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 College
Y. Y. Lee, Auburn University
Mohammed Moniruzzaman, Genencor International
Bruce E. Dale, Michigan State University
Richard T. Elander, National Renewable Energy Laboratory
Michael R. Ladisch, Purdue University
Mark T. Holtzapple, Texas A&M University
John N. Saddler, University of British Columbia
Biomass Refining CAFI
Presentation Outline
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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
technologies
Biomass Refining CAFI
Hydrolysis Stages
Cellulase enzyme
Biomass
Chemicals
Stage 1
Pretreatment
Solids: cellulose,
hemicellulose,
lignin
Dissolved sugars,
oligomers, lignin
Biomass Refining CAFI
Stage 2
Enzymatic
hydrolysis
Dissolved sugars,
oligomers
Residual solids:
cellulose,
hemicellulose,
lignin
Mass Balance Approach: AFEX Example
Enzyme (15 FPU/g of Glucan)
Ammonia
Stover
Hydrolyzate
Liquid
99.0 lb
AFEX Treated
Hydrolysis
Wash
38.5 lb glucose
System Stover
100 lb
(dry basis)
36.1 lb glucan
21.4 lb xylan
Residual 18.9 lb xylose
Solids
101.0 lb
Solids washed out
2 lb
(Ave. of 4 runs)
39.2 lb
Very few solubles from pretreatment—about 2% of inlet stover
95.9% glucan conversion to glucose, 77.6% xylan conversion to xylose
99% mass balance closure includes:
(solids + glucose + xylose + arabinose )
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
Increasing pH
Pretreatment
system
Xylose yields*
Glucose yields*
Total sugars*
Stage 1
Stage 2
Total
xylose
Stage
1
Stage 2
Total
glucose
Stage 1
Stage 2
Combined
total
Maximum
possible
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
34.6/29.3
34.6/29.3
59.8
59.8
94.4/89.1
94.4/89.1
56.1
56.1
17.8/0
71.6
89.4/71.6
57.0
58.0/57.3
10.2/0.6
76.6
86.8/77.2
AFEX
ARP
17.8/0
15.5
33.3/15.5
Lime
9.2/0.3
19.6
28.8/19.9
1.0/0.3
*Cumulative soluble sugars as total/monomers. Single number = just monomers.
Biomass Refining CAFI
St
ag
e
St 1
ag
e2
St
ag
e
St 1
ag
e2
St
ag
e
St 1
ag
e2
0
Maximum possible
Lime
ARP
AFEX
Controlled pH
Flowthrough
50
St
ag
e
St 1
ag
e2
Dilute acid
25
St
ag
e
St 1
ag
e2
St
ag
e
St 1
ag
e2
Sugar yields, % of max total -
Pretreatment Yields at 15 FPU/g Glucan
100
75
Oligoxylose
Monoxylose
Oligoglucose
Monoglucose
St
ag
e
St 1
ag
e2
St
ag
e
St 1
ag
e2
St
ag
e
St 1
ag
e2
0
Maximum possible
Lime
ARP
AFEX
Controlled pH
Flowthrough
50
St
ag
e
St 1
ag
e2
Dilute acid
25
St
ag
e
St 1
ag
e2
St
ag
e
St 1
ag
e2
Sugar yields, % of max total -
Pretreatment Yields at 15 FPU/g Glucan
100
75
Oligoxylose
Monoxylose
Oligoglucose
Monoglucose
St
ag
e
St 1
ag
e2
St
ag
e
St 1
ag
e2
St
ag
e
St 1
ag
e2
0
Maximum possible
Lime
ARP
AFEX
Controlled pH
Flowthrough
50
St
ag
e
St 1
ag
e2
Dilute acid
25
St
ag
e
St 1
ag
e2
St
ag
e
St 1
ag
e2
Sugar yields, % of max total -
Pretreatment Yields at 15 FPU/g Glucan
100
75
Oligoxylose
Monoxylose
Oligoglucose
Monoglucose
St
ag
e
St 1
ag
e2
St
ag
e
St 1
ag
e2
St
ag
e
St 1
ag
e2
0
Maximum possible
Lime
ARP
AFEX
Controlled pH
Flowthrough
50
St
ag
e
St 1
ag
e2
Dilute acid
25
St
ag
e
St 1
ag
e2
St
ag
e
St 1
ag
e2
Sugar yields, % of max total -
Pretreatment Yields at 15 FPU/g Glucan
100
75
Oligoxylose
Monoxylose
Oligoglucose
Monoglucose
St
ag
e
St 1
ag
e2
St
ag
e
St 1
ag
e2
St
ag
e
St 1
ag
e2
0
Maximum possible
Lime
ARP
AFEX
Controlled pH
Flowthrough
50
St
ag
e
St 1
ag
e2
Dilute acid
