Fractionation Of Lignin-carbohydrate Complexes Research Articles

Extraction of super high-yield lignin-carbohydrate complexes from rice straw without compromising cellulose hydrolysis

Lignin-carbohydrate complexes (LCC) that exhibit both structural advantages of lignin and carbohydrates are promising amphiphilic biopolymers, but the extraction is challenged by its liable chemical bond cleavage between lignin and carbohydrates. This work proposed a facile chemical route to integrating the production of water-insoluble (WIS LCC) and water-soluble LCC (WS LCC) into the emerging deep eutectic solvent (DES) biorefinery at mild conditions. The tailored mechanochemical fractionation process of ball milling assisted aqueous alkaline DES could extract 24.2 % LCC in total, with the co-production of a highly hydrolysable cellulose fraction (98.7 % glucose conversion). The resulting LCC exhibited considerably high contents of β-O-4, phenyl glycoside, and ferulic acid linkage bonds. When 100 g starting straw was subjected to this technique route, 9.1 g WIS LCC, 15.1 g WS LCC and 45.5 g glucose were cascaded produced. It was proposed that the selective disruption of hydrogen bonding entangled network and the quasi-state dissolution of the whole biomass allowed the subsequent cascade fractionation of WIS LCC, WS LCC and highly hydrolysable cellulose through solution property adjustment. This work showed a promising approach for LCC production with high yield without compromising cellulose conversion potential, which has been challenging in the current lignocellulose biorefinery.

Structural variations of lignin and lignin-carbohydrate complexes from the fruit shells of Camellia oleifera during ripening

Camellia oleifera fruit shell (CFS), a waste lignocellulosic biomass resulting from Camellia oleifera oil production industry, is abundantly available in Southern China. Herein, to understand the structural variations of CFS lignins and lignin-carbohydrate complexes (LCC) during ripening, the native lignin and LCC fractions from CFS (harvested every seven days from October 1 to 30, 2022) were isolated and characterized systematically. The molecular weights of both MWL and DEL fractions steadily increased during ripening. CFS lignins contained abundance of β-O-4′ linkages (maximum of 58.6 per 100Ar in DEL-2), and had low S/G ratios (S/G < 0.6). Moreover, the amounts of β-O-4′ linkages in MWL, DEL, and LCC-AcOH fractions increased first and then decreased during ripening. The main lignin-carbohydrate linkages in the LCC-AcOH fractions were benzyl-ether (7.0–9.4 per 100Ar) and phenyl-glycoside (4.5–5.2 per 100Ar) bonds. Based on the quantitative results, the potential structural diagrams of lignins from different ripening stages of CFS were proposed. Additionally, the LCC-AcOH fractions exhibited pronounced antioxidant capacity and were promising as natural antioxidants. The properties and functions of lignin in plant cell walls, as well as its further appreciation, are crucial for the design and selection of feasible pretreatment strategies for the lignocellulosic materials.

Structural characterization of lignin-carbohydrate complexes from Chinese quince fruits extracted after enzymatic hydrolysis pretreatment

Chinese quince fruit (CQF) contains abundant pectin; however, the pectin cannot be efficiently separated by conventional approaches because of strong lignin-carbohydrate complexes (LCC). In this study, to elucidate the structural characteristics of the original LCC formed by lignin and pectin in CQF, single and multiple enzymatic hydrolysis pretreatments were innovatively performed, and the resulting LCC preparations were comprehensively characterized using a series of techniques. The enzymatic hydrolysis pretreatments significantly increase the LCC yield, releasing LCC fractions with low molecular weights (Mw = 4660–8288 Da). LCC-4, isolated by pretreatment with cellulase plus xylanase, had the highest galacturonic acid content (15.5 %), followed by LCC-2 (isolated by xylanase pretreatment) of 14.0 %. In CQF, lignin develops lignin-carbohydrate (LC) bonds with pectin to form LCC, with phenyl-glycoside bond being the dominant linkage. Although the pectinase pretreatment reduced the pectin content, signals of the LC linkages in the 2D-HSQC spectra were enhanced. LCC-4 could be considered as the most representative of the original LCC in CQF due to its high pectin content and multiple LCC signals in the 2D-HSQC spectrum. The structural understanding of the original LCC in CQF will lay a foundation for designing appropriate methods for extracting pectin from CQF.

