1. Ethanol Production from Lignocellulosic Biomass March 23, 2009 Shinichi Yano Biomass Technology Research Center, National Institute for Advanced Industrial Science and Technology (AIST), Japan

2. An Independent Administrative Institution (IAI),in association with the Ministry of Economy, Trade, and Industry (METI) Covers wide areas of industrial technologies: Life science & technology Information technology & Electronics Nanotechnology, materials & manufacturing Environment & energy Geological survey & applied geoscience Metrology & measuremetn technology The National Institute of Advanced Industrial Science and Technology (AIST)

3. Research bases of AIST AIST Chugoku (Kure city, Hiroshima Pref.) ● Biomass Technology Research Center Relocate to Hiroshima Central Science Park (close to Hiroshima university) in March of 2010

4. ● Established in 2003 as Biomass Technology Research Laboratory in AIST Chugoku ● Developed into Biomass Technology Research Center in October 2005 (Main site :AIST Chugoku + Tsukuba, Kyushu) ● Main research areas 1. Fuel ethanol production from lignocellulosic biomass with enzymatic saccharification methods 2. Diesel fuel production from woody biomass via synthetic gas and Fischer-Tropsch synthesis (BTL ： biomass to liquid) 3. Studies on biomass total system (economics, LCA, etc.) Biomass Technology Research Center of AIST

5. Biodiesel fuel (BDF) for diesel engines (High cetane number) Feedstock: Vegetable oil, or animal fats Ethanol for Otto cycle engines (High octane number) Feedstock: sugars or starch Converted by conventional technologies First-generation biofuels Problems 1. Competition with food usage (supplies and costs) 2. Bumper crops or poor harvest → instability 3. Can be real energy production? → LCA analysis is necessary. Biofuels: present feedstock

6. Lignocellulosic biomass (Wood wastes, Agricultural residues, Energy crops) ● Cellulosic ethanol ● BTL （ Biomass to Liquid) diesel fuels Second-generation biofuels However, the production technology from Lignocellullosic biomass is not established. Further R & D’s are required. Feedstock for the future

7. Hemicellulose (hexose ) Hemicellulos e (pentose) Cellulose Lignin Protein, ash etc. Softwoods Hardwoods Oil palm EFB 46%16% 6% 30% 48%12%17%23% Lignin in softwoods is more stable than that of hardwoods. 42% 26%20%11% Composition of lignocellulosic biomass 1%

8. To brake lignin seal, an appropriate pretreatment is essential. Cellulose Hemi- cellulose Hemi- cellulose Lignin Schematic structure of lignocellulosic biomass

9. Pretreatments Enzymatic saccharification Acid hydrolysis Fermentation Distillation/Purification Ethanol ETBE Lignocellulosic biomass (woods, bagasse, rice straw etc.) Lignocellulosic biomass (woods, bagasse, rice straw etc.) Scheme for ethanol production from lignocellulosic biomass

10. CHO H OH OH H H OH CH ２ OH Hydrolyzed products → mainly D - xylose Pentose sugars cannot be metabolized by Saccharomyces cerevisiae. Technologies to overcome this problem are required. CHO H OH OH H H OH CH ２ OH Lignin Hydrolyzed product → D - glucose Easily fermented to ethanol with conventional systems Hemicellulose ・ Amorphous ・ Relatively easy to hydrolyze Hemicellulose ・ Amorphous ・ Relatively easy to hydrolyze The major challenges for the ethanol production from lignocellulosic biomass Cellulose ・ Crystalline ・ Difficult to hydrolyze Efficient and low-cost technologies are required. Cellulose ・ Crystalline ・ Difficult to hydrolyze Efficient and low-cost technologies are required.

11. 1. Physical treatment Pulverization (Milling) Irradiation (micro wave, gamma ray, etc.) Hydrothermal treatment Steam explosion 2. Chemical treatment Acids Alkali Ozone, Hydrogen peroxide Organic solvents １ 3. Biological treatment Lignin degrading enzymes or MOs Various kinds of pretreatment technology

12. ×100 100μm ×20005μm Mechanical milling treatment ・ Pulverize to fine particles ・ Decreasing degree of crystalinity of cellulose → Increasing reactivity of enzymes ・ Pulverize to fine particles ・ Decreasing degree of crystalinity of cellulose → Increasing reactivity of enzymes Eucalyptus powder before and after MM treatment Pretreatment technology of AIST

