Structural modifications of cellulose and lignin 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 pulp 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 (acetic acid 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 substrate 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