Aspen Lignin Research Articles

Functional Expression of an Arabidopsis cDNA Clone Encoding a Flavonol 3′-O-Methyltransferase and Characterization of the Gene Product

We report that the cDNA clone (Accession No. U70424), previously isolated from Arabidopsis thaliana as encoding a caffeic acid/5-hydroxyferulic acid O-methyltransferase (OMT) (1), has now been overexpressed in Escherichia coli BL21 and its recombinant protein identified as a novel flavonol 3′-OMT. It is, therefore, renamed AtOMT1. This cDNA clone has previously been identified on the basis of its 88% amino acid sequence similarity and 80% identity to the aspen bispecific lignin OMT (2), the type member of the group involved in lignin biosynthesis. Our data indicate that this novel OMT uses the flavonol quercetin as the preferred substrate, but neither of the hydroxycinnamic acids, caffeic or 5-hydroxyferulic, to any significant extent. This indicates that the high sequence similarity/identity of AtOMT1 to that of the aspen lignin OMT (2) is not sufficient to assign the function of this gene product.

Distribution oferythro andthreo forms of different types of β-O-4 structures in aspen lignin by13C NMR using the 2D INADEQUATE experiment

Carbon–carbon connectivity spectra of 13C-enriched aspen lignin recorded using the 2D INADEQUATE experiment revealed cross peaks which can be assigned to four types of arylglycerol β-aryl ethers (β-O-4 structures): erythro forms of arylglycerol β-syringyl ethers, threo forms of arylglycerol β-syringyl ethers, erythro forms arylglycerol β-guaiacyl ethers and threo forms of arylglycerol β-guaiacyl ethers. The intensities of the cross peaks suggest larger amounts of β-syringyl ethers than β-guaiacyl ethers. The erythro isomers dominate among the β-syringyl ethers. Erythro and threo forms of β-guaiacyl ethers are present in similar amounts. © 1998 John Wiley & Sons Ltd.

Distribution of erythro and threo forms of different types of β‐O‐4 structures in aspen lignin by 13C NMR using the 2D INADEQUATE experiment

Carbon–carbon connectivity spectra of 13C-enriched aspen lignin recorded using the 2D INADEQUATE experiment revealed cross peaks which can be assigned to four types of arylglycerol β-aryl ethers (β-O-4 structures): erythro forms of arylglycerol β-syringyl ethers, threo forms of arylglycerol β-syringyl ethers, erythro forms arylglycerol β-guaiacyl ethers and threo forms of arylglycerol β-guaiacyl ethers. The intensities of the cross peaks suggest larger amounts of β-syringyl ethers than β-guaiacyl ethers. The erythro isomers dominate among the β-syringyl ethers. Erythro and threo forms of β-guaiacyl ethers are present in similar amounts. © 1998 John Wiley & Sons Ltd.

Destruction of aspen wood by the fungusPhanerochaete sanguinea quantitative1H and13C NMR spectroscopies of biologically degraded lignin

The lignin of mechanically ground aspen wood and lignins isolated from aspen wood attacked by the fungusPhanerochaete sanguinea have been investigated by quantitative1H and13C NMR spectroscopies. It has been shown that the biodestruction of the lignin takes place through the cleavage of alkyl-aryl and aryl-aryl bonds and is accompanied by demethylation (demethoxylation) reactions, and the oxidation of Cα and Cγ atoms. In addition to reactions in which the C—C bonds are cleaved, the formation of ether bonds has been observed. An interconnection has been shown between the variations in the amount of functional groups, fragments, and the bonds in biolignins and the loss in mass of the wood. A method is proposed for evaluating the carbohydrate content in lignin preparations using the NMR method.

The Distribution of Phenolic Hydroxyl Groups in Hardwood Lignins

Abstract Periodate oxidation, because of its high selectivity in the degradation of free-phenolic units, has been used along with the combined phenyl nucleus exchange and nitrobenzene oxidation techniques to estimate the distribution of phenolic hydroxyl groups in hardwood lignins. Analyses of the aspen and white birch wood lignin in situ indicate that the percentage of uncon-densed structures having a phenolic hydroxyl group was considerably higher for guaiacyl (∼20%) than for syringyl (−5%) units. Thus, the total phenolic hydroxyl groups of hardwood lignins are largely associated with the guaiacyl propane units.

