Ethanol from lignocellulose overview. Neue Krafstoffe Berlin, 6. Mai 2008

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2 Ethanol from lignocellulose overview Neue Krafstoffe Berlin, 6. Mai 2008

3 Content I. Introduction II. Ethanol from lignocellulosic feedstocks i. Pretreatment ii. Enzymatic hydrolysis iii. Fermentation III. Concluding remarks 3

4 Royal Nedalco Producer of potable and technical alcohol since 1899 and subsidiary of Royal Cosun. Production capacity of ~ 1,8 Mhl with an annual turnover of 120 mln and EU market leader in potable alcohol. Recent investments for the traditional market in Sas van Gent (2005) and Manchester (2007). Manchester Production (2007) Sas van Gent Production Bergen op Zoom Head ffice & Production Future growth is targeted in fuel ethanol. Heilbronn Sales & Rectification 4

5 Competitive environment 5

6 Content I. Introduction II. Ethanol from lignocellulosic feedstocks i. Pretreatment ii. Enzymatic hydrolysis iii. Fermentation III. Concluding remarks 6

7 Technology market The driver behind lignocellulosic ethanol is political support for reasons of energy independence (US) and sustainability (EU). Driven by public and private investments the US R&D market is developing fast > 25 announced projects on a variety of feedstocks (corn fiber, corn cobs, straw, corn stover, paper pulp, switchgrass, wood residues etc.). Majority of these projects are based on enzymatical hydrolysis. Distinction could be made between: existing producers (like Abengoa, PET, ICM) that focus on residue streams (corn fiber, corn cobs, wheat bran); and new players (like Mascoma, Verenium, Range Fuels) who focus on dedicated soft biomass (e.g. switchgrass) & hard biomass (e.g. straw). 7

8 Feedstock categories Second generation has become somewhat of a catch-all definition for bioethanol other than from starch or sugar crops. In understanding the technological challenges that need to be taken, a more differentiated approach is helpful. simple and cheap starch-degrading enzymes & use of conventional S. cerevisiae pretreatment required to a certain extent + complex polymerdegrading enzymes + C5/C6 yeast pretreatment required to a very substantial extent resulting in toxic byproducts + complex and expensive polymerdegrading enzymes + C5/C6 yeast that is resistant against toxic compounds Category 1 Sugar and starch streams» sucrose in beet and cane including molasses, glucose from starch in wheat and corn, glucose from starch-containing side streams Category 2 (Hemi)cellulosic side streams + dedicated soft biomass a) side streams (wheat bran, beet pulp, corn fiber) containing the difficult to degrade polymers hemicellulose (into xylose and arabinose) and cellulose (into glucose) apart from starch. Category 3 Hard biomass b) dedicated soft biomass (grasses, engineered crops) containing difficult to degrade polymers and some lignin» wheat straw, corn stover and woody materials containing substantial amounts of lignin which is seriously frustrating the hydrolysis of (hemi)cellulose 8

9 C5 technology learning curve Complexity First generation Second generation Hard biomass Category 3 (b) Dedicated soft biomass Sugar and starch streams (a) Wheat bran Corn fiber, etc Category 2 (a and b) Category Commercial Implementation Time 9

10 Technological challenges Average cellulose biomass structure 10% other 20% lignin 45% cellulose 25% hemicellulose 10

11 Pretreatment & enzymatic hydrolysis Efficiency hydrolysis approx. 80 % n Site Enzyme production Pretreatment 37 C, Low ph Remaining polymers 11

12 Category 2a feedstocks wheat wheat bran Starch Cellulose Arabinoxylan corn other sugars (galactose, glucuronic acid, mannose) corn fiber Lignin Protein Lipid Ash 12

13 Content I. Introduction II. Ethanol from lignocellulosic feedstocks i. Pretreatment ii. Enzymatic hydrolysis iii. Fermentation III. Concluding remarks 13

14 Wheat bran heat-pretreatment lab-scale 1 L reactor Electric heating, but also steam (20 bar)! -> heating up time: within 5 min to 180 C steam (20 bar) through spiral 14

15 Wheat bran heat-pretreatment lab-scale Temperature ( C) Example of heating curve start cold water through spiral stop steam start steam through spiral Time (min)

16 Twenty pretreatments: HMF and furfural Pretreatments (20 Experiments): H2S4 (%): 0, 1 or 2 % w/w (based on dry weight feedstock) Temp ( C): 120, 140, 160, 180 C Time (min): 5, 10, 15 minutes All: 12% dry weight wheat bran in water time T ( C) % H2S4 16

17 Content I. Introduction II. Ethanol from lignocellulosic feedstocks i. Pretreatment ii. Enzymatic hydrolysis iii. Fermentation III. Concluding remarks 17

18 Enzymes and cellulose cellulose -> -1,4-glucan Enzymes and Cellulose Enzymes needed for degradation of cellulose: Crystalline parts: difficult degradation Endo-glucanase (EC ) 1 2 etc Cellobiohydrolase (CBH) (EC ) glucose cellobiase = -glucosidase (EC ) 18

19 Press release 2007 GENENCR LAUNCHES FIRST EVER CMMERCIAL ENZYME PRDUCT FR CELLULSIC ETHANL Accellerase 1000 is launched at the Cellulosic Ethanol Summit in Washington, DC RCHESTER, NY. ctober 15, Genencor, a division of Danisco A/S, today announced a new product, Accellerase 1000, the first ever commercially available biomass enzyme developed specifically for second generation biorefineries. AccelleraseTM 1000 contains a potent complex of enzymes that reduces complex lignocellulosic biomass into fermentable sugars -- an indispensable step for the production of cellulosic ethanol. 19

