Comparative Study on Enzymatic Digestibility of Upland and Lowland Switchgrass Varieties

Processed by Leading Pretreatment TechnologiesYoungmi Kim, Nathan S. Mosier, Michael R. Ladisch

Purdue UniversityV. Ramesh Pallapolu and Y. Y. Lee

Auburn UniversityBonnie Hames

CeresSteven R. Thomas

DOE - Golden Field OfficeRebecca Garlock, Venkatesh Balan, and Bruce E. Dale

Michigan State UniversityBryon S. Donohoe, Todd B. Vinzant, and Richard T. Elander

NRELMatthew Falls, Rocio Sierra, and Mark T. Holtzapple

Texas A&M UniversityJian Shi, Mirvat A. Ebrik, Tim Redmond, Bin Yang, and Charles E. Wyman

University of California Riverside

Ryan E. WarnerGenencor, A Danisco Division

2009 AIChE Annual Meeting, Nashville, Nov 08-13, 2009

Material in this work supported by US Department of Energy Office of the Biomass Program,Contract DE-FG36-04GO14017

For gifts of enzymesGenencor, a Danisco Division

Switchgrass Ceres

CAFI Team Collaborators

LORRE StaffLinda Liu, Rick Hendrickson

Acknowledgments

Switchgrass

Photos courtesy of Department of Agronomy, Purdue University

A plentiful, warm-season perennial grass

Low fertility requirement

Tolerant of poor soils

High yield

(6-8 ton/acre)

Switchgrass VarietyMorphological type•Lowland type:

Adapted to poor drainage Tall, coarse, thicker stems, Grows faster and higher yield potential than uplandAlamo, Kanlow

•Upland type: Short, fine stems, tolerant to cold temperature and droughtShawnee, Dacotah, Trailblazer, Cave-in-Rock, Caddo

Physiological type (based on latitude-of-origin)• Southern ecotype:

Higher biomass productivity, higher risk of winterkill• Northern ecotype:

Earlier flowering, shorter, lower yield, lower cell wallconcentration, better winter survival than southern ecotypes

Cassida et al., Crop Sci. (2005)

Factors Affecting Biomass Yields and Composition

Cassida et al., Crop Sci. (2005); Adler et al., Crop Sci. (2006); Wiselogel et al., Bioresource Tech. (1996)

Morphological type Latitude-of-origin Harvest Season

Lowland Upland Southern Northern FallAfter-

Winter

Biomass Yield > > >Moisture level > > >

Cellulose > > <Lignin > > <

Soluble Carbohydrates

unknown unknown >Storage

Polysaccharidesunknown unknown >

Ash < < >N < < >

Other factors, such as N fertilizer, precipitation, storage method, are not considered in this study.

CAFI3-Allotted Switchgrass

Alamo 1 Alamo 2 Shawnee DacotahLatitude-of-Origin 29 °N 38 °N 46 °N

Ecotype Southern Lowland Northern UplandMorphology Thick stems Thin stems

Harvest LocationArdmore, OK

34°N (Elev. 870 ft)

Stillwater, OK36°N

(Elev. 960 ft)

Pierre, SD44

(Elev. 1420 ft)Plant date June, 2005 June, 2007 June, 2005 December, 1999

Harvest dateDecember,

2006November,

2007December,

2006

May, 2008

(Plot was allowed to stand over the

winter)

Post processing and Storage

Small square bales stored in a buildingDried at 50 C to less than 10% Moisture

Milled using a Knife mill or hammer mill to 2-6 mm size

Objectives

• Compare compositions of CAFI3-allotted switchgrass

• Apply different pretreatment technologies and compare enzymatic digestibility

% Water-Extractable Free Sugars in CAFI3 Switchgrass

9.6%

6.9%8.2%

0.8%0%

2%

4%

6%

8%

10%

12%

Alamo 1 Alamo 2 Shawnee Dacotah

% W

ater

-Ext

ract

able

Free

Sug

ars i

n Ra

w

Switc

hgra

ss

Cell Wall Composition of CAFI3 Switchgrass(Samples were hot-water washed by x10 v/w DI water to remove water-soluble free sugars)

59.7% 60.5% 58.9% 61.4%

20.8% 20.9% 21.5%22.8%

6.0% 4.9% 4.7%1.2%

4.3% 4.6% 4.6% 3.3%

2.5% 3.0% 3.1% 3.6%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Alamo 1 Alamo 2 Shawnee Dacotah

% D

ry M

ass o

f Pre

-Was

hed

Swit

chgr

ass

Acetyl

Ash

Protein

Lignin

Structural Carbohydrates

Effect of Ecotype and Harvest Season on CompositionLowland vs Upland, Late-Fall Harvest vs Spring Harvest

0%

5%

10%

15%

20%

25%

30%

35%

40%

25 30 35 40 45 50

Latitude-of-origin (oN)

Cellulose

Lignin

Water Extractable Sugars

Protein

Ash

Lowland (A)Late-fall

Upland (S)Late-fall

Upland (D)Spring

Ecotype:Harvest:

Pretreatment Conditions

Pretreatment Method

SubstrateChemicals Loading(per g dry biomass)

Temperature (oC)

DurationMoisture Level

(per g dry biomass)

