Lignin-carbohydrate Complex Linkages Research Articles

Lignin's complex role in lignocellulosic biomass Recalcitrance: A case study on bamboo

Lignin is widely recognized as a key factor influencing the recalcitrance of lignocellulosic biomass, yet the specifics of this relationship remain complex and not fully understood due to the complex interplay between lignin content variation and cell wall structure. Bamboo, which exhibits greater recalcitrance compared to wood in biorefinery processing, was chosen as the focus of investigation in this study. Three variants of Dendrocalamus farinosus with high, middle and low lignin contents were systematically analyzed and compared, in terms of molecular structure of lignin, hemicelluloses and cellulose, cell wall polymer spatial orientation, lignin-carbohydrate complex (LCC) linkages, cell wall geometrical size, as well as deconstruction efficiency, including delignification, alkaline extraction, and cellulose enzymatic hydrolysis efficiency. Findings reveal that an increase in lignin content generally lowers bamboo's deconstruction efficiency, but the mechanisms are more nuanced than previously thought. While lignin content variations do not significantly affect the molecular structure of xylan, cellulose and the chemical linkage type of LCC linkages, they do impact cellulose crystallinity, xylan orientation and notably, the thickness of fiber cell wall in the free fiber stands of bamboo. These findings highlight that lignin's role extends beyond mere physical protection of the cell wall, but also induces subtle variations in polymer structure and cell wall morphologies, which collectively influence the deconstruction efficiency of bamboo cell walls.

Facile fractionation of bamboo hydrolysate and characterization of isolated lignin and lignin-carbohydrate complexes

Abstract An efficient separation technology for hydrolysates towards a full valorization of bamboo is still a tough challenge, especially regarding the lignin and lignin-carbohydrate complexes (LCCs). The present study aimed to develop a facile approach using organic solvent extraction for efficiently fractionating the main components of bamboo hydrolysates. The high-purity lignin with only a trace of carbohydrates was first obtained by precipitation of the bamboo hydrolysate. The water-soluble lignin (WSL) fraction was extracted in organic solvent through a three-stage organic solvent extraction process, and the hemicellulosic sugars with increased purity were also collected. Furthermore, a thorough characterization including various NMR techniques (31P, 13C, and 2D-HSQC), GPC, and GC-MS was conducted to the obtained lignin-rich-fractions. It was found that the WSL fraction contained abundant functional groups and tremendous amount of LCC structures. As compared to native LCC of bamboo, the WSL fraction exhibited more typical LCC linkages, i.e. phenyl glycoside linkage, which is the main type of chemical linkage between lignin and carbohydrate in both LCC samples. The results demonstrate that organic phase extraction is a highly efficient protocol for the fractionation of hydrolysate and the isolation of LCC-rich streams possessing great potential applications.

Structural characterization and antioxidant activity of water-soluble lignin-carbohydrate complexes (LCCs) isolated from wheat straw

Lignin-carbohydrate complex (LCC) was proven to possess antioxidant activities. To understand structures and antioxidant activities of LCC, water-soluble LCC-stalk, LCC-sheath and LCC-leaf were isolated from ball-milled wheat straw stalk, sheath and leaf by successive dissolution in LiCl/DMSO solvent, water extraction and purification. LCCs were later structurally characterized by wet chemistry, chromatography and spectroscopy respectively. Their antioxidant activities were evaluated by ferric reducing antioxidant power (FRAP) and DPPH radical scavenging assays. The results showed that three LCCs were carbohydrate-rich (≈70%) and presented relatively narrow molecular weight distribution (PI < 2.0). The lignin moieties of LCCs were mainly connected with β-O-4′ structures, and phenyl glycoside and γ-ester linkages were main LCC linkages in LCCs. However, guaiacyl units were the predominant lignin units in LCC-stalk and LCC-sheath, while syringyl units were predominant in LCC-leaf. Intermolecular cross-linkages were mainly pCA-bridges in LCC-stalk and FA-bridges in LCC-sheath and LCC-leaf. Besides, LCC-sheath featured higher polysaccharide content exhibited higher molecular weight, fewer LCC linkages and better antioxidant activities (DPPH radical scavenging rate up to 74.91%) than both LCC-stalk (74.55%) and LCC-leaf (64.52%). This work helped to know LCCs in wheat straw well and inspire the application of LCC as potential antioxidants.

