Wood Deterioration and Preservation - American Chemical Society

Chapter 5 Overview of White-Rot Research: Where We are Today 1

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Downloaded by UNIV LAVAL on September 16, 2015 | http://pubs.acs.org Publication Date: March 31, 2003 | doi: 10.1021/bk-2003-0845.ch005

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Kurt Messner, Karin Fackler, Pongsak Lamaipis, Wolfgang Gindl , Ewald Srebotnik, and Takashi Watanabe 1

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Institute for Biochemical Technology and Microbiology, University of Technology Vienna, Getreidema 125rkt 9, A-1060 Vienna, Austria Wood Research Institute, University of Agricultural Science, Gregor Mendel-Strasse 33, 1180 Vienna, Austria Wood Research Institute, Kyoto University, Gokasho, JP-611-0011 Kyoto, Uji, Japan 2

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White-rot fungi are the most active lignin degrading organisms and thus, they play a key role in the carbon cycle on earth. Some species are known to cause heavy damage to wood construction and building materials, requiring that this damage be prevented by wood preservatives. Chemical products to increase wood durability currently in use are very effective but in addition, a strong demand to develop new products with less environmental impact cannot be overlooked. Biotechnological processes have been successfully implemented in the pulp and paper industry during the last decade. In the past, developments were driven mainly by environmental considerations. In the future however, the main driving force for research and development will be reductions in manufacturing costs using new, low investment delignification processes. The application of white -rot fungi, or their ligninolytic systems, is one option for this. Understanding the microbial mechanisms leading to wood -and especially lignin degradation is a prerequisite for

© 2003 American Chemical Society In Wood Deterioration and Preservation; Goodell, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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understanding both the development of new wood preservatives as well as wood biotechnology processes. This paper briefly reviews what is known about the effects of simultaneous and selective white-rot fungi with regard to the morphological and ultrastructural changes in wood. A n overview is given on reactions mediated by ligninolytic enzymes. It is generally accepted that low molecular weight compounds, smaller than enzymes, are the agents responsible for selective white-rot delignification. A new mechanism involving a powerful lignin degrading system based on coordinated Cu and peroxide, either hydrogen peroxide or organic peroxides, is proposed to be the agent involved at least in the initial depolymerization and degradation of lignin. Hypothetical pathways for both reaction types, involving H 2 O 2 or organic hydroperoxides, respectively, are presented. The capacity of the Cu system to degrade native wood lignin was evaluated by two methods: 1) a newly developed method employing section staining, and 2) UV-microscopy. It was shown that the cell walls of a hardwood species were almost completely delignified and the middle lamella was degraded after treatment with coordinated Cu and organic hydroperoxides. The treatment matches the effect of selective white-rot fungal degradation. With H2O2 only, the cell walls of the hardwood were degraded to some extent, while the middle lamella was not attacked. The likelihood of the coordinated Cu system functioning as the agent primarily responsible for selective white-rot is discussed and a hypothetical scheme of wood degradation is presented. The Cu system mechanism, both transition metals. chance to develop applying the Cu discussed.

seems to be related to the brown-rot of which employ reactions catalysed by Inhibiting this mechanism may offer a new wood preservatives. The possibility of system in pulp and paper production is

Introduction The defining character of white-rot fungi in the conversion of lignocellulose in nature is their strong capacity to degrade lignin, the second most abundant biopolymer in nature. In principle both softwoods and hardwoods are colonized

In Wood Deterioration and Preservation; Goodell, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.


75 by white-rot fungi but generally these fungi degrade the hardwoods, whereas the brown-rot fungi preferentially attack softwood species. Although white-rot fungi may play a secondary role in the decay of wood used for construction and building materials, wood preservatives designed to prevent fungal decay must be effective against both types of fungi. The wood preservatives currently in use are very effective; however, the trend to products with improved environmental performance cannot be overlooked. Understanding the biochemical mechanisms involved in decay may enable the development of products acting in a more targeted manner to control decay fungi. Besides its use as construction and building material, wood is also a primary substrate for the pulp and paper industry. In the past decade, biotechnological processes have been successfully implemented that contribute to the constant improvement of technology of this industrial sector. Several new applications of enzymes have reached, or are approaching, the stage of commercial use. These include enzyme-aided bleaching with xylanases, direct delignification with oxidative enzymes, energy saving refining with cellulases, pitch removal with lipases, freeness enhancement with cellulases and hemicellulases as well as enzymatic slime control in the paper machine (7). Besides enzymes, biopulping -the use of white-rot fungi to treat wood chips - is close to mill application. While environmental aspects have been a major driving force in the past, the need for significant reduction in manufacturing costs has become a primary goal of the pulp and paper industry. This is expected to be reached only by a major redesign of the industry's core technologies. New revolutionary breakthrough technologies may be developed in bio-manufacturing and involve lignin degradation processes such as bio-pulping and bio-bleaching. Implementation of these technologies could result in an estimated 50% reduction in capital cost requirements (2). Reduction of investment and energy costs by low temperature cooking processes allowed by implementation of biopulping processes is just one of the technologies that offers this potential. To reach this goal, new approaches in research are needed and the known concepts of delignification by white-rot fungi must be re-evaluated. Ligninolytic enzymes and their catalytic cycle, and the degradation of lignin model compounds and pulp lignin have been studied in detail. However, this work has not been able to explain the degradation of native wood lignin, and we still do not know how lignin is depolymerized by white-rot fungi in nature. This chapter is divided into two sections. The first section provides a brief overview of white-rot degradation processes whereas the second section focuses on a coordinated Cu system for the oxidation of lignin that the authors have researched. The coordinated Cu system includes components of naturally occurring white-rot biochemical degradation mechanisms and its study offers insight into how certain white rot fungi may degrade lignocellulosic materials.


