1Department of Chemical & Environmental Engineering, Center for Environmental Research and Technology, University of California, Riverside,

BioEnergy Science Center, Riverside, CA

2Environmental Sciences Division and Chemical Sciences Division,

Oak Ridge National Laboratory,

BioEnergy Science Center, Oak Ridge, TN

3School of Chemistry and Biochemistry,

Georgia Institute of Technology,

BioEnergy Science Center, Atlanta, GA

October 25, 2011

Solubilization of lignin and hemicellulose during hydrothermal pretreatment

Heather L. McKenzie1, Nancy L. Engle2, Joshua F. Emory2, Marcus B.

Foston3, Arthur Ragauskas3, Bruce A. Tomkins2, Timothy

Tschaplinski2, Gary J. Van Berkel2, Charles E. Wyman1

2

Acknowledgments

ArborGen LLCBrookhaven National Laboratory

Verenium Corporation

Georgia Institute of TechnologyGeorgia Institute of TechnologyMascoma Corporation

National Renewable Energy Laboratory

Oak Ridge National LaboratoryOak Ridge National LaboratoryDartmouth College

Samuel Roberts Noble FoundationUniversity of Georgia

University of TennesseeWashington State University

North Carolina State UniversityVirginia Polytechnic Institute and State

UniversityCornell University

University of CaliforniaUniversity of California--RiversideRiversideUniversity of Minnesota

The BioEnergy Science Center is a U.S. Department of Energy Bioenergy

Research Center supported by the Office of Biological and Environmental

Research in the DOE Office of Science.

3

Outline

• Introduction and objectives

• Materials and methods

• Results and discussion

– Influence of flow rate

– Release of lignin and phenols from P. trichocarpaand isolated lignin

– Changes in molecular weight and composition of lignin

– Release of xylan and xylooligomers from P. trichocarpa and Birchwood xylan

• Summary

4

Introduction and objectives

5

Introduction

• Ethanol from cellulosic biomass is one option for renewable, low GHG emission transportation energy.

Overcome

natural recalcitrance

of biomass

Make enzymes.

Breakdown polymers

glucan and xylan to

sugars, glucose and

xylose.

Ferment all sugars

Process boundaries

Process heat,

electricity

Cellulosic

biomass

Pretreatment

Utilities Fuel ethanol

Exported

electricity

Lignin, etc

Ethanol recovery

Residue processing

Process effluents

Biological steps:Enzyme production

Hydrolysis

Fermentation

Overcome

natural recalcitrance

of biomass

Make enzymes.

Breakdown polymers

glucan and xylan to

sugars, glucose and

xylose.

Ferment all sugars

Process boundaries

Process heat,

electricity

Cellulosic

biomass

Pretreatment

Utilities Fuel ethanol

Exported

electricity

Lignin, etc

Ethanol recovery

Residue processing

Process effluents

Biological steps:Enzyme production

Hydrolysis

Fermentation

Cellulose (primarily glucan)

Hemicellulose (primarily xylan)

Lignin

H-lignin

G-lignin

S-lignin

Chemical structures from Fengel and

Wegener (1984).

Block diagram courtesy of Dr. Wyman.

• Release of hemicellulose and lignin during

pretreatment is poorly understood.

6

Objectives

• Perform hydrothermal flowthrough pretreatment in order to:

– compare the release of lignin and lignin derivatives from poplar and isolated lignin.

– compare the release of xylose and xylooligomers from poplar and birchwood xylan.

• These comparisons will provide indications of the role of lignin-carbohydrate interactions.

7

Materials and methods

8

Materials• Substrates:

- Populus trichocarpa (Pt).- Lignin isolated from Pt by J. Seokwon and Dr. S.

Cao (IL).

- Birchwood xylan (BX).• Left: Fluidized sand bath (Techne, Princeton, NJ).

• Middle: Custom 10 mL batch reactor.• Right: Custom flowthrough reactor system.

9

Methods: Pretreatment

• Solids were loaded and sealed into reactor.

• Pretreatment was conducted with 180oC water at conditions summarized below.

• Liquid samples were collected continuously during run.

• Residual solids were collected by filtration and massed at the end of a run.

MaterialMass per

run (g)Flow rate (ml/min)

Temperature (oC)

Time(min)

Sampling Period (min)

Pt 1.00 20-25 180 0-10 2

BX 1.00 25 180 0-10 2

IL 0.71 0-20 180 12-192 3

10

Methods: Analytical procedures

Measurement Procedure Performed by

Sugar monomer and K-lignin content of

hydrolysate and the residual solids.