Structural variation of lignin-carbohydrate complexes (LCC) in Chinese quince (Chaenomeles sinensis) fruit as it ripens

Chinese quince (Chaenomeles sinensis) fruits are rich in lignin, and too sour, astringent and woody to be eaten raw. More than 50 % of lignin in plant cell walls is covalently associated with carbohydrates to form lignin-carbohydrate complexes (LCC). In this study, LCC preparations were extracted from fruits harvested on the 15th day of the month from May–October 2019. A variety of chemical and instrumental analytical approaches were used to characterize the LCC fractions, including HPAEC, TGA, GPC, FT-IR, and 2D HSQC NMR. Antioxidant activities were evaluated by DPPH radical scavenging assays. Results showed that the LCC fractions from October fruits had better thermal stability and homogeneity. NMR results revealed that the lignin-lignin linkages in LCC-AcOH preparations included β-O-4′, β-β' and β-5′, but β-5′ linkages were not present in LCC preparations. And the NMR signals of carbohydrate confirmed the presence of lignin-pectin complexes, which was consistent with sugar analysis. All LCC preparations showed good antioxidant activity, among which Björkman LCC from October fruits showed best. This study will facilitate understanding the chemical bonds of LCC macromolecules in the plant cell wall. More specifically, it provides information critical for specific industrial applications of quince fruits.

Lignin-carbohydrate complexes suppress SCA3 neurodegeneration via upregulating proteasomal activities

Lignin-carbohydrate complexes (LCCs) represent a group of macromolecules with diverse biological functions such as antioxidative properties. Polyglutamine (polyQ) diseases such as spinocerebellar ataxia type 3 (SCA3) comprise a set of neurodegenerative disorders characterized by the formation of polyQ protein aggregates in patient neurons. LCCs have been reported to prevent such protein aggregation. In this study, we identified a potential mechanism underlying the above anti-protein aggregation activity. We isolated and characterized multiple LCC fractions from bamboo and poplar and found that lignin-rich LCCs (BM-LCC-AcOH and PR-LCC-AcOH) effectively eliminated both monomeric and aggregated mutant ataxin-3 (ATXN3polyQ) proteins in neuronal cells and a Drosophila melanogaster SCA3 disease model. In addition, treatment with BM-LCC-AcOH or PR-LCC-AcOH rescued photoreceptor degeneration in vivo. At the mechanistic level, we demonstrated that BM-LCC-AcOH and PR-LCC-AcOH upregulated proteasomal activity. When proteasomal function was impaired, the ability of the LCCs to suppress ATXN3polyQ aggregation was abolished. Thus, we identified a previously undescribed proteasome-inducing function of LCCs and showed that such activity is indispensable for the beneficial effects of LCCs on SCA3 neurotoxicity.

Structural characterization of the lignin-carbohydrate complex in biomass pretreated with Fenton oxidation and hydrothermal treatment and consequences on enzymatic hydrolysis efficiency

In this study, lignin-carbohydrate complexes (LCCs) were isolated from biomass (raw and pretreated) to investigate the structural changes in biomass pretreated by Fenton oxidation and hydrothermal treatment, and their effect on enzymatic hydrolysis. The composition and structure of the LCCs fractions were investigated via carbohydrate analysis, XRD, FT-IR, and 2D HSQC NMR. The biomass degradation rate of yellow poplar and larch during Fenton oxidation and hydrothermal treatment was approximately 30%. Most of the hemicellulose was degraded during pretreatment, while xylan remained in the yellow poplar, and galactan, mannan, and xylan remained in the larch. The fractional yield of glucan-rich LCC (LCC1) in the yellow poplar (raw and pretreated biomass) was high, while that of glucomannan-rich LCC (LCC3) in larch was higher than the yield yellow poplar. Phenyl glycoside, γ-ester, and benzyl ether linkages were observed in the LCCs of yellow poplar, while phenyl glycoside and γ-ester were detected in those of larch. Following pretreatment, the frequencies of β–β′, β-5, and γ-ester in the LCCs of larch were found to be higher than in those of yellow poplar. The efficiencies of enzymatic hydrolysis for the pretreated yellow poplar and larch were 93.53% and 26.23%, respectively. These finding indicated that the β–β′, β-5, and γ-ester linkages included in the pretreated biomass affected the efficiency of enzymatic hydrolysis.

The coordinated action of glucuronoyl esterase and α-glucuronidase promotes the disassembly of lignin-carbohydrate complexes.

