Enzyme technology for cellulosic ethanol production - bioenergybaltic

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Enzyme technology for cellulosic ethanol production - bioenergybaltic

Enzyme technology for cellulosicethanol productionBiomass & Bioenergy Conference 200827-29.2.2008 TallinnMatti Siika-ahoVTT/Bioprocessing

VTT TECHNICAL RESEARCH CENTRE OF FINLANDContents Introduction Raw materials Ethanol process and Process concepts Drivers and targets for enzyme development Economics and feasibility Ongoing activities2

VTT TECHNICAL RESEARCH CENTRE OF FINLANDIntroduction – BioethanolGlobally Demand and markets for ethanol increasing rapidly; USA and Brazil leading thedevelopment Ethanol from cellulose is necessary for improved GHG balance Oil prize increasing rapidly Countries are pursuing for energy self-sufficiencyIn Europe EU Biofuel directive 2003: 5.75% of transport fuels substituted with biofuels by2010; EU Comission strategy 2007: binding minimum 10 % of transport fuelssubstituted with biofuels by 2020 Variable local raw materials for cellulosic ethanolMarkets Estimated global biofuel production 45 billion litres/year Production will possibly double from this by 2011 In the range of at least 100 new plants (mainly 1st generation) would start within 5years in USA3

VTT TECHNICAL RESEARCH CENTRE OF FINLANDKey topics with biofuels production First generation biofuels Raw materials - sustainability - conversion efficiency Energy balance, CO 2 /GHG emissions - side streamutilization Integration with second generation fuel production Second generation (e.g. from cellulose) biofuels Improved conversion technologies for various raw materials Reduction of production costs - integrated process concepts LCA, CO 2 /GHG - energy balances Product qualitySource: Prof. L. Viikari4


VTT TECHNICAL RESEARCH CENTRE OF FINLANDMajor raw materials for bioethanol production 1 st generation raw materials Sugar cane juice (sucrose) Corn starch (glucose) Wheat and barley starch (glucose) Sugar beet juice (sucrose) 2 nd generation raw materials Sugar cane bagasse Sugar cane trash Corn stover Wheat and barley straw Sugar beet pulp Energy crops Forest industry side streams Municipal waste streams6

VTT TECHNICAL RESEARCH CENTRE OF FINLANDTypical raw material composition (% of DM)and theoretical maximum ethanol yield (L/ton DM)SoftwoodHardwoodCorn stoverSugarcane(spruce(willow)bagasseGlucan45423743Mannan143--Galactan2230.4Xylan7152224Arabinan1252Lignin28252322Other components411108Ethanol from C6420330280300Ethanol from C557120200190Data from Lund University7


VTT TECHNICAL RESEARCH CENTRE OF FINLANDMajor steps of typical ethanol process (2 nd generation)RenewablelignocellulosematerialsPhysical pretreatmentand fractionation byeg. steam explosionFermentationPretreatmentSimultaneous or separatesaccharificationand fermentationEnzymatichydrolysisHydrolysis of celluloseand hemicelluloseby enzymes (or acid)Biotechnical conversionof sugars (hexoses andpentoses) to productSolid residueDistillationorseparationProduct(Ethanol)Concentration andseparation ofproduct (ethanol)9

VTT TECHNICAL RESEARCH CENTRE OF FINLANDDrivers and targets forenzyme development10

VTT TECHNICAL RESEARCH CENTRE OF FINLANDMajor bottlenecks of the present conversion technologies1. Resistant structure of (ligno)cellulose: efficient pretreatmenttechnologies are needed2. Efficiency of the enzymatic hydrolysis of cellulose is too low3. Fermentation of pentoses is restricted on real substrates4. Too low yield and concentration of product (ethanol)5. High energy demand in the production process11

VTT TECHNICAL RESEARCH CENTRE OF FINLANDFactors affecting the efficiency and economics ofenzymatic hydrolysisComposition and accessibility of substrate (cellulose,hemicellulose, lignin), improved by pre-treatmentProperties of enzymes: specific activity, stability, end-productinhibition, unproductive binding, role of CBD’s etc.Composition of enzyme mixtures (cellulase mixtures for optimalsynergy), role of additional enzymes (hemicellulases, ligninmodifying etc.)Hydrolysis technologies: separate/simultaneous/stepwise,temperature, mixing (affecting e.g. diffusion), recycling of enzymesEnzyme prize; efficient production systems12


