kth royal institute of technology stockholm wheat straw lignin – successive fractionation and...
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KTH Royal Institute of Technology
Stockholm
Wheat straw lignin – Successive Fractionation and Structural
Characterization Florian Zikeli
Vienna University of TechnologyKTH Stockholm
COST action FP1105 meeting San Sebastian, May 26-27, 2015
Grass lignin: HGS-Typ; p-hydroxyphenyl (H), coniferyl (G) und syringyl (S) Units
Special features: - different/additional Lignin-Carbohydrate Complexes (LCCs) than in trees
incorporation of ferulic acid bridges
- monolignols acylated by p-hydroxycinnamic acids (pHCAs)
can be p-coumaric acid (pCA) or ferulic acid (FA)
- ester/ether ratio:
pCA > 2:1FA < 1:2
ether bond ester bond
KTH Royal Institute of Technology
Stockholm
Buranov and Mazza (2008)
Grass lignin: HGS-Typ; p-hydroxyphenyl (H), coniferyl (G) und syringyl (S) Units
Special features: - recent research identified a Flavon (Tricin) in a MWL preparation from wheat straw ; (Prinsen et al. (2012))
suggested incorporation into the lignin macromolecule via beta-O-4 bonds
- wide spectrum of functions in the plant:
- UV protection- defensive compound against herbivores - inhibitory activity against pathogenic organisms
- potential positive effect on human health:
- anti-allergic, anti-inflammatory, anti-microbial, anti-cancer- very high anti-oxidant potential!
KTH Royal Institute of Technology
Stockholm
Wheat straw lignin isolation and characterization:
Fractions: - Dioxane lignin (DL): Free lignin (FL) 0h BMDioxane lignin (DL) 8h BMNetDL deducted: DL(8h) - FL
- Acidolysis lignin (AL) 8h BM- Residual lignin (RL)
Gravimetric analysis: - yieldsKL – carbohydrates analysis: - compositionHP-SEC (alkaline): - molar mass2D HSQC NMR: - monomeric ratio S:G:H
- interunit linkages ratio- estimated pHCAs, Tricin contents
31P-NMR: - functional groups
KTH Royal Institute of Technology
Stockholm
Residual lignin (RL)
wheat straw ball-milling dioxane-water
Free lignin (FL) / Dioxane lignin (DL)
dioxane-water Acidolysis lignin (AL)0 h, 4 h, 8 h, 12 h, 16 h
Soxhlet 0.1 M HCl incut to 40 mesh
KTH Royal Institute of Technology
Stockholm
Results: fractions FL, DL/NetDL, AL, RL
Wheat straw lignin isolation and characterization:
Klason lignin recovery: - based on KL: main lignin fraction is AL!
- DL already accessible w/o BM!and after structure opening: double yield of DL!
hint that differentiation is needed between Free lignin (FL) and DL
- in total ca. 70-80% of KL recovered in the isolated fractions
AND: constant KL recovery from 4-8 h BM for DL and AL!
Ball-milling (h)
Klason lignin (%) recovery
FL/DL AL RL total
(relative% of the starting material)
0 2.2 (13.8)
6.3 (39.4)
6.1 (38.1)
14.6 (91.3%)
4 5.1 (13.8+18.1)
5.9 (37.5)
2.9 (18.4)
13.9 (86.9%)
8 5.1 (13.8+18.1)
6.0 (37.5)
2.0 (12.5)
13.1 (81.9%)
KTH Royal Institute of Technology
Stockholm
Wheat straw lignin isolation and characterization:
Ball-milling
[h]
Dioxane lignin (FL/DL) Acidolysis lignin (AL) Residual lignin (RL)
Klason lignin
[%]
Relative sugar composition [%]Klason lignin
[%]
Relative sugar composition [%]Klason lignin
[%]
Relative sugar composition [%]
Ara Gal Glu Xyl Ara Gal Glu Xyl Ara Gal Glu Xyl
0 46.0 10 15 63 12 88.3 34 4 13 49 11.8 0 0 74 26
4 52.2 14 12 46 28 87.1 33 2 16 49 9.1 0 0 92 8
8 54.8 12 5 27 56 87.5 24 3 24 49 7.3 0 0 93 7
8 (Net) 64.0 >12 <<5 <<27 >>56
Composition: Klason lignin: FL/DL << AL
RL very low KL content! … hardly a „lignin fraction“
Carbohydrates: FL Glucose-lignin
DL/NetDL Xylan-lignin (after 8h BM) with low Arabinose content („linear“ xylan)
AL Xylan-lignin with high Arabinose content („branched“ xylan)
RL „Glucose-lignin“
