Possible mechanisms for acid-catalyzed condensation/depolymerization of lignin

(Li et al., 2007. Biores.Tech. 98; 3061-68.

Structural changes in lignin and cellulose resulting from the two-

step dilute acid pretreatment of Loblolly pine

Poulomi Sannigrahi1, Arthur J. Ragauskas1, Stephen J. Miller2

1. School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA

2. Chevron Energy Technology Company, Richmond, CA

Funding from Chevron is gratefully acknowledged

BIOMASS FEEDSTOCK

Cellulose: Solid-state CP/MAS 13C NMR

CONCLUSIONS�Effects of acid pretreatment on cellulose

�Increase in cellulose crystallinity�Preferential degradation of amorphous cellulose

�Increase in proportion cellulose Iβ�May lead to inhibition of enzymatic hydrolysis

�Concurrent decrease in Iα and Iα+β�Possible conversion of cellulose Iα to Iβ

�Effects of acid pretreatments on lignin�Increase in condensed aromatic C

�Decrease in β-O-4 linkages

�Depolymerization by fragmentation of β-O-4

�Pretreatment conditions should be optimized to�Retain/increase the proportion of reactive cellulose

�Prevent lignin condensation

ABSTRACTTwo-step dilute sulfuric acid pretreatment was performed on Loblolly pine to enhance the overall efficiency of the enzymatic conversion of lignocellulosic biomass to monomeric sugars

prior to their fermentation to bioethanol. Lignin, cellulose and hemicellulose the major

components of lignocellulosic biomass, are closely associated with each other at the plant cell level. This close association, together with the partly crystalline nature of cellulose

protects it from enzymatic hydrolysis of native biomass. In the overall conversion of biomass to bioethanol, the structure of lignin is also of importance as it may physically hinder cellulase

access to cellulose microfibrils and participate in non-productive binding to enzymes. Detailed structural characterization of cellulose and milled wood lignin isolated

from Loblolly pine before and after the two-step dilute sulfuric acid pretreatment elucidates

the modifications taking place in these biomolecules as a result of this pretreatment. Solid-state 13C NMR spectroscopy coupled with line shape analysis has been used to determine

cellulose crystallinity and ultrastucture. The results indicate an increase in the degree of crystallinity and reduced relative proportion of less ordered cellulose allomorphs following

acid pretreatment. These changes may be attributed to a preferential degradation of

amorphous cellulose and less ordered crystalline forms during the high temperature treatment. Milled wood lignin structural elucidation by quantitative 13C and 31P NMR reveals

an increase in the degree of condensation of lignin due to the pretreatment. This is accompanied by a decrease in the number of β-O-4 linkages which are fragmented and

subsequently recondensed during high temperature acid-catalyzed reactions. The impact of these changes on pine recalcitrance and enzymatic deconstruction will be reviewed.

Reactor used for pretreatments

Distribution map of Loblolly Pine

� Widely prevalent softwood species in

Eastern US

� Mature tree from Baldwin Co., GA

sectioned, debarked and chipped� Chips stored at < -5 °C� Composite sample used for all analyses

Atlanta

Baldwin Co.

CAUSES OF BIOMASS RECALCITRANCE

� Close association of cellulose, hemicellulose and

lignin at the plant cell wall level

� Partly crystalline nature of cellulose

� Lignin-carbohydrate complexes

� Non-productive binding of lignin to enzymes

PRETREATMENTS ARE NECESSARY FOR THE EFFICIENT

CONVERSION OF BIOMASS TO ETHANOL

Results from Scifinder Web searchon “Biomass Pretreatment” journal articles and patents in English

Increasing efforts to understand andovercome biomass recalcitrance

MOTIVATION

� Dilute sulfuric acid pretreatment is an established

method for the pretreatment of softwoods

� Effect of this pretreatment on Cellulose and

Lignin structure is not known

� Effect of cellulose crystallinity on rates of

enzymatic hydrolysis is debated

FEEDSTOCK COMPOSITIONLIGNIN: 29 %CELLULOSE: 54 %HEMICELLLULOSE: 14%

Two-step dilute sulfuric acid pretreatment

Step 1

� 0.5 % sulfuric acid (soaked overnight); 1:8 (solid to liquid); 180 °C; 10 min

Step 2

� 1.0 % sulfuric acid (soaked overnight); 200 °C; 2 min

COMPOSITION OF PRETREATED MATERIALLIGNIN: 31 % CELLULOSE: 30 % HEMICELLLULOSE: 7%

Internal std.Cyclohexanol

Carboxylic OH

Aliphatic OH

Para-hydroxyl phenylGuaiacyl

OCH3

Cα, Cβ, Cγ

Substituted aryl C

C3, C4 C1

Unsubstituted aryl C

C5C6, C2

Quantitative 13C NMR

O (OH)

R-O-CH

CHO

CH2OH

OCH3

H+

-ROH

O (OH)

CHO

CH2OH

OCH3

HC+

O (OH)

OCH3

OCH3

CH2

C=O

CH2OH

OH

H3CO

+

CH

CH-O

CH2OH

OCH3

O (OH)

H3CO

Depolymeriz

ation

Repolymerization

Quantitative 31P NMR

C4

C2,3,5

C6

C1

O

O H

O

C H 2O H

O

O H O

C H 2O H

O H

O H

6

4

3

2

1

1

Untreated

Pretreated

� Cellulose isolated from untreated and acid treated wood by

holocellulose pulping followed by treatment with 2.5 M HCl at 100 °C for 4 h.

� Cellulose hydrated to ~40 % moisture content for NMR

� CP/MAS pulse sequence; 5 sec delay; 8k scans

� Line shape analysis of C4 region to determine structure

NMR spectrum

Sum of fitted curves

Fitted curves

Milled wood lignin: Quantitative 13C and 31P NMR

� Milled wood lignin isolated from untreated and acid treated wood

� Quantitative 13 C NMR

� 100 mg lignin dissolved in 0.50 ml DMSO-d6

� Spectra acquired at 50 C; inverse gated decoupling; 12 sec pulse

delay; 10k scans

� Quantitative 31P NMR

� 25 mg lignin derivatized with TMDP

� Waltz-16 pulse sequence; 25 sec delay; 150 scans

Crystallinity Index = δ 86-92/ δ 80-92

Untreated: 62.5 %; Pretreated: 69.9 %

0.02.55.07.5

10.012.515.017.520.022.525.027.530.032.535.037.540.042.5

Cellulose Iα

Cellulose Iα+β

para-crystalline

Cellulose Iβ

Accessible

fibril surfa

ces

Inaccessible

fibril surfa

cesAccessible

fibril surfa

ces

Untreated

Acid pretreated

Rela

tive p

rop

ort

ion

(%

)

Crystalline cellulose

Para-crystalline cellulose

Inaccessible fibril surface

Accessible fibril surface

Line fitting results from the C4 region

Methoxyl0.0

0.5

1.0

1.5

2.0

2.5

3.0

# p

er

aro

ma

tic

rin

g

Condensed aromatic

Degree of condensation

Untreated

Acid pretreated

Protonated aromatic

Oxygenated aromatic

-0.023) Carboxylic acid OH133.6 – 136.6

0.060.12

2e) Para–hydroxyl–

phenyl

137.3 – 138.2

0.200.052d) Catechol138.2 – 139.0

1.620.522c) Guaiacyl 139.0 – 140.0

0.340.082b) C5 substituted

"condensed"

140.0 – 144.7

1.940.33

2a) Combined para–

OH–phenyl and guaiacyl

137.3 – 140.0

3.741.032) Phenols136.6 – 144.7

0.370.40

IS) Cyclohexanol (Internal

standard)

144.7 – 145.5

3.424.161) Aliphatic OH150.0 – 145.5

Untreated Acid

mmol/g ligninAssignmentChemical shift

range

δδδδ31P–NMR

Calculated from 13C NMR

31P NMR integration results