclusions The LignoRef project has gathered central Norwegian players along the value chain from biomass to bioproduct. Fundamental knowledge about central processes involved in the development of cost-effective conversion of lignocelluloses has been established. Pretreatment and separation Enzymatic hydrolysis Fermentation and thermochemical conversion processes The project has promoted national collaboration and progress in the biorefinery area.

Acknowledgement We gratefully acknowledge The Research Council of Norway (grant no. 190965/S60), Statoil ASA, Borregaard AS, Allskog BA, Cambi AS, Xynergo AS/Norske Skog, Hafslund ASA and Weyland AS for financial support. My co-authors: PFI: Ingvild A. Johnsen, Kai Toven NTNU: Størker Moe, Swarnima Agnihotri (postdoc) UMB: Vincent Eijsink, Al MacKenzie (postdoc) UoB: Tanja Barth, Bjarte Holmelid (postdoc), Mikel Oregui (PhD) Statoil: Ingvar Eide