Effects of Two-Stage Dilute Acid Pretreatment on the Structure and Composition of Lignin and Cellulose in Loblolly Pine

A standard two-step dilute sulfuric acid pretreatment was performed on Loblolly pine to enhance the overall efficiency of enzymatic deconstruction of woody biomass to monomeric sugars. The structure of milled wood lignin and cellulose isolated from the untreated and acid-treated biomass was studied in detail. Solid-state ¹³C NMR spectroscopy coupled with line shape analyses has been employed to elucidate cellulose crystallinity and ultrastructure. The results indicate an increase in the degree of crystallinity and reduced relative proportion of less ordered cellulose allomorphs following the acid pretreatment. This increase was attributed to a preferential degradation of amorphous cellulose and less ordered crystalline forms during the high temperature pretreatment. Milled wood lignin structural elucidation by quantitative ¹³C and ³¹P NMR reveals an increase in the degree of condensation of lignin due to the pretreatment. The increase in degree of condensation is accompanied by a decrease in β-O-4 linkages which were fragmented and recondensed during the high temperature acid-catalyzed reactions.     

Structural characterization of lignin during Pinus taeda wood treatment with Ceriporiopsis subvermispora

Pinus taeda wood chips were biotreated with Ceriporiopsis subvermispora under solid-state fermentation for periods varying from 15 to 90 days. Milled wood lignins extracted from sound and biotreated wood samples were characterized by wet-chemical and spectroscopic techniques. Treatment of the lignins by derivatization followed by reductive cleavage (DFRC) made it possible to detect DFRC monomers and dimers that are diagnostic of the occurrence of arylglycerol-beta-O-aryl and beta-beta, beta-5, beta-1, and 4-O-5 units in the lignin structure. Quantification of these DFRC products indicated that beta-O-aryl cleavage was a significant route for lignin biodegradation but that beta-beta, beta-5, beta-1, and 4-O-5 linkages were more resistant to the biological attack. The amount of aromatic hydroxyls did not increase with the split of beta-O-4 linkages, suggesting that the beta-O-4 cleavage products remain as quinone-type structures as detected by UV and visible spectroscopy. Nuclear magnetic resonance techniques also indicated the formation of new substructures containing nonoxygenated, saturated aliphatic carbons (CH(2) and CH(3)) in the side chains of lignins extracted from biotreated wood samples.     

Biodegradation of lignin-carbohydrate complexes

Covalent lignin-carbohydrate (LC) linkages exist in lignocellulose from wood and groups herbaceous plants. In wood, they consist of ester and ether linkages through sugar hydroxyl to the -carbanol of phenylpropane subunits in lignin. In grasses, ferulic and p-coumaric acids are esterified to hemicelluloses and lignin, respectively. Hemicelluloses also contain substitutents and side groups that restrict enzymatic attack. Watersoluble lignin-carbohydrate complexes (LCCs) often precipitate during digestion with polysaccharidases, and the residual sugars are more diverse than the bulk hemicellulose. A number of microbial esterases and hemicellulose polysaccharidases including acetyl xylan esterase, ferulic acid esterase, and p-coumaric esterase attack hemicellulose side chains. Accessory hemicellulases include -l-arabinofuranosidase and -methyl-glucuranosidase. Both of these side chains are involved in LC bonds. -Glucosidase will attach sugar residues to lignin degradation products and when carbohydrate is attached to lignin, lignin peroxidase will depolymerize the lignin more readily.Abbreviations APPL acid precipitable polymeric lignin - CBQase cellobioquinone oxidoreductase - LC lignincarbohydrate - LCC(s) lignin-carbohydrate complex - DHP Dehydrogenative polymerisate - DMSO dimethylsulfoxide - DP degree of polymerisation - MWEL milled wood enzyme lignin - MWL milled wood lignin (not digested with carbohydrases)     

<Emphasis Type="Italic">Phoma herbarum</Emphasis>, a soil fungus able to grow on natural lignin and synthetic lignin (DHP) as sole carbon source and cause lignin degradation

