Condensed Lignin Structures Research Articles

Application of lithium cationization tandem mass spectrometry for structural analysis of lignin model oligomers with β-β' and β-O-4' linkages.

Structural elucidation of lignin degradation products is a requirement for successfully developing lignin valorization technology. Most of mass spectrometry-based techniques have utilized negative ion mode mass spectrometry for structural elucidation of lignin-derived compounds. Unfortunately, simple deprotonation can lead to in-source fragmentation and may not be suitable for condensed lignin structures without acidic moieties. Herein, we present a lithium cationization methodology for mass spectrometry sequencing of advanced lignin oligomers having β-β' and β-O-4' bonding motifs. To do so, two advanced lignin oligomers were first synthesized through a step-by-step synthetic route, and then subjected to two different ESI mass spectrometry techniques in positive ion mode using lithium cations for ionization. An orbitrap mass spectrometer was used to obtain exact mass information, and higher-energy collisional dissociation (HCD) was used to sequence the lignin model oligomers. Based on the sequence-specific fragment ions, sequence rules were proposed. Multi-stage (MSn) collision-induced dissociation (CID) using an ion trap mass spectrometer provided data to investigate the origin of each fragment ion and to further confirm proposed fragmentation pathways. In addition to β-O-4' bond cleavage, the presented lithium cationization approach led to cleavage of β-β' bonds on the model oligomers in both ion trap and orbitrap mass spectrometry experiments. Additionally, MSn experiments were used to investigate possible lithium cationization sites on the model oligomers. Lithium cationization in positive ion mode mass spectrometry proved to be a robust tool for characterization and sequencing of advanced lignin oligomers with different bonding motifs.

Alkaline sulfonation and thermomechanical pulping pretreatment of softwood chips and pellets to enhance enzymatic hydrolysis

To assess the impact of alkalinity on sulfonation and the enzyme-mediated hydrolysis of softwood cellulose, Lodgepole pine chips were impregnated with 8% sodium sulfite and increasing loadings of sodium carbonate before thermomechanical pulping. It was apparent that alkali addition enhanced lignin sulfonation with an additional 4% loading of sodium carbonate proving optimal. TEM indicated that sulfonation predominantly occurred within the secondary-cell-wall lignin, increasing cellulose accessibility to the cellulase enzymes. Although increasing alkalinity did not significantly enhance lignin sulfonation, likely due to the lower acetyl content of the softwood chips, it increases mannan solubilization. Despite their smaller particle size, softwood pellets were more poorly sulfonated, probably due to their higher lignin content and lower amount of acid groups. This more condensed lignin structure was confirmed by 2D-NMR and GPC analyses which indicated that the EMAL derived from softwood pellets contained less native β-O-4 linkages and had a higher molecular weight.

Similarities in Recalcitrant Structures of Industrial Non-Kraft and Kraft Lignin.

This work compares the structure of industrially isolated lignin samples from kraft pulping and three alternative processes: butanol organosolv, supercritical water hydrolysis, and sulfur dioxide/ethanol/water fractionation. Kraft processes are known to produce highly condensed lignin, with reduced potential for catalytic depolymerization, whereas the alternative processes have been hypothesized to impact the lignin less. The structural properties most relevant to catalytic depolymerization are characterized by elemental analysis, quantitative 13 C and 2 D HQSC NMR spectroscopy, gel permeation chromatography, and thermogravimetric analysis. Quantification of the β-O-4 ether bond content shows partial depolymerization, with all samples having less than 12 bonds per 100 aromatic units. This results in theoretical monomer yields of less than 5 %, strongly suggesting the alternative fractionation processes generate highly condensed lignin structures that are no more suitable for catalytic depolymerization than kraft lignin. However, the different thermal degradation profiles suggest there are physicochemical differences that could be leveraged in other valorization strategies.

