Enzymology of Kraft Pulp Bleaching by

Chapter 11

Enzymology of Kraft Pulp Bleaching by Trametes versicolor 1

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M. G. Paice , F. S. Archibald , R. Bourbonnais , L. Jurasek , I. D. Reid , T. Charles , and T. Dumonceaux 1

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Downloaded by GEORGETOWN UNIV on February 19, 2015 | http://pubs.acs.org Publication Date: November 21, 1996 | doi: 10.1021/bk-1996-0655.ch011

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Pulp and Paper Research Institute of Canada, 570 boulevard Saint Jean, Pointe-Claire, Québec H9R 3J9, Canada Department of Natural Resource Sciences, Macdonald Campus, McGill University, Sainte Anne de Bellevue, Québec H9X 1C0, Canada 2

Trametes versicolor and other white-rot fungi can lower the residual lignin content (kappa number) and increase brightness of kraft pulps without diminishing the pulps' strength or yield. Experiments with C-labelled lignin indicate that the residual lignin is solubilized but not extensively mineralized by T. versicolor. Laccase, manganese peroxidase (MnP) and cellobiose dehydrogenase (CDH) are produced by the fungus during bleaching, but based on molecular modelling studies, they are unable to freely access residual lignin in the kraft pulp fibre wall. Two isozymes of laccase were purified and compared: both enzymes required a mediator such as ABTS for pulp delignification, and under optimum conditions could produce up to 55% lignin removal. However, several lines of evidence indicate that MnP is the key enzyme required for fungal bleaching. C D H has several potential roles in delignification, including generation of complexing agents and Μn(II) for MnP. 14

Kraft pulps are brown, and must be bleached to acquire the high and stable brightness desired in fine writing and printing papers. Bleaching is achieved by removing the residual ligninfromthe pulp. Traditionally this was done with C l , but the production of organochlorine by-products has made chlorine bleaching an unpopular technology. Alternative bleaching chemicals, such as oxygen, ozone, and peroxide, are being adopted, but they are more expensive, or more likely to damage the strength of the pulp, than chlorine was. Biological delignification with fungi and their enzymes is an alternative approach which has shown promise, but has not yet attracted as much development effort as have the bleaching chemicals. 2

Fungal Bleaching The lignin-degrading fungi are called white rots because of their characteristic bleaching effect as they decay wood. The first attempt to bleach kraft pulps with these fungi was 0097-6156/96/0655-0151$15.00/0 © 1996 American Chemical Society

In Enzymes for Pulp and Paper Processing; Jeffries, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


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made by Kirk and Yang with Phanerochaete chrysosporium (1). That fungus lowered the lignin content (kappa number) of the pulp, but also attacked the cellulose and significantly decreased the pulp strength. Subsequently, other white-rot fungi have been found to more selectively delignify kraft pulps. Paice et al. (2) found that Trametes versicolor markedly increased the brightness of dilute suspensions of hardwood kraft pulp, and lowered their lignin content. Japanese scientists have extensively screened for fungi that bleach kraft pulps; Nishida isolated an unidentified strain, called IZU-154, that delignifles and brightens kraft pulps under solid-state fermentation conditions (3-6). Kondo has selected an isolate, YK-624, of Phanerochaete sordida which is also very effective (7-9). The highest brightness that has been achieved by fungal treatment is 80%, after a two-stage treatment of hardwood pulp with YK-624 (9). Usually the pulp brightness after fungal treatment is 50-60%, but it can be increased to 80-90% by post-treatment with chlorine dioxide (2) or peroxide (5,10). Fungal treatment often measurably decreases the viscosity and zero-span tensile strength of pulps (2,77), indicating that some cellulose depolymerization is taking place. Whether this attack on cellulose is hydrolysis by low levels of cellulase, or oxidation by radical by-products of lignin degradation, has not yet been clarified. In any case, the fungal treatment seems to improve inter-fiber bonding, since the sheet strength properties of the fungally delignifled pulps are equal to or better than those of chemically bleached pulps (2,5,6,11). Because the residual lignin is immobilized inside the pulp fiber walls, its removal must involve diffusible agents released by the fungal hyphae. Experiments in which the fungus was separated from the pulp by membranes have confirmed that delignification does not require direct contact between fungus and fiber (7,72). Extensive bleaching does require the presence of living fungus, however. Apparently one or more components of the delignifying system are labile or consumed, and need to be replenished constantly. One obvious candidate for this factor is H 0 , but just supplying H 0 is not sufficient to replace the fungus. As shown in Figure 1, a filtrate from a bleaching culture of T. versicolor supplemented.with glucose and glucose oxidase to provide H 0 could demethylate kraft pulp almost as extensively as and faster than an intact culture. The methanol is released from methoxyl groups on lignin aromatic rings with a free phenolic hydroxyl by manganese peroxidase (13); its appearance indicates that conditions were favourable for MnP activity. But the brightening effect of the culture fluid was shortlived and much less than that of the whole culture. This shows that some other factor, not yet identified, may be required for optimal activity of the Trametes bleaching system. The enzymes laccase and manganese peroxidase, found in pulp bleaching cultures, can effectively delignify pulp under conditions optimized for their action (see below). Their measured activities in the cultures, however, do not account for the delignification and brightening produced by the fungus. This suggests that other enzymes contribute to pulp bleaching. The residual lignin in kraft pulps is heavily modified during pulping; the alkyl-aryl ethers characteristic of lignin are depleted and diphenylmethane linkages that do not occur in native lignins are formed (for a review see Ref.74). Little is known about the chemical changes that white-rot fungi produce to remove this modified lignin from the 2

