KTH Royal Institute of Technology Stockholm Wheat straw

KTH Royal Institute of Technology Stockholm Wheat straw lignin – Successive Fractionation and Structural Characterization Florian Zikeli Vienna University of Technology KTH Stockholm COST action FP 1105 meeting San Sebastian, May 26 -27, 2015

KTH Royal Institute of Technology Stockholm 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 (p. HCAs) can be p-coumaric acid (p. CA) or ferulic acid (FA) - ester/ether ratio: p. CA > 2: 1 FA < 1: 2 Buranov and Mazza (2008) ether bond ester bond

KTH Royal Institute of Technology Stockholm 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: Free lignin (FL) / Dioxane lignin (DL) wheat straw cut to 40 mesh ball-milling dioxane-water 0 h, 4 h, 8 h, 12 h, 16 h Soxhlet 0. 1 M HCl in dioxane-water Acidolysis lignin (AL) Residual lignin (RL) Fractions: - Dioxane lignin (DL): Free lignin (FL) 0 h BM Dioxane lignin (DL) 8 h BM Net. DL deducted: DL(8 h) - FL - Acidolysis lignin (AL) 8 h BM - Residual lignin (RL) Gravimetric analysis: KL – carbohydrates analysis: HP-SEC (alkaline): 2 D HSQC NMR: 31 P-NMR: - yields - composition - molar mass - monomeric ratio S: G: H - interunit linkages ratio - estimated p. HCAs, Tricin contents - functional groups

KTH Royal Institute of Technology Stockholm Wheat straw lignin isolation and characterization: Results: fractions FL, DL/Net. DL, AL, RL 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) 0 Klason lignin (%) recovery FL/DL AL RL total (relative% of the starting material) 2. 2 6. 3 (13. 8) (39. 4) 6. 1 (38. 1) 14. 6 (91. 3%) 13. 9 (86. 9%) 4 5. 1 (13. 8+18. 1) 5. 9 (37. 5) 2. 9 (18. 4) 8 5. 1 (13. 8+18. 1) 6. 0 (37. 5) 2. 0 13. 1 (12. 5) (81. 9%)

KTH Royal Institute of Technology Stockholm Wheat straw lignin isolation and characterization: Results: fractions FL, DL/Net. DL, AL, RL Composition: Klason lignin: FL/DL << AL RL very low KL content! … hardly a „lignin fraction“ Carbohydrates: FL Glucose-lignin DL/Net. DL Xylan-lignin (after 8 h BM) with low Arabinose content („linear“ xylan) AL Xylan-lignin with high Arabinose content („branched“ xylan) RL „Glucose-lignin“ Dioxane lignin (FL/DL) Acidolysis lignin (AL) Residual lignin (RL) Ballmilling Klason Relative sugar composition [%] [%] lignin [h] [%] 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

KTH Royal Institute of Technology Stockholm Wheat straw lignin isolation and characterization: Results: fractions FL, DL/Net. DL, AL, RL Composition: Klason lignin: FL/DL << AL RL very low KL content! … hardly a „lignin fraction“ Carbohydrates: FL Glucose-lignin DL/Net. DL Xylan-lignin (after 8 h BM) with low Arabinose content („linear“ xylan) AL Xylan-lignin with high Arabinose content („branched“ xylan) RL „Glucose-lignin“ Dioxane lignin (FL/DL) Acidolysis lignin (AL) Residual lignin (RL) Ballmilling Klason Relative sugar composition [%] [%] lignin [h] [%] 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

KTH Royal Institute of Technology Stockholm Wheat straw lignin isolation and characterization: Results: 4 fractions FL, DL/Net. DL, AL Composition: Klason lignin: FL/DL << AL RL very low KL content! … hardly a „lignin fraction“ Carbohydrates: FL Glucose-lignin DL/Net. DL Xylan-lignin (after 8 h BM) with low Arabinose content („linear“ xylan) AL Xylan-lignin with high Arabinose content („branched“ xylan) Dioxane lignin (FL/DL) Acidolysis lignin (AL) Residual lignin (RL) Ballmilling Klason Relative sugar composition [%] [%] lignin [h] [%] 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

KTH Royal Institute of Technology Stockholm Wheat straw lignin isolation and characterization: Results: 4 fractions FL, DL/Net. DL, AL Molar mass analysis: FL: - smallest MM! - very high- and very low-molar mass parts - p. HCAs seem dominant! 31 P-NMR: highest content of free COOHs monomeric or ether-bound p. HCAs: p. CA and FA identified as monomers by preparative HPSEC and RP-HPLC Net. DL: - similar molar mass as AL - but: stronger p. HCAs-peak! AL: - most symmetric/uniform MMD - p. HCAs-peak: just a shoulder, so no signs of p. HCAs with free COOHs (31 P-NMR: lowest COOHs!) Ballmilling [h] 0 8 8 (Net) Dioxane lignin (DL) Acidolysis lignin (AL) Mw Mn Pd [k. Da] 1520 900 1. 69 2300 690 3. 33 3000 1100 2. 73 3300 770 4. 29 4600 1310 3. 51

KTH Royal Institute of Technology Stockholm Wheat straw lignin isolation and characterization: Results: 4 fractions FL, DL/Net. DL, AL Molar mass analysis: FL: - smallest MM! - very high- and very low-molar mass parts - p. HCAs seem dominant! 31 P-NMR: highest content of COOHs monomeric or etherbound p. HCAs: p. CA and FA identified as monomers by preparative HPSEC and RP-HPLC Net. DL: - similar molar mass as AL - but: stronger p. HCAs-peak! AL: - most symmetric/uniform MMD - p. HCAs-peak: just a shoulder, so no signs of p. HCAs with free COOHs (31 P-NMR: lowest COOHs!) Ballmilling [h] 0 8 8 (Net) Dioxane lignin (DL) Acidolysis lignin (AL) Mw Mn Pd [k. Da] 1520 900 1. 69 2300 690 3. 33 3000 1100 2. 73 3300 770 4. 29 4600 1310 3. 51