Molecular weight distribution of aspen lignins from conventional gel permeation chromatography, universal calibration and sedimentation equilibrium

Molecular weight distributions of ball-milled, organosolv, alkali-extracted/mild acid-hydrolyzed and alkali-extracted/steam-exploded aspen lignins in tetrahydrofuran were compared using conventional gel permeation chromatography (GPC), universal calibration and sedimentation equilibrium. Molecular weight averages reported in the literature from universal calibration for the four low-molecular-weight lignins agreed more closely with values found in this study from sedimentation equilibrium experiments than from conventional GPC. This result supports the suggestion that these acetylated lignins fit universal calibration.

Lignin and Related Compounds XII. Catalytic Degradation of Proto and Isolated Aspen Lignins Under Initially Alkaline Conditions

Abstract Catalytic degradation of aspen lignin under initially alkaline conditions has been studied as a method of selective conversion of the lignin polymer into potentially valuable phenolic chemicals. In contrast to initially neutral conditions, degradation is not dependent on the presence of molecular hydrogen and relatively larger amounts of C6-C-C over C6-C-C-C degradation products are obtained. The addition of anthraquinone to a typical soda pulping process results in a twofold increase in the yield of monomeric lignin products.

Electrochemistry of Biomass-Derived Materials I. Characterization, Practionation, and Reductive Electrolysis of Ethanol-Extracted Explosively-Depressurized Aspen Lignin

Abstract The chemical characterization (functional group distribution) and spectroscopic properties (13C, 1H, IR, UV-Vis) of a number of lignins obtained from ethanol extraction of explosively-decompressed aspen (Populus tremuloides) lignin (EEEDAL) are described. In this process mod is heated to ∼240°C by direct steam and fiberized by sudden decompression. Both mechanical and chemical degradation occur which lead to a decrease of the molecular weight of all of the natural polymers. The lignin fraction is soluble in alcohols; it has a low number average molecular weight (925) and is very reactive. EEEDAL was subjected to fractionation by differential solubility prior to and after electrolyses on mercury cathodes at −2.6 V vs. Ag/AgCl in methanol/tetraethyl-ammonium perchlorate. After recovery of 85–90% of the original weight of material used by precipitation with acid, followed by a series of extractions, the chemical and spectroscopic properties of each fraction were determined. The acid-insoluble fractions are more condensed than the acid-soluble fractions and have lower phenolic OH and lower aliphatic OH contents than the acid-soluble fractions. The acid-soluble fractions phenolic OH content approaches one per phenylpropane (5) unit of the Lignin. Electrolyses increase the amount of lower molecular weight, high phenolic content, acid-soluble lignins and changes the molecular weight distribution by decreasing the polydispersity of the resulting lignins. These low-molecular-weight lignins are chemically reactive (for instance, with formaldehyde) and can be used in the partial replacement of phenol in phenol-formaldehyde thermosetting resins.

High-performance size exclusion chromatography of low-molecular-weight lignins and model compounds

An experimental high-performance size exclusion chromatographic procedure to obtain apparent molecular weights and their distribution for low-molecular-weight lignins was developed. Polystyrenes and lignin model compounds were utilized for calibration of the molecular weights. Values found in this study for weight and number average molecular weights for steam-exploded aspen lignins agree well with values reported by other workers using different solvent systems and calibration methods for similar lignins. In addition, possible associative effects were estimated by using one model compound with a free phenolic group and a carbonyl group, acetovanillone. The mobile phase and concentration range were optimized to minimize associative effects. The procedure used proved to be reproducible and to give good resolution of paucidisperse components. An examination of the application of Kirkwood-Riseman theory to the elution behavior of low-molecular-weight polystyrenes and lignin model compounds, is also included.