Characterization and Application of Lignin–Carbohydrate Complexes from Lignocellulosic Materials as Antioxidants for Scavenging In Vitro and In Vivo Reactive Oxygen Species

Lignin–carbohydrate complexes (LCCs) have shown antioxidant ability to scavenge the individual free radicals in vitro, while little work has been carried out to show if the LCCs can efficiently scavenge the intracellular and endogenous reactive oxygen species (ROS), which are the multiple radicals derived from the reduction of molecular oxygen during the metabolism process. In this work, carbohydrate-rich LCCs from bamboo (LCCs–B-B) and poplar (LCCs–B-P) were isolated according to the classical method, and their antioxidant activities were evaluated by scavenging intracellular ROS in RAW 264.7 cells in vitro and endogenous ROS in zebrafish in vivo. Results from composition analysis show that both LCC preparations possess similar contents of carbohydrate (52.2% and 51.2%) and lignin (44.1% and 47.8%). However, NMR analysis revealed that the LCCs–B-B contain 16.1/100C9 LCCs linkages, higher than that in LCCs–B-P (12.3/100C9). Antioxidant assays indicated that LCCs–B-B exhibited better antioxidant activities...

The influence of lignin content and structure on hemicellulose alkaline extraction for non-wood and hardwood lignocellulosic biomass

The extractability of hemicellulose from different lignocellulosics depends on the source of biomass. Differences in hemicellulose extractability are believed to be due to plant-specific hemicellulose arrangement alongside lignin within the cell wall. In this research, six biomasses were used to probe hemicellulose alkaline extractability as a function of the native lignin within the biomasses. Quantitative 2D-HSQC and 13C NMR analysis were performed to determine the S/G (S: syringyl, G: guaiacyl) and lignin-carbohydrate complex (LCC) linkages of milled wood lignin isolated from these biomasses. A strong negative correlation was observed between total lignin content and hemicellulose extractability, demonstrating that a greater presence of lignin in the original material results in lower xylan solubilization. In addition, a correlation between S/G of lignin and xylan dissolution was found within a group of hardwoods and within a group of non-woods. This suggests that monomeric constituency also influences xylan’s propensity for dissolution in 10% NaOH. Although there is some uncertainty in the quantification of LCC linkages, both non-woods and hardwoods exhibited negative correlations between alkaline-stable LCC linkages content and xylan extractability. This suggests that alkaline-stable LCC structures are associated with a decrease in the alkaline extractability of hemicellulose.

Structural characterization of lignin and its carbohydrate complexes isolated from bamboo (Dendrocalamus sinicus)

Isolation of earth abundant biopolymer, Lignin, from Dendrocalamus sinicus and their structural properties were investigated to achieve its large-scale practical applications in value-added products. Two lignin fractions (MWL, DSL) were isolated with successive treatments of dioxane and dimethylsulfoxide (DMSO) from dewaxed and ball milled bamboo (D. sinicus) sample. The two-step treatments yielded 52.1% lignin based on the total lignin content in the dewaxed bamboo sample. Spectroscopy analyses indicated that the isolated bamboo lignin was a typical grass lignin, consisting of p-hydroxyphenyl, guaiacyl, and syringyl units. The major interunit linkages presented in the obtained bamboo lignin were β-O-4′ aryl ether linkages, together with lower amounts of β-β', β-5′, and β-1′ linkages. The tricin was detected to be linked to lignin polymer through the β-O-4′ linkage in the bamboo. In addition, phenyl glycoside and benzyl ether lignin-carbohydrate complexes (LCC) linkages were clearly detected in bamboo (D. sinicus), whereas the γ-ester LCC linkages were ambiguous due to the overlapping NMR signals with other substructures. The detailed structural properties of the obtained lignin fraction together with the light-weight will benefit efficient utilization of natural polymers as a possibly large-scale bio-based precursor for making polymeric materials, biochemicals, functional carbon and biofuels, and multifunctional polymer nanocomposites.

