1 May, 2014 36 SIM SBFC - Chemical Engineering and ... · throughput pretreatment‐hydrolysis (iWALL) Correlation Coefficients between CellWall Properties 35 40 45 50

Correlating cell wall properties in diverse h lk lgrasses to their response to alkaline

pretreatment and enzymatic hydrolysis

Feedstock II – Biomass Physicochemical Analysis

1 May, 2014

36th SIM SBFC36th SIM SBFC

Muyang Li, Daniel Williams, and David Hodge

www.glbrc.org

Scope of WorkScope of Work

• Introduction to alkaline pretreatment• Introduction to alkaline pretreatment

• Correlating cell wall properties to g p puntreated and pretreated hydrolysis yieldsyields

• Chemometric models for composition and hydrolysis yield prediction





Soda Pulping of Non‐Wood Feedstocks

Tamil Nadu Newsprint and Papers Limited:p p4 million tonnes/yr sugar cane bagasse to bleached pulpWorld’s largest non‐wood pulp and paper mill O2 delignification

Sappi’s Stanger Mill, KwaZulu‐Natal, South Africa O2 delignification

Kimberly Clark’s Orizaba Mill,Veracruz, Mexico

NaOH Pretreatment of Corn Stover

100 kg corn stover

8 kg NaOH500 kg water

2.5 kg H2O2

Make‐up NaOH (2 kg)C‐Tec2, H‐Tec218.75 mg/g glc

NaOHPretreatment 80°C, 1 h

Oxidative Delignification

50°C, 3 h

Enzymatic Hydrolysis50°C, 72 h

corn stover

100 kg dry wt36.1 kg glucan22 4 kg xylan

76 kg insoluble33.8 kg glucan18 3 kg xylan


8.5 kg insoluble0.9 kg glucan2 0 kg xylan22.4 kg xylan

18.2 kg lignin18.3 kg xylan7.7 kg ligninSolubles: 2.8 kg glucan5.3 kg xylan9.4 kg lignin

16.0 kg xylan4.9 kg ligninSolubles: 1.9 kg glucan3.0 kg xylan3.0 kg lignin

2.0 kg xylan5.0 kg ligninSolubles: 30.5 kg glucose2.7 kg glc olig13.6 kg xylose

~20% solids (wt/wt total)


g g g g5.1 kg xyl olig

• 70% of solubles removed by /L/S separation

• Recovered alkali‐extracted corn stover lignin+xylang y

NaOH Pretreatment of Corn Stover

100 kg corn stover

8 kg NaOH500 kg water

2.5 kg H2O2

Make‐up NaOH (2 kg)C‐Tec2, H‐Tec218.75 mg/g glc

8.5 kg insoluble0.9 kg glucan2 0 kg xylan

NaOHPretreatment 80°C, 1 h

Oxidative Delignification

50°C, 3 h

Enzymatic Hydrolysis50°C, 72 h

corn stover

100 kg dry wt36.1 kg glucan22 4 kg xylan


70 kg insoluble31.9 kg glucan16 0 kg xylan 2.0 kg xylan

5.0 kg ligninSolubles: 30.5 kg glucose2.7 kg glc olig13.6 kg xylose

22.4 kg xylan18.2 kg lignin





5.1 kg xyl olig

Techno‐economic model based 20 2 l

Recovery of alkali by recausticizationPretreatment and chemical recovery:

g g g g

on 2012 NREL Aspen Plus model for 2000 ton per day plant (Andrea Orjuela, Bruce Dale Group, MSU)

Pretreatment and chemical recovery:Capital costs $50MNet water use in pretreatment 2 m3/ton biomassNet thermal energy use ‐200 MJ/ton biomass p, )MESP $2.07 / gal EtOH

High‐Sugar Hydrolysate Fermentation• High‐solids alkali pre‐extraction oxidative post‐High solids alkali pre extraction, oxidative posttreatment, and hydrolysis

