Fractionation and Characterization of Wood Cell Wall Components of Fagus crenata Blume Using LiCl/DMSO Solvent System

Abstract

The fractionation of wood cell wall components was achieved by the application of the LiCl/DMSO solvent system developed in our previous study. Various soluble and insoluble fractions were prepared from beech milled wood by extractions with DMSO containing different amounts of lithium chloride (LiCl) for the study of a lignin-carbohydrate complex (LCC). Nitrobenzene oxidation (NO) analyses demonstrated that the lignin in the soluble fractions always has lower yields of NO products consisting of syringaldehyde + syringic acid (S_y) and vanillin + vanillic acid (V_a). The syringyl ratios, S_y/(S_y+V_a), were also lower than in insoluble fractions. Accordingly, lignins with lower syringyl ratios are better soluble than those with higher syringyl ratios. The former is typical in the primary wall and the latter in the secondary wall. Solubilization of glucan is significantly dependent on the LiCl concentration in DMSO. In the absence of LiCl, only about 6% of glucan was found in the soluble fraction, but about 40% of lignin and xylan were solubilized. The additional 40% of lignin and xylan became soluble together with glucan solubilization, which increased at 3% LiCl concentration. However, a fraction amounting to 13% (based on the whole wood) still remained as a residue under these conditions. Glucan solubilization could not be the reason for this observation as ball-milled cellulose is soluble in 3% LiCl/DMSO. It is likely that strong interactions between lignin and carbohydrates prevent solubilization of this fraction, which has never been isolated and analyzed in previous studies.     

How Well Do MWL and CEL Preparations Represent the Whole Hardwood Lignin?

Hardwood lignin preparations were isolated using classical milled wood lignin (MWL) and cellulolytic enzyme lignin (CEL) protocols. Furthermore, we managed to produce a lignin preparation of a very high yield, above 90%, with high purity and minimal structural degradation. This was achieved by dissolution and regeneration of milled wood prior to enzymatic hydrolysis, along with the use of 80% dioxane for lignin extraction. This preparation (RCEL-80) yield was about 4.5 and 2.5 times higher than the yields of the traditional MWL and CEL preparations, correspondingly, at the same milling time. The preparations obtained were comprehensively analyzed with state-of-the-art quantitative NMR techniques and wet chemistry methods. CELs were representative preparations for hardwood lignins as the S/G ratios in the CELs were equivalent to those in lignin in situ. Degradation of the main lignin subunits was very low. Importantly, the structures of CELs were independent of the preparation yield and were very similar to the structure of the high-yield lignin, RCEL-80. In contrast, the structures of MWLs were noticeably dependent on the preparation yield, especially when the yield was below 15–20% of total wood lignin. In particular, the S/G ratio increased with increasing MWL yield, but was still lower than the S/G ratios of the whole lignins in situ, even at high MWL yields. The amounts of β-O-4 lignin units in MWL were lower than those in the corresponding CEL preparations. It has been concluded that CEL preparations were representative of the whole wood lignin whereas MWLs represent a fraction with a lower S/G ratio.     

An NMR Comparison of the Whole Lignin from Milled Wood,MWL, and REL Dissolved by the DMSO/NMI Procedure

Abstract

Lignins isolated from pine milled wood, milled wood lignin (MWL), and residual enzyme lignin (REL) were compared using modified thioacidolysis, modified DFRC, gel permeation chromatography (GPC), two‐dimensional Heteronuclear Multiple Quantum Coherence (HMQC) NMR, and quantitative ¹³C NMR. Dissolution of the lignin for solution‐state NMR was accomplished by utilizing the recently reported DMSO/N‐methylimidazole/acetic anhydride solvent system. Contrary to previous reports, comparison of the lignin preparations by thioacidolysis indicated that REL was more structurally similar to the lignin in the milled wood and Wiley wood meal than MWL. Total monomer yields indicated that the MWL was lower in β‐aryl ether content than the other preparations, and this was verified by quantitative ¹³C NMR. NMR analysis indicated that the inter‐unit linkages present in all the lignin preparations are consistent with the present knowledge about lignin biosynthesis. The contribution of minor end group structures in the MWL are further decreased in the milled wood, indicating that they are preferentially isolated as low molecular weight material, possibly generated during the milling process. All other structural moieties were similar in all preparations. GPC data indicated that the milled wood and REL both contain a portion of lignin with a molecular weight of 55,000 g/mol. Data indicate that the inefficiency of the DFRC method may be related to molecular mobility or accessibility in higher molecular weight portions of the lignin polymer.     