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.

Effect of Lignin Carbohydrate Complexes of Hardwood Hybrids on the Kraft Pulping Process

Lignin-carbohydrates complexes influence many chemical properties in the wood, such as difficult-to-remove lignin from Kraft pulps at the end of pulping due to the occurrence of lignin carbohydrates bonds. Therefore, this study aimed to study the influence of lignin-carbohydrate complexes on eucalyptus Kraft pulping. Spectroscopic techniques (13C NMR and HSQC-2D) were applied for the determination and quantification of lignin-carbohydrate complex (LCC) structures, and then evaluated the effect of LCC on Kraft pulping of eucalyptus hybrids. The analytical tools allowed the identification and quantification of the benzyl ether, γ-ester, and phenyl glucoside linkages of the lignin-carbohydrate complexes in eucalyptus hybrid wood. The glycosidic phenyl and γ-ester linkages are, respectively, more and less significant from the quantitative point of view. Analysis of 13C NMR of the samples showed that the eucalyptus hybrid GxGL contained high β-O-4 linkages content and also higher pulping yield than the other samples, suggesting that the linkages between lignin are more important than LCC linkages in pulping.

Determination of Lignin-Carbohydrate Complexes Structure of Wheat Straw using Carbon-13 Isotope as a Tracer

To maximize the use of wheat straw as a feedstock for biofuels and other biorefinery products, the structure of lignin-carbohydrate complexes (LCCs) was characterized by injection of 13C isotope-labeled xylose into living wheat straw. Afterwards, lignin-carbohydrate complexes were extracted from the harvested straw by the Bjorkman method. The extracted LCCs were chemically characterized by Fourier transform-infrared spectroscopy (FT-IR), sugar composition, molecular weight analysis, 13C-NMR, and HSQC. The results showed that LCCs in wheat straw were particularly enriched with xylan and exhibited narrow polydispersity (Mw/Mn < 1.5). NMR analysis showed that the lignin was linked with the carbohydrates through γ-ester, phenyl glycoside, and benzyl ether bonds. In addition to S, G, and H lignin units, p-coumarate and ferulate were also in the LCCs. The substructures in lignin were β-O-4', β-β', and β-5'. Quantitative data analysis of 13C-NMR combined with HSQC showed that the lignin in the LCCs of wheat straw contained guaiacyl (G), syringyl (S), and p-hydroxyphenyl (H) units in the proportion of 5:4:1 (S:G:H). The main lignin substructure, β-O-4', comprised 71.64% of the isolated lignin. The total LCC linkages (the sum of phenyl glycoside, γ-ester and benzyl ether bonds) were 210.86 /100C9 in 13C-LCC, which was dominated by phenyl glycoside linkages, followed by γ-ester bonds and minor amounts of benzyl ether bonds. Lignin and xylan in the LCCs of wheat straw were mainly linked by benzyl ether bonds and phenyl glycoside linkages.

Comparative study of lignin characteristics from wheat straw obtained by soda-AQ and kraft pretreatment and effect on the following enzymatic hydrolysis process

To understand the structural changes of lignin after soda-AQ and kraft pretreatment, milled straw lignin, black liquor lignin and residual lignin extracted from wheat straw were characterized by FT-IR, UV, GPC and NMR. The results showed that the main lignin linkages were β-aryl ether substructures (β-O-4′), followed by phenylcoumaran (β-5′) and resinol (β-β′) substructures, while minor content of spirodienone (β-1′), dibenzodioxocin (5-5′) and α,β-diaryl ether linkages were detected as well. After pretreatment, most lignin inter-units and lignin-carbohydrate complex (LCC) linkages were degraded and dissolved in black liquor, with minor amount left in residual pretreated biomass. In addition, through quantitative 13C and 2D-HSQC NMR spectral analysis, lignin and LCC were found to be more degraded after kraft pretreatment than soda-AQ pretreatment. Furthermore, the subsequent enzymatic hydrolysis results showed that more cellulose in wheat straw was converted to glucose after kraft pretreatment, indicating that LCC linkages were important in the enzymatic hydrolysis process.

