Milled Wood Lignin Research Articles

Lignin Structural Changes During Liquefaction in Acidified Ethylene Glycol

Beech (Fagus Sylvatica) milled-wood lignin was used as a model substrate in a study of lignin-catalyzed liquefaction in the presence of p-toluene sulfonic acid monohydrate (PTSA) or sulphuric acid as the catalysts. The structural changes that lignin undergoes during the treatment were studied by NMR spectroscopy, FTIR, size-exclusion chromatography, and high-performance liquid chromatography. For the sulphuric acid-catalyzed liquefaction, it was shown that the greater hydronium ion concentration in the reaction mixture induced formation of more condensed structures compared to the ones obtained after PTSA-catalyzed liquefaction. In addition, lignin during the PTSA-catalyzed liquefaction suffered degradation and was functionalized by the ethylene glycol. Gradual introduction of the ethylene glycol moieties into the lignin structure formed a condensed lignin-based polymeric material with predominant aromatic hydroxyl groups. HPLC and NMR analysis of the liquefied lignin with low-molecular mass fraction confirmed the presence of lignin monomers and further conversion of initially identified products into the aliphatic, aromatic (syringyl- and guaiacyl-based) esters and acids.

Effect of ionic liquid/organic solvent pretreatment on the enzymatic hydrolysis of corncob for bioethanol production. Part 1: Structural characterization of the lignins

Corncob was submitted to pretreatments with 1-ethyl-3-methylimadazolium acetate and water/organic solvents (DMSO, DMF, and DMAc) followed by alkaline extraction to isolate lignin. The lignin fractions obtained were comprehensively characterized by alkaline nitrobenzene oxidation, HPAEC, GPC, TGA, UV, FT-IR and HSQC spectra. The results showed that a maximum yield of 85.04% (based on the original lignin) was achieved for LDMSO prepared with the EMIMAc/DMSO pretreatment. The fractions prepared with EMIMAc/organic solvents contained lower amounts of carbohydrates (0.48–1.40%) than milled wood lignin (MWL, 8.73%) and had similar molecular weights (Mw, 2050–2430g/mol) to MWL. On the other hand, the fraction LH2O prepared with EMIMAc/H2O contained relatively abundant carbohydrates (11.19%) and had a high Mw (4310g/mol) than MWL. HSQC spectra analysis revealed that corncob lignin consisted of β-O-4, β-β, β-5, β-1 linkages, and cinnamyl alcohol end groups. In addition, the amounts (Ar/100Ar) of β-O-4 units increased in the order of LH2O<LDMSO<MWL.

Accurate and Reproducible Determination of Lignin Molar Mass by Acetobromination

The accurate and reproducible determination of lignin molar mass by using size exclusion chromatography (SEC) is challenging. The lignin association effects, known to dominate underivatized lignins, have been thoroughly addressed by reaction with acetyl bromide in an excess of glacial acetic acid. The combination of a concerted acetylation with the introduction of bromine within the lignin alkyl side chains is thought to be responsible for the observed excellent solubilization characteristics acetobromination imparts to a variety of lignin samples. The proposed methodology was compared and contrasted to traditional lignin derivatization methods. In addition, side reactions that could possibly be induced under the acetobromination conditions were explored with native softwood (milled wood lignin, MWL) and technical (kraft) lignin. These efforts lend support toward the use of room temperature acetobromination being a facile, effective, and universal lignin derivatization medium proposed to be employed prior to SEC measurements.

Evaluation of chemical composition and lignin structural features of Simarouba versicolor wood on its pulping performance

Simarouba versicolor wood was evaluated relative to its kraft pulping ability and compared with Eucalyptus urograndis wood. Comprehensive chemical analysis of wood and milled wood lignin (MWL) was performed, aiming to correlate wood and lignin structural features with kraft pulping response. Wood characterization of S. versicolor revealed higher lignin content (37.3%) and lower cellulose content (45.1%) than E. urograndis. 13C NMR spectroscopy was performed to characterize MWL, and the results showed a lower syringyl to guaiacyl ratio (S/G), higher degree of condensation, and lower β-O-4 linkages for S. versicolor. The gross heating value of S. versicolor was slightly higher than that for E. urograndis. Significant variations were observed in chemical charge demand and in pulping yield for the two species. This behavior was attributed mainly to the S/G ratio and degree of condensation of the lignin, although total and insoluble lignin, as well as cellulose contents may have affected pulping efficiency too.

