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Integration of Leading Biomass Pretreatment Technologies with Enzymatic Digestion and
Hydrolyzate Fermentation DOE OBP Pretreatment Core R&D Gate Review Meeting
June 9-10, 2005
Charles E. Wyman, Dartmouth CollegeY. Y. Lee, Auburn University
Mohammed Moniruzzaman, Genencor InternationalBruce E. Dale, Michigan State University
Richard T. Elander, National Renewable Energy LaboratoryMichael R. Ladisch, Purdue University
Mark T. Holtzapple, Texas A&M UniversityJohn N. Saddler, University of British Columbia
Biomass Refining CAFI
Presentation Outline
• Project background• Technical feasibility and risks• Biomass Refining CAFI• Competitive advantage• History and accomplishments• Project overview• Plan/Schedule and recent results• Critical issues and show stoppers• Summary and caveats• Plans and resources for next stage
Biomass Refining CAFI
Project Background: Pretreatment Needs
• High cellulose accessibility to enzymes
• High sugar yields from hemicellulose• Low capital cost – low pressure,
inexpensive materials of construction• Low energy cost• Low degradation• Low cost and/or recoverable
chemicals
Biomass Refining CAFI
Technical Feasibility and Risks
• Dilute acid pretreatment is often favored based on more extensive development
• Many other options have been studied, but only a few are promising
• Pretreatment is most expensive single operation• Difficult to compare leading pretreatments based on data
available• Limited knowledge of pretreatment mechanisms slows
commercial use of all options
Biomass Refining CAFI
Project Background: CAFI
• Biomass Refining Consortium for Applied Fundamentals and Innovation organized in late 1999
• Included top researchers in biomass hydrolysis from Auburn, Dartmouth, Michigan State, Purdue, NREL, Texas A&M, UBC, 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
Competitive Advantage
• 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
technologiesBiomass Refining CAFI
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
Mass Balance Approach: AFEX Example
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
CAFI USDA IFAFS Project Overview
• 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
Biomass Refining CAFI
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
Pretreatment 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
Pretreatment 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
Pretreatment 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
Pretreatment 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
Pretreatment 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
Pretreatment 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
Pretreatment 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
Pretreatment 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
Pretreatment Yields at 15 FPU/g Glucan
General PFD for Cost Estimates
Biomass Refining CAFI
Boiler +
Generator
Different Pretreatments
Hydrolysis +
Fermentation
Feed Handling Recovery
DifferentPretreatments
Stover
Syrup + Solids
Chemicals Water
Enzymes CO2 Water
EtOH
Steam
Power
Minimum Ethanol Selling Price (MESP)
Biomass Refining CAFI
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
Effect of Oligomer Conversion on MESP
Biomass Refining CAFI
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
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
Biomass Refining CAFI
CAFI Project Advisory Board
1. Quang Nguyen, Abengoa Bioenergy2. Mat Peabody, formerly Applied
CarboChemicals3. Gary Welch, Aventinerei4. Greg Luli, BC International5. Paris Tsobanakis, Cargill6. Robert Wooley, Cargill Dow7. James Hettenhaus, CEA8. Lyman Young, ChevronTexaco9. Kevin Gray, Diversa10. Paul Roessler, Dow11. Susan M. Hennessey, DuPont12. Michael Knauf, Genencor
13. Don Johnson, GPC (Retired)14. Dale Monceaux, Katzen
Engineers15. Kendall Pye, Lignol16. Farzaneh Teymouri, MBI17. Richard Glass, National
Corn Growers Association18. Bill Cruickshank, Natural
Resources Canada19. Joel Cherry, Novozymes20. Ron Reinsfelder, Shell 21. Carl Miller, Syngenta22. Carmela Bailey, USDA23. Don Riemenschneider,
USDA
Serve as extension agents for technology transferProvide feedback on approach and results
Meet with team every 6 months
Tasks for the DOE OBP Project
Biomass Refining CAFI
• Pretreat corn stover and poplar by leading technologies to improve cellulose accessibility to enzymes
• Develop conditioning methods as needed to maximize fermentation yields by a recombinant yeast, determine the cause of inhibition, and model fermentations
