charles e. wyman, dartmouth college y. y. lee, auburn university

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 CollegeY. Y. Lee, Auburn University

Mohammed Moniruzzaman, Genencor InternationalBruce E. Dale, Michigan State University

Richard T. Elander, National Renewable Energy LaboratoryMichael R. Ladisch, Purdue University

Mark T. Holtzapple, Texas A&M UniversityJohn 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


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


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


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.


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

technologiesBiomass Refining CAFI

Hydrolysis Stages


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

Mass Balance Approach: AFEX Example

Hydrolysis

Enzyme (15 FPU/g of Glucan)

ResidualSolids

HydrolyzateLiquidAFEX

SystemTreatedStover

Ammonia

Stover

101.0 lb100 lb

(dry basis)36.1 lb glucan21.4 lbxylan 39.2 lb

95.9% glucan conversion to glucose, 77.6% xylan conversion to xylose

99% mass balance closure includes:(solids + glucose + xylose + arabinose )

Wash

2 lb

99.0 lb

Solids washed out

38.5 lb glucose18.9 lb xylose (Ave. of 4 runs)

Very few solubles from pretreatment—about 2% of inlet stover

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


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 %


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%.


Pretreatment Yields 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


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


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


General PFD for Cost Estimates


Boiler +

Generator

Different Pretreatments

Hydrolysis +

Fermentation

Feed Handling Recovery

DifferentPretreatments

Stover

Syrup + Solids

Chemicals Water

Enzymes CO2 Water

EtOH

Steam

Power

Minimum Ethanol Selling Price (MESP)


0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

Dilute Acid Hot Water AFEX ARP Lime Ideal

Net Stover Other Variable Fixed w/o Depreciation Depreciation Income Tax Return on Capital

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

CashCostPlantLevel

MESP

Effect of Oligomer Conversion on MESP


1.00

1.25

1.50

1.75

Dilute Acid Hot Water AFEX ARP Lime

ME

SP

, $/

gal

EtO

H

w/o Oligomer Credit w/ Oligomer Credit

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


CAFI Project Advisory Board

1. Quang Nguyen, Abengoa Bioenergy2. Mat Peabody, formerly Applied

CarboChemicals3. Gary Welch, Aventinerei4. Greg Luli, BC International5. Paris Tsobanakis, Cargill6. Robert Wooley, Cargill Dow7. James Hettenhaus, CEA8. Lyman Young, ChevronTexaco9. Kevin Gray, Diversa10. Paul Roessler, Dow11. Susan M. Hennessey, DuPont12. Michael Knauf, Genencor

13. Don Johnson, GPC (Retired)14. Dale Monceaux, Katzen

Engineers15. Kendall Pye, Lignol16. Farzaneh Teymouri, MBI17. Richard Glass, National

Corn Growers Association18. Bill Cruickshank, Natural

Resources Canada19. Joel Cherry, Novozymes20. Ron Reinsfelder, Shell 21. Carl Miller, Syngenta22. Carmela Bailey, USDA23. Don Riemenschneider,

USDA

Serve as extension agents for technology transferProvide feedback on approach and results

Meet with team every 6 months

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

CAFI 2 Stover


Component Composition (wt %)

Sucrose 2.2Glucan 34.4Xylan 22.8

Arabinan 4.2Mannan 0.6Galactan 1.4

Lignin 11.0Protein 2.3Acetyl 5.6Ash 6.1

Uronic Acids 3.8Extractives 8.5

• 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 ¼ inch round screen

• Feedstock: USDA-supplied hybrid poplar (Alexandria, MN)– Debarked, chipped, and milled to pass

¼ inch round screen


Component Composition (wt %)Glucan 43.8Xylan 14.9

Arabinan 0.6Mannan 3.9Galactan 1.0

Lignin 29.1Protein ndAcetyl 3.6Ash 1.1

Uronic Acids ndExtractives 3.6

CAFI 2 Poplar

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


Log (Ro) Glucose (%) Xylose (%)

3.02 99 83

3.35 100 64

4.03 88 45

3.02 58 62

3.35 88 60

4.03 88 45

3.02 73 70

3.35 95 64

4.03 88 45

Pretreatment

Hydrolysis15 FPU

Hydrolysis60 FPU

SO2 Pretreatment of Corn Stover

AFEX Pretreated Poplar

AFEX pretreated samples

1 g dry biomass : 0.8 g NH3

0.0%

2.0%

4.0%

6.0%

8.0%

10.0%

12.0%

14.0%

16.0%

18.0%

20.0%

80 90 100 110 120

Temperature (°C)

Pe

rce

nt

co

nv

ers

ion

24 hour Glucan

72 hour Glucan

24 hour Xylan

72 hour Xylan

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


Enzymatic Hydrolysis of Dilute Acid Pretreated Poplar

2% glucan concentration50 FPU/g glucan, no β-glucosidase supplementation

Model Predictions of Effect of Lignin

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


0.9% (w/v) consistency, corn stover-190oC, 5min, 3% S02, 0.0417g Spezyme SP, 0.0073g cocktail BG-X-001

0 5 10 15 20 250

10

20

30

40

50

60

70

80

cellulase cellulase+xylanase cellulase+BSA xylanase

Xyl

an c

onve

rsio

n (%

)

Time (hours)

0 5 10 15 20 250

10

20

30

40

50

60

70

80

Xyl

an c

onve

rsio

n (%

)

Time (hours)


0 5 10 15 20 250

10

20

30

40

50

60

70

80


Xyl

an c

onve

rsio

n (%

)

Time (hours)

0.006g of protein/g of cellulose 0.03g of protein/g of cellulose 0.06g of protein/g of cellulose

12%

21% 31%

Method: High Throughput Microassay

Xylanase Supplementation of SO2 Treated Stover

Dilute Acid Pretreated Corn Stover Hydrolyzate Fermentation (resin conditioned)

0

10

20

30

40

50

60

70

80

0 24 48 72 96 120 144 168Fermentation Time (hr)

Xyl

ose

(g/L

) .

0

5

10

15

20

25

30

Eth

anol

(g/

L)

.


Initial Fermentation Results after 144 hours

Control Overlime XAD4 Overlime + XAD4

Xylose Consumed ( %) 54.1 42.4 44.5 41.3

Ethanol Yield (% theoretical for

glucose + xylose consumed)

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


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


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


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 as initiallly shown for poplar


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 will be important to better account for performance differences

• Consideration should be given to taking advantage of differences among pretreatment options


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


Insanity is doing what you always have always been doing and

expecting different results


Questions?


charles e. wyman, dartmouth college y. y. lee, auburn university

Documents

energy balance methodsthe

risksdilute acid pretreatment

leading pretreatments

cost of sugars

glucan conversion

generation technologies

mass balance closure

future agriculture