VTT TECHNICAL RESEARCH CENTRE OF FINLANDProduction costs of cellulose ethanol In several moderate estimations, production costs of 2 nd generationethanol are ca. 0.4-0.5 €/L. But "Iogen ready to sell by 0.25 €/L" Cost structure (European estimate; TIME project, 2004; by courtesy ofPer Sassner, Lund University): Production costs:0.45 – 0.6 €/L, depending onraw material and process Raw materials: total ca. 40 % Chemicals (incl. enzymes): 20 - 25 % Residue (energy): income 20 - 25 % (i.e. -20 - -25 %) Utilities and other costs: ca. 15 % Capital costs: 40 - 45 %14



VTT TECHNICAL RESEARCH CENTRE OF FINLANDExample 1: Development of more thermophilic enzymesRelative FPU activity (FPU/ml)25020015010050030 35 40 45 50 55 60 65 70 75Assay temperatureEconase+Novozym188Celluclast+Novozym188MIXTURE 2MIXTURE 2XMIXTURE 3XIn EU-project TIME severalthermophilic cellulases werecharacterized, cloned,overproduced and evaluatedin bench-scale hydrolysisand fermentation processThe work will continue inFP7 Project HYPE17

VTT TECHNICAL RESEARCH CENTRE OF FINLANDExample 2: Development of helper enzymes6Pructs by HPLC after 72h (mg/ml)543210MannoseProducts from xylan as xyloseProducts from cellulose as glucoseWithout AXE With AXE Without AXE With AXE Without AXE With AXEHemicellulases and otherhelper enzymes have majorrole in the efficient hydrolysisof technical agro or forestbasedmaterials10.0 % Xylan 7.4 % xylan 3.3% xylanHydrolysis products in 48 h (mg/ml)543210Productsfrom from xylan xylan as monosaccharides as mannoseProductsfrom from cellulose as glucose as glucoseCBH's +CBH's +CBH's +CBH's +CBH's +EGIIEGIEGI + XYLEGIIEGICBH's +EGI + XYLCBH's +EGIICBH's +EGICBH's +EGI + XYLThese enzymes have beenstudied and their role hasbeen evaluated in severalprojects, including TIME andEU-project NILESE spruce SE willow WO corn stover18

VTT TECHNICAL RESEARCH CENTRE OF FINLANDExample 3: Development of more efficient enzymesComparison of the Michaelis-Menten, cellobiose inhibition constants and the temperatureoptimum of the four cellobiohydrolasesEnzymeOn CNPLacOn MULack cat(min -1 )K m(µM)k cat/K m(min -1 M -1 )K i(Glc 2)(µM)Type ofinhibitionT opt(°C)k cat(min -1 )K m(µM)k cat/K m(min -1 M -1 )Ct Cel7A19 ±12000 ±2009.5 10 339 ±14comp.6569 ±3.2221 ±353.1 10 5Ta Cel7A1.7 ±0.1990 ±701.7 10 3107 ±14comp.6519.5±0.8268 ±387.3 10 4At Cel7A2.8 ±0.12100 ±1501.3 10 3141 ±25comp.6011.3±0.7220 ±447.3 10 4Tr Cel7A2.6 ±0.05520 ±305.0 10 319± 4comp.6028.7±1.0287 ±311.0 10 5Ref. Voutilainen et al., Biotechnol. Bioeng, in press.Catalytic efficiency of the key cellulase enzymes,cellobiohydrolases is of extreme importance.New and more efficient CBH enzymes havebeen found characterized in our earlier projects.This work will be continued in our on-goingprojects (e.g. EU-project DISCO, coordinatedby VTT).19

VTT TECHNICAL RESEARCH CENTRE OF FINLANDAcknowledgements VTT Kristiina Kruus Niklas von Weymarn Anu Koivula Sanni Voutilainen et al. Lund University Per Sassner University of Helsinki Liisa Viikari Budapest University ofTechnology and Economics Kati Réczey’s students European Union Roal Oy Terhi Puranen Jarno Kallio Jari Vehmaanperä20

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