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 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

Stage 2 Enzymatic hydrolysis

Residual solids: cellulose, hemicellulose, lignin

Dissolved sugars, oligomers, lignin

Dissolved sugars, oligomers

Biomass Refining CAFI

Mass Balance Approach: AFEX Example

Ammonia

Enzyme (15 FPU/g of Glucan) Hydrolyzate Liquid

(Ave. of 4 runs)

Stover

100 lb (dry basis) 36.1 lb glucan 21.4 lb xylan

99.0 lb AFEX Treated Hydrolysis Wash 38.5 lb glucose System Stover

101.0 lb
Solids washed out

Residual 18.9 lb xylose Solids

2 lb

39.2 lb

Very few solubles from pretreatmentabout 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 inch round screen
Glucan Xylan Arabinan Mannan Galactan Lignin Protein Acetyl Ash Uronic Acid Non-structural Sugars 36.1 % 21.4 % 3.5 % 1.8 % 2.5 % 17.2 % 4.0 % 3.2 % 7.1 % 3.6 % 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

Pretreatment system

Xylose yields* Stage 1 Stage 2

37.7

Glucose yields* Total xylose

37.7

Total sugars* Total glucose

62.3

Stage 1
62.3

Stage 2
62.3

Stage 1
100.0

Stage 2
100.0

Combined total
100.0

Maximum possible Dilute acid

37.7

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

Increasing pH

Flowthrough Controlled pH AFEX

36.3/1.7 21.8/0.9

0.6/0.5 9.0

36.9/2.2 30.8/9.9

4.5/4.4 3.5/0.2

55.2 52.9

59.7/59.6 56.4/53.1

40.8/6.1 25.3/1.1

55.8/55.7 61.9

96.6/61.8 87.2/63.0

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

Sugar yields, % of m ax total 100 25 50 75 0

e1 St ag

Dilute acid
St ag e2 St ag e1

Flowthrough

Controlled pH
St ag e2 St ag e1

AFEX

ARP
St ag e2

Lime

Maximum possible
Oligoxylose Monoxylose Oligoglucose Monoglucose

Pretreatment Yields at 15 FPU/g Glucan

Sugar yields, % of m ax total 100 25 50 75 0

e1 St ag

Dilute acid
St ag e2 St ag e1

Flowthrough

Controlled pH
St ag e2 St ag e1

AFEX

ARP
St ag e2

Lime

Maximum possible
Oligoxylose Monoxylose Oligoglucose Monoglucose

Pretreatment Yields at 15 FPU/g Glucan

Sugar yields, % of m ax total 100 25 50 75 0

e1 St ag

Dilute acid
St ag e2 St ag e1

Flowthrough

Controlled pH
St ag e2 St ag e1

AFEX

ARP
St ag e2

Lime

Maximum possible
Oligoxylose Monoxylose Oligoglucose Monoglucose

Pretreatment Yields at 15 FPU/g Glucan

Sugar yields, % of m ax total 100 25 50 75 0

e1 St ag

Dilute acid
St ag e2 St ag e1

Flowthrough

Controlled pH
St ag e2 St ag e1

AFEX

ARP
St ag e2

Lime

Maximum possible
Oligoxylose Monoxylose Oligoglucose Monoglucose

Pretreatment Yields at 15 FPU/g Glucan

Sugar yields, % of m ax total 100 25 50 75 0

e1 St ag

Dilute acid
St ag e2 St ag e1

Flowthrough

Controlled pH
St ag e2 St ag e1

AFEX

ARP
St ag e2

Lime

Maximum possible
Oligoxylose Monoxylose Oligoglucose Monoglucose

Pretreatment Yields at 15 FPU/g Glucan

Sugar yields, % of m ax total 100 25 50 75 0

e1 St ag

Dilute acid
St ag e2 St ag e1

Flowthrough

Controlled pH
St ag e2 St ag e1

AFEX

ARP
St ag e2

Lime

Maximum possible
Oligoxylose Monoxylose Oligoglucose Monoglucose

Pretreatment Yields at 15 FPU/g Glucan

Sugar yields, % of m ax total 100 25 50 75 0

e1 St ag

Dilute acid
St ag e2 St ag e1

Flowthrough

Controlled pH
St ag e2 St ag e1

AFEX

ARP
St ag e2

Lime

Maximum possible
Oligoxylose Monoxylose Oligoglucose Monoglucose

Pretreatment Yields at 15 FPU/g Glucan

Pretreatment Yields at 15 FPU/g Glucan

100 Sugar yields, % of m ax total -

75

Oligoxylose S1 Monoxylose S1 Monoxylose S2 Oligoglucose S1 Monoglucose S1 Monoglucose S2

