Structural modifications of and in LblllLoblolly pi ne ari iising f rom th e eth ano l organoso lv pretreatment

School of Chemistry and Biochemistry, GiGeorgia ThAtltTech, Atlanta, GA Backggground: Lignin and cellulose in biomass

 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 biopolymer 9  Annual biosynthesis rate of 20 x 10 metric tons  Structure and chemistry of lignin is species and process dependant

 Can be manipulated by chemical and enzymatic pathways

Ethanol Organosolv pretreatment of 2 Loblolly pine Backggpround: Biomass pretreatments

 Pretreatments necessary to overcome inherent recalitlcitrance o flif lignoce lllllulos ic biomass towar ds enzymatic deconstruction

 Steam explosion, dilute acid, AFEX, organosolv

 Most current pretreatments produce co-products of low economilic value

 Lignin is used as heat/energy source for the process Lign in Hemicellulose

Cellulose www. genomics. energy. gov

Ethanol Organosolv pretreatment of 3 Loblolly pine Orggpanosolv pretreatment

 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 High-qqyguality lignin Hemicellulose-rich liquid

Ethanol Organosolv pretreatment of 4 Loblolly pine Objectives

 Investigate structural changes in Loblolly pine cellu lose a fter organoso lv pre trea tment and enzymatic hydrolysis  Determine lignin composition in untreated Loblolly pine, residual lignin after organosolv pretreatment and organosolv lignin  Understand the mechanisms of changgges in lignin structure during organosolv pretreatment

Ethanol Organosolv pretreatment of 5 Loblolly pine Biomass Feedstock: Loblolly pine

 Mature (15 yr. old) tree from Baldwin Co., GA

 Sections debarked and chipped, stored at -5 °C  Composite sample used for study  Lignin: 30%; Cellulose:42%; Hemicellulose: 21%

Baldwin Co.

Atlanta

Ethanol Organosolv pretreatment of 6 Loblolly pine Ethanol Orggpanosolv pretreatment Loblolly Pine biomass

• 65 % ethanol/water

•1.1 %H2SO4 Unwashed Heat at 170 Volatile effluents °C, 60 min (HMF + Furfural) Warm ethanol washing Liquor with lignin

Washed pulp Dilution with water Filtration

Warm water washing Precipitate Filtrate Water-soluble fraction

 hemicellulose Etha n ol o rga noso lv lig nin  dldepolymeri idliized lignin Organosolv treated biomass  HMF and furfural . Cellulose Enzymatic Glucose . Residual lignin hydrolysis Pan et al. Ind. Eng. Chem. Res. 46, 2609-2619 Ethanol Organosolv pretreatment of 7 Loblolly pine Pretreatment results

Untreated (100g) Organosolv treated Organosolv lignin .Ligggnin: 30g .Ligggnin: 12g .Ligggnin: 6g .Cellulose: 42g .Cellulose: 33g .Hemicellulose: 21g .Hemicellulose: 1g Liquid fraction .Cellulose: 3g .Hemicellulose:15g .Lignin: 6g Ethanol Organosolv pretreatment of 8 Loblolly pine Enzymatic hydrolysis of organosolv treated Loblolly pine

Cellulase: 8 FPU/g cellulose β-glucosidase: 16 IU/g cellulose pH 484.8, 50 mM acetttate b bffuffer 50 ºC, 150 rpm

Ethanol Organosolv pretreatment of 9 Loblolly pine Cellulose

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

Ethanol Organosolv pretreatment of 10 Loblolly pine Cellulose NMR

C2, 3, 5 C1 C4 C6 OS-enzyme

Organosolv

UtUntreat tded

Ethanol Organosolv pretreatment of 11 Loblolly pine Cellulose NMR

 Line shappye analysis of the C4 re gion to determine cellulose ultra structure

 Cryyystallinity Index =  86-92/  80-92

NMR spec trum Sum of fitted curves Fitted curves

Ethanol Organosolv pretreatment of 12 Loblolly pine Cellulose structure and crystallinity

Crystallinity Index =  86-92/  80-92 Accessible surface Inaccessible surface Para-crystalline cellu lose Crystalline cellulose

Ethanol Organosolv pretreatment of 13 Loblolly pine Changes in cellulose structure

 Cellulose crystallinity

 Decreases after organosolv pretreatment  Pretreatment capable of decreasing ordering of cellulose  Together with delignification, produces amenable to enzymatic hydrolysis

 Increases after enzymatic hydrolysis  Enzymes selectively degrading less ordered forms of cellulose  Cellulose structure after pretreatment