Relationships between Hemicellulose Composition and Lignin Structure in Woods

The composition and absolute amount of neutral sugars were determined for 48 hardwood species (including 17 hardwoods of genera Acacia, 14 hardwoods of genera Eucalyptus, and 17 hardwoods of other genera) and 14 softwood species by alditol-acetate method, and their relationships to the syringyl ratio (syringyl/(syringyl+guaiacyl)) of lignin, which was determined by nitrobenzene oxidation, was investigated. In the hardwood species, an increase in the syringy ratio of lignin correlated with a tendency toward increased xylose/glucose, rhamnose/glucose, and arabinose/glucose ratios. However, the absolute amount of glucose in hardwood was maintained in a small range (0.4–0.5 g in 1 g sample), independent of changes in the syringyl ratio. In the softwood species, with increasing lignin content, the mannose/glucose ratio decreased, but the absolute amount of glucose remained almost constant. In both hardwood and softwood species, a strong correlation was suggested between lignin, indicated by higher syringy ratio, and hemicellulose, indicated by higher xylan/mannan ratio.     

Analytical Pyrolysis of Hardwood and Softwood Lignins and Its use in Lignin-Type Determination of Hardwood Vessel Elements

Abstract

Pyrolysis-gas chromatography-mass spectrometry was performed on milled wood lignins (MWLs) and wood samples. Among the major pyrolysis products identified from loblolly pine lignin were guaiacol, 4-methylguaiacol, 4-vinylguaiacol, vanillin, coniferal-dehyde, and coniferyl alcohol. White oak lignin pyrolysis products also included guaiacol, 4-methylguaiacol, and vanillin and additionally 2,6-dimethoxyphenol, 4-methyl-2,6-dimethoxyphenol, syringaldehyde, and sinapaldehyde. By identification of these pyrolysis products from either wood or MWL it is possible to classify lignins as either guaiacyl-type or syringyl/guaiacyl-type. Pyrolysis of isolated vessel elements from white oak, white birch, and American elm indicated that vessel lignin is of the syringyl/ guaiacyl-type.     

Analysis of Lignin Aromatic Structure in Wood Fractions Based on IR Spectroscopy

In our previous studies, a method of quantitative analysis of lignin structure by IR spectroscopy with direct use of wood meal was developed which can be apply to a wide range of wood species. Lignin structure deposition in different fractions in the same wood sample also has characteristic diversity and differences. In this study, quantitative analysis of lignin structure by IR spectroscopy was used to determine whether it can be applied to different fractions in the same wood sample. Finely milled beech wood was fractionated by stepwise extraction using aqueous dioxane and followed by subsequent dimethyl sulfoxide (DMSO)/LiCl solvents with increasing LiCl concentrations. The results showed that fractions of the peak area ratio of log(1595/1509) fit a linear relationship with different wood species very well. Therefore, log(1595/1509) could be used not only with different wood species but also with different fractions from the same wood. For 1509/1460, log(1275/1220), 1130/1030 and 835/(835+815), different fractions deviated from the original relationship obtained from different wood species.     