Water-soluble products from the degradation of aspen lignin by Phanerochaete chrysosporium

When Phanerochaete chrysosporium degrades [14C] lignin-labelled aspen wood in static liquid culture, it releases almost as much 14C in water-soluble form as in 14CO2. Water-soluble ultraviolet-absorbing materials also accumulate in cultures that are degrading lignin. The water-soluble radioactive materials appear to be intermediates in lignin degradation, because they can be further metabolized to CO2 and because the soluble materials labelled during [14C] lignin degradation are distinct from the materials labelled during [14C] glucose metabolism. These lignin degradation products can be fractionated by gel chromatography and are mostly acidic.

ChemInform Abstract: LIGNIN AND RELATED COMPOUNDS. IX. THE ISOLATION OF A DIMER WITH A BENZYLISOCHROMAN STRUCTURE FROM THE HYDROGENOLYSIS PRODUCTS OF ASPEN LIGNIN

A dimer with a benzylisochroman structure, 1, has been isolated from the products of the catalytic (Rh—C) hydrogenolysis of aspen lignin.

Lignin and related compounds. IX. The isolation of a dimer with a benzylisochroman structure from the hydrogenolysis products of aspen lignin

A dimer with a benzylisochroman structure, 1, has been isolated from the products of the catalytic (Rh—C) hydrogenolysis of aspen lignin.

Characterization of autohydrolysis aspen (P. tremuloides) lignins. Part 3. Infrared and ultraviolet studies of extracted autohydrolysis lignin

Infrared and ultraviolet studies of the lignins extracted from extractive-free aspen wood meal after autohydrolysis at 195 °C for periods varying from 5 min to 2 h indicated that these lignins were functionally modified and different from aspen milled wood lignin. The extracted lignins changed from a guaiacyl–syringyl type lignin to a syringyl-deficient type lignin with increasing autohydrolysis time. Extracted lignins were also observed to contain unconjugated β-ketone groups and conjugated carboxylic acid groups. It is proposed that the unconjugated β-ketone groups resulted from the depolymerization reactions of the lignin macromolecule under acidic conditions which formed monomeric, dimeric, and oligomeric/polymeric lignin fragments with Hibbert's ketone side chains. The conjugated carboxylic acid group observed to be present was attributed to p-hydroxybenzoic acid. p-Hydroxybenzoic acid has been postulated as contributing to the extractability of aspen lignin by acting as a blocking agent in the repolymerization of lignin fragments to form insoluble lignin.

Characterization of autohydrolysis aspen (P. tremuloides) lignins. Part 1. Composition and molecular weight distribution of extracted autohydrolysis lignin

Extractive-free aspen wood meal was subjected to autohydrolysis at 195 °C for 5 min to 2 h, and the lignin extracted with 90% dioxane. Extracted autohydrolysis lignin was found to be higher in carbon but lower in hydrogen and oxygen content than aspen milled wood lignin. The methoxyl content was also lower than the reference lignin. These differences have been attributed to condensation and incorporation into the lignin of non-lignin components. A lignin extractability curve with a maximum delignification at autohydrolysis time of 30–40 min was found. From molecular weight distribution studies the ratio of high molecular weight to low molecular weight materials varied for the different extracted lignins and reached a maximum at autohydrolysis time of 40 min. A mechanism of depolymerization/repolymerization of the lignin via carbonium ions has been proposed. p-Hydroxybenzoic acid is postulated as contributing to the extractability of aspen lignin by acting as a blocking agent.

Lignin and related compounds. V. The hydrogenolysis of aspen wood lignin using rhodium-on-charcoal as catalyst

The hydrogenolysis of aspen lignin using rhodium-on-charcoalas catalyst has been studied. The maximum yield of chloroform-soluble lignin degradation products was obtained at a reaction temperature of 195 °C. Analysis of this chloroform-soluble fraction revealed that the monomeric fraction was composed essentially of the four compounds, 1–4, only, and in a total yield representing at least 40% of the original lignin.Solvent extraction was shown to be preferred over thermal distillation as a preparative procedure for the separation of these phenolic derivatives. The significance of the isolation of these lignin degradation products is discussed.

