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LignocellulosicBiomasConversiontoBiofuelsGLBRC: Nirmal Uppugundla, Shishir Chundawat, Leonardo Sousa,
Dahai Gao, Bruce Dale and Venkatesh Balan*
BESC : Charles Wyman and Xiadi Gao
JBEI : Seema Singh, Dean Dibble and Simmons Blake
Reaction 2012, Chennai
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www.glbrc.org
Fundamental science to sustainably convert cellulosic plant biomass intoethanol & next generation fuels
BioenergyResearchCentersMissioninUS 2
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www.glbrc.org
Pretreatment
Hydrolysis
Fermentation
Lignocellulosic Biorefinery Sugar Platform3
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www.glbrc.org
1
2
34
1
Feedstock
Woody biomass Corn stover/cob Swithchgrass Miscanthus Sorghum
Rice/wheat straw Sugar cane bagasse
2
Pretreatment
Steam explosion Dilute/Conc. acid Lime NaOH/peroxide Ammonia
Mechanical Ionic liquid
3
Hydrolysis
Novozyme (Fungal) Danisco (Fungal) DSM (T. Bacteria)
4
Fermentation
Yeast (Native)Yeast (GM) Bacteria (Native) Bacteria (GM) CBP organism
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www.glbrc.org
Other Major Players
Dupont-DDCE, ICM Inc, Iogen, POET, Mascoma, Rangefuel, Pacific ethanol and Verenium
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Contents 6
Comparison of three different pretreatment processcarried out by three bioenergy centers
Composition of different pretreated biomass andresponse to purified and commercial enzymes duringhydrolysis (both 0.6% and 18% solid loadings)
Ethanol Fermentation profile for high solid loadinghydrolysate using S. cerevisiae 424A strain
Complete mass balance during pretreatment, hydrolysisand fermentation process
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ComparisonofThreePretreatmentProcesses
Method
Pretreatment PostWash
Temp
(C)Residence
Time
Catalyst
Type
Catalyst
Loadinga
Catalyst
Re
cyclable?
Water
Loading
During
Pretreat
mentb
Post
Wash
Water
Useb
Water
Temp
(C)
AFEXTM
(GLBRC)140 15mins
Anhydrous
Ammonia100Kg Yes(97%) 60 N/R NA
Dilute
Acid
(BESC)160
20
mins
0.5%
Sulfuricacid 5kg No 895 3000
Room
temp
Ionic
Liquid
(JBEI)
140 180mins
IonicLiquid
(1ethyl3
methyl
imidazolium
acetate)
900kg Yes(89%) N/R 3000Room
temp
aCatalystLoading:kg/100kgDBPbWaterUse:L/100kgDBP
N/RNotrequired,NA NotApplicable
7
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AFEXTMPretreatmentProcess 8
REACTORBiomass Ammonia Expansion
PretreatedBiomass (dry)
Liquid /Gaseous
Ammonia
Gaseous
Ammonia
AmmoniaRecovery
Ammonia recovery 97%
Water
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8/28/2012 9
DiluteAcidPretreatmentProcess
PreWash
&Drying
DiluteAcid
PretreatmentBiomass
DApretreatedSolidResidue
Pretreatment
WaterWash
(Conditioning)
Solid/Liquid
Separation
AcidPost
Hydrolysis
Liquid
PentoseSugars Post-Washing
Liquid
SlurryWater/Chemicals
SolubleSugars DiluteH2SO49
Acid recovery 0%
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IonicLiquidbasedPretreatmentProcess
10
Aliphatic-Rich Product
Lignin-Rich Product
Recovered IL
Precipitation& Wash B SL
VacuumDistillation
WaterPhase
Separator
Distillation B
2-StageExtraction
Distillation ADissolutionPrecipitation& Wash A
Biomass
Ionic Liquid Pretreated Biomass
1st
2nd
ResidueS
L
Pretreated BiomassGlucan-Rich Product
Xylan-Rich Product
Water Wash
S
L
1
0
IL recovery
89%
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Reactor +
Dilute Acid AFEXBiomassAcid
Additional water
BiomassAmmonia
Solids(C6 sugars
+ Lignin)
Liquid(C5 sugars)
+Acid
Water to wash Ammonia
Solids(C6/C5 sugars
+ Lignin)
Detoxify
Ammonia
Reactor Reactor
BM Water(L) NH4Cl(Kg)
100kg 3835 5
1Ton 38,350 50
10Ton 3,38,500 500
100Ton 33,85,000 5000
BM Water(L) NH4Cl(Kg)
100kg
60
2
1Ton 600 20
10Ton 6,000 200
100Ton 60,000 2000
DA Vs AFEX 11
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CompositionData(Pretreatedsolids,waterwashed)
