FEDERAL UNIVERSITY OF RIO DE JANEIRO SCHOOL OF CHEMISTRY LABORATORY OF BIOPROCESS DEVELOPMENT

LIGNOCELLULOSIC BIOMASS FOR THE PRODUCTION BIOETHANOL WITHIN THE CONCEPT OF BIOREFINERY

Nei Pereira Jr., PhD Full Professor [email protected] LADEBIO-EQ/UFRJ Website: www.ladebio.org.br The Brazilian Context

BIOMASS is an attractive candidate as an alternative to petroleum, since:

 The only abundant source of renewable carbon;

 Lower CO2 footprint than petroleum;

 Domestically available. Source Share of Primary Energy in Brazil (2005)

Renewable % Non-renewable %

Hydropower 14.4 petroleum 39,1

Sugar cane 13.5 Natural gas 8.9

Wood and vegetal coal 13.2 Mineral coal 6.7

Others 2.7 Nuclear 1.5 . Total 43.9 56.1 THE COUNTRY HAS ONE OF THE CLEANEST ENERGY MATRIX IN THE WORLD

Fonte: BEN/MME CO CELLULOSE 2 LIGHT What are Biomasses and Why to SUGAR WATER use them in substitution and/or in association to fossil sources ? O2 STARCH

 ORGANIC MATTER from PLANT or ANIMAL sources;  Direct or indirect origin from the PHOTOSYNTHESIS PROCESS;  They are RENEWABLE.

Plant Biomass  Natural Biomass

 Food Biomass

 Energetic Plantation Biomass

 Residual Biomass Plant Biomasses

CARBOHYDRATESSUGARY STARCHY LIGNOCELLULOSIC

SUCROSE STARCH=CELLULOSE HEMICELLULOSE SUGARS

FRUCTOSE GLUCOSE= XYLOSE MANNOSE SACCHARIDESGALACTOSE ARABINOSE Ranking dos Países Produtores de Etanol

80

60

34 bilhões de litros

40 Etanol (milhões m3) de (milhões Etanol 20 22,5 bilhões de litros

0 2003 2004 2005 2006 2007 2008 Brasil USA China EU Índia

Fonte: LMC International (2008). TECHNOLOGIES FOR ETHANOL PRODUCTION FROM BIOMASSES

SUGAR STARCH LIGNOCELLULOSE

RESIDUE PRETREATMENT PRETREATMENT Animal Starch solubilization PRETREATMENT: Juice Extraction feedingst EXTRACTION/nd 1 xylose 2HEMICELLULOSE HYDROLYSIS STARCH arabinose RESIDUE co HYDROLYSIS Animal feeding 2 Generationgalactose Energy productionGeneration glucose glucose CELLULOSE sucrose HYDROLYSIS glucose FERMENTATION fructose glucose • BATCH • FED BATCH TECHNOLOGICAL• CONTINUOUS COMPLEXITY LIGNIN Energy production

ETHANOL PRODUCT SEPARATION/ MATURE PURIFICATION EMERGING TECHNOLOGIES • DISTILLATION TECHNOLOGY • SOLVENT EXTRATION • ADSORPTION VINASSE ETHANOL AND ITS DIFFERENT GENERATIONS SUGAR CANE &  1st GENERATION ETHANOL GRAINS/CEREALS

 Fuels vs Food

 2nd GENERATION ETHANOL LIGNOCELLULOSIC BIOMASS

 Does not compete with food production

 3rd GENERATION ETHANOL ALGAE BIOMASS

 Faster growth than traditional crops;  Does not compete with agricultural cultures. Importance of Bioetanol and its Production from Lignocelulosic Biomass

 The oil barrel prices reached values which make unfeasible the self- sustainable growth of the nations;

 Increasing interest for alternative sources of energy;

 Necessity in aggregating value to residual biomass, whose generation tends to grow;

 Possibility for increasing the ethanol production without the need to expand the availability of agricultural area for feedstock production. RESIDUAL BIOMASS OF LIGNOCELLULOSIC COMPOSITION Benefits of Lignocellulose Conversion Technologies through Biological and/or Chemical Routes for the Producition of Fuels & Chemicals  Abundant and cheap renewable resources;  Their generation does not compete with the use of land for food production;  No necessity of expanding the agricultural land for feedstock production;  Opportunity for industrial development based on the concept of BIOREFINERY;  Reduction in gas emissions that cause the so called “green house effect”;  Cleaner technologies;  Macroeconomic benefits for rural communities and for Society as a whole;  They are within the perspective of Sustainable Development. The Lignocellulosic Complex