• 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
• Estimate capital and operating costs for each integrated pretreatment, hydrolysis, and fermentation system and use to guide research
CAFI 2 Stover
Biomass Refining CAFI
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
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 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
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
Log (Ro) Glucose (%) Xylose (%)
3.02 99 83
3.35 100 64
4.03 88 45
3.02 58 62
3.35 88 60
4.03 88 45
3.02 73 70
3.35 95 64
4.03 88 45
Pretreatment
Hydrolysis15 FPU
Hydrolysis60 FPU
SO2 Pretreatment of Corn Stover
AFEX Pretreated Poplar
AFEX pretreated samples
1 g dry biomass : 0.8 g NH3
0.0%
2.0%
4.0%
6.0%
8.0%
10.0%
12.0%
14.0%
16.0%
18.0%
20.0%
80 90 100 110 120
Temperature (°C)
Pe
rce
nt
co
nv
ers
ion
24 hour Glucan
72 hour Glucan
24 hour Xylan
72 hour Xylan
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 CAFI
Enzymatic Hydrolysis of Dilute Acid Pretreated Poplar
2% glucan concentration50 FPU/g glucan, no β-glucosidase supplementation
Model Predictions of Effect of Lignin
20
30
40
50
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
Biomass Refining CAFI
0.9% (w/v) consistency, corn stover-190oC, 5min, 3% S02, 0.0417g Spezyme SP, 0.0073g cocktail BG-X-001
0 5 10 15 20 250
10
20
30
40
50
60
70
80
cellulase cellulase+xylanase cellulase+BSA xylanase
Xyl
an c
onve
rsio
n (%
)
Time (hours)
0 5 10 15 20 250
10
20
30
40
50
60
70
80
Xyl
an c
onve
rsio
n (%
)
Time (hours)
cellulase cellulase+xylanase cellulase+BSA xylanase
0 5 10 15 20 250
10
20
30
40
50
60
70
80
cellulase cellulase+xylanase cellulase+BSA xylanase
Xyl
an c
onve
rsio
n (%
)
Time (hours)
0.006g of protein/g of cellulose 0.03g of protein/g of cellulose 0.06g of protein/g of cellulose
12%
21% 31%
Method: High Throughput Microassay
Xylanase Supplementation of SO2 Treated Stover
Dilute Acid Pretreated Corn Stover Hydrolyzate Fermentation (resin conditioned)
0
10
20
30
40
50
60
70
80
0 24 48 72 96 120 144 168Fermentation Time (hr)
Xyl
ose
(g/L
) .
0
5
10
15
20
25
30
Eth
anol
(g/
L)
.
Biomass Refining CAFI
Initial Fermentation Results after 144 hours
Control Overlime XAD4 Overlime + XAD4
Xylose Consumed ( %) 54.1 42.4 44.5 41.3
Ethanol Yield (% theoretical for
glucose + xylose consumed)
76.8
63.4 79.0 72.0
Biomass Refining CAFI
CAFI Presentations/Publications
• Team presentations at – 2004 Annual Meeting of the American Institute of Chemical
Engineers, Austin, Texas, November 11 – 2003 Annual Meeting of the American Institute of Chemical
Engineers, San Francisco, California, November 20 – 25th Symposium on Biotechnology for Fuels and Chemicals,
Breckenridge, Colorado, May 7, 2003 – 2002 Annual Meeting of the American Institute of Chemical
Engineers, Indianapolis, Indiana, November 4 – 24th Symposium on Biotechnology for Fuels and Chemicals,
Gatlinburg, Tennessee, April 28, 2002 • Mosier N, Wyman CE, Dale B, Elander R, Lee YY, Holtzapple M,
Ladisc1 M. 2005. “Features of Promising Technologies for Pretreatment of Lignocellulosic Biomass,” BioResource Technology 96(6): 673-686
• Special issue of Bioresource Technology in progress to report USDA IFAFS findings in several papers including joint papers to introduce project and summarize results
Biomass Refining CAFI
Critical Issues and Show Stoppers
• Must assure that all pretreatments realize near maximize possible yields
• Include both pretreatment and subsequent enzymatic hydrolysis• Evaluate effect of enzymes on yields of both xylose and glucose• Characterize well hydrolyzate fermentability and conditioning
demands• Biggest concern is unknown challenges that prove too time
consuming to resolve
Biomass Refining CAFI
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
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 as initiallly shown for poplar
Biomass Refining CAFI
Plans and Resources for Next Stage
• The results from this project will provide a basis for industry to select technologies to commercialize
• Results should also suggest new enzyme and organism strategies
• Further research will be important to better account for performance differences
• Consideration should be given to taking advantage of differences among pretreatment options
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 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
Biomass Refining CAFI
Insanity is doing what you always have always been doing and
expecting different results
Biomass Refining CAFI
Questions?
Biomass Refining CAFI