The fungus Phoma herbarum isolated from soil showed growth on highly pure lignin extracted from spruce wood and on synthetic lignin (DHP). The lignin remaining after cultivation was shown to have a lower molecular weight. The reduction in the numbers of ether linkages of the extracted lignins was also observed by derivatization followed by reductive cleavage (DFRC) in combination with ³¹P NMR studies. The fungal strain showed an ability to degrade synthetic lignin by extracellular catalysts. GC–MS was applied to study the evolution of low molar mass adducts, e.g., monolignols and it was shown that a reduced coniferyl alcohol product was produced from DHP in a cell-free environment. The work has demonstrated the ability of soil microbes to grow on lignin as sole carbon source. The potential impact is in the production of low molar mass renewable phenols for material application.     

A novel and efficient oxidative functionalization of lignin by layer-by-layer immobilised Horseradish peroxidase

Horseradish peroxidase (HRP) was chemically immobilised onto alumina particles and coated by polyelectrolytes layers, using the layer-by-layer technique. The reactivity of the immobilised enzyme was studied in the oxidative functionalisation of softwood milled wood and residual kraft lignins and found higher than the free enzyme. In order to investigate the chemical modifications in the lignin structure, quantitative (31)P NMR was used. The immobilised HRP showed a higher reactivity with respect to the native enzyme yielding extensive depolymerisation of lignin.     

Structural Characterization of Lignin during Pinus taeda Wood Treatment with Ceriporiopsis subvermispora

Pinus taeda wood chips were biotreated with Ceriporiopsis subvermispora under solid-state fermentation for periods varying from 15 to 90 days. Milled wood lignins extracted from sound and biotreated wood samples were characterized by wet-chemical and spectroscopic techniques. Treatment of the lignins by derivatization followed by reductive cleavage (DFRC) made it possible to detect DFRC monomers and dimers that are diagnostic of the occurrence of arylglycerol-β-O-aryl and β-β, β-5, β-1, and 4-O-5 units in the lignin structure. Quantification of these DFRC products indicated that β-O-aryl cleavage was a significant route for lignin biodegradation but that β-β, β-5, β-1, and 4-O-5 linkages were more resistant to the biological attack. The amount of aromatic hydroxyls did not increase with the split of β-O-4 linkages, suggesting that the β-O-4 cleavage products remain as quinone-type structures as detected by UV and visible spectroscopy. Nuclear magnetic resonance techniques also indicated the formation of new substructures containing nonoxygenated, saturated aliphatic carbons (CH₂ and CH₃) in the side chains of lignins extracted from biotreated wood samples.     

Multidimensional NMR analysis reveals truncated lignin structures in wood decayed by the brown rot basidiomycete Postia placenta

Lignocellulose biodegradation, an essential step in terrestrial carbon cycling, generally involves removal of the recalcitrant lignin barrier that otherwise prevents infiltration by microbial polysaccharide hydrolases. However, fungi that cause brown rot of wood, a major route for biomass recycling in coniferous forests, utilize wood polysaccharides efficiently while removing little of the lignin. The mechanism by which these basidiomycetes breach the lignin remains unclear. We used recently developed methods for solubilization and multidimensional (1) H-(13) C solution-state NMR spectroscopy of ball-milled lignocellulose to analyse aspen wood degraded by Postia placenta. The results showed that decay decreased the content of the principal arylglycerol-β-aryl ether interunit linkage in the lignin by more than half, while increasing the frequency of several truncated lignin structures roughly fourfold over the level found in sound aspen. These new end-groups, consisting of benzaldehydes, benzoic acids and phenylglycerols, accounted for 6-7% of all original lignin subunits. Our results provide evidence that brown rot by P. placenta results in significant ligninolysis, which might enable infiltration of the wood by polysaccharide hydrolases even though the partially degraded lignin remains in situ. Recent work has revealed that the P. placenta genome encodes no ligninolytic peroxidases, but has also shown that this fungus produces an extracellular Fenton system. It is accordingly likely that P. placenta employs electrophilic reactive oxygen species such as hydroxyl radicals to disrupt lignin in wood.     