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White-rot The literature reviewed in this paper focuses mainly on decay mechanisms. A comprehensive, general description of degradative fungi is presented elsewhere (3). The primary path of colonisation of wood by all types of fungi is usually the rays where easy access to non-structural nutrients contained in parenchyma cells is provided. From there, hyphae spread into fibres and vessels, and further colonization takes place via pits or less often by bore hyphae. Electron microscopic studies have demonstrated that an extracellular mucilage or slime layer (4-9) covers the hyphae. This extracellular slime produced by Phanerochaete chrysosporium has been characterized as a glucan. When wood degraded by white- and brown-rot fungi was investigated in T E M after ruthenium red staining, extracellular tripartite membranous structures were found covering the hyphae and slime layers throughout all stages of wood decay (10). The extracellular structures of Phanerochaete chrysosporium were isolated by enzymatic digestion of the fungal cell walls. They were found to be composed of equal amounts of carbohydrates, lipids (none of these were phospholipids), and proteins, including five fractions with molecular weights between 30,000 and 200,000 (77). The extracellular slime layer may cover the entire surface of the wood cell wall. As the optimum moisture content of wood for white-rot fungal activity is far above the fibre saturation point, the slime layer permits a film of liquid water to surround the wood cell wall. This water film presumably contains the slime components and the watery matrix they form may decrease evaporation when wood dries. Furthermore it is very likely that this layer is involved in regulating cell wall degradation by regulating the glucose level in the medium (72). Production of glucane polymers may either occur directly via the involvement of extracellular enzymes localised in the extracellular membrane, or by metabolism of carbohydrates and de novo synthesis of glucanses. The slime layer may also form a microenvironment where H 0 needed for lignin degradation is maintained. As the glucane polymers are also depolymerised by extracellular laminarinase (12) it is most likely that slime fractions of a molecular weight, low enough to penetrate the pores of the wood cell wall are created. The chemical structure of the extracellular slime layer and its viscosity might also have an effect on diffusion of degrading agents into the cell wall as well as affecting the passage of degradation compounds to and from the hyphae. Unfortunately, the functions of the slime layer and of the proteins localized in the extracellular membrane are not understood and further study must confirm the concepts discussed. Taxonomically, most white-rot fungi belong to the order of Aphyllophorales within the basidiomycetes and comprise a large number of species. Interestingly, some species causing white-rot and brown-rot are taxonomically closely related to each other. One example is the genus Tyromyces, with T. chioneus and T. 2

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77 placenta causing white-rot and most other species causing brown-rot. This might lead to the assumption that although white- and brown-rot decay are very different from each other in appearance, and they attack different chemical components of the wood cell wall, the mechanisms of decay of both types might be closely related to each other. The mechanism of white-rot with its preference for lignin degradation may be based on Cu reactions while Fenton reactions based on Fe have been suggested to be involved in brown-rot. The types of wood cell wall attack caused by white-rot fungi can be diverse, and macroscopic as well as microscopic differences have been reported. The unifying feature of most white- rots is the extensive degradation of lignin resulting in a bleached appearance. Several types of white-rots were characterised by macroscopic and microscopic differences as early as 1878 by Robert Hartig (3). White- rots have been classified by macroscopic characteristics into different sub-categories such as white-pocket, white-mottled, and white-stringy. The types of decay produced are affected by the fungal species, wood species, and ecological conditions among other things. From the microscopic and ultrastructural observation, two main types of white-rot have been distinguished. These types are classified with regard to the order in which different amounts of components are degraded (13,14) as follows: • •

Simultaneous white-rot: lignin, cellulose and hemicellulose are lost more or less simultaneously. Selective white-rot: preferential removal of lignin and hemicellulose. In most cases, cellulose is also degraded to some extent.

Simultaneous White-Rot Degradation of the wood cell wall starts by erosion of the lumen surface, sometimes around hyphae, and progresses from the cell lumen to the middle lamella. A l l components are degraded, the middle lamella is degraded last and only at exposed areas after complete cell wall degradation. Some fungi like Fomes fomentarius are not able to degrade the middle lamella. Walls of the surrounding cells remain lignified, and the lignin degrading agent does not diffuse into adjacent cell walls. The cell corners remain in an undegraded state for a long period and sometimes are left after decay. Losses of crystalline cellulose have been examined using polarized light microscopy (15,16). Distribution of lignin was determined by bromination and X-ray microanalysis by Saka and Thomas (17) as well as by UV-microscopy (18). It was demonstrated that lignin was removed from the inner circumference of the secondary wall near the lumen before cellulose was degraded, and lignin was removed continