Linkages In Lignin Research Articles

Effects of Enzyme Loading, Incubation Time and Incubation Temperature on Sawdust Hydrolysis by Locally Produced Bacterial Xylanase

The present study aims to investigate the enzymatic hydrolysis conditions of bacterial xylanase on alternative cheaper substrate which is hardwood sawdust (SD) in order to produce reducing sugars (xylose). The bacterial xylanase was produced and secreted from the Bacillus sp. The wood industry in Malaysia has become a major source of foreign exchange across the globe for developing the countries. Therefore, more wood residues (sawdust) are produced during the logging and processing of wood. Most of the sawdust will be disposed into the landfills. In actual, the sawdust can be utilised into more valuable products such as in producing reducing sugars. Thus, previous researches have studied on xylose production from wooden sawdust using commercial xylanases, but only few with the bacterial xylanase. Therefore, a study on the best conditions of enzymatic hydrolysis in producing xylose from sawdust using bacterial xylanase is essential. Prior to the enzymatic hydrolysis, the hardwood sawdust was pre-treated by autoclave at 121°C for 20 min in order to breakdown the lignin linkage and obtain the hemicellulosic xylan (delignification). The enzymatic hydrolysis conditions such as enzyme loading, incubation time and incubation temperature were experimented by One-Factor-At-Time (OFAT) method. Based on the experiment, the fifth cycle pre-treated autoclaved sawdust showed 5.5-fold higher than the untreated sawdust. The best enzymatic hydrolysis conditions for xylose production were enzyme loading of 1.4%, incubation time of 30 min, and incubation temperature of 56.9 °C. These conditions also succeeded in producing 2.5-fold higher xylose than the one without the enzymatic hydrolysis. 

1-Ethyl-3-methylimidazolium acetate ionic liquid as simple and efficient catalytic system for the oxidative depolymerization of alkali lignin

The oxidative depolymerization of alkali lignin (AL) in 1-ethyl-3-methylimidazolium acetate ([C2C1im]OAc) system without additional catalyst was investigated under mild conditions (initial O2 pressure of 1.5 MPa, 80 °C–100 °C). Compared with other ionic liquids (ILs), the cooperation of imidazolium cation and acetate anion successfully enhanced AL conversion. Among the investigated imidazolium acetate ILs with ethyl- to octyl-side chains, [C2C1im]OAc presented the best catalytic capacity for AL oxidative depolymerization. Adding an appropriate amount of water to [C2C1im]OAc can further improve the reaction efficiency. In the [C2C1im]OAc system with the addition of 0.10–0.25 mL of water, approximately 77 wt% AL was depolymerized into small molecule soluble products at 100 °C for 2 h. The extracted oil was composed mainly of phenolic derived compounds. With the use of the [C2C1im]OAc-based system, the specific inter-unit linkages of lignin were broken down, and residual lignin with low molecular weight and narrow polydispersity index (1.88–1.96) was obtained. Compared with that in AL conversion with fresh [C2C1im]OAc, only a minimal decrease (~3.2%) was observed with the recovered IL until the fifth cycle. These findings revealed that [C2C1im]OAc–based system is a simple and efficient catalytic system for lignin oxidative depolymerization.

Fractionation of Poplar Wood Using a Bifunctional Aromatic Acid under Mild Conditions

The development of an energy-efficient fractionation process as well as the preservation of the fractionated cellulose, hemicellulose sugars, and lignin are the key to the valorization of lignocellulose. This study presents a mild-condition fractionation process based on a recyclable and bifunctional 4-chlorobenzenesulfonic acid (4-Cl-BSA). The aqueous (e.g., 72%) 4-Cl-BSA solution near-completely fractionated unmilled poplar chips at 50–80 °C for 18–180 min and successively preserved the theoretical maximum yields and key structures of the fractionated cellulose, lignin, and hemicellulose sugars. Around 21.3–27.8% lignin was hydrotropically dissolved at a mesoscale level through accumulation by and complexation with 4-Cl-BSA and its aggregates. The solubilized lignin preserved about 24.7–50.7% of the 61% β-O-4 linkages in the native lignin and about 48.3–82% aromatic units uncondensed. About 72.2–78.7% lignin was insolubilized and quickly deposited on the surfaces of cellulose fibers. Remarkably, the deposited lignin preserved about 61.9–81.1% of the β-O-4 linkages in the native lignin and about 78.2–86.2% aromatic units uncondensed. Hemicellulose sugars and cellulose (millimeter-size, CrI: 71–75, DPᵥ: 910–1022) had high purity and high quality. Compared to the other selected aryl sulfonic acids whether they have or do not have substituents (dichloro, bromo, hydroxyl, and methyl) and mineral acids, 4-Cl-BSA performed better in fractionating unmilled poplar chips and preserving the β-O-4 linkages and aromatic units of lignin. The results indicate that both acidity and hydrophobicity of aryl sulfonic acid greatly influence its fractionation and preservation performances.