Effects of one-step alkaline and two-step alkaline/dilute acid and alkaline/steam explosion pretreatments on the structure of isolated pine lignin

Biological valorization of biomass most often depends on the efficient reduction of plant cell wall recalcitrance and conversion of lignin – the most recalcitrant constituent – to fuels, chemicals and/or value-added substances. Lignin conversion to fuels and value-added chemicals requires a sound understanding of the structure of lignin before and after different pretreatments. In the current work, an effort has been made to compare the structural differences in isolated pine lignin after one- (alkaline) and two-step (alkaline/dilute acid and alkaline/steam explosion) pretreatments. Our results indicate removal of the low molecular weight fraction of lignin after an initial alkaline pretreatment. A subsequent dilute acid pretreatment resulted in the loss of lignin inter-unit linkages such as β-O-4’ aryl ethers. However, with a steam explosion pretreatment, lignin exhibited a competing condensation process leading to increased condensed lignin structures.

Simultaneous pyrolysis and trimethylsilylation with N-methyl-(trimethylsilyl) trifluoroacetamide for the characterisation of lignocellulosic materials from kraft pulping

AbstractSimultaneous pyrolysis and trimethylsilylation (SPyT) withN-methyl-(trimethylsilyl)trifluoroacetamide (MSTFA) was used to characterise lignocellulosic materials from pine and eucalyptus obtained by kraft pulping. This approach protects the carboxyl groups and helps preserve the original structure of phenolic products during analytical pyrolysis, and so that pyrolysis and derivatisation occur simultaneously and the fragments of underivatised compounds are also silylatated. The SPyT products are representative for the structures present in carbohydrates and lignin of the investigated materials. The method is also suited to semi-quantitative evaluation also in terms of detection of condensed lignin structures in pulps and kraft lignins.

Spectroscopic analysis of hot-water- and dilute-acid-extracted hardwood and softwood chips

Hot-water and dilute sulfuric acid pretreatments were performed prior to chemical pulping for silver/white birch (Betula pendula/B. pubescens) and Scots pine (Pinus sylvestris) chips to determine if varying pretreatment conditions on the original wood material were detectable via attenuated total reflectance (ATR) infrared spectroscopy. Pretreatment conditions varied with respect to temperature (130°C and 150°C) and treatment time (from 30min to 120min). The effects of the pretreatments on the composition of wood chips were determined by ATR infrared spectroscopy. The spectral data were compared to those determined by common wood chemistry analyses to evaluate the suitability of ATR spectroscopy method for rapid detection of changes in the wood chemical composition caused by different pretreatment conditions. In addition to determining wood species-dependent differences in the wood chemical composition, analytical results indicated that most essential lignin- and carbohydrates-related phenomena taking place during hot-water and acidic pretreatments could be described by applying this simple spectral method requiring only a small sample amount and sample preparation. Such information included, for example, the cleavage of essential lignin bonds (i.e., mainly β-O-4 linkages in guaiacyl and syringyl lignin) and formation of newly condensed lignin structures under different pretreatment conditions. Carbohydrate analyses indicated significant removal of hemicelluloses (especially hardwood xylan) and hemicelluloses-derived acetyl groups during the pretreatments, but they also confirmed the highly resistant nature of cellulose towards mild pretreatments.

Revealing the Molecular Structural Transformation of Hardwood and Softwood in Dilute Acid Flowthrough Pretreatment

To understand better the intrinsic recalcitrance of lignocellulosic biomass, the main hurdle to its efficient deconstruction, the effects of dilute acid flowthrough pretreatment on the dissolution chemistry of hemicellulose, cellulose, and lignin for both hardwood (e.g., poplar wood) and softwood (e.g., lodgepole pine wood) were investigated at temperatures of 200 to 270 °C and a flow rate of 25 mL/min with 0.05% (w/w) H2SO4. Results suggested that the softwood cellulose was more readily degraded into monomeric sugars than that of hardwood under same pretreatment conditions. However, while the hardwood lignin was completely removed into hydrolysate, ∼30% of the softwood lignin remained as solid residues under identical conditions, which was plausibly caused by vigorous C5-active recondensation reactions (C–C5). Effects of molecular structural features (i.e., lignin molecular weight, cellulose crystallinity, and condensed lignin structures) on the recalcitrance of hardwood and softwood to dilute acid pretreatment were identified for the first time in this study, providing important insights to establish the effective biomass pretreatment.

