A modified ionization difference UV-vis method for fast quantitation of guaiacyl-type phenolic hydroxyl groups in lignin.

An ionization difference UV-Vis method (Δε-spectrum method) is the most potentially simple method for fast quantitation of phenolic hydroxyl groups (ph-OH) in lignin. However, the underestimated results were calculated from the conventional Δε-spectrum method using one- or two-point wavelengths measurement. In this study, a modified Δε-spectrum method using multi-point wavelengths measurement was developed and the negative absorbance was also considered. Four main typical lignin models, e.g. vanilla alcohol, 5-5 biphenyl, stilbenoid and vanillin, were applied as the guaiacyl-type (G-type) phenolic models for the determination of ph-OH by the modified Δε-spectrum method. The 2-methoxyethanol/water/acetic acid = 8/2/0.2 (V/V/V) was used as the acidic solvent system and the 2-methoxyethanol/0.2 M NaOH solution = 1/9 (V/V) was used as the alkaline solvent system. The ph-OH contents in the spruce milled wood lignin (SMWL) and the spruce Kraft lignin (SKL) were respectively quantified by the modified Δε-spectrum method as 1.078 and 4.348 mmol/g, which were comparable to the counterparts determined by 31P Nuclear Magnetic Resonance Spectroscopy (31P NMR). The results revealed that the modified Δε-spectrum method can provide more accurate and reliable results compared to the conventional method.

Understanding pulp delignification by laccase-mediator systems through isolation and characterization of lignin-carbohydrate complexes.

The effects and mechanism of pulp delignification by laccases in the presence of redox mediators have been investigated on unbleached eucalyptus kraft pulp treated with laccases from Pycnoporus cinnabarinus (PcL) and Myceliophthora thermophila (MtL) and 1-hydroxybenzotriazole (HBT) and methyl syringate (MeS) as mediators, respectively. Determination of the corrected κ number in eucalyptus pulps after the enzymatic treatments revealed that the PcL-HBT system exhibited a more remarkable delignification effect than the MtL-MeS system. To obtain further insight, lignin-carbohydrate complexes were fractionated and subsequently characterized by nuclear magnetic resonance, thioacidolysis (followed by gas chromatography and size exclusion chromatography), and pyrolysis-gas chromatography-mass spectrometry (pyrolysis-GC-MS) analyses before and after the enzymatic treatments and their controls. We can conclude that the laccase-mediator treatments altered the lignin structures in such a way that more lignin was recovered in the xylan-lignin fractions, as shown by Klason lignin estimation, with smaller amounts of both syringyl (S) and guaiacyl (G) uncondensed units, as shown by thioacidolysis and gas chromatography, especially after the PcL-HBT treatment. The laccase-mediator treatment produced oxidation at Cα and cleavage of Cα and Cß bonds in pulp lignin, as shown by pyrolysis-GC-MS. The general mechanism of residual lignin degradation in the pulp by laccase-mediator treatments is discussed in light of the results obtained.

Universal fractionation of lignin-carbohydrate complexes (LCCs) from lignocellulosic biomass: an example using spruce wood.

It is of both theoretical and practical importance to develop a universally applicable approach for the fractionation and sensitive lignin characterization of lignin-carbohydrate complexes (LCCs) from all types of lignocellulosic biomass, both natively and after various types of processing. In the present study, a previously reported fractionation approach that is applicable for eucalyptus (hardwood) and flax (non-wood) was further improved by introducing an additional step of barium hydroxide precipitation to isolate the mannan-enriched LCC (glucomannan-lignin, GML), in order to suit softwood species as well. Spruce wood was used as the softwood sample. As indicated by the recovery yield and composition analysis, all of the lignin was recovered in three LCC fractions: a glucan-enriched fraction (glucan-lignin, GL), a mannan-enriched fraction (GML) and a xylan-enriched fraction (xylan-lignin, XL). All of the LCCs had high molecular masses and were insoluble or barely soluble in a dioxane/water solution. Carbohydrate and lignin signals were observed in (1) H NMR, (13) C CP-MAS NMR and normal- or high-sensitivity 2D HSQC NMR analyses. The carbohydrate and lignin constituents in each LCC fraction are therefore believed to be chemically bonded rather than physically mixed with one another. The three LCC fractions were found to be distinctly different from each other in terms of their lignin structures, as revealed by highly sensitive analyses by thioacidolysis-GC, thioacidolysis-SEC and pyrolysis-GC.

