Maximum Total Time for Talk = 25 minutes

Maximum Total Time for Talk = 25 minutes

Comparative Sugar Recovery Data from Application of Leading Pretreatment

Technologies to Corn Stover and Poplar

Charles E. Wyman, Dartmouth College/University of CaliforniaBruce E. Dale, Michigan State University

Richard T. Elander, National Renewable Energy LaboratoryMark T. Holtzapple, Texas A&M University

Michael R. Ladisch, Purdue UniversityY. Y. Lee, Auburn University

Mohammed Moniruzzaman, Genencor InternationalJohn N. Saddler, University of British Columbia

28th Symposium on Biotechnology for Fuels and ChemicalsNashville, Tennessee

May 1, 2006Biomass Refining CAFI

• Biomass Refining Consortium for Applied Fundamentals and Innovation organized in late 1999 and early 2000

• Included top researchers in biomass hydrolysis from Auburn, Dartmouth, Michigan State, Purdue, NREL, Texas A&M, U. British Columbia, U. Sherbrooke

• Mission: • Develop information and a fundamental understanding of

biomass hydrolysis that will facilitate commercialization, • Accelerate the development of next generation

technologies that dramatically reduce the cost of sugars from cellulosic biomass

• Train future engineers, scientists, and managers.

Biomass Refining CAFI

CAFI Background

• Developing data on leading pretreatments using:– Common feedstocks– Shared enzymes– Identical analytical methods– The same material and energy balance methods– The same costing methods

• Goal is to provide information that helps industry select technologies for their applications

• Also seek to understand mechanisms that influence performance and differentiate pretreatments– Provide technology base to facilitate commercial use– Identify promising paths to advance pretreatment

technologies


CAFI Approach

USDA IFAFS Project Overview: CAFI 1• Multi-institutional effort funded by USDA Initiative for Future

Agriculture and Food Systems Program for $1.2 million to develop comparative information on cellulosic biomass pretreatment by leading pretreatment options with common source of cellulosic biomass (corn stover) and identical analytical methods– Aqueous ammonia recycle pretreatment - YY Lee, Auburn University– Water only and dilute acid hydrolysis by co-current and flowthrough

systems - Charles Wyman, Dartmouth College– Ammonia fiber explosion (AFEX) - Bruce Dale, Michigan State University– Controlled pH pretreatment - Mike Ladisch, Purdue University– Lime pretreatment - Mark Holtzapple, Texas A&M University– Logistical support and economic analysis - Rick Elander/Tim Eggeman,

NREL through DOE Biomass Program funding• Completed in 2004


Hydrolysis Stages


Stage 2Enzymatichydrolysis

Dissolved sugars, oligomers

Solids: cellulose, hemicellulose,

lignin

Chemicals

Biomass Stage 1 Pretreatment

Dissolved sugars, oligomers, lignin

Residual solids: cellulose,

hemicellulose,lignin

Cellulase enzyme

Pretreatment system

Temperature, oC

Reaction time,

minutes

Chemical agent used

Percent chemical

used

Other notes

Dilute acid1 160 20 Sulfuric acid 0.49 25% solids concentration during run in batch tubes

Flowthrough2 200 24 none 0 Continuously flow just hot water at 10mL/min for 24minutes

Partial flow pretreatment2

200 24 none 0 Flow hot water at 10mL/min from 4-8 minutes, batch otherwise

Controlled pH3 190 15 none 0 16% corn residue slurry in water

AFEX4 90 5 Anhydrous ammonia

100 62.5% solids in reactor(60% moisture dry weight basis), 5 minutes at temperature

ARP5,6 170 10 ammonia 15 Flow aqueous ammonia at 5 mL/min without presoaking

Lime7 55 4 weeks lime 0.08 g CaO/g biomass

Purged with air.