Results: fractions FL, DL/NetDL, AL, RL
KTH Royal Institute of Technology
Stockholm
Wheat straw lignin isolation and characterization:
Ball-milling
[h]
Dioxane lignin (FL/DL) Acidolysis lignin (AL) Residual lignin (RL)
Klason lignin [%]
Relative sugar composition [%] Klason
lignin [%]
Relative sugar composition [%] Klason
lignin [%]
Relative sugar composition [%]
Ara Gal Glu Xyl Ara Gal Glu Xyl Ara Gal Glu Xyl
0 46.0 10 15 63 12 88.3 34 4 13 49 11.8 0 0 74 26
4 52.2 14 12 46 28 87.1 33 2 16 49 9.1 0 0 92 8
8 54.8 12 5 27 56 87.5 24 3 24 49 7.3 0 0 93 7
8 (Net) 64.0 >12 <<5 <<27 >>56
Composition: Klason lignin: FL/DL << AL
RL very low KL content! … hardly a „lignin fraction“
Carbohydrates: FL Glucose-lignin
DL/NetDL Xylan-lignin (after 8h BM) with low Arabinose content („linear“ xylan)
AL Xylan-lignin with high Arabinose content („branched“ xylan)
RL „Glucose-lignin“
Results: fractions FL, DL/NetDL, AL, RL
KTH Royal Institute of Technology
Stockholm
Wheat straw lignin isolation and characterization:
Ball-milling
[h]
Dioxane lignin (FL/DL) Acidolysis lignin (AL) Residual lignin (RL)
Klason lignin [%]
Relative sugar composition [%]Klason lignin [%]
Relative sugar composition [%]Klason lignin [%]
Relative sugar composition [%]
Ara Gal Glu Xyl Ara Gal Glu Xyl Ara Gal Glu Xyl
0 46.0 10 15 63 12 88.3 34 4 13 49 11.8 0 0 74 26
4 52.2 14 12 46 28 87.1 33 2 16 49 9.1 0 0 92 8
8 54.8 12 5 27 56 87.5 24 3 24 49 7.3 0 0 93 7
8 (Net) 64.0 >12 <<5 <<27 >>56
Composition: Klason lignin: FL/DL << AL
RL very low KL content! … hardly a „lignin fraction“
Carbohydrates: FL Glucose-lignin
DL/NetDL Xylan-lignin (after 8h BM) with low Arabinose content („linear“ xylan)
AL Xylan-lignin with high Arabinose content („branched“ xylan)
Results: 4 fractions FL, DL/NetDL, AL
Molar mass analysis:
FL: - smallest MM!- very high- and very low-molar mass
parts- pHCAs seem dominant! 31P-NMR: highest content of
free COOHs monomeric or ether-bound
pHCAs: pCA and FA identified as monomers by preparative HP-
SEC and RP-HPLC
NetDL: - similar molar mass as AL- but: stronger pHCAs-peak!
AL: -most symmetric/uniform MMD
- pHCAs-peak: just a shoulder, so no signs of pHCAs with free COOHs
(31P-NMR: lowest COOHs!)
KTH Royal Institute of Technology
Stockholm
Wheat straw lignin isolation and characterization:
Results: 4 fractions FL, DL/NetDL, AL
Ball-milling
[h]
Dioxane lignin (DL) Acidolysis lignin (AL)
Mw [kDa]
Mn [kDa]
Pd [kDa]
Mw [kDa]
Mn [kDa]
Pd [kDa]
0 1520 900 1.69 2300 690 3.338 3000 1100 2.73 3300 770 4.29
8 (Net) 4600 1310 3.51
Molar mass analysis:
FL: - smallest MM!- very high- and very low-molar mass
parts- pHCAs seem dominant! 31P-NMR: highest content of
COOHs monomeric or ether-
bound pHCAs: pCA and FA identified as monomers by preparative HP-
SEC and RP-HPLC
NetDL: - similar molar mass as AL- but: stronger pHCAs-peak!
AL: -most symmetric/uniform MMD
- pHCAs-peak: just a shoulder, so no signs of pHCAs with free COOHs
(31P-NMR: lowest COOHs!)
KTH Royal Institute of Technology
Stockholm
Wheat straw lignin isolation and characterization:
Results: 4 fractions FL, DL/NetDL, AL
Ball-milling
[h]
Dioxane lignin (DL) Acidolysis lignin (AL)
Mw [kDa]
Mn [kDa]
Pd [kDa]
Mw [kDa]
Mn [kDa]
Pd [kDa]
0 1520 900 1.69 2300 690 3.338 3000 1100 2.73 3300 770 4.29
8 (Net) 4600 1310 3.51
Molar mass analysis:
FL: - smallest MM!- very high- and very low-molar mass
parts- pHCAs seem dominant! 31P-NMR: highest content of
COOHs monomeric or ether-
bound pHCAs: pCA and FA identified as monomers by preparative HP-
SEC and RP-HPLC
NetDL: - similar molar mass as AL- but: stronger pHCAs-peak!
AL: -most symmetric/uniform MMD
- pHCAs-peak: just a shoulder, so no signs of pHCAs with free COOHs
(31P-NMR: lowest COOHs!)
