Structural modifications of cellulose and lignin in Loblolly i L bl ll pine arising from the ethanol organosolv ii f h h l l pretreatment

School of Chemistry and Biochemistry, Georgia T h Atl t GA G i Tech, Atlanta,

Background: Lignin and cellulose in biomass g g

2nd generation biofuels are cellulose-based

Lignin is generated as a by-product Can be modified during biofuels processing Annual biosynthesis rate of 20 x 109 metric tons

Cellulose structure varies with species

Lignin is the second most abundant biopolymer

Structure and chemistry of lignin is species and process dependant

Can be manipulated by chemical and enzymatic pathways

Ethanol Organosolv pretreatment of Loblolly pine

Background: Biomass pretreatments g p

Pretreatments necessary to overcome inherent recalcitrance of lignocellulosic bi l it f li ll l i biomass t towards d enzymatic deconstruction

Steam explosion, dilute acid, AFEX, organosolv Most current pretreatments produce co-products of low economic value i l Lignin is used as heat/energy source for the process
Lignin Li i

Hemicellulose

Cellulose
www.genomics.energy.gov www genomics energy gov

Ethanol Organosolv pretreatment of Loblolly pine

Organosolv pretreatment g p

Developed in Canada as the Alcell process 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
Ragauskas et al. Science 311, 484-489

Cellulose-rich solid
4

High-quality lignin g q y g

Hemicellulose-rich liquid q

Ethanol Organosolv pretreatment of Loblolly pine

Objectives j

Investigate structural changes in Loblolly pine cellulose after organosolv pretreatment and ll l ft l t t t d 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 g structure during organosolv pretreatment

Ethanol Organosolv pretreatment of Loblolly pine

Biomass Feedstock: Loblolly pine p

Mature (15 yr. old) tree from Baldwin Co., GA Sections debarked and chipped stored at -5 C chipped, 5 C Composite sample used for study Lignin: 30%; Cellulose:42%; Hemicellulose: 21%
Baldwin Co. Atlanta

Ethanol Organosolv pretreatment of Loblolly pine

Ethanol Organosolv pretreatment g p

Loblolly Pine biomass
65 % ethanol/water 1.1 %H2SO4 Unwashed pulp Heat at 170 C, 60 min Liquor with lignin Volatile effluents (HMF + Furfural)

Warm ethanol washing

Washed pulp

Dilution with water

Filtration

Warm water washing

Precipitate

Filtrate

Water-soluble fraction
hemicellulose d depolymerized lignin l i d li i HMF and furfural

Ethanol organosolv lignin t a o o ga oso g Organosolv treated biomass

Cellulose Residual lignin

Enzymatic hydrolysis

Glucose
Pan et al Ind Eng Chem Res 46 2609 2619 al. Ind. Eng. Chem. Res. 46, 2609-2619

Ethanol Organosolv pretreatment of Loblolly pine

Pretreatment results

Untreated (100g) Lignin: 30g g g Cellulose: 42g Hemicellulose: 21g

Organosolv treated Lignin: 12g g g Cellulose: 33g Hemicellulose: 1g

Organosolv lignin Lignin: 6g g g

Liquid fraction Cellulose: 3g Hemicellulose:15g Lignin: Lignin: 6g

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Ethanol Organosolv pretreatment of Loblolly pine

Enzymatic hydrolysis of organosolv treated Loblolly pine

Cellulase: 8 FPU/g cellulose -glucosidase: 16 IU/g cellulose pH 4 8 50 mM acetate buffer H 4.8, M t t b ff 50 C, 150 rpm

Ethanol Organosolv pretreatment of Loblolly pine

Cellulose

Biomass samples delignified by holocellulose pulping (acetic acid and sodium chlorite) Cellulose isolated by hydrolyzing holocellulose with 2 M HCl i h 2.5 Analyzed with solid-state 13C CP/MAS NMR Structure determined by line fitting analysis of g C4 region

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Ethanol Organosolv pretreatment of Loblolly pine

Cellulose NMR
C2, 3, 5 C1 C4 C6 OS-enzyme

Organosolv

Untreated U t t d

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Ethanol Organosolv pretreatment of Loblolly pine

Cellulose NMR

Line shape analysis of the C4 region to determine p y g cellulose ultra structure Crystallinity Index = 86-92/ 80-92 y y
NMR spectrum t Sum of fitted curves Fitted curves

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Ethanol Organosolv pretreatment of Loblolly pine

Cellulose structure and crystallinity

Crystallinity Index = 86-92/ 80-92
Accessible surface Inaccessible surface Para-crystalline cellulose ll l Crystalline cellulose

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Ethanol Organosolv pretreatment of Loblolly pine

Changes in cellulose structure g

Cellulose crystallinity

Decreases after organosolv pretreatment

Pretreatment capable of decreasing ordering of cellulose Together with delignification, produces substrate amenable to enzymatic hydrolysis Enzymes selectively degrading less ordered forms of cellulose