Key Features of CAFI Pretreatments

CAFI 1 Feedstock: Corn Stover

• NREL supplied corn stover to all project participants (source: BioMass AgriProducts, Harlan IA)

• Stover washed and dried in small commercial operation, knife milled to pass ¼ inch round screen

Glucan 36.1 %

Xylan 21.4 %

Arabinan 3.5 %

Mannan 1.8 %

Galactan 2.5 %

Lignin 17.2 %

Protein 4.0 %

Acetyl 3.2 %

Ash 7.1 %

Uronic Acid 3.6 %

Non-structural Sugars 1.2 %


Consistent Mass Balance Approach as Applied to AFEX

Hydrolysis

Enzyme (15 FPU/g of Glucan)

ResidualSolids

HydrolyzateLiquidAFEX

SystemTreatedStover

Ammonia

Stover

101.0 lb100 lb

(dry basis)36.1 lb glucan21.4 lbxylan 39.2 lb

95.9% glucan conversion to glucose, 77.6% xylan conversion to xylose

99% mass balance closure includes:(solids + glucose + xylose + arabinose )

Wash

2 lb

99.0 lb

Solids washed out

38.5 lb glucose18.9 lb xylose (Ave. of 4 runs)

Very few solubles from pretreatment—about 2% of inlet stover


Calculation of Sugar Yields• Comparing the amount of each sugar monomer or oligomer

released to the maximum potential amount for that sugar would give yield of each

• However, most cellulosic biomass is richer in glucose than xylose

• Consequently, glucose yields have a greater impact than for xylose

• Sugar yields in this project were defined by dividing the amount of xylose or glucose or the sum of the two recovered in each stage by the maximum potential amount of both sugars– The maximum xylose yield is 24.3/64.4 or 37.7%– The maximum glucose yield is 40.1/64.4 or 62.3%– The maximum amount of total xylose and glucose is 100%.


Overall Sugar Yields from Corn Stover at 60 FPU/g Glucan

Pretreatment system

Xylose yields* Glucose yields* Total sugars*

Stage 1 Stage 2 Totalxylose

Stage 1

Stage 2 Totalglucose

Stage 1 Stage 2 Combinedtotal

Maximumpossible

37.7 37.7 37.7 62.3 62.3 62.3 100.0 100.0 100.0

Dilute acid 32.1/31.2 3.3 35.4/34.5 3.9 53.3 57.2 36.0/35.1 56.6 92.6/91.7

Flowthrough 36.3/1.7 0.8/0.7 37.1/2.4 4.5/4.4 57.0 61.5/61.4 40.8/6.1 57.8/57.7 98.6/63.8

Controlled pH

21.8/0.9 9.0 30.7 3.5/0.2 54.7 58.2 25.3/1.1 63.6 88.9

AFEX ND/30.2 ND/30.2 61.8 61.8 ND/92.0 ND/92.0

ARP 17.8/0 17.0 34.8/17.0 59.4 59.4 17.8/0 76.4 94.2/76.4

Lime 9.2/0.3 20.2 29.4/20.5 1.0/0.3 59.5 60.5/59.8 10.2/0.6 79.7 89.9/80.3

*Cumulative soluble sugars as total/monomers. Single number = just monomers.

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Sugar Yields from Corn Stover at 15 FPU/g Glucan


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Monoxylose S1

Monoxylose S2

Oligoglucose S1

Monoglucose S1

Monoglucose S2



CAFI Economic Estimates

Pretreatment ModelAspen Plus

Bioethanol Plant Model 2001 NREL Design Case

2000 Metric Tons Stover (dry)/Day Stover Cost: $35/ton

Enzyme Cost: ~$0.15/gal ethanol

Thermodynamics ProcessAnalogies

Design Methods

Chemistry

CAFIResearcher


Updated Model Basis and

Feedstock Basis in “CAFI 2” Project

General Process Flow Diagram


Boiler +

Generator

Hydrolysis +

Fermentation

Feed Handling Recovery Pretreatment

Stover

Syrup + Solids

Chemicals Water

Enzymes CO2 Water

EtOH

Steam

Power

Poplar

Capital Cost Estimates


PretreatmentSystem

Pretreatment Direct Fixed

Capital ($MM)