Unmasking the structural features and property of lignin from bamboo

In this study, a simple method based on DMSO/NMI (dimethylsulfoxide/N-methylimidazole) dissolved system for the isolation of dissolved lignin (DL) from bamboo was presented. The lignin extracted was studied as compared to milled wood lignin (MWL) and alkali lignin (AL). Quantitative 13C and 2D-HSQC NMR analysis indicated that all lignin preparations are HGS-type and partially acylated (below 12%) at the γ-carbon of the side chain by p-coumarate. In addition, some LCC linkages and acetylated β-d-xylopyranoside were distinguished from HSQC spectra in the specific lignin preparations. The abundances (Ar/100Ar) of β-O-4, β-β, and β-5, S/G ratios, acylation degree, phenolic hydroxyl, aliphatic hydroxyl and methoxy groups (mmol/g) were also observed in the lignin preparations. Furthermore, the amount of β-O-4 coupling and molecular weight of the lignin decreased in the order of DL<MWL<AL. The in vitro antioxidant activity results showed that the radical scavenging index (RSI) is correlated to the phenolic hydroxyl groups (Ar-OH) involved in the lignin preparations.

Preliminary Results on an Approach for the Quantification of Lignin-Carbohydrate Complexes (LCC) in Hardwood Pulps

The literature on biomass research contains many references to lignin-carbohydrate complexes (LCC) decreasing the rate of delignification in chemical pulp production, decreasing the yield of cellulosic ethanol via fermentation, and decreasing forage digestibility. However, it is difficult to find correlations between rates of the processes above and initial LCC concentration. One of the main reasons for the lack of such correlations is the absence of methods for accurate quantification of LCC. In this investigation, repeatable and reproducible determinations of bound sugars at monomeric concentrations as low as 0.3 wt% on enzymatic lignin (EL) have been achieved. The bound sugars are hydrolyzed by H2SO4, most likely as low molecular weight oligomers. In the same H2SO4 treatment, the oligomers are hydrolyzed to monomers which are subsequently quantified by 1H NMR analyses. A significant enrichment of bound arabinan was previously reported when a crude milled wood lignin (MWL) was compared to the starting wood meal. A similar arabinan enrichment was observed for ELs from kraft and soda-AQ (SAQ) pulps in the present study. Also, well-resolved cross-peaks have been obtained in 2D HSQC NMR analyses of ELs. It has so far been confirmed that the EL from a 30.6 kappa number SAQ pulp from sugar maple contained ∼30% more benzyl ethers linked to primary-OH groups in sugar units than the corresponding EL from a 33.7 kappa number kraft pulp.

Microwave-enhanced extraction of lignin from birch in formic acid: Structural characterization and antioxidant activity study

A rapid and mild extraction protocol for the preparation of lignin was achieved by microwave-assisted heating in formic acid at 101°C under atmospheric pressure. In this case, birch lignin was extracted with microwave heating process (ML) in formic acid and characterized by elemental analysis, FTIR, GPC, 1H NMR and 13C–1H HSQC. In addition, the antioxidant activity of the samples was investigated. For comparative study, milled wood lignin (MWL) and lignin extracted with oil bath heating process (OL) were prepared. The results showed that the lignin yield under microwave heating was much higher than that under oil bath heating. A maximal delignification degree (89.77%) was achieved when microwave heating time was 30min. When double time (60min) was used under oil bath heating, the delignification degree was 66.11%. The structural characterization showed that the lignin structure of ML did not change dramatically, which is a mixture of GS-type with β-O-4′ ether bond as the major inter-unit linkage. As for antioxidant activity against DPPH, the radical scavenging index (RSI) of ML was 1.20, which was higher than that of MWL (0.53), suggesting that ML exhibited much higher antioxidant activity than MWL.