Development of a Highly Efficient Pretreatment Sequence for the Enzymatic Saccharification of Loblolly Pine Wood

The efficient pretreatment of lignocellulosic materials for bioenergy production is a critical step upon which efficient saccharification is highly dependent, particularly in softwoods due to both their high lignin content and condensed lignin structures. In the present study, preliminary pretreatment steps (e.g., Wiley milling, acetone extraction, autohydrolysis, and disc refining) and economical subsequent/core-pretreatment steps (e.g., reagents immersion, hydrothermolysis, dilute acid hydrolysis, and ionic liquids treatment) were systematically investigated to identify which combinations led to effective enzymatic saccharification of loblolly pine wood, the dominant softwood resource in the US. The results demonstrated that 85% phosphoric acid based immersions were highly efficient for both cellulose crystallinity degradation and enzymatic hydrolysis, and thus can be included as core pretreatment steps. The highest glucan recovery yield obtained was 93.0% after enzymatic hydrolysis when a pretreatment ...

Structural features of lignin-rich solid residues obtained from two-step acid-hydrolysis of Miscanthus biomass (Miscanthus sacchariflorus Benth.)

Miscanthus was subjected to two-step hydrolysis under different concentrations of sulfuric-acid (first-step: 72–18%; second-step: 36−2%) for conversion to monomeric sugars and lignin-rich solid residues. Compared to native lignin isolated from Miscanthus, there were obvious reductions of phenolic hydroxyl(0.5–2.6% vs. 5.9%) and methoxyl groups(3.0–13.9% vs. 16.5%) in solid residues. TGA confirmed that thermal stability of solid residues was proportional to greater abundance of condensed lignin structures and inversely proportional to remaining carbohydrate amount. Py-GC/MS revealed that sum of lignin derivatives (H-,G-,S-type phenols) amounted to 23–76% of the levels of MWL, meaning that lignins in solid residues seem to be highly condensed.

Condensed lignin structures and re-localization achieved at high severities in autohydrolysis of Eucalyptus globulus wood and their relationship with cellulose accessibility.

Eucalyptus globulus wood was subjected to autohydrolysis pretreatment at different severity factors. The pretreated materials were enzymatically saccharified at a substrate load of 10% (w/v) using a cellulase enzyme complex. Around 82-95% of original glucans were retained in the pretreated material, and the enzymatic hydrolysis yields ranged from 58% to 90%. The chemical and structural changes in the pretreated materials were investigated by microscopic (SEM, LSCM) and spectroscopic (2D-HSQC NMR and FT-IR) techniques. 2D-NMR results showed a reduction in the amounts of β-O-4 aryl-ether linkages and suggested the presence of newly condensed structures of lignin in the biomass pretreated at the more severe conditions. Furthermore, the microscopic analysis showed that lignin migrates out of the cell wall and re-deposits in certain regions of the fibers at the more severe conditions to form droplet-like structures and expose the cellulose surface. These changes improved the glucose yield up to 69%, on dry wood basis.

Unraveling the structural characteristics of lignin in hydrothermal pretreated fibers and manufactured binderless boards from Eucalyptus grandis

