Mikhail Balakshin - Academia.edu (original) (raw)

Papers by Mikhail Balakshin

Research paper thumbnail of Derivatives of native lignin

Research paper thumbnail of Isolation and Analysis of Lignin-Carbohydrate Complexes (LCC) Preparations with Traditional and Advanced Methods

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

Journal of Wood Chemistry and Technology, 2015

Factors affecting the accuracy of the analysis of lignin hydroxyl and carboxyl groups with 31 P N... more Factors affecting the accuracy of the analysis of lignin hydroxyl and carboxyl groups with 31 P NMR have been further elucidated. Two modifications of 31 P 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 31 P NMR standard protocol with PR-II underestimated OH groups by about 30%, whereas the 31 P 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 13 C NMR protocol in the quantification of hydroxyl groups as average, with the variability between the methods in the range of 5-15%. However, the 31 P NMR protocols report COOH content that is twice as low as that of 13 C NMR data. Finally, the best approach for the use of the 31 P and 13 C NMR methods in lignin analysis is discussed.

Research paper thumbnail of Recent Advances in the Isolation and Analysis of Lignins and Lignin–Carbohydrate Complexes

Characterization of Lignocellulosic Materials, 2008

Research paper thumbnail of Isolation and Analysis of Lignin–Carbohydrate Complexes Preparations with Traditional and Advanced Methods

Studies in Natural Products Chemistry, 2014

Research paper thumbnail of Quantification of lignin–carbohydrate linkages with high-resolution NMR spectroscopy

Planta, 2011

A quantitative approach to characterize lignincarbohydrate complex (LCC) linkages using a combina... more A quantitative approach to characterize lignincarbohydrate complex (LCC) linkages using a combination of quantitative 13 C NMR and HSQC 2D NMR techniques has been developed. Crude milled wood lignin (MWLc), LCC extracted from MWLc with acetic acid (LCC-AcOH) and cellulolytic enzyme lignin (CEL) preparations were isolated from loblolly pine (Pinus taeda) and white birch (Betula pendula) woods and characterized using this methodology on a routine 300 MHz NMR spectrometer and on a 950 MHz spectrometer equipped with a cryogenic probe. Structural variations in the pine and birch LCC preparations of different types (MWL, CEL and LCC-AcOH) were elucidated. The use of the high field NMR spectrometer equipped with the cryogenic probe resulted in a remarkable improvement in the resolution of the LCC signals and, therefore, is of primary importance for an accurate quantification of LCC linkages. The preparations investigated showed the presence of different amounts of benzyl ether, c-ester and phenyl glycoside LCC bonds. Benzyl ester moieties were not detected. Pine LCC-AcOH and birch MWLc preparations were preferable for the analysis of phenyl glycoside and ester LCC linkages in pine and birch, correspondingly, whereas CEL preparations were the best to study benzyl ether LCC structures. The data obtained indicate that pinewood contains higher amounts of benzyl ether LCC linkages, but lower amounts of phenyl glycoside and c-ester LCC moieties as compared to birch wood.

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

Holzforschung, 2000

A preparation enriched in lignin-carbohydrate fragments (LCC-AcOH) was isolated in the course of ... more 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 1 H-13 C (HMQC and HMBC) correlation NMR techniques and quantitative 13 C 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 gposition of the side chain.

Research paper thumbnail of Inhibition of cellulase, xylanase and β-glucosidase activities by softwood lignin preparations

Journal of Biotechnology, 2006

The conversion of lignocellulosic biomass to fuel ethanol typica11y involves a disruptive pretrea... more The conversion of lignocellulosic biomass to fuel ethanol typica11y involves a disruptive pretreatment process followed by enzyme-catalyzed hydrolysis of the cellulose and hemicellulose components to fermentable sugars. Attempts to improve process economics include protein engineering of cellulases, xylanases and related hydrolases to improve their specific activity or stability. However, it is recognized that enzyme performance is reduced during lignocellulose hydrolysis by interaction with lignin or lignin-carbohydrate complex (LCC), so the selection or engineering of enzymes with reduced lignin interaction offers an alternative means of enzyme improvement. This study examines the inhibition of seven cellulase preparations, three xylanase preparations and a ~-glucosidase preparation by two purified, particulate lignin preparations derived from softwood using an organosolv pretreatment process followed by enzymatic hydrolysis. The two lignin preparations had similar particle sizes and surface areas but differed significantly in other physical properties and in their chemical compositions determined by a 2D correlation HSQC NMR technique and quantitative !3C NMR spectroscopy. The various cellulases differed by up to 3.5-fold in their inhibition by lignin, while the xylanases showed less variability (:::: 1.7-fold). Of all the enzymes tested, ~-glucosidase was least affected by lignin.