Pretreatment Breakdown,

(% Reactor/ % Other)

Total Fixed Capital ($MM)

Ethanol Production

(MM gal/yr)

Total Fixed Capital ($/gal

Annual Capacity)

Dilute Acid 25.0 64/36 208.6 56.1 3.72

Controlled pH Hot Water

4.5 100/0 200.9 44.0 4.57

AFEX 25.7 26/74 211.5 56.8 3.72

ARP 28.3 25/75 210.9 46.3 4.56

Lime 22.3 19/81 163.6 48.9 3.35

No Pretreatment

0 - 200.3 9.0 22.26

Ideal Pretreatment

0 - 162.5 64.7 2.51

Basis: 2000 metric tons (dry basis) corn stover/day, assumes only monomers fermented

Minimum Ethanol Selling Price (MESP)


0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

Dilute Acid Hot Water AFEX ARP Lime Ideal

Net Stover Other Variable Fixed w/o Depreciation Depreciation Income Tax Return on Capital

Proof Year: 4th Year of Operation$/gal EtOH

CashCostPlantLevel

MESP

Assumptions: 2.5 years construction, 0.5 years start up, 20 year plant life, zero net present value when cash flows are discounted at 10% real after tax rate

Effect of Oligomer Conversion


1.00

1.25

1.50

1.75

Dilute Acid Hot Water AFEX ARP Lime

ME

SP

, $/

gal

EtO

H

w/o Oligomer Credit w/ Oligomer Credit

Observations for Corn Stover

• All pretreatments were effective in making cellulose accessible to enzymes

• Lime, ARP, and flowthrough remove substantial amounts of lignin and achieved somewhat higher glucose yields from enzymes than dilute acid or controlled pH

• However, AFEX achieved slightly higher yields from enzymes even though no lignin was removed

• Cellulase was effective in releasing residual xylose from all pretreated solids during enzymatic hydrolysis in Stage 2

• Xylose release by cellulase was particularly important for the high-pH pretreatments by AFEX, ARP, and lime, with about half being solubilized by enzymes for ARP, two thirds for lime, and essentially all for AFEX

• The projected costs were similar due to the high yields and similar capital costs for the overall processes


Publication of Results from CAFI 1• Bruce Dale of the CAFI Team arranged for and edited a special December 2005 issue of Bioresource

Technology entitled “Coordinated Development of Leading Biomass Pretreatment Technologies” to document these results:

– Wyman CE, Dale BE, Elander RT, Holtzapple M, Ladisch MR, Lee YY. 2005. “Coordinated Development of Leading Biomass Pretreatment Technologies,” Bioresource Technology 96(18): 1959-1966, invited.

– Lloyd TA, Wyman CE. 2005. “Total Sugar Yields for Pretreatment by Hemicellulose Hydrolysis Coupled with Enzymatic Hydrolysis of the Remaining Solids,” Bioresource Technology 96(18): 1967-1977, invited.

– Liu C, Wyman CE. 2005. "Partial Flow of Compressed-Hot Water Through Corn Stover to Enhance Hemicellulose Sugar Recovery and Enzymatic Digestibility of Cellulose,” Bioresource Technology 96(18): 1978-1985, invited.

– Mosier N, Hendrickson R, Ho N, Sedlak M, Ladisch MR. 2005. “Optimization of pH Controlled Liquid Hot Water Pretreatment of Corn Stover,” Bioresource Technology 96(18): 1986-1993, invited.

– Kim S, Holtzapple MT. 2005. “Lime Pretreatment and Enzymatic Hydrolysis of Corn Stover,” Bioresource Technology 96(18): 1994-2006, invited.