Upgrade the torrefaction process of bamboo based on autohydrolysis pretreatment

Torrefaction is a routine and effective method for recovering energy from biomass. In this study, autohydrolysis pretreatment was proposed to upgrade the torrefaction process of moso bamboo, and their relationship was evaluated. Results showed that about 80.24 % of bamboo hemicellulose were dissolved during autohydrolysis pretreatment. Compared with main chain, arabinan side chain of hemicellulose was much easier to be degraded, and more uniform hemicellulose could be obtained at 160 °C. The O/C values of autohydrolysis solid residues and the mass yield of torrefied bamboo decreased gradually with the enhancement of reaction severity (autohydrolysis and/or torrefaction), while the HHV values increased. The higher torrefaction temperature or autohydrolysis pretreatment severity was, the better thermostability of torrefied bamboo could be observed. 2D-HSQC of milled wood lignin revealed that autohydrolysis pretreatment can effectively accelerate the degradation of β-O-4 linkage in lignin and increase G-type lignin content during torrefaction process. Compared with direct torrefaction, prehydrolysis can greatly facilitate the torrefaction process and enhance the quality of final products.

LsdD has a critical role in the dehydrodiconiferyl alcohol catabolism among eight lignostilbene α,β-dioxygenase isozymes in Sphingobium sp. strain SYK-6

Lignin is the most abundant aromatic bioresource in nature, and elucidation of its biodegradation system is essential for understanding the carbon cycling on earth and its effective usage. The β−5 bond is one of the intermolecular linkages in lignin. Sphingobium sp. strain SYK-6 can assimilate various lignin-derived aromatic compounds, including a β−5 bond-containing dimer (phenylcoumaran-type), dehydrodiconiferyl alcohol (DCA). In the catabolic pathway of DCA, a stilbene-type compound, 3-(4-hydroxy-3-(4-hydroxy-3-methoxystyryl)-5-methoxyphenyl)acrylate (DCA-S), is produced. DCA-S is subjected to the cleavage of the interphenyl double bond by lignostilbene α,β-dioxygenase (LSD) to generate 5-formylferulate and vanillin. Among the eight LSD genes (lsdA−lsdH) found in the SYK-6 genome, the gene products of lsdA, lsdC, lsdD, and lsdG exhibited DCA-S conversion activity. The DCA-S conversion activity of SYK-6 was induced (6.8-fold) by vanillate produced as an intermediate metabolite of DCA-S. Of the LSD genes mentioned above, only the transcriptions of lsdA, lsdD, and lsdG were induced (2.6–10-fold) in the presence of vanillate. Analyses of lsdA, lsdD, and lsdG mutants showed that an lsdD mutant lost the DCA-S conversion activity under vanillate-induced conditions. These results demonstrate that lsdD plays a critical role in converting DCA-S during the DCA catabolism in SYK-6.

Properties versus application requirements of solubilized lignins from an elm clone during different pre-treatments

Kraft pulping, organosolv process and acid hydrolysis were applied on an elm clone. The solubilized lignins were recovered and analyzed. Kraft pulping and acid hydrolysis led to lignins with higher phenolic OH content as result of extensive cleavage of β-O-4′ linkages, as revealed by 13C solid state and 13C−1H heteronuclear single quantum coherence nuclear magnetic resonance. This depolymerization also yielded lower molecular weight lignins inferred by size exclusion chromatography. Contrarily, organosolv process gave rise to a lignin with a more preserved structure, maintaining a large number of β-O-4′ linkages. Consequently, organosolv lignin presented lower phenolic OH content and higher molecular weight. Moreover, the high content of the labile native β-O-4′ linkages in organosolv lignin resulted in a lower thermostability as compared to the kraft and acid lignins. On the other hand, the solubilized lignins from kraft and acid processes displayed an enrichment of S-units, whereas lignin from organosolv process was slightly enriched in G-units, containing all of them different native as well as pre-treatment derived units. These results could help to increase the inventory of lignin sources available for future lignin-based products, for which knowledge of the lignin properties versus application requirements is crucial.

