pseudo-lignin chemistry in dilute acid pretreatment …...pseudo-lignin chemistry in dilute acid...
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Pseudo-lignin Chemistry in Dilute Acid Pretreatment (DAP)
Fan Hu and Art J. Ragauskas
BioEnergy Science CenterInstitute of Paper Science & TechnologySchool of Chemistry and BiochemistryGeorgia Institute of Technology, 500 10th St., Atlanta, GA 30332, USA
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The Behaviors of Lignin during DAP• Lignin coalesces into larger molten bodies that migrate within and out of the cell
wall, and then redeposit as droplets on the surface of biomass cell walls.• Lignin is depolymerized and repolymerized caused by the formation of carbonium
ion during DAP.
• An increase in acid-insoluble (Klason) lignin content after DAP has been hypothesized due to repolymerization of polysaccharides degradation products and/or polymerization with lignin to form a lignin-like material termed pseudo-lignin.
Pu, Y.; Hu, F.; Huang, F.; Davison, B. H.; Ragauskas, A. J., Assessing the molecular structure basis for biomass recalcitrance during dilute acid and hydrothermal pretreatments. Biotechnology for biofuels 2013, 6 (1), 15.
Hu, F.; Ragauskas, A., Pretreatment and Lignocellulosic Chemistry. BioEnergy Research 2012, 5 (4), 1043-1066.
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Extraction Yield and Molecular Weight Analysis of Pseudo-lignin
Pseudo-lignin isolated from Mn (g/mol) Mw (g/mol) Polydispersity index (PDI)
α-Cellulose B 1.08 x 103 3.44 x 103 3.17
Holocellulose A 1.24 x 103 5.08 x 103 4.09
Holocellulose B 1.19 x 103 5.97 x 103 5.00
2.33 4.686.61
37.47
19.95
86.93
0102030405060708090
100
α-Cellulose Holocellulose
Perc
enta
ge
Acid-insoluble Lignin %
Untreated
Pretreatment A
Pretreatment B
0
51.3
44.58
33.71
0
10
20
30
40
50
60
α-Cellulose Holocellulose
Pseudo-lignin Extraction %
Pretreatment A
Pretreatment B
Pretreatment A for α-cellulose: 170 ºC, 0.1 M H2SO4, 20 min; Pretreatment B for α-cellulose: 180 ºC, 0.1 M H2SO4, 40 minPretreatment A for holocellulose: 180 ºC, 0.1 M H2SO4, 40 min; Pretreatment B for holocellulose: 180 ºC, 0.1 M H2SO4, 40 min
Hu, F.; Jung, S.; Ragauskas, A., Pseudo-lignin formation and its impact on enzymatic hydrolysis. Bioresour Technol 2012, 117, 7-12.
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Pretreated Cellulose Samples from UC Riverside
Substrate Pretreatment conditions CSF Glucan % Xylan % Pseudo-lignin % Acid-soluble
lignin %
Avicel PH 101 (C)
170 ºC, 1 wt% H2SO4, 40 min 2.95
93.20 0 3.07 0.46
Avicel PH 101 + Beechwood
xylan* (C + Xyn)
89.80 0 8.10 0.51
Avicel PH 101 + Xylose* (C +
Xys)89.10 0 10.60 0.55
C
180 ºC, 2 wt% H2SO4, 40 min 3.56
11.50 0 85.70 0.93
C + Xyn* 9.25 0 88.20 0.80
C + Xys* 5.06 0 94.40 0.82
*Avicel to xylan/xylose weight ratio 2 : 1CSF = log [t exp [(T - Tref)/14.7]] - pH, where t is the pretreatment time (min), T is the pretreatment temperature (ºC) and Tref is 100 ºC
Pseudo-lignin content increased moderately
Pseudo-lignin content increased dramatically
Kumar, R.; Hu, F.; Sannigrahi, P.; Jung, S.; Ragauskas, A. J.; Wyman, C. E., Carbohydrate derived-pseudo-lignin can retard cellulose biological conversion. Biotechnol Bioeng 2013, 110 (3), 737-53.
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Characterization of Pseudo-lignin
Wavenumber (cm-1) Assignment
3238 O-H stretching in alcohols, phenols or carboxylic acids
2923 Aliphatic C-H stretching
1697 C=O stretching in carboxylic acids, conjugated aldehydes or ketones
1611, 1512 Aromatic C=C stretching
1360 Aliphatic C-H rocking
1299, 1203, 1020 C-O stretching in alcohols, ethers or carboxylic acids
867, 800 Aromatic C-H out-of-plane bending
*Pretreatment conditions for poplar cellulose and holocellulose (0.1M): 180 ºC, 0.1M H2SO4, 40 min (CSF: 3.25)Pretreatment conditions for poplar holocellulose (0.2M): 180 ºC, 0.2M H2SO4, 60 min (CSF: 3.73)
Hu, F.; Jung, S.; Ragauskas, A., Pseudo-lignin formation and its impact on enzymatic hydrolysis. Bioresour Technol 2012, 117, 7-12.