Research paper thumbnail of Chemical properties in CAD-deficient pine and their effect on pulping

Incremental cores of 200 selected trees were collected from a 10 years old open pollinated family... more Incremental cores of 200 selected trees were collected from a 10 years old open pollinated family trail growing under two nutrition treatment in Scotland County, North Carolina. We analyzed wood density, chemical and pulping properties. Analysis of the samples with ...

Research paper thumbnail of How Well Do MWL and CEL Preparations Represent the Whole Hardwood Lignin?

Journal of Wood Chemistry and Technology, 2014

Hardwood lignin preparations were isolated using classical milled wood lignin (MWL) and celluloly... more 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.

Research paper thumbnail of Oxidative Ammonolysis of Technical Lignins. Part 4. Effects of the Ammonium Hydroxide Concentration and pH

Journal of Wood Chemistry and Technology, 2006

The effects of ammonium hydroxide concentration and pH on the kinetics and reaction mechanism of ... more The effects of ammonium hydroxide concentration and pH on the kinetics and reaction mechanism of oxidative ammonolysis of Repap organosolv lignin were studied. The reactions were carried out at 1008C with an oxygen pressure of 8 bar (116 psi) and 0.4-1.6 M [NH 4 OH] and 9 -12.7 pH. The resulting N-modified lignins were analyzed for elemental composition and methoxyl group content. An increase in ammonium hydroxide concentration increased the rate of nitrogen incorporation, oxygen consumption, CO 2 formation, and lignin dissolution. The rate of nitrogen incorporation was 0.5 order with respect to NH 4 OH concentration. The amount of oxygen consumed, oxygen incorporated into the lignin, CO 2 formed, and OMe groups eliminated per mole of nitrogen incorporated decreased with increasing ammonium hydroxide concentration indicating that the increase in [NH 4 OH] accelerated nitrogen incorporation more than lignin oxidation. The dependence of the rate of nitrogen incorporation on the reaction pH went through a maximum leading to the This article is dedicated to the memory of Josef S. Gratzl.

Research paper thumbnail of Polyoxometalates as mediators in the laccase catalyzed delignification

Journal of Molecular Catalysis B: Enzymatic, 2001

The polyoxometalate (POM)-laccase catalytic system was applied for the first time to aerobic deli... more The polyoxometalate (POM)-laccase catalytic system was applied for the first time to aerobic delignification of kraft pulps at moderate (40-60 • C) temperatures. Laccase was found to readily catalyze the re-oxidation of different kinds of polyoxometalates, including those, which cannot be re-oxidized by dioxygen even at high temperatures (PMo 11 V 1 , SiW 11 V 1 , etc.). This allows a sequence of catalytic redox cycles similar to that in the laccase-mediator system (LMS) where electrons are transferred from the substrate (lignin) via POM and laccase to oxygen. Results obtained showed that the POM-laccase system could decrease κ number of eucalypt kraft pulp from 13.7 to 8.5 though the reaction rate is relatively slow. Among different POM used, SiW 11 V showed the best results. The effect of the process variables on the delignification was studied. The best results in delignification of eucalypt pulp were obtained at 60 • C, oxygen pressure of 5 bar, pH 6.3, SiW 11 V concentration of 4.2 mM and laccase concentration of 0.65 U/ml. The reaction temperature appears to be one of the crucial factors in the achievement of a delignification rate acceptable for practical application.