• Capable of reaching >5% (w/v) ethanol• Y128 more inhibited by high ethanol

/ l h

Liu et al., 2014. BiotechnolBiofuels 7(1), 48

4.580 4.580

XR/XDH pathway:S. cerevisiae GLBRC Y73

Xylose Isomerase pathway:S. cerevisiae GLBRC Y128

2

2.5

3

3.5

4

40

50

60

70

mass (g/L)

oncentration

(g/L)

Glucose XyloseEthanol XylitolGlycerol Biomass

2

2.5

3

3.5

4

40

50

60

70

ass (g/L)

oncentration

(g/L)

0

0.5

1

1.5

2

0

10

20

30 Biom

Metabolite Co

0

0.5

1

1.5

2

0

10

20

30 Biom

Metabolite C o

www.glbrc.org

0 20 40 60 80 100 120 140

time (h)0 20 40 60 80 100 120 140

time (h)





Cell Wall Properties Impacting Pretreatability, Ruminant Digestibility, and Cellulolytic Enzyme Hydrolyzability

Structural DifferencesGenotype Phenotype

– Ratio of cell types? • Epidermis• SclerenchymaEnvironmental/agronomic Sclerenchyma• Vascular bundle zone cells• Pith parenchyma

– Cell wall thickness

Environmental/agronomic– Harvest time/maturity

– N, water, environment,… – Cell wall thickness, composition, accessibility,…

Composition80

90

100

Unassigned

Ash

– Lignin, structural polysaccharides

pCA and FA30

40

50

60

70

Compo

sition

(wt %)

Ash

Water+EtOH ExtractivesAcetate

Lignin

Uronic Acids

Galactan

Mannan

Arabinan

MaizeSwitchgrass

– pCA and FA

– Acetyl 0

10

20

Corn stover Switchgrass

Arabinan

Xylan

Glucan

(Pioneer hybrid 36H56) (cv. Cave-in-Rock)

Maize (and Grass) Diversity

“Genetic diversity in maize results in wide phenotypic

Image: USDAImage: USDA‐‐ARSARS

results in wide phenotypic variation across strains”– High digestibility phenotype?

Substantial literature on– Substantial literature on ruminant digestibility

– Common themes: lignin content, ferulate content, , ,lignin structures?

• Differences in cell wall response to pretreatments?response to pretreatments?

Grass Diversity Studies GoalsGoals

Correlate compositional and structural properties to:structural properties to: (1) enzymatic hydrolysis yields and (2) response to pretreatments

Corn Stovers (Zea mays)52220 A632xM16S5A659 B14A

NC290A NC368 NK807 No.380

(2) response to pretreatments

– “Diversity panel” 30 maize lines

Other Grasses

B47 B66 B7 CH157 F431

Oh7B PHG86 W611S W64A W64A[8] bm1 s6– Switchgrass (Panicum virgatum) – 2 cultivars

– Miscanthus (Miscanthus x giganteus)

Sideoats grama (Bouteloua curtipendula)

F431 HuanyaoKy226 LH143 LP1NR HT

W64A[8] bm1 s6 W64A[8] bm2 s6W64A[8] bm3 s6 W64A[7] bm4 s8 Wf9 – Sideoats grama (Bouteloua curtipendula)

– Big bluestem (Andropogon gerardii)Mo5 N215

YE4

Grass Diversity Studies GoalsGoals

Correlate compositional and structural properties to:structural properties to: (1) enzymatic hydrolysis yields and (2) response to pretreatments

Corn Stovers (Zea mays)

(2) response to pretreatments

– “Diversity panel” 30 maize lines

Other Grasses– Switchgrass (Panicum virgatum) – 2 cultivars

– Miscanthus (Miscanthus x giganteus)

Sideoats grama (Bouteloua curtipendula)– Sideoats grama (Bouteloua curtipendula)

– Big bluestem (Andropogon gerardii)

Maize Cell Wall Digestibility Screening, Property Correlation, and Yield PredicationProperty Correlation, and Yield Predication

Diverse Mild Alkaline Pretreatment

Enzymatic Hydrolysis (30 mg protein/gSolids

GrassesPretreatment

(0.08 g NaOH/g, 80˚C, 1hr )