Lignin and Other Aromatic Substances Released from Spruce Wood During Pressurized Hot-Water Extraction,Part 1: Extraction,Fractionation and Physico-Chemical Characterization

Ground spruce wood was extracted with pressurized hot water in an accelerated solvent extractor (ASE) at 170°C during 20, 60, and 100 min. Released aromatic substances (from 2.2 to 2.5% on wood basis) were isolated on XAD-7 resin and fractionated into lignin, oligomeric aromatic substances (OAS), and compounds insoluble in methanol (IMC). The separated aromatic fractions, and the ground wood before and after extraction, were characterized by physico-chemical methods (GC, GC-MS, HPLC-SEC, TG, DSC). The major part of the aromatic substances was oligomeric aromatic substances (OAS). This fraction increased significantly with the extraction time, while the yield of dissolved lignin increased only slightly with extraction time. Isolated lignins had a lower molar mass and differed considerably from spruce milled wood lignin (MWL). The isolated lignins were more stable against oxidative thermodegradation than spruce MWL. The UV-extinction coefficients at 280 nm were lower for the isolated lignins than for MWL.     

Effects of Solvation and Ionization on 13C NMR Spectra of Lignin Model Compounds,Spruce Milled Wood Lignin and Kraft Lignin

The effects of alkali and polar aprotic solvent on the aromatic carbons signals in ¹³C NMR (Carbon-13 nuclear magnetic resonance) spectra of lignin model compounds and spruce milled wood lignin (MWL) were studied. It was found that in 1 M aqueous NaOH signal shifts of C-1 and C-4 carbon atoms in the aromatic ring were the most noticeable in lignin models with free phenolic hydroxyl groups, which are ionized under the conditions. A similar effect in the spectra of the studied model compounds was observed in 0.5 M aqueous NaOD-deuterated dimethyl sulfoxide (DMSO) mixture (DMSO: water ratio 3:7 v/v). The model data help explaining changes in the ¹³C NMR spectra of MWL and lignin in situ dissolved in spruce kraft black liquor caused by ionization. In the ¹³C NMR spectra of spruce black liquor the signals of phenolic and non-phenolic lignin units are clearly separated and do not overlap with the signals of the carbon atoms of carbohydrates and other aliphatic products of wood degradation. The data obtained are useful in understanding the important role of solvation and ionization processes leading to lignin solubilization.     

Separation and Structural Characterization of Lignin from Hybrid Poplar Based on Complete Dissolution in DMSO/LiCl

In this paper, the physicochemical properties and structural features of six lignin preparations separated with a total dissolution of ball-milled wood in dimethyl sulfoxide and lithium chloride (DMSO/LiCl) followed by extraction with ethanol/water were investigated. These isolated lignin fractions were characterized using wet chemical analysis, FT-IR, and ¹H and¹³C-NMR techniques. Experimental results showed that separated lignin preparations were relatively free of associated polysaccharides. These lignin fractions were classified as guaiacyl-syringyl lignin type: mainly composed of guaiacyl units with noticeable amounts of syringyl units and fewer p-hydroxyphenyl units. The molar ratio of non-condensed guaiacyl units to syringyl units (G/S) decreased as the ratio of LiCl to poplar weight increased. The results also showed that these lignin preparations consisted mainly of β-O-4 ether bonds combined with small quantities of β-β′ and β-5 carbon-carbon linkages. Furthermore, considerable amounts of esterified p-hydroxybenzoic acids and minor amounts of esterified p-coumaric acid were also detected in these lignin fractions.     

桉木木素的付立叶变换红外光谱研究

付立叶变换红外光谱是分析木素结构的一种有效手段。本研究用付立叶变换红外光谱法分析了桉木分离木素和桉木原本木素，并对两者进行了比较。结果表明桉木磨木木素由愈创木基和紫丁香基组成，其中紫丁香基占40％～45％，愈创木基占55％～60％，甲氧基含量为每C9单元1．40个左右。与杉木和山毛榉相比，桉木磨木木素中含有较多的乙酸基和共轭羰基，并且含有一定量的芳香酸的酯类。用差示光谱法对桉木原本木素进行了研究，结果表明原本木素比磨木木素含有更多的氢键联接和乙酸基，但原本木素中的α-羰基含量较少，紫丁香基结构单元的比例也较小，结果还表明磨木木素可能更多地来源于木材纤维细胞次生壁。     