Research paper thumbnail of Elucidation of the Structures of Residual and Dissolved Pine Kraft Lignins Using an HMQC NMR Technique

Journal of Agricultural and Food Chemistry, 2003

Comparative studies on the structures of residual and dissolved lignins isolated from pine kraft ... more Comparative studies on the structures of residual and dissolved lignins isolated from pine kraft pulp and pulping liquor have been undertaken using the 1 H-13 C HMQC NMR technique, GPC, and sugar analysis to elucidate the reaction mechanisms in kraft pulping and the lignin reactivity. A modified procedure for the isolation of enzymatic residual lignins has resulted in an appreciable decrease in protein contaminants in the residual lignin preparations (N content < 0.2%). The very high dispersion of HMQC spectra allows identification of different lignin moieties, which signals appear overlapped in 1D 13 C NMR spectra. Elucidation of the role of condensation reactions indicates that an increase in the degree of lignin condensation during pulping results from accumulation of original condensed lignin moieties rather than from the formation of new alkyl-aryl structures. Among aryl-vinyl type moieties, only stilbene structures are accumulated in lignin in appreciable amounts. Benzyl ether lignin-carbohydrate bonds involving primary hydroxyl groups of carbohydrates have been detected in residual and dissolved lignin preparations. Structures of the R-hydroxyacid type have been postulated to be among the important lignin degradation products in kraft pulping. The effect of the isolation method on the lignin structure and differences between the residual and dissolved lignins are discussed.

Research paper thumbnail of An improved procedure for isolation of residual lignins from hardwood kraft pulps

Holzforschung, 2000

Residual lignin preparations were isolated from birch, aspen and Eucalyptus grandis kraft pulp by... more Residual lignin preparations were isolated from birch, aspen and Eucalyptus grandis kraft pulp by enzymatic hydrolysis of the pulps with cellulase:hemicellulase mixture. Residual lignin preparations were characterized by investigation of nitrogen content, carbohydrate composition and molecular mass distribution. The use of enzyme with high activity and optimization of enzyme charge resulted in significant decrease in protein contaminants in residual lignin preparations as compared to previously published results. A second order law correlation between enzyme mass charge and nitrogen content in birch residual lignin preparations indicates a strong effect of enzyme charge on the amount of protein contaminants. However, the enzyme charge in the range studied does not appreciably affect either the yields of the residual lignin preparations or percentage and composition of carbohydrates in these preparations. The optimal enzyme charge was highest for Eucalyptus grandis pulp and lowest for birch pulp. It has been suggested that a significant part of the hardwood residual lignin in pulps, especially in E. grandis pulp, consists of low molecular mass lignin fragments bonded to pulp carbohydrates. Higher amount of glucose in E. grandis residual lignin preparation compared to those from birch and aspen implied a higher frequency of lignin-cellulose bonds in eucalypt pulp. Different fractions of birch residual lignin have rather similar molecular mass distribution, which was not affected by the charge of the enzyme. Eucalypt residual lignin preparation had higher molecular mass than birch residual lignin. a This paper was partially present at the 7 th Brazilian Symposium on the Chemistry of Lignin and Other Wood Components, Belo Horizonte, MG, Brazil, 2001. pp. 61-68. Isolation of hardwood residual lignins 465

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

Holzforschung, 2000

Structural analysis was conducted on residual lignin from pine Kraft AQ pulp, Eucalyptus Kraft li... more Structural analysis was conducted on residual lignin from pine Kraft AQ pulp, Eucalyptus Kraft lignin from Eucalyptus globulus and Repap Organosolv lignin by 2D 13 C-1 H 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 b-O-4', pino-and syringayresinol (b-b') and phenylcoumarane (b-5') structures. Stilbene structures were also identified. Vinyl ether structures were present only in Eucalyptus Kraft lignin. All the lignins contain a-carbonyl groups conjugated to aromatic moieties as terminal side chains rather than involving b-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.

Research paper thumbnail of Dion Chen-Loung Chen (1930–2009)

Research paper thumbnail of Structural analysis of hardwood native lignins by quantitative 13C NMR spectroscopy

Holzforschung

Milled wood lignins from alkaline pretreated wood with very low sugar content and a wide range of... more Milled wood lignins from alkaline pretreated wood with very low sugar content and a wide range of syringyl-to-guaiacyl (S/G) ratio between 1.2 and 3.0 were isolated from 12 industrially valuable hardwood (HW) species. The lignin preparations were investigated by means of a comprehensive 13C nuclear magnetic resonance (NMR) methodology to address the possibilities and limitations of this approach for HW native lignins and to estimate the structural variations within HW lignins. Good correlations were found for different independent methods for the quantification of major lignin moieties. The results were reliable at the C6 level and not only for relative comparison. The correlation was good between methoxyl group determinations by wet chemistry and those by 13C NMR spectroscopy. The limitations of the 13C NMR method were also pointed out. The differences in the S/G ratios can be large, but other structural deviations are less significant. Strong correlations between the S/G ratios an...

