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

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

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

Estimate for 2008 (April 29 th ) Biggest production ever Brazil will produce billion liters of ethanol From to million tons of sugarcane will be harvested Figures are 14.9% to 19.4% higher in comparison to billion liters will be exported (2.5 billion liters to the USA) Source:

Biomass Ethanol

MechanizedStraw Haverst Non mechanized Sugarcane Plantation

Ethanol from Biomass Residue Straw and/or Bagasse  ETHANOL

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

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

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

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

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

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

Enzymes Production Area

 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

Sugarcane bagasse enzymatic hydrolysis using Trichoderma reesei cellulase

Sugarcane Bagasse Treated Sugarcane Bagasse BIOMASS

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

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

HYDROLYSIS EXPERIMENTS USING Eucalyptus

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

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

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

Sugarcane biomass hydrolysis using the ENZITEC enzyme blend

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 rpm

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

Obrigada!