Strong acid hydrolysis with HPLC

H. McKenzie, UCR

Mn and Mw of ligninGel permeation

chromatography (GPC)Dr. Foston, GIT

Functional groups in lignin

Heteronuclear single quantum coherence (HSQC)

Dr. Foston, GIT

Phenols in liquid hydrolysate

Gas chromatography-mass spectrometry (GCMS)

N. Engle and Dr. Tschaplinski,

ORNL.

Xylooligomers in liquid hydrolysate

Ultra-high pressure liquid chromatography (UPLC)

Drs. Emory, Tomkins, and Van

Berkel, ORNL

11

Results and discussion

12

0 2 4 6 8 100

10

20

30

40

50

60

70

80

90

100Xylan Yield

25 mL/min

20 mL/min

Lignin Removal

20 mL/min

25 mL/min

Glucan Yield

25 mL/min

20 mL/min

Glu

can a

nd X

yla

n Y

ield

(w

t%)

Pretreatment Time (min)

0

10

20

30

40

50

60

70

80

90

100

Influence of flow rate

• Pt was pretreated at 180oC

for 10 minutes with water at 20 ml/min (triplicate) and 25 ml/min.

• Glucan and xylan yields and percent lignin removal

are plotted as a function of time.

• Reducing the flowrate from 25 ml/min to 20 ml/min did not affect final yields.

13

0

10

20

30

40

50

Pt

25

180

10

3.36F

ractio

n S

olu

bili

ze

d (

wt%

)

Material

Q(ml/min)

T(oC)

t(min)

IL

20

180

12

3.43

IL

20

180

192

4.64

IL

0

180

12

3.43

• More lignin is removed from

IL during flowthrough pretreatment than during batch pretreatment.

• This indicates that lignin fragments are removed from

the reactor before they can return to the solid phase.

• The flowthrough reactor improves understanding of

product evolution as a function of time.

Lignin removal from P. trichocarpa and Isolated lignin

IL

Batch180oC

12 min

IL

20 ml/min180oC

12 min

14

0

10

20

30

40

50

Pt

25

180

10

3.36

Fra

ctio

n S

olu

bili

zed

(w

t%)

Material

Q(ml/min)

T(oC)

t(min)

IL

20

180

12

3.43

IL

20

180

192

4.64

IL

0

180

12

3.43

Lignin removal from P. trichocarpa and Isolated lignin

• At approximately equal severity, more lignin is removed from Pt than from IL.

Pt

25 ml/min180oC

10 min

log(Ro)=3.46

IL20 ml/min

180oC

12 min

log(Ro)=3.43

15

• Greater release of monomers from Pt. • This may be due to extractives in raw Pt.

Release of phenols per gram of raw lignin

0 2 4 6 8 10 12

0.000

0.025

0.050

0.075

0.100

0.125

0.150

0.175

0.200

0.225

0.250

Cu

mu

lative

re

lea

se

of p

he

no

l m

eta

bo

lites

(g/g

ra

w lig

nin

)

Time (t, min)

sinapyl alcohol

hydroquinone

coniferyl alcohol

4-hydroxybenzoic acid

Total

mp/m

raw=0.067+0.018t

R2=0.97

0 2 4 6 8 10 12

0.000

0.025

0.050

0.075

0.100

0.125

0.150

0.175

0.200

0.225

0.250

vanillin

syringaldehyde

hydroquinone

sinapyl alcohol

coniferyl alcohol

4-hydroxybenzoic acid

Cu

mu

lative

re

lea

se

of p

he

no

l m

eta

bo

lite

s

(g/g

ra

w lig

nin

)

Time (t, min)

Total

mp/m

raw=0.016+0.012t

R2=0.94

GCMS performed by:

N. Engle and Dr. Tschaplinski, ORNL.

from P. trichocarpa from Isolated lignin

16

• IL’s rate of monomer production from per gram of raw

lignin is 2/3 of that of Pt.