UntreatedCS AFEXTM DiluteAcid IonicLiquid
Glucan
33.4%
33.5%
59.1%
46.9%
Xylan 24.9% 24.8% 6.5% 29.8%
Arabinan 3.7% 3.3% 3.6% 0.3%
Acetyl 2.1% 0.0% 0.6% 1.5%
Insoluble
Lignin17.2% 17.2% 22.2% 2.7%
Ash
3.6%
4.3%
2.5%
1.3%
Total
Extractives10.4% 24.8% 15.4% 13.1%
12
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Enzymes in Action During Hydrolysis
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CommercialEnzymes 14
Ctec 2 (Novozyme)
A blend of cellulases, high level of G, and hemicellulases
*Protein concentration: 138 mg/mL
Effective at high solid concentrationspH optimum: 4.5 to 5.0 and temp opt: 45 - 50oC
Htec 2 (Novozyme)
A blend of hemicellulases and cellulase background
*Protein concentration: 157 mg/mL
pH optimum: 4.5 to 5.0 and temp opt: 45 - 50oC
Multifect Pectinase (Genencor)
Contains pectinase, cellulase and hemicellulase activity
*Protein concentration: 72 mg/mL
*protein assay done using Kjeldahl micro analyzer. Non-protein nitrogen has been subtracted
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TernaryEnzymeCombinationStudies
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
Percentagecomposition(%)
Mixtures
MultifectPectinase Htec
2 Ctec
2
Ternary enzyme mixture optimization using DOE (Minitab)
15
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EnzymaticHydrolysisConditions
Reference: Chundawat et. al., Biotechnol. Bioeng. 99, 1281-1294 (2008)
FreezeDried
Milled
Pretreated
biomass
Ground
Pretreated
biomass
BiomassSlurry
PipettingWorkstation
Multi aspirateSlurry suspension
Sugarquantification
SpectrophotometricSugar Bio-Assay
HydrolysateFiltration
PlateIncubation
Enzyme MixAddition
DispenseBiomass Slurry
16
Pretreated biomass was freeze dried and milled to 250 m,
Biomass suspension was delivered to micro plate at 0.2% glucan loadings
Hydrolysis was done at 50oC, 24 hrs, 250 rpm, pH 4.5 Citric Buffer 50 mM
Enzyme loading 15 and 30 mg/g glucan
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Pretreatment Ctec2 Htec2Multifect
Pectinase
AFEXTM 67% 16.5% 16.5%
DiluteAcid 67% 32% 0%
IonicLiquid 39% 33% 28%
17Optimum Enzyme Mixtures for Best Conversions
Protein Loading Used: 30mg/g glucan
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HighSolidLoadingEnzymeHydrolysis
18
Substrate :AFEXTM,DiluteAcid,Ionic
LiquidTreatedCS
Substrateloading:6%glucan (18%solid)
ReactionTime :72hr
Enzymeloading
:30
mg/g
of
glucan
Reactionvessel :3Lfermentor
Reactionvolume :1.5L
0
10
20
30
40
24 48 72Concentration(G/L)
Time(hrs)
XyloseAfex
DiluteAcid
IonicLiquid
0
20
40
60
80
24 48 72Concentration(G/L)
Time(hrs)
GlucoseAfex
DiluteAcid
IonicLiquid
18
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Fermentation 19
Saccharomyces cerevisiae 424A (capable of fermenting
both glucose and xylose)
With/without nutrient supplementation*
50 mL fermentations done in duplicate in shake flask
Conditions: pH 5.5, 30oC, 150 rpm, 5 days
Initial O.D: 0.5
Time Profiling* Nutrients: Peptone and Yeast extract
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FermentationEffectofNutrientSupplementation
Without nutrient supplementation
With nutrient supplementation
20
0
20
40
60
80
0 50 100 150Concentration(G/L)
Time(hrs)
AFEXTMGlucose
Xylose
Ethanol
O.D
20
0
20
40
60
80
0 50 100 150Conce
ntration(G/L)
Time(hrs)
AFEXTM GlucoseXylose
Ethanol
O.D
20
0
20
40
60
80
0 50 100 150Concentration(G/L)
Time(hrs)
DiluteAcidGlucose
Xylose
Ethanol
O.D
20
020
40
60
80
0 50 100 150Concen
tration(G/L)
Time(hrs)
DiluteAcidGlucoseXylose
Ethanol
O.D
20
20
020
40
60
80
0 50 100 150Concentration(G/L)
Time(hrs)
IonicLiquidGlucose
Xylose
Ethanol
O.D
20
0
20
40
60
80
0 50 100 150Conce
ntration(G/L)
Time(hrs)
IonicLiquidGlucose
Xylose
Ethanol
O.D
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OverallMassBalanceAFEXTM 21
Ethanol Metabolic Yield : 95%
