Differences in residual lignin properties between Betula verrucosa and Eucalyptus urograndis kraft pulps

By comparing the ultrastructural features of two oxygen delignified hardwood kraft pulps (Eucalyptus urograndis and Betula verrucosa), we have demonstrated a marked difference in their residual lignin properties. In this study, properties such as crystallinity and crystal size of cellulose, molecular weights, carboxyl group contents, and carbohydrate compositions of the two kraft pulps were compared. The examined pulps were in our observations relatively similar. A significant difference, however, was observed in the size exclusion chromatography measurements, which indirectly suggested that a significant portion of residual lignin in eucalyptus pulp was associated with cellulose. Birch pulp, in contrast, exhibited a more conventional tendency for hardwood pulps: lignin mainly associated with hemicelluloses.     

Enzymatic accessibility of xylans in lignocellulosic materials

The hydrolysis of fibre-bound and isolated xylans from both birch and pine wood and kraft pulps was studied using purified xylanolytic enzymes of Trichoderma reesei. Despite high enzyme loading, the degree of hydrolysis of fibre-bound substrates did not exceed 20% of the theoretical value, apparently due to limited accessibility of the substrates. The fibre-bound xylans were as equally accessible in softwood as in hardwood pulps. The isolated xylans of wood and kraft pulps could be solubilized more extensively, with a hydrolysis yield of 50–65%. The substitution degree of the isolated xylan substrates was reflected in the different hydrolysis yields obtained by the two xylanases, with isoelectric point (pI) values of 9.0 and 5.5. On the more substituted substrates, i.e. pine kraft xylan and pine wood xylan, the two enzymes acted almost similarly, whereas on the less substituted xylan substrates, such as isolated birch kraft xylan, the pI-9.0 enzyme was more efficient. The side-group-cleaving enzymes increased only moderately the solubilization of the substrates.Correspondence to: L. Viikari     

Pulp properties and their influence on enzymatic degradability

Endoglucanase treatment of pulp for the adjustment of viscosity and the increase in pulp reactivity is a promising step in the concept for the beneficial production of dissolving pulps from paper grade pulps. To promote the commercial applicability of these enzymes, the influence of pulp properties such as carbohydrate composition, pulp type and cellulose morphology on the enzymatic degradability of a pulp was examined. High contents of hemicelluloses and lignin were shown to impair the accessibility of the cellulose to the enzymes. Due to the elevated swelling capacity of cellulose II, conversion of the cellulose morphology from I to II upon alkaline treatments showed a large increasing effect on the cellulose accessibility, and enzymatic degradability. Reactivity measurements of softwood sulfite pulps after enzymatic degradation and acid-catalyzed hydrolysis, respectively, revealed elevated reactivity for the pulp after acid treatment. This is in contrast to effects of enzyme treatments reported for CCE treated kraft pulps.     

Application of thermostable xylanase of Dictyoglomus sp. in enzymatic treatment of kraft pulps

Enzymatic treatment of pine and birch kraft pulps with a xylanase preparation from a thermophilic anaerobic bacterium Dictyoglomus sp. strain B1 was studied in order to improved pulp bleachability. Maximal solubilization of pulp xylan was obtained at 90°C and pH 6.0–7.0. The enzyme was also active in the alkaline pH range; at pH 9.0 xylan hydrolysis was decreased by only 18% from the maximum at pH 7.0. The positive effect of xylanase pretreatment at 80°C and pH 6.0 or 8.0 on bleachability of pine kraft pulp was demonstrated. The brightness was increased by two ISO units in one-stage peroxide delignification, which corresponds well to values obtained with other enzymes at lower temperatures and pH values. Thus, the Dictyoglomus xylanase is well suited for pulp treatments at elevated temperatures in neutral and alkaline conditions.Correspondence to: M. Rättö     

Application of the affinity binding of xylanases to oat-spelt xylan in the purification of endoxylanase CM-2 from Streptomyces chattanoogensis CECT 3336

bstract The use of the insoluble polysaccharides Avicel and oat-spelt xylan for the binding and subsequent purification of active xylanases from Streptomyces chattanoogensis was investigated. Maximum recovery of xylanases was achieved with oat-spelt xylan, using NaCl (2 M) to remove active protein. The application of this technique to the purification of xylanases resulted in the purification of an endoxylanase (CM-2) with high specific activity (729.5 U mg⁻¹). The properties of the purified enzyme, exhibiting activity and stability between 40 °C and 60 °C and between pH 5 and 8, suggest a potential role for both the enzyme and the rapid purification protocol in the removal of hemicelluloses from kraft pulp prior to bleaching. Received: 6 April 1998 / Accepted: 8 May 1998     

Do the non-catalytic polysaccharide-binding domains and linker regions enhance the biobleaching properties of modular xylanases?