Glucuronoxylans represent a significant fraction of woody biomass, and its decomposition is complicated by the presence of lignin–carbohydrate complexes (LCCs). Herein, LCCs from birchwood were used to investigate the potential coordinated action of a glucuronoyl esterase (TtCE15A) and two α‐glucuronidases (SdeAgu115A and AxyAgu115A). When supplementing α‐glucuronidase with equimolar quantities of TtCE15A, total MeGlcpA released after 72 h by SdeAgu115A and AxyAgu115A increased from 52% to 67%, and 61% to 95%, respectively. Based on the combined TtCE15A and AxyAgu115A activities, ~ 34% of MeGlcpA in the extracted birchwood glucuronoxylan was occupied as LCCs. Notably, insoluble LCC fractions reduced soluble α‐glucuronidase concentrations by up to 70%, whereas reduction in soluble TtCE15A was less than 30%, indicating different tendencies to adsorb onto the LCC substrate.

Lignin-carbohydrate complexes from coconut (Cocos nucifera) coir: Fractionation, structural elucidation, and potential applications

Coconut coir, a major type of tropical lignocellulosic waste, has been restricted from higher-value applications due to its chemical complexity and inherent variability. To better disclose the chemical relationships between polysaccharides and lignin in coir and further exploit its high value-added bio-based materials, two lignin-carbohydrate complexes (LCCs) designated as glucan-lignin (GL) and xylan-lignin (XL) were successively isolated from coir via a simplified and quantitative fractionation method. The characterization of chemical composition, molecular weight distribution, and constituent substructures of the isolated LCC fractions were examined, and the lignin-carbohydrate (LC) chemical linkages were revealed as γ-esters by a 2D heteronuclear singular quantum correlation (HSQC) NMR technique. Furthermore, XL was demonstrated as a more competitive candidate than GL for 2,2-diphenyl-1-picryl-hydrazyl (DPPH) removal in comparison with 2,6-di-tert-butyl-4-methylphenol (BHT, a commercial antioxidant), and was also featured as a more promising reinforcing agent for elevating the adsorption property of polyacrylamide-based hydrogels via the synergistic effect of physical and hydrogen bonding adsorptions.

New insights on structure of lignin‐carbohydrate complex from hot water pretreatment liquor

To understand the lignin-carbohydrate linkages and the structure changes in hot water pretreatment process, lignin-carbohydrate complex (LCC) fraction was extracted from hot water pretreatment liquor of poplar for the first time. Various structural analyses of LCC fraction were performed by different methods including high performance anion exchange chromatography, gel permeation chromatography, ultraviolet-visible spectroscopy, Fourier transform infrared spectroscopy and two-dimensional nuclear magnetic resonance spectroscopy. The results show that the lignin in LCC fraction mainly composed of syringyl unit, which was connected with β-O-4´ linkage. The above lignin linked to glucomannan in the initial phase of hot water pretreatment, then gradually connected with glucuronoxylan. Compared with ball-milling LCC, LCC fraction from hot water pretreatment liquor presented advantageous hydrophilia and biocompatibility based on higher contents of acid-soluble lignin and galactose. Various LCCs would be obtained by hot water pretreatment design, which was attractive for boosting its applicability in the field of biomaterial.

Facile Isolation of LCC-Fraction from Organosolv Lignin by Simple Soxhlet Extraction.

A new fractionation protocol for wheat straw organosolv lignin was developed on the basis of the dominating H-bonding orientations of its components. Acetone as H-bond accepting aprotic polar solvent and methanol as H-bond donating and accepting protic polar solvent were used in sequence. Obtained fractions were structurally and thermally analysed. The protocol allowed for the generation of purified lignin fractions and the isolation of a novel, yet unobserved lignin carbohydrate complex (LCC) fraction. This LCC fraction was found to contain exclusively phenyl glycosides and γ-esters as LCC motifs.