Lignin depolymerisation in supercritical carbon dioxide/acetone/water fluid for the production of aromatic chemicals.

Valorisation of lignin plays a key role in further development of lignocellulosic biorefinery processes the production of biofuels and bio-based materials. In the present study, organosolv hardwood and wheat straw lignins were converted in a supercritical fluid consisting of carbon dioxide/acetone/water (300-370°C, 100bar) to a phenolic oil consisting of oligomeric fragments and monomeric aromatic compounds with a total yield of 10-12% based on lignin. These yields are similar to the state-of-the-art technologies such as base-catalysed thermal processes applied for lignin depolymerisation. Addition of formic acid increases the yield of monomeric aromatic species by stabilizing aromatic radicals. Supercritical depolymerisation of wheat straw and hardwood lignin yielded monomeric compounds in different compositions with a maximum yield of 2.0% for syringic acid and 3.6% for syringol, respectively. The results of the present study showed that under the applied conditions competition occurred between lignin depolymerisation and recondensation of fragments.

On hexenuronic acid (HexA) removal and mediator coupling to pulp fiber in the laccase/mediator treatment.

Flax soda/AQ pulps were treated with different fungal laccase-mediator combinations followed by physical and chemical characterization of the pulps to obtain a thorough understanding of the laccase/mediator effects on hexenuronic acid (HexA) removal and the coupling of mediator onto pulps for fiber functionalization. Large differences were found and the presence of lauryl gallate (LG) during Trametes villosa laccase (TvL) treatment (TvL+LG) resulted in a much larger reduction of pulp-linked HexA than the combination of p-coumaric acid (PCA) and Pycnoporus cinnabarinus laccase (PcL). A major portion of LG became attached to the pulp as revealed by an increase in the kappa number and further confirmed by thioacidolysis and (1)H NMR analysis of solubilized pulp fractions. Additional experiments with other chemical pulps and isolated pulp xylan and lignin revealed that HexA seems to be the sole pulp component attacked by TvL+LG. As a substrate for TvL, the reaction preference order is PCA>HexA>LG.

Investigation of the molecular weight increase of commercial lignosulfonates by laccase catalysis.

Lignosulfonates are by-products from the sulfite pulping process. During this process, lignin is liberated from pulp fibers through sulfonation and washed away. As a consequence, the lignosulfonate molecules contain both hydrophobic and hydrophilic moieties. Lignosulfonates are low-value products with limited performance and are used as such as binders, surfactants, and plasticizers in concrete. Lignosulfonates face strong competition from synthetic petroleum-based plasticizers with superior quality. Therefore, increasing the performance of lignosulfonates is desirable not only from a sustainability point of view but also to expand their usage. One important aspect that describes how well lignosulfonates can act as plasticizers is the molecular weight. In this paper, the molecular weight of four commercial lignosulfonates is increased through oxidation by two laccases without utilization of mediators. Different parameters to obtain maximal molecular weight increase were identified and the technical significance of the experiments is discussed.

Polymerization of lignosulfonates by the laccase-HBT (1-hydroxybenzotriazole) system improves dispersibility.

