Structural changes in lignin and cellulose resulting from...
Transcript of Structural changes in lignin and cellulose resulting from...
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