Release of phenols per gram of raw lignin

0 2 4 6 8 10 12

0.000

0.025

0.050

0.075

0.100

0.125

0.150

0.175

0.200

0.225

0.250

Cu

mu

lative

re

lea

se

of p

he

no

l m

eta

bo

lites

(g/g

ra

w lig

nin

)

Time (t, min)

sinapyl alcohol

hydroquinone

coniferyl alcohol

4-hydroxybenzoic acid

Total

mp/m

raw=0.067+0.018t

R2=0.97

0 2 4 6 8 10 12

0.000

0.025

0.050

0.075

0.100

0.125

0.150

0.175

0.200

0.225

0.250

vanillin

syringaldehyde

hydroquinone

sinapyl alcohol

coniferyl alcohol

4-hydroxybenzoic acid

Cu

mu

lative

re

lea

se

of p

he

no

l m

eta

bo

lite

s

(g/g

ra

w lig

nin

)

Time (t, min)

Total

mp/m

raw=0.016+0.012t

R2=0.94

from P. trichocarpa from Isolated lignin

mp/mraw=0.067+0.018t mp/mraw=0.016+0.012t

17

P. Trichocarpa vs. Isolated lignin

• Given:

• greater lignin removal from Pt than IL,

• greater phenol production from Pt than IL, and

• greater rate of phenol production from Pt than IL,

• it seems that lignin-carbohydrate interactions promote the release of lignin.

18

0 1 2 3

0

1000

2000

3000

4000

5000

6000

7000

We

igh

t A

ve

rag

ed

Mo

lecu

lar

We

igh

t (M

w, g

/mo

l)

Q(ml/min)

T(oC)

t(min)

Raw 20

180

12

20

180

192

0

180

12

0 1 2 3

0

500

1000

1500

2000

2500

3000

Nu

mb

er

Ave

rag

ed

Mo

lecu

lar

We

igh

t (M

n, g

/mo

l)

RawQ(ml/min)

T(oC)

t(min)

20

180

12

20

180

192

0

180

12

• The decrease in Mn and Mw indicates that IL undergoes depolymerization reactions.

Average molecular weight of lignin

Weight averaged

molecular weight

Number averagedmolecular weight

Raw

FlowB

atc

h

Raw

Flow

Batc

h

GPC by Dr. Foston, GIT

19

Raw lignin- aliphatic region

Bonds in isolated lignin

B

β-5/α-O-4 phenyl-coumararan

Cspirodienone

(Samuel et al., 2010)

50

60

70

80

2.43.24.04.85.6

1H (ppm)

13C

(ppm

)

Methoxy

Bβ

Aα

Aγ

Bγ

Carbohydrates

AβCα

Bβ

Methoxy group

13C

(ppm

)1H(ppm)

HSQC by Dr. M. Foston,

GIT.

A

β-O-4 ether

20

Bonds in isolated lignin

20 ml/min

180oC

12 min

Batch

180oC

12 min

50

60

70

80

2.43.24.04.85.6

1H (ppm)

13C

(ppm

)

Methoxy

Aα

Aγ

Bγ

AβCα

Bβ

50

60

70

80

2.43.24.04.85.6

1H (ppm)1

3C

(ppm

)

Methoxy

Bβ

Aα

Aγ

Bγ

Carbohydrates

AβCα

Bβ

Raw

50

60

70

80

2.43.24.04.85.6

1H (ppm)

13C

(ppm

)

Methoxy

Aα

Aγ

Bγ

20 ml/min

180oC

192 min

50

60

70

80

2.43.24.04.85.6

1H (ppm)

13C

(ppm

)

Methoxy

Bβ

Aα

Aγ

Bγ

AβCα

Bβ

21

Lignin functional groups

Raw Lignin- Aromatic Region

GGuaiacyl unit

SSyringyl unit

S’Oxidized syringyl unit,

Cα=O

(Samuel et al., 2010)

100

120

110

140

130

7.08.0 6.0

1H (ppm)

13C

(ppm

)

PB2,6

G6

G5

G2

S2,6S’2,6

13C

(ppm

)1H(ppm)

OH

ORO

6 2

35P

PBp-Hydroxybenzoate

unit

HSQC by Dr. M. Foston,

GIT.

