1. 01/07/2008 JOINT RESEARCH AGREEMENT AIST - Biomass Technology Research Center and UFRJ – Chemistry Institute

2. 01/07/2008 COLLABORATION AREA Research on Sustainable Biofuel Production from Lignocellulosic Biomass Resources

3. PRINCIPLES  Sustainable zero-carbon emission total system of biofuels production  No competition with food production  Bioethanol from lignocellulosic biomass resources  Mainly from sugarcane biomass residue (bagasse)  Using non- acidic pretreatments  Enzymatic hydrolysis  Ethanol fermentation

4. Estimate for 2008 (April 29 th ) Biggest production ever Brazil will produce 26.4 - 27.4 billion liters of ethanol From 607.8 to 631.5 million tons of sugarcane will be harvested Figures are 14.9% to 19.4% higher in comparison to 2007 4.2 billion liters will be exported (2.5 billion liters to the USA) Source: www.conab.gov.br

5. Biomass Ethanol

6. MechanizedStraw Haverst Non mechanized Sugarcane Plantation

7. Ethanol from Biomass Residue Straw and/or Bagasse  ETHANOL

8. ENZYMATIC Saccharification Buffer treated corn stover Enzyme treated corn stover Acknowledgement NREL - USA

9. Total sugarcane production600.000.000.000 Total bagasse production (28% of sugarcane - 50% moisture) 168.100.000.000 Total dry bagasse production84.000.000.000 Surplus bagasse (12% dry bagasse)10.000.000.000 Theoretical ethanol yield from sugarcane biomass cellulose 1 Kg of bagasse:0.24L 10.000.000.000 Kg of bagasse: 2.400.000.000 L Bagasse production and availability/year (kg)

10. 01/07/2008 ETHANOL PRODUCTION VIA ENZYMATIC HYDROLYSIS OF SUGAR-CANE BAGASSE AND STRAW BRAZILIAN BIOETHANOL PROJECT Elba P. S. Bon - Scientific Coordinator Chemistry Institute Federal University of Rio de Janeiro - Brazil elba1996@iq.ufrj.br

11. RESEARCH NETWORK to develop in Brazil the technology for the conversion of the sugarcane biomass (bagasse and straw) into fuel ethanol using enzymatic hydrolysis. The BIO-ETHANOL Project - Synopsis

12. Development of biomass pre-treatment processes for sugar cane bagasse and straw Raw and pre-treated biomass characterization Cellulases / xylanases production Enzymatic hydrolysis Sugars syrups characterization Ethanol fermentation (C 6 ) C 5 sugars and lignin uses Energy optimisation Effluents and water Main Research Areas

13. Pre-treatment Harvest Pre treatment Enzymatic hydrolysis Fermentation Destilation Sugarcane biomass Ethanol Enzyme Production Steam explosion Milling Trichoderma reesei RUT C30 and Aspergillus awamori Process Overview

14. Enzymes Production Area

15.  Enzyme cost contribution and effectiveness depends on the biomass source and pre- treatment conditions  Development of “tailored made” enzyme blends for sugarcane biomass  Use of crude “cellulase/xylanase /accessory enzymes” preparations  “In house” production to reduce cost Principles

16. Sugarcane bagasse enzymatic hydrolysis using Trichoderma reesei cellulase

17. Sugarcane Bagasse Treated Sugarcane Bagasse BIOMASS

18. HYDROLYSIS RESULTS Yield em 48 h LAC71% GC 22068% Spezyme CP68% Hydrolysis of STEAM TREATED bagasse in presence of an ACID CATALYST – Lund University (STBA - 53% cellulose) ENZYMES GC 220 (Genencor) Spezyme CP (Genencor) Produced on lactose (LAC) FPU/BGU LAC: 1 CG 220: 0,70 Spezyme CP: 0,75 HYDROLYSIS EXPERIMENTS Volume: 100 mL Bagasse conc.: 25 g/L ENZYME LOAD: 10 FPU/G

19. Preliminary hydrolysis experiments using milled Eucalyptus provided by the AIST Biomass Research Centre JAPAN

20. HYDROLYSIS EXPERIMENTS USING Eucalyptus

21. MILLING DOES NOT USE WATER, HIGH TEMPERATURE OR PRESSURE ALTER THE BIOMASS COMPONENTS CHEMICALLY (ADVANTAGE FOR BIOREFINARY) GENERATE INHIBITORS FOR THE HYDROLYSIS AND FERMENTATION STEPS GENERATE POLLUTANT WATER STREAMS AND SALT  MAY BE EASIER TO SCALE UP IN COMPARISON TO STEAM EXPLOSION  IT IS SAFER TO OPERATE  ENERGY CONSUMPTION AND MAINTENANCE ARE THE SENSITIVE ASPECTS

22. MILLED EUCALYPTUS HYDROLYSIS RESULTS  Milled Eucalyptus particle size: 25 micrometer  Eucalyptus contains 42% of cellulose  Hydrolysis: biomass 25g/L, 10 FPU/g, 50ºC, 200 rpm  Hydrolysis yields: 98% using the ENZYTEC blend and 92% using the Acremonium enzyme  Faster process using the ENZYTEC enzyme

23. HYDROLYSIS RESULTS Yield em 48 h LAC71% GC 22068% Spezyme CP68% Hydrolysis of STEAM TREATED bagasse in presence of an ACID CATALYST – Lund University (STBA - 53% cellulose) ENZYMES GC 220 (Genencor) Spezyme CP (Genencor) Produced on lactose (LAC) FPU/BGU LAC: 1 CG 220: 0,70 Spezyme CP: 0,75 HYDROLYSIS EXPERIMENTS Volume: 100 mL Bagasse conc.: 25 g/L ENZYME LOAD: 10 FPU/G

24. Sugarcane biomass hydrolysis using the ENZITEC enzyme blend

25. HYDROLYSIS EXPERIMENTS (STBA – 53% cellulose) 130 g/L of treated sugarcane bagasse ENZITEC blend – 10 FPU/g Sodium citrate buffer pH 4.8 Temperature - 50ºC Agitation - 200 rpm

26. STBA HYDROLYSIS RESULTS STBA + ENZITEC Enzyme Glucose Syrup 60 g/L (87%Yield) Ethanol fermentation Lignin Solid hydrophobic fuel

27. Obrigada!