(30 mg protein/g glucan C‐Tec2, pH 5.0, 50˚C, 6 or 72 h)

Solids

Liquor

Water Retention Value lignin

Solubilized pCAand FA

Solids Hydrolysis Yields

Water Retention Value, lignin,xylan, pCA, FA, acetate

Multiple Linear Regression Models

Py‐MBMSg

Chemometric Models

18

pCA

Ferulate (FA) and p‐coumarate (pCA)GLBRC

10

12

14

16

nten

t (mg/g)

FA

14

16

18

GLBRCSwitchgrass

GLBRCMiscanthus

GLBRC Corn Stover

4

6

8

Cinn

amate Co

n

6

8

10

12

pCA (m

g/g)

Switchgrass “Cave‐In‐Rock”

Big Bluestem

0

2

W64A

F431 B47

Ky226

8]bm

1 S6

B66

LP1N

R HT

Mo5

7] bm4 S8

PHG86

8]bm

2 S6

CH157

01LFY/LFY …

NC2

90A B7

Wf9

NC3

68Oh7

BNo.380

A659

NK8

07N215

8]bm

3 S7

52220

UAN YAO

LH143

B14A

W611S YE4

0

2

4

0 5 10 15 20

Maize Diversity Set

Other Grasses

Sideoats Grama

y = 0.2743x + 8.0551R² = 0.1425

12

14

16

al biomass)W

64A[8 L

W64A[7

W64A[8

INB 10

W64A[8 HU

FA (mg/g)

• Range of pCA and FA contentsW k l i b CA d

4

6

8

10

tent (m

g/g origin

No Treatment

NaOH Treatment

• Weak correlation between pCA and FA contents

• Both are removed with mild NaOHy = 0.4911x + 2.6247

R² = 0.41030

2

4

0 5 10 15 20

FA co

n

pCA content (mg/g original biomass)

Both are removed with mild NaOHtreatment

40

45

50(m

g/g)

0.05

Acetyl and Arabinosyl Content

20

25

30

35

cetate Con

tent

0.035

0.04

0.045

ntent (g/g)

0

5

10

15

B 7 9 5 0 7 T 8 1 A S 6 9 7 6 7 A 6 8 3 6 5 O 0 4 A

Cell Wall A

c

0.02

0.025

0.03

Acetate Co

Range of substitutions from

Diverse range of acetyl contents

Oh7

BCH

157

A659

Mo5

52220

B47

LP1N

R HT

W64A[7] b

m4 S8

F431

INB 101LFY/LFY

W64A

W611S

B66

Wf9 B7

PHG86

NK8

07NC2

90A

Ky226

NC3

68LH

143

W64A[8]bm2 S6

N215

HUAN YAO

No.380

YE4

B14A

N l ti b t t l

0.0150.1 0.15 0.2 0.25 0.3

Xylan Content (g/g)0.06

0.25 – 0.60 Ac:Xyl (mol:mol)

• No correlation between acetyl content and xylose content

COOHMeOC

HO

HO

0.04

0.05

tent (g/g)

Monocots (grasses)

o

OHHO

o o

o

CH2OH

OH

OH

o

o o

HO

o

o

CH2OH

OH

OH

o

-(1→4)-β-D-Xylp-(1→4)-β-D-Xylp-(1→4)-β-D-Xylp-(1→4)-β-D-Xylp-(1→4)-β-D-Xylp-(1→4)-β-D-Xylp-

y = 0.1004x + 0.0262R² = 0.2775

0.02

0.03

rabino

se Con

t

Range of substitutions from

0

0.01

0.1 0.15 0.2 0.25 0.3

Ar

Xylan Content (g/g)

Range of substitutions from 0.19 – 0.28 Ara:Xyl (mol:mol)