Xylanase A (XylA) from Pseudomonas fluorescens subsp. cellulosa consists of an N-terminal non-catalytic cellulose-binding domain joined to a functionally independent C-terminal catalytic domain by a sequence rich in serine residues. Xylanase D (XylD) from Cellulomonas fimi also exhibits a modular structure comprising an N-terminal catalytic domain linked to an internal non-catalytic xylan-binding domain and a C-terminal cellulose-binding domain. To determine the importance of the non-catalytic polysaccharide-binding domains and linker sequences of XylA and XylD in relation to their capacity to hydrolyse pulp xylan and enhance bleachability, purified full-length and modified derivatives of both enzymes were incubated with a hardwood kraft pulp. Deletion of the cellulose-binding domain or linker region from XylA decreased the activity of the enzyme against pulp xylan, but had no significant effect on the capacity of the enzyme to facilitate delignification and reduce pulp kappa number. While full-length and truncated forms of XylD, lacking either the cellulose-binding or the cellulose- and xylan-binding domains, were equally effective in hydrolysing pulp xylan, enzyme derivatives containing a polysaccharide-binding domain were marginally more efficient in reducing pulp kappa number. The reduction in kappa number elicited by full-length and isolated catalytic domains of XylA and XylD was reflected in an increase in the brightness of paper handsheets derived from pretreated pulps. Thus, the polysaccharide-binding domains of XylA and XylD did not appear to confer any advantage in terms of the ability of the enzymes to improve pulp bleachability. However, XylA and XylD, which belong to different glycosyl hydrolase families, differed in their ability to hydrolyse pulp xylan and facilitate the delignification of kraft pulp. Received: 21 March 1996 / Received revision: 11 July 1996 / Accepted: 19 July 1996     

Xylanases in bleaching: From an idea to the industry

Abstract: The utilization of hemicellulases in bleaching of kraft (sulphate) pulp is considered as one of the most important new large-scale industrial applications of enzymes. This is partly due to the great potential of an environmentally safe method. This method has in a short period also proven to be economically realistic. The main enzymes needed in the enzyme-aided bleaching have been shown to belong to the group of endo-/gb-xylanases. Xylanases act mainly on the relocated, reprecipitated xylan on the surface of the pulp fibres. Enzymatic hydrolysis of this specific type of xylan renders the structure of the fibres more permeable. The hydrolysis of xylan or mannan in the inner fibre layers may also enhance the bleachability. In practical process conditions, properties of the enzymes such as substrate specificity and the pH and temperature optima are of utmost importance. The benefits obtained by enzymes are dependent on the chemical bleaching sequence used as well as on the residual lignin content of pulp. The main goals in the enzyme-aided bleaching of kraft pulps have been the reduction of consumption of chlorine chemicals in the bleaching process and consequently lowering the AOX of the effluents. Enzymes have been applied as a pretreatment both in conventional (C/D)EDED and in ECF (elementary chlorine-free) bleaching sequences. In the production of TCF (totally chlorine-free) pulps, enzymes have also been successfully used for increasing the brightness of pulp.     

Synergistic action of xylanase and mannanase improves the total hydrolysis of softwood

The impact of xylan and glucomannan hydrolysis on cellulose hydrolysis was studied on five pretreated softwood substrates with different xylan and glucomannan contents, both varying from 0.2% to 6.9%, using mixtures of purified enzymes.The supplementation of pure cellulase mixture with non-specific endoglucanase TrCel7B and xylanase TrXyn11 enhanced the hydrolysis of all substrates, except the steam pretreated spruce, by more than 50%. The addition of endo-β-mannanase increased the overall hydrolysis yield by 20-25%, liberating significantly more glucose than theoretically present in glucomannan.When supplemented together, xylanolytic and mannanolytic enzymes acted synergistically with cellulases. Moreover, a linear correlation was observed between the hydrolysis of polysaccharides, irrespective of the composition, indicating that glucomannan and xylan form a complex network of polysaccharides around the cellulosic fibres extending throughout the lignocellulosic matrix. Both hemicellulolytic enzymes are crucial as accessory enzymes when designing efficient mixtures for the total hydrolysis of lignocellulosic substrates containing both hemicelluloses.     