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FT-IR Characterization
4000 3500 3000 2500 2000 1500 1000 50030
40
50
60
70
80
90
100
% T
rans
mit
tanc
e
Wavenumbers ( cm-1)
UT Avicel cellulose Pretreated C @ CSF 2.95 Pretreated C+XYn @ CSF 2.95 Pretreated C+XYs @ CSF 2.95 Xylose derived humins
4000 3500 3000 2500 2000 1500 1000 50030
40
50
60
70
80
90
100
% T
rans
mit
tanc
e
Wavenumbers ( cm-1)
UT Avicel cellulose Pretreated C @ CSF 3.56 Pretreated C+XYn @ CSF 3.56 Pretreated C+XYs @ CSF 3.56 Xylose derived humins
CSF-2.95; Pseudo-lignin > 8 wt% CSF-3.56; Pseudo-lignin > 80 wt%
No structural change up until CSF-3.56
CSF- combined severity factor; Pure xylose derived pseudo-lignin - 180 ºC- 5wt% H2SO4- 180 minKumar, R.; Hu, F.; Sannigrahi, P.; Jung, S.; Ragauskas, A. J.; Wyman, C. E., Carbohydrate derived-pseudo-lignin can retard cellulose biological conversion.
Biotechnol Bioeng 2013, 110 (3), 737-53.
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13C CP/MAS NMR AnalysisCSF-2.95; Pseudo-lignin >8 wt% CSF-3.56; Pseudo-lignin > 80 wt%
No structural change up until CSF-3.56
CSF- combined severity factor; Pure xylose derived pseudo-lignin - 180 ºC- 5wt% H2SO4- 180 minKumar, R.; Hu, F.; Sannigrahi, P.; Jung, S.; Ragauskas, A. J.; Wyman, C. E., Carbohydrate derived-pseudo-lignin can retard cellulose biological conversion.
Biotechnol Bioeng 2013, 110 (3), 737-53.
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Structural ComparisonPseudo-lignin vs. Lignin
EMAL DAP: enzymatic mild acidolysis lignin from poplar treated at 170 ºC, 0.5 wt% H2SO4, 8 minPseudo-lignin: pseudo-lignin from poplar holocellulose treated at 180 ºC, 1.0 wt% H2SO4, 40 min
102030405060708090100110120130140150160170180190200f1 (ppm )
DMSO
p - dioxane
Pseudo-lignin
EMAL DAP
Pseudo-lignin contains more C=O groups
Different aromatic structures
Pseudo-lignin contains more C=O groups
Hydroxylatedmethylene
Methoxy
Pseudo-lignin possesses more aliphatic structures
Pseudo-lignin is NOT derived from native lignin
Hu, F.; Jung, S.; Ragauskas, A., Impact of Pseudolignin versus Dilute Acid-Pretreated Lignin on Enzymatic Hydrolysis of Cellulose. ACS Sustainable Chemistry & Engineering 2013, 1, 62-65.
Ar C-O Ar C-C Ar C-H
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Enzymatic HydrolysisPseudo-lignin vs. Lignin
Condition: 1% (w/v) consistency in 50 mM citrate buffer (pH 4.8) with cellulase and β-glucosidase loadings of 20 FPU/g and 40 CBU/g, respectively
SEM images of holocellulose (left) and the pseudo-lignin on holocelluose sample (right). All scale bars are equivalent to 1 µm.
0
10
20
30
40
50
60
70
80
0 20 40 60 80
Glu
cose
yie
ld %
Time (h)
a:holocellulose
b: 12% EMAL DAP
c:22% EMAL DAP
d:36% EMAL DAP
e:12% plignin/EMAL DAP
f:22% plignin/EMAL DAP
g:36% plignin/EMAL DAP
h:12% pseudo-lignin
i:22% pseudo-lignin
j:36% pseudo-lignin
Pseudo-lignin is more detrimental to enzymatic hydrolysis of cellulose when compared to lignin, indicating its formation should be avoided.
Hu, F.; Jung, S.; Ragauskas, A., Pseudo-lignin formation and its impact on enzymatic hydrolysis. Bioresour Technol 2012, 117, 7-12.Hu, F.; Jung, S.; Ragauskas, A., Impact of Pseudolignin versus Dilute Acid-Pretreated Lignin on Enzymatic Hydrolysis of Cellulose. ACS
Sustainable Chemistry & Engineering 2013, 1, 62-65.
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Pseudo-lignin Formation Reduction
Cellulosic Biomass
Cellulose Hemicellulose (Xylan) Lignin
Glucose Xylose/XOs
HMF/LA/FA Furfural/ FA
Pseudo-lignin Lignin
HMF: 5-Hydroxymethylfurfural; LA: Levulinic acid; FA: Formic acid
At high severity, it is difficult to prevent carbohydrates from forming furfural and HMF
Efforts will thus focus on preventing furfural and HMF from further reactions
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Proposed Reaction Pathways
Cellulose
Hemicellulose(Xylan)
Glucose
Xylose
OHOO
HMF
Hydrolysis O O
Furfural
OOH
OOH
3,8-dihydroxy-2-methylchromone
HDehydration HO O
O
OH HO
HO O
1,2,4-benzenetriol
O OOHO
O
OOH
OOH
HO OH
OH
Oxidation, Polymerization, PolycondensationPseudo-lignin
2-oxopentanedial
Hydrolysis Dehydration H HOH
OH
HO
Key Intermediates
Bio
mas
s Po
lysa
ccha
rides
Efforts will focus on preventing furfural and HMF from further reactions
Hu, F.; Jung, S.; Ragauskas, A., Pseudo-lignin formation and its impact on enzymatic hydrolysis. Bioresour Technol 2012, 117, 7-12.