– Kim TH, Lee YY. 2005. “Pretreatment and Fractionation of Corn Stover by Ammonia Recycle Percolation Process,” Bioresource Technology 96(18): 2007-2013, invited.

– Teymouri F, Laureano-Perez L, Alizadeh H, Dale BE. 2005. “Optimization of the Ammonia Fiber Explosion (AFEX) Treatment Parameters for Enzymatic Hydrolysis of Corn Stover,” Bioresource Technology 96(18): 2014-2018, invited.

– Eggeman T, Elander RT. 2005. “Process and Economic Analysis of Pretreatment Technologies,” Bioresource Technology 96(18): 2019-2025, invited.

– Wyman CE, Dale BE, Elander RT, Holtzapple M, Ladisch MR, Lee YY. 2005. “Comparative Sugar Recovery Data from Laboratory Scale Application of Leading Pretreatment Technologies to Corn Stover,” Bioresource Technology 96(18): 2026-2032, invited.


DOE OBP Project: April 2004 Start

• Funded by DOE Office of the Biomass Program for $1.88 million through a joint competitive solicitation with USDA

• Using identical analytical methods and feedstock sources to develop comparative data for corn stover and poplar

• Determining more depth information on– Enzymatic hydrolysis of cellulose and hemicellulose in solids– Conditioning and fermentation of pretreatment hydrolyzate liquids– Predictive models

• Added University of British Columbia to team through funding from Natural Resources Canada to– Capitalize on their expertise with xylanases for better

hemicellulose utilization– Evaluate sulfur dioxide pretreatment along with those previously

examined: dilute acid, controlled pH, AFEX, ARP, lime• Augmented by Genencor to supply commercial and advanced

enzymes


Tasks for the DOE OBP Project


• Pretreat corn stover and poplar by leading technologies to improve cellulose accessibility to enzymes

• Enzymatically hydrolyze cellulose and hemicellulose in pretreated biomass, as appropriate, and develop models to understand the relationship between pretreated biomass features, advanced enzyme characteristics, and enzymatic digestion results

• Develop conditioning methods as needed to maximize fermentation yields by a recombinant yeast, determine the cause of inhibition, and model fermentations

• Estimate capital and operating costs for each integrated pretreatment, hydrolysis, and fermentation system and use to guide research

CAFI 2 Corn Stover


Component Composition (wt %)

Sucrose 2.2Glucan 34.4Xylan 22.8

Arabinan 4.2Mannan 0.6Galactan 1.4

Lignin 11.0Protein 2.3Acetyl 5.6Ash 6.1

Uronic Acids 3.8Extractives 8.5

• 2nd pass harvested corn stover from Kramer farm (Wray, CO)– Collected using high rake setting to avoid soil pick-up

– No washing

– Milled to pass ¼ inch round screen

• Feedstock: USDA-supplied hybrid poplar (Alexandria, MN)– Debarked, chipped, and milled to pass