The ability of laccases from Trametes villosa (TvL), Myceliophthora thermophila (MtL), Trametes hirsuta (ThL) and Bacillus subtilis (BsL) to improve the dispersion properties of calcium lignosulfonates 398 in the presence of HBT as a mediator was investigated. Size exclusion chromatography showed an extensive increase in molecular weight of the samples incubated with TvL and ThL by 107% and 572% from 28400 Da after 17h of incubation, respectively. Interestingly, FTIR spectroscopy, (13)C NMR and Py-GC/MS analysis of the treated samples suggested no substantial changes in the aromatic signal of the lignosulfonates, a good indication of the ability of TvL/ThL-HBT systems to limit their effect on functional groups without degrading the lignin backbone. Further, the enzymatic treatments led to a general increase in the dispersion properties, indeed a welcome development for its application in polymer blends.

Structural characterization of the lignin from jute (Corchorus capsularis) fibers.

The structural characteristics of the lignin from jute (Corchorus capsularis ) fibers, which are used for high-quality paper pulp production, were studied. The lignin content (13.3% Klason lignin) was high compared to other nonwoody bast fibers used for pulp production. The lignin structure was characterized by pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS), 2D-NMR, and thioacidolysis. Upon Py-GC/MS, jute fibers released predominantly products from syringylpropanoid units with the S/G ratio being 2.1 and a H/G/S composition of 2:33:65. 2D-NMR of the milled wood lignin (MWL) isolated from jute fibers showed a predominance of beta-O-4' aryl ether linkages (72% of total side chains), followed by beta-beta' resinol-type linkages (16% of total side chains) and lower amounts of beta-5' phenylcoumaran (4%) and beta-1' spirodienone-type (4%) linkages and cinnamyl end groups (4%). The high predominance of the S-lignin units, together with the high proportion of beta-O-4' aryl ether linkages, which are easily cleaved during alkaline cooking, are advantageous for pulping. On the other hand, a small percentage (ca. 4%) of the lignin side chain was found to be acetylated at the gamma-carbon, predominantly over syringyl units. The analysis of desulphurated thioacidolysis dimers provided additional information on the relative abundances of the various carbon-carbon and diaryl ether bonds and the type of units (syringyl or guaiacyl) involved in each of the above linkage types. Interestingly, the major part of the beta-beta' dimers included two syringyl units, indicating that most of the beta-beta' substructures identified in the HSQC spectra were of the syringaresinol type (pinoresinol being absent), as already observed in the lignin of other angiosperms.

Steam explosion lignins; their extraction, structure and potential as feedstock for biodiesel and chemicals.

In the present study, a steam explosion wood pre-treatment process, optimized earlier with respect to ethanol production, has been applied to both softwoods (Picea abies and Pinus sylvestris) and hardwoods (Betula verrucosa and Populus tremula). The alkaline extractable lignins have then been isolated to investigate lignin separation efficiency and lignin structure and to evaluate their potential for producing value-added products, such as biodiesel components or chemicals, in terms of the purity, molecular size, functional groups, beta-O-4' inter-unit linkage content, and degradability in a subsequent processing treatment. The mechanism of lignin modification and possible improvements to the steam explosion pre-treatment process are discussed.

Lignin biosynthesis in transgenic Norway spruce plants harboring an antisense construct for cinnamoyl CoA reductase (CCR).

An attractive objective in tree breeding is to reduce the content of lignin or alter its composition, in order to facilitate delignification in pulping. This has been achieved in transgenic angiosperm tree species. In this study we show for the first time that changes in lignin content and composition can be achieved in a conifer by taking a transgenic approach. Lignin content and composition have been altered in five-year-old transgenic plants of Norway spruce (Picea abies [L.] Karst) expressing the Norway spruce gene encoding cinnamoyl CoA reductase (CCR) in antisense orientation. The asCCR plants had a normal phenotype but smaller stem widths compared to the transformed control plants. The transcript abundance of the sense CCR gene was reduced up to 35% relative to the transformed control. The corresponding reduction in lignin content was up to 8%, which is at the lower limit of the 90-99% confidence intervals reported for natural variation. The contribution of H-lignin to the non-condensed fraction of lignin, as judged by thioacidolysis, was reduced up to 34%. The H-lignin content was strongly correlated with the total lignin content. Furthermore, the kappa number of small-scale Kraft pulps from one of the most down-regulated lines was reduced 3.5%. The transcript abundances of the various lignin biosynthetic genes were down-regulated indicating co-regulation of the biosynthetic pathway.

