enzymatic hydrolysis of cellulose and hemicellulose in solids prepared by leading pretreatment...
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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
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
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
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
Pretreatment Yield Comparisons 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 Yield Comparisons 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 Yield Comparisons 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 Yield Comparisons 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 Yield Comparisons 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 Yield Comparisons 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 Yield Comparisons 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 Yield Comparisons 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
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
Biomass Refining CAFI
• 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
Biomass Refining CAFI
• 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
Biomass Refining CAFI
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 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
Biomass Refining CAFI
• 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).
Biomass Refining CAFI
Predictions of Effect of Lignin by Selected Models
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
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
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