Interactions Between Water and Pretreated Biomass

80%

100%

sion R2=0.900

Water Retention Value (g/g) 40%

Bou

nd

AHP Corn Stover

Does not distinguish f ifi

Yield

40%

60%

Glu

can

Con

vers R² = 0.995

20%

30%

ion

Enz

ymes

B from non-specific binding

e Hydrolysis

0%

20%7 D

ay G AHP SG

LHW-AHP SG

AHP CS

LHW-AHP CSCS Corn StoverSG Switchgrass

Williams and Hodge (2014). Cellulose. 21(1):221‐235

10%

Frac

ti

Glucose

0%1.4 2.0 2.6 3.2 3.8 4.4

Water Swelling

• WRV is a very good predictor of hydrolysis yields

0%2.0 2.6 3.2 3.8

Water SwellingWRV

WRV is a very good predictor of hydrolysis yields and cellulase adsorption for some conditions

• Potential for WRV to incorporate many structural• Potential for WRV to incorporate many structural features of the cell wall into single parameter

Water Retention Value (WRV) for Untreated and Mild‐NaOH Pretreated Samples

3.1

3.3

3.5

NaOH Treatment

No Treatment

and Mild NaOH Pretreated Samples

4

2.5

2.7

2.9

WRV

(g/g)

y = 1.2424x + 0.1559R² = 0.4509

3

3.5

reatmen

t

1.9

2.1

2.3W

2

2.5

WRV

, NaO

H Tr

GLBRCSwitchgrass

1.5

1.7

[8]bm256

7]bm

4 S8

HUANYA

OLH

143

52220

WF9

NO. 380

NC2

90N215

B66

B14A B7

A659

CH157

YE 4

Oh7

BNK8

07Mo5

PHG86

F431

LP1N

RB4

701LFY/LFY

Ky226

NC3

68W64A

1.51.5 1.7 1.9 2.1 2.3 2.5

WRV, No Treatment

g

GLBRC Miscanthus

W64A[

W64A[7 H

INB1

0 Weak correlation between WRV in untreated and pretreated

• Substantial increase in magnitude and range for WRV g gfollowing pretreatment

0.9NaOH Treatment

Enzymatic Hydrolysis Yields (72 h)

0.7

0.8

eld (g/g)

No Treatment

0.9

1

0 5

0.6

matic Glucose Yie

y = 0.0174x + 0.547R² = 0.5116

0.5

0.6

0.7

0.8

0.9

Yield (g/g)

0.4

0.5

Enzym

0.1

0.2

0.3

0.4

0 5

Glucose Y

0.3

W61

15

Mo5

5222

0

PHG86

B14A

NC3

68

CH15

7

HUANYA

O

B47

W64

A

W64

A[7]bm4 S8

W64

A[8]bm25

6

A65

9

N21

5

Ky22

6

NC2

90

WF9

LP1N

R

B66

INB1

01LFY/LFY

B7

LH14

3

NK8

07

Oh7

B

NO. 380

00 5 10 15 20

Glucose Release (mM) ‐‐‐‐ iWALL

W W I

• Wide range of responses to mild NaOH pretreatment

• Measurement independently validated by μ‐scale, high‐throughput pretreatment‐hydrolysis (iWALL)

