Lignin- evergeen biopolymer

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1 Lignin- evergeen biopolymer Jussi Sipilä University of Helsinki Department of Chemistry Laboratory of Organic Chemistry

2 What is lignin? The cell walls of vascular plants consists of macromolecular substances: cellulose ( ca 50% of dry wood weight) hemicellulose ( ca 20%) lignin (ca 30%) While cellulose and hemicelluloses are polysaccharides, lignin is an amorphic polyphenol formed in plant secondary metabolism in the presence of carbohydrates

3 Prominent structures in softwood lignin

4 Lignin Definition Lignins are heterogenic aromatic polymers formed in a random dehydrogenative polymerization of three primary precursors during plant cell wall biosynthesis and always formed in the pricense of carbohydrates: Lignin acts as a binding material for the cell walls and give them rigidity to resist external forces like gravity and wind. By prohibiting water transportation trough the cell walls, lignin also plays important role in the transportation system of living trees.

5 Lignin in the cell wall

6 Why lignin research? Academic: Lignin is an essential component of vascular plants Pulp and paper industry: it is lignin that is removed in pulping and bleaching processes!! Present interests: - development of chlorine free paper making processes - treatment of waste effluents of pulping industry - applying biotechnology in pulping processes Biorefineries: Utilization of lignin markedly boosts the economy of the processes Chances of affecting the cource of cell wall biosynthesis by means of gene technology: better timber and/or trees with better pulping properties by modifying the structure of lignin

7 Most important tools in lignin research Lignin prepaprates: MWL (milled wood lignin) % of total lignin, obtained by milling extractive free wood in toluene. Mostly slightly modified secondary cell wall lignin. Extra Cellular ligins -lignins released by cell cultures by treatment with elicitors or hormones Analytical tools: Chemical degradations: - potassium permanganate, nitrobenzene oxidations - acidolysis and thioacidolysis - degradation by ozone - reductive cleavage after derivatization (DFRC) NMR and IR

8 Most important tools in lignin research... Study of lignin structure and biosynthesis by model compounds: Studies on synthetic lignins, DHP s Studies on lignin reactivity by oligomeric model compounds Information obtained from materials extracted from pulping and bleaching processes In 1990 s, NMR has begun a cornerstone in structural determination of lignins

9 Abundance of main structural elements in MWL Hans Önnerud: Dissertation 2002, KTH, Sweden β-o-4 β-5 spruce spruce comp. birch β-o β β-β trace β trace β-1 β-β 5-5-O O-4 s are the newest major structural types in lignins, found in 1994 at the laboratory of organic chemistry at Helsinki University Chemistry Department

10 Other structures in softwood lignins α-o-4 (1%) 5 5 (23%?) anillin (3%) cyclohexadienone(3%) 5-O-4 (4%) coniferyl alcohol (2%) dihydroconiferyl alcohol (1%) coniferyl aldehyde (3%)

11 However, due to the structural irregularity and intractable nature of lignins, model studies are of crucial importance in lignin structural determinations! How is lignin chemically formed?

12 Lignin is formed in a dehydrogenative polymerization of its precursors

13 Main elements in the phenylpropanoid pathway

14 Coniferyl alcohol makes first dilignols

15 Predominant mode of lignin formation: Endwise polymerization! The growth of the lignin polymer predominantly takes place in a process called end-wise polymerization

16 Relevant questions concerning lignin biosynthesis Problems: medium? oxidant? oxidation potentials? intoduction of momomers? molecular weight? LCC?

17 Relevant questions concerning lignin biodegradation and its application Basic problems: shuttle system? natural mediators? Technical questions: demethoxylating enzymes? strip to simple monomers?

18 Relevant questions concerning lignin utilization Problems: Reactivity? Homogenity? Difficult Tg? Technical solutions: simple derivatization? Adjustment of Mw? SULFUR! In Kraft lignin

19 Wee need model compounds!!

20 We need to study the formation of in vitro lignins! coniferyl alcohol in dioxane hydrogen peroxide in buffer HRP in dioxane-water 1:4 end-wise : slow, simultaneous addition of coniferyl alcohol and hydrogen peroxide

21 Reaction medium! DHPs prepared at low ph generate material that has higher amount of arylglycerol β-ether structures than conventional DHP prepared at ph 6. However, at abundance much below than that observed in MWL The presence of carbohydrates has no effect on the ratio of the main structural patterns Even at low ph: We need a catalysts!

22 Oxidant!

23 Other problems in biosynthesis: Studies on DHP have classically been made using horseradish peroxidase (HRP) as a catalysts. HRP isn t natural peroxidase for lignin biosynthsis and may give erroneous results by exhibiting wrong substrate specificity and oxidation potentials. Promising results in DHP formation has recently been obtained using Mn-peroxidase enabled redox shuttles (H. Önnerud et al. 2002). It is also obvious that small molecular weight phenolics participates as mediators in oxidation processes (Nousiainen et al. 2012)

24 Oxidation potentials! Do we need a robust oxidant?

25 Diffusion of monomers! Using a very slow (diffusing) addition of lignin precursors the amount of β-o-4 linked dimers seem to increase compared to conventional DHPs (Brunow, Syrjänen, Karhunen, 2001) 10 : 1 dimers in DHP 1 : 1 DHPs closer to MWL has been obtained using similar diffusion idea in works by Terashima (coniferin + β-glucosidase + glucose oxidase + HRP) and Higuchi (HRP)