酶解木素制备过程中的影响因素

系统研究了酶解木素的制备方法,在酶木素的制备过程中,加强对试样机械作用,会使酶解得率大幅度地提高;酶解底物的质量分数以0.06—0.08为宜;酶解过程中前48小时的酶解得率提高较快;酶的活性大小直接影响酶解效果;当酶用量超过一定范围后再增加用量也不能较大地提高酶解得率;多次酶解能有效地提高酶解得率;高的酶解得率会使木素提取率增大。本实验还对酶木素中的残糖组成及其含量作了分析测定。     

The isolation and characterization of lignin of kenaf fiber

In this article, milled wood lignin (MWL) was isolated and purified from retted kenaf fiber, the lignin obtained was characterized by elemental analysis, FTIR, ¹H‐NMR, and ¹³C‐NMR spectroscopy. The C₉ formula is calculated for kenaf fiber MWL as C₉H_9.32O_3.69(OCH₃)_1.30. The spectra of FTIR, ¹H‐NMR, and ¹³C‐NMR show the kenaf fiber lignin to be of the G/S type with high proportion of syringyl (S) unit. The numbers of phenolic and aliphatic hydroxyl groups in the kenaf fiber MWL are estimated to be 0.14 and 1.31, respectively, per C₉ unit. The OH_aliph is 90.3% in total numbers of hydroxyl groups of kenaf fiber MWL, and the OH_ph is 9.7%. It is evident that the β‐O‐4 structures are mainly linkage in the MWL of kenaf fiber, which contain more erythro stereochemistry type in β‐O‐4 units than thero stereochemistry type. In general, the characteristics of lignin of kenaf fiber are similar to that of hardwood. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

杨木MWL和LCC制备多孔生物载体及其在人体肝细胞培养中的应用

为了比较磨木木质素（MWL）与木质素-碳水化合物复合体（LCC）与人体肝细胞的生物相容性,从杨木中提取MWL和LCC,制备水凝胶,冷冻干燥形成多孔生物载体。利用红外光谱和扫描电子显微镜对多孔生物载体的成分及表面形态进行了分析,采用多孔生物载体对人肝细胞进行体外培养,并对肝细胞形态及代谢活性进行检测。结果表明：LCC基载体中木质素含量较低而碳水化合物含量较高,因此其两亲性较MWL基载体好。以MWL和LCC制备的多孔生物载体,孔隙率达到60%左右,平均孔径约为30 μm。细胞形态、白蛋白分泌和葡萄糖代谢的分析结果显示,相比MWL基载体,LCC基多孔生物载体能更好地促进肝细胞长期生长,具有更好的生物相容性。     

Simulated autohydrolysis of aspen milled wood lignin in the presence of aromatic additives. Changes in molecular weight distribution

The reactions between aspen milled wood lignin (MWL) and 2-naphthol, resorcinol, and p-hydroxybenzoic acid were studied under simulated autohydrolysis conditions. The material after the reaction was separated into a dioxane-insoluble fraction (DI), a dioxane-soluble but ether-insoluble fraction (DSEI), and an ether-soluble fraction (ES). In the absence of additive, the lignin first depolymerized and remained solvent soluble, then repolymerized to become insoluble. With 2-naphthol as additive, the amount of DI lignin decreased proportionally to the amount of additive present. When more than 0.1 mole of 2-naphthol/C-9 unit was present, no DI lignin was formed, that is, all the lignin was soluble. Gel permeation chromatography studies indicated that the additive was acting effectively as a blocking agent, preventing lignin repolymerization. Small amounts of resorcinol acted as a repolymerizing agent since two or more fragments of lignin could readily condense with a molecule of the very active additive. As a result of this bridging effect, the amount of DI lignin increased. As the amount of resorcinol was increased, it behaved more like a blocking agent and a reduction in the amount of DI lignin was observed. The presence of a large excess of resorcinol resulted in the formation of soluble lignin only. The molecular weight distributions supported the proposed dual role of resorcinol as a bridging or blocking agent. The use of an excess of p-hydroxybenzoic acid prevented the formation of DI material and resulted in low molecular weight soluble lignin.     