Correlation Coefficients between Cell Wall Properties

35

40

45

50

55

Cell Wall PropertiesInitial: Untreated

Final: Alkaline pretreated

7 10 8 4 5 3 9 11 16 15 1 2 12 13 14 6

25

30

35

X 8

X 10

X 7 Positive correlation

X7. Initial acetate

X10. Solubilized pCA

X8 Initial pCA

p

X 3

X 5

X 4

X 8X8. Initial pCA

X4. Final xylan

X5. Initial lignin

X3. Initial xylan

X9 I iti l FA

Y 3

Y 4

X 11

X 9X9. Initial FA

X11.Solubilized FA

Y4. Final 72‐hr glucose yield


Y 1

X 12

X 2

X 1 Negative correlation

X1. Initial WRV

X2. Final WRV

X12. S/G Ratio

Y1. Initial 6‐hr glucose yield

X 7 X 10 X 8 X 4 X 5 X 3 X 9 X 11 Y4 Y3 X 1 X 2 X 12 Y1 Y2 X 6

X 6

Y 2

Y . Initial 6 hr glucose yield


X6. Final lignin

Correlation Coefficients between Cell Wall Properties

35

40

45

50

55

Cell Wall PropertiesInitial: Untreated


7 10 8 4 5 3 9 11 16 15 1 2 12 13 14 6

25

30

35

X 8

X 10


X7. Initial acetate


X8 Initial pCA

p

X 3

X 5

X 4

X 8X8. Initial pCA

X4. Final xylan

X5. Initial lignin

X3. Initial xylan

X9 I iti l FA

Y 3

Y 4

X 11

X 9X9. Initial FA

X11.Solubilized FA



Y 1

X 12

X 2


X1. Initial WRV

X2. Final WRV

X12. S/G Ratio



X 6

Y 2



X6. Final lignin

WRV vs. 6‐hr Untreated Hydrolysis Yields

35

40

45

50

55Initial: Untreated


7 10 8 4 5 3 9 11 16 15 1 2 12 13 14 6

25

30

35

X 8

X 10


X7. Initial acetate


X8 Initial pCA

p

X 3

X 5

X 4

X 8X8. Initial pCA

X4. Final xylan

X5. Initial lignin

X3. Initial xylan

X9 I iti l FA

Y 3

Y 4

X 11

X 9X9. Initial FA

X11.Solubilized FA



Y 1

X 12

X 2


X1. Initial WRV

X2. Final WRV

X12. S/G Ratio



X 6

Y 2



X6. Final lignin

Acetyl vs. 6‐hr Untreated Hydrolysis Yields

35

40

45

50



7 10 8 4 5 3 9 11 16 15 1 2 12 13 14 6

25

30

35

X 8

X 10


X7. Initial acetate


X8 Initial pCA

p

X 3

X 5

X 4

X 8X8. Initial pCA

X4. Final xylan

X5. Initial lignin

X3. Initial xylan

X9 I iti l FA

Y 3

Y 4

X 11

X 9X9. Initial FA

X11.Solubilized FA



Y 1

X 12

X 2


X1. Initial WRV

X2. Final WRV

X12. S/G Ratio



X 6

Y 2



X6. Final lignin

pCA vs. 6‐hr Untreated Hydrolysis Yields

35

40

45

50



7 10 8 4 5 3 9 11 16 15 1 2 12 13 14 6

25

30

35

X 8

X 10


X7. Initial acetate


X8 Initial pCA

p

X 3

X 5

X 4

X 8X8. Initial pCA

X4. Final xylan

X5. Initial lignin

X3. Initial xylan

X9 I iti l FA

Y 3

Y 4

X 11

X 9X9. Initial FA

X11.Solubilized FA



Y 1

X 12

X 2


X1. Initial WRV

X2. Final WRV

X12. S/G Ratio



X 6

Y 2



X6. Final lignin

Lignin vs. 6‐hr Untreated Hydrolysis Yields

35

40

45

50



7 10 8 4 5 3 9 11 16 15 1 2 12 13 14 6

25

30

35

X 8

X 10


X7. Initial acetate


X8 Initial pCA

p

X 3

X 5

X 4

X 8X8. Initial pCA

X4. Final xylan

X5. Initial lignin

X3. Initial xylan

X9 I iti l FA

Y 3

Y 4

X 11

X 9X9. Initial FA

X11.Solubilized FA



Y 1

X 12

X 2


X1. Initial WRV

X2. Final WRV

X12. S/G Ratio



X 6

Y 2



X6. Final lignin