Characterization of lignin-rich residues remaining after continuous super-critical water hydrolysis of poplar wood (Populus albaglandulosa) for conversion to fermentable sugars

Poplar wood flour (Populous albaglandulosa) was treated with sub- and super-critical water (subcritical: 325, 350 °C; super-critical: 380, 400, 425 °C) for 60 s at 220 ± 10 atm. Hydrochloric acid (0.05% v/v) was added to samples as acidic catalyst. The final products were separated into water soluble fraction and undegraded solids. The yields of undegraded solids were thoroughly dependent on temperature severity and mainly composed of lignin fragments. Average molecular weights of the lignins were between 1500 and 4400 Da, which was only 1/3-1/8-fold of poplar milled wood lignin (13,250 Da). DFRC (Derivatization Followed by Reductive Cleavage) analysis revealed that C6C3 phenols (coniferyl and sinapyl alcohol) were rarely detected in the lignins, indicating occurrence of two probable lignin reactions during SCW hydrolysis: lignin fragmentation via splitting of β-O-4 linkage and loss of propane side chains. These results were also confirmed by ¹H and ¹³C NMR spectroscopic analysis.     

Styrene-spaced copolymers including anthraquinone and β-O-4 lignin model units: synthesis, characterization and reactivity under alkaline pulping conditions

A series of random copoly(styrene)s has been synthesized via radical polymerization of functionalized anthraquinone (AQ) and β-O-4 lignin model monomers. The copolymers were designed to have a different number of styrene spacer groups between the AQ and β-O-4 lignin side chains aiming at investigating the distance effects on AQ/β-O-4 electron transfer mechanisms. A detailed molecular characterization, including techniques such as size exclusion chromatography, MALDI-TOF mass spectrometry, and (1)H, (13)C, (31)P NMR and UV-vis spectroscopies, afforded quantitative information about the composition of the copolymers as well as the average distribution of the AQ and β-O-4 groups in the macromolecular structures. TGA and DSC thermal analysis have indicated that the copolymers were thermally stable under regular pulping conditions, revealing the inertness of the styrene polymer backbone in the investigation of electron transfer mechanisms. Alkaline pulping experiments showed that close contact between the redox active side chains in the copolymers was fundamental for an efficient degradation of the β-O-4 lignin model units, highlighting the importance of electron transfer reactions in the lignin degradation mechanisms catalyzed by AQ. In the absence of glucose, AQ units oxidized phenolic β-O-4 lignin model parts, mainly by electron transfer leading to vanillin as major product. By contrast, in presence of glucose, anthrahydroquinone units (formed by reduction of AQ) reduced the quinone-methide units (issued by dehydration of phenolic β-O-4 lignin model part) mainly by electron transfer leading to guaiacol as major product. Both processes were distance dependent.     

Incorporation of p-cresol into lignins via peroxidase-catalysed copolymerization in nonaqueous media

Lignin, the second most abundant biopolymer after cellulose, is a low value by-product of agricultural and wood conversion processes, including wood pulp manufacture. Copolymerization with phenols has the potential to convert by-product lignins to higher-value phenolic resins. In this initial investigation, we have studied the use of horseradish peroxidase (HRP) in aqueous dioxane to catalyse the grafting of p-cresol (p-methylphenol) onto milled wood lignin, kraft lignin, and a lignin selectively o-demethylated by a brown-rot fungus. Advantages of this system are (1) the mild reaction conditions employed and (2) the ability of HRP to function in the dioxane: water solutions which solubilize lignin. The reaction is monitored by gel permeation chromatography using a reaction system of [¹⁴C]-p-cresol with unlabeled lignins. We have found that optimal incorporation of cresol into high-molecular-weight polymer occurs at 50–70% dioxane in water under the conditions used; a maximum incorporation of ca. 4 mol% (e.g., p-cresol incorporated per C₉ lignin unit) was obtained. Blocking the phenolic hydroxyl groups of the lignin inhibits copolymerization, consistent with the proposed mechanism of phenoxy radical copolymerization for this reaction.     

