1. KTH Royal Institute of Technology Stockholm Wheat straw lignin – Successive Fractionation and Structural Characterization Florian Zikeli Vienna University of Technology KTH Stockholm COST action FP1105 meeting San Sebastian, May 26-27, 2015

2. 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  > 2:1 FA  < 1:2 ether bond ester bond KTH Royal Institute of Technology Stockholm Buranov and Mazza (2008)

3. 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

4. Wheat straw lignin isolation and characterization: Fractions:- Dioxane lignin (DL): Free lignin (FL)0h BM Dioxane lignin (DL)8h BM NetDLdeducted: DL(8h) - FL - Acidolysis lignin (AL)8h BM - Residual lignin (RL) Gravimetric analysis:- yields KL – carbohydrates analysis:- composition HP-SEC (alkaline):- molar mass 2D HSQC NMR:- monomeric ratio S:G:H - interunit linkages ratio - estimated pHCAs, Tricin contents 31 P-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 in cut to 40 mesh

5. 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/DLALRLtotal (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%)

6. 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 [%] AraGalGluXylAraGalGluXylAraGalGluXyl 046.01015631288.3344134911.8007426 452.21412462887.133216499.100928 854.8125275687.5243 497.300937 8 (Net)64.0>12<<5<<27>>56 Composition:Klason lignin:FL/DL << AL RL very low KL content! … hardly a „lignin fraction“ Carbohydrates:FLGlucose-lignin DL/NetDL Xylan-lignin (after 8h BM) with low Arabinose content („linear“ xylan) ALXylan-lignin with high Arabinose content („branched“ xylan) RL„Glucose-lignin“ Results: fractions FL, DL/NetDL, AL, RL

7. 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 [%] AraGalGluXylAraGalGluXylAraGalGluXyl 046.01015631288.3344134911.8007426 452.21412462887.133216499.100928 854.8125275687.5243 497.300937 8 (Net)64.0>12<<5<<27>>56 Composition:Klason lignin:FL/DL << AL RL very low KL content! … hardly a „lignin fraction“ Carbohydrates:FLGlucose-lignin DL/NetDL Xylan-lignin (after 8h BM) with low Arabinose content („linear“ xylan) ALXylan-lignin with high Arabinose content („branched“ xylan) RL„Glucose-lignin“ Results: fractions FL, DL/NetDL, AL, RL

8. 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 [%] AraGalGluXylAraGalGluXylAraGalGluXyl 046.01015631288.3344134911.8007426 452.21412462887.133216499.100928 854.8125275687.5243 497.300937 8 (Net)64.0>12<<5<<27>>56 Composition:Klason lignin:FL/DL << AL RL very low KL content! … hardly a „lignin fraction“ Carbohydrates:FLGlucose-lignin DL/NetDL Xylan-lignin (after 8h BM) with low Arabinose content („linear“ xylan) ALXylan-lignin with high Arabinose content („branched“ xylan) RL„Glucose-lignin“ Results: fractions FL, DL/NetDL, AL, RL

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 [%] AraGalGluXylAraGalGluXylAraGalGluXyl 046.01015631288.3344134911.8007426 452.21412462887.133216499.100928 854.8125275687.5243 497.300937 8 (Net)64.0>12<<5<<27>>56 Composition:Klason lignin:FL/DL << AL RL very low KL content! … hardly a „lignin fraction“ Carbohydrates:FLGlucose-lignin DL/NetDL Xylan-lignin (after 8h BM) with low Arabinose content („linear“ xylan) ALXylan-lignin with high Arabinose content („branched“ xylan) Results: 4 fractions FL, DL/NetDL, AL

10. Molar mass analysis: FL:- smallest MM! - very high- and very low-molar mass parts -pHCAs seem dominant! 31 P-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 ( 31 P-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) M w [kDa] M n [kDa ] P d [kDa] M w [kDa] M n [kDa] P d [kDa] 015209001.6923006903.33 8300011002.7333007704.29 8 (Net)460013103.51

11. Molar mass analysis: FL:- smallest MM! - very high- and very low-molar mass parts -pHCAs seem dominant! 31 P-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 ( 31 P-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) M w [kDa] M n [kDa ] P d [kDa] M w [kDa] M n [kDa] P d [kDa] 015209001.6923006903.33 8300011002.7333007704.29 8 (Net)460013103.51

12. Molar mass analysis: FL:- smallest MM! - very high- and very low-molar mass parts -pHCAs seem dominant! 31 P-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 ( 31 P-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) M w [kDa] M n [kDa ] P d [kDa] M w [kDa] M n [kDa] P d [kDa] 015209001.6923006903.33 8300011002.7333007704.29 8 (Net)460013103.51

13. Monomeric ratio, pHCAs content: FLlow S units and highest in G units; low pHCAs content and pCA > FA highest Tricin content DL also low S units and highest H units highest pHCAs content (NetDL) and FA >> pCA ALhigh S units ratio high pHCAs content and pCA >> FA very 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 unitsG unitsH units total pHCAs FApCA ratio FA/pCA [ ] DL 0346155.82.43.30.714.9 83556913.48.94.52.010.6 8 (Net)36511322.016.06.02.76.0 AL84252613.14.28.90.56.8

14. Monomeric ratio, pHCAs content: FLlow S units and highest in G units; low pHCAs content and pCA > FA highest Tricin content DL also low S units and highest H units highest pHCAs content (NetDL) and FA >> pCA ALhigh S units ratio high pHCAs content and pCA >> FA very 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 unitsG unitsH units total pHCAs FApCA ratio FA/pCA [ ] DL 0346155.82.43.30.714.9 83556913.48.94.52.010.6 8 (Net)36511322.016.06.02.76.0 AL84252613.14.28.90.56.8

15. Monomeric ratio, pHCAs content: FLlow S units and highest in G units; low pHCAs content and pCA > FA highest Tricin content DL also low S units and highest H units highest pHCAs content (NetDL) and FA >> pCA ALhigh S units ratio high pHCAs content and pCA >> FA very 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 unitsG unitsH units total pHCAs FApCA ratio FA/pCA [ ] DL 0346155.82.43.30.714.9 83556913.48.94.52.010.6 8 (Net)36511322.016.06.02.76.0 AL84252613.14.28.90.56.8

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

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

18. 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 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)

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