이온성액체(1-ethyl-3-methylimidazolium acetate)로 추출한 리기다소나무(pitch pine) 리그닌의 화학·구조 특성

1-Ethyl-3-methylimidazolium acetate known as efficient biomass pretreatment reagent was used for the extraction of lignin from rigida pine wood (pitch pine), which was called to ionic liquid lignin (ILL), and chemical structural features of ILL were compared with the corresponding milled wood lignin (MWL). The amounts of phenolic hydroxyl groups (Phe-OH) was determined to 10.0% for ILL and 7.2% for MWL, respectively, where those of methoxyl groups (OMe) were 4.9% for ILL and 11.0% for MWL, respectively. The weight average molecular weight (Mw) of ILL (3,995) were determined to ca. 1/2 of that of MWL (8,438) and polydispersity index (PDI: Mw/Mn) suggested that the lignin fragments were more uniform in the ILL (PDI 1.36) than in the MWL (PDI 2.64). The temperature (Tm) corresponding to maximum decomposition rate (Vm) of ILL () was ca. lower than that of MWL (), suggesting that ILL was thermally unstable than MWL, as evidence from the lower Tm for ILL. Moreover, the structural characteristics of ILL and MWL were confirmed by spectroscopic analyses (FT-IR and -NMR), and these results indicated ionic liquid (1-ethyl-3-methylimidazolium acetate) was chemically or physically bound to ILL.

Lignin Structural Variation in Hardwood Species

A comprehensive lignin structure analysis of ten industrially relevant hardwood species is presented. Milled wood lignin (MWL) was isolated from each species using a modified protocol and all milled wood lignin preparations were analyzed through quantitative (13)C NMR spectroscopy, elemental analysis, methoxyl analysis, sugar analysis, and nitrobenzene oxidation. Nitrobenzene oxidation and ozonation were carried out on extractive-free wood, alkali-extracted wood, milled wood lignin, and alkali-extracted lignin. Milled wood lignin isolated by the modified protocol was found to be representative of the total lignin in alkali-extracted wood. Significant variations in lignin structures, such as syringylpropane/guaiacylpropane ratio (S/G ratio), arylglycerol-β-aryl ether (β-O-4), degree of condensation, and elemental and methoxyl contents, were found among the hardwood species studied. These structural variations among species appear to be correlated to a single factor, the syringyl/guaiacyl ratio. A new method to predict the S/G ratio of total lignin in wood was developed, using a calibration line established by the syringaldehyde/vanillin (S/V) ratio (nitrobenzene oxidation) and the S/G ratio ((13)C NMR) of milled wood lignin (MWL).

Fractionation and characterization of lignin carbohydrate complexes (LCCs) of Eucalyptus globulus in residues left after MWL isolation. Part I: Analyses of hemicellulose-lignin fraction (HC-L)

Abstract The residual wood meal left after milled wood lignin (MWL) isolation (MWR) was extracted with the cellulose solvent lithium chloride/N,N-dimethylacetamide (LiCl/DMAc) to obtain a soluble fraction (C-L) and an insoluble fraction (C-L-residue). The C-L-residue was further extracted with the hemicellulose solvent 3 M NaOH to give a soluble fraction named hemicellulose-lignin fraction (HC-L) with 21.3% yield based on MWR. It was found that HC-L was composed of xylan, cellulose and lignin with abundant S-type β-O-4 substructures. HC-L lignin was bonded to HC-L cellulose or HC-L hemicelluloses or both. The method, which comprised acetylation for hardwood xylan (by acetic anhydride/pyridine/formamide) and extraction with chloroform, was found to be effective for selective xylan acetate fractionation. HC-L was further fractionated by the same method and subsequent deacetylation to give a xylan-lignin fraction (X-L) in 11.3% yield based on HC-L. X-L was composed mainly of xylan and lignin with abundant S-type β-O-4 substructures, and bonded to X-L xylan. X-L is considered as a promising fraction for elucidation of the structure of lignin-carbohydrate linkages.

