Lignin distribution in spruce (Picea abies) determined by mercurization with SEM-EDXA technique

Summary The lignin distribution between the middle lamella and the cell wall of spruce fibers has been determined by a new technique based on a mercurization of the lignin and a concomitant determination of mercury by the SEM-EDXA technique. The ratio of lignin in the middle lamella at the cell corners to the lignin in the secondary wall was 2.5±0.6 for latewood and 2.4±0.6 for earlywood. This gives a lignin content of 55–58% in the true middle lamella in the cell corners. The reactivity to mercuric acetate of different wood elements was determined in separate experiments. Fractions enriched in ray cells, middle lamella, and compression wood all reacted at the same rate as the whole wood; about one mole of mercury was incorporated per mole of lignin (C₉-unit).     

Lignin distribution in birch (Betula verrucosa) as determined by mercurization with SEM- and TEM-EDXA

Summary A new technique suitable for determination of lignin distribution in hardwoods has been developed. The technique is based on the mercurization of the aromatic nucleus of the lignin and a concomitant determination of the mercury content by the SEM- or TEM-EDXA technique. An incorporation of one mole mercury per mole lignin (C₉-unit) was obtained for the birch lignin. The concentration of lignin was found to be about 3 times higher in the cell corners of the middle lamella than in the secondary wall of the birch fibers. The cell wall of the vessels and the ray cells were almost equal in lignin concentration and had about 1.5–1.6 times higher concentration than the secondary wall of the fibers. No specific mass loss of the organically bound mercury during the electron beam bombardment was found under the conditions used in this work.     

Chemical characterization of tissue fractions from the middle lamella and secondary wall of black spruce tracheids

Summary Elemental and functional group analyses were carried out on tissue fractions from the secondary wall and middle lamella of black spruce tracheids. The secondary wall lignin was found to contain 1.7 times as much methoxyl per C₉ as the middle lamella lignin, indicating a substantial proportion of unmethylated para-hydroxyphenylpropane residues in the middle lamella. The content of carbonyl groups was at least three times larger in middle lamella lignin than in secondary wall lignin. The carboxyl content of the middle lamella was found to be about three times as large as that of the secondary wall. Elemental analyses show a higher carbon and lower oxygen content in the middle lamella than in the secondary wall.     

Heterogeneity in formation of lignin

Summary The formation of lignin in the cell wall of compression wood of Pinus thunbergii was examined by selective radio-labeling of specific structural units in the lignin and visualization of the label in the different morphological regions by microautoradiography. Deposition of lignin in the tracheid cell wall of compression wood occurred in the order: p-hydroxyphenyl, guaiacyl and syringyl lignin, which is the same order as observed in normal wood. However, the period of lignification in the compression wood was quite different from those of normal and opposite woods. The p-hydroxyphenyl units were deposited mainly in the early stage of cell wall formation in compound middle lamella in normal and opposite woods, while in compression wood, they were formed in both the compound middle lamella and the secondary wall. The most intensive lignification was observed during the formation of the S₂ layer, proceeding from the outer to inner S₂ layers for a long period in compression wood. In the normal or opposite woods, in contrast, the lignification became active after formation of S₃ had begun, then proceeded uniformly in the secondary wall and ended after a short period.A part of this report was originally presented at the 1989 International Symposium on Wood and Pulping Chemistry at Raleigh, NC, U.S.A.     

Comparative studies on lignin distribution by UV microscopy and bromination combined with EDXA

Summary In order to elucidate a previously reported discrepancy in the ratio of the lignin concentration in the middle lamella to that in the secondary wall as determined by ultraviolet (UV) microscopy and bromination combined with EDXA, the ultraviolet absorptivity of the lignin and the lignin reactivity towards bromination were compared for black spruce wood (Picea mariana Mill.). In addition, UV microscopy and EDXA techniques were applied to the determination of lignin distribution in the tracheids in order to establish the relationship between the two techniques. The results indicated that, although the ultraviolet absorptivity in different morphological regions is essentially the same, the secondary wall lignin was 1.70 times more reactive towards bromination than the middle lamella lignin. By applying the value of 1.70 as a correction to the EDXA results, the estimated lignin distribution by EDXA was in fairly good agreement with that from UV microscopy.The authors would like to thank Dr. J.-F. Revol and Dr. M. Tsuji for their assistance during this study     

A study of lignification in loblolly pine tracheids by the SEM-EDXA technique

Summary The lignification process in different morphological regions of loblolly pine tracheids was studied by the SEM-EDXA technique. Prior to S₂ layer formation, lignification was initiated in the cell corner middle lamella and compound middle lamella regions. Subsequently a rapid lignin deposition was observed in both regions, whereas secondary wall lignification was a more gradual process and initiated when the middle lamella lignin concentration was approximately 50% of maximum. Within the secondary wall, the S₁ layer is lignified first. Then, lagging just behind cell wall formation, lignification of the S₂ layer is initiated adjacent to the S₁ layer and extends toward the lumen. Finally, the S₃ layer lignified. Upon completion of lignification, the cell walls had a higher concentration of lignin in both the S₁ and S₃ layers than in the S₂ layer.This Paper is an excerpt from the Ph.D. dissertation of Shiro Saka     

Changes in structural and chemical components of wood delignified by fungi

Summary Cerrena unicolor, Ganoderma applanatum, Ischnoderma resinosum and Poria medulla-panis were associated with birch wood that had been selectively delignified in the forest. Preferential lignin degradation was not uniformly distributed throughout the decayed wood. A typical white rot causing a simultaneous removal of all cell wall components was also present. In the delignified wood, 95 to 98% of the lignin was removed as well as substantial amounts of hemicelluloses. Scanning and transmission electron microscopy were used to identify the micromorphological and ultrastructural changes that occurred in the cells during degradation. In delignified areas the compound middle lamella was extensively degraded causing a defibration of cells. The secondary wall, especially the S₂ layer, remained relatively unaltered. In simultaneously white-rotted wood all cell wall layers were progressively removed from the lumen toward the middle lamella causing erosion troughs or holes to form. Large voids filled with fungal mycelia resulted from a coalition of degraded areas. Birch wood decayed in laboratory soil-block tests was also intermittently delignified. Selective delignification, sparsely distributed throughout the wood, and a simultaneous rot resulting in the removal of all cell wall components were evident. Scanning electron microscopy appears to be an efficient technique for examining decayed wood for fungi with the capacity to selectively delignify wood.The authors would like to thank Kathy Zuzek for technical assistance and Dr. M. Larsen, Forest Prod. Lab., Madison, for identifying the sporophores of Poria medulla-panis. This research was founded in part by a grant from the USDA Forest Service, Forest Products Laboratory and from the Graduate School, University of Minnesota     

Recent progress in the chemistry and topochemistry of compression wood

Summary A review of the chemistry and topochemistry of compression wood with 200 references. Compression wood contains on the average 30% cellulose, 35–40% lignin, 10% galactan, 9% galactoglucomannan, 8% xylan, and 2% of a 1,3-glucan (laricinan). The cellulose is less crystalline, and the xylan has fewer arabinose side chains than in normal wood. The lignin is composed of guaiacylpropane and p-hydroxyphenylpropane units. It is more condensed, has a higher proportion of carbon-carbon bonds, and contains fewer arylglycerol--aryl ether structures than a normal conifer lignin. The ray cells and the primary wall of the tracheids have the same chemical composition in normal and compression woods. The galactan is largely located in the outer region of the secondary wall. Only 5–10% of the lignin in compression wood tracheids is extracellular. The middle lamella is less lignified than in normal wood, while the S₁ and inner S₂ layers have a lignin concentration of 30–40% which is twice as high as in normal wood. The lignin content of the S₂ (L) layer is equal to or higher than that of the intercellular region along the wall. The review is concluded with a brief reference to areas where present information is incomplete or lacking.A portion of an Academy Lecture of the International Academy of Wood Science, presented at the International Symposium on Wood and Pulping Chemistry (Ekmandagarna 1981), held in Stockholm, Sweden, June 9–12, 1981. Reprints of the unabridged review, published under the title Recent Progress in the Chemistry, Ultrastructure, and Formation of Compression Wood in the preprints of the symposium (SPCI Report 38, Vol. 1, p. 99–147) are available from the author. I wish to express my gratitude to my colleague Professor Robert A. Zabel for generous travel assistance     

Cellular UV-microspectrophotometric investigations on pine wood ( Pinus taeda and Pinus elliottii) delignification during biopulping with Ceriporiopsis subvermispora (Pilát) Gilbn. & Ryv. and alkaline sulfite/anthraquinone tr

Scanning UV-microspectrophotometry was used to investigate the topochemistry of lignin removal from pine wood (Pinus taeda and P. elliottii) chips during biopulping involving wood treatment with Ceriporiopsis subvermispora (Pilát) Gilbn. & Ryv. followed by alkaline sulfite/anthraquinone delignification. A delignification front starting from the lumen towards the compound middle lamella was clearly observed in micrographs recorded from individual cell wall layers of wood samples biotreated for 30 days. Lignin was removed without cell wall erosion. UV-micrographs of wood samples cooked for a short time (90 min pulping) showed that the S2 of biotreated samples are more homogeneously delignified compared to the S2 of the undecayed controls. Similarly, the compound middle lamella and cell corners are also more delignified in biotreated samples. On the other hand, UV-micrographs of samples cooked for a long time (150 min pulping at 170°C) showed that there are no significant differences in the contents of residual lignin retained in the S2 of undecayed and biotreated wood samples.     

Seasonal changes in lignin distribution during tracheid development in Pinus radiata D. Don

Summary Lignin distribution in developing tracheids of Pinus radiata was studied throughout the growth' season using quantitative interference microscopy. The pattern of lignification remained constant although the number of lignifying cells varied reaching a maximum in summer. Lignification of the secondary wall of latewood tracheids was incomplete at the onset of winter. Each stage of lignification was preceded by deposition of carbohydrates with lignification of the middle lamella starting after S1 formation and lignification of the secondary wall starting after S3 formation. Lignification of the middle lamella was completed before the start of lignin deposition in the secondary wall. In one of the trees examined, the secondary wall lignified concurrently with the middle lamella and this was associated with a low lignin concentration in the middle lamella of mature cells. The secondary wall reached a mature lignin concentration of 21–22% v/v except in one specimen containing severe compression wood which reached 28% v/v. The cell corner middle lamella reached a mature lignin concentration of 74–87% v/v.     

Topochemistry of delignification in Douglas-fir wood with soda,soda-anthraquinone and karft pulping as determined by SEM-EDXA

Summary Delignification studies on 0.5 m sections of Douglas-fir earlywood tracheids pulped by soda, soda-anthraquinone (soda/AQ) and kraft pulping processes were performed by determining bromine concentrations in various morphological regions with SEM-EDXA technique. Soda/AQ pulping was much more selective in removing lignin from the middle lamella regions than either soda or kraft pulping. However, up to 50% delignification, more lignin was removed from the secondary wall by soda or kraft, compared to soda/AQ pulping. The kinetics of lignin removal in the various morphological regions were established. Addition of AQ and sodium sulfide resulted in an earlier transition from a slow initial to a rapid bulk delignification, particularly in the middle lamella, and in an enhanced bulk delignification in the secondary wall. Anthraquinone was also found to promote residual delignification in the secondary wall, where sodium sulfide was not effective. The opposite was observed for the bulk delignification in the middle lamella, where only sodium sulfide addition improved the rate significantly. The great differences observed in the bulk delignification rates between middle lamella and secondary wall in soda pulping as well as their response to additives suggest structural differences between middle lamella and secondary wall lignins.Paper No. 6712 in the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina. The paper, an excerpt from the Ph. D. dissertation of Shiro Saka, was selected for the second place Wood Award for 1980 cosponsored by Forest Products Research Society and the Kirby Forest Industries, Inc., Houston, Texas, and presented at the American Chemical Society Annual Meeting, Las Vegas, Nevada, August, 1980     

Heterogeneity in formation of lignin—XI: An autoradiographic study of the heterogeneous formation and structure of pine lignin

Summary Selective labeling of p-hydroxyphenyl-, guaiacyl-and syringylpropane moieties in protolignin was achieved by administration of corresponding ³H-labeled monolignol glucosides to differentiating xylem of pine. The growing process of the protolignin macromolecule in the specific morphological region was visualized by application of high resolution microautoradiography to the selectively labeled wood tissue.p-Hydroxyphenyl lignin is formed mainly in the compound middle lamella and cell corner in an early stage of cell wall differentiation. There are two peaks of deposition of guaiacyl lignin in the compound middle lamella at an early stage and in the secondary wall at a late stage. The content of condensed guaiacyl units is higher in the middle lamella than in the secondary wall lignin. Syringyl lignin is formed mainly in the inner layer of the secondary wall in a late stage as a minor structural moiety. During the formation of the cell wall, protolignin grows under definite biological regulations to a heterogeneous macromolecule which consists of various structural moieties arranged in a regular manner. The origin of the heterogeneous structure was explained as a result of the biogenesis of protolignin in the cell wall.     