AbstractBackgroundEucalyptus grandisis one of the most abundant biomass from plantation in many parts of the world. The binderless board were manufactured from hydrothermal pretreated fibers ofEucalyptuswood and characterized for the chemical analyses and mechanical strengths in order to assess the mechanism of self-bonding. To make clear the self-bonding mechanism of these binderless boards, the structural characteristics of cellulolytic enzyme lignin (CEL) isolated fromEucalyptuswood, its hydrothermal pretreated fibers, and binderless boards were thoroughly investigated by chemical and spectroscopic methods.ResultsThe result revealed that hydrothermal pretreatment and hot pressing process could change cellulose crystalline structures by disrupting inter/intra hydrogen bonding of cellulose chains. During the hydrothermal pretreatment ofEucalyptuswood, acid-catalyzed cleavage of β-O-4′ linkages and ester bonds were the major mechanisms of lignin cleavage. This degradation pathway led to a more condensed lignin which has a high average molecular weight and more phenolic hydroxyl groups than the control. The hot pressing process resulted in the binderless boards with reduced lignin contents and decreased the glass transition temperature, thus making the lignin more accessible to the fiber surface. CEL isolated from the binderless boards showed an increased syringyl to guaiacyl propane (S/G) ratio but a lower molecular weight than those of the untreatedEucalyptuswood and the hydrothermal pretreated fibers.ConclusionsBased on the finding of this study, it is suggested that the combination of hydrothermal pretreatment and hot pressing process is a good way for conditioning hardwood sawdust for the production of binderless boards. The thermal softening of lignin, rich in phenolic hydroxyl groups, and increased condensed lignin structure contributed to the self-bonding formation of lignocellulosic materials.

Structural Modification of Lignin and Characterization of Pretreated Wheat Straw by Ozonation

Ozonolysis is potentially an effective method for pretreating lignocellulosic biomass to improve the production of fermentable sugars via enzymatic hydrolysis. Further understanding of the ozonolysis process and identifying specific lignin structural changes are crucial for improving the pretreatment process. Investigation into pretreatment of wheat straw using ozonolysisis is reported in this paper, with special emphasis on selective modification/degradation of lignin subunits. The ozonolysis was performed for 2 h with less than 60 mesh particles in order to achieve maximum lignin oxidation. The results showed that the lignin structure was significantly modified under these conditions, leading to higher sugar recovery of more than 50% which increased from 13.11% to 63.17% corresponding to the control and ozone treated samples, respectively. Moisture content was found to be an important parameter for improving sugar recovery. Ninety percent (w/w) moisture produced the highest sugar recovery. The concentration of acid soluble lignin in the ozone treated sample increased from 4% to 11% after 2 h treatment. NMR analysis revealed that the S2/6 and G2 lignin units in the wheat straw were most prone to oxidation by ozone as the concentration of aromatic units decreased while the carboxylic acids became more abundant. The experimental data suggest the degradation of β-O-4 moieties and aromatic ring opening in lignin subunits. The pyrolysis-gas chromatography/mass spectrometry results revealed that the rate of lignin unit degradation was in the following order: syringyl > guaiacyl > p-hydroxyphenyl. Long ozone exposure resulted in few condensed lignin structure formation. In addition, the formation of condensed units during this process increased the activation energy from ASTM-E, 259.74 kJ/mol; Friedman-E, 270.08 kJ/mol to ASTM-E, 509.29 kJ/mol; Friedman-E, 462.17 kJ/mol. The results provide new information in overcoming lignin barrier for lignocellulose utilization.

Improvement of kraft pulp bleaching by treatments with laccase, urea, and refining

Abstract Unbleached Eucalyptus kraft pulps were treated by three methods and the bleaching effects have been evaluated. The treatments were performed by a laccase-mediator system (LMS), refining (R), and urea (U) alone and in combination of these methods. The bleached pulps were characterized in terms of κ number, hexenuronic acid content, brightness, and viscosity. In addition, the lignin-carbohydrate complexes (LCCs) were fractionated and characterized with regard to the fractionation yield, lignin content, and carbohydrate composition. Moreover, the 1H nuclear magnetic resonance spectra were recorded and the thioacidolysis-gas chromatography and thioacidolysis-size exclusion chromatography analyses were performed. The LMS treatment resulted mainly in a slightly better delignification as manifested by a κ number (K no.) reduction of 0.8 units. The effects of the U treatment included delignification (1.4 κ units), hexenuronic acid removal (3 μmol g-1), and improved pulp brightness (3.4 ISO units). The R treatment darkened the pulp (3 ISO units), whereas the bleaching of the R-pulp by subsequent LMS or U treatment enhanced the brightness (0.5 unit more) or delignification (0.3 unit more). The residual lignin in the pulp samples was present mainly as xylan-lignin. The Klason lignin content from the xylan-lignin fraction decreased as the degree of delignification increased. The pulp darkening followed by the R treatment was interpreted as the result of the formation of condensed lignin structures, but these condensed lignins were substantially removed by the subsequent LMS or U treatment.