New insights into lignin electrolysis on nickel-based electrocatalysts: Electrochemical performances before and after oxygen evolution

The production of H2 via electrolysis seems to be the best alternative to traditional catalytic reforming processes. The electrolysis of lignin allows to produce pure H2 while valorizing a low-price biomass waste with (theoretically) lower energy requirements than water electrolysis. Previous studies were published in the last years dealing with the electro-oxidation of lignin on Ni-based catalysts. Ni is a robust non-expensive metal that presents a wide variety of oxidation states and phase transitions. To this date, it is still not completely clear how these states/transitions activate the lignin molecule. The aim of this study is to get more insights into the cleavage of the main chemical bonds present in the complex lignin structure over different Ni oxidation states. A Ni/C catalyst, prepared by the polyol method, was thoroughly characterized and tested for the electro-oxidation of both, lignin and 2-phenoxyethanol, where the latter is a model molecule representative of the quintessential β-O-4 linkage in lignin.

Comparison of methodologies used to determine aromatic lignin unit ratios in lignocellulosic biomass

BackgroundMultiple analytical methods have been developed to determine the ratios of aromatic lignin units, particularly the syringyl/guaiacyl (S/G) ratio, of lignin biopolymers in plant cell walls. Chemical degradation methods such as thioacidolysis produce aromatic lignin units that are released from certain linkages and may induce chemical changes rendering it difficult to distinguish and determine the source of specific aromatic lignin units released, as is the case with nitrobenzene oxidation methodology. NMR methods provide powerful tools used to analyze cell walls for lignin composition and linkage information. Pyrolysis-mass spectrometry methods are also widely used, particularly as high-throughput methodologies. However, the different techniques used to analyze aromatic lignin unit ratios frequently yield different results within and across particular studies, making it difficult to interpret and compare results. This also makes it difficult to obtain meaningful insights relating these measurements to other characteristics of plant cell walls that may impact biomass sustainability and conversion metrics for the production of bio-derived fuels and chemicals.ResultsThe authors compared the S/G lignin unit ratios obtained from thioacidolysis, pyrolysis-molecular beam mass spectrometry (py-MBMS), HSQC liquid-state NMR and solid-state (ss) NMR methodologies of pine, several genotypes of poplar, and corn stover biomass. An underutilized approach to deconvolute ssNMR spectra was implemented to derive S/G ratios. The S/G ratios obtained for the samples did not agree across the different methods, but trends were similar with the most agreement among the py-MBMS, HSQC NMR and deconvoluted ssNMR methods. The relationship between S/G, thioacidolysis yields, and linkage analysis determined by HSQC is also addressed.ConclusionsThis work demonstrates that different methods using chemical, thermal, and non-destructive NMR techniques to determine native lignin S/G ratios in plant cell walls may yield different results depending on species and linkage abundances. Spectral deconvolution can be applied to many hardwoods with lignin dominated by S and G units, but the results may not be reliable for some woody and grassy species of more diverse lignin composition. HSQC may be a better method for analyzing lignin in those species given the wealth of information provided on additional aromatic moieties and bond linkages. Additionally, trends or correlations in lignin characteristics such as S/G ratios and lignin linkages within the same species such as poplar may not necessarily exhibit the same trends or correlations made across different biomass types. Careful consideration is required when choosing a method to measure S/G ratios and the benefits and shortcomings of each method discussed here are summarized.

Staged biorefinery of Moso bamboo by integrating polysaccharide hydrolysis and lignin reductive catalytic fractionation (RCF) for the sequential production of sugars and aromatics