Research paper thumbnail of Bioenergy Research: Advances and Applications

Research paper thumbnail of EFFECT OF HARDWOODS CHARACTERISTICS ON KRAFT PULPING PROCESS: EMPHASIS ON LIGNIN STRUCTURE

…, 2011

In an attempt to explain variations in delignification behaviors among different hardwood species... more In an attempt to explain variations in delignification behaviors among different hardwood species, the kraft pulping delignification rates of Eucalyptus urograndis, E. nitens, E. globulus, sweet gum, maple, red oak, birch, red alder, cottonwood, and acacia were ...

Research paper thumbnail of Label-free in situ imaging of lignification in the cell wall of low lignin transgenic Populus trichocarpa

Planta, 2009

Chemical imaging by confocal Raman microscopy has been used for the visualization of the cellulos... more Chemical imaging by confocal Raman microscopy has been used for the visualization of the cellulose and lignin distribution in wood cell walls. Lignin reduction in wood can be achieved by, for example, transgenic suppression of a monolignol biosynthesis gene encoding 4-coumarate-CoA ligase (4CL). Here, we use confocal Raman microscopy to compare ligniWcation in wild type and lignin-reduced 4CL transgenic Populus trichocarpa stem wood with spatial resolution that is sub-m. Analyzing the lignin Raman bands in the spectral region between 1,600 and 1,700 cm ¡1 , diVerences in lignin signal intensity and localization are mapped in situ. Transgenic reduction of lignin is particularly pronounced in the S2 wall layer of Wbers, suggesting that such transgenic approach may help overcome cell wall recalcitrance to wood sacchariWcation. Spatial heterogeneity in the lignin composition, in particular with regard to ethylenic residues, is observed in both samples.

Research paper thumbnail of Derivatives of native lignin

Research paper thumbnail of Isolation and Analysis of Lignin-Carbohydrate Complexes (LCC) Preparations with Traditional and Advanced Methods

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

Journal of Wood Chemistry and Technology, 2015

Factors affecting the accuracy of the analysis of lignin hydroxyl and carboxyl groups with 31 P N... more Factors affecting the accuracy of the analysis of lignin hydroxyl and carboxyl groups with 31 P NMR have been further elucidated. Two modifications of 31 P 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 31 P NMR standard protocol with PR-II underestimated OH groups by about 30%, whereas the 31 P 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 13 C NMR protocol in the quantification of hydroxyl groups as average, with the variability between the methods in the range of 5-15%. However, the 31 P NMR protocols report COOH content that is twice as low as that of 13 C NMR data. Finally, the best approach for the use of the 31 P and 13 C NMR methods in lignin analysis is discussed.

Research paper thumbnail of Recent Advances in the Isolation and Analysis of Lignins and Lignin–Carbohydrate Complexes

Characterization of Lignocellulosic Materials, 2008

Research paper thumbnail of Isolation and Analysis of Lignin–Carbohydrate Complexes Preparations with Traditional and Advanced Methods

Studies in Natural Products Chemistry, 2014

Research paper thumbnail of Quantification of lignin–carbohydrate linkages with high-resolution NMR spectroscopy

