Enzymatic Digestion of Corn Stover and Poplar Wood after Pretreatment by Leading Technologies

Enzymatic Digestion of Corn Stover and Poplar Wood after Pretreatment

by Leading TechnologiesCharles E. Wyman, Dartmouth College/University of California

Rajeev Kumar, Dartmouth CollegeBruce 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

BIO MeetingChicago, IllinoisApril 12, 2006

Biomass Refining CAFI

• 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

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

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 %


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 Yields 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.

Incr

easi

ng p

H



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

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

easi

ng p

H


0

25

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Suga

r yi

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Oligoxylose

Monoxylose

Oligoglucose

Monoglucose

Dil

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Flo

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Monoglucose

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Oligoxylose

Monoxylose

Oligoglucose

Monoglucose

Dil

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Oligoxylose S1

Monoxylose S1

Monoxylose S2

Oligoglucose S1

Monoglucose S1

Monoglucose S2

Total Yields at 15 FPU/g Glucan

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

• 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


Tasks for the DOE OBP CAFI 2 Project


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

• Enzymatically hydrolyze cellulose and hemicellulose in pretreated biomass (corn stover and poplar), 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

• 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

Arabinan 0.6Mannan 3.9Galactan 1.0

Lignin 29.1Protein ndAcetyl 3.6Ash 1.1

Uronic Acids ndExtractives 3.6

CAFI 2 Standard Poplar

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

SO2/Poplar August 2005

Ammonia Fiber Explosion/Poplar September 2005

Ammonia Recycled Percolation/Poplar October 2005

Flowthrough/Poplar March 2006

Lime/Poplar April 2006


Effect of Enzyme Loading on Hydrolysis of SO2 Pretreated Corn Stover

SO2 pretreated corn stover at 1% glucan concentration

Protein

( mg)

FPU/gm

(SP-CP)

6.4 3.0

16.1 7.5

32.2 15

42.9 20

107.4 50

128.9 60

CBU:FPU = 2.0Digestion time =72hr

0

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0 20 40 60 80 100 120 140

Protein loading, mg/gm cellulose

Glu

cose

yie

ld, %

GC220 + beta-g


Effect of Enzyme Loading on Hydrolysis of SO2 Pretreated Corn Stover

SO2 pretreated corn stover at 1% glucan concentration

Protein

( mg)

FPU/gm

(SP-CP)

6.4 3.0

16.1 7.5

32.2 15

42.9 20

107.4 50

128.9 60


0

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Protein loading, mg/gm cellulose

Glu

cose

yie

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Spezyme CP+ beta-g

GC220 + beta-g


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

0

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

Glu

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yie

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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


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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0 20 40 60 80 100 120 140 160Protein loading (mg/gm original cellulose)

Glu

cose

yie

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Dilute Acid

ARP

Neutral pH



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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 Composition (wt %)GlucanXylan

ArabinanMannanGalactan

LigninProteinAcetylAsh

Uronic AcidsExtractives

CAFI 2 Initial Poplar

AFEX Optimization for High/Low Lignin Poplar

0

20

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100168h

72h

High Lignin Poplar

% G

lucan

Co

nvers

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ylan

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Low Lignin Poplar

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an c

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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 Poplar Standard

•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

Observations• 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

• All pretreatments gave similar results for corn stover

• Initial performance for poplar is not as good, with one source more recalcitrant than other

• Yields can be further increased for some pretreatments with enzymes a potential key


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


Questions?


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.


Documents

Enzymatic Digestion of Corn Stover and Poplar Wood after Pretreatment by Leading Technologies