Formation of free radicals in photoirradiated cellulose. VI. Effect of lignin

AbstractThe effect of lignin on free‐radical formation in photoirradiated pulp was studied by means of electron spin resonance spectroscopy. Samples were irradiated with light of wavelength longer than 3400 Å as well as longer than 2537 Å. Radical formation in aspen lignin was observed before and after irradiation with light of wavelength longer than 3400 Å. Upon irradiation, free radicals were formed in a pulp sample with 0% lignin content only when oxygen was present. On irradiation with light longer than 2537 Å, in the presence of nitrogen, oxygen, and under vacuum, free‐radical formation was found in all cases. The largest concentration of radicals was obtained under vacuum, the smallest in oxygen. It is evident that cellulose was protected from radiation by the presence of lignin. Increased aspen lignin content in pulp led to a decrease in the relative signal intensity of ESR spectrum, that is, a decrease of the yield of free radicals in the cellulose fraction in pulp. Irradiation with light in the presence of oxygen caused significant yellowing of the pulp sample, and these photooxidation and discoloration reactions occurred primarily on the surface of the sample irradiated.

Investigation of phenols prepared from aspen lignin by its decomposition with a solution of sodium in liquid ammonia

The decomposition of aspen lignin with sodium in liquid ammonia gave a mixture of phenols, of which dihydroeugenol, 1-(4-hydroxy-3-methoxyphenyl)-1- and -3-propanols, and 1-(4-hydroxy-3,5-dimethoxyphenyl)propane were identified by paper chromatography.

THE ISOLATION AND PROPERTIES OF LIGNINS OBTAINED BY THE ACIDOLYSIS OF SPRUCE AND ASPEN WOODS IN DIOXANE–WATER MEDIUM

A study has been made of the isolation of a lignin fraction from spruce and aspen wood meals using a low temperature (90–95 °C) acidolysis involving a dioxane–water (9:1) solvent medium containing the equivalent of 0.2 N hydrochloric acid. The effect of extraction time on the yield of precipitated lignin and ether-soluble material, and on the methoxyl content of these products, was investigated. For both species prolonged extraction times led to greater yields of lignin with lower methoxyl content.Further acidolysis of isolated spruce lignin led to no significant loss of methoxyl indicating the stability of this group to these reaction conditions. With increasing times of acidolysis of the aspen wood, a slow rise in the carbon–methyl/methoxyl ratio was observed for the isolated lignins. This is in agreement with earlier findings for the spruce lignin. The yields of vanillin and syringaldehyde from aspen lignin on alkaline nitrobenzene oxidation were much less than those from wood meal, and the relative yield of syringaldehyde was higher.It is suggested that this type of lignin may be only mildly modified during extraction and hence suitable for subsequent chemical investigation. The significance of the findings is discussed.

THE ALKALINE NITROBENZENE OXIDATION OF ASPEN WOOD AND LIGNIN MODEL SUBSTANCES

The yields of vanillin and syringaldehyde obtained by the alkaline nitrobenzene oxidation of aspen wood meal have been determined at various temperatures for various times. The maximum yield of each of these aldehydes, ca. 15 and 36% respectively, was obtained under the same conditions. Similar maximum yields result at 130 ± 5 °C. as at 170 ± 5 °C. if the reaction time is markedly increased. Treatment of the wood meal with sodium hydroxide at 160 °C. for two and one half hours prior to the addition of nitrobenzene and subsequent heating under the same conditions decreases, by over 30%, the yields of aldehydes. Samples of 3,4,5-trimethoxybenzaldehyde, β-D-glucovanillin, and β-D-glucosyringaldehyde were oxidized by alkaline nitrobenzene at 160 °C. for two and one half hours and yields of the corresponding phenolic aldehydes of 10.7, 69.6, and 71.9% respectively were obtained. These results are discussed with respect to the chemistry of aspen lignin.