Jonas Hafrén

The distribution of acidic and esterified pectin in cambium, developing xylem and mature xylem of Pinus sylvestris

Homogalacturonans with low and high degree of methyl esterification have been immuno-localised in the cambium, differentiating xylem and mature xylem of Pinus sylvestris, by monoclonal antibodies JIM5 and JIM7. In the unlignified cambial tissue the antibodies revealed a similar distribution for acidic and esterified pectin in the compound middle lamella, ray cell walls and pit membranes. In the lignified xylem tissue, pectin was also found in the compound middle lamella, although dominantly in the methylesterified form. Lignification seemed to coincide with a decrease in the presence of acidic pectin in the compound middle lamella. Both antibodies indicated labelling in pit membranes and ray cell walls in partially and fully lignified wood fibres.

Distribution of methyl-esterified galacturonan in chemical and mechanical pulp fibers

The effects of chemithermomechanical pulping (CTMP), high-temperature (HT)-CTMP pulping, and kraft pulping on the distribution of the pectin polymer, methyl-esterified polygalacturonan have been qualitatively evaluated using immunocytochemistry. Pectin was immunolocalized using an antibody (JIM7) specific for partly methyl-esterified polygalacturonan. A fluorescent antibody was linked to JIM7 and analyzed by epifluorescence microscopy. Pectin was found in both chemithermomechanical pulps in similar uneven patterns: Some fibers showed no labeling, whereas others showed extensive labeling in patches restricted to the fiber surfaces. Pectin labeling of bleached and unbleached kraft pulps did not show any presence of pectin. Labeling was correlated to the presence of compound middle lamella tissue left on the CTMP-treated surfaces. Pectin on pulp-fiber surfaces may affect the interfiber bonds and thereby the pulp properties.

Micromorphology and topochemistry of extractives in Scots pine and Norway spruce thermomechanical pulps: a cytochemical approach

Due to the increasing demand for Norway spruce as prime raw material for high-yield pulping, recent interest has focused on Scots pine as an alternative. However, the intrinsic properties of Scots pine, particularly the high amounts of extractives and the fiber properties, have been considered a disadvantage for thermomechanical pulping. A study was therefore conducted on the variations in the spatial distribution and redistribution of lipophilic extractives in spruce and pine wood and thermomechanical pulp (TMP) using cytochemical staining methods and chemical analysis. Chemical analyses showed chips from pine thinnings and sawmill slabs to contain three to five and two to three times, respectively, more extractives than found in spruce; in particular, the amount of triglycerides differed significantly. Results from staining techniques on the abundance and distribution of extractives (i.e., fats) between pine and spruce correlated with amounts detected by Fourier transform infrared spectroscopy and gel permeation chromatography. Cytochemical observations revealed information pertaining to species-specific distribution and redistribution of extractives among TMP fines and fibers and indicated the presence of a molecular film of extractives. Results indicate that the high concentrations of extractives in pine ray parenchyma are released during TMP processing and are redistributed onto the surfaces of the pulps, negatively affecting energy usage during primary refining.

Nondestructive analysis of lignin structure by NMR spectroscopy of specifically 13C-enriched lignins: Part 1. Solid state study of ginkgo wood

Summary Guaiacyl-type lignin is the major component of ginkgo lignin. Specific 13c-enrichment of α, β and γ-carbons of the guaiacylpropane side chains was achieved by administration of coniferin-[α13C], coniferin-[β-13C] and coniferin-[γ13C], respectively, to growing stems of ginkgo trees. Unenriched coniferin was administered as a control. The xylem tissues containing specifically 13C-enriched lignins or unenriched lignin were subjected to analysis by solid state NMR. Subtraction of the spectrum of tissue containing unenriched lignin from the spectra of tissues containing 13C-enriched lignins gave difference spectra exclusively assigned to the enriched side chain carbons of intact protolignin in the cell walls. From the signal intensities determined under quantitative conditions and an estimate of corresponding standard uncertainties, the percentage ranges of the major inter-unit lignin bonds originating from the β-carbon of the coniferin precursor were estimated to be: β-O-4/α-O-R (R = H, polysaccharides or lignols) including β-OH, 53% to 57%; combined β-5, β-β and β-1, 32% to 36%; coniferaldehyde end groups, 2% to 4%; and coniferyl alcohol end groups, 4% to 6%.

Formation of 13C-enriched cell-wall DHP using isolated soft xylem from Picea abies

Summary Cell-wall dehydrogenation polymers (CW-DHP) were prepared by incubating a mixture of soft xylem (differentiating xylem) from Picea abies, coniferin 13C-enriched at the side-chain β-carbon and unenriched coniferin, both with and without the addition of β-glucosidase and glucose oxidase, which causes an in situ polymerisation of the coniferyl alcohol in the cell wall. From difference solid state 13C-NMR spectra between 13C-enriched CW-DHP and unenriched CW-DHP, the bond frequencies involving specifically 13C-enriched carbon can be quantitatively determined. The sub-structures in CW-DHP prepared without the addition of β-glucosidase and glucose oxidase showed more similarity to protolignin than CW-DHP prepared with the addition of extraneous enzymes. The CW-DHP obtained without the addition of enzymes contained 36% β-O-4-derived sub-structures, 44% combined β-β, β-5 and β-1 sub-structures and 20% coniferyl alcohol/coniferaldehyd end groups. After acetone/water extraction of the CW-DHP, the content of β-β, β-5 and β-1 structures decreased by 4%, and the β-O-4 dominating peak increased by 4%. The 13C-enriched CW-DHP material can be used to study lignin reactions in a solid wood matrix, and is also a powerful system for detailed studies on in vivo lignification mechanisms and the effects on lignification conditions on lignin structure.