Structural elucidation of lignin-carbohydrate complexes (LCCs) from Chinese quince (Chaenomeles sinensis) fruit

The lignin-carbohydrate complexes (LCCs) of the mesocarp (MS) and near the endocarp (NE) of Chinese quince (Chaenomeles sinensis) fruits were analyzed using three different methods of fractioning: milled wood lignin (MWL), LCC extracted from crude MWL with acetic acid (LCC-AcOH), and Bjorkman LCC. The MWL and LCC fractions were characterized by carbohydrate composition analysis, SEC, FT-IR, Py-GC/MS, thermal analysis and 2D HSQC NMR. Notably, large amounts of arabinose and galactose remained in the Björkman LCC fractions suggesting a chemical bond between the lignin and pectin. MWL and LCC-AcOH fractions showed better thermal stability than the Björkman LCC fractions. The structure of MS lignin was similar to that of NE lignin; however, fractions from the different fractionation methods revealed differences. The MWL fractions were rich in benzyl ether and γ-ester linkages, while the Björkman LCC fractions contained phenyl glycoside and γ-ester linkages, and the LCC-AcOH fractions contained phenyl glycoside and benzyl ether linkages. These findings are helpful in understanding the nature of lignin and LCC in Chinese quince fruits and provide a theoretical support for their potential application.

Selective precipitation and characterization of lignin-carbohydrate complexes (LCCs) from Eucalyptus.

Six types of lignin-carbohydrate complex (LCC) fractions were isolated from Eucalyptus. The acidic dioxane treatment applied significantly improved the yield of LCCs. The extraction conditions had a limited impact on the LCC structures and linkages. Characterization of the lignin-carbohydrate complex (LCC) structures and linkages promises to offer insight on plant cell wall chemistry. In this case, Eucalyptus LCCs were extracted by aqueous dioxane, and then precipitated sequentially by 70% ethanol, 100% ethanol, and acidic water (pH=2). The composition and structure of the six LCC fractions obtained by selective precipitation were investigated by sugar analysis, molecular weight determination, and 2D HSQC NMR. It was found that the acidic (0.05-M HCl) dioxane treatment significantly improved the yield of LCCs (66.4% based on Klason lignin), which was higher than the neutral aqueous dioxane extraction, and the extraction condition showed limited impact on the LCC structures and linkages. In the fractionation process, the low-molecular-weight LCCs containing a high content of carbohydrates (60.3-63.2%) were first precipitated by 70% ethanol from the extractable solution. The phenyl glycoside (PhGlc) bonds (13.0-17.0 per 100Ar) and highly acetylated xylans were observed in the fractions recovered by the precipitation with 100% ethanol. On the other hand, such xylan-rich LCCs exhibited the highest frequency of β-O-4 linkages. The benzyl ether (BE) bonds were only detected in the fractions obtained by acidic water precipitation.

Structural Basis for the Formation and Regulation of Lignin–Xylan Bonds in Birch

The covalent connectivity between lignin and polysaccharides forming the so-called lignin–carbohydrate complexes (LCCs) is important to obtain fundamental knowledge on wood formation and may shed light on molecular aspects of wood processing. Although widely studied, unequivocal proofs of their existence in native-state biomass are still lacking, mainly because of harsh preanalytical fractionation conditions that could cause artifacts. In the present study, we applied a mild protocol for quantitative fractionation of LCCs and performed detailed structural studies using 2D HSQC NMR spectroscopy, 31P NMR spectroscopy, and thioacidolysis in combination with GC–MS and GC with flame ionization detection. The detailed structural analysis of LCCs, including both lignin and the carbohydrate skeleton, unveiled insights into the role of molecular structure of xylan on the type of lignin–carbohydrate (LC) bonds formed. More specifically, it is shown that xylan LCCs differ in the degree of substitution of hydroxyl functionality on the xylan skeleton by the presence of acetyl- or 4-O-methylglucuronic acid. The highly substituted xylan had a lower prevalence of phenyl glycosidic and benzyl ether LC bond types than the lowly substituted xylan. In addition, structural differences in the lignin part of the LCCs were observed. On the basis of the results, it is suggested that acetylation on xylan regulates the type and frequency of LC bonds.

Lignin-carbohydrate complexes from sisal (Agave sisalana) and abaca (Musa textilis): chemical composition and structural modifications during the isolation process.

