organosolv pine
TRANSCRIPT
Structural modifications of cellulose and lignin in L bl ll i i i f h h l lLoblolly pine arising from the ethanol organosolv pretreatment
School of Chemistry and Biochemistry, G i T h Atl t GAGeorgia Tech, Atlanta, GA
Background: Lignin and cellulose in biomassg g
2nd generation biofuels are cellulose-based Lignin is generated as a by-product
Cellulose structure varies with species Can be modified during biofuels processing
Lignin is the second most abundant biopolymerLignin is the second most abundant biopolymer Annual biosynthesis rate of 20 x 109 metric tons
Structure and chemistry of lignin is species and Structure and chemistry of lignin is species and process dependant Can be manipulated by chemical and enzymatic Can be manipulated by chemical and enzymatic
pathways
2Ethanol Organosolv pretreatment of
Loblolly pine
Background: Biomass pretreatmentsg p
Pretreatments necessary to overcome inherent l it f li ll l i bi t drecalcitrance of lignocellulosic biomass towards
enzymatic deconstruction Steam explosion, dilute acid, AFEX, organosolv Most current pretreatments produce co-products of low
i leconomic value Lignin is used as heat/energy source for the process
Li iLigninHemicellulose
Cellulosewww genomics energy gov
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www.genomics.energy.gov
Ethanol Organosolv pretreatment of Loblolly pine
Organosolv pretreatmentg p Developed in Canada as the Alcell® process
Biomass treated with water and organic solvent Biomass treated with water and organic solvent Acid or base added as catalysts
Has been optimized for softwood, hardwood and agricultural waste
Acts like a biorefinery
Lignocellulosic biomass
Organosolv pretreatment
High-quality ligninCellulose-rich solid Hemicellulose-rich liquid
Organosolv pretreatmentRagauskas et al. Science 311, 484-489
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g q y g q
Ethanol Organosolv pretreatment of Loblolly pine
Objectivesj
Investigate structural changes in Loblolly pine ll l ft l t t t dcellulose after organosolv pretreatment and
enzymatic hydrolysis Determine lignin composition in untreated
Loblolly pine, residual lignin after organosolv pretreatment and organosolv lignin
Understand the mechanisms of changes in lignin g gstructure during organosolv pretreatment
5Ethanol Organosolv pretreatment of
Loblolly pine
Biomass Feedstock: Loblolly pinep Mature (15 yr. old) tree from Baldwin Co., GA
Sections debarked and chipped stored at 5 °C Sections debarked and chipped, stored at -5 C Composite sample used for study Lignin: 30%; Cellulose:42%; Hemicellulose: 21%
Baldwin Co.
Atlanta
Baldwin Co.
6Ethanol Organosolv pretreatment of
Loblolly pine
Ethanol Organosolv pretreatmentg pLoblolly Pine biomass
• 65 % ethanol/water
Heat at 170 °C, 60 min
•1.1 %H2SO4
Unwashed pulp Volatile effluents(HMF + Furfural)
Warm ethanol washing Liquor with lignin
Dilution with water FiltrationWashed pulp
Precipitate Filtrate Water-soluble fraction hemicellulose
d l i d li iEthanol organosolv lignin
Warm water washing
depolymerized lignin HMF and furfural
t a o o ga oso gOrganosolv treated biomass Cellulose Residual lignin
Pan et al Ind Eng Chem Res 46 2609 2619
Enzymatic hydrolysis
Glucose
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Pan et al. Ind. Eng. Chem. Res. 46, 2609-2619
Ethanol Organosolv pretreatment of Loblolly pine
Pretreatment results
Untreated (100g)Lignin: 30g
Organosolv treatedLignin: 12g
Organosolv ligninLignin: 6g g g
Cellulose: 42gHemicellulose: 21g
g gCellulose: 33gHemicellulose: 1g
Liquid fraction
g g
Liquid fractionCellulose: 3gHemicellulose:15gLignin: 6g
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Lignin: 6g Ethanol Organosolv pretreatment of
Loblolly pine
Enzymatic hydrolysis of organosolv treated Loblolly pine
Cellulase: 8 FPU/g celluloseβ-glucosidase: 16 IU/g cellulose
H 4 8 50 M t t b ffpH 4.8, 50 mM acetate buffer50 ºC, 150 rpm
9Ethanol Organosolv pretreatment of
Loblolly pine
Cellulose Biomass samples delignified by holocellulose
pulping (acetic acid and sodium chlorite)pulping (acetic acid and sodium chlorite) Cellulose isolated by hydrolyzing holocellulose
i h 2 M HClwith 2.5 M HCl Analyzed with solid-state 13C CP/MAS NMR Structure determined by line fitting analysis of
C4 regiong
10Ethanol Organosolv pretreatment of
Loblolly pine
Cellulose NMRC2, 3, 5
C1C4
C1C6
OS-enzyme
OrganosolvOrganosolv
U t t dUntreated
Ethanol Organosolv pretreatment of Loblolly pine11
Cellulose NMR
Line shape analysis of the C4 region to determine p y gcellulose ultra structure
Crystallinity Index = 86-92/ 80-92
NMR t
y y 86-92 80-92
NMR spectrumSum of fitted curvesFitted curves
Ethanol Organosolv pretreatment of Loblolly pine12
Cellulose structure and crystallinity
Accessible surface
Crystallinity Index = 86-92/ 80-92
Para-crystalline ll l
Inaccessible surface
surface
Crystalline cellulosecellulose
Ethanol Organosolv pretreatment of Loblolly pine13
Changes in cellulose structureg
