Enzymatic Digestion of Corn Stover and Poplar Wood after Pretreatment by Leading Technologies

1
Enzymatic Digestion of Corn Stover and Poplar
Wood after Pretreatment by Leading Technologies

• Charles E. Wyman, Dartmouth College/University of
  California
• Rajeev Kumar, Dartmouth College
• Bruce E. Dale, Michigan State University
• Richard T. Elander, National Renewable Energy
  Laboratory
• Mark T. Holtzapple, Texas AM University
• Michael R. Ladisch, Purdue University
• Y. Y. Lee, Auburn University
• Mohammed Moniruzzaman, Genencor International
• John N. Saddler, University of British Columbia
• BIO Meeting
• Chicago, Illinois
• April 12, 2006

Biomass Refining CAFI
2
CAFI Approach

• 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
3
Hydrolysis Stages
Cellulase enzyme
Stage 2 Enzymatic hydrolysis
Stage 1 Pretreatment
Residual solids cellulose, hemicellulose, ligni
n
Biomass
Solids cellulose, hemicellulose, lignin
Chemicals
Dissolved sugars, oligomers
Dissolved sugars, oligomers, lignin
Stage 3 Sugar fermentation
Biomass Refining CAFI
4
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
5
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
6
Overall Yields 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.
Biomass Refining CAFI
7
Overall Yields 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.
Biomass Refining CAFI
8
Overall Yields at 15 FPU/g Glucan
Maximum possible
Dilute acid
Controlled pH
Flowthrough
ARP
Lime
AFEX
9
Overall Yields at 15 FPU/g Glucan
Maximum possible
Dilute acid
Controlled pH
Flowthrough
ARP
Lime
AFEX
10
Overall Yields at 15 FPU/g Glucan
Maximum possible
Dilute acid
Controlled pH
Flowthrough
ARP
Lime
AFEX
11
Overall Yields at 15 FPU/g Glucan
Maximum possible
Dilute acid
Controlled pH
Flowthrough
ARP
Lime
AFEX
12
Total Yields at 15 FPU/g Glucan
13
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
14
Tasks for the DOE OBP CAFI 2 Project

• Pretreat corn stover and poplar by leading
  technologies to improve cellulose accessibility
  to enzymes
• Enzymatically hydrolyze cellulose and
  hemicellulose in pretreated biomass (corn stover
  and poplar), as appropriate, and develop models
  to understand the relationship between pretreated
  biomass features, advanced enzyme
  characteristics, and enzymatic digestion results
• Develop conditioning methods as needed to
  maximize fermentation yields by a recombinant
  yeast, determine the cause of inhibition, and
  model fermentations
• Estimate capital and operating costs for each
  integrated pretreatment, hydrolysis, and
  fermentation system and use to guide research

Biomass Refining CAFI
15
CAFI 2 Standard Poplar

• Feedstock USDA-supplied hybrid poplar
  (Alexandria, MN)
• Debarked, chipped, and milled to pass ¼ inch
  round screen

Biomass Refining CAFI
16
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
17
Effect of Enzyme Loading on Hydrolysis of SO2
Pretreated Corn Stover
Protein ( mg) FPU/gm (SP-CP)
6.4 3.0
16.1 7.5
32.2 15
42.9 20
107.4 50
128.9 60
CBUFPU 2.0 Digestion time 72hr
SO2 pretreated corn stover at 1 glucan
concentration
Biomass Refining CAFI
18
Effect of Enzyme Loading on Hydrolysis of SO2
Pretreated Corn Stover
Protein ( mg) FPU/gm (SP-CP)
6.4 3.0
16.1 7.5
32.2 15
42.9 20
107.4 50
128.9 60
CBUFPU 2.0 Digestion time 72hr
SO2 pretreated corn stover at 1 glucan
concentration
Biomass Refining CAFI
19
Effect of Pretreatment Severity on Enzymatic
Hydrolysis of Dilute Acid Pretreated Poplar
For 50 FPU, Total Protein ( mg/gm original glucan) For 50 FPU, Total Protein ( mg/gm original glucan)
POP1 122.2
POP2 122.0
POP3 142.0
POP4 160.3
CBUFPU 2.0 Digestion time 72hr
2 glucan concentration 50 FPU/ gm original glucan
Biomass Refining CAFI
20
Effect of Protein Loadings on Cellulose
Hydrolysis of Poplar Solids
Digestion time 72hr
21
Effect of Protein Loadings on Cellulose
Hydrolysis of Poplar Solids
Digestion time 72hr
22
Effect of Protein Loadings on Cellulose
Hydrolysis of Poplar Solids
Digestion time 72hr
23
CAFI 2 Initial Poplar