Characterization of Lignins Isolated with Alkaline Ethanol from the Hydrothermal Pretreated Tamarix ramosissima

The objective of this study was to characterize the changes in lignin structure during hydrothermal pretreatment of shrub Tamarix ramosissima. Lignins in residual wood meal were isolated with alkaline ethanol solution and recovered with acid precipitation. A comparison between the recovered lignin fractions with milled wood lignin has been made in terms of yield, purity, gel permeation chromatography, Fourier transform infrared spectroscopy, pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS), 1D ¹³C and 2D heteronuclear single quantum coherence nuclear magnetic resonance (HSQC NMR) spectroscopic techniques. Semiquantitative HSQC NMR showed that the relative amounts of β-O-4′ (around 76 % side chains) and resinol type substructures (16 %) of lignins were significantly modified during hydrothermal pretreatment. Py-GC/MS analyses brought direct evidences of these lignin samples with high S/G ratios ranging from 1.7 to 2.6. Moreover, the results indicated that an increase in the severity of the hydrothermal pretreatment enhanced the degradation of lignin unit side chains and the condensation of lignin and decreased the molecular weight of the recovered lignin fractions. This study demonstrated that the combination of autohydrolysis and alkaline ethanol process could potentially turn the recovered lignin fractions into value added products being in accordance with the “biorefinery” concept.     

Biological activity of phenolic compounds. Hepatic cytochrome P-450, cytochrome b5, and NADPH cytochrome c reductase in chicks and rats fed phenolic monomers, polymers, and glycosides

Eight experiments were conducted to determine effects of a phenolic polymer (Kraft wood lignin, Indulin), phenolic glycosides (cane molasses and wood molasses), and phenolic monomers (vanillin, vanillic acid, ferulic acid, and p-coumaric acid) on liver cytochromes P-450, cytochrome b5, and NADPH cytochrome c reductase in chicks and rats. Chicks fed 6.0% lignin had a higher (P less than 0.01) cytochromes P-450 content than did chicks fed 0% fiber, 6.0% wood cellulose (Solka Floc), or 6.0% arenaceous flour. NADPH cytochrome c reductase activity was not affected by treatment. Chicks fed 12.0% wood molasses had a higher (P less than 0.06) cytochromes P-450 level than did chicks fed 0% fiber or 6.0% wood molasses. Cane molasses incorporated at both 6.0 and 12.0% of the diet induced (P less than 0.05) cytochromes P-450 content over those of control-fed birds. Chicks fed 6.0% lignin, with or without antibiotic (bacitracin:neomycin sulfate, 2:1), had a higher (P less than 0.01) cytochromes P-450 level than did chicks fed control diets, with or without antibiotic. Additionally, chicks fed 6.0% lignin had lower (P less than 0.01) intestinal diaminopimelic acid (DAP) levels than did chicks fed 0% fiber. Rats fed 0% fiber, 6.0% wood cellulose, 6.0% arenaceous flour, or 6.0% lignin exhibited no difference in cytochrome level or activity among treatments. Chicks fed 0.5% vanillin, 0.5% vanillic acid, 0.5% ferulic acid, or 0.5% p-coumaric acid had comparable cytochromes level and activity compared with chicks fed no phenolics. Chicks fed 0.5% p-coumaric acid had lower (P less than 0.05) rates of gain than did chicks fed control or other phenolic-containing diets. Rats fed these phenolics had similar cytochromes P-450 content among treatments.     