Family-10 and Family-11 xylanases differ in their capacity to enhance the bleachability of hardwood and softwood paper pulps

Enzyme-aided bleaching of softwood and hardwood kraft pulps by glycosyl hydrolase family-10 and -11 xylanases and a family-26 mannanase was investigated. The ability to release reducing sugar from pulp xylan and to enhance bleachability is not a characteristic shared by all xylanases. Of the six enzymes tested, two xylanases belonging to family 11 were most effective at increasing bleachability and improving final paper brightness. None of the enzymes had a deleterious effect on pulp fibre integrity. The efficiency of individual xylanases as bleach enhancers was not dependent on the source microorganism, and could not be predicted solely on the basis of the quantity or nature of products released from pulp xylan. Cooperative interactions between xylanase/xylanase and xylanase/mannanase combinations, during the pretreatment of softwood and hardwood pulps, were investigated. Synergistic effects on reducing-sugar release and kappa number reduction were elicited by a combination of two family-10 xylanases. Pretreatment of kraft pulp with mannanase A from Pseudomonas fluorescens subsp. cellulosa and any one of a number of xylanases resulted in increased release of reducing sugar and a larger reduction in kappa number than obtained with the xylanases alone, confirming the beneficial effects of family-26 mannanases on enzyme-aided bleaching of paper pulp. Received: 6 January 1997 / Received revision: 10 April 1997 / Accepted: 19 April 1997     

Effect of Residual Lignin Type and Amount on Bleaching of Kraft Pulp by Trametes versicolor

The white rot fungus Trametes (Coriolus) versicolor can delignify and brighten unbleached hardwood kraft pulp within a few days, but softwood kraft pulps require longer treatment. To determine the contributions of higher residual lignin contents (kappa numbers) and structural differences in lignins to the recalcitrance of softwood kraft pulps to biobleaching, we tested softwood and hardwood pulps cooked to the same kappa numbers, 26 and 12. A low-lignin-content (overcooked) softwood pulp resisted delignification by T. versicolor, but a high-lignin-content (lightly cooked) hardwood pulp was delignified at the same rate as a normal softwood pulp. Thus, the longer time taken by T. versicolor to brighten softwood kraft pulp than hardwood pulp results from the higher residual lignin content of the softwood pulp; possible differences in the structures of the residual lignins are important only when the lignin becomes highly condensed. Under the conditions used in this study, when an improved fungal inoculum was used, six different softwood pulps were all substantially brightened by T. versicolor. Softwood pulps whose lignin contents were decreased by extended modified continuous cooking or oxygen delignification to kappa numbers as low as 15 were delignified by T. versicolor at the same rate as normal softwood pulp. More intensive O₂ delignification, like overcooking, decreased the susceptibility of the residual lignin in the pulps to degradation by T. versicolor.     

Relationship between lignin structure and delignification degree in Pinus pinaster kraft pulps

This study examines the structure of residual and dissolved lignins from Pinus pinaster pulps obtained at different degrees of delignification by laboratory conventional kraft pulping. The cooking H factor was varied from 85 to 8049. The residual and dissolved lignin samples were characterised by elemental analysis, residual carbohydrate content, permanganate oxidation and 13C NMR spectroscopy. The reflectance factor of the pulps was also determined in order to tentatively correlate the delignification degree and residual lignin structure with the pulp colour. The obtained results confirmed that the delignification degree increases the condensation of the lignin structure, which might have an influence upon the observed increased pulp colour. The lack of selectivity of kraft pulping process in the case of more delignified pulps was also shown.     