22

Lignin functional groups

Run 6

Batch

180oC

12 min

Run 4

20 ml/min

180oC

12 min

Run 5

20 ml/min

180oC

192 min

Raw100

120

110

140

130

7.08.0 6.0

1H (ppm)13C

(ppm

)

PB2,6

G6

G5

G2

S2,6S’2,6

100

120

110

140

130

7.08.0 6.0

1H (ppm)

13C

(ppm

)

PB2,6

G6

G5

G2

S2,6

S’2,6

100

120

110

140

130

7.08.0 6.01H (ppm)

13C

(ppm

)

PB2,6

G6

G5

G2

S2,6

100

120

110

140

130

7.08.0 6.0

1H (ppm)

13C

(ppm

)

PB2,6

G6

G5

G2

S2,6

S’2,6

23

100

120

110

140

130

7.08.0 6.0

1H (ppm)13C

(ppm

)

PB2,6

G6

G5

G2

S2,6S’2,6

100

120

110

140

130

7.08.0 6.0

1H (ppm)

13C

(ppm

)

PB2,6

G6

G5

G2

S2,6

S’2,6

0 1 2 3

0

500

1000

1500

2000

2500

3000

Nu

mb

er

Ave

rag

ed

Mo

lecu

lar

We

igh

t (M

n, g

/mol)

RawQ(ml/min)

T(oC)

t(min)

log(Ro)

20

180

12

3.43

20

180

192

4.64

0

180

12

3.43

Evidence of lignin reactions

20 ml/min

180oC

12 min

Raw

• Based on drop in functional group signal intensity, expect Mn and Mw

to decrease substantially.

• The molecular weight drops but not as much as expected from HSQC

spectra.

24

Evidence of lignin reactions

• The retention of molecular weight, poor solubility, and low signal to noise ratio in the spectra all suggest large polymers in pretreated lignin solids.

• These observations coupled with the low intensity of characteristic lignin bonds suggest formation of new,

unknown bonds in the solids.

25

0 2 4 6 8 100.0

0.2

0.4

0.6

0.8

1.0

Norm

aliz

ed m

ass o

f re

leased x

yla

n

(mx,

l/mx,

raw,

g/g

raw

xyla

n)

Pretreatment time (minutes)

Xylan released from

Birchwood xylan

Xylan released from

P. trichocarpa

Release of xylan from P. trichocarpa and Birchwood xylan

• Pt and BX were pretreated by flowing water at 25 ml/min at 180oC through the solids.

• After 10 minutes, 71% and 100% of the xylan from Ptand BX had been released to liquid phase, respectively.

26

Distribution of xylooligomers from P. trichocarpa and Birchwood xylan

HP 0-2 2-4 4-6 6-88-10HP 0-2 2-4 4-6 6-88-100

10

20

30

40

50

60

70

80

90

100

Dis

trib

utio

n o

f xylo

olig

om

ers

(w

t% x

ylo

se

)

Sampling period (minutes)

• Oligomers with DP<6 account for a larger fraction of the xylooligomers released from poplar than from xylan.

From poplar:

DP=1

DP=2

DP=3

DP=4

DP=5

DP=6

DP>7

From birchwood xylan:

DP=1

DP=2

DP=3

DP=4

DP=5

DP=6

DP>7

UPLC performed by:

Drs. Emory, Tomkins, and

Van Berkel, ORNL

27

P. Trichocarpa vs. Birchwood xylan

• Given:

• the reduction in xylan removal from Pt, and

• the high fraction of low DP oligomers in xylan released from Pt,

• it seems that the lignin-carbohydrate interactions limit the release of high DP xylooligomers.

• Consistent with the hypothesis that lignin-carbohydrate

bonds reduce xylooligomer solubility (Yang and Wyman, 2008).

28

Summary

29

Summary

• In order to better understand the release of xylan and lignin from biomass, P. trichocarpa, isolated lignin, and birchwood xylan were subjected to batch and

flowthrough pretreatment with water at 180oC.

• The liquid hydrolysate and residual solids were

analyzed with numerous techniques.

30

Summary, continued

• Comparison of lignin removal from Pt and IL:

–More lignin was removed from Pt.

–Greater phenol production from Pt.

–Greater rate of phenol production in Pt.

• Strong evidence of lignin reactions during pretreatment:

–Release of phenol monomers.

–Loss of characteristic bonds and functional groups.

–Retention of molecular weight.

31

Summary, continued

• Comparison of xylan removal from Pt and BX:

–Less xylan was removed from Pt.

–More xylooligomers with DP<6 were released from Pt.

• The substantial differences in lignin and xylan removal from the native biomass and model substrates suggest that lignin-carbohydrate interactions appear to enhance

lignin removal while limiting the release of xylan, especially large xylooligomers.

32

Thank you.Questions?