Structural elucidation of lignin–carbohydrate complex (LCC) preparations and lignin from Arundo donax Linn

Lignin carbohydrate complex (LCC) impedes the industrial application of plant fibers but also displays diverse pharmacological activities. In the present study, lignin chemical linked LCC preparations were subsequently isolated from energy crop Arudo donax Linn., and chemically characterized by using GPC, HPAEC, FT-IR, and 2D HSQC NMR spectroscopy. The results indicated that the isolated LCC preparations were xylan-rich and exhibited relatively narrow molecular weight distribution (PI<2.0). 2D HSQC NMR analysis showed direct evidences that the carbohydrate and lignin constituents in each LCC preparation were chemically bonded. Specially, Björkman LCC preparation was rich in phenyl glycoside, benzyl ether, and benzyl ester LCC linkages, indicating that the classic Björkman LCC preparation was preferable for the analysis of LCC linkages in A. donax. Moreover, lowest “concentration” of benzyl ether LCC linkage was found in CEL preparation. Further lignin characterization demonstrated that β-O-4′ alkyl ether linkages (77–100%) were predominant. With respect to the hydroxycinnamates, p-coumarate was incorporated into all LCC preparations, whereas ferulate existed mainly in Björkman LCC and may ether link to lignin. These findings will provide a theoretical basis for biofuels and biomedicine production.

The influence of lignin–carbohydrate complexes on the cellulase-mediated saccharification I: Transgenic black cottonwood (western balsam poplar, California poplar) P. trichocarpa including the xylan down-regulated and the lignin down-regulated lines

The influence of the putative lignin–carbohydrate complexes (LCCs) on enzymatic saccharification was elucidated for the first time by examining two groups of transgenic black cottonwood (P. trichocarpa) comprised of the lignin down-regulated and the xylan down-regulated lines. Any adventitious contaminants that could affect the characterization of LCCs and the enzymatic saccharification were removed by performing a thorough extraction on the samples. The crude milled wood lignin was subsequently isolated from which the phenyl glycoside, benzyl ether and γ-ester linkages representative of the LCCs were identified and quantified with the combination of 13C and 1H–13C Heteronuclear Single Quantum Coherence (HSQC) NMR. The result indicated that the samples showed different levels of the three LCC linkages, depending on the xylan and/or lignin content. The correlation between the LCCs and enzymatic saccharification nearly conclusively demonstrated that the LCCs accounting for the recalcitrance of lignocellulosic biodegradation.

The influence of lignin–carbohydrate complexes on the cellulase-mediated saccharification II: Transgenic hybrid poplars (Populus nigra L. and Populus maximowiczii A.)

Twelve transgenic hybrid poplars (Populus nigra L. and Populus maximowiczii A.) were used to demonstrate the influence of the lignin–carbohydrate complexes (LCCs) on enzymatic saccharification. The samples have different levels of the syringaldehyde (S) to vanillin (V) ratio from 0.1 to 2.6 and the lignin content from 10.5% to 24.3%, compared to the control (the S/V ratio 1.7 and the lignin content 22.0%). Any adventitious contaminants that could affect the final enzymatic saccharification were removed by performing thorough extraction on the samples. The crude milled wood lignins were subsequently isolated from which the phenyl glycoside, benzyl ether and γ-ester linkages representative of the LCC were identified and quantified by 13C and 1H–13C HSQC NMR. It was found that the samples showed different levels of the three LCC linkages, depending on the lignin content and/or the S/V ratio. The correlation between the LCCs and enzymatic saccharification nearly conclusively demonstrated that the LCCs accounted for the recalcitrance of lignocellulosic biodegradation.