Cross-sectional analysis of the polysaccharide composition in cellulosic fiber materials by enzymatic peeling/high-performance capillary zone electrophoresis

A combined enzymatic, chemical, and analytical approach was used to determine the cross-sectional carbohydrate composition in cellulosic fibers. The outer surface of cellulosic fibers was enzymatically removed layer-by-layer with precise quantitative control, and the monosaccharides in the peelings were subsequently analyzed by high-performance capillary electrophoresis (HPCE) after precolumn derivatization with a UV label. This method was applied to dissolving pulps and regenerated cellulose fibers, with special emphasis on the cross-sectional distribution of hemicelluloses. Commercially available enzyme solutions were used, resulting in a reproducible peeling. Significant differences were found in the hemicellulose distribution across the fiber of different dissolving pulps, dependent on both natural source (beech or spruce) and preparation process (acidic sulfite cook or prehydrolysis kraft cook). Among the dissolving pulps, beech prehydrolysis kraft pulp showed the highest enrichment of surface xylan. Similar, albeit smaller, differences were noticed between various regenerated fibers (viscose, viscose Modal, and Lyocell): a thin hemicellulose-rich outermost layer was found in all the regenerated fibers studied.     

Biokraft pulping of European black pine with Ceriporiopsis subvermispora

European black pine (Pinus nigra Arn.) chips were treated with the white-rot fungus Ceriporiopsis subvermispora for periods ranging from 20 to 100 days. The effects of pretreatment on the chemical composition of wood and kraft pulping were investigated. The results showed that fungal pretreatment reduced the lignin and extractive content of wood chips. Also, weight losses occurred. Kappa number, viscosity, and reject ratio of biokraft pulps decreased. Biokraft pulps gave better response to beating, which led to significant energy saving during refining. The tear index, burst index, and tensile index of biokraft pulps were found to be lower than those of kraft pulps. However, the tensile index and burst index of 20-day biotreated and unbeaten pulp was higher than those of kraft pulp. Also, the tear index of 20-day biotreated and beaten pulp was higher than that of kraft pulp. The brightness of biokraft pulps decreased irregularly with increasing incubation time.     

Laccase-initiated cross-linking of lignocellulose fibres using a ultra-filtered lignin isolated from kraft black liquor

In this work, the effect of Trametes pubescens laccase (TpL) used in combination with a low-molecular-weight ultra-filtered lignin (UFL) to improve mechanical properties of kraft liner pulp and chemi-thermo-mechanical pulp was studied. UFL was isolated by ultra-filtration from the kraft cooking black liquor obtained from softwood pulping. This by-product from the pulp industry contains an oligomeric lignin with almost twice the amount of free phenolic moieties than residual kraft pulp lignin. The reactivity of TpL on UFL and kraft pulp was studied by nuclear magnetic resonance spectroscopy and size exclusion chromatography. Laccase was shown to polymerise UFL and residual kraft pulp lignin in the fibres, seen by the increase in their average molecular weight and in the case of UFL as a decrease in the amount of phenolic hydroxyls. The laccase initiated cross-linking of lignin, mediated by UFL, which gives rise to more than a twofold increase in wet strength of kraft liner pulp handsheets without loosing other critical mechanical properties. Hence, this could be an interesting path to decrease mechano-sorptive creep that has been reported to lessen in extent as wet strength is given to papers. The laccase/2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) mediator system showed a greater increase in wet tensile strength of the resulting pulp sheets than the laccase/UFL system. However, other mechanical properties such as dry tensile strength, compression strength and Scott Bond internal strength were negatively affected by the laccase/ABTS system.     

Fate of Residual Lignin during Delignification of Kraft Pulp by Trametes versicolor

The fungus Trametes versicolor can delignify and brighten kraft pulps. To better understand the mechanism of this biological bleaching and the by-products formed, I traced the transformation of pulp lignin during treatment with the fungus. Hardwood and softwood kraft pulps containing ¹⁴C-labelled residual lignin were prepared by laboratory pulping of lignin-labelled aspen and spruce wood and then incubated with T. versicolor. After initially polymerizing the lignin, the fungus depolymerized it to alkali-extractable forms and then to soluble forms. Most of the labelled carbon accumulated in the water-soluble pool. The extractable and soluble products were oligomeric; single-ring aromatic products were not detected. The mineralization of the lignin carbon to CO₂ varied between experiments, up to 22% in the most vigorous cultures. The activities of the known enzymes laccase and manganese peroxidase did not account for all of the lignin degradation that took place in the T. versicolor cultures. This fungus may produce additional enzymes that could be useful in enzyme bleaching systems.     