KTH Royal Institute of Technology Stockholm Wheat straw lignin isolation and characterization: Results: 4 fractions FL, DL/Net. DL, AL Molar mass analysis: FL: - smallest MM! - very high- and very low-molar mass parts - p. HCAs seem dominant! 31 P-NMR: highest content of COOHs monomeric or etherbound p. HCAs: p. CA and FA identified as monomers by preparative HPSEC and RP-HPLC Net. DL: - similar molar mass as AL - but: stronger p. HCAs-peak! AL: - most symmetric/uniform MMD - p. HCAs-peak: just a shoulder, so no signs of p. HCAs with free COOHs (31 P-NMR: lowest COOHs!) Ballmilling [h] 0 8 8 (Net) Dioxane lignin (DL) Acidolysis lignin (AL) Mw Mn Pd [k. Da] 1520 900 1. 69 2300 690 3. 33 3000 1100 2. 73 3300 770 4. 29 4600 1310 3. 51

KTH Royal Institute of Technology Stockholm Wheat straw lignin isolation and characterization: Results: 4 fractions FL, DL/Net. DL, AL Monomeric ratio, p. HCAs content: FL low S units and highest in G units; low p. HCAs content and p. CA > FA highest Tricin content DL also low S units and highest H units highest p. HCAs content (Net. DL) and FA >> p. CA AL high S units ratio high p. HCAs content and p. CA >> FA very little free COOH groups found! p. CA-esters! DL AL Monomeric ratio [%] Ballmilling S units G units H units [h] 0 34 61 5 8 35 56 9 8 (Net) 36 51 13 8 42 52 6 p. HCAs [%] total p. HCAs FA p. CA ratio FA/p. CA [ ] 5. 8 13. 4 22. 0 13. 1 2. 4 8. 9 16. 0 4. 2 3. 3 4. 5 6. 0 8. 9 0. 7 2. 0 2. 7 0. 5 Tricin [%] 14. 9 10. 6 6. 0 6. 8

KTH Royal Institute of Technology Stockholm Wheat straw lignin isolation and characterization: Results: 4 fractions FL, DL/Net. DL, AL Monomeric ratio, p. HCAs content: FL low S units and highest in G units; low p. HCAs content and p. CA > FA highest Tricin content DL also low S units and highest H units highest p. HCAs content (Net. DL) and FA >> p. CA AL high S units ratio high p. HCAs content and p. CA >> FA very little free COOH groups found! p. CA-esters! DL AL Monomeric ratio [%] Ballmilling S units G units H units [h] 0 34 61 5 8 35 56 9 8 (Net) 36 51 13 8 42 52 6 p. HCAs [%] total p. HCAs FA p. CA ratio FA/p. CA [ ] 5. 8 13. 4 22. 0 13. 1 2. 4 8. 9 16. 0 4. 2 3. 3 4. 5 6. 0 8. 9 0. 7 2. 0 2. 7 0. 5 Tricin [%] 14. 9 10. 6 6. 0 6. 8

KTH Royal Institute of Technology Stockholm Wheat straw lignin isolation and characterization: Results: 4 fractions FL, DL/Net. DL, AL Monomeric ratio, p. HCAs content: FL low S units and highest in G units; low p. HCAs content and p. CA > FA highest Tricin content DL also low S units and highest H units highest p. HCAs content (Net. DL) and FA >> p. CA AL high S units ratio high p. HCAs content and p. CA >> FA very little free COOH groups found! p. CA-esters! DL AL Monomeric ratio [%] Ballmilling S units G units H units [h] 0 34 61 5 8 35 56 9 8 (Net) 36 51 13 8 42 52 6 p. HCAs [%] total p. HCAs FA p. CA ratio FA/p. CA [ ] 5. 8 13. 4 22. 0 13. 1 2. 4 8. 9 16. 0 4. 2 3. 3 4. 5 6. 0 8. 9 0. 7 2. 0 2. 7 0. 5 Tricin [%] 14. 9 10. 6 6. 0 6. 8

KTH Royal Institute of Technology Stockholm Wheat straw lignin isolation and characterization: Results: 4 fractions FL, DL/Net. DL, AL proposed arrangement of isolated lignin fractions: FL p. HCA/Tricin --- FL --- Cellulose(OS) plus monomeric p. CA, FA and cinnamyl alcohols DL Net. DL AL p. CA --- AL --- (FA) --- Ara --- Xylan (branched) (p. CA)/FA --- DL --- FA-(FA) --- (Ara) --- Xylan (linear)

KTH Royal Institute of Technology Stockholm Wheat straw lignin isolation and characterization: Results: 4 fractions FL, DL/Net. DL, AL proposed arrangement of isolated lignin fractions: FL p. HCA/Tricin --- FL --- Cellulose(OS) plus monomeric p. CA, FA and cinnamyl alcohols high G … ML DL Net. DL AL p. CA --- AL --- (FA) --- Ara --- Xylan (branched) (p. CA)/FA --- DL --- FA-(FA) --- (Ara) --- Xylan (linear) high H, FA … ML high S, p. CA-ester … SW Donaldson 2001 Fukushima and Terashima, 1990 He and Terashima, 1989, 1990

KTH Royal Institute of Technology Stockholm Wheat straw lignin isolation and characterization: Results: 4 fractions FL, DL/Net. DL, AL Conclusions: - unique fraction FL accessible already w/o BM and AH origin? 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)

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