Dissolved Lignin and Other Aromatic Substances in Thermomechanical Pulp Waters

Abstract

Dissolved aromatic substances in water suspensions of unbleached and peroxide‐bleached spruce thermomechanical pulp (TMP) were isolated by sorption on XAD‐8 resin and fractionated into lignin and oligomeric aromatic substances (OAS). The fractions were characterized by chromatographic (GC and HP‐SEC) and spectrometric (FT‐IR, ¹³C‐NMR, GC‐MS) methods, as well as by GC after direct on‐line pyrolysis and thermally assisted hydrolysis and methylation. The dissolved lignin in unbleached TMP water was structurally similar to spruce milled‐wood lignin (MWL), but its average molar‐mass was lower and the mass distribution more narrow. The oligomeric aromatic substances included phenolic dimers, trimers, and tetramers that were structurally different from those of MWL. After peroxide bleaching the amount of dissolved semipolar (MTBE) extractives and oligomeric aromatic substances was much lower. The amount of dissolved lignin from wood fibers was, on the contrary, much higher. The lignin released from TMP after bleaching was extensively oxidized and had a slightly higher average molar‐mass than lignin released from unbleached TMP.     

球磨大豆秸秆在LiCI／DMSO全溶体系中的溶解-再生性能

对大豆秸秆纤维形态、化学成分及球磨预处理后秸秆原料在8％LiCl／DMSO全溶体系中的溶解一再生性能进行了详细研究。大豆秸秆纤维长度明显较短,宽度较大,长宽比比其他秸秆纤维小;其灰分和二氧化硅含量比多数木材原料高,比麦草、玉米秆等原料低很多;其不同部位木质素、综纤维素、抽出物、蛋白质及各种无机元素等成分相差较大。利用8％LiCl／DMSO溶剂体系溶解球磨茎秆和荚组分后发现,球磨时间延长,溶解性能越好,球磨时间达4h,可完全溶解形成澄清透亮的均一溶液。溶解后样品经水再生,产物得率随球磨时间的延长而降低,且在溶解一再生过程中木质素和多糖会有较多损失,多糖损失大于木质素的损失,灰分和硅能得到较好保留。     

Characteristics and Reactivity of Lignin in Acacia and Eucalyptus Woods

The chemical characteristics of lignin, including the Klason lignin content, acid-soluble lignin content, proportion of the aromatic ring types [syringyl ratio = syringyl/(syringyl + guaiacyl)], and proportion of the diastereomeric forms for the β-O-4 structure [erythro ratio = erythro/(erythro + threo)], and pulpability were investigated for 28 wood samples belonging to the genera Acacia and Eucalyptus. Although the lignin characteristics of these 28 woods varied widely, the chemical characteristics of the two genera could be categorized in two clearly distinguished groups on the basis of the syringyl ratio. Clear negative correlations were observed between both the syringyl and erythro ratios and the total lignin content (sum of the Klason and acid-soluble lignin contents) within each genus. In addition, the syringyl ratio correlated positively with the erythro ratio and acid-soluble lignin content, regardless of the genus. The existence of a clear, high correlation between the syringyl and erythro ratios supports the hypothesis that the aromatic ring type (syringyl ratio) is a decisive factor for controlling the diastereomeric forms of the β-O-4 structure (erythro ratio). Each of the wood samples was also subjected to Kraft pulping, and it was demonstrated that the woods with higher syringyl ratios were easier to delignify. This tendency is reasonably attributed to the high reactivity of the erythro-rich and syringyl-rich β-O-4 structures, and the low lignin content of these syringyl-rich woods.     