Planta, 2011

A quantitative approach to characterize lignincarbohydrate complex (LCC) linkages using a combina... more A quantitative approach to characterize lignincarbohydrate complex (LCC) linkages using a combination of quantitative 13 C NMR and HSQC 2D NMR techniques has been developed. Crude milled wood lignin (MWLc), LCC extracted from MWLc with acetic acid (LCC-AcOH) and cellulolytic enzyme lignin (CEL) preparations were isolated from loblolly pine (Pinus taeda) and white birch (Betula pendula) woods and characterized using this methodology on a routine 300 MHz NMR spectrometer and on a 950 MHz spectrometer equipped with a cryogenic probe. Structural variations in the pine and birch LCC preparations of different types (MWL, CEL and LCC-AcOH) were elucidated. The use of the high field NMR spectrometer equipped with the cryogenic probe resulted in a remarkable improvement in the resolution of the LCC signals and, therefore, is of primary importance for an accurate quantification of LCC linkages. The preparations investigated showed the presence of different amounts of benzyl ether, c-ester and phenyl glycoside LCC bonds. Benzyl ester moieties were not detected. Pine LCC-AcOH and birch MWLc preparations were preferable for the analysis of phenyl glycoside and ester LCC linkages in pine and birch, correspondingly, whereas CEL preparations were the best to study benzyl ether LCC structures. The data obtained indicate that pinewood contains higher amounts of benzyl ether LCC linkages, but lower amounts of phenyl glycoside and c-ester LCC moieties as compared to birch wood.

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

Holzforschung, 2000

A preparation enriched in lignin-carbohydrate fragments (LCC-AcOH) was isolated in the course of ... more 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 1 H-13 C (HMQC and HMBC) correlation NMR techniques and quantitative 13 C 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 gposition of the side chain.

Research paper thumbnail of Inhibition of cellulase, xylanase and β-glucosidase activities by softwood lignin preparations

Journal of Biotechnology, 2006

The conversion of lignocellulosic biomass to fuel ethanol typica11y involves a disruptive pretrea... more The conversion of lignocellulosic biomass to fuel ethanol typica11y involves a disruptive pretreatment process followed by enzyme-catalyzed hydrolysis of the cellulose and hemicellulose components to fermentable sugars. Attempts to improve process economics include protein engineering of cellulases, xylanases and related hydrolases to improve their specific activity or stability. However, it is recognized that enzyme performance is reduced during lignocellulose hydrolysis by interaction with lignin or lignin-carbohydrate complex (LCC), so the selection or engineering of enzymes with reduced lignin interaction offers an alternative means of enzyme improvement. This study examines the inhibition of seven cellulase preparations, three xylanase preparations and a ~-glucosidase preparation by two purified, particulate lignin preparations derived from softwood using an organosolv pretreatment process followed by enzymatic hydrolysis. The two lignin preparations had similar particle sizes and surface areas but differed significantly in other physical properties and in their chemical compositions determined by a 2D correlation HSQC NMR technique and quantitative !3C NMR spectroscopy. The various cellulases differed by up to 3.5-fold in their inhibition by lignin, while the xylanases showed less variability (:::: 1.7-fold). Of all the enzymes tested, ~-glucosidase was least affected by lignin.

Research paper thumbnail of Chemical properties in CAD-deficient pine and their effect on pulping

Incremental cores of 200 selected trees were collected from a 10 years old open pollinated family... more Incremental cores of 200 selected trees were collected from a 10 years old open pollinated family trail growing under two nutrition treatment in Scotland County, North Carolina. We analyzed wood density, chemical and pulping properties. Analysis of the samples with ...

Research paper thumbnail of How Well Do MWL and CEL Preparations Represent the Whole Hardwood Lignin?

Journal of Wood Chemistry and Technology, 2014

Hardwood lignin preparations were isolated using classical milled wood lignin (MWL) and celluloly... more 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.

Research paper thumbnail of Oxidative Ammonolysis of Technical Lignins. Part 4. Effects of the Ammonium Hydroxide Concentration and pH

Journal of Wood Chemistry and Technology, 2006

The effects of ammonium hydroxide concentration and pH on the kinetics and reaction mechanism of ... more The effects of ammonium hydroxide concentration and pH on the kinetics and reaction mechanism of oxidative ammonolysis of Repap organosolv lignin were studied. The reactions were carried out at 1008C with an oxygen pressure of 8 bar (116 psi) and 0.4-1.6 M [NH 4 OH] and 9 -12.7 pH. The resulting N-modified lignins were analyzed for elemental composition and methoxyl group content. An increase in ammonium hydroxide concentration increased the rate of nitrogen incorporation, oxygen consumption, CO 2 formation, and lignin dissolution. The rate of nitrogen incorporation was 0.5 order with respect to NH 4 OH concentration. The amount of oxygen consumed, oxygen incorporated into the lignin, CO 2 formed, and OMe groups eliminated per mole of nitrogen incorporated decreased with increasing ammonium hydroxide concentration indicating that the increase in [NH 4 OH] accelerated nitrogen incorporation more than lignin oxidation. The dependence of the rate of nitrogen incorporation on the reaction pH went through a maximum leading to the This article is dedicated to the memory of Josef S. Gratzl.

