Ewellyn A. Capanema

MWL fraction with a high concentration of lignin- carbohydrate linkages: Isolation and 2D NMR spectroscopic analysis

Abstract A preparation enriched in lignin-carbohydrate fragments (LCC-AcOH) was isolated in the course of purification of loblolly pine crude milled wood lignin (MWL). The preparation contained approximately equal amounts of lignin and carbohydrates, with high amounts of arabinose and galactose compared to their levels in wood. LCC-AcOH was investigated by 2D 1H-13C (HMQC and HMBC) correlation NMR techniques and quantitative 13C NMR. The HMQC spectra allowed direct detection of phenyl glycoside, ester and benzyl ether lignin-carbohydrates linkages in high amounts. The assignment of these structures was supported by the HMBC technique. It is noteworthy that in the ester lignin-carbohydrate linkages, a uronic acid residue was attached not to the benzylic position of lignin, as commonly believed, but to the γ-position of the side chain.

Lignin Changes after Steam Explosion and Laccase-Mediator Treatment of Eucalyptus Wood Chips

Eucalyptus globulus chips were steam exploded followed by treatment with a laccase-mediator system (LMS) under different experimental conditions. Removal of hemicelluloses and, to a lesser extent, lignin was observed. Thermogravimetic analyses of whole meal obtained from chips before and after steam explosion indicated an increase in lignin degradation temperature due to lignin condensation. In contrast, application of LMS treatment caused a reduction in lignin and polysaccharide degradation temperatures. Lignins were isolated from wood samples before and after each treatment and analyzed by 2D NMR and (13)C NMR. An increase in carboxyl and phenolic hydroxyl groups and a significant decrease in β-O-4 structures were found in steam-exploded samples. The most relevant changes observed after laccase treatment were increased secondary OH and degree of condensation.

Structural Analysis of Residual and Technical Lignins by 1H-13C Correlation 2D NMR-Spectroscopy

Summary Structural analysis was conducted on residual lignin from pine Kraft AQ pulp, Eucalyptus Kraft lignin from Eucalyptus globulus and Repap Organosolv lignin by 2D 13C-1H correlation NMR spectroscopic techniques such as HMQC sequence. These lignins contain a rather wide variety of saturated aliphatic groups. The HMQC NMR spectra of the lignins do not verify the presence of diarylmethane moieties in any lignin investigated. The type and amount of other condensed structures depend on the nature of lignin preparation. All the lignins investigated still contained β-O-4′, pino- and syringayresinol (β-β′) and phenylcoumarane (β-5′) structures. Stilbene structures were also identified. Vinyl ether structures were present only in Eucalyptus Kraft lignin. All the lignins contain α-carbonyl groups conjugated to aromatic moieties as terminal side chains rather than involving β-O-4′ structures. No coniferyl alcohol and coniferyl aldehyde type structures are detected in the lignins after pulping. The spectra of kraft lignins show some new signals, the origin of which is discussed.

Lignin structural variation in hardwood species.

A comprehensive lignin structure analysis of ten industrially relevant hardwood species is presented. Milled wood lignin (MWL) was isolated from each species using a modified protocol and all milled wood lignin preparations were analyzed through quantitative (13)C NMR spectroscopy, elemental analysis, methoxyl analysis, sugar analysis, and nitrobenzene oxidation. Nitrobenzene oxidation and ozonation were carried out on extractive-free wood, alkali-extracted wood, milled wood lignin, and alkali-extracted lignin. Milled wood lignin isolated by the modified protocol was found to be representative of the total lignin in alkali-extracted wood. Significant variations in lignin structures, such as syringylpropane/guaiacylpropane ratio (S/G ratio), arylglycerol-β-aryl ether (β-O-4), degree of condensation, and elemental and methoxyl contents, were found among the hardwood species studied. These structural variations among species appear to be correlated to a single factor, the syringyl/guaiacyl ratio. A new method to predict the S/G ratio of total lignin in wood was developed, using a calibration line established by the syringaldehyde/vanillin (S/V) ratio (nitrobenzene oxidation) and the S/G ratio ((13)C NMR) of milled wood lignin (MWL).

Biobleaching of pulp with dioxygen in the laccase-mediator system — reaction mechanisms for degradation of residual lignin

Abstract Pine Kraft-AQ pulp was biobleached with pressurized dioxygen at 40°C in laccase-mediator system (LMS), i.e. in acetate buffer (pH 4.5) containing Coriolus-laccase and 1-hydroxy-benzotriazole (HOBT), the latter being as a mediator. The LMS-treatment was followed by alkaline extraction (E) under standard conditions. The structures of the residual lignins before and after the biobleaching did not differ appreciably. This indicates that only a part of the residual lignin in the pulp undergoes oxidative degradation in the LMS treatment. In contrast, the treatment resulted in strong changes in the structure of the lignin isolated from E-effluents. The 2D HMQC (1H13C correlation) spectra showed the disappearance of β-O-4′, β-β′ and β-5′ bonds in the structure of the alkaline soluble lignin (ASL) from E-effluents, which are present in the 2D spectrum of the original residual lignin (RKL). In addition, the spectra exhibited new signals that are assigned to ArCOOH in biphenyl (5-5′) moieties. This implies that oxidative cleavage of side chains plays an important role in the delignification of pulp. The NMR studies also indicated that intensive degradation of aromatic ring has occurred in the biobleaching. However, premethylation of neither benzyl alcohol nor phenolic hydroxyl groups of the residual lignin in pulp before the biobleaching affected the rate of delignification. The latter indicates that phenolic moieties participate not only in oxidative degradation but also dehydrogenative polymerization reactions in the biobleaching. This is consistent with an appreciable increase in the proportion of fractions with higher molecular mass in lignin isolated from E-effluents.