KTH Royal Institute of Technology
Stockholm
Wheat straw lignin isolation and characterization:
Results: 4 fractions FL, DL/NetDL, AL
Ball-milling
[h]
Dioxane lignin (DL) Acidolysis lignin (AL)
Mw [kDa]
Mn [kDa]
Pd [kDa]
Mw [kDa]
Mn [kDa]
Pd [kDa]
0 1520 900 1.69 2300 690 3.338 3000 1100 2.73 3300 770 4.29
8 (Net) 4600 1310 3.51
Monomeric ratio, pHCAs content:
FL low S units and highest in G units; low pHCAs content and pCA > FA highest Tricin content
DL also low S units and highest H unitshighest pHCAs content (NetDL) and FA >> pCA
AL high S units ratio high pHCAs content and pCA >> FAvery little free COOH groups found! pCA-esters!
KTH Royal Institute of Technology
Stockholm
Wheat straw lignin isolation and characterization:
Results: 4 fractions FL, DL/NetDL, AL
Monomeric ratio [%] pHCAs [%]Tricin [%]
Ball-milling
[h]S units G units H units total
pHCAs FA pCA ratio FA/pCA [ ]
DL0 34 61 5 5.8 2.4 3.3 0.7 14.98 35 56 9 13.4 8.9 4.5 2.0 10.6
8 (Net) 36 51 13 22.0 16.0 6.0 2.7 6.0AL 8 42 52 6 13.1 4.2 8.9 0.5 6.8
Monomeric ratio, pHCAs content:
FL low S units and highest in G units; low pHCAs content and pCA > FA highest Tricin content
DL also low S units and highest H unitshighest pHCAs content (NetDL) and FA >> pCA
AL high S units ratio high pHCAs content and pCA >> FAvery little free COOH groups found! pCA-esters!
KTH Royal Institute of Technology
Stockholm
Wheat straw lignin isolation and characterization:
Results: 4 fractions FL, DL/NetDL, AL
Monomeric ratio [%] pHCAs [%]Tricin [%]
Ball-milling
[h]S units G units H units total
pHCAs FA pCA ratio FA/pCA [ ]
DL0 34 61 5 5.8 2.4 3.3 0.7 14.98 35 56 9 13.4 8.9 4.5 2.0 10.6
8 (Net) 36 51 13 22.0 16.0 6.0 2.7 6.0AL 8 42 52 6 13.1 4.2 8.9 0.5 6.8
Monomeric ratio, pHCAs content:
FL low S units and highest in G units; low pHCAs content and pCA > FA highest Tricin content
DL also low S units and highest H unitshighest pHCAs content (NetDL) and FA >> pCA
AL high S units ratio high pHCAs content and pCA >> FAvery little free COOH groups found! pCA-esters!
KTH Royal Institute of Technology
Stockholm
Wheat straw lignin isolation and characterization:
Results: 4 fractions FL, DL/NetDL, AL
Monomeric ratio [%] pHCAs [%]Tricin [%]
Ball-milling
[h]S units G units H units total
pHCAs FA pCA ratio FA/pCA [ ]
DL0 34 61 5 5.8 2.4 3.3 0.7 14.98 35 56 9 13.4 8.9 4.5 2.0 10.6
8 (Net) 36 51 13 22.0 16.0 6.0 2.7 6.0AL 8 42 52 6 13.1 4.2 8.9 0.5 6.8
KTH Royal Institute of Technology
Stockholm
proposed arrangement of isolated lignin fractions:
FL pHCA/Tricin --- FL --- Cellulose(OS) plus monomeric pCA, FA and cinnamyl alcohols
DL (pCA)/FA --- DL --- FA-(FA) --- (Ara) --- Xylan (linear)NetDL
AL pCA --- AL --- (FA) --- Ara --- Xylan (branched)
Wheat straw lignin isolation and characterization:
Results: 4 fractions FL, DL/NetDL, AL
KTH Royal Institute of Technology
Stockholm
proposed arrangement of isolated lignin fractions:
FL pHCA/Tricin --- FL --- Cellulose(OS) plus monomeric pCA, FA and cinnamyl alcohols
DL (pCA)/FA --- DL --- FA-(FA) --- (Ara) --- Xylan (linear)NetDL
AL pCA --- AL --- (FA) --- Ara --- Xylan (branched)
high G … ML
high H, FA … ML
high S, pCA-ester … SW
Donaldson 2001Fukushima and Terashima, 1990
He and Terashima, 1989, 1990
Wheat straw lignin isolation and characterization:
Results: 4 fractions FL, DL/NetDL, AL
KTH Royal Institute of Technology
Stockholm
Wheat straw lignin isolation and characterization:
Results: 4 fractions FL, DL/NetDL, AL
Conclusions: - unique fraction FL accessible already w/o BM and AHorigin? partly from living cells filled with lignin precursors
and: ML lignin oligomers (young!)
- BM disrupts fibre structure and release ML lignin (DL)
- AH is needed to depolymerize SW polysaccharides and release SW lignin (AL)
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