Enzymatic Hydrolysis of Cellulose and
Hemicellulose in Solids Prepared by Leading
Pretreatment Technologies
Charles E. Wyman, Dartmouth College
Y. Y. Lee, Auburn University
Mohammed Moniruzzaman, Genencor International
Bruce E. Dale, Michigan State University
Tim Eggeman, Neoterics International
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
Bioprocessing of Agricultural Feedstocks:
Report on Pretreatment for Biomass Refining
2nd World Congress on Industrial Biotechnology and Bioprocessing
Orlando, Florida
Biomass Refining CAFI
April 20, 2005
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
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
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 Yield Comparisons at
60 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
3.3
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
ND/30.2
ND/30.2
61.8
61.8
ND/92.0
ND/92.0
59.4
59.4
17.8/0
76.4
94.2/76.4
59.5
60.5/59.8
10.2/0.6
79.7
89.9/80.3
AFEX
ARP
17.8/0
17.0
34.8/17.0
Lime
9.2/0.3
20.2
29.4/20.5
1.0/0.3
*Cumulative soluble sugars as total/monomers. Single number = just monomers.
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 Yield Comparisons 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 Yield Comparisons 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 Yield Comparisons 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 Yield Comparisons 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 Yield Comparisons 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 Yield Comparisons 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 Yield Comparisons at
15 FPU/g Glucan
100
75
Oligoxylose
Monoxylose
Oligoglucose
Monoglucose
Pretreatment Yield Comparisons 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
m
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
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
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
Biomass Refining CAFI
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
Biomass Refining CAFI
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
Biomass Refining CAFI
Enzymatic Hydrolysis Plan
• 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
Biomass Refining CAFI
Enzymatic Hydrolysis of Cellulose from
Pretreated Poplar Wood
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
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
Expected Date
September 2004
October 2004
December 2004
March 2005
May 2005
August 2005
September 2005
October 2005
Flowthrough/Poplar
March 2006
Lime/Poplar
April 2006
Biomass Refining CAFI
Kinetic Models*
•
Non-mechanistic (NM): 2
– Based on data correlation without an explicit calculation of
adsorbed enzyme concentration.
•
Semi-mechanistic (SM): 8
– Based on single enzyme activity and single substrate feature
(concentration).
•
Functionally-based (FB): 3
– Featuring an adsorption model, multiple enzyme activities, and
substrate variables.
•
Structurally-based (SB): 0
– Structural features of cellulase and interaction between
substrate and enzyme.
Biomass Refining CAFI
*Zhang and Lynd ( in press)
Predictions of Effect of Lignin by
Selected Models
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
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
Questions?
Stop
Pretreatment Yield Comparisons 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.