LIGNIN

Basic Composition :

 Cellulose (40-60%)

 Hemicellulose (20-40%)

 Lignin (10-25%) CELLULOSE HEMICELLULOSE Main Differences between Cellulose and Hemicelluloses

CELLULOSE HEMICELLULOSES Homopolysaccharide composed Heteropolysaccharides composed of of GLUCOSE units several units of PENTOSES and HEXOSES High degree of polymerization Low degree of polymerization (2,000 to 18,000) (50 to 300) Produces fibrous arrangements Do not produce fibrous arrangements Presents crystalline and Present only amorphous regions amorphous regions Slowly hydrolyzed by diluted Rapidly attacked by diluted inorganic inorganic acid in high acid in high temperatures temperatures Is alkaline insoluble Are alkaline soluble Composition of Some Lignocellulosic Residues

Corn Wheat Rice Sugar Cotton Newspaper Urban cobs straw straw cane seed residues bagasse Carbohydrate (%) Glucose 39.0 36.6 41.0 38.1 20.0 64.4 40.0 Mannose 0.3 0.8 1.8 n.d. 4.1 16.6 8.0 Galactose 0.8 4.4 0.4 1.1 0.1 n.d. n.d. Xylose 14.8 19.2 14.8 24.3 4.6 4.6 14.0 Arabinose 4.2 4.4 4.5 4.5 4.3 0.5 4.0 Non-carbohydrate (%) Lignin 15.1 14.5 9.9 18.4 17.6 21.0 20.0 Ashes 4.3 9.6 4.4 4.8 14.8 0.4 1.0 Proteins 4.0 4.0 n.d. 4.0 4.0 n.d. n.d.

~ 70% carbohydrate Source: Lee (1997) What is a Biorefinery?

BIOREFINERIES are similar to petroleum refineries in concept; however, biorefineries use biological matter (as opposed to petroleum or other fossil sources) to produce TRANSPORTATION FUELS, CHEMICALS, and HEAT and POWER.

INDUSTRIAL BIOREFINERIES have been identified as the most promising route to the creation of a new industry based on renewable resources.

SUGARS BIOCHEMICAL PLATFORM intermediates and • HYDROLYSIS (Chemical/Enzymatic) lignin derivatives • LIGNIN CONVERSION BIOFUELS (Ethanol; DMF; Gren Diesel; Gasoline and Kerosene)

LIGNOCELLULOSIC BIOMASS CHEMICALS

POWER

THERMOCHEMICAL PLATFORM GAS and LIQUID • PYROLYSIS (Bio-oil) intermediates • GASIFICATION BTL LC BIOREFINERY: Products from Celulose Alcohol-chemistry SYNTETIC POLYESTERS RUBBER POLY-HYDROXY-ALCANOATES BIOPOLYMERS VITAMIN C (BIO)POLYMERS BUTANOL ACETONE FURANIC POLYMERS LEVULINIC ACID BUTADIENE GLYCEROL ESTERS CITRIC ACID BUTIRIC ACID ACETIC DMF ETHYLENE ACID GLUTAMIC ACID LACTIC ACID REGENERATED ENZYMES CELLULOSE HMF GLUCONIC ACID SORBITOL ETHANOL SCP SUCCINIC ACID ETHERS

ESTERS GLUCOSE

CELLULOSE

BIOMASS LCF: Lignocelulose Feedstock NYLON LC BIOREFINERY: Products from Hemicellulose TETRA HYDRO FURAN

RESINS and GLUTAMIC FURAN LYSINA PLASTICS ACID

XYLITOL PHA’s SORBITOL DMF ENZYMES MANITOL GALACTOL ORGANIC ACIDS FURFURAL ACETONE BUTANOL ARABITOL SCP ETHANOL

MONOSSACHARIDES (SUGARS) ACETIC ACID URONIC ACID XYLOSE; GLUCOSE; ARABINOSE; GALACTOSE; MANNOSE

HEMICELLULOSE CELLULOSE

BIOMASS LCF: Lignocelulose Feedstock LC BIOREFINERY: Products from Lignin

liquid hydrocarbons similar to those of petroleum crude oil ENERGY

LIQUID CHEMICAL PRODUCTS FUELS SOLID FUEL PHENOL, VANILLIN, PHENOLIC OXIDIZED LIGNIN RESINS BIO-OILS METHANOL

PYROLYSIS OXIDIZING PROCESSES SYNGAS

GASIFICATION

HEMICELLULOSE CELLULOSE LIGNIN

BIOMASS LCF: Lignocelulose Feedstock Recently-awarded Projects (Small Biorefineries)