Two types of lignins occurred in different lignin-carbohydrate fractions, a lignin enriched in syringyl units, less condensed, preferentially associated with xylans, and a lignin with more guaiacyl units, more condensed, associated with glucans. Lignin-carbohydrate complexes (LCC) were isolated from the fibers of sisal (Agave sisalana) and abaca (Musa textilis) according to a plant biomass fractionation procedure recently developed and which was termed as "universally" applicable to any type of lignocellulosic material. Two LCC fractions, namely glucan-lignin (GL) and xylan-lignin (XL), were isolated and differed in the content and composition of carbohydrates and lignin. In both cases, GL fractions were enriched in glucans and comparatively depleted in lignin, whereas XL fractions were depleted in glucans, but enriched in xylans and lignin. Analysis by two-dimensional Nuclear Magnetic Resonance (2D-NMR) and Derivatization Followed by Reductive Cleavage (DFRC) indicated that the XL fractions were enriched in syringyl (S)-lignin units and β-O-4' alkyl-aryl ether linkages, whereas GL fractions have more guaiacyl (G)-lignin units and less β-O-4' alkyl-aryl ether linkages per lignin unit. The data suggest that the structural characteristics of the lignin polymers are not homogeneously distributed within the same plant and that two different lignin polymers with different composition and structure might be present. The analyses also suggested that acetates from hemicelluloses and the acyl groups (acetates and p-coumarates) attached to the γ-OH of the lignin side chains were extensively hydrolyzed and removed during the LCC fractionation process. Therefore, caution must be paid when using this fractionation approach for the structural characterization of plants with acylated hemicelluloses and lignins. Finally, several chemical linkages (phenylglycosides and benzyl ethers) could be observed to occur between lignin and xylans in these plants.

Structural Elucidation of Whole Lignin in Cell Walls of Triploid of Populus tomentosa Carr.

To protect adequately the structural features and characterize whole lignin structure of triploid of Populus tomentosa Carr., milled wood lignin (MWL), lignin–carbohydrate complex (LCC), cellulolytic enzyme lignin (CEL), and enzymatic hydrolysis residual enzyme lignin (EHREL) fractions were sequentially isolated from ball-milled wood with mild conditions. The total pure yield of the four lignin fractions could reach up to 92.6% based on Klason lignin content. The structures of the four lignin fractions were quantitatively analyzed. Results showed that the main substructures in the four lignin fractions were β-O-4′ aryl ether and resinol. The LCC fraction contained a high percentage of xylose (96.2%) and numerous β-O-4′ aryl ether linkages (84.4 per 100Ar). The relative high content of phenyl glycoside (PhGlc) linkages between lignin and xylan in the LCC fraction (4.39 per 100Ar) was clearly detected. In addition, some low-molecular-weight lignin fractions were found in the secondary wall. A panorama of whole lignin structure of poplar was proposed.

Universal fractionation of lignin–carbohydrate complexes (LCCs) from lignocellulosic biomass: an example using spruce wood

It is of both theoretical and practical importance to develop a universally applicable approach for the fractionation and sensitive lignin characterization of lignin-carbohydrate complexes (LCCs) from all types of lignocellulosic biomass, both natively and after various types of processing. In the present study, a previously reported fractionation approach that is applicable for eucalyptus (hardwood) and flax (non-wood) was further improved by introducing an additional step of barium hydroxide precipitation to isolate the mannan-enriched LCC (glucomannan-lignin, GML), in order to suit softwood species as well. Spruce wood was used as the softwood sample. As indicated by the recovery yield and composition analysis, all of the lignin was recovered in three LCC fractions: a glucan-enriched fraction (glucan-lignin, GL), a mannan-enriched fraction (GML) and a xylan-enriched fraction (xylan-lignin, XL). All of the LCCs had high molecular masses and were insoluble or barely soluble in a dioxane/water solution. Carbohydrate and lignin signals were observed in (1) H NMR, (13) C CP-MAS NMR and normal- or high-sensitivity 2D HSQC NMR analyses. The carbohydrate and lignin constituents in each LCC fraction are therefore believed to be chemically bonded rather than physically mixed with one another. The three LCC fractions were found to be distinctly different from each other in terms of their lignin structures, as revealed by highly sensitive analyses by thioacidolysis-GC, thioacidolysis-SEC and pyrolysis-GC.

SEPARATION OF GALACTOGLUCOMANNANS, LIGNIN, AND LIGNIN-CARBOHYDRATE COMPLEXES FROM HOT-WATER-EXTRACTED NORWAY SPRUCE BY CROSS-FLOW FILTRATION AND ADSORPTION CHROMATOGRAPHY

