Room Temperature hydrophilic Ionic Liquids (RTILs), hydrolases and their combination with other pretreatments for the valorization of biomasses

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1 Room Temperature hydrophilic Ionic Liquids (RTILs), hydrolases and their combination with other pretreatments for the valorization of biomasses Eric Husson, Catherine Sarazin* Génie Enzymatique et Cellulaire UMR 7025 CNRS - Université de Picardie Jules Verne 33 rue Saint-Leu, Amiens Cedex, France * eric.husson@u-picardie.fr; catherine.sarazin@u-picardie.fr 1/19

2 BIOREFINERY CONCEPT Toward the valorization of the whole plant Synthesis of sugar esters from C 5 mono/oligomers: SURFACTANTS Hemicellulose (20-30 %) Lignin (15-30 %) FELIX project - SFR Condorcet 2012 Others (5 %) Cellulose (30-45 %) Proteins, lipids, polyphenols, etc. Synthesis of ester derivatives of lignins: partially biosourced COMPOSITES Hulin et al. Molecules (2015). 20, Glucose/cellobiose as PLATFORM MOLECULES or FERMENTABLES SUGARS Auxenfans et al. Energ. Conv. Manag.(2014). 88, Auxenfans et al. Biomass Bioenergy (2014). 62, Lignocellulose constituents: some examples of application Innovative and ecological strategies for valorization 2/19

3 PRETREATMENT: THE KEY TO UNLOCKING Disorganization, compositional or structural changes? Example no 1 Single disorganization Hemicellulose Disorganization + fractionation Cellulose Lignin Pretreatment(s)* Example no 2 + Ex: delignification Simplified view of recalcitrant LCB *All possible effects are not presented Example no 3 P. Mäki-Arvela et al. Industrial Crops and Products, (2010). 32: p Chandra, R., et al. (2007). Advances in Biochemical Engineering/Biotechnology. 108: Tomás-Pejó, E. et al. (2011). Chapter 7 - Biofuels. Amsterdam, Academic Press: Disorganization + fractionation + structural changes of biopolymers + Ex: delignification 3/19

4 ROOM TEMPERATURE IONIC LIQUIDS (RTILs) Organic salt: organic cation and organic or inorganic anion A rational selection Interesting properties: non-volatil solvent, low melting point, low toxicity, capacity to solubilize various biomolecules, recycling, etc. Solubilization of biopolymers Eco-friendly properties Corrosive effect Biocompatibility Hydrophilic a Imidazolium-based b (alkyl chain lenght < 4C) No c Yes d Cellulases / Xylanases / S. cerevisiae / K. marxianus H 3 C N N CH 3 H 3 C C O O H 3 C N N CH 3 H 3 C H O P O O 1-ethyl-3-methylimidazolium acetate [C2mim][OAc] 1-ethyl-3-methylimidazolium methylphosphonate [C2mim][MeO(H)PO 2 ] a. C. Froschauer et al. Biomacromolecules (2013), 14: ; Y. Fukaya et al. Green Chem., (2008), 10: b. Egorova, K.S et al. (2014). ChemSuschem, 7(2), c. ECORBIO project Region Haut-de-France - leader: INERIS. d. T. Auxenfans et al. (2017). Biochemical Engineering Journal. 117, N. Mehmood et al. (2015). Biotechnology for Biofuels /19

5 FLOWCHART Schematic of the overall sequential process Lignocellulosic biomass Incubation in RTIL Temperature duration Structural properties (ssnmr, FTIR, XRD) Regeneration step by adding water Filtration and washing * Morphological & textural properties (ESEM) Chemical composition (Van Soest method, NREL, etc) Solid fraction Enzymatic hydrolysis Sugar monomers/oligomersrich liquid fraction *Containing lignin and others compounds RTIL-water mixture * Water evaporation Recovered RTIL * Lignin extraction (patent protection) Characterization, separation and/or quantification (HPAEC-PAD or UV) 5/19

6 APPLICATION TO LIGNOCELLULOSIC BIOMASSES Agricultural residues Wheat straw Rape straw (pellets) Sunflower seed shells Wheat bran Forest residues Dedicated crops Spruce sawdust (Softwood) Oak sawdust (Hardwood) Miscanthus Large representative panel of lignocellulosic biomasses 6/19