Xylan vs. 6‐hr Untreated Hydrolysis Yields

35

40

45

50



7 10 8 4 5 3 9 11 16 15 1 2 12 13 14 6

25

30

35

X 8

X 10


X7. Initial acetate


X8 Initial pCA

p

X 3

X 5

X 4

X 8X8. Initial pCA

X4. Final xylan

X5. Initial lignin

X3. Initial xylan

X9 I iti l FA

Y 3

Y 4

X 11

X 9X9. Initial FA

X11.Solubilized FA



Y 1

X 12

X 2


X1. Initial WRV

X2. Final WRV

X12. S/G Ratio



X 6

Y 2



X6. Final lignin

S/G Ratio vs. 6‐hr Untreated Hydrolysis Yields

35

40

45

50

55

Hydrolysis YieldsInitial: Untreated


7 10 8 4 5 3 9 11 16 15 1 2 12 13 14 6

25

30

35

X 8

X 10


X7. Initial acetate


X8 Initial pCA

p

X 3

X 5

X 4

X 8X8. Initial pCA

X4. Final xylan

X5. Initial lignin

X3. Initial xylan

X9 I iti l FA

Y 3

Y 4

X 11

X 9X9. Initial FA

X11.Solubilized FA



Y 1

X 12

X 2


X1. Initial WRV

X2. Final WRV

X12. S/G Ratio



X 6

Y 2



X6. Final lignin

S/G ratio vs. 72‐hr Pretreated Hydrolysis Yields

35

40

45

50

55



7 10 8 4 5 3 9 11 16 15 1 2 12 13 14 6

25

30

35

X 8

X 10


X7. Initial acetate


X8 Initial pCA

p

X 3

X 5

X 4

X 8X8. Initial pCA

X4. Final xylan

X5. Initial lignin

X3. Initial xylan

X9 I iti l FA

Y 3

Y 4

X 11

X 9X9. Initial FA

X11.Solubilized FA



Y 1

X 12

X 2


X1. Initial WRV

X2. Final WRV

X12. S/G Ratio



X 6

Y 2



X6. Final lignin

Final Lignin vs. 72‐hr Pretreated Hydrolysis Yields

35

40

45

50

55



7 10 8 4 5 3 9 11 16 15 1 2 12 13 14 6

25

30

35

X 8

X 10


X7. Initial acetate


X8 Initial pCA

p

X 3

X 5

X 4

X 8X8. Initial pCA

X4. Final xylan

X5. Initial lignin

X3. Initial xylan

X9 I iti l FA

Y 3

Y 4

X 11

X 9X9. Initial FA

X11.Solubilized FA



Y 1

X 12

X 2


X1. Initial WRV

X2. Final WRV

X12. S/G Ratio



X 6

Y 2



X6. Final lignin

Solubilized FA vs. 72‐hr Pretreated Hydrolysis Yields

35

40

45

50

55



7 10 8 4 5 3 9 11 16 15 1 2 12 13 14 6

25

30

35

X 8

X 10


X7. Initial acetate


X8 Initial pCA

p

X 3

X 5

X 4

X 8X8. Initial pCA

X4. Final xylan

X5. Initial lignin

X3. Initial xylan

X9 I iti l FA

Y 3

Y 4

X 11

X 9X9. Initial FA

X11.Solubilized FA



Y 1

X 12

X 2


X1. Initial WRV

X2. Final WRV

X12. Predicted S/G



X 6

Y 2



X6. Final lignin

Solubilized pCA vs. 72‐hr Pretreated Hydrolysis Yields

35

40

45

50

55



7 10 8 4 5 3 9 11 16 15 1 2 12 13 14 6

25

30

35

X 8

X 10


X7. Initial acetate


X8 Initial pCA

p

X 3

X 5

X 4

X 8X8. Initial pCA

X4. Final xylan

X5. Initial lignin

X3. Initial xylan

X9 I iti l FA

Y 3

Y 4

X 11

X 9X9. Initial FA

X11.Solubilized FA



Y 1

X 12

X 2


X1. Initial WRV

X2. Final WRV

X12. S/G Ratio



X 6

Y 2



X6. Final lignin

Correlation between Individual Cell Wall Properties and Hydrolysis YieldsProperties and Hydrolysis Yields

p‐values of individual linear regressions between single cell wall properties and hydrolysis yieldinitialWRV

FinalWRV

InitialXylan

FinalXylan

InitialLignin

FinalLignin

InitialAcet.