26 N. Lewis, L. Davin and S. Sarkanen Chemical & Engeneering News 2000

27 Some hypothesis of Lewis et al. random coupling notation doesn t make any biochemical sense. Comparatively small dirigent proteins are involved in lignan biosynthesis recognizing specific substrates and controlling regio- and stereochemistry in radical coupling. Why not in lignin biosynthesis? Lignin formation is conrolled by proteins containing an array of dirigent (monomeric-specific binding) sites located near the lignin initiation sites of specific cell types. The proteins dictate the monomers and the type of lingages that will be formed trough the specificity and orientation of their binding sites. A progenitor macromolecule will be formed which then will be replicated by template mechanism.

28 The opponents The differences in composition of lignin in different parts of cell wall can be explained by the plant shifting production from one monolignol to another. Plants direct what goes on in the wall, and there is no need to select anything. Plants lacking enzymes in their monolignol synthetic pathways often incorporate phenolics other than the traditional monolignols Isn t the cell wall itself a template? Nothing now known about lignin requires invoking the new model

29 Lignin are not optically active but can learn something by studying brancing of lignin macromolecule: most important are biphenylic structures According to Pew, total amount of biphenyls in softwood lignins is around 25%!!!!!

30 Biphenyls also form via radical coupling According to the rigidity of the polymeric radicals the probability term should be unfavourable?

31 Can we approach the problem via biomimetic oxidation experiments with model compounds!

32 Formation of dibenzodiocoxins with simple biphenyls Simple biphenyls give dibenzodioxocins in reasonable yields.

33 Cross-coupling of 5-5-O-4 DHDVA HPLC chromatogram of the oxidation mixture

34 Dehydro-bis-coniferyl alcohol, Do they exist? Freudenberg (Constitution and Biosynthesis of Lignin): Dehydrobisconiferyl alcohol is a very labile substance? Zutropf, HRP/H 2 O 2 ph 4 (acetone-water)

35 HSQC-NMR of cross-coupling experiment of dehydro-bis-coniferyl alcohol with coniferyl alcohol α β? We also found that dehydro-bis-coniferyl alcohol is stabe in acetone at least for three days!!!

36 More problems - LCC? Laboratory experiment : yield 20 % The nature of LCCs (covalent or physically bound) has been extensively discussed for decades because of the lack of unambiguous analytical results. What short of information on LCC we get from pulping?

37 Here we need 2D-NMR techniques

38 HSQC-spectrum of milled wood lignin (MWL) α in β-o-4 α in β-5 α in β-β β in 5 5 Ο 4 2D NMR connected to 13C NMR has become one of the most important tools in lignin research - for example, detection of the 5-5-O-4 structures in softwood MWL 1994

39 Release of lignin fron the cell walls during pulping reveals interesting aspects on LCC s FT-SLL 60 min. FT-SLL 90 min. Milled-wood lignin - like material is released in the initial phase

40 HSQC of FT-SLL FT-SLL 180 FT-SLL 240 significant amounts of carbohydtares are found in the SLL s released at later stages of cook.

41 KRAFT RESIDUAL LIGNIN AND KRAFT SPENT LIQUOR LIGNIN OF PINE K-SLL K-RL from enol-ether SAQ-SLL contains less enol-ether moieties than K-SLL

42 Problems in Mw-determination!

43 GPC chromatograms of MWL and CEL from: Holtman, K. M., Chang, H-M and Kadla, J. F. Solution -State Nuclear Magnetic Resonance Study of the Similarities between Milled Wood Lignin and Cellulolytic Enzyme Lignin J. Agric. Food. Chem. 2004, 52,

44 Concluding remarks At present, the structure of secondary cell wall lignin is well established Despite intensive investigation spanning over decades, the bio-organic chemistry of lignin formation is still rather poorly understood. For plant biomass utilization, much more detailed information is needed for example, on the enzymatic assistance in lignin polymerization and formation and nature of lignin-carbohydrate complexes.

45 ACKNOWLEDGEMENTS The author also wish to thank the members and students of Wood Chemistry Group at Chemistry Department at Helsinki University, especially Gösta Brunow, Paula Nousiainen, Anssi Haikarainen, Pirkko Karhunen, Ilari Filpponen, Camilla Wiik and Kaisa Koli, Jussi Kontro, Helmiina Manner and many, many others in Finland and abroad.. Also financial support to our lignin studies at UH from TEKES, the National Technology Agency of Finland, Kemira Oyj, M-Real Oyj, Stora Enso Oyj and UPM-Kymmene Oyj, Andriz-Ahlström Oy, EU Shared Cost project FAIR-CT , EU-project Biorenew and Finnish Academy (Pro-Lignin, LuomuCat) is gratefully acknowledged.

FORMATION AND STRUCTURE OF LIGNIFIED PLANT CELL WALL - FACTORS CONTROLLING LIGNIN STRUCTURE DURING ITS FORMATION

69 FORMATION AND STRUCTURE OF LIGNIFIED PLANT CELL WALL - FACTORS CONTROLLING LIGNIN STRUCTURE DURING ITS FORMATION Noritsugu Terashima and Rajai H. Atalla USDA Forest Products Laboratory, One Gifford