Structural Characterization of Lignin in the Process of Cooking of Cornstalk with Solid Alkali and Active Oxygen

A novel, efficient, and environmentally friendly technology is used in cornstalk cooking, active oxygen (O₂ and H₂O₂) cooking with solid alkali (MgO). After the cooking, the milled wood lignin in the raw material and pulp and the water-soluble and insoluble lignin in the yellow liquor were all characterized by attenuated total reflectance Fourier transform infrared spectroscopy and two-dimensional heteronuclear single-quantum coherence NMR. The results showed that the cooking procedure with solid alkali and active oxygen had a high selectivity for delignification, which could remove 85.5% of the lignin from the raw material. The syringyl (S/S'/S') units could be dissolved preferentially because of their high reactivity, and a novel guaiacyl unit with a carbonyl group (G') was generated in the cooking process. Moreover, during the cooking, the β-O-4' (A/A'/A″) structures as the main side-chain linkages in all the lignins could be partly broken and the β-O-4' (A') with a ring-conjugated structure was readily attacked by oxygen, whereas the H unit and β-5' and β-β' structures were found to stay stable without characteristic reaction.

Identification of Grass-specific Enzyme That Acylates Monolignols with p-Coumarate

Lignin is a major component of plant cell walls that is essential to their function. However, the strong bonds that bind the various subunits of lignin, and its cross-linking with other plant cell wall polymers, make it one of the most important factors in the recalcitrance of plant cell walls against polysaccharide utilization. Plants make lignin from a variety of monolignols including p-coumaryl, coniferyl, and sinapyl alcohols to produce the three primary lignin units: p-hydroxyphenyl, guaiacyl, and syringyl, respectively, when incorporated into the lignin polymer. In grasses, these monolignols can be enzymatically preacylated by p-coumarates prior to their incorporation into lignin, and these monolignol conjugates can also be "monomer" precursors of lignin. Although monolignol p-coumarate-derived units may comprise up to 40% of the lignin in some grass tissues, the p-coumarate moiety from such conjugates does not enter into the radical coupling (polymerization) reactions of lignification. With a greater understanding of monolignol p-coumarate conjugates, grass lignins could be engineered to contain fewer pendent p-coumarate groups and more monolignol conjugates that improve lignin cleavage. We have cloned and expressed an enzyme from rice that has p-coumarate monolignol transferase activity and determined its kinetic parameters.

Mild Acetosolv Process To Fractionate Bamboo for the Biorefinery: Structural and Antioxidant Properties of the Dissolved Lignin

Fractionation of lignocellulosic material into its constitutive components is of vital importance for the production of biofuels as well as other value-added chemicals. The conventional acetosolv processes are mainly focused on the production of pulp from woody lignocelluloses. In this study, a mild acetosolv process was developed to fractionate bamboo under atmospheric pressure to obtain cellulosic pulp, water-soluble fraction, and acetic acid lignin. The structural features of the lignins obtained under various conditions were characterized with elemental analysis, sugar analysis, alkaline nitrobenzene oxidation, gel permeation chromatography (GPC), (1)H nuclear magnetic resonance ((1)H NMR), and heteronuclear single-quantum coherence (HSQC) spectroscopy. As compared to milled wood lignin (MWL) of bamboo, acetic acid lignins had low impurities (carbohydrates 2.48-4.56%) mainly due to the cleavage of linkages between lignin and carbohydrates. In addition, acetic acid lignins showed a low proportion of syringyl (S) units. Due to the cleavage of linkages between lignin units, acetic acid lignins had weight-average molecular weights ranging from 4870 to 5210 g/mol, less than half that of MWL (13000 g/mol). In addition, acetic acid lignins showed stronger antioxidant activity mainly due to the significant increase of free phenolic hydroxyls. The lignins obtained with such low impurities, high free phenolic hydroxyls, and medium molecular weights are promising feedstocks to replace petroleum chemicals.