A simple method to simultaneously recover polymeric carbohydrates, mainly galactoglucomannans (GGM), lignin, and lignin-carbohydrate complex (LCC) from hot-water-extracted Norway spruce wood is presented. The isolation method consists of cross-flow filtration, where high and low molecular mass species are removed, followed by fixed-bed adsorption on a hydrophobic polymeric resin (XAD-16) to remove lignins and lignans. In the second step of fixed-bed adsorption, a phenylic reversed-phase analytical chromatography column, where mass transport resistance is minimized and a very high selectivity towards aromatic compounds have been observed, was used to separate LCC from GGM. The isolated LCC fraction contained about 10% aromatics, whereas the upgraded GGM fraction contained about 1.5% aromatics and the lignin fraction contained about 56% aromatics. Polymeric xylan was accumulated in the GGM fraction, while mannose was the dominant sugar found in the LCC fraction. As products, approximately 7% was recovered in the lignin fraction in the first adsorptive step, 5% was recovered as LCC, and 88% as upgraded hemicelluloses.

Structural Differences between the Lignin−Carbohydrate Complexes Present in Wood and in Chemical Pulps

Lignin-carbohydrate complexes (LCCs) were prepared in quantitative yield from spruce wood and from the corresponding kraft and oxygen-delignified pulps and were separated into different fractions on the basis of their carbohydrate composition. To obtain an understanding of the differences in lignin structure and reactivity within the various LCC fractions, thioacidolysis in combination with gas chromatography was used to quantify the content of beta-O-4 structures in the lignin. Periodate oxidation followed by determination of methanol was used to quantify the phenolic hydroxyl groups. Furthermore, size exclusion chromatography (SEC) of the thioacidolysis fractions was used to monitor any differences between the original molecular size distribution and that after the delignification processes. Characteristic differences between the various LCC fractions were observed, clearly indicating that two different forms of lignin are present in the wood fiber wall. These forms are linked to glucomannan and xylan, respectively. On pulping, the different LCCs have different reactivities. The xylan-linked lignin is to a large extent degraded, whereas the glucomannan-linked lignin undergoes a partial condensation to form more high molecular mass material. The latter seems to be rather unchanged during a subsequent oxygen-delignification stage. On the basis of these findings, a modified arrangement of the fiber wall polymers is suggested.

Differential scanning calorimetric analysis of the lignin-carbohydrate complex degraded by wood-rotting fungi

Differential scanning calorimetric (DSC) analysis was applied to the lignin-carbohydrate complex (LCC) fractions degraded by the wood-rotting fungiTyromyces palustris andCoriolus versicolor. Based on changes of the thermogram, it is assumed that the brown-rot fungusT. palustris split the hemicellulose fragment from the LCC during incubation. On the other hand, in the case of the white-rot fungusC. versicolor, the peak temperature of the LCC fraction became higher and then lower, indicating that this fungus first degraded the hemicellulose portion and then decomposed the lignin polymer. Furthermore, the appearance of a lignin-rich fraction was found in the fraction fromC. versicolor, which was not determined by gel-permeation chromatographic analysis. These results demonstrated that DSC analysis can be used to analyze the decay mechanism of solid lignocellulosic samples.

Molecular properties of lignin-carbohydrate complexes from beech (Fagus crenata) and pine (Pinus densiflora) woods

Lignin-carbohydrate complexes (LCC) were isolated from pine (Pinus densiflora) and beech (Fagus crenata) milled-wood lignins. The LCCs were treated with enzyme to obtain precipitates (A-P, B-P) and water soluble fractions. The water soluble fraction from beech LCC was subjected to gel filtration to give LCC fragments (B-E-I). In order to protect the phenolic hydroxyl group, B-E-I and B-P were methylated with diazomethane, resulting in nonphenolic LCC fractions B-E-Ip and B-Pp, respectively. On treatment of B-E-I and B-P with sodium hydroxide, a remarkable amount of xylose and a trace of arabinose were detected as monomeric sugars. However, the same alkaline treatment of B-E-Ip and B-Pp gave only a faint trace of xylose. With DDQ treatment of acetylated B-E-I and B-P, monomeric sugars were released in the same amount as those obtained on alkaline treatment. Methylation of the monomeric sugars gave monomethylated xylose. From these results, it was concluded that xylose residues participate in lignin-carbohydrate linkages, and that lignin is linked to xylose at the 0–2 or 0–3 positions through an alkali-labile benzyl ether bond. The molecular-weight distributions of the lignin moieties, measured by HPLC, indicate that the lignin moieties of beech LCC are 100 times larger but less frequent than those of pine LCC. This nonuniformity of distribution of lignin and carbohydrate moieties in hardwood LCC molecules would result in less hydrophobic interaction in aqueous solutions.