enzymatic digestion of corn stover and poplar wood after pretreatment by leading technologies...
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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
Biomass Refining CAFI
CAFI Approach
Hydrolysis Stages
Biomass Refining CAFI
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 %
Biomass Refining CAFI
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%.
Biomass Refining CAFI
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
Biomass Refining CAFI
Overall Yields 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
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
Biomass Refining CAFI
0
25
50
75
100
Suga
r yi
elds
, % o
f max
tota
l -
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
e
Overall Yields at 15 FPU/g Glucan
0
25
50
75
100
Suga
r yi
elds
, % o
f max
tota
l -
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
e
Overall Yields at 15 FPU/g Glucan
0
25
50
75
100
Suga
r yi
elds
, % o
f max
tota
l -
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
e
Overall Yields at 15 FPU/g Glucan
0
25
50
75
100
Suga
r yi
elds
, % o
f max
tota
l -
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
e
Overall Yields at 15 FPU/g Glucan
0
25
50
75
100
Suga
r yi
elds
, % o
f max
tota
l -
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
Biomass Refining CAFI
Tasks for the DOE OBP CAFI 2 Project
Biomass Refining CAFI
• 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
Biomass Refining CAFI
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
Biomass Refining CAFI
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
10
20
30
40
50
60
70
80
90
100
0 20 40 60 80 100 120 140
Protein loading, mg/gm cellulose
Glu
cose
yie
ld, %
GC220 + beta-g
Biomass Refining CAFI
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
10
20
30
40
50
60
70
80
90
100
0 20 40 60 80 100 120 140
Protein loading, mg/gm cellulose
Glu
cose
yie
ld, %
Spezyme CP+ beta-g
GC220 + beta-g
Biomass Refining CAFI
Effect of Pretreatment Severity on Enzymatic Hydrolysis of Dilute Acid Pretreated Poplar
0
10
20
30
40
50
60
70
80
90
100
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
20
40
60
80
100
0 20 40 60 80 100 120 140 160Protein loading (mg/gm original cellulose)
Glu
cose
yie
ld, %
Dilute Acid
ARP
Neutral pH
Digestion time =72hr
Effect of Protein Loadings on Cellulose Hydrolysis of Poplar Solids
0
20
40
60
80
100
0 20 40 60 80 100 120 140 160Protein loading (mg/gm original cellulose)
Glu
cose
yie
ld, %
Dilute Acid
ARP
Neutral pH
Digestion time =72hr
Effect of Protein Loadings on Cellulose Hydrolysis of Poplar Solids
0
20
40
60
80
100
0 20 40 60 80 100 120 140 160Protein loading (mg/gm original cellulose)
Glu
cose
yie
ld, %
Dilute Acid
ARP
Neutral pH
• Feedstock: USDA-supplied hybrid poplar (Arlington, WI)– Debarked, chipped, and milled to pass ¼
inch round screen
– Not enough to meet needs
Biomass Refining CAFI
Component Composition (wt %)GlucanXylan
ArabinanMannanGalactan
LigninProteinAcetylAsh
Uronic AcidsExtractives
CAFI 2 Initial Poplar
AFEX Optimization for High/Low Lignin Poplar
0
20
40
60
80
100168h
72h
High Lignin Poplar
% G
lucan
Co
nvers
ion
0
20
40
60
80
100168h
72h
% X
ylan
Con
vers
ion High Lignin Poplar
0
20
40
60
80
100168h
72h
%
Glu
can
co
nvers
ion
Low Lignin Poplar
0
20
40
60
80
100168h
72h
%
Xyl
an c
on
vers
ion
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
Biomass Refining CAFI
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
Biomass Refining CAFI
Questions?
Biomass Refining CAFI
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.
Biomass Refining CAFI
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