Quantitative Molecular Structure–Pyrolytic Energy Correlation for Hardwood Lignins

The molecular structures of the milled wood lignins (MWLs) and technical lignins (TLs) obtained from four hardwood species (Eucalyptus urograndis, Eucalyptus nitens, Eucalyptus globulus, and Populus trichocarpa) were quantified by 13C nuclear magnetic resonance (NMR) in tandem with their thermal responses, as obtained by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). Both MWLs and TLs showed similar DSC curves with two exothermic peaks (referred to as peaks 1 and 2). Also, maximum temperatures for MWL peaks were higher than for TL peaks, with an average of 20 and 10 °C for the first peak and second peak, respectively. Even though calculated enthalpies for MWLs were higher because of their purities, TLs had reasonable relationships between heat values and lignin substructures. TL had a positive correlation between condensed structures and the enthalpy value between 413 and 428 °C, but negative relationships were found at the latter temperature window for lignin substructures,...

Investigation of structure of milled wood and dioxane lignins of Populus nigra and Cupressus sempervirens using the DFRC method

AbstractIn this study, derivatisation followed by reductive cleavage (DFRC) were used to investigate milled wood lignin (MWL) and dioxane lignin (DL) structures of Populus nigra and Cupressus sempervirens. After the DFRC reactions, the constituents obtained from these two kinds of lignin were recognised structurally using several chromatographic and spectral methods such as 13C NMR, GC-MS, and GPC. Comparative results showed that the dominant structural components of the two kinds of lignin are obtained from the cleavage of β-O-4 bonds. The main component of DL and MWL of P. nigra is 4-hydroxy-3,5-dimethoxy-1-phenyl-γ-hydroxypropene (syringyl structures). Also, some guaiacyl structures were observed. The dominant component identified in both lignins of C. sempervirens is 4-hydroxy-3-methoxy-1-phenyl-γ-hydroxypropene (guaiacyl structures). The cleavage method has a good performance for both P. nigra and C. sempervirens and the results obtained are in good agreement with previously published data.

A novel xylogenic suspension culture model for exploring lignification in Phyllostachys bamboo

BackgroundSome prominent cultured plant cell lines, such as the BY-2 cell line of tobacco (Nicotiana tabacum cv. ‘Bright Yellow 2’) and the T87 cell line of Arabidopsis (Arabidopsis thaliana L. Heynh., ecotype Columbia) are used as model plant cells. These suspension cell culture systems are highly applicable for investigating various aspects of plant cell biology. However, no such prominent cultured cell lines exist in bamboo species.ResultsWe standardized a novel xylogenic suspension culture model in order to unveil the process of lignification in living bamboo cells. Initial signs of lignin deposition were able to be observed by a positive phloroglucinol-HCl reaction at day 3 to 5 under lignification conditions (LG), i.e., modified half-strength Murashige and Skoog medium (m1/2MS) containing 10 μM 6-benzyladenine (BA) and 3% sucrose. Two types of xylogenic differentiation, both fiber-like elements (FLEs) with cell wall thickening and tracheary elements (TEs) with formation of perforations in the cell wall, were observed under these conditions. The suspension cells rapidly formed secondary cell wall components that were highly lignified, making up approximately 25% of the cells on a dry weight basis within 2 weeks. Detailed features involved in cell growth, differentiation and death during lignification were characterized by laser scanning microscopic imaging. Changes in transcript levels of xylogenesis-related genes were assessed by RT-PCR, which showed that the transcription of key genes like PAL1, C4H, CCoAOMT, and CCR was induced at day 4 under LG conditions. Furthermore, interunit linkage of lignins was compared between mature bamboo culms and xylogenic suspension cells by heteronuclear single quantum coherence (HSQC) NMR spectroscopy. The presence of the most common interunit linkages, including β-aryl ether (β-O-4), phenylcoumaran (β-5) and resinol (β-β) structures was identified in the bamboo cultured cell lignin (BCCL) by HSQC NMR. In addition to these common features of lignin, several differences in lignin substructures were also found between the BCCL and the bamboo milled wood lignin (BMWL).ConclusionsOur xylogenic suspension culture model could be used for detailed characterization of physiological and molecular biological events in living bamboo cells.