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Kraft Pulp Bleaching by Trametes versicolor

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fiber walls. Demethylation of the phenolic rings occurs early in pulp delignification by T. versicolor, and seems to lead to an increase in the alkali extractability of the demethylated lignin (75). We have prepared kraft pulps containing C-labelled lignin. Some of the lignin carbon is mineralized, but more of it is solubilized by the fungus (16). Detailed comparison of the effects of laccase and manganese peroxidase and intact fungal cultures on this labelled pulp should provide more clues to the nature of the enzymes involved in pulp delignification. 14


Molecular Architecture of Kraft Pulp Fibres Enzymatic bleaching of kraft fibres take place in the complex three-dimensional environment of the fibre wall. Therefore it is not only the chemical reactivity of lignin and other components of the wall that determine the course of the biological bleaching, but also the configuration of the cell wall components. The three-dimensional structure of the cell wall is difficult to study, and as a result our knowledge is rather limited. In order to gain a better understanding of the structure, we have applied molecular modelling to construct a representative sample of the wall. A model of a small piece of the secondary wall structure was produced using the method of coarse lignin model building developed previously (77). This structure was then subjected to simulated kraft pulping as described by Archibald et al. (14). The simulation was designed to provide a simultaneous partial removal of lignin and hemicellulose. The residual lignin was much richer in condensed structures than the native lignin, due to the preferential breakage of non-condensed bonds during the simulated kraft pulping. Thus a model of unbleached pulp fibre structure was obtained (Figure 2A). Although the removal of some fibre components leads to an increased porosity, the diffusion of relatively large redox enzyme molecules would still be strongly hindered. Fortunately neither manganese peroxidase nor laccase has to contact residual lignin in the kraft fibre directly, but rather can effect the delignification via low molecular weight intermediates such as, for example, ABTS (with laccase) or complexed manganese (ΓΠ) ions (with manganese peroxidase). The sizes of these mediators are such that their diffusion deep into the kraft fibre walls appears feasible. These mediators can then either reduce the size of the residual lignin, and thus accelerate leaching, or achieve a similar effect by increasing solubility through oxidation or other modifications. The sizes of the residual lignin molecules, as shown in Figure 2A, appear to be such that only a small change in size would be required to allow their leaching from the cell wall. The present molecular model has imperfections but none of them appear insurmountable. Our current focus is on the refinement of the atomic coordinates in order to obtain a model that could be subjected to further study using computational chemistry methods. An example of the refinement is shown in Figure 2, where a small part of the coarse model of lignin (Figure 2A) has been translated into atomic coordinates (Figure 2B). Part of a lignin macromolecule is shown in the vicinity of manganese peroxidase (18) together with hydrogen peroxide and manganic oxalate, both of which are required for the enzyme function.


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Culture

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Enzymes

CD

Enzymology of Kraft Pulp Bleaching by

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