Silica-cast replicas for morphology studies on spruce and birch xylem

Sol-gel mineralization has been used and evaluated as a tool for morphological studies on Picea abies and Betula verrucosa. Wood specimens and a pulped spruce sample were impregnated with a silica sol-gel and subsequently heated (calcined) to condense the surfactant-templated polysilicic acid into structured mesoporous silica. During this calcination process, the wood substance and the surfactant were removed and a silica-cast replica of the sample was obtained. The sol-gel mineralization method produced replicas that were studied by environmental scanning and transmission electron microscopy (ESEM, TEM) without additional sample preparation. The calcination induced some shrinkage above the fiber level, but the ultrastructural dimensions were not discernibly affected. The silica-cast replica method may therefore be a useful tool for studies of the wood ultrastructure, including the cell-wall pore structure (microcavities), down to the nanometer level.

Proteins in Norway spruce thermomechanical pulp

Two methods of cytochemical staining using Coomassie dye and Cu+-bicinchoninic acid, respectively, showed that there are proteins in thermomechanical pulp (TMP) of Norway spruce. Protein isolated from TMP was analyzed for amino acid composition. There was about twice the amount of acidic amino acid material compared with basic amino acids, and the presence of glucosamine indicated that the isolated polypeptides also contained glycoproteins. The presence of proteins in ray cells and fiber tracheids in TMP adds to the chemical heterogeneity of the structurally complex high-yield pulp.

Excitation wavelength-specific changes in lignocellulosic autofluorescence

Lignocellulose (paper sheets of thermomechanical pulp) was irradiated with specific wavelengths in an epifluorescence microscope equipped with filter cubes with excitation and emission filters commonly used for excitation of fluorophores with blue, green, and red emissions. The irradiation was shown to affect the relative autofluorescence intensity over time. Shorter wavelength excitation (λex = 340–380 nm) induced photoyellowing that increased the autofluorescence. Filter cubes allowing irradiation at longer wavelengths (λex = 450–490 nm and λex = 515–560, respectively) caused photobleaching and a decrease in lignocellulosic autofluorescence. The increase and decrease in autofluorescence was reversible; prephotoyellowed samples could be photobleached and prephotobleached samples regained autofluorescence by shorter wavelength irradiation. Thus, the specificity of the excitation wavelength and the time-dependent autofluorescence might affect long-term imaging experiments of lignocellulose-based samples.

Antibody-based assay for galacturonan deesterification on wood pulp fibers during bleaching

Antibodies recognizing galacturonan were used in immunoassays for industrial unbleached and hydrogen peroxide-bleached chemithermomechanical pulp (CTMP). The assays were based on a colorimetric method using specific monoclonal antibody recognition of acidic and methylated homogalacturonan, respectively, on the pulp fiber surfaces. Alkaline phosphatase-conjugated antibodies were linked to the galacturonan specific antibodies, and an alkaline phosphatase substrate (p-nitrophenylphosphate) was used to develop a yellow reaction product that can be followed spectrophotometrically. Together the newly developed immunoassays were able to describe the deesterification, specifically, of surface-localized galacturonan on CTMP, induced by alkaline hydrogen peroxide bleaching. Unbleached CTMP showed relatively more labeling by methylated galacturonan recognizing antibodies, whereas bleached CTMP was relatively more labeled for acidic galacturonan. The increase in negative charge of the bleached pulp was also shown by polyelectrolyte titration; the negative surface charge was 9.3 µeq g−1 in unbleached CTMP and 21.7 µeq g−1 in bleached pulp.

Fiber- and fine fractions-derived effects on pulp quality as a result of mechanical pulp refining consistency

High-yield pulping of wood chips using low-consistency (LC) refining in combination with primary-stage high-consistency (HC) refining has previously been shown to produce paper with quality parameters (tensile strength and light-scattering coefficient) commonly targeted for newsprint with significantly less refining energy input than using only HC refining. However, questions remain on the differences in the refining action between the two refiner types and for high-yield pulping, the refiner energy demand is a crucial process parameter. Therefore, fines- and fiber-fraction development in HC and LC refining has been studied in detail using Bauer-McNett fractionation, and the respective tensile strengths of the different fractions have been compared. Quantitative and qualitative (morphological) characteristics of the isolated fine fractions have also been analyzed in detail using a newly developed automated fluorescence microscopy method and scanning electron microscopy. The results suggest the difference in LC/HC pulp properties (strength and optical) is partly derived from deviating fiber and fines morphologies and mass balances. The quality of the fines generated during HC and LC refining also differs. LC-refined pulps contain thinner fibrillar fines (thread-like) and HC-refined pulps broader fibrils such as lamellae-type fines. Flake-like fines from the outer fiber wall decreased in relative amount with energy input.