Ethanol Production from Lignocellulosic Biomass

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. The National Institute of Advanced Industrial Science and Technology (AIST) 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

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. Biomass Technology Research Centerof AIST ● 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.)

5. Biofuels: present feedstock 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.

6. Feedstock for the future 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.

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

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

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

10. The major challenges for the ethanol production from lignocellulosic biomass CHO Cellulose ・Crystalline ・Difficult to hydrolyze Efficient and low-cost technologies are required. HOH Hydrolyzed product　　　　　　　 →D-glucose Easily fermented to ethanol with conventional systems OHH HOH HOH CH２OH Hydrolyzed products　　　　　 →mainly D-xylose Pentose sugars cannot be metabolized by Saccharomyces cerevisiae. Technologies to overcome this problem are required. CHO Hemicellulose ・ Amorphous ・ Relatively easy to hydrolyze HOH OHH HOH CH２OH Lignin

11. Various kinds of pretreatment technology 1. Physical treatment Pulverization (Milling) Irradiation (micro wave, gamma ray, etc.) Hydrothermal treatment Steam explosion 2. Chemical treatment Acids Alkali Ozone, Hydrogen peroxide Organic solvents１　　　　　　　　　　　　　　　　　　　 3. Biological treatment Lignin degrading enzymes or MOs

12. Pretreatment technology of AIST 100μm ×100 Mechanical milling treatment ・ Pulverize to fine particles ・ Decreasing degree of crystalinity of cellulose →Increasing reactivity of enzymes ×2000 5μm Eucalyptus powder before and after MM treatment

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

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

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

16. Improvement of enzyme productivity of A. Cellulolyticus 18 SF 4.0% + Lac 1.0% 16 14 SF 4.5% + Lac0.5% SF 4.0% + TH 1.0% 12 FPA [U/mL] FPU 10 SF 5.0 % SF 4.5% + TH 0.5% Mutant 8 100% 200% β-gluco-sidase SF 5.0 % 6 CMCase parent 4 2 0 Avicelase 0 2 4 6 8 10 0 2 4 6 8 Time [day] 1.Mutation 2. Inducer SF:Solka Floc Lac:Lactose

17. Strategies for xylose utilization 1．Introduce pentose metabolizing ability to Saccharomyces cerevisiae or Zymomonas mobilis Xylose cannot be metabolized, but xylulose can be. Introduce the conversion ability from xylose to xylulose 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.)

18. The conversion of xylose into xylulose is the most important process for the xylose utilization The main pathway of fungi CH２OH The pathway of bacteria HOH OHH Xylitoldehydrogenase Xylose reductase NAD+ HOH NADP+ CH２OH NADH NADPH Xylitol CHO CH２OH HOH O OHH OHH Xylose isomerase HOH HOH CH２OH CH２OH D-Xylose D-Xylulose Xylulose kinase(XK) Xylulose-5-phosphate Ethanol Pentose phosphate pathway

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

20. Fermentation of xylose into ethanol with genetically-engineered yeasts XR/XDH XR/modifiedXDH XR/XDH/XK XR/modifiedXDH/XK Substrates：15 g/L xylose＋5 g/L glucose 9.0 8.0 7.0 Control 6.0 5.0 Ｅｔｈａｎｏｌ (g/l) 4.0 3.0 2.0 1.0 0 60 0 12 24 36 48 72 Time (h)

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

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