AFEXA 1.5 g NH3 140 20 min 2 g H2O

S, D 1.5 g NH3 150 30 min 2 g H2O

DA A, S, D 0.005 g H2SO4 160 10 min 19 g H2O

LHW A, S, D 6.7 g H2O 200 10 min 6.7 g H2O

Lime A, S, D 1 g Ca(OH)2 120 4 hr None

SAAA, S 0.15 g NH4OH 90 24 hr 9 g H2O

D 0.15 g NH4OH 160 1 hr 9 g H2O

SO2 D 0.05 g SO2 180 10 min 9 g H2O

Fate of Solids During Pretreatment of Dacotah Switchgrass

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Untreated AFEX DA LHW Lime SAA SO2

% T

otal

Init

ialD

ry M

assLignin (acid insoluble)

Arabinan

Xylan

Cellulose

Composition of AFEX Pretreated Solids of CAFI3 Switchgrass

(Pretreatment conditions: 1.5 g NH3, 2 g H2O:g dry solids140 oC/20 min for Alamo, 150 oC/30 min for Shawnee and Dacotah)

0%

20%

40%

60%

80%

100%

% D

ry W

eigh

tProtein

Acetyl

Lignin

Arabinan

Xylan

Cellulose

Untreated AFEX

Composition of Dilute Acid Pretreated/Hot-Washed Solids of CAFI3 Switchgrass

(Pretreatment conditions: 0.005 g H2SO4, 19 g H2O:g dry solids160 oC/10 min)

0%

20%

40%

60%

80%

100%

% D

ry W

eigh

tLignin

Xylan

Cellulose

Untreated DA

Composition of LHW Pretreated/Hot-Washed Solids of CAFI3 Switchgrass

(Pretreatment conditions: 6.7 g H2O: g dry solids, 200 C, 10 min)

0%

20%

40%

60%

80%

100%

% D

ry W

eigh

tAcetyl

Lignin

Arabinan

Xylan

Cellulose

Untreated LHW

Composition of Lime Pretreated/Hot-Washed Solids of CAFI3 Switchgrass

(Pretreatment conditions: 1 g Ca(OH)2 : g dry solids, 120 oC/4 hr)

0%

20%

40%

60%

80%

100%

% D

ry W

eigh

tLignin

Arabinan

Xylan

Cellulose

Untreated Lime

Composition of SAA Pretreated/Hot-Washed Solids of CAFI3 Switchgrass

(Pretreatment conditions: 0.15 g NH4OH, 9 g H2O : g dry solids, 90oC/24 hr for Alamo and Shawnee, 160 oC/1 hr for Dacotah)

0%

20%

40%

60%

80%

100%

% D

ry W

eigh

tAcetyl

Lignin

Arabinan

Xylan

Cellulose

Untreated SAA

Enzymatic Hydrolysis Conditions• Substrate loading:

DA, LHW, Lime, SAA, SO2 - Pretreated and hot-washed solids at 1% glucan loadingAFEX - Pretreated solids at 1% glucan loading

• Enzyme dose: Spezyme CP and Novo188 at 15 FPU and 30 CBU per g glucan in the prewashed, untreated switchgrass(~27 mg protein/g glucan)

• Hydrolysis conditions: 150 RPM, 50 0CpH 4.8, 0.05 M citrate buffer

• Sampling: 1 hr (for initial rate), 24 hr, and at the end of hydrolysis (168 hr)

Glucose Yields after 1 hr Hydrolysis

Yields calculated based on glucan in pretreated/hot washed solids for all pretreatments except for AFEX

0%

20%

40%

60%

80%

100%

AFEX DA LHW Lime SAA

% G

luco

se Y

ield

Alamo

Shawnee

Dacotah

Glucose Yields after 24 hr Hydrolysis

Yields calculated based on glucan in pretreated/hot washed solids for all pretreatments except for AFEX

0%

20%

40%

60%

80%

100%

AFEX DA LHW Lime SAA

% G

luco

se Y

ield

Alamo

Shawnee

Dacotah

Glucose Yields after 168 hr Hydrolysis

Yields calculated based on glucan in pretreated/hot washed solids for all pretreatments except for AFEX

0%

20%

40%

60%

80%

100%

AFEX DA LHW Lime SAA

% G

luco

se Y

ield

Alamo

Shawnee

Dacotah

Xylose Yields after 168 hr Hydrolysis

Yields calculated based on glucan in pretreated/hot washed solids for all pretreatments except for AFEX

0%

20%

40%

60%

80%

100%

AFEX DA LHW Lime SAA

% X

ylos

e Yi

eld

Alamo

Shawnee

Dacotah

Effect of Supplementary Xylanase(Multifect Pectinase) on Sugar Yields

LHW pretreated Dacotah, No hot-water washing after pretreatment15 FPU Spezyme CP+ 200 OSX M. Pectinase/g glucan= 27 mg protein/g glucan

0%

20%

40%

60%

80%

100%

0 30 60 90 120 150 180

% G

luco

se Y

ield

Hydrolysis Time (hr)

No Pectinase

With Pectinase

Yields calculated based on glucan and xylan in untreated biomass

0%

20%

40%

60%

80%

100%

0 30 60 90 120 150 180%

Xyl

ose

Yiel

d

Hydrolysis Time (hr)

Summary

• Harvest season and possibly age of stand are more important factors than ecotype of switchgrass in terms of affecting switchgrass saccharifaction.

• Spring harvest contains more lignocellulose and less ash, protein and water extractable free-sugars than fall/late fall harvest.

• After pretreatment, compositional variability between switchgrass batches becomes less related to ecotype and/or harvest season.

• Enzymatic digestibility of Dacotah switchgrass, which is upland variety and was harvested in spring, was the lowest among the switchgrass samples, regardless of the pretreatment methods applied.

• The recalcitrant nature of upland cultivar and spring harvest of switchgrass may require more severe pretreatment conditions.