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Pseudo-lignin Reduction under DAP conditions
Sample DAP condition Solid recovery % K-lignin % Xylan % Glucan %
Holocellulose N/A N/A 4.3 2.9 93.4
Control DAP 180 ºC, 0.1MH2SO4, 40 min 18.9 42.0 0 54.4
A (O2)180 ºC, 0.1M
H2SO4, 40 min @100 Psi O2
18.1 89.2 0 4.3
B (N2)180 ºC, 0.1M
H2SO4, 40 min @100 Psi N2
19.1 48.7 0 47.1
C (DMSO)
180 ºC, 0.1MH2SO4
(H2O/DMSO: 8/2, v/v), 40 min
37.8 14.7 0 86.2
D (Tween)
180 ºC, 0.1MH2SO4, 40 min with 5% (w/w)
Tween-80
18.3 52.1 0 45.0
Hu F and Ragauskas AJ. Suppression of Pseudo-lignin Formation under Dilute Acid Pretreatment Conditions. RSC Adv. 2014, 4, 4317-4323
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The FT-IR spectrum of sample C (DMSO) resembles that of holocellulose
FT-IR characterization of solids recovered after DAP
Hu F and Ragauskas AJ. Suppression of Pseudo-lignin Formation under Dilute Acid Pretreatment Conditions. RSC Adv. 2014, 4, 4317-4323
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HMF in H2O/DMSO Mixture
• (a): The angular distribution of water molecules around carbonyl oxygen atom do not compete for the same space with the DMSO molecule interacting with the C1 carbon atom, showing that the water molecules hydrogen-bonded to the C1 carbonyl oxygen would not hamper the coordination of DMSO around the C1 carbon.
• (b): High patches of DMSO can be seen near the hydrogen atom of the HMF hydroxyl group due to stronger hydrogen bond than water molecules.
• (c): The highest density region of DMSO around HMF is around the C1 atom and for water molecules, it is around the only hydroxyl group of HMF.
• The preferential arrangement of DMSO in the vicinity of the C1 carbon strongly indicates that it protects the C1 carbon of the HMF molecule from further reactions to form pseudo-lignin.
Hu F and Ragauskas AJ. Suppression of Pseudo-lignin Formation under Dilute Acid Pretreatment Conditions. RSC Adv. 2014, 4, 4317-4323S. H. Mushrif, S. Caratzoulas and D. G. Vlachos, Physical chemistry chemical physics : PCCP, 2012, 14, 2637-2644.
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Summary and Perspectives
• Pseudo-lignin can be formed from biomass polysaccharides without significant contribution from native lignin during DAP process, accounting for the additional acid-insoluble lignin content.
• Pseudo-lignin is polymeric and contains carbonyl, carboxylic, aromatic, methoxyand aliphatic structures, which are produced from both dilute acid-treated cellulose and hemicellulose.
• Although pseudo-lignin is not derived from native lignin, it is even more detrimental to enzymatic deconstruction of cellulose compared to dilute acid-treated lignin.
• Addition of DMSO to DAP reaction medium can effectively increase solid recovery yield and suppress pseudo-lignin formation, even at high-severity pretreatment conditions, which prevents sugar degradation during DAP and increases the enzymatic digestibility of cellulose after DAP.
• Future work will focus on developing solid catalyst which not only can decrease pseudo-lignin formation yield, but also can be recovered and recycled.
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Back-up Slides
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Extraction Yield and Molecular Weight Analysis of Pseudo-lignin
Sample Acid-insolublelignin % Xylan % Glucan % Recovery % from
pretreatment
Pseudo-lignin extraction yield (isolated pseudo-
lignin/total acid-insoluble lignin x 100%)
α-Cellulose 2.33 1.01 99.42 NA NA
α-Cellulose A 6.61 0 94.01 62.67 0
α-Cellulose B 19.95 0 80.41 31.73 44.58
Holocellulose 4.68 22.00 68.09 NA NA
Holocellulose A 37.47 0 65.31 28.81 51.30
Holocellulose B 86.93 0 6.95 19.19 33.71
Pseudo-lignin isolated from Mn (g/mol) Mw (g/mol) Polydispersity index (PDI)
α-Cellulose (180 ºC, 0.1 M H2SO4, 40 min) 1.08 x 103 3.44 x 103 3.17
Holocellulose (180 ºC, 0.1 M H2SO4, 40 min) 1.24 x 103 5.08 x 103 4.09
Holocellulose (180 ºC, 0.2 M H2SO4, 60 min) 1.19 x 103 5.97 x 103 5.00