An improved methodology for the quantification of uronic acid units in xylans and other polysaccharides.

Uronic acids can be quantified either by a colorimetric determination after treatment with concentrated sulfuric acid and carbazole or by gas chromatography after methanolysis and subsequent acetylation. Both methods suffer from incomplete hydrolysis, an unavoidable degradation of the products to be analysed, and an inability to separate and quantify different types of uronic acids. In the present work, the fundamental chemistry involved in the two methods has been evaluated, and some modifications to increase their accuracy are suggested. By combining the two methods, a complete quantification of all individual types of uronic acids present in a sample can be achieved.

Quantitative 2D HSQC NMR determination of polymer structures by selecting suitable internal standard references.

A new analytical method based on the 2D HSQC NMR sequence is presented, which can be applied for quantitative structural determination of complicated polymers. The influence of T1 and T2 relaxations, off-resonance effects, coupling constants and homonuclear couplings are discussed. It was found that the T2 values measured on polymeric samples with the conventional HSQC-CPMG sequence could not be used to correct the errors caused by T2 relaxations during the polarization transfer delay. A unique way of selecting the proper internal standard reference signal(s) is therefore proposed to eliminate the major errors caused by T2 relaxations, resonance offsets, coupling constant deviations and homonuclear couplings. Two polymer samples, a cellulose triacetate and an acetylated lignin, have been used to illustrate the principles. The methodology developed in this work is robust to instrument miss-setting and it can find wide-spread applications in areas where a quantitative analysis of structurally complicated polymers is necessary.

Lignin depolymerization/repolymerization and its critical role for delignification of aspen wood by steam explosion.

Steam explosion is an important process for the fractionation of biomass components. In order to understand the behaviour of lignin under the conditions encountered in the steam explosion process, as well as in other types of steam treatment, aspen wood and isolated lignin from aspen were subjected to steam treatment under various conditions. The lignin portion was analyzed using NMR and size exclusion chromatography as major analytical techniques. Thereby, the competition between lignin depolymerization and repolymerization was revealed and the conditions required for these two types of reaction identified. Addition of a reactive phenol, 2-naphthol, was shown to inhibit the repolymerization reaction strongly, resulting in a highly improved delignification by subsequent solvent extraction and an extracted lignin of uniform structure.

Smooth model surfaces from lignin derivatives. I. Preparation and characterization.

Lignin model surfaces were prepared from aqueous alkaline solutions by spin-coating on silica wafers. Films of thicknesses between 20 and 140 nm were easily made by variations in the spinning rate or in the lignin concentration. The roughnesses of the lignin surfaces were relatively low, approximately 1.1 nm (rms) on an area of 25 microm2, as determined by atomic force microscopy imaging. The stability of the lignin films in aqueous solutions was found to be excellent. No changes in the thickness of model surfaces immersed in slightly alkaline solutions (pH 9.2) could be detected even after 5 h soaking. A 10 percent reduction in the thickness of the lignin film was observed after 5 h of exposure to a solution containing 0.1 M NaCl. This novel preparation method opens great possibilities for further fundamental studies, where interactions between lignin and other substances are of interest to investigate.

Structural differences between the lignin-carbohydrate complexes present in wood and in chemical pulps.