Evaluation of the quantitative assay of lignin distribution by SEM-EDXA-technique

Summary A method has been developed for the determination of lignin distribution in wood cell walls by studying bromine concentrations in 0.5 m sections with scanning electron microscopy coupled with energy dispersive X-ray analysis (SEM-EDXA) technique. Evaluation of backscattering, absorption and fluorescence effects on quantitative assay of lignin distribution indicated that these effects may all be ignored, if relative comparisons are made. Thus, a relative ratio of the measured Br-L X-ray intensities directly provides bromine concentration ratio, from which the ratio of lignin concentrations in different morphological regions can be estimated. Additionally, the effect of electron beam bombardment on bromine was found to be negligible. Therefore, the SEM-EDXA technique provides quantitative information of lignin distribution with relatively high accuracy. The potential and limitations of this technique were also elucidated.Paper No. 6861 in the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina. This paper is an excerpt from the Ph.D. dissertation of Shiro Saka     

The diffusion, size and location of added silver grains in the cell walls of Swedish pine, Pinus sylvestris

The size and location of silver particles in K-glycerate/AgNO₃ impregnated Swedish pine, green wood as well as high temperature dried, have been studied using TEM micrographs. The diameter of the silver particles was found to be 2–20 nm in the impregnated green wood and as large as 1000 nm (major axis) for the ellipsoid-shaped silver clusters in the impregnated dried wood. Studying the projected area of the silver particles in impregnated green wood indicated that there are a lot of particles (40%) in the compound middle lamella with fewer particles in the S₂ (6–8%), S₁ (4%) and S₃ (2%) layers. The average distance between the silver particles, 50 nm (S₂-layer), in impregnated green wood shows that the impregnant is distributed in the cell wall at the microfibrilar level. Experimental results show that the fastest diffusion path into the cell wall is from the lumen over the pit membrane through the compound middle lamella and not from the lumen through the secondary wall layer S₃. Received 11 January 1998     

Ultraviolet microspectrophotometric investigation of the distribution of lignin in Prunus jamasakura differentiated on a three-dimensional clinostat

To examine the effect of gravity on lignin content and deposition in plant cells, we used ultraviolet (UV) microspectrophotometry and chemical methods to investigate the secondary xylem of Prunus jamasakura grown on a three-dimensional (3D) clinostat, which simulates microgravity. The stem of the 3D-clinostat specimens elongated with bending and the width of their secondary phloem increased. The UV absorbance of the 3D-clinostat specimens at 278 nm was higher than that of the control specimens, which were grown on the ground, in the wood fiber cell corner middle lamella, compound middle lamella, and fiber secondary wall; the UV absorbance in the vessel secondary wall did not differ between the specimens. The lignin content in the stem, including the bark, of the 3D-clinostat specimens, as determined using an acetyl bromide method, was less than that of the control specimens. In the specimens that differentiated on a 3D clinostat, the amount of lignin in the wood fibers increased, while the proportion of the lignified xylem in the stem decreased relative to control values.     

The occurrence of p-hydroxyphenylpropane units in the middle-lamella lignin of spruce (Picea abies)

Summary A sieving technique has been developed for the separation of middle-lamella fragments. The middle-lamella fraction as well as the whole wood and compression wood from Picea abies have been analysed by nitrobenzene oxidation and acidolysis in order to determine the content of p-hydroxyphenylpropane units in the middle-lamella lignin. These analyses revealed only traces of p-hydroxyphenylpropane units in the whole wood and in the middle-lamella fraction but considerable amounts were found in compression-wood lignin. This points to the fact that middle-lamella lignin is of guaiacyl nature and that earlier results reporting high proportions of p-hydroxyphenylpropane units in the middle lamella-lignin may be due to the inclusion of compression wood in the fraction studied. The acidolysis experiments further indicate that the middle-lamella lignin has fewer uncondensed -0-4 aryl ether structures than the whole wood lignin.The skilful technical assistance of Mrs. Britta Samuelsson and Mr. Johan Lindberg is highly appreciated. The author also thanks Dr. Knut Lundquist for supplying some of the reference compounds, and Dr. Hanne-Lise Hardell for help with the microscopic pictures     