20 Wheat bran arabinoxylans and enzymes Arabinoxylan -> -1,4-xylan -1,4-endo-xylanases (EC ) Enzymes needed for degradation of wheat xylan: xylose arabinose glucuronic acid -acetyl AXH-d3 (EC ) -(4--methyl)glucuronosidases xylan-acetylesterase (EC ) AXH-s1,2 (EC ) -xylosidase (EC ) Extra: Feruloyl esterase (EC ) 20

21 Example arabinoxylanase enzymes Enzymatic Hydrolysis of Wheat Arabinoxylan by a Recombinant Minimal Enzyme Cocktail Containing β-xylosidase and Novel endo-1,4-β-xylanase and α-l-arabinofuranosidase Activities Hanne R. Sørensen, Sven Pedersen, Christel T. Jørgensen, and Anne S. Meyer Novozymes A/S, Krogshøjvej 36, DK-2880 Bagsværd, Denmark, and Department of Chemical Engineering, Technical University of Denmark, DK Kgs. Lyngby, Denmark American Chemical Society and American Institute of Chemical Engineers Published on Web 01/09/

22 Enzymes strategy Test both commercial and experimental enzymes on Nedalco substrates. Detect effectiveness. Select best enzyme or devise cocktail of enzymes. Iterative process with enzyme companies. 22

23 Twenty pretreatments: enzymes and release arabinose time T ( C) % H2S4 23

24 Twenty pretreatments: enzymes and release xylose time T ( C) % H2S4 24

25 Corn versus wheat bran Comparison in situations of almost no pretreatment Experimental enzymes + Celluclast + Novozyme188 Release of monomers (%): Xylose Arabinose Corn Wheat bran Why these differences? 25

26 Corn versus wheat bran Corn hemicellulose structure: Calculated from molar composition: Per 100 xyloses: - 66 Arabinoses - 20 GlcA(me) - 24 Acetyl - 14 Galactoses Wheat bran hemicellulose structure: Calculated from molar composition: Per 100 xyloses: - 58 Arabinoses - 7 GlcA(me) - 7 Acetyl - 5 Galactoses 26

27 Content I. Introduction II. Ethanol from lignocellulosic feedstocks i. Pretreatment ii. Enzymatic hydrolysis iii. Fermentation III. Concluding remarks 27

28 Fermentation by S. cerevisiae cellulose starch sucrose hemicellulose xylose and arabinose glucose fructose 28

29 S. cerevisiae RWB 218 H Kuyper, M, Toirkens, M.J., Diderich, J.A., Winkler, A.A., Van Dijken, J.P. and Pronk, J.T. Evolutionary engineering of mixed-sugar utilization by a xylose-fermenting Saccharomyces cerevisiae strain. FEMS Yeast Research 5, (2005). H H H H xylitol H H H H H H H Xylose isomerase H D-xylulose D-xylose H Ethanol 29

30 Fermentation by RWB 218 cellulose starch sucrose hemicellulose xylose and arabinose xylose and arabinose glucose fructose 30

31 Wheat bran (SSF) fermentation labscale 31

32 Wheat bran pretreated; addition of xylose; labscale Effect addition of xylose on fermentation of wheat bran hydrolysate (10 % dm) 200,0 Xylose added (52 g/l) 180,0 Xylose added (78 g/l) Xylose added (104 g/l) Flow C2 [ml/h] 160,0 140,0 Xylose added 131 g/l 120,0 100,0 80,0 60,0 40,0 Xylose added (158 g/l) 20,0 0,0 0,0 20,0 40,0 60,0 80,0 100,0 120,0 Elapsed Time [hours] 32

33 Wheat bran at scale 2 m3 Enzymes liberate free sugars WHEAT BRAN ENZYMES Amylases Cellulase SLURRY 18% Dry Weight Experimental arabinoxylanases FERMENTATIN (2100 Liter) Nedalco Yeast Yeast produces bioethanol Material made accessible to enzymes PRETREATMENT 150 C ph = 3 BIETHANL 33

34 Free monomers or Ethanol (g/l) Wheat bran at scale 2 m3 45 Fermentation 2200L monomers/ ethanol 40 Glucose 35 Ethanol 30 Theory: 32.0 g/l (calculated from amount of monomers at t0) Xylose Arabinose Fructose confidential fermentation time (h) 40 34

35 Arabinose fermentation Based on bacterial enzymes expressed in S. cerevisiae J. Becker and E. Boles A modified Saccharomyces cerevisiae strain that consumes L-arabinose and produces ethanol Applied and Environmental Microbiology 69, 4144 (2003) H.W. Wisselink, M.J. Toirkens, M. del Rosario Franco Berriel, A.A. Winkler, J.P. van Dijken, J.T. Pronk and A.J.A. van Maris Engineering of Saccharomyces cerevisiae for efficient anaerobic alcoholic fermentation of L-arabinose Applied and Environmental Microbiology, June

36 Content I. Introduction II. Ethanol from lignocellulosic feedstocks i. Pretreatment ii. Enzymatic hydrolysis iii. Fermentation III. Concluding remarks 36

37 Concluding remarks Deployment of lignocellulosic ethanol technology is a stepwise process. An integrated approach for pretreatment, enzymes and fermentation is necessary to account for the specific characteristics of each feedstock. Commercial availability of enzymes (cellulases and arabinoxylanases) is crucial. An industrial pentose fermenting yeast strain is no longer a show stopper. 37