25
St
ag
e
St 1
ag
e2
St
ag
e
St 1
ag
e2
Sugar yields, % of max total -
Pretreatment Yields at 15 FPU/g Glucan
100
75
Oligoxylose
Monoxylose
Oligoglucose
Monoglucose
St
ag
e
St 1
ag
e2
St
ag
e
St 1
ag
e2
St
ag
e
St 1
ag
e2
0
Maximum possible
Lime
ARP
AFEX
Controlled pH
Flowthrough
50
St
ag
e
St 1
ag
e2
Dilute acid
25
St
ag
e
St 1
ag
e2
St
ag
e
St 1
ag
e2
Sugar yields, % of max total -
Pretreatment Yields at 15 FPU/g Glucan
100
75
Oligoxylose
Monoxylose
Oligoglucose
Monoglucose
St
ag
e
St 1
ag
e2
St
ag
e
St 1
ag
e2
St
ag
e
St 1
ag
e2
0
Maximum possible
Lime
ARP
AFEX
Controlled pH
Flowthrough
50
St
ag
e
St 1
ag
e2
Dilute acid
25
St
ag
e
St 1
ag
e2
St
ag
e
St 1
ag
e2
Sugar yields, % of max total -
Pretreatment Yields at 15 FPU/g Glucan
100
75
Oligoxylose
Monoxylose
Oligoglucose
Monoglucose
Pretreatment Yields at 15 FPU/g Glucan
75
Oligoxylose S1
Monoxylose S1
Monoxylose S2
Oligoglucose S1
50
Monoglucose S1
Monoglucose S2
25
le
os
s ib
M
ax
p
Li
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e
AR
P
AF
EX
nt
ro
lle
d
pH
h
Co
th
ro
ug
Fl
ow
ea
cid
0
Di
lu
t
Sugar yields, % of max total -
100
General PFD for Cost Estimates
Enzymes
Stover
Feed
Handling
Different
Pretreatments
CO2
Hydrolysis
+
Fermentation
Water
Recovery
EtOH
Syrup +
Solids
Chemicals
Different
Pretreatments
Biomass Refining CAFI
Water
Boiler
+
Generator
Steam
Power
Minimum Ethanol Selling Price (MESP)
$/gal EtOH
Proof Year: 4th Year of Operation
1.75
1.50
1.25
1.00
0.75
MESP
0.50
Cash
Cost
0.25
Plant
Level
0.00
Dilute Acid
Net Stover
Hot Water
Other Variable
Biomass Refining CAFI
AFEX
Fixed w/o Depreciation
ARP
Depreciation
Lime
Income Tax
Ideal
Return on Capital
Effect of Oligomer Conversion on MESP
MESP, $/gal EtOH
1.75
1.50
1.25
1.00
Dilute Acid
Hot Water
w/o Oligomer Credit
Biomass Refining CAFI
AFEX
ARP
w/ Oligomer Credit
Lime
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
Serve as extension agents for technology transfer
Provide feedback on approach and results
Meet with team every 6 months
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
Quang Nguyen, Abengoa
Bioenergy
Mat Peabody, formerly
Applied CarboChemicals
Gary Welch, Aventinerei
Greg Luli, BC International
Paris Tsobanakis, Cargill
Robert Wooley, Cargill Dow
James Hettenhaus, CEA
Lyman Young,
ChevronTexaco
Kevin Gray, Diversa
Paul Roessler, Dow
Susan M. Hennessey,
DuPont
Michael Knauf, Genencor
13. Don Johnson, GPC (Retired)
14. Dale Monceaux, Katzen
Engineers
15. Kendall Pye, Lignol
16. Farzaneh Teymouri, MBI
17. Richard Glass, National
Corn Growers Association
18. Bill Cruickshank, Natural
Resources Canada
19. Joel Cherry, Novozymes
20. Ron Reinsfelder, Shell
21. Carl Miller, Syngenta
22. Carmela Bailey, USDA
23. Don Riemenschneider,
USDA
Tasks for the DOE OBP Project
• 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
Biomass Refining CAFI
CAFI 2 Stover
• 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
Component
Composition (wt %)
Sucrose
2.2
34.4
22.8
4.2
0.6
1.4
11.0
2.3
5.6
6.1
3.8
8.5
Glucan
Xylan
Arabinan
Mannan
Galactan
Lignin
Protein
Acetyl
Ash
Uronic Acids
Extractives
Biomass Refining CAFI
CAFI 2 Poplar
• Feedstock: USDA-supplied hybrid
poplar (Alexandria, MN)
– Debarked, chipped, and milled to pass
¼ inch round screen
Component
Glucan
Xylan
Arabinan
Mannan
Galactan
Lignin
Protein
Acetyl
Ash
Uronic Acids