Characterization of Milled Wood Lignin (MWL) in Loblolly Pine Stem Wood, Residue, and Bark

Milled wood lignin samples from Loblolly pine stem wood, forest residue, and bark were isolated and characterized by quantitative (13)C and (31)P nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FT-IR), and gel permeation chromatography (GPC) for molecular weight determination. Results from (13)C NMR show the stem wood and forest residue samples have similar functional group contents. However, the bark has fewer methoxyl groups, β-O-4 structures, dibenzodioxocin, and side chains than the other two lignins. The bark lignin has the highest amounts of p-hydroxyphenyl (h) and C-5 condensed lignin, stem wood has the lowest, and the residue lies between. (31)P NMR analysis indicates that bark lignin contains more C-5 substituted phenolics and fewer aliphatic hydroxyl groups than the lignin isolated from stem wood or residue. The molecular weight distribution analysis indicates the bark lignin has higher weight-average molecular weight (M(w)) and polydispersity index than the lignin recovered from stem wood or residue.

A comparative study of bamboo (Phyllostachys incarnata Wen) milled wood lignin and the successively alkali-fractionated lignins

The chemical composition and structural characterization of the milled wood lignin (MWL) and successively alkali-fractionated lignins from bamboo (Phyllostachys incarnata Wen) were comparatively investigated using Fourier transform infrared (FT-IR), gel permeation chromatography (GPC), 2D heteronuclear single quantum coherence (HSQC), and alkaline nitrobenzene oxidation. It was found that bamboo MWL (S/G ~ 1.3) is of SGH type with a considerable amount of esterified p-coumaric acid and etherified ferulic acid. Molecular weights of 3,320 g/mol for MWL and varying weights between 970 and 3,430 g/mol for the alkali-fractionated lignins were obtained. Moreover, 2D HSQC of MWL showed a predominance of β-O-4′ linkages (65%), followed by β–β′ linkages (15%) and lower amounts of β-5′ (7%), β-1′ (4%), 5–5′ (4%) linkages, and cinnamyl acetate end groups (5%). In addition, a percentage (~45%) of the lignin side chain was found to be acylated at the γ-carbon. However, the alkali-fractionated lignins lost parts of these linkages during successive alkaline fractionation processes.

Understanding the Degree of Condensation of Phenolic and Etherified C-9 Units of in Situ Lignins

A novel approach for the quantification of the degree of condensation at the C(5) position of etherified and phenolic phenylpropane (C-9) units of in situ lignin is described. This is achieved by degrading unmethylated and methylated wood by thioacidolysis and analyzing the resultant product mixtures by quantitative (31)P NMR spectroscopy. Applying this new method to compression wood and normal wood from Pinus radiata showed that, whereas 41-47% of etherified guaiacyl C-9 units are condensed at the C(5) position, almost all phenolic guaiacyl C-9 units exist as uncondensed moieties. Analysis of milled wood lignin (MWL) isolated from the same wood by (31)P NMR spectroscopy before and after thioacidolysis showed that the phenolic guaiacyl C-9 units were more condensed than those in the in situ lignin. This is likely due to partial cleavage of the more condensed etherified linkages during the lignin isolation, leading to a relative increase in condensed phenolic guaiacyl C-9 units.

Formic acid based organosolv pulping of bamboo ( Phyllostachys acuta): Comparative characterization of the dissolved lignins with milled wood lignin

Bamboo ( Phyllostachys acuta) was pulped with aqueous formic acid (F), aqueous formic acid with the addition of HCl (FH), aqueous formic acid with the addition of H 2O 2 (FP), and three-stage Milox, respectively. The results indicated that the Milox method achieved the highest delignification (88.9%) followed by FP (87.9%), F (80.4%) and FH (79.1%). The dissolved lignins were separated from the spent liquors by precipitation with water followed by further purification, and their structures were characterized compared to milled wood lignin (MWL). The decrease in intensities of aryl ether linkages in formic acid lignins as compared to MWL indicated that the degradation of lignin during the pulping process. As compared to MWL, formic acid lignins had relatively lower molecular weights (Mw 7300–10,020) and lower impurities (i.e., carbohydrates content 3.9–6.4% and ash 0–0.1%). These results suggested that formic acid pulping can effectively delignify bamboo and produced relatively pure and high-quality lignins.