 Increase in cellulose Iβ , accessible fibril surfaces

 Decrease in cellulose Iα+β

Ethanol Organosolv pretreatment of 14 Loblolly pine Lignin

 Milled wood lignin (MWL)

 Isolated from very finely milled, extractives free biomass

 Extracted with dioxane/water and purified  MWL before and after organosolv pretreatment and Ethanol Organosolv Lignin studied with: 13  QtittiQuantitative CNMRC NMR -- quantifie d as func tiona l groups/aromatic ring 31  PNMRP NMR-- Lignin OH groups quantified after phosphitylation

 Gel permeation chromatography-- Molecular weight distribution

Ethanol Organosolv pretreatment of 15 Loblolly pine Quantitative 13C NMR spectra

DMSO EOL

OCH3

Substituted aryl C Unsubstituted aryl C OS-MWL

HO

HO C O

OCH3 C C3, C4 OCH3 C6, C2 C O C1 C5 β-O-4 MWL

Ethanol Organosolv pretreatment of 16 Loblolly pine 13C NMR results 303.0

 β-O-4 linkages most abundant 2.5 MWL OS-MWL  OS-MWL and EOL EOL

 Decrease in β-O-4 2.0 g  Acid catalyzed scission

 Decrease in protonated 1.5

aromatic C rin aromatic

 Increase in condensed # per # per aromatic C 1.0  Increased condensation

 Increase in carboxylic 0.5 acids  Ester hydrolysis

0.0 Methoxyl Oxygenated Condensed Protonated Degree of aromatic aromatic aromatic condensation

Ethanol Organosolv pretreatment of 17 Loblolly pine Ligggpnin OH groups by 31P NMR

Aliphatic OH

Internal std. Cyclohexanol Guaiacyl

Para-hydroxy phenyl CbCarboxy liOHlic OH MWL

Ethanol Organosolv pretreatment of 18 Loblolly pine 31P NMR results

4.5  Aliphatic OH dominant 4.0  OS-MWL and EOL:

 Lower aliphatic OH 3.5 MWL  β-O-4 cleavage 3.0 OS-MWL  Higher phenolic OH EOL 2.5  Aids in organosolv l/g lignin l/g

delignification oo 202.0

 Higher condensed mm phenolic 1.5

 Hig her car boxy lic OH 1.0

0.5

0.0 s d l l l OH ol te cy ho ny OH c en itu ia ec he d ati h st ua at p ci h P ub G C y– a lip s ox lic A 5 dr xy C y bo –h ar ra C Pa

Ethanol Organosolv pretreatment of 19 Loblolly pine Ligggnin molecular weight distribution

M  Lignin samples were acetylated Mn w  Analyzed by Gel Permeation Chromatography

 THF as solvent

Mn (g/mol)Mw (g/mol) Mw / Mn

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

Ethanol Organosolv pretreatment of 20 Loblolly pine Structural changgpes with pretreatment

 Decrease in aliphatic groups  DiDecrease in β-O-4lik4 linkages  Increase in phenolic groups, condensation

 Higher polydispersity in OS-MWL (lower Mn, higher Mw)

a. Deppyolymerization

b. Repolymerization

Ethanol Organosolv pretreatment of 21 Loblolly pine Potential uses of orggganosolv lignin

 Low molecular weight of EOL

 Suitable for various industrial applications due to increased solubility

 Also has low S con ten t

 Substitute for polymeric materials; precursor for chemicals; dispersant  Coupled with the higher phenolic content

 Higher radical scavenging potential

 Imparts anti-oxidant activity by inhibiting oxidation of ldittilow density proteins

 Anti-inflammatory, anti-carcinogenic Pan et al. Biotech. Bioeng. 94, 851-861 Ethanol Organosolv pretreatment of 22 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 h ydrol y sis of less ordered cellu lose

 There is a need to develop enzymes capable of hydrolyzing crystalline cellulose

Ethanol Organosolv pretreatment of 23 Loblolly pine Summary and Conclusions

 Acid catalyzed cleavage of β-O-4 and ester are majhifliibkdjor mechanisms of lignin breakdown  Residual lignin after organosolv pretreatment is more condensed

 Also shows evidence of repolymerization  Ethanol organosolv lignin

 Low molecular weight

 Higher phenol and carboxylic acid content

 May be suitable as anti-oxidants or other value-added uses

Ethanol Organosolv pretreatment of 24 Loblolly pine Ethanol Organosolv pretreatment of 25 Loblolly pine