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)
Thank you for your attention!!
KTH Royal Institute of Technology
Stockholm
Wood Chemistry and Pulp TechnologyDocent Jiebing Li
Working Group Bioresource TechnologyProf. Ewald Srebotnik
Acknowledgements
Austrian Research Promotion Agency Annikki GmbH, Graz, Austria