Research paper thumbnail of Polyoxometalates as mediators in the laccase catalyzed delignification

Journal of Molecular Catalysis B: Enzymatic, 2001

The polyoxometalate (POM)-laccase catalytic system was applied for the first time to aerobic deli... more The polyoxometalate (POM)-laccase catalytic system was applied for the first time to aerobic delignification of kraft pulps at moderate (40-60 • C) temperatures. Laccase was found to readily catalyze the re-oxidation of different kinds of polyoxometalates, including those, which cannot be re-oxidized by dioxygen even at high temperatures (PMo 11 V 1 , SiW 11 V 1 , etc.). This allows a sequence of catalytic redox cycles similar to that in the laccase-mediator system (LMS) where electrons are transferred from the substrate (lignin) via POM and laccase to oxygen. Results obtained showed that the POM-laccase system could decrease κ number of eucalypt kraft pulp from 13.7 to 8.5 though the reaction rate is relatively slow. Among different POM used, SiW 11 V showed the best results. The effect of the process variables on the delignification was studied. The best results in delignification of eucalypt pulp were obtained at 60 • C, oxygen pressure of 5 bar, pH 6.3, SiW 11 V concentration of 4.2 mM and laccase concentration of 0.65 U/ml. The reaction temperature appears to be one of the crucial factors in the achievement of a delignification rate acceptable for practical application.

Research paper thumbnail of Elucidation of the Structures of Residual and Dissolved Pine Kraft Lignins Using an HMQC NMR Technique

Journal of Agricultural and Food Chemistry, 2003

Comparative studies on the structures of residual and dissolved lignins isolated from pine kraft ... more Comparative studies on the structures of residual and dissolved lignins isolated from pine kraft pulp and pulping liquor have been undertaken using the 1 H-13 C HMQC NMR technique, GPC, and sugar analysis to elucidate the reaction mechanisms in kraft pulping and the lignin reactivity. A modified procedure for the isolation of enzymatic residual lignins has resulted in an appreciable decrease in protein contaminants in the residual lignin preparations (N content < 0.2%). The very high dispersion of HMQC spectra allows identification of different lignin moieties, which signals appear overlapped in 1D 13 C NMR spectra. Elucidation of the role of condensation reactions indicates that an increase in the degree of lignin condensation during pulping results from accumulation of original condensed lignin moieties rather than from the formation of new alkyl-aryl structures. Among aryl-vinyl type moieties, only stilbene structures are accumulated in lignin in appreciable amounts. Benzyl ether lignin-carbohydrate bonds involving primary hydroxyl groups of carbohydrates have been detected in residual and dissolved lignin preparations. Structures of the R-hydroxyacid type have been postulated to be among the important lignin degradation products in kraft pulping. The effect of the isolation method on the lignin structure and differences between the residual and dissolved lignins are discussed.

Research paper thumbnail of An improved procedure for isolation of residual lignins from hardwood kraft pulps

Holzforschung, 2000

Residual lignin preparations were isolated from birch, aspen and Eucalyptus grandis kraft pulp by... more Residual lignin preparations were isolated from birch, aspen and Eucalyptus grandis kraft pulp by enzymatic hydrolysis of the pulps with cellulase:hemicellulase mixture. Residual lignin preparations were characterized by investigation of nitrogen content, carbohydrate composition and molecular mass distribution. The use of enzyme with high activity and optimization of enzyme charge resulted in significant decrease in protein contaminants in residual lignin preparations as compared to previously published results. A second order law correlation between enzyme mass charge and nitrogen content in birch residual lignin preparations indicates a strong effect of enzyme charge on the amount of protein contaminants. However, the enzyme charge in the range studied does not appreciably affect either the yields of the residual lignin preparations or percentage and composition of carbohydrates in these preparations. The optimal enzyme charge was highest for Eucalyptus grandis pulp and lowest for birch pulp. It has been suggested that a significant part of the hardwood residual lignin in pulps, especially in E. grandis pulp, consists of low molecular mass lignin fragments bonded to pulp carbohydrates. Higher amount of glucose in E. grandis residual lignin preparation compared to those from birch and aspen implied a higher frequency of lignin-cellulose bonds in eucalypt pulp. Different fractions of birch residual lignin have rather similar molecular mass distribution, which was not affected by the charge of the enzyme. Eucalypt residual lignin preparation had higher molecular mass than birch residual lignin. a This paper was partially present at the 7 th Brazilian Symposium on the Chemistry of Lignin and Other Wood Components, Belo Horizonte, MG, Brazil, 2001. pp. 61-68. Isolation of hardwood residual lignins 465

