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

3

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

4

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