Comparison between 10,000-year old and contemporary spruce lignin

Wood from white spruce Picea glauca that had been preserved by rapid burial in lake sediments 10,000 years ago, was investigated and compared to a contemporary reference white spruce wood. The 10,000-year old sample appeared to have an intact primary cell wall and middle lamella, whereas the carbohydrate monomer distribution, and microscopic images showed that the secondary wall was at least partially removed, indicating that this structure had been selectively attacked by bacteria. The Klason lignin amount in the aged spruce was found to be 60%. The relative lignin monomer content in the aged spruce was 9% lower than that of the reference wood, showing that there were fewer β-O-4′ linkages in the aged sample. This finding was supported by SEC analysis of the thioacidolysed samples as a larger proportion of lignin oligomers were observed in the aged spruce than in the reference material. This indicates a somewhat greater number of condensed bonds in the aged spruce than in the reference spruce sample. Quantitative ¹³C NMR analysis and HSQC techniques applied on milled wood lignins (MWL) revealed no significant structural differences between the aged spruce and the reference.     

Reactions between Cr(VI) and wood and its model compounds

Wood, macromolecular and simple model compounds, were reacted with CrO₃ or K₂CrO₄ aqueous solutions. Extracted lignin, guaiacol, vanillin, vanillyl alcohol and homovanillyl alcohol were chosen as model compounds for lignin, whilst cellulose, gum Ghatti, xylan, extracted hemicellulose from pine, methyl-β-D-glucopyranoside and methyl-β-cellobioside were used as models for wood polysaccharides. The kinetics of the reduction reactions of Cr(VI) were monitored using UV-Vis spectroscopy and the results obtained for several temperatures are discussed. In general terms, wood, lignin and lignin model compounds reduced Cr(VI) faster and to a greater extent than polysaccharides or simple sugar molecules. Moreover, lignin model compounds were reduced even faster than lignin. Simple sugars showed a reduction pattern similar to that of cellulose. Extracted hemicellulose revealed to be a poorer reductant while gum Ghatti was the strongest among the polysaccharides. As expected, CrO₃ aq. behaved as a more powerfull oxidant than K₂CrO₄ aq. for these substances. Even at 100 °C, sugars or polysaccharides did not seem to be oxidised by K₂CrO₄ aq. 0.01 M. These results suggest that, because of the differences in reactivity, lignin reacts preferentially when wood is treated with Cr(VI)-containing formulations, like those which are applied in wood preservation treatments.     

Lignin distribution across tracheid cell walls of poorly lignified wood from deformed copper deficient Pinus radiata (D. Don)

Summary Lignin topochemistry of tracheid walls from a deformed, copper deficient Pinus radiata (D. Don) tree was examined by linescan and point analyses using a Scanning Electron Microscope and Energy Dispersive Spectrometry. Both opposite and compression wood had abnormal lignin distributions compared to those observed in normal wood from a straight tree. Lignin contents in the compound middle lamella were lower than lignin contents in the secondary wall in both opposite and compression wood tracheids.One of us (G. D.) held a Commonwealth Forestry Postgraduate Research Award during this study. The research was supported in part by a grant from the Reserve Bank of Australia Rural Credits Development Fund, the Pine Fund, and members of the forest industry     

Coloring characteristics of in situ lignin during heat treatment

To investigate the effects of lignin on the discoloration of Black locust (Robinia pseudoacacia) wood during heat treatment, chromatic indexes of the extractive-free wood samples are examined at different moisture contents (MC) under oxygen and nitrogen environment, respectively. The organic acids are produced during heat treatment, resulting in pH decrease in the samples. Components absorbing visible light are formed during heat treatment, and oxygen and moisture contents have obvious effects on the decrease in L*, increase in a* value, yellowness (b*) and total color difference (ΔE) of the samples. It is found that the β-5, C _α C _β unsaturated bond, the conjugated carbonyl group, quinones structures, α, β-unsaturated ketone and α-C?=?O in lignin increased after heat treatment. The formation of condensation products, the low-molecular-weight phenolic substances and the oxidation products in lignin result in the increment of the light absorption within the entire visible region.