Towards a waste-free biorefinery was of the lignocellulose valuable and sustainable conversion. In this study a staged biorefinery for lignocellulose conversion to maximum valuable products was proposed and experimentally demonstrated by using Moso bamboo (Phyllostachys heterocycla cv. Pubescens). It is an integrated sequential process including hemicellulose acidolysis with FeCl3 as the catalyst, reductive catalytic fractionation (RCF) of lignin with Pd/C and sulfuric acid as catalysts, and enzymatic hydrolysis of cellulose. The influence of acid FeCl3 catalyzed hemicellulose acidolysis on the subsequent RCF of lignin and enzymatic hydrolysis of cellulose were investigated and the optimized conditions for hemicellulose acidolysis were obtained. The yield of pentose, aromatic monomers, and glucose was respectively 85.8 wt%, 28.5 wt%, and 64.3 wt% relative to the initial hemicellulose, lignin, and cellulose contained in the Moso bamboo. The conversion and the products were analyzed and characterized using gas chromatography mass spectrometry (GCMS), Fourier transform infrared spectroscopy (FT-IR), gel permeation chromatography (GPC), and heteronculear single quantum coherence nuclear magnetic resonance (HSQC-NMR) spectrometry. The results indicated that hemicellulose acidolysis with FeCl3 resulted in the dissolution of some low molecular weight lignin and the cleavage of lignin-carbohydrate linkages of 2-O-Ac-β-d-xylopyranoside, while the connections of 3-O-Ac-β-d-xylopyranoside, phenyl glycoside and the β-ether linkages in lignin were remained. However, excessive removal of hemicellulose negatively influenced the stability of the lignin structure and delignification. RCF of lignin resulted to the hydrolysis of lignocellulose, yielded cellulose and aromatic monomers via the cleavage of β-ether bonds.

Pathway discovery and engineering for cleavage of a β-1 lignin-derived biaryl compound

Lignin biosynthesis typically results in a polymer with several inter-monomer bond linkages, and the heterogeneity of linkages presents a challenge for depolymerization processes. While several enzyme classes have been shown to cleave common dimer linkages in lignin, the pathway of bacterial β-1 spirodienone linkage cleavage has not been elucidated. Here, we identified a pathway for cleavage of 1,2-diguaiacylpropane-1,3-diol (DGPD), a β-1 linked biaryl representative of a ring-opened spirodienone linkage, in Novosphingobium aromaticivorans DSM12444. In vitro assays using cell lysates demonstrated that RS14230 (LsdE) converts DGPD to a lignostilbene intermediate, which the carotenoid oxygenase, LsdA, then converts to vanillin. A Pseudomonas putida KT2440 strain engineered with lsdEA expression catabolizes erythro-DGPD, but not threo-DGPD. We further engineered P. putida to convert DGPD to a product, cis,cis-muconic acid. Overall, this work demonstrates the potential to identify new enzymatic reactions in N. aromaticivorans and expands the biological funnel of P. putida for microbial lignin valorization.

Extraction of lignin from Chinese quince fruit by acetic acid solution at above atmospheric pressure: Yield distribution, structural characterization, and antioxidant activities

Acetic acid at atmospheric pressure is an alternative solvent that can be used for high-efficiency separation of lignin. However, few studies reported the isolation of lignin using acetic acid at above atmospheric pressure. In this study, acetic acid solution at above atmospheric pressure was used to extract lignin from Chinese quince fruit. The influences of different extraction conditions on the lignin yields were firstly investigated. Among the five extraction parameters (extraction temperature/pressure, acetic acid concentration, extraction time, catalyst dosage, and solid/liquid ratio), catalyst dosage had the most important influence on lignin yields. Five acetic acid lignin (AAL80, AAL100, AAL120, AAL140, and AAL160) samples obtained at different extraction temperatures/pressures (80 °C–160 °C/0.03–0.35 MPa) were used to analyze the influence of extraction temperatures/pressures on their structural features. Comparing to lignins extracted with acetic acid at atmospheric pressure, lignins obtained by acetic acid extraction at above atmospheric pressures (AAL120, AAL140, and AAL160) had higher purity. The acetic acid could degrade lignin at temperatures above 120 °C. When extracted at 120 °C–160 °C, the depolymerization degree of lignins increased with rising temperatures. AAL160 with more condensed structures was the most thermally stable. As the extraction temperature rose, the proportion of β–5 linkages in lignins gradually increased, while the proportion of β–O–4 and β–β linkages decreased. AAL80 had the highest DPPH, while AAL120 showed the highest ferric reducing activity. The study clarifies the lignins isolated with acetic acid at above atmospheric pressure from Chinese quince fruits and contributes to their potential application.

Kraft Lignin Ethanolysis over Zeolites with Different Acidity and Pore Structures for Aromatics Production

To utilize its rich aromatics, lignin, a high-volume waste and environmental hazard, was depolymerized in supercritical ethanol over various zeolites types with different acidity and pore structures. Targeting at high yield/selectivity of aromatics such as phenols, microporous Beta, Y, and ZSM-5 zeolites were first examined in lignin ethanolysis, followed by zeolites with similar micropore size but different acidity. Further comparisons were made between zeolites with fin-like and worm-like mesoporous structures and their microporous counterparts. Despite depolymerization complexity and diversified ethanolysis products, strong acidity was found effective to cleave both C–O–C and C–C linkages of lignin while mild acidity works mainly in ether bond breakdown. However, when diffusion of gigantic molecules is severe, pore size, particularly mesopores, becomes more decisive on phenol selectivity. These findings provide important guidelines on future selection and design of zeolites with appropriate acidity and pore structure to promote lignin ethanolysis or other hydrocarbon cracking processes.

