Balan Biomass

7/28/2019 Balan Biomass

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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


pH optimum: 4.5 to 5.0 and temp opt: 45 - 50oC

Multifect Pectinase (Genencor)

Contains pectinase, cellulase and hemicellulase activity


*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


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


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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