Enzymatic Hydrolysis of Cellulose and Hemicellulose in Solids Prepared by Leading Pretreatment Technologies Charles E. Wyman, Dartmouth College Y. Y. Lee,

Enzymatic Hydrolysis of Cellulose and Hemicellulose in Solids Prepared by Leading

Pretreatment TechnologiesCharles E. Wyman, Dartmouth College

Y. Y. Lee, Auburn UniversityMohammed Moniruzzaman, Genencor International

Bruce E. Dale, Michigan State UniversityTim Eggeman, Neoterics International

Richard T. Elander, National Renewable Energy LaboratoryMichael R. Ladisch, Purdue University

Mark T. Holtzapple, Texas A&M UniversityJohn N. Saddler, University of British Columbia

Bioprocessing of Agricultural Feedstocks: Report on Pretreatment for Biomass Refining

2nd World Congress on Industrial Biotechnology and Bioprocessing Orlando, FloridaApril 20, 2005Biomass Refining CAFI

USDA IFAFS Project Tasks

• Apply leading pretreatment technologies to prepare biomass for conversion to products

• Characterize resulting fluid and solid streams• Close material and energy balances for each

pretreatment process • Determine cellulose digestibility and liquid

fraction fermentability• Compare performance of pretreatment

technologies on corn stover

Biomass Refining CAFI

Pretreatment and Enzymatic 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

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%.


Pretreatment Yield Comparisons 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

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0

25

50

75

100

Suga

r yi

elds

, % o

f max

tota

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Oligoxylose

Monoxylose

Oligoglucose

Monoglucose

Dil

ute

aci

d

Flo

wth

rou

gh

Con

trol

led

pH

Max

imu

m p

ossi

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AR

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AF

EX

Lim

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Suga

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Oligoxylose

Monoxylose

Oligoglucose

Monoglucose

Dil

ute

aci

d

Flo

wth

rou

gh

Con

trol

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pH

Max

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AR

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EX

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Suga

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, % o

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Oligoxylose

Monoxylose

Oligoglucose

Monoglucose

Dil

ute

aci

d

Flo

wth

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gh

Con

trol

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pH

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ble

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P

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EX

Lim

e


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25

50

75

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Suga

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, % o

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Oligoxylose

Monoxylose

Oligoglucose

Monoglucose

Dil

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Flo

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Con

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pH

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Suga

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Oligoxylose

Monoxylose

Oligoglucose

Monoglucose

Dil

ute

aci

d

Flo

wth

rou

gh

Con

trol

led

pH

Max

imu

m p

ossi

ble

AR

P

AF

EX

Lim

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0

25

50

75

100

Suga

r yi

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, % o

f max

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Oligoxylose

Monoxylose

Oligoglucose

Monoglucose

Dil

ute

aci

d

Flo

wth

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Con

trol

led

pH

Max

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m p

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ble

AR

P

AF

EX

Lim

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0

25

50

75

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Suga

r yi

elds

, % o

f max

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Oligoxylose

Monoxylose

Oligoglucose

Monoglucose

Dil

ute

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d

Flo

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gh

Con

trol

led

pH

Max

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m p

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ble

AR

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EX

Lim

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25

50

75

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Suga

r yi

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, % o

f max

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Oligoxylose S1

Monoxylose S1

Monoxylose S2

Oligoglucose S1

Monoglucose S1

Monoglucose S2

Observations from IFAFS Project 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


Caveats• 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

• The conditioning and fermentability of the sugar streams must be assessed

• These results are only for corn stover, and performance with other feedstocks will likely be different


Tasks for the DOE OBP Project


• Corn stover and poplar pretreated by leading technologies to improve cellulose accessibility to enzymes

• Conditioning methods developed as needed to maximize fermentation yields by a recombinant yeast, the cause of inhibition determined, and fermentations modeled

• Cellulose and hemicellulose in pretreated biomass enzymatically hydrolyzed, as appropriate, and models developed to understand the relationship between pretreated biomass features, advanced enzyme characteristics, and enzymatic digestion results

• Capital and operating costs estimated for each integrated pretreatment, hydrolysis, and fermentation system and used to direct research

Tasks for the DOE OBP Project


• Corn stover and poplar pretreated by leading technologies to improve cellulose accessibility to enzymes

• Conditioning methods developed as needed to maximize fermentation yields by a recombinant yeast, the cause of inhibition determined, and fermentations modeled

• Cellulose and hemicellulose in pretreated biomass enzymatically hydrolyzed, as appropriate, and models developed to understand the relationship between pretreated biomass features, advanced enzyme characteristics, and enzymatic digestion results

• Capital and operating costs estimated for each integrated pretreatment, hydrolysis, and fermentation system and used to direct research

• Measure enzymatic hydrolysis of cellulose and hemicellulose as a function of cellulase and xylanase loadings and beta glucosidase and beta xylosidase supplementation

• Apply fractional factorial experimental design to determine key trends and interactions

• Characterize enzyme and substrate features for each feedstock and pretreatment

• Develop kinetic models to better understand key factors impacting performance

• Define routes to improve cellulose and hemicellulose conversion with less enzyme

Enzymatic Hydrolysis Plan


0

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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


Enzymatic Hydrolysis of Cellulose from Pretreated Poplar Wood

2% glucan concentration50 FPU/g glucan, no β-glucosidase supplementation

Pretreated Substrate SchedulePretreatment/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


• Non-mechanistic (NM): 2– Based on data correlation without an explicit calculation of

adsorbed enzyme concentration.

Kinetic Models*

• Functionally-based (FB): 3– Featuring an adsorption model, multiple enzyme activities, and

substrate variables.

*Zhang and Lynd ( in press)

• Structurally-based (SB): 0– Structural features of cellulase and interaction between

substrate and enzyme.

• Semi-mechanistic (SM): 8– Based on single enzyme activity and single substrate feature

(concentration).


Predictions of Effect of Lignin by Selected Models

20

30

40

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60

70

80

90

100

0 10 20 30 40 50 60

Lignin concentration ( g/l)

Cel

lulo

se c

onve

rsio

n, %

South et al.

Phillipidis et al.

Holtzapple et al.

100 g substrate/L, 50% cellulose, 10 FPU cellulase/g cellulose, 2 CBU/FPU

NM, 5 FPU/gm


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 Our team from Dartmouth College; Auburn,

Michigan State, Purdue, and Texas A&M Universities; the University of British Columbia; Genencor International; and the National Renewable Energy Laboratory


Questions?

Stop


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

Enzymatic Hydrolysis of Cellulose and Hemicellulose in Solids Prepared by Leading Pretreatment Technologies Charles E. Wyman, Dartmouth College Y. Y. Lee,

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