Mechanisms of Caromatic-C bonds cleavage in lignin over NbOx-supported Ru catalyst

Efficient and selective cleavage of Caromatic-C linkages in lignin to enhance the productivity of monocyclic aromatic hydrocarbons is an extremely challenging and attractive task. Previously, we reported a Ru/NbOPO4 catalyst that realized the catalytic cleavage of both interunit C-C and C-O bonds in one-pot lignin conversions [Chem, 2019, 5, 1521–1536]. However, the roles of NbOx species in the selective Caromatic-C cleavage are not clear and need to be extensively studied. Herein, we focus on the exploration of the catalytic mechanisms of Caromatic-C bond cleavage over NbOx-supported Ru catalyst, by comparing with those of Ru/TiO2, Ru/ZrO2, and Ru/Al2O3 catalysts. It shows that the Ru/Nb2O5 catalyst exhibits high activity and remarkable selectivity to monocyclic hydrocarbons. A combined diffuse reflectance infrared Fourier Transform spectra (DRIFTS) and density functional theory (DFT) calculation analysis unambiguously confirmed that (i) NbOx not only can strongly adsorb phenylcyclohexane via benzene ring, but also selectively activate Caromatic-C bond between benzene ring and cyclohexane; (ii) NbOx can promote the protonation of phenylcyclohexane with the assistance of H3PO4; and (iii) NbOx can significantly reduce the barrier of Caromatic-C bonds cleavage of the carbocation intermediate (C6H5(H)C6H11+). A detail underlying mechanism has been established, which would have further implications to promote the design of new, high efficient catalysts.

Enhancing the multi-functional properties of renewable lignin carbon fibers via defining the structure–property relationship using different biomass feedstocks

Lignin from different biomass feedstock has been explored to make quality carbon fiber. The mechanistic study revealed the β-O-4 linkages of lignin linearly correlated to both mechanical and electroconductive performance of carbon fibers.

Visible-light photoredox-catalyzed C\u2013O bond cleavage of diaryl ethers by acridinium photocatalysts at room temperature

Cleavage of C–O bonds in lignin can afford the renewable aryl sources for fine chemicals. However, the high bond energies of these C–O bonds, especially the 4-O-5-type diaryl ether C–O bonds (~314 kJ/mol) make the cleavage very challenging. Here, we report visible-light photoredox-catalyzed C–O bond cleavage of diaryl ethers by an acidolysis with an aryl carboxylic acid and a following one-pot hydrolysis. Two molecules of phenols are obtained from one molecule of diaryl ether at room temperature. The aryl carboxylic acid used for the acidolysis can be recovered. The key to success of the acidolysis is merging visible-light photoredox catalysis using an acridinium photocatalyst and Lewis acid catalysis using Cu(TMHD)2. Preliminary mechanistic studies indicate that the catalytic cycle occurs via a rare selective electrophilic attack of the generated aryl carboxylic radical on the electron-rich aryl ring of the diphenyl ether. This transformation is applied to a gram-scale reaction and the model of 4-O-5 lignin linkages.

Insights into Structural Transformations of Lignin Toward High Reactivity During Choline Chloride/Formic Acid Deep Eutectic Solvents Pretreatment

Understanding lignin structural transformations during DES pretreatment would facilitate to produce lignin with tailor-made properties based on intended applications. To unravel the structural variant of lignin in the DES, the alkali lignin (AL) was pretreated in choline chloride/formic acid (ChCl/FA) (the ratio of 1:2) at 80-120 oC. Characterization of the AL and regenerated lignin samples were determined to understand lignin structure, such as linkage in lignin, phenolic OH content, molecular weight, and aromatic products. ChCl/FA DES pretreatment resulted in large amounts of the dissociation of β-O-4' bonds, partial cleavage of carbon-carbon bonds (i.e., β-β′, β-5′), dehydration of hydroxyl radicals in lignin’s side-chain, and demethoxylation reaction. Furthermore, various monomeric phenols derived from AL were also determined in spite of low combined yields, which facilitate the production of fine chemicals in the future. Detailed unmasking of lignin structural transformation during the DES process is conducive to the optimizing preparation of homogeneous lignin with low molecular weight.