APPLICANT TOTAL COST DOE SHARE ANNUAL PROJECT FEEDSTOCK Technology PRODUCTION LOCATION CAPACITY VERENIUM 91,347,330 76,000,000 1,500,000 Jennings, LA Bagasse, Biochemical (granted 2007) energy crops, agricultural wastes, wood residues FLAMBEAU LLC 84,000,000 30,000,000 6,000,000 Park Falls, WI Forest residue BTL

ICM 86,030,900 30,000,000 1,500,000 St. Joseph, MO Switchgrass, Biochemical forage sorghum, stover LIGNOL 88,015,481 30,000,000 2,500,000 Commerce City, Woody biomass, Biochemical INNOVATIONS CO agricultural Organosolve residue PACIFIC 73,040,000 24,340,000 2,700,000 Boardman, OR Wheat straw, Biogasol ETHANOL stover, poplar (ETOH, biogas, residuals solid fuels) NEW PAGE 83,653,212 30,000,000 5,500,000 Wisconsin, WI Wood biomass – BTL mill residues RSE PULP 90,000,000 30,000,000 2,200,000 Old Town, Wood chips Biochemical Maine (mixed hardwood) ECOFIN, LLC 77,000,000 30,000,000 1,300,000 Washington Corn cobs Biochemical Country, KY (Solid State Fermentation) MASCOMA 135,000,000 25,000,000 2,000,000 Monroe, TN Swichgrass and Biochemical hardwoods TOTAL 808,086,923 305,340,000 25.2 M gallons = 95.4 M liters LIQUID BIOFUELS FROM LIGNOCELLULOSIC BIOMASS

POTENTIAL CANDIDATES

BIOFUELS Process Technological maturity BIOETHANOL Biochemical Emerging

BIOBUTANOL Biochemical Embrionary DMF Hybrid Embrionary

FOSSIL LIKE GREEN FUELS Themochemical Growing FRACTIONING LIGNOCELLULOSIC BIOMASS FOR BIOCHEMICAL PLATFORM

LIGNOCELLULOSIC FEEDSTOCK

PRETREATMENT HEMICELLULOSE (PRE-HYDROLYSIS/AUTO-HYDROLYSIS) (PENTOSES + HEXOSES) + H OH- (HYDROLYSIS) (DELIGNIFICATION) SOLUBLE CELLULOSE + LIGNIN GLUCOSE LIGNIN (CELLULIGNIN)

LIGNIN CELLULOSE  Enzymatic Hydrolysis  Inhibitors of Biological Processes GLUCOSE TENDENCY Pretreatment of Lignocellulosic Feedstock

Aims at disorganizing the lignocellulosic complex, resulting in an increase of CELLULOSE DIGESTABILITY (enzymes accessibility to cellulose molecules)

Lignin Cellulose

Pretreatment

Hemicellulose MAIN PRETREATMENT TECHNOLOGIES (for solubilization and hydrolysis of HEMICELLULOSE)

 STEAM EXPLOSION (needs further hydrolysis)  CATALYZED STEAM EXPLOSION (pretreatment associated to hydrolysis)  DILUTED ACID HYDROLYSIS IN MILD CONDITIONS (more used)  THERMOHYDROLYSIS (needs further hydrolysis)

 AMMONIA FIBER EXPANSION (needs separation of HEM from Lignin) Simplified Scheme of the Steam Explosion Process

Humid material with the lignocellulosic complex disorganized and with its XYLAN CONTAINING digestibility increased LIGNOCELULOSIC High Steam Pressure MATERIALS

Liquid extracted by explosion, composed of: Sudden Decompression • Xylose ~ 10% (to flash cool the biomass) • Xylooligosaccharides (DP 2 - 10) ~ 90% • Uronic and acetic acids Characteristics of the Main Pretreatment Technologies for LC Feedstsocks (Biochemical Platform)

Characteristics Pretreatment Technology SE CSE DAH TH (LHW) AFEX Typical operational Batch or Batch or Continuous Batch or Batch Batch conditions Continuous 2 to 10 min Continuous 5 to 60 min 5-15% amônia 2 to 10 min 5 to 30 min 10-30 min Temperature 190-270oC 160-200oC 150-180oC 170-230oC 100-180oC