Cellulose crystallinity Decreases after organosolv pretreatment
Pretreatment capable of decreasing ordering of cellulose Together with delignification, produces substrate
amenable to enzymatic hydrolysisIncreases after enzymatic hydrolysis Increases after enzymatic hydrolysis Enzymes selectively degrading less ordered forms of
cellulosecellulose
Cellulose structure after pretreatment Increase in cellulose I accessible fibril surfaces Increase in cellulose Iβ , accessible fibril surfaces Decrease in cellulose Iα+β
Ethanol Organosolv pretreatment of Loblolly pine14
Ligning Milled wood lignin (MWL)
Isolated from very finely milled extractives free biomass Isolated from very finely milled, extractives free biomass Extracted with dioxane/water and purifiedMWL before and after organosolv pretreatment MWL before and after organosolv pretreatment and Ethanol Organosolv Lignin studied with:
Q tit ti 13C NMR tifi d f ti l Quantitative 13C NMR -- quantified as functional groups/aromatic ring31P NMR Lignin OH groups quantified after 31P NMR-- Lignin OH groups quantified after phosphitylation
Gel permeation chromatography-- Molecular weight Gel permeation chromatography-- Molecular weight distribution
15Ethanol Organosolv pretreatment of
Loblolly pine
Quantitative 13C NMR spectrap
EOLDMSO
OCH3
OS-MWLSubstituted aryl C Unsubstituted aryl C
HO
HO
OC
MWL
CC3, C4
C1 C5 C6, C2O
OCH3
OOCH3
CMWLβ-O-4
16Ethanol Organosolv pretreatment of
Loblolly pine
13C NMR results3 0
β-O-4 linkages most abundant 2.5
3.0
MWLabundant OS-MWL and EOL
Decrease in β-O-4 2.0
g
OS-MWLEOL
Acid catalyzed scission Decrease in protonated
aromatic C1.5
arom
atic
rin
Increase in condensed aromatic C Increased condensation
1.0#
per
Increase in carboxylic acids Ester hydrolysis
0.5
Ester hydrolysis0.0
Methoxyl Oxygenatedaromatic
Condensedaromatic
Protonatedaromatic
Degree ofcondensation
17Ethanol Organosolv pretreatment of
Loblolly pine
Lignin OH groups by 31P NMRg g p
Aliphatic OH
Internal std.Cyclohexanol
Guaiacyl
C b li OH
Para-hydroxy phenyl
Guaiacyl
Carboxylic OHMWL
18Ethanol Organosolv pretreatment of
Loblolly pine
31P NMR results Aliphatic OH dominant OS MWL and EOL: 4.0
4.5
OS-MWL and EOL: Lower aliphatic OH
β-O-4 cleavage 3.0
3.5
MWL
Higher phenolic OH Aids in organosolv
delignification 2 0
2.5
3.0
ol/g
lign
in
OS-MWLEOL
delignification Higher condensed
phenolicHi h b li OH
1.5
2.0
mm
o
Higher carboxylic OH
0.5
1.0
0.0
Aliphatic OH
Phenols
C5 substituted
Guaiacyl
Catechol
hydroxy–phenyl
rboxylic acid OH
19Ethanol Organosolv pretreatment of
Loblolly pine
C
P
ara–hy Carbo
Lignin molecular weight distributiong g
Lignin samples were acetylated MnMw
Analyzed by Gel Permeation Chromatography THF as solvent THF as solvent
(g/mol) (g/mol)wM nw MM /nM
MWL 7.6 x103 1.4 x104 1.8
OS-MWL 6.5 x103 1.7 x104 2.6
EOL 3.1 x103 5.4 x103 1.8
20Ethanol Organosolv pretreatment of
Loblolly pine
Structural changes with pretreatmentg p
Decrease in aliphatic groupsD i β O 4 li k Decrease in β-O-4 linkages
Increase in phenolic groups, condensation Higher polydispersity in OS-MWL (lower Mn, higher Mw)
a. Depolymerizationp y
b. Repolymerization
21Ethanol Organosolv pretreatment of
Loblolly pine
Potential uses of organosolv ligning g
Low molecular weight of EOL Suitable for various industrial applications due to
increased solubilityAl h l S t t Also has low S content
Substitute for polymeric materials; precursor for chemicals; dispersantchemicals; dispersant
Coupled with the higher phenolic content Higher radical scavenging potential Imparts anti-oxidant activity by inhibiting oxidation of
l d it t ilow density proteins Anti-inflammatory, anti-carcinogenic
Pan et al Biotech Bioeng 94 851 861
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Pan et al. Biotech. Bioeng. 94, 851-861
Ethanol Organosolv pretreatment of Loblolly pine
Summary and conclusions
Organosolv pretreatment produces a substrate Organosolv pretreatment produces a substrate with reduced cellulose crystallinity and lignin contentcontent Highly amenable to enzymatic deconstruction
Cellulose crystallinity increases after enzyme Cellulose crystallinity increases after enzyme hydrolysis
Preferential h drol sis of less ordered cell lose Preferential hydrolysis of less ordered cellulose There is a need to develop enzymes capable of
hydrolyzing crystalline cellulosehydrolyzing crystalline cellulose
Ethanol Organosolv pretreatment of Loblolly pine23
Summary and Conclusions
Acid catalyzed cleavage of β-O-4 and ester are j h i f li i b kdmajor mechanisms of lignin breakdown
Residual lignin after organosolv pretreatment is more condensed Also shows evidence of repolymerization
Ethanol organosolv lignin Low molecular weight Low molecular weight Higher phenol and carboxylic acid content May be suitable as anti-oxidants or other value-added May be suitable as anti oxidants or other value added
uses
24Ethanol Organosolv pretreatment of
Loblolly pine
25Ethanol Organosolv pretreatment of
Loblolly pine