NMR studies on Fraser fir Abies fraseri (Pursh) Poir. lignins

Abstract The composition of mature, juvenile uninfested and juvenile infested Fraser fir wood (Rotholz) and the structures of lignins isolated from these woods were elucidated to verify differences between juvenile and mature wood and the effect of balsam woolly adelgid (BWA) infestation. Milled wood lignin (MWL) isolated from mature, juvenile and Rotholz wood were comprehensively characterized using heteronuclear multiple quantum coherence (HMQC) and quantitative 13C NMR techniques. The Rotholz wood was found to have ∼13% higher lignin content and more than five-fold the amount of galactans than that of the uninfested wood. Rotholz lignin possesses higher amounts of p-hydroxyphenyl units and aliphatic OH groups and a lower amount of alkyl-O-alkyl linkages and dibenzodioxocin moieties. The degree of condensation of the Rotholz lignin was rather similar to that of normal wood. Only small differences in the structure of mature and juvenile wood components were found.

On the Quantification of Lignin Hydroxyl Groups With 31P and 13C NMR Spectroscopy

Factors affecting the accuracy of the analysis of lignin hydroxyl and carboxyl groups with 31P NMR have been further elucidated. Two modifications of 31P NMR analysis of lignin, namely the protocols using 1,3,2-dioxaphospholane (PR-I) and 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane (PR-II) as phosphorylation reagents with different internal standards, were studied. The previous 31P NMR standard protocol with PR-II underestimated OH groups by about 30%, whereas the 31P NMR standard protocol with PR-I tended to produce overestimated data. It has been shown that cholesterol is not an appropriate internal standard, resulting in underestimated values for OH groups due to incomplete baseline resolution. The best internal standard has been found to be endo-N-hydroxy-5-norbornene-2,3-dicarboximide. Strong care should be taken related to the stability of the internal standards to avoid inflated results due to IS degradation. Under modified optimized conditions, both methods show a good correlation with the 13C NMR protocol in the quantification of hydroxyl groups as average, with the variability between the methods in the range of 5–15%. However, the 31P NMR protocols report COOH content that is twice as low as that of 13C NMR data. Finally, the best approach for the use of the 31P and 13C NMR methods in lignin analysis is discussed.

Comprehensive structural analysis of biorefinery lignins with a quantitative 13C NMR approach

The advance in analytical methodology is critical for the progress in the biorefinery and lignin commercialization. This paper reports a comprehensive approach (more than 30 common structural characteristics along with moieties specific for various lignin types) for the analysis of biorefinery lignins with quantitative 13C NMR spectroscopy, which has been demonstrated to be significantly different from the analysis of native lignins. Experimentals required for high precision NMR spectra are highlighted. The statistic data allowed for evaluating the accuracy in the quantification of different lignin units for the first time. The analysis of various lignins originated from the key biorefinery processes of different types of biomass clearly demonstrated that, in general, lignin degradation was always accompanied with decreases in aliphatic OH (primary and especially secondary ones), oxygenated aliphatic moieties, specifically β-O-4 units, and increasing amounts of phenolic OH, COOR, saturated aliphatic moieties and the degree of condensation. However, the differences in the quantity of different functionalities between the lignins investigated were very significant. Hardwood steam explosion lignins were the less degraded ones whereas aspen kraft lignin underwent the most severe structural modification. Finally, this report presents a comprehensive database on the structure of reference biorefinery lignins that is of primary importance for their commercialization.

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.

Isolation and Analysis of Lignin–Carbohydrate Complexes Preparations with Traditional and Advanced Methods: A Review

Abstract This review discusses various methods for the isolation and characterization of lignin–carbohydrate complexes (LCC) with a focus on the structures and quantities of the main linkages between lignin and carbohydrates. The advantages and limitations of different wet chemistry methods and nuclear magnetic resonance (NMR) spectroscopic techniques are indicated, and the optimal use of these techniques is suggested. The identification and quantification of the main LCC linkages in softwoods and hardwoods has recently become possible due to the application of high-resolution quantitative two-dimensional and 13 C NMR techniques in the analysis of LCC. However, the NMR data should be complemented with analytical information generated by appropriate wet chemistry techniques, which provide critical insights into the carbohydrate sites involved in LCC linkages. Achievements in the LCC analysis are summarized, and important issues to be addressed in further studies are indicated.