50

25

0
gh AF EX pH P cid e ea ed Li m AR ou th r Di lu t oll po M ax ss ib le

Fl ow

Co

nt r

General PFD for Cost Estimates

Enzymes

CO2

Water

Stover

Feed Handling

Different Pretreatments

Hydrolysis + Fermentation

Recovery
Syrup + Solids

EtOH

Chemicals

Water

Different Pretreatments

Boiler + Generator

Steam Power

Biomass Refining CAFI

Minimum Ethanol Selling Price (MESP)

$/gal EtOH 1.75
Proof Year: 4th Year of Operation

1.50

1.25

1.00

0.75
MESP

0.50
Cash Cost
Plant Level

0.25

0.00 Dilute Acid

Net Stover

Hot Water

AFEX

ARP
Depreciation

Lime
Income Tax

Ideal
Return on Capital

Other Variable

Fixed w/o Depreciation

Biomass Refining CAFI

Effect of Oligomer Conversion on MESP

1.75

MESP, $/gal EtOH

1.50

1.25

1.00 Dilute Acid Hot Water w/o Oligomer Credit AFEX ARP w/ Oligomer Credit Lime

Biomass Refining CAFI

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

Serves as extension agents for technology transfer Provides feedback on approach and results Meets 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 inch round screen
Component Sucrose Glucan Xylan Arabinan Mannan Galactan Lignin Protein Acetyl Ash Uronic Acids Extractives Composition (wt %)

2.2 34.4 22.8 4.2 0.6 1.4 11.0 2.3 5.6 6.1 3.8 8.5

Biomass Refining CAFI

CAFI 2 Poplar
Feedstock: USDA-supplied hybrid poplar (Alexandria, MN)
Debarked, chipped, and milled to pass inch round screen
Component Glucan Xylan Arabinan Mannan Galactan Lignin Protein Acetyl Ash Uronic Acids Extractives Composition (wt %) 43.8 14.9 0.6 3.9 1.0 29.1 nd 3.6 1.1 nd 3.6

Biomass Refining CAFI

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

Corn stover
36.1 g glucan 21.4 g xylan

Liquid phase

Sugars
1.2 g glucose 8.7 g xylose

Pretreatment
(190oC, 5min, 3% SO2)

Solid phase

Hydrolysis
(60FPU/g of glucan)

Sugars
36.9 g glucose 6.8 g xylose

95% conversion of glucan to glucose 64% conversion of xylan to xylose 83% overall yield of sugars

AFEX Pretreated Poplar

AFEX pretreated samples 1 g dry biomass : 0.8 g NH3
20.0% 18.0% 16.0% Percent conversion 14.0% 12.0% 10.0% 8.0% 6.0% 4.0% 2.0% 0.0% 80 90 100 Temperature (C) 110 120 24 hour Glucan 72 hour Glucan 24 hour Xylan 72 hour Xylan

Enzymatic Hydrolysis of Dilute Acid Pretreated Poplar

100 90 80

Glucose yield, %

70 60 50 40 30 20 10 0 0 10 20 30
POP-1-Severity -3.01 POP-3-Severity -3.31 POP-2-Severity -3.25 POP-4-Severity -3.55

Time, hours

40

50

60

70

80

Biomass Refining CAFI

2% glucan concentration 50 FPU/g glucan, no -glucosidase supplementation

Model Predictions of Effect of Lignin

100 g substrate/L, 50% cellulose, 10 FPU cellulase/g cellulose, 2 CBU/FPU

100 90

NM, 5 FPU/gm

Cellulose conversion, %

80 70 60 50 40 30 20 0 10 20 30 40 50 60

Phillipidis et al.

South et al.

Holtzapple et al.

Biomass Refining CAFI

Lignin concentration ( g/l)

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
0.006g of protein/g of cellulose
80 70 80 70

0.03g of protein/g of cellulose

80 70

0.06g of protein/g of cellulose 31%

Xylan conversion (%)

Xylan conversion (%)

60 50 40 30 20 10 0 0 5 10 15

60 50 40 30 20 10 0 0 5 10 15

21%

Xylan conversion (%)

60 50 40 30 20 10 0 0 5 10 15

12%

cellulase cellulase+xylanase cellulase+BSA xylanase

20 25

cellulase cellulase+xylanase cellulase+BSA xylanase

20 25

cellulase cellulase+xylanase cellulase+BSA xylanase

20 25

Time (hours)

Time (hours)

Time (hours)

Method: High Throughput Microassay

Dilute Acid Pretreated Corn Stover Hydrolyzate Fermentation (resin conditioned)

80 70 60 Xylose (g/L) . 50 40 30 20 10 0 0 24 48 5 Ethanol (g/L) . 20 30

25

15

10

Biomass Refining CAFI

72 96 Fermentation Time (hr)

120

144

0 168

Initial Fermentation Results after 144 hours

Control

Overlime XAD4 Overlime + XAD4

Xylose Consumed ( %) Ethanol Yield (% theoretical for glucose + xylose consumed)

Biomass Refining CAFI

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 characterized Initial results are for corn stover, and performance with other feedstocks will likely be different as initially 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 is important to understand reasons 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