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22OverallMassBalanceDiluteAcid
Ethanol Metabolic Yield : 92%
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Conclusions
24
Composition analysis of the pretreated biomass solids shows thefollowing
no change in composition for AFEXTM-CS
about 85% of hemicellulose is solubilized for DA-CSabout 90% of lignin is removed from pretreated solids for IL-CS
change in cellulose crystallinity noticed for IL-CS
Enzyme desorption for IL > AFEXTM > DA
The optimal commercial enzyme combination was found to bedifferent for three different substrates
Because of loss of nutrients due to pretreatment and wash steps,DA and IL pretreated hydrolysate required nutrient supplementationto ferment glucose and xylose efficiently compared to AFEXTM
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Acknowledgements25
Prof. Nancy Ho, Purdue University - for providing 424Ayeast strain
Novozyme and Genencor for providing CommercialEnzymes for this work
Charles Donald, Margaret Magyar, Christa Gunawan,Rebecca Garlock, Mingjie J in (BCRL, MSU) For helpingto coordinate this work.
MBI International For access to their 5 Gallon batchreactor
DOE Great Lakes Bioenergy Center (DE-FC02-07ER64494) For funding this project
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Page 26
MBI: AResourcefortheBiobasedEconomy
Notforprofit,entrepreneurial
organization;
established
in
1981
AffiliatedwithMichiganStateUniversity
Cross
disciplinary
expertise
in
science,engineering,
business;
35
professionalstaff
Expertiseinbiomassprocessing,fermentation,derisking/scaleup,technology
transfer,
licensing
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Integrated
Process
Development
&
Derisking
HighThrough
Put
Screening
Robotics
Miniaturization
Informatics
AnalyticalChemistry
ProcessMonitoring
ImpurityProfile
DiverseTechniques
Recovery&
Purification
SolidLiquidSeparation
DiverseTechniques
ProductSpecification
ChemicalReactions
andModification
HigherValueProducts
Catalysis
ApplicationsInterface
Lignocellulosic
Processing
HighSolidsProcessing
Chem/EnzymaticTreatment
FermentationInterface
ScaleUp
SeedTrain
Mixing/mass
transfer
Processintegration
Fermentation
MediaDevelopment
Optimization
ProcessControl
Strain/MetabolicEngineering
Enzymology
Microbialgenomics
Fluxanalysis
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SuccessfulTrackRecord
PolyLacticacid ProcessintegrationandscaleupofPLAtechnologyfromfermentation,recoverythrough
polymerization:300MMlbsNatureWorksplantinBlaire,NE,designed,builtandcommercializedbasedontechnologypackagederiskedatMBI/MSU
3hydroxy
butyrolactone
Chemicalroutetoaversatilechiralintermediate,developedandcommercializedbyMSU/MBIstartupSynthon
Succinicacid Integratedprocessdevelopedandscaledupwithnovel,engineeredbacteriumforthe
productionofaversatilebuildingblockchemical;recentlycompletedcommercialdeal
AmmoniaFiber
Expansion
(AFEX)
Pilotscaledevelopmentofabiomasspretreatmentprocessforlignocellulosicsugars;MBI/MSUwithgovernment(DOE,NREL)andindustrialcollaborators;received$4.3MDOEawardforscaleup
Fumaricacid Technologypatentedanddemonstratedatpilotscale,seekinglicensingpartners
Derisking
Services
Collaborativederiskingandscaleupofproprietarybiobasedprocesses;clientsincludeDuPontAppliedBioSciencesandGenomatica
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ReactorReaction Expansion
Ammonia
Recovery
Treated
Biomass
Raw
Biomass
Densification
Biomass
Commodity
Multipleagriculturalfeedstocks Increasesdigestibilityto75+%
9fold
dense
biomass
commodity
EnablingDecentralizedProcessing:
AFEXTMPretreatment
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Rumen
digestibility
correlateswith
enzyme
hydrolysis
Displacecorn,silage,alfalfa
75%+
sugars
releasedwith
enzyme
Hydrolysis
Convertible
sugars
AFEXBiomassCommodity:
Versatile,conversionready
feedstock
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