Increases after enzymatic hydrolysis

Cellulose structure after pretreatment

Increase in cellulose I , accessible fibril surfaces Decrease in cellulose I+

Ethanol Organosolv pretreatment of Loblolly pine

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Lignin g

Milled wood lignin (MWL)

Isolated from very finely milled extractives free biomass milled, Extracted with dioxane/water and purified

MWL before and after organosolv pretreatment and Ethanol Organosolv Lignin studied with:

Quantitative 13C NMR -- quantified as f Q tit ti tifi d functional ti l groups/aromatic ring 31P NMR Lignin OH groups quantified after NMR-phosphitylation Gel permeation chromatography-- Molecular weight distribution
Ethanol Organosolv pretreatment of Loblolly pine

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Quantitative 13C NMR spectra p

DMSO EOL

OCH3 Substituted aryl C Unsubstituted aryl C OS-MWL

HO HO O OCH3 OCH3 O

C
C C3, C4 C1 C5 C6, C2

C
MWL

-O-4

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Ethanol Organosolv pretreatment of Loblolly pine

13C

NMR results

3.0 30

Acid catalyzed scission

Decrease in protonated aromatic C Increase in condensed aromatic C

# per aromatic ring

-O-4 linkages most abundant OS-MWL and EOL Decrease in -O-4

2.5

MWL OS-MWL EOL

2.0

1.5

1.0

Increased condensation
0.5

Increase in carboxylic acids

Ester hydrolysis
0.0 Methoxyl Oxygenated aromatic Condensed aromatic Protonated aromatic Degree of condensation

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Ethanol Organosolv pretreatment of Loblolly pine

Lignin OH groups by 31P NMR g g p

Aliphatic OH
Internal std. Cyclohexanol

Guaiacyl Para-hydroxy phenyl MWL Carboxylic OH C b li

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Ethanol Organosolv pretreatment of Loblolly pine

31P

NMR results
4.5 4.0 3.5 3.0 MWL OS-MWL EOL

Aliphatic OH dominant OS-MWL OS MWL and EOL: Lower aliphatic OH

-O-4 cleavage
mmo lignin ol/g

Higher phenolic OH

Aids in organosolv delignification Higher condensed phenolic

2.5 2.0 20 1.5 1.0 0.5 0.0

OH t ic ha lip A

Higher Hi h carboxylic OH b li

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Ethanol Organosolv pretreatment of Loblolly pine

ls yl ol yl ted OH no en ch iac it u h id t a te he p P ac bs Ca Gu y su ylic rox C5 ox yd h arb ra C Pa

Lignin molecular weight distribution g g

Lignin samples were acetylated Analyzed by Gel Permeation Chromatography

Mn

Mw

THF as solvent

Mn (g/mol)
MWL OS-MWL EOL
20

Mw (g/mol)
1.4 x104 1.7 x104 5.4 x103

Mw / Mn
1.8 2.6 1.8

7.6 x103 6.5 x103 3.1 x103

Ethanol Organosolv pretreatment of Loblolly pine

Structural changes with pretreatment g p

Decrease in aliphatic groups Decrease i O 4 li k D in -O-4 linkages Increase in phenolic groups, condensation Higher polydispersity in OS-MWL (lower Mn, higher Mw)

a. Depolymerization p y b. Repolymerization

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Ethanol Organosolv pretreatment of Loblolly pine

Potential uses of organosolv lignin g g

Low molecular weight of EOL

Suitable for various industrial applications due to increased solubility Also has low S content Al h l t t Substitute for polymeric materials; precursor for chemicals; dispersant Higher radical scavenging potential Imparts anti-oxidant activity by inhibiting oxidation of low d l density proteins it t i Anti-inflammatory, anti-carcinogenic
Pan et al Biotech Bioeng 94 851 861 al. Biotech. Bioeng. 94, 851-861

Coupled with the higher phenolic content

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Ethanol Organosolv pretreatment of Loblolly pine

Summary and conclusions

Organosolv pretreatment produces a substrate with reduced cellulose crystallinity and lignin content

Highly amenable to enzymatic deconstruction

Cellulose crystallinity increases after enzyme hydrolysis

Preferential hydrolysis of less ordered cell lose h drol sis cellulose There is a need to develop enzymes capable of hydrolyzing crystalline cellulose

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Ethanol Organosolv pretreatment of Loblolly pine

Summary and Conclusions

Acid catalyzed cleavage of -O-4 and ester are major mechanisms of li i b kd j h i f lignin breakdown Residual lignin after organosolv pretreatment is more condensed

Also shows evidence of repolymerization Low molecular weight Higher phenol and carboxylic acid content May be suitable as anti-oxidants or other value-added anti oxidants value added uses
Ethanol Organosolv pretreatment of Loblolly pine

Ethanol organosolv lignin

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Ethanol Organosolv pretreatment of Loblolly pine