Structural change in wood by brown rot fungi and effect on enzymatic hydrolysis

The effects of biological pretreatment on Pinus radiata and Eucalyptus globulus, were evaluated after exposure to two brown rot fungi Gloephylum trabeum and Laetoporeus sulphureus. Changes in chemical composition, structural modification, and susceptibility to enzymatic hydrolysis in the degraded wood were analyzed. After eight weeks of biodegradation, the greatest loss of weight and hemicellulose were 13% and 31%, respectively, for P. radiata with G. trabeum. The content of glucan decreased slightly, being the highest loss of 20% for E. globulus with G. trabeum. Consistent with degradation mechanism of these fungi, lignin was essentially undegraded by both brown rot fungi. Both brown rot fungi cause a sharp reduction in the cellulose degree of polymerization (DP) in the range between 58% and 79%. G. trabeum depolymerized cellulose in both wood faster than L. sulphureus. Also, structural characteristic of crystalline cellulose were measured by using two different techniques - X-ray diffraction (XRD) and infrared spectroscopy (FT-IR). The biological pretreatments showed an effect on cellulose crystallinity structure, a decrease between 6% and 21% was obtained in the crystallinity index (CrI) calculated by IR, no changes were observed in the XRD. Material digestibility was evaluated by enzymatic hydrolysis, the conversion of cellulose to glucose increased with the biotreatment time. The highest enzymatic hydrolysis yields were obtained when saccharification was performed on wood biopretreated with G. trabeum (14% P. radiata and 13% E. globulus). Decreasing in DP and CrI, and hemicellulose removal result in an increase of enzymatic hydrolysis performance. Digestibility was better related to DP than with other properties. G. trabeum can be considered as a potential fungus for biological pretreatment, since it provides an effective process in breaking the wood structure, making it potentially useful in the development of combined pretreatments (biological-chemical). A viable alternative to pretreatment process that can be used is a bio-mimetic system, similar to low-molecular complexes generated by fungi such as G. trabeum combined pretreatments (biological-chemical).     

Lignin radicals in the plant cell wall probed by Kerr-gated resonance Raman spectroscopy

Lignin radicals are crucial intermediates for lignin biosynthesis in the cell wall of vascular plants. In this work they were for the first time, to our knowledge, selectively observed in wood cell walls by laser-based Kerr-gated resonance Raman spectroscopy, and the observations were supported by density functional theory prediction of their vibrational properties. For dry wood cells a lignin radical Raman band is observed at 1,570 cm(-1) irrespective of species. For wet beech cells they were generated in situ and observed at 1,606 cm(-1). DFT/B3LYP/6-31+G(d) modeling results support that in beech they are formed from syringyl (S) phenolic moieties and in spruce from guaiacyl (G) phenolic moieties. The observed lignin radical band is predicted as G is approximately 1,597 cm(-1) and S is approximately 1,599 cm(-1), respectively, and is assigned the (Wilson notation) nu(8a) phenyl ring mode. The RR band probes lignin radical properties, e.g., spin density distribution, and these respond to charge polarization or hydrogen bonding to proximate water molecules. These observations can be crucial for an understanding of the factors that control cell wall structure during biosynthesis of vascular plants and demonstrate the unique potential of RR spectroscopy of lignin radicals.     

Linoleic acid peroxidation and lignin degradation by enzymes produced by Ceriporiopsis subvermispora grown on wood or in submerged liquid cultures

Ceriporiopsis subvermispora is a white-rot fungus used in biopulping processes and seems to use the fatty acid peroxidation reactions initiated by manganese-peroxidase (MnP) to start lignin degradation. The present work shows that C. subvermispora was able to peroxidize unsaturated fatty acids during wood biotreatment under biopulping conditions. In vitro assays showed that the extent of linoleic acid peroxidation was positively correlated with the level of MnP recovered from the biotreated wood chips. Milled wood was treated in vitro by partially purified MnP and linoleic acid. UV spectroscopy and size exclusion chromatography (SEC) showed that soluble compounds similar to lignin were released from the milled wood. SEC data showed a broad elution profile compatible with low molar mass lignin fractions. MnP-treated milled wood was analyzed by thioacidolysis. The yield of thioacidolysis monomers recovered from guaiacyl and syringyl units decreased by 33% and 20% in MnP-treated milled wood, respectively. This has suggested that lignin depolymerization reactions have occurred during the MnP/linoleic acid treatment.     