Lignin oxidation by laccase isozymes from Trametes versicolor and role of the mediator 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) in kraft lignin depolymerization.

Two laccase isozymes (I and II) produced by the white-rot fungus Trametes versicolor were purified, and their reactivities towards various substrates and lignins were studied. The N-terminal amino acid sequences of these enzymes were determined and compared to other known laccase sequences. Laccase II showed a very high sequence similarity to a laccase which was previously reported to depolymerize lignin. The reactivities of the two isozymes on most of the substrates tested were similar, but there were some differences in the oxidation rate of polymeric substrates. We found that the two laccases produced similar qualitative effects on kraft lignin and residual lignin in kraft pulp, with no evidence of a marked preference for depolymerization by either enzyme. However, the presence of the mediator 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) prevented and reversed the polymerization of kraft lignin by either laccase. The delignification of hardwood and softwood kraft pulps with the two isozymes and the mediator was compared; either laccase was able to reduce the kappa number of pulp, but only in the presence of 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate).     

The assessment of chromophores in bleached cellulosic pulps employing UV-Raman spectroscopy

UV-Resonance Raman (UV-RR) coupled with UV-visible Diffuse Reflectance (UV-vis DR) spectroscopy was applied to a solid-state study of chromophores in Eucalyptus globulus kraft cellulosic pulps bleached by chlorine dioxide and hydrogen peroxide. The UV-RR spectra were acquired at 325 nm laser beam excitation, which was shown to be appropriate for selective analysis of chromophore structures in polysaccharides. The proposed approach allowed the monitoring of chromophores in pulps and to track the extent of polysaccharide oxidation. However, precaution was suggested while performing a quantitative analysis of chromophores at the characteristic band of ∼1600 cm⁻¹ because of charge transfer complexes (CTCs) that exist in the pulp. These CTCs can affect the intensity of the aforementioned band by diminishing the conjugate state in the chromophore moieties. The amount of carbonyl and carboxyl groups in polysaccharides correlated with the intensity of the band at 1093 cm⁻¹. The analysis of UV-RR spectra revealed xylan as an important source of chromophores in eucalypt kraft pulp.     

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.     

Lignin-hemicellulose complexes restrict enzymatic solubilization of mannan and xylan from dissolving pulp

Mannan and xylan present in bleached softwood dissolving pulp were found to be partially resistant to hemicellulases even after repeated enzyme treatment. Despite the additional effect of an endoglucanase from Gloeophyllum sepiarium, which increased the␣accessibility of mannan and xylan to a mannanase from Sclerotium rolfsii and to a xylanase from Thermomyces lanuginosus, the enzyme mixture solubilized only half of the hemicellulose present in the pulp. Half of the remaining hemicellulose present in the pulp appeared to be entrapped within the cellulose matrix while the other half was associated with lignin-carbohydrate complexes. The latter hemicellulose portion was isolated and characterized. Chromatography and spectroscopic techniques revealed the presence of two types of lignin-carbohydrate complex, a galactoglucomannan-lignin complex (degree of polymerization DP 50–60) and a xylan-lignin complex (DP >200). Received: 8 December 1997 / Received revision: 30 April 1998 / Accepted: 8 May 1998     

Arundo donax L. reed: new perspectives for pulping and bleaching. Part 4. Peroxide bleaching of organosolv pulps

A comparative study on TCF (totally chlorine-free) bleachability of organosolv pulps from the annual fibre crop Arundo donax L. (giant reed) was carried out using a simple three-stage peroxide bleaching sequence without oxygen pre-bleaching. ASAM (alkali-sulfite-anthraquinone-methanol), Organocell (alkali-anthraquinone-methanol) and ethanol-soda organosolv pulps were bleached and compared with kraft pulp, as a reference. The final brightness of 76-78% ISO was attained for all tested pulps. The chemical charge required to reach this level of brightness varied for different pulps (despite the equal initial content of the residual lignin) and directly related to starting brightness values. No direct correlation between brightness improvement and lignin removal during bleaching was found, indicating the influence of the specific pulp properties introduced by pulping process on bleaching chemistry. The general higher bleaching response of organosolv pulps from A. donax was noted in comparison with kraft.