13. Sugar yields from milled wood after enzymatic saccharification Eu ： eucalyptus, Oa ： Oak, Be ： beech 、 JCy ： Japanese cypress, Df ： Douglas-fir Sugar yields from milled wood after enzymatic hydrolysis

14. OOOOO O O OOO OOOOOOOOOO O OO OO OOOOOOOO OO Endoglucanase （ for amorphous region ） Exoglucanase （ for crystalline region ） β-glucosidase (produce glucose ） Cellulase: consists of three kinds of enzymes Synergetic effects of three enzymes are observed. O O OO O O ････････ ･･････ ････････ ･･････ OOOOOOOO OOO For the cost reduction of cellulase ● Appropriate pretreatments ● Selection of enzymes and combination of enzymes ● On-site production of enzymes Enzymatic saccharification: Cellulase

15. Acremonium cellulolyticus : A fungus isolated from soil in Japan by AIST researchers Industrially produced by Meiji Seika Co. LTD., mainly for silage preparation Acremonium cellulase has higher beta- β- glucosidase activity than Trichoderma cellulase. suitable for ethanol production The research for elevating enzyme productivity AIST original cellulase: Acremonium cellulase

16. 1.Mutation2. Inducer SF:Solka Floc Lac:Lactose FPU CMCase β-gluco- sidase Avicelase 100%200% Mutant parent Improvement of enzyme productivity of A. Cellulolyticus

17. 2 ． Introduce ethanol fermenting ability to pentose- utilizing microorganisms 2 ． Introduce ethanol fermenting ability to pentose- utilizing microorganisms e.g. Introduction pdc and ald genes into E.coli. (KO11:developed by Florida Univ., licensed to Verenium Inc.) Xylose cannot be metabolized, but xylulose can be. Introduce the conversion ability from xylose to xylulose 1 ． Introduce pentose metabolizing ability to Saccharomyces cerevisiae or Zymomonas mobilis Strategies for xylose utilization

18. CHO H OH OH H H OH CH ２ OH H OH OH H H OH CH ２ OH O OH H H OH CH ２ OH D -Xylose Xylitol D -Xylulose Xylose isomerase Xylose reductase Xylitol dehydrogenase Xylulose-5-phosphate Pentose phosphate pathway Xylulose kinase(XK) Ethanol The main pathway of fungi The pathway of bacteria NADPH NADP + NAD + NADH The conversion of xylose into xylulose is the most important process for the xylose utilization

19. XR: mainly require NADPH XDH: require NAD + Accumulation of xylitol, from imbalance of coenzymes Xylose reductase NADPH NADP + Xylitol dehydrogenase NAD + NADH Xylose Xylitol Xylulose Prof. Kosuke Makino, Kyoto University, has developed NADP-dependant xylitol dehydrogenase by protein engineering. NADPHNADP + AIST is developing practical yeast strains with this new enzyme in collaboration with Kyoto University. (XR)(XDH) Overcoming imbalance of coenzymes

20. Ｅｔｈａｎｏｌ (g/l) 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0 8.0 9.0 Time (h) 012243648 60 72 XR/XDH XR/modifiedXDH XR/XDH/XK XR/modifiedXDH/XK Substrates ： 15 g/L xylose ＋ 5 g/L glucose Control Fermentation of xylose into ethanol with genetically-engineered yeasts

21. Integration of AIST Technology :New ethanol production mini-pilot plant in AIST Chugoku Capacity: 0.2t biomass per one operation Started operation in February, 2009 Course milling Hot- compressed Water treatment Fine milling Enzymatic hydrolysis Fermentation Distillation/ Dehydration Enzyme Production Pretreatment Biomass Ethanol

22. Pretreatment: Mechano-chemical (Ball-millig): 240min Enzymatic saccharification: Enzymes cocktail containing Acremonium Cellulase, Novozyme188, OPTIMASH BG Reaction: 50 ℃, 72hr Preliminary experiments for oil palm biomass Sugar yields were lower than other biomass.

23. Why low sugar recoveries? Materials used in Japan were completely dried and very rigid. Actual EFBs are obtained in wet condition after steam sterilization. Sugar yields may improve when fresh EFBs are used for saccharification. Experiments with fresh materials are required.

24. UPM-KIT-AIST collaboration JRA for Research on utilization of palm biomass as feedstock for biofuels and biomaterials (Concluded on November 7, 2008) Established a common laboratory in UPM campus (MTDC) Experiments with fresh materials