Effect of pulping conditions on lignin structure from maritime pine kraft pulps

Residual and dissolved lignins from maritime pine pulps obtained by laboratory batch reactor (conventional and modified with surfactant) and flow-through reactor kraft cookings, were studied and compared. The same experimental conditions were used in order to achieve the same degree of delignification. The lignin samples were characterised by elemental analysis, residual carbohydrate content, permanganate oxidation and quantitative 13C NMR analysis. These studies confirmed that the pulping procedure influences the lignin structure, namely the lateral chain degradation, the content of hydroxyl groups and condensed lignin units. It was also shown that the amount of condensed lignin structures was lower in the case of flow-through reactor and batch modified cookings when compared with batch conventional cooking.

Can the Biochemical Features and Histology of Wheat Residues Explain their Decomposition in Soil?

The biochemical characteristics or quality of crop residues is an important factor governing soil residue decomposition. To improve C and N biotransformation models the process underlying this decomposition needs to be better understood and new quality criteria found to describe it. The aims of this explorative study were to (i) improve our understanding of residue decomposition from detailed studies of cell wall biochemical compositions and tissue architecture (ii) find new ways of exploring generic indicators of organic matter quality. To do this, the cell wall composition and tissue architecture of wheat leaves, internodes and roots, before and after their incorporation into soil were determined. Results showed that leaves which were poorly lignified decomposed faster in soil than internodes and roots. Cellulose was the most degraded polysaccharide irrespective of wheat residue. However, cellulose was much more degraded in the case of leaves as compared to internodes and roots. Leaves also presented a highly condensed lignin structure and the extent to which uncondensed leaf lignin was affected by soil decomposition suggests that the contribution of leaf lignin to C mineralization during incubation was very low. Roots which contained similar amounts of lignin than the internodes decomposed more slowly. Roots were enriched in phenolic acids, and more particularly p-coumaric acid (pCA) and presented a more condensed lignin structure than internodes. Phenolic acids are involved in the formation of lignin–polysaccharide complexes known to be recalcitrant to enzymatic attack. Microscopic investigations confirmed that the vessels were the most resistant tissues to decomposition in soil and this could be related either to their lignin content or to the quality of this lignin (condensed-like type lignin). Therefore, cell wall biochemical analyses have revealed that phenolic acids, which in their esterified form represent only 0.1–1% of plant dry matter, have cross link functions within the cell walls that could be of major interest in estimating soil residue degradability. Lignin quality (monomers, level of condensation) was another crucial criterion that could explain why residues with similar amounts of lignin decomposed at different rates in soil (roots vs. aerial parts). Visualization of residue cell walls before and after decomposition in soil underlined the interest of a microscopic approach coupled with image analysis. This study, corroborated by the extensive literature on forage digestibility, confirmed that the proportions of vascular tissue and sclerenchyma cells in plant material are determinant factors affecting plant degradability. In the future, classification of plant material based on these criteria could lead to the definition of new quality parameters for models of C and N biotransformation in soil.

Characterisation of thermally modified hard- and softwoods by 13C CPMAS NMR

The changes induced by thermal modification in the chemical structure of spruce [ Picea abies (L.) Karst.], birch ( Betula pendula), aspen ( Populus tremula) and oak ( Quercus robur) were studied by 13C CPMAS NMR spectroscopy. Spruce, birch and aspen were thermally modified at ∼195 °C and oak at ∼160 °C, under steam, according to an industrial-scale heat treatment process. In both hard- and softwood samples, 13C CPMAS NMR measurements revealed a degradation of less ordered carbohydrates (i.e. hemicelluloses and amorphous cellulose) in the thermally modified wood, which resulted in an increase in the cellulose crystallinity. Furthermore, thermal modification induced changes in the lignin structure by a cleavage of the β-O-4 linkages. In the softwood lignin, a decrease also occurred in the methoxyl group content leading to a more condensed lignin structure.