Extractives
Biomass Refining CAFI
Composition (wt %)
43.8
14.9
0.6
3.9
1.0
29.1
nd
3.6
1.1
nd
3.6
Pretreated Substrate Schedule
Pretreatment/Substrate
Dilute Acid/Corn Stover
Dilute Acid/Poplar (Bench Scale)
Dilute Acid/Poplar (Pilot Plant)
SO2/Corn Stover
Controlled pH/Poplar
SO2/Poplar
Ammonia Fiber Explosion/Poplar
Ammonia Recycled Percolation/Poplar
Flowthrough/Poplar
Lime/Poplar
Biomass Refining CAFI
Expected Date
September 2004
October 2004
December 2004
March 2005
May 2005
August 2005
September 2005
October 2005
March 2006
April 2006
Pretreated Substrate Schedule
Pretreatment/Substrate
Dilute Acid/Corn Stover
Dilute Acid/Poplar (Bench Scale)
Dilute Acid/Poplar (Pilot Plant)
SO2/Corn Stover
Controlled pH/Poplar
SO2/Poplar
Ammonia Fiber Explosion/Poplar
Ammonia Recycled Percolation/Poplar
Flowthrough/Poplar
Lime/Poplar
Biomass Refining CAFI
Expected Date
September 2004
October 2004
December 2004
March 2005
May 2005
August 2005
September 2005
October 2005
March 2006
April 2006
SO2 Pretreatment of Corn Stover
Pretreatment
Hydrolysis
15 FPU
Hydrolysis
60 FPU
Log (Ro)
3.02
Glucose (%)
99
Xylose (%)
83
3.35
4.03
100
88
64
45
3.02
58
62
3.35
4.03
88
88
60
45
3.02
3.35
73
95
70
64
4.03
88
45
AFEX Pretreated Poplar
AFEX pretreated samples
1 g dry biomass : 0.8 g NH3
20.0%
18.0%
Percent conversion
16.0%
14.0%
24 hour Glucan
12.0%
72 hour Glucan
10.0%
24 hour Xylan
8.0%
72 hour Xylan
6.0%
4.0%
2.0%
0.0%
80
90
100
Temperature (°C)
110
120
Enzymatic Hydrolysis of
Dilute Acid Pretreated Poplar
100
90
Glucose yield, %
80
70
60
50
40
30
20
POP-1-Severity -3.01
POP-2-Severity -3.25
POP-3-Severity -3.31
POP-4-Severity -3.55
10
0
0
10
Biomass Refining CAFI
20
30
40
Time, hours
50
60
70
80
2% glucan concentration
50 FPU/g glucan, no β-glucosidase supplementation
Model Predictions of Effect of Lignin
100 g substrate/L, 50% cellulose, 10 FPU cellulase/g cellulose, 2 CBU/FPU
100
Cellulose conversion, %
90
NM, 5 FPU/gm
80
70
Phillipidis et al.
60
50
South et al.
40
30
Holtzapple et al.
20
0
10
Biomass Refining CAFI
20
30
40
Lignin concentration ( g/l)
50
60
Xylanase Supplementation of SO2 Treated Stover
0.9% (w/v) consistency, corn stover-190oC, 5min, 3% S02,
0.0417g Spezyme SP, 0.0073g cocktail BG-X-001
0.03g of protein/g of cellulose
0.06g of protein/g of cellulose
80
80
70
70
70
60
12%
50
40
30
20
cellulase
cellulase+xylanase
cellulase+BSA
xylanase
10
0
0
5
10
15
Time (hours)
20
25
21%
60
50
40
30
20
cellulase
cellulase+xylanase
cellulase+BSA
xylanase
10
0
0
5
10
15
20
25
Xylan conversion (%)
80
Xylan conversion (%)
Xylan conversion (%)
0.006g of protein/g of cellulose
31%
60
50
40
30
20
cellulase
cellulase+xylanase
cellulase+BSA
xylanase
10
0
0
5
Time (hours)
Method: High Throughput Microassay
10
15
Time (hours)
20
25
Dilute Acid Pretreated Corn Stover Hydrolyzate
Fermentation (resin conditioned)
80
30
70
25
20
50
40
15
30
10
20
5
10
0
0
24
Biomass Refining CAFI
48
72
96
Fermentation Time (hr)
120
144
0
168
Ethanol (g/L) .
Xylose (g/L) .
60
Initial Fermentation Results
after 144 hours
Control
Xylose
Consumed ( %)
Ethanol Yield
(% theoretical for
glucose + xylose
consumed)
Biomass Refining CAFI
Overlime XAD4 Overlime
+ XAD4
54.1
42.4
44.5
41.3
76.8
63.4
79.0
72.0
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-FG3604GO14017
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