¼ inch round screen


Component Composition (wt %)Glucan 43.8Xylan 14.9


Lignin 29.1Protein ndAcetyl 3.6Ash 1.1

Uronic Acids ndExtractives 3.6

CAFI 2 Standard Poplar

Hydrolysis Stages


Stage 2Enzymatichydrolysis

Dissolved sugars, oligomers

Solids: cellulose, hemicellulose,

lignin

Chemicals

Biomass Stage 1 Pretreatment

Dissolved sugars, oligomers, lignin

Residual solids: cellulose,

hemicellulose,lignin

Cellulase enzyme

Stage 3Sugar

fermentation

CAFI 2 Pretreated Substrate Schedule

Pretreatment/Substrate Expected Date

Dilute Acid/Corn Stover September 2004

Dilute Acid/Poplar (Bench Scale) October 2004

Dilute Acid/Poplar (Pilot Plant) December 2004

SO2/Corn Stover March 2005

Controlled pH/Poplar May 2005

Ammonia Fiber Explosion/Poplar September 2005

Ammonia Recycled Percolation/Poplar October 2005

Flowthrough/Poplar March 2006

SO2/Poplar April 2006

Lime/Poplar April 2006


Overall Yields for Corn Stover at 15 FPU/g Glucan

Pretreatment system

Xylose yields* Glucose yields* Total sugars*

Stage 1 Stage 2 Totalxylose

Stage 1

Stage 2 Totalglucose

Stage 1 Stage 2 Combinedtotal

Maximumpossible

37.7 37.7 37.7 62.3 62.3 62.3 100.0 100.0 100.0

Dilute acid 32.1/31.2 3.2 35.3/34.4 3.9 53.2 57.1 36.0/35.1 56.4 92.4/91.5

SO2 Steamexplosion

14.7/1.0 20.0 34.7/21.0 2.5/0.8 56.7 59.2/57.5 17.2/1.8 76.7 93.9/78.5

Flowthrough 36.3/1.7 0.6/0.5 36.9/2.2 4.5/4.4 55.2 59.7/59.6 40.8/6.1 55.8/55.7 96.6/61.8

Controlled pH

21.8/0.9 9.0 30.8/9.9 3.5/0.2 52.9 56.4/53.1 25.3/1.1 61.9 87.2/63.0

AFEX 34.6/29.3 34.6/29.3 59.8 59.8 94.4/89.1 94.4/89.1

ARP 17.8/0 15.5 33.3/15.5 56.1 56.1 17.8/0 71.6 89.4/71.6

Lime 9.2/0.3 19.6 28.8/19.9 1.0/0.3 57.0 58.0/57.3 10.2/0.6 76.6 86.8/77.2

*Cumulative soluble sugars as total/monomers. Single number = just monomers.

Incr

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Effect of Pretreatment Severity on Enzymatic Hydrolysis of Dilute Acid Pretreated Poplar

0

10

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30

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0 10 20 30 40 50 60 70 80Time, hours

Glu

cose

yie

ld, %

POP-1-Severity -3.01 POP-2-Severity -3.25

POP-3-Severity -3.31 POP-4-Severity -3.55

Biomass Refining CAFI2% glucan concentration50 FPU/ gm original glucan

CBU:FPU = 2.0Digestion time =72hr

Increasing severity

For 50 FPU, Total Protein ( mg/gm original glucan)

POP1 122.2

POP2 122.0

POP3 142.0

POP4 160.3

Digestion time =72hr

Effect of Protein Loadings on Cellulose Hydrolysis of Poplar Solids

0

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• Feedstock: USDA-supplied hybrid poplar (Arlington, WI)– Debarked, chipped, and milled to pass ¼

inch round screen

– Not enough to meet needs


Component Wt %Glucan 45.1Xylan 17.8




CAFI 2 Initial Poplar

• Feedstock: USDA-supplied hybrid poplar (Arlington, WI)– Debarked, chipped, and milled to pass ¼

inch round screen

– Not enough to meet needs


Component Wt %Glucan 45.1Xylan 17.8




CAFI 2 Initial Poplar

AFEX Optimization for High/Low Lignin Poplar

0

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High Lignin Poplar

% G

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ylan

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Low Lignin Poplar

C - Cellulase(31.3 mg/g glucan)X - Xylanase(3.1 mg/g glucan)A - Additive (0.35g/g glucan)