Consumption of No Yes Yes No Yes chemical inputs Removal of Yes Yes Yes Yes No, only after hemicelullose washing Xylose yield 10% Xylose; 90% 70-90% 85-95% 50% 60% HDS after Xylosaccharides Hemicellulose washing and removal derived sugars of lignin Removal of Lignin Minor effect Moderate effect Moderate effect Minor effect Major effect

Formation of Yes, under severe Yes, under severe Yes, under severe Few Yes, under severe inhibitors conditions conditions conditions conditions Reduction of the Medium Medium High Medium High required particle size Efficiency of the 80-90% 80-85 % 80-85 % 80-90% 50-90 % Cellulose Enz. Hydrol. Waste Generation Less significant Moderate Significant Less significant Significant

Corrosivity of the Low Low to moderate Moderate to high Low Low to moderate medium Simplicity of the High Moderate to high Moderate Not evaluated Moderate process (potential) State of arte Pilot plants Pilot plants Pilot and demo Bench scale Pilot plant plants

SE: Steam-explosion; CSE: Catalysed Steam-explosion; DAH: Diluted Acid hydrolysis; TH: Thermohydrolysis (liquid hot water pretreatment); AFEX: Ammonia fiber explosion. Sources: Adapted from LYND (1996); OGIER et al. (1999) and Balat et al. (2008); Pérez et. Al. (2007) Substances that are commonly reported as inhibitors of the metabolic activity, originated from the acid hydrolysis/pretreatment of lignocelulosic materials

CELLULOSE HEMICELLULOSE LIGNIN

p-hydroxibenzoic acid hydroxy-methyl furfural furfural acetic acid m-hydroxibenzoic acid acetaldehyde vanilinic acid siringic acid HOH C 2 CHO CHO p-hydroxibenzaldehyde vanilin cinamic acid siringaldehyde coniferyl alcohol sinapyl alcohol Parajó et al. (1998) Enzymatic Hydrolysis of Cellulose

Cellulose AMORPHOUS REGION 2 3 CELLOBIOSE

CRYSTALLINE REGION

GLUCOSE 1

CELLODEXTRINS

1: ENDOGLUCANASE 2: EXOGLUCANASE 3: -GLUCOSIDASE

 CELLULASES ARE INHIBITED BY CELLOBIOSE E GLUCOSE Why hydrolyse cellulose enzymatically?

 Milder conditions of pressure, temperature and pH;

 High specificity;

 Elimination of hydroxymethyl furfural, amongst other toxic substances (lignin derivatives);

 Low energy consumption;

 Low material costs with construction of equipments, differently of those processes which utilize acid hydrolysis. However,  High production costs; R & D & I Strategies for Ethanol Production from Lignocelulosics Following the Biochemical Platform SEPARATE HYDROLYSIS AND FERMENTATION

CELLULASE PRODUCTION

PRETREATMENT solids (HEMICELLULOSE (cellulignin) SHF HYDROLYSIS) ENZYMATIC HYDROLYSIS C6 OF CELLULOSE FERMENTATION

SOLUBLE SUGARS C5 FERMENTATION

However, cellulases are inhibited by their final hydrolysis products DISTILLATION (CELOBIOSE and GLUCOSE) ETHANOL SIMULTANEOUS SACCHARIFICATION AND FERMENTATION

CELLULASE PRODUCTION

PRETREATMENT solids (HEMICELLULOSE (cellulignin) SSF HYDROLYSIS) ENZYMATICENZYMATIC HYDROLYSIS HYDROLYSISOF CELLULOSE &C6 FERMENTATION OF CELLULOSEC6 FERMENTATION

SOLUBLE SUGARS C5 FERMENTATION

Two Stream Fermentation DISTILLATION

Model ETHANOL SIMULTANEOUS SACCHARIFICATION AND COFERMENTATION

CELLULASE PRODUCTION

PRETREATMENT solids (HEMICELLULOSE (cellulignin) SSCF HYDROLYSIS) HIDRÓLISE ENZIMÁTICAENZYMATIC HYDROLYSISFERMENTAÇÃO DE CELULOSEOF CELULOSE C6 & C6 FERMENTATION & SOLUBLE SUGARS C5 FERMENTATION

Molecular Biology Integrated DISTILLATION Fermentation Model ETHANOL CONSOLIDATED BIOPROCESSING