Biosynthesis and incorporation of side-chain-truncated lignin monomers to reduce lignin polymerization and enhance saccharification

Lignocellulosic biomass is utilized as a renewable feedstock in various agro‐industrial activities. Lignin is an aromatic, hydrophobic and mildly branched polymer integrally associated with polysaccharides within the biomass, which negatively affects their extraction and hydrolysis during industrial processing. Engineering the monomer composition of lignins offers an attractive option towards new lignins with reduced recalcitrance. The presented work describes a new strategy developed in Arabidopsis for the overproduction of rare lignin monomers to reduce lignin polymerization degree (DP). Biosynthesis of these ‘DP reducers’ is achieved by expressing a bacterial hydroxycinnamoyl‐CoA hydratase‐lyase (HCHL) in lignifying tissues of Arabidopsis inflorescence stems. HCHL cleaves the propanoid side‐chain of hydroxycinnamoyl‐CoA lignin precursors to produce the corresponding hydroxybenzaldehydes so that plant stems expressing HCHL accumulate in their cell wall higher amounts of hydroxybenzaldehyde and hydroxybenzoate derivatives. Engineered plants with intermediate HCHL activity levels show no reduction in total lignin, sugar content or biomass yield compared with wild‐type plants. However, cell wall characterization of extract‐free stems by thioacidolysis and by 2D‐NMR revealed an increased amount of unusual C₆C₁ lignin monomers most likely linked with lignin as end‐groups. Moreover the analysis of lignin isolated from these plants using size‐exclusion chromatography revealed a reduced molecular weight. Furthermore, these engineered lines show saccharification improvement of pretreated stem cell walls. Therefore, we conclude that enhancing the biosynthesis and incorporation of C₆C₁ monomers (‘DP reducers’) into lignin polymers represents a promising strategy to reduce lignin DP and to decrease cell wall recalcitrance to enzymatic hydrolysis.     

Functional redundancy of the two 5-hydroxylases in monolignol biosynthesis of Populus trichocarpa: LC–MS/MS based protein quantification and metabolic flux analysis

Flowering plants have syringyl and guaiacyl subunits in lignin in contrast to the guaiacyl lignin in gymnosperms. The biosynthesis of syringyl subunits is initiated by coniferaldehyde 5-hydroxylase (CAld5H). In Populus trichocarpa there are two closely related CAld5H enzymes (PtrCAld5H1 and PtrCAld5H2) associated with lignin biosynthesis during wood formation. We used yeast recombinant PtrCAld5H1 and PtrCAld5H2 proteins to carry out Michaelis-Menten and inhibition kinetics with LC-MS/MS based absolute protein quantification. CAld5H, a monooxygenase, requires a cytochrome P450 reductase (CPR) as an electron donor. We cloned and expressed three P. trichocarpa CPRs in yeast and show that all are active with both CAld5Hs. The kinetic analysis shows both CAld5Hs have essentially the same biochemical functions. When both CAld5Hs are coexpressed in the same yeast membranes, the resulting enzyme activities are additive, suggesting functional redundancy and independence of these two enzymes. Simulated reaction flux based on Michaelis-Menten kinetics and inhibition kinetics confirmed the redundancy and independence. Subcellular localization of both CAld5Hs as sGFP fusion proteins expressed in P. trichocarpa differentiating xylem protoplasts indicate that they are endoplasmic reticulum resident proteins. These results imply that during wood formation, 5-hydroxylation in monolignol biosynthesis of P. trichocarpa requires the combined metabolic flux of these two CAld5Hs to maintain adequate biosynthesis of syringyl lignin. The combination of genetic analysis, absolute protein quantitation-based enzyme kinetics, homologous CPR specificity, SNP characterization, and ER localization provides a more rigorous basis for a comprehensive systems understanding of 5-hydroxylation in lignin biosynthesis.     