UT - UntreatedAFEX condition24 h water soaked1:1 (Poplar:NH3)10 min. res. time

Differences Among Poplar Species*

Original Poplar - Low Lignin Poplar Standard - High Lignin

•Arlington, WI near Madison

•Very rich, loamy soil

•Demonstrated some of best growth rates

•Harvested and shipped in February 17, 2004

•Planted in 1995, probably in spring but possibly in fall

•Alexandria, Minnesota

•Lower growth rate than Arlington

•Slightly shorter growing season

•Harvested and shipped in August 2004

•Planted in spring 1994

* Based on information provided by Adam Wiese, USDA Rheinlander, WI


Fermentation of Dilute Acid Treated Corn Stover

cornstover 175C

Fermentation

30C Ethanol

H2SO4

Cells

pH 6.0pH 1.2A

Fermentation at 0 hr at 48 hr

Ethanol 0 20

Glucose 24 0

Xylose 75 50

Acetic Acid 13 13.3

Furfural 2.0 0

HMF 3.0 0

Stream g/L g/L

Inhibitor

Liquid

Ca(OH)2

SolidspH 1.2

A

consumedconsumed

B

B

S. cerevisiae 424A(LNH-ST)

80% of theoretical

Fermentation of Hot Water Treated Corn Stover

cornstover 190C 50C

Fermentation

30C Ethanol

Water Enzyme

Cells + Solids

pH 6.0pH 4.5


Ethanol 0 22

Glucose 32 0

Xylose 18 5

Acetic Acid 1.2 1.2

Furfural 0.4 0

HMF 0.1 0

Stream g/L g/L

No Xylanase

A

A

B

B

consumed

consumed

Ca(OH)2 S. cerevisiae 424A(LNH-ST)

95% of theoretical

below threshold

Fermentation of SO2 Treated Corn Stover

cornstover


Ethanol 0 29

Glucose 27 0

Xylose 37 5

Acetic Acid 4.6 4.6

Furfural 0.2 0

HMF 0.3 0

Stream g/L g/L

below threshold

A

consumedconsumed

180C 50C Fermentation

30C Ethanol

SO2 Enzyme S. cerevisiae 424A(LNH-ST)

CellspH 6.0~pH 1

ApH 4.8

Solids

No Xylanase

B

B

Ca(OH)2 Ca(OH)2

96% of theoretical

Observations• The yields can be further increased for some pretreatments

with enzymes a potential key• Mixed sugar streams will be better used in some processes

than others• Oligomers may require special considerations, depending

on process configuration and choice of fermentative organism

• Initial data on conditioning and fermentation shows mostly good yields

• All pretreatments gave similar results for corn stover• Initial hydrolysis results for poplar are not as good, with

one variety more recalcitrant than other


Planned Work

• Maximize yields with standard poplar for each pretreatment

• Evaluate differences with initial poplar at optimal conditions for standard poplar

• Develop fermentation data with hydrolyzate for each material

• Upgrade technoeconomic model with corn stover and poplar

• Identify key features that distinguish performance of different pretreatments


Acknowledgments US Department of Agriculture Initiative for

Future Agricultural and Food Systems Program, Contract 00-52104-9663

US Department of Energy Office of the Biomass Program, Contract DE-FG36-04GO14017

Natural Resources Canada All of the CAFI Team members, students,

and others who have been so cooperative


CAFI DOE Project A&I Advisory Board:Meetings Every 6 Months

Quang Nguyen, Abengoa Bioenergy

Mat Peabody, Applied CarboChemicals

Gary Welch, AventinereiGreg Luli, BC InternationalParis Tsobanakis, CargillRobert Wooley, Cargill DowJames Hettenhaus, CEASteve Thomas, CERESLyman Young, ChevronTexacoKevin Gray, DiversaPaul Roessler, DowJulie Friend, DuPontJack Huttner, Genencor

Don Johnson, GPC (Retired)Dale Monceaux, Katzen EngineersKendall Pye, LignolFarzaneh Teymouri, MBIRichard Glass, National Corn

Growers AssociationBill Cruickshank, Natural

Resources CanadaRobert Goldberg, NISTJoel Cherry, NovozymesRon Reinsfelder, Shell Andrew Richard, SunoptaCarl Miller, SyngentaCarmela Bailey, USDADon Riemenschneider, USDA

Questions??

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Documents

Maximum Total Time for Talk = 25 minutes