InitialpCA

InitialFA

Solub. pCA

Solub.FA

S/G Ratio

X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12

Raw

Biom

ass 6‐hr

yieldY1 0.039 0.729 0.028 0.116 0.037 0.596 0.004 0.002 0.594 0.001 0.206 0.005

72‐hryield

Y2 0.376 0.937 0.358 0.192 0.130 0.886 0.084 0.078 0.551 0.128 0.579 0.087

NoCorrelation

B yield

Pretreated

Biom

ass 6‐hr

yieldY3 0.052 0.550 0.646 0.198 0.478 0.548 0.540 0.994 0.943 0.924 0.876 0.966

72‐hr yield

Y4 0.934 0.637 0.496 0.158 0.373 0.004 0.260 0.635 0.114 0.022 0.013 0.469 95% CL Correlation

Untreated maize:• 6‐hr yield (hydrolysis rate): highly correlated with the initial composition• 72‐hr yield (hydrolysis extent): less correlated with the initial composition72 hr yield (hydrolysis extent): less correlated with the initial composition

NaOH pretreated maize:6 h i ld (h d l i ) l d i h ll ll i• 6‐hr yield (hydrolysis rate): not correlated with any cell wall properties

• 72‐hr yield (hydrolysis extent, pretreatment efficiency): correlated with the removal of lignin, pCA and FA.

Multiple Linear Regression ModelsModels based on Cp and BIC criteria and the modified models.Models based on Cp and BIC criteria and the modified models.

Untreated Biomass Pretreated Biomass6‐hr yield 72‐hr yield 6‐hr yield 72‐hr yield

Top 3 models b d

X3+X6+X7+X9+X11+X12 X5+X12 X1 X5+X6+X10+X11+X12based on Cpand BIC criteria

X3+X5+X6+X7+X9+X11+X12 X8 X4 X1+X5+X6+X10+X11+X12X3+X5+X10 X3+X5+X12 X1+X5 X1+X5+X10+X11+X12

Example model X1+X3+X5+X10 X5+X12 N/A X6+X10+X11+X12

S l bili d CA

0.8

1

Yields

No Treatment, 6‐hr

No Treatment, 72‐hr

NaOH Treatment, 72‐hr

Solubilized pCA

Solubilized FA

0 4

0.6

d Glucose Y

Linear model:

Y = Xβ + ε0.2

0.4

Pred

icted

WRV

S/G RatioY Xβ ε

00 0.2 0.4 0.6 0.8 1

Measured Glucose Yields Initial XylanInitial Lignin

Final Lignin

Summary: Correlation Models• Diverse range of properties for maize lines• Many correlations between properties and y p pbetween properties and yields

• No treatment: WRV Initial Initial

hWRVXylan Lignin

Initial Acetate

Initial pCA

S/G Ratio

6‐hr yield

• NaOH Pretreatment: Solub. pCA

Final Lignin

Solub. S/G

72‐hr yield

• Capable of developing linear models to correlate

Solub.FA

S/G Ratio

yield

p p gproperties to hydrolysis yields





Pyrolysis Molecular Beam Mass Spectrometry (py‐MBMS)Spectrometry (py MBMS)

• Collaboration with Robert Sykes at NREL

• All pyrolysis products directly to MS (no GC separation)All pyrolysis products directly to MS (no GC separation)

• Chemometric models to extract information from datasets

Principle component analysis partial least squares– Principle component analysis, partial least squares,…

– Used in the past for S/G ratio, composition

15

20

25

Aspen

Hybrid poplar

Switchgrass

Corn StoverSyringyl‐

Example data

bund

ance

5

10

15 Corn StoverFerulatep‐coumarate derived monomers

malized

ab

025 50 75 100 125 150 175 200 225 250

m/z

Nor

Chemometric AnalysisPyrolysis Cell wallPyrolysis‐MBMS spectra

PCA analysis

Principle components

PLS regression

Cell wall properties(Lignin, pCA, FA, Hydrolysis Yields)

# Sample 51 m/z …… 450 m/z

Biomass 1 … ……. …

# Sample PC1 PC2 … PC10

Biomass1 … … … …

# Sample Lignin content

Biomass1

Py‐MBMS spectra Principle components (PCs) Measurements

p Hydrolysis Yields)

Biomass 2.....

… …… ... Biomass 2.....

… … … …PCA analysis

Biomass 2.....