Lignins: Natural polymers from oxidative coupling of 4-hydroxyphenyl- propanoids

Lignins are complex natural polymers resulting from oxidative coupling of, primarily, 4-hydroxyphenylpropanoids. An understanding of their nature is evolving as a result of detailed structural studies, recently aided by the availability of lignin-biosynthetic-pathway mutants and transgenics. The currently accepted theory is that the lignin polymer is formed by combinatorial-like phenolic coupling reactions, via radicals generated by peroxidase-H₂O₂, under simple chemical control where monolignols react endwise with the growing polymer. As a result, the actual structure of the lignin macromolecule is not absolutely defined or determined. The ``randomness'' of linkage generation (which is not truly statistically random but governed, as is any chemical reaction, by the supply of reactants, the matrix, etc.) and the astronomical number of possible isomers of even a simple polymer structure, suggest a low probability of two lignin macromolecules being identical. A recent challenge to the currently accepted theory of chemically controlled lignification, attempting to bring lignin into line with more organized biopolymers such as proteins, is logically inconsistent with the most basic details of lignin structure. Lignins may derive in part from monomers and conjugates other than the three primary monolignols (p-coumaryl, coniferyl, and sinapyl alcohols). The plasticity of the combinatorial polymerization reactions allows monomer substitution and significant variations in final structure which, in many cases, the plant appears to tolerate. As such, lignification is seen as a marvelously evolved process allowing plants considerable flexibility in dealing with various environmental stresses, and conferring on them a striking ability to remain viable even when humans or nature alter ``required'' lignin-biosynthetic-pathway genes/enzymes. The malleability offers significant opportunities to engineer the structures of lignins beyond the limits explored to date. Abbreviations: 4CL – 4-coumarate:CoA ligase; C3H –p-coumarate 3-hydroxylase; HCT –p-hydroxycinnamoyl-CoA: quinate shikimate p-hydroxycinnamoyltransferase; CCoAOMT – caffeoyl-CoA O-methyltransferase; CCR – cinnamoyl-CoA reductase; F5H – ferulate 5-hydroxylase; CAld5H – coniferaldehyde 5-hydroxylase; COMT – caffeic acid O-methyltransferase; AldOMT – (5-hydroxyconifer)aldehyde O-methyltransferase; CAD – cinnamyl alcohol dehydrogenase; NMR – nuclear magnetic resonance (spectroscopy); DFRC – derivatization followed by reductive cleavage; TIZ – tosylation, iodination, zinc (a DFRC method); DHP – dehydrogenation polymer.     

Structural features of lignin macromolecules extracted with ionic liquid from poplar wood

1-Ethyl-3-methylimidazolium acetate ([Emim][CH?COO]) was used for the extraction of lignin from poplar wood (Populus albaglandulosa), which was called to ionic liquid lignin (ILL) and structural features of ILL were compared with the corresponding milled wood lignin (MWL). Yields of ILL and MWL were 5.8±0.3% and 4.4±0.4%, respectively. The maximum decomposition rate (V(M)) and temperature (T(M)) corresponding to V(M) were 0.25%/ °C and 308.2 °C for ILL and 0.30%/ °C and 381.3 °C for MWL. The amounts of functional groups (OMe and phenolic OH) appeared to be similar for both lignins; approximately 15.5% and 6.7% for ILL and 14.4% and 6.3% for MWL. However, the weight average molecular weight (M(w)) of ILL (6347 Da) was determined to be 2/3-fold of that of MWL (10,002 Da) and polydispersity index (PDI: M(w)/M(n)) suggested that the lignin fragments were more uniform in the ILL (PDI 1.62) than in the MWL (PDI 2.64).