CBP

PREATRETMENT CELLULASE PRODUCTION (HEMICELLULOSE & HYDROLYSIS) CELLULOSE HYDROLYSIS solids & (cellulignin) C6 FERMENTATION & C5 FERMENTATION SOLUBLE SUGARS

Molecular Consolidated Biology Bioprocess DISTILLATION

Model ETHANOL Empresas que empregam tecnologias para a produção de etanol de segunda geração (escala demonstrativa) e características de processo

Empresa País de origem Características de processo Localização Capacidade (m3/ano) AE Biofuels EUA Hidrólise enzimática Montana 567 Blue Fire Ethanol EUA/Japão Hidrólise com ácido Califórnia 12.110 concentrado Izumi n.d. Chempolis Ou Finlândia Hidrólise com ácido diluído Oulu n.d. Iogen Canadá Hidrólise enzimática Ontario 4.000 KL Energy EUA Hidrólise enzimática Wyoming 5.680 Lignol Energy Canadá Pré-tratamento Organosolv Vancouver 2.500 Mascoma EUA n.d. Nova Iorque 1.890 Poet EUA n.d. Dakota do Sul 75 Sekab Suécia Hidrólise enzimática n.d. n.d. ST1 Finlândia n.d. Lappeenranta 1.000 Hamina 1.000 Närpiö 1.000 St. Petersburg Rússia Hidrólise c/ácido diluído 13 unidades no n.d State Forest- país Technical Academy Sun Opta Canadá Hidrólise enzimática China n.d. Universidade da EUA Hidrólise enzimática com E. Flórida 7.570 Flórida coli recombinantes (modelo integrado) Verenium EUA Hidrólise enzimática (modelo Louisiana 5.300 de duas correntes) Japão 4.920 BAGAÇO PETROBRAS PILOT PLANT

CaO Alcohol HEMICELLULOSE Fermentation HYDROLYSATE Utilidade Fria Pichia stipitis

26/10/2007

CaSO 4 ETANOL

P-2 CELLULIGNIN Utilidade Quente

Água Cellulase

Production Pretreatment Utilidade fria

VINHOTO

Utilidade quente UFRJ

SSF (Saccharomyces cerevisiae) Patent PI0505299-8 (11/2005) Patent PI0605017-4 (12/2006) (PATENT nb. 1000) Patent PI0200801-58 (12/2008) CENPES-PETROBRAS

2nd Generation Ethanol Pilot Plant

2006 – 2009

 Building of the pilot plant;

 1st in Latin America;

 More than 80 runs with bagasse;

 Process reproductibity (bench scale  pilot plant);

 New projects in partnership to implement this technology in an industrial level. Mapa tecnológico do tema “Biorrefinarias: Rota Bioquímica” no Mundo e no Brasil (2010 – 2030)

Desenvolvimento do tema “biorrefinarias: rota bioquímica” Desenvolvimento do tema “biorrefinarias: rota bioquímica” Estágios no mundo Estágios no Brasil 2010 - 2015 2016 - 2025 2026 - 2030 2010 - 2015 2016 - 2025 2026 - 2030

Comercialização Comercialização T1b T1d T1a T1d T1e T1b T1c T1a T1e T1c T1e T1b T1c Produção/ T1a Produção/ T1d processo processo T1e T1b T1d T1c T1a

T1a Inovação/ Inovação/ T1d implantação implantação T1a T1d T1c T1b T1c T1b

Scale-up Scale-up T1d T1c T1d T1c T1b T1a T1a Fase demonstração Fase demonstração T1d T1d T1b T1c T1c T1a T1a Fase piloto Fase piloto T1d T1b T1c T1d T1a T1c T1a Pesquisa em bancada Pesquisa em bancada T1d T1b T1c T1a T1c

Notação: T1a – Pré-tratamento; T1b – Produção de celulases; T1c – Biologia molecular; T1d – Produção de biocombustíveis de segunda geração e de outras moléculas; T1e – Produção de energia e Integração energética de processo. Challenges for efficient ethanol production from lignocellulosic biomass

 Cost-effective pretreatment technology, with minimum generation of toxic substances (fermentation inhibitors);

 Dedicated (in plant) cellulase production;

 Enzyme engineering for efficient biomass hydrolysis;

 Genetically modified microorganisms and process optimization for efficient fermentation of C5 and C6;

 Process energy integration (valorization of lignin). Thanks to my “Army”, and Thanks for you attention !

Laboratories of Bioprocess Development Federal University of Rio de Janeiro