Solution-State Nuclear Magnetic Resonance Study of the Similarities between Milled Wood Lignin and Cellulolytic Enzyme Lignin

The structures of milled wood lignin (MWL) and cellulolytic enzyme lignin (CEL) have been analyzed using traditional chemical methods and solution-state NMR techniques. Comparisons of the results obtained reveal that subtle differences exist between the two lignin preparations. Thioacidolysis produced higher monomer yields from CEL than MWL, suggesting MWL has a more condensed structure. Quantitative (13)C NMR determined the degree of condensation in MWL to be 0.43 unit per aromatic moiety as compared to 0.36 in CEL. The MWL also contained a lower amount of beta-O-4' substructures per aromatic ring than CEL, 0.41 versus 0.47, respectively. Carbohydrate analysis revealed that the MWL may contain a higher proportion of middle lamella material as compared to the CEL. Because the middle lamella is considered to have a more condensed lignin structure, on the basis of the bulk polymerization theory, these results could explain the differences in beta-O-4' and degree of condensation.

Difference of condensed lignin structures in eucalyptus species

Difference of condensed lignin structures in eucalyptus species

Decomposition pattern of lignin in forest litters and soils

Abstract To characterize the degree of alteration of the lignin molecule during biodegradation, CuO oxidation has been used in litters and soils. Condensed lignin structures are not attacked by the relatively‐mild alkaline CuO oxidation, for this reason the oxidation degradation with CuO is considered a specific method for the characterization of intact lignin structures in forest litters and soils. In this paper we examine two natural lignins, one from a gymnosperm litter, the other from angiosperm litter and from respective soils under air pollution conditions. The sum of phenolic CuO oxidation products gives an overall pattern of lignin decomposition. The degree of alteration of remnant lignin is described by the acid‐to‐aldehyde ratio (AC:A1) of syringyl and vanillyl units (S:V) for angiosperm lignin. The air pollution conditions do not influenced the degree of lignin degradation.

PERMANGANATE OXIDATION OF HUMIC ACIDS, FULVIC ACIDS, AND HUMINS EXTRACTED FROM Ah HORIZONS OF A BLACK CHERNOZEM, A BLACK SOLOD, AND A BLACK SOLONETZ SOIL

Humic acid, fulvic acid, and humin fractions extracted from a Black Chernozem, a Black Solod, and a Black Solonetz soil were methylated with diazomethane and oxidized with permanganate solution. The oxidation products were extracted into ethyl acetate, separated by preparative gas chromatography, and identified by comparing their mass and micro-IR spectra with those of authentic specimens. Total yields of oxidation products followed the order humic acids > humins > fulvic acids. The products resulting from the oxidation of humic and fulvic acids averaged 63% benzenecarboxylic, 32% phenolic, and 5% aliphatic carboxylic acids. The oxidation products from humins averaged 76% benzenecarboxylic but only 20% phenolic and 4% aliphatic carboxylic acids, indicating some differences in the chemical structure of humins from those of humic and fulvic acids. The most prominent compounds produced by the oxidation of the humic acids were hydroxy benzenepentacarboxylic and benzenetetracarboxylic acids. In general, differences in the distribution of the major oxidation products between the three major fractions were greater than those within individual humic fractions. Thus, the chemical structures of humic acids extracted from three different soils appeared to be more similar to each other than to those of fulvic acid and humin fractions from the same soil. The same was true for fulvic acids and humins extracted from the different soils. Between 79 and 95% of the oxidation products were identified. The oxidation products may have originated from (a) condensed lignin structures, (b) complex structures of microbiological origin, and/or (c) polymeric structures consisting of benzenecarboxylic and phenolic acids held together by hydrogen-bonding.