Unveiling the Structural Heterogeneity of Bamboo Lignin by In Situ HSQC NMR Technique

One of the primary challenges for efficient utilization of lignocellulosic biomass is to clarify the complicated structure of lignin. In this study, in situ heteronuclear single quantum coherence nuclear magnetic resonance (NMR) characterization of the structural heterogeneity of lignin polymers during successively treated bamboo was emphatically performed without componential separation. Specially, the NMR spectra were successfully obtained by dissolving the acetylated and non-acetylated bamboo samples in appropriate deuterated solvent (CDCl3 and DMSO-d 6). The heterogeneous lignin polymers in bamboo samples were demonstrated to be HGS-type and partially acylated at the γ-carbon of the side chain by p-coumarate and acetate groups. The major lignin linkages (β–O–4, β–β, and β–5, etc.) and various lignin–carbohydrate complex linkages (benzyl ether and phenyl glycoside linkages) can be assigned, and the frequencies of the major lignin linkages were quantitatively obtained. In particular, the residual enzyme lignin (REL) contained a higher amount of syringyl units and less condensed units as compared to other samples. Inspiringly, the method gives us a vision to track the structural changes of plant cell wall (e.g., lignin polymers) during the different pretreatments.

Characterization of Lignin Structures and Lignin–Carbohydrate Complex (LCC) Linkages by Quantitative 13C and 2D HSQC NMR Spectroscopy

To characterize the lignin structures and lignin-carbohydrate complex (LCC) linkages, milled wood lignin (MWL) and mild acidolysis lignin (MAL) with a high content of associated carbohydrates were sequentially isolated from ball-milled poplar wood. Quantification of their structural features has been achieved by using a combination of quantitative (13)C and 2D HSQC NMR techniques. The results showed that acetylated 4-O-methylgluconoxylan is the main carbohydrate associated with lignins, and acetyl groups frequently acylate the C2 and C3 positions. MWL and MAL exhibited similar structural features. The main substructures were β-O-4' aryl ether, resinol, and phenylcoumaran, and their abundances per 100 Ar units changed from 41.5 to 43.3, from 14.6 to 12.7, and from 3.7 to 4.0, respectively. The S/G ratios were estimated to be 1.57 and 1.62 for MWL and MAL, respectively. Phenyl glycoside and benzyl ether LCC linkages were clearly quantified, whereas the amount of γ-ester LCC linkages was ambiguous for quantification.

Quantification of lignin–carbohydrate linkages with high-resolution NMR spectroscopy

A quantitative approach to characterize lignin-carbohydrate complex (LCC) linkages using a combination of quantitative ¹³C NMR and HSQC 2D NMR techniques has been developed. Crude milled wood lignin (MWLc), LCC extracted from MWLc with acetic acid (LCC-AcOH) and cellulolytic enzyme lignin (CEL) preparations were isolated from loblolly pine (Pinus taeda) and white birch (Betula pendula) woods and characterized using this methodology on a routine 300 MHz NMR spectrometer and on a 950 MHz spectrometer equipped with a cryogenic probe. Structural variations in the pine and birch LCC preparations of different types (MWL, CEL and LCC-AcOH) were elucidated. The use of the high field NMR spectrometer equipped with the cryogenic probe resulted in a remarkable improvement in the resolution of the LCC signals and, therefore, is of primary importance for an accurate quantification of LCC linkages. The preparations investigated showed the presence of different amounts of benzyl ether, γ-ester and phenyl glycoside LCC bonds. Benzyl ester moieties were not detected. Pine LCC-AcOH and birch MWLc preparations were preferable for the analysis of phenyl glycoside and ester LCC linkages in pine and birch, correspondingly, whereas CEL preparations were the best to study benzyl ether LCC structures. The data obtained indicate that pinewood contains higher amounts of benzyl ether LCC linkages, but lower amounts of phenyl glycoside and γ-ester LCC moieties as compared to birch wood.

Lignin-carbohydrate complexes in forages: structure and consequences in the ruminal degradation of cell-wall carbohydrates.

Lignin-carbohydrate complexes (LCCs) are recognised as key structures in forage degradability. Apart from ester bonds involving phenolic acids, which seem to play a major role in grasses, little is known about the other types of linkages that must exist but have proved difficult to demonstrate. The chemical nature of possible LCC linkages is presented and the various mechanisms through which LCCs in the cell-wall architecture may interfere with carbohydrate utilisation by rumen microorganisms are discussed.