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

Holzforschung, 2000

Structural analysis was conducted on residual lignin from pine Kraft AQ pulp, Eucalyptus Kraft li... more Structural analysis was conducted on residual lignin from pine Kraft AQ pulp, Eucalyptus Kraft lignin from Eucalyptus globulus and Repap Organosolv lignin by 2D 13 C-1 H 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 b-O-4', pino-and syringayresinol (b-b') and phenylcoumarane (b-5') structures. Stilbene structures were also identified. Vinyl ether structures were present only in Eucalyptus Kraft lignin. All the lignins contain a-carbonyl groups conjugated to aromatic moieties as terminal side chains rather than involving b-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.

Research paper thumbnail of Dion Chen-Loung Chen (1930–2009)

Research paper thumbnail of Structural analysis of hardwood native lignins by quantitative 13C NMR spectroscopy

Holzforschung

Milled wood lignins from alkaline pretreated wood with very low sugar content and a wide range of... more Milled wood lignins from alkaline pretreated wood with very low sugar content and a wide range of syringyl-to-guaiacyl (S/G) ratio between 1.2 and 3.0 were isolated from 12 industrially valuable hardwood (HW) species. The lignin preparations were investigated by means of a comprehensive 13C nuclear magnetic resonance (NMR) methodology to address the possibilities and limitations of this approach for HW native lignins and to estimate the structural variations within HW lignins. Good correlations were found for different independent methods for the quantification of major lignin moieties. The results were reliable at the C6 level and not only for relative comparison. The correlation was good between methoxyl group determinations by wet chemistry and those by 13C NMR spectroscopy. The limitations of the 13C NMR method were also pointed out. The differences in the S/G ratios can be large, but other structural deviations are less significant. Strong correlations between the S/G ratios an...

Research paper thumbnail of Bioenergy Research: Advances and Applications

Research paper thumbnail of EFFECT OF HARDWOODS CHARACTERISTICS ON KRAFT PULPING PROCESS: EMPHASIS ON LIGNIN STRUCTURE

…, 2011

In an attempt to explain variations in delignification behaviors among different hardwood species... more In an attempt to explain variations in delignification behaviors among different hardwood species, the kraft pulping delignification rates of Eucalyptus urograndis, E. nitens, E. globulus, sweet gum, maple, red oak, birch, red alder, cottonwood, and acacia were ...

Research paper thumbnail of Label-free in situ imaging of lignification in the cell wall of low lignin transgenic Populus trichocarpa

Planta, 2009

Chemical imaging by confocal Raman microscopy has been used for the visualization of the cellulos... more Chemical imaging by confocal Raman microscopy has been used for the visualization of the cellulose and lignin distribution in wood cell walls. Lignin reduction in wood can be achieved by, for example, transgenic suppression of a monolignol biosynthesis gene encoding 4-coumarate-CoA ligase (4CL). Here, we use confocal Raman microscopy to compare ligniWcation in wild type and lignin-reduced 4CL transgenic Populus trichocarpa stem wood with spatial resolution that is sub-m. Analyzing the lignin Raman bands in the spectral region between 1,600 and 1,700 cm ¡1 , diVerences in lignin signal intensity and localization are mapped in situ. Transgenic reduction of lignin is particularly pronounced in the S2 wall layer of Wbers, suggesting that such transgenic approach may help overcome cell wall recalcitrance to wood sacchariWcation. Spatial heterogeneity in the lignin composition, in particular with regard to ethylenic residues, is observed in both samples.