PLS regression

.

.

.

.

.Biomass N

.

.

.

.

.Biomass N

.

.

.

.

.Biomass N

Nobservations

400 variables

Nobservations

10 variables

Nobservations

1 variable

observations

Prediction of Lignin Content across Diverse Species and Maize LinesDiverse Species and Maize Lines

0.25

nt

MiscanthusSide oats grama Big bluestem

Aspen

0 20

0.25

ent

R2 = 0.97

0.15

0.20

Lignin Con

ten

biom

ass)hybrid stover

Switchgrass

bm1 stover

bm2 stoverbm3 stoverbm4 stover

g

0.15

0.20

d Lignin Con

teg biom

ass)

R2 = 0.56

0.10

Pred

icted

(g/g

Maize diversity set

0.10

Pred

icted

(g/g

Diverse Grasses (and Aspen)

0.050.05 0.10 0.15 0.20 0.25

Measured Lignin Content (g/g biomass)

0.050.05 0.10 0.15 0.20 0.25

Measured Lignin Content (g/g biomass)

• Good prediction of lignin content across species

• Less effective prediction for maize diversity setLess effective prediction for maize diversity set– Measured lignin: Klason lignin corrected by ash

Prediction of pCA and FA Content

15

20

ontent

ss)

15

20

nten

t ss)

10

15

icted pC

ACo

mg/g biom

a

10

15

dicted

FA Co

mg/g biom

as

55 10 15 20

Pred( m

55 10 15 20

Pred (m

M d l bl di CA FA

5 10 15 20

Measured pCA Content (mg/g biomass)

5 10 15 20

Measured FA Content (mg/g biomass)

• Model able to predict pCA, FA content

• Mass peaks at 120 and 150 m/z are strongly correlated to pCA and FA content, respectively

Prediction of Hydrolysis YieldsUntreated Maize NaOH‐Pretreated Maize

6‐hr hydrolysis yield 72‐hr hydrolysis yield 72‐hr hydrolysis yield

NaOH Pretreated Maize

R² = 0.7513 R² = 0.4239 R² = 0 37450.6

can)

0 9

1

an)

0.6

eld R 0.4239 R = 0.3745

0.5

2‐hr Yield (g/g gluc

0.7

0.8

0.9

‐hr Yield (g/g gluca

0.3

0.4

0.5

ed 6‐hr Glucose Yie

0.3

0.4Pred

icted 72

0.4

0.5

0.6

Pred

icted 72

‐

0

0.1

0.2

0 0 2 0 4 0 6

Pred

icte

• Accurate prediction on 6‐hr yield and less accurate

0.3 0.4 0.5 0.6

Measured 72‐hr Glucose Yield0.4 0.5 0.6 0.7 0.8 0.9 1

Measured 72‐hr Glucose Yield

0 0.2 0.4 0.6

Measured 6‐hr Glucose Yield

prediction on 72‐hr yield for untreated maize:– Composition sets the initial recalcitrance for hydrolysis

• Poor prediction on pretreated maize– Removal of pCA and FA

Summary: py‐MBMS models• Model prediction of cell wall composition:

– Lignin content

–pCA, FA content

• Model prediction of hydrolysis yields with or without alkaline pretreatments:

–Accurate prediction for 6‐hr yield

– Less accurate prediction for 72‐hr yield forLess accurate prediction for 72 hr yield for untreated and pretreated maize

AcknowledgementsResearch Group: Collaborators:Research Group:

Dr. Tongjun Liu Ryan StoklosaMuyang Li Zhenglun LiD Willi Ch l Ch

Collaborators:Natalia de Leon, U. WisconsinShawn Kaeppler, U. Wisconsin

Dan Williams Charles ChenJacob Crowe John Groetsch

Robert Sykes, NREL

Funding:Funding:• DOE, BER DE‐FC02‐07ER64494• NSF CBET‐1336622

Documents

1 May, 2014 36 SIM SBFC - Chemical Engineering and ... · throughput pretreatment‐hydrolysis (iWALL) Correlation Coefficients between CellWall Properties 35 40 45 50