Enzymatic Hydrolysis of Cellulose and Hemicellulose in Solids Prepared by Leading Pretreatment Technologies

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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 AM 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
• April 20, 2005

Biomass Refining CAFI
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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
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Pretreatment and Enzymatic Hydrolysis Stages
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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.

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Pretreatment Yield Comparisons at 60 FPU/g Glucan
Pretreatment system Xylose yields Xylose yields Xylose yields Glucose yields Glucose yields Glucose yields Total sugars Total sugars Total sugars
Pretreatment system 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
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.
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Pretreatment Yield Comparisons at 15 FPU/g Glucan
Maximum possible
Dilute acid
Controlled pH
Flowthrough
Lime
AFEX
ARP
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Pretreatment Yield Comparisons at 15 FPU/g Glucan
Maximum possible
Dilute acid
Controlled pH
Flowthrough
Lime
AFEX
ARP
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Pretreatment Yield Comparisons at 15 FPU/g Glucan
Maximum possible
Dilute acid
Controlled pH
Flowthrough
Lime
AFEX
ARP
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Pretreatment Yield Comparisons at 15 FPU/g Glucan
Maximum possible
Dilute acid
Controlled pH
Flowthrough
Lime
AFEX
ARP
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Pretreatment Yield Comparisons at 15 FPU/g Glucan
Maximum possible
Dilute acid
Controlled pH
Flowthrough
Lime
AFEX
ARP
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Pretreatment Yield Comparisons at 15 FPU/g Glucan
Maximum possible
Dilute acid
Controlled pH
Flowthrough
Lime
AFEX
ARP
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Pretreatment Yield Comparisons at 15 FPU/g Glucan
Maximum possible
Dilute acid
Controlled pH
Flowthrough
Lime
AFEX
ARP
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Pretreatment Yield Comparisons at 15 FPU/g Glucan
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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

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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

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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

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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

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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

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Enzymatic Hydrolysis of Cellulose from Pretreated
Poplar Wood
2 glucan concentration 50 FPU/g glucan, no
ß-glucosidase supplementation
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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
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Kinetic Models

• Non-mechanistic (NM) 2
• Based on data correlation without an explicit
  calculation of adsorbed enzyme concentration.

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Zhang and Lynd ( in press)
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Predictions of Effect of Lignin by Selected Models
100 g substrate/L, 50 cellulose, 10 FPU
cellulase/g cellulose, 2 CBU/FPU
Phillipidis et al.
South et al.
Holtzapple et al.
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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 AM Universities the
  University of British Columbia Genencor
  International and the National Renewable Energy
  Laboratory

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Questions?
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Stop
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Pretreatment Yield Comparisons at 15 FPU/g Glucan
Pretreatment system Xylose yields Xylose yields Xylose yields Glucose yields Glucose yields Glucose yields Total sugars Total sugars Total sugars
Pretreatment system 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
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.