7 Agricultural residues Forest residues Dedicated crops PRODUCTION OF GLUCOSE Toward fermentable sugars or platform molecules Miscanthus Spruce sawdust (56 %)* (67 %)* Biomass loading: 2 % w/v Cellulase from T. reesei (250 IU / g of biomass) Acetate buffer (50 mm, ph 4.8) 48h at 40 C Oak sawdust (70 %)* Untreated Sunflower seed shell (97 %)* [C2mim][MeO(H)PO 2 ] Rape straw (50 %)* [C2mim][OAc] Destarched Wheat bran (50 %)* *Yield relative to cellulose content (%) Wheat straw (89 %)* Glucose yield (g / 100 g of dry matter)* Significative improvement of enzymatic saccharification [C2mim][OAc] as promising RTIL for efficient pretreatment Summary of data extracted from projects in biomass area since /19

8 Glucose yield (g / 100 g of dry matter) Water content of RTIL (% w/w) Lignin content in RTIL (g/l) PRODUCTION OF GLUCOSE Focus on Spruce sawdust [C2mim][OAc] reuse and lignin accumulation * Reuses of recovered RTIL *Obtained with new RTIL Reuses number Recycling and reuse until 7 times without loss of performances despite significative accumulation of lignin in the RTIL T. Auxenfans et al. (2014). Energy Conversion and Management. 88, /19

9 IMPACT ON CHEMICAL COMPOSITION OF SOLID FRACTION RTIL-pretreatment: fractioning or disorganization? 70 Cellulose (% w/w) Lignin (% w/w) Xylan (% w/w) Wheat straw Spruce sawdust Oak sawdust 0 Wheat straw Spruce sawdust Oak sawdust 0 Wheat straw Spruce sawdust Oak sawdust Untreated [C2mim][OAc]-pretreated [C2mim][MeO(H)PO 2 ]-pretreated Specific interest of RTIL to apply soft pretreatment on biomass in minimizing fractioning and depolymerization of polysaccharidic fractions T. Auxenfans et al. (2014). Energy Conversion and Management. 88, E. Husson et al (2018). Bioresources Technology, 251, /19

10 Percentage of Transmission (%) Cristallinity index (%) IMPACT ON STRUCTURAL PROPERTIES OF BIOPOLYMERS What is happening about changes? XRD Spruce sawdust (a) (b) (c) d e f g h i j k 30 Oak sawdust Miscanthus Rape straw ssnmr FTIR Sunflower seed shells Wheat straw Wave number (1/cm) Glucose yield (g / 100 g of dry matter) Digestibility of cellulosic fraction not exclusively related to CrI depending on biomasses Mild RTIL pretreatment: toward the preservation of structural integrity of each polymer T. Auxenfans et al. (2014). Biomass and Bioenergy. 62, T. Auxenfans et al. (2014). Energy Conversion and Management. 88, E. Husson et al. (2018). Bioresources Technology, 251, /19

11 IMPACT ON TEXTURAL PROPERTIES ON SOLID FRACTION Focus on Miscanthus SEM analyses for a better understanding 100 µm [C2mim][OAc] pretreatement (110 C 40 min) 50 µm 300 µm 300 µm A complex organization: highly fibrillar morphology of strongly agglomerated submicrometric particles Disorganization resulting in an expanded material with irregular and more porous texture RTIL pretreatment for a drastic disorganization of lignocellulosic matrix: toward a better accessibility for enzymes T. Auxenfans et al. (2014). Biomass and Bioenergy. 62, T. Auxenfans et al. (2014). Energy Conversion and Management. 88, /19

12 SCALE-UP OF RTIL PRETREATMENT Application to lignocellulosic wastes from oleaginous crops [C2mim][OAc] 1 L RTIL recycling/reuse Liquid fraction [Lignin] = 4,2 g/l Rape straw 10 % (w/v) Batch stirred reactor / Volume = 5 L 110 C 40 min Solid fraction Disorganization of lignocellulosic fraction Partial delignification Cellulosic and hemicellulosic fractions preserved Improvment of enzymatic saccharification : 40 % versus 11 % for untreated Data extracted from COPIBIOM project Genesys Pivert Lignin extraction (patent protection) Characterized by FTIR 12/19