• Feedstock USDA-supplied hybrid poplar
  (Arlington, WI)
• Debarked, chipped, and milled to pass ¼ inch
  round screen
• Not enough to meet needs

Biomass Refining CAFI
24
C - Cellulase (31.3 mg/g glucan) X -
Xylanase (3.1 mg/g glucan) A - Additive (0.35g/g
glucan)
UT - Untreated AFEX condition 24 h water
soaked 11 (PoplarNH3) 10 min. res. time
AFEX Optimization for High/Low Lignin Poplar
25
Differences Among Poplar Species
Original Poplar Poplar Standard
Arlington, WI near Madison Very rich, loamy soil Demonstrated some of best growth rates Harvested and shipped in February 17, 2004 Planted in 1995, probably in spring but possibly in fall Alexandria, Minnesota Lower growth rate than Arlington Slightly shorter growing season Harvested and shipped in August 2004 Planted in spring 1994
Based on information provided by Adam Wiese,
USDA Rheinlander, WI
26
Observations

• 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
• All pretreatments gave similar results for corn
  stover
• Initial performance for poplar is not as good,
  with one source more recalcitrant than other
• Yields can be further increased for some
  pretreatments with enzymes a potential key

Biomass Refining CAFI
27
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

Biomass Refining CAFI
28
Questions?
Biomass Refining CAFI
29
Publication of Results from CAFI 1

• Bruce Dale of the CAFI Team arranged for and
  edited a special December 2005 issue of
  Bioresource Technology entitled Coordinated
  Development of Leading Biomass Pretreatment
  Technologies to document these results
• Wyman CE, Dale BE, Elander RT, Holtzapple M,
  Ladisch MR, Lee YY. 2005. Coordinated
  Development of Leading Biomass Pretreatment
  Technologies, Bioresource Technology 96(18)
  1959-1966, invited.
• Lloyd TA, Wyman CE. 2005. Total Sugar Yields for
  Pretreatment by Hemicellulose Hydrolysis Coupled
  with Enzymatic Hydrolysis of the Remaining
  Solids, Bioresource Technology 96(18)
  1967-1977, invited.
• Liu C, Wyman CE. 2005. "Partial Flow of
  Compressed-Hot Water Through Corn Stover to
  Enhance Hemicellulose Sugar Recovery and
  Enzymatic Digestibility of Cellulose,
  Bioresource Technology 96(18) 1978-1985,
  invited.
• Mosier N, Hendrickson R, Ho N, Sedlak M, Ladisch
  MR. 2005. Optimization of pH Controlled Liquid
  Hot Water Pretreatment of Corn Stover, Bioresourc
  e Technology 96(18) 1986-1993, invited.
• Kim S, Holtzapple MT. 2005. Lime Pretreatment
  and Enzymatic Hydrolysis of Corn
  Stover, Bioresource Technology 96(18)
  1994-2006, invited.
• Kim TH, Lee YY. 2005. Pretreatment and
  Fractionation of Corn Stover by Ammonia Recycle
  Percolation Process, Bioresource Technology
  96(18) 2007-2013, invited.
•  Teymouri F, Laureano-Perez L, Alizadeh H, Dale
  BE. 2005. Optimization of the Ammonia Fiber
  Explosion (AFEX) Treatment Parameters for
  Enzymatic Hydrolysis of Corn Stover, Bioresource
  Technology 96(18) 2014-2018, invited.
• Eggeman T, Elander RT. 2005. Process and
  Economic Analysis of Pretreatment Technologies,
  Bioresource Technology 96(18) 2019-2025,
  invited.
• Wyman CE, Dale BE, Elander RT, Holtzapple M,
  Ladisch MR, Lee YY. 2005. Comparative Sugar
  Recovery Data from Laboratory Scale Application
  of Leading Pretreatment Technologies to Corn
  Stover, Bioresource Technology 96(18)
  2026-2032, invited.

Biomass Refining CAFI