Preparation of Enzymatically Liberated Lignin From Naturally Brown-Rotted Wood

Abstract

Brown rots from various conifer species were sieved (&lt60 mesh) and alcohol and water extracted to yield crude preparations of “naturally enzymatically liberated lignin” (NEL) containing 61.4–91.7Z of Klason plus soluble lignin and 0.5–2.6% ash. The <200 mesh fractions were generally best represented in <60 mesh preparations and contained highest Klason plus soluble lignin percentages (86.6–92.2Z). Carbohydrates varied in these fractions between 3.7 and 8.0% and contained glucose, mannose, galactose, xylose, and arablnose, decreasing generally in that order. Methoxyl contents were lower and oxygen contents higher than in milled wood lignin (MWL) suggesting some oxidative demethylation. Treatment with Cuoxaa increased Klason plus soluble lignin content to 93.0–95.8%, decreased carbohydrates to 1.7–5.0%, increased methoxyl and decreased oxygen contents by removal of a part of cellulose and hemicelluloses and of more degraded lignin fractions. Infrared spectra of the preparations showed a small increase in carboxyls and possibly in phenyl conjugated double bonds and a decrease in aromatic structures, as compared with MWL. In thermo-gravimetric analysis the curves for MWL and Cuoxam treated NEL preparations (CuNEL) were very similar when run in nitrogen. In air, however, MWL lost weight appreciably slower. In differential scanning calorimetry in air and oxygen, MWL exhibited less intensive exotherms below 500°C than NEL and left higher amounts of char. The different behaviour of MWL in thermal analysis in oxidative atmosphere was explained by its lower molecular weight and corresponding low glass transition temperature (Tg). It was concluded that preparation of enzymatically liberated lignin from natural brown rot represents a convenient procedure where larger amounts of lignin are required; such lignins are somewhat more degraded than MWL in terms of functional groups present, but are possibly closer to protolignin in terms of molecular weight.     

Monomeric Products Formed from the Lignin of Sugi Wood During Alcohol Sulfite Pulping

Abstract

Isopropanol bisulfite treatment of sugi (Cryptomeria japonica D. Don) MWL yielded a significant amount of lipophilic low-molecular-weight products as well as water-soluble high-molecular-weight lignin whereas with an aqueous bisulfite treatment a very small amount of lipophilic products was formed and about half of the MWL was changed to insoluble and probably condensed products.

Seven monomeric and one dimeric lignin degradation products as well as two carbohydrate-derived products were found in the spent liquor from isopropanol bisulfite pulping of sugi wood of which eugenol and isoeugenol were the major products. After cooking for 50–100 minutes at 165°C, the combined yield of these two products reached a maximum of 4.5% based on lignin, which is ten times as high as the yield of a phenolic fraction previously obtained from aqueous bisulfite pulping of Pinus radiata. Formation of the other products such as stilbene and coniferaldehyde increased gradually after 100 minutes and the combined yield was less than 0.5%. The high yield of eugenol and isoeugenol as byproducts may increase the viability of the alcohol sulfite pulping process.     

Effects of Green Liquor (GL) and Sodium Carbonate (SC) Pretreatment on Structural Characteristics of Wheat Stem Lignin

The content and structure of lignin have been considered as important factors that affect both pretreatment and enzymatic hydrolysis of lignocellulosic biomass. In this work, wheat stems (WS) were pretreated using mild alkali including green liquor (GL) and sodium carbonate (SC). The results indicate that GL pretreatment exhibits better delignification selectivity and higher enzymatic digestibility than SC pretreatment. Analysis of ¹H–¹³C HSQC NMR and FTIR on cellulolytic enzyme lignin (CEL) preparations isolated from untreated and pretreated WS also proves that a certain amount of lignin degrades which leads to a decrease of β-O-4′ linkages. Under mild alkaline conditions, more guaiacyl units in lignin are removed than syringyl units, which results in a higher condensation degree and S/G ratio of CELs isolated from GL- and SC-pretreated stems. Compared with p-coumarate structures, ferulates in lignin are more stable under mild alkaline conditions.