13 PRODUCTION OF C 6 & C 5 SUGARS MONOMERS Wheat straw RTIL, cellulases and xylanases [C2mim][OAc] pretreatment (110 C 40 min) 100 µm RTIL Solid fraction no1 RTIL recycling Xylanases Xylanase a -catalyzed hydrolysis Solid fraction no2 Xylose-rich liquid fraction Y xylose ~ 98 % Cellulase b -catalyzed hydrolysis Glucose-rich liquid fraction Y glucose ~ 98 % a Endo-xylanases from Thermobacillus xylanilyticus (UMR FARE) b Cellulases from Trichoderma reesei (commercial) Toward well-separated fibers of cellulose Promising complementarity of [C2mim][OAc] xylanase and cellulase for total depolymerisation of carbohydrate polymers E. Husson et al. (2018). Bioresources Technology, 251, /19

14 RTIL & SUBCRITICAL WATER COMBINATION Application to lignocellulosic wastes from oleaginous crops Sunflowers seed shells or rape straw (from 2% to 10 % w/v) RTIL (4 % v/v) [C2mim][OAc] or [C2mim][MeOHPO 2 ] Solid fraction no1 Cellulase-catalyzed hydrolysis Liquid fraction no1 Water evaporation Recovered RTIL Glucose-rich liquid fraction Reactor volume: 560 ml 200 C 120 min Pressure 5-10 bars. Lignin extraction (patent protection) C 5 sugars from hemicellulosic fraction PATENT FR17/ E. Husson, C. Sarazin, H. Ducatel, I. Murrieta-Pazos, G. Ravot (2017). Pretreatment process of biomass. (Focus Transfert CNRS FIST S.A.) 14/19

15 RTIL & SUBCRITICAL WATER COMBINATION Focus on Rape straw Impact on chemical composition Comparison with single RTIL pretreatment Impact on enzymatic saccharification *(%) Yield relative to cellulose content [C2mim][OAc] - Subc water (79 %)* [C2mim][OAc] (67 %)* Untreated (11 %)* Untreated [C2mim][OAc] [C2mim][OAc] Subc water Glucose yield (g/100 g of dry matter) PATENT FR17/ Better improvment of saccharification / single RTIL Selective depolymerisation of hemicellulose Cost-saving in minimizing the required RTIL amount RTIL recycling without loss of efficiency Recovery of lignin fraction from the recovered RTIL 15/19

16 CONCLUSIONS Versatility toward the varieties of lignocellulosic biomasses C 6 sugars from cellulosic fraction C 5 sugars from hemicellulosic fraction Lignin fraction with promising purity for further valorization 16/19

17 EXPAND OUR APPROACH TO OTHER POLYSACCHARIDES From lignocellulosic to animal biorefinery Focus on chitin Proof of concept: [C2mim][OAc] as suitable solvent to affect supramolecular structure of chitin and improve selective chitinase-catalyzed depolymerisation into mono- or disaccharides E. Husson et al. (2017). Green Chemistry, 19, /19

18 ACKNOWLEDGEMENT Isabelle Gosselin (Lecturer) Monica Araya-Farias (Post-doc) Virginie Lambertyn (Technician) Ranim Alayoubi (PhD student) Caroline Rémond (Professor) Harivoni Rakotoarivonina (Lecturer) Domnique Larcher (Professor) Jean-Pierre Bonnet (Lecturer) Tatjana Stevanovic (Professor) Albert Nguyen Van Nhien (Professor) Caroline Hadad (Lecturer) Sylvain Laclef (Research engineer) Gael Huet (PhD student) Eugène Vorobiev (Professor) Nabil Grimi (Lecturer) Nadia Boussetta (Lecturer) Olivier Bals (Lecturer) Industrial samples from PFA & PME Hélène Ducatel (Scientific resp.) Pierre FERCHAUD (Project manager) Loic Dupont (Professor) Arash Jamali (Research engineer) Romain Roulard (Post-doc) Dominique Cailleu (Research engineer) Funding 18/19

19 THANK YOU FOR YOUR ATTENTION