Lignin-carbohydrate complexes (LCCs) were prepared in quantitative yield from spruce wood and from the corresponding kraft and oxygen-delignified pulps and were separated into different fractions on the basis of their carbohydrate composition. To obtain an understanding of the differences in lignin structure and reactivity within the various LCC fractions, thioacidolysis in combination with gas chromatography was used to quantify the content of beta-O-4 structures in the lignin. Periodate oxidation followed by determination of methanol was used to quantify the phenolic hydroxyl groups. Furthermore, size exclusion chromatography (SEC) of the thioacidolysis fractions was used to monitor any differences between the original molecular size distribution and that after the delignification processes. Characteristic differences between the various LCC fractions were observed, clearly indicating that two different forms of lignin are present in the wood fiber wall. These forms are linked to glucomannan and xylan, respectively. On pulping, the different LCCs have different reactivities. The xylan-linked lignin is to a large extent degraded, whereas the glucomannan-linked lignin undergoes a partial condensation to form more high molecular mass material. The latter seems to be rather unchanged during a subsequent oxygen-delignification stage. On the basis of these findings, a modified arrangement of the fiber wall polymers is suggested.

Carbohydrate reactions during high-temperature steam treatment of aspen wood.

Aspen wood was treated with steam at different time-temperature severity factors. Analysis of the amounts of acids released revealed a relationship between the acidity and the formation of furfural and hydroxymethyl furfural as degradation products from carbohydrates. It is suggested that two concurrent or consecutive mechanisms are responsible for the observed results: a homolytic cleavage and an acid hydrolysis of glucosidic linkages in the polysaccharides. By preimpregnating the wood with alkali, hydrolysis can be eliminated, resulting in a much cleaner depolymerization of the polysaccharides without any further acid-catalyzed degradation. The enzymatic digestibility of the steam-treated wood material for the formation of glucose was compared with that of steam-exploded wood. A more efficient route for glucose production from steam-exploded wood was found as long as the biomass-pretreated material was homogeneous and without shives.

Towards a new concept of lignin condensation in kraft pulping. Initial results.

In kraft pulping, a comparatively large amount of lignin remains in the fibres after the cook. Based on various analytical techniques for lignin, such as thioacidolysis, GPC and NMR, it is suggested that lignin condensation reactions take place during the cook. The reaction seems, however, not to involve ionic intermediates as has been suggested previously but rather a one-electron mechanism with elemental sulphur as the initiator. Support for such a reaction has been found through kraft cooks in the presence of an added phenol, 2,6-xylenol, as well as through NMR analysis of kraft lignin. It was found that the added phenol is incorporated in the pulp lignin with linkages indicative of radical coupling. Furthermore, kraft lignin was found to contain substantial amounts of chemically linked fatty acids. By complementary analyses of sulphur and polysulphide in an industrial black liquor, the presence of these compounds throughout a kraft cook was demonstrated.

The formation of beta-beta structures in lignin biosynthesis--are there two different pathways?

Based on results from 2D NMR studies, both pinoresinol and secoisolariciresinol structures were found to be present in native lignin from spruce wood as well as in spruce kraft lignin and residual kraft pulp lignin. These two structures constitute the major types of beta-beta inter-unit linkages present in spruce lignin, but their formation in the lignin polymer may follow different pathways leading to their different bonding patterns with the rest of the lignin polymer. The mechanisms involved are discussed.

Polymerization of monolignols by redox shuttle-mediated enzymatic oxidation: a new model in lignin biosynthesis I.

Lignin is one of the most abundant biopolymers, and it has a complex racemic structure. It may be formed by a radical polymerization initiated by redox enzymes, but much remains unknown about the process, such as how molecules as large as enzymes can generate the compact structure of the lignified plant cell wall. We have synthesized lignin oligomers according to a new concept, in which peroxidase is never in direct contact with the lignin monomers coniferaldehyde and coniferyl alcohol. Instead, manganese oxalate worked as a diffusible redox shuttle, first being oxidized from Mn(II) to Mn(III) by a peroxidase and then being reduced to Mn(II) by a simultaneous oxidation of the lignin monomers to radicals that formed covalent linkages of the lignin type. Furthermore, a high molecular mass polymer was generated by oxidation of coniferyl alcohol by Mn(III) acetate in a dioxane and water mixture. This polymer was very similar to natural spruce wood lignin, according to its NMR spectrum. The possible involvement of a redox shuttle/peroxidase system in lignin biosynthesis is discussed.