Catalytic hydrogenolysis of lignin in ethanol/isopropanol over an activated carbon supported nickel-copper catalyst

Lignin is the renewable and abundant source of aromatics on earth, and the depolymerization of lignin provides significant potential for producing valuable monophenols. In this work, catalytic hydrogenolysis of organosolv poplar lignin (OPL) in ethanol/isopropanol solvent over monometallic and bimetallic nonprecious catalysts was investigated. Ni/C and a series of NiCu/C catalyst with different Cu loadings were prepared and applied for depolymerization of OPL. The highest yield of phenolic monomers was 63.4 wt% achieved over the Ni10Cu5/C catalyst at 270 °C without external H2. The introduction of Cu in catalysts further promoted the hydrogen donor process of ethanol/isopropanol solvent and facilitated the cleavage of lignin linkages, resulting in the decreased molecular weight of bio-oil. The possible lignin dimer type structures, such as diphenylethane (β-1) type, phenylcoumaran (β-5) type, and pinoresinol (β-β) type structures, were proposed and identified by MALDI-TOF MS, giving a better understanding of the NiCu/C catalyzed lignin depolymerization.

Selective Cleavage of Ester Linkages in Lignin Catalyzed by La-Doped Ni/MgO

Novel strategies for converting renewable lignin to bulk petrochemicals are highly desirable for achieving sustainable practices in the age of environmental awareness. Herein, we propose an efficie...

Nucleophilic Thiols Reductively Cleave Ether Linkages in Lignin Model Polymers and Lignin

Lignin may serve as a renewable feedstock for the production of chemicals and fuels if mild, scalable processes for its depolymerization can be devised. The use of small organic thiols represents a bioinspired strategy to cleave the β‐O‐4 bond, the most common linkage in lignin. In the present study, synthetic β‐O‐4 linked polymers were treated with organic thiols, yielding up to 90 % cleaved monomer products. Lignin extracted from poplar was also treated with organic thiols resulting in molecular weight reductions as high as 65 % (M n) in oxidized lignin. Thiol‐based cleavage of other lignin linkages was also explored in small‐molecule model systems to uncover additional potential pathways by which thiols might depolymerize lignin. The success of thiol‐mediated cleavage on model dimers, polymers, and biomass‐derived lignin illustrates the potential utility of small redox‐active molecules to penetrate complex polymer matrices for depolymerization and subsequent valorization of lignin into fuels and chemicals.

Possible mechanisms for the generation of phenyl glycoside-type lignin-carbohydrate linkages in lignification with monolignol glucosides.

The existence and formation of covalent lignin-carbohydrate (LC) linkages in plant cell walls has long been a matter of debate in terms of their roles in cell wall development and biomass use. Of the various putative LC linkages proposed to date, evidence of the native existence and formation mechanism of phenyl glycoside (PG)-type LC linkages inplanta is particularly scarce. The present study aimed to explore previously overlooked mechanisms for the formation of PG-type LC linkages through the incorporation of monolignol glucosides, which are possible lignin precursors, into lignin polymers during lignification. Peroxidase-catalyzed lignin polymerization of coniferyl alcohol in the presence of coniferin and syringin invitro resulted in the generation of PG-type LC linkages in synthetic lignin polymers, possibly via nucleophilic addition onto quinone methide (QM) intermediates formed during polymerization. Biomimetic lignin polymerization of coniferin via the β-glucosidase/peroxidase system also resulted in the generation of PG-type as well as alkyl glycoside-type LC linkages. This occurred via non-enzymatic QM-involving reactions and also via enzymatic transglycosylations involving β-glucosidase, which was demonstrated by in-depth structural analysis of the synthetic lignins by two-dimensional NMR. We collected heteronuclear single-quantum coherence (HSQC) NMR for native cell wall fractions prepared from pine (Pinus taeda), eucalyptus (Eucalyptus camaldulensis), acacia (Acacia mangium), poplar (Populus×eurarnericana) and bamboo (Phyllostachys edulis) wood samples, which exhibited correlations, albeit at low levels, that were well matched with those of the PG-type LC linkages in synthetic lignins incorporating monolignol glucosides. Overall, our results provide a molecular basis for feasible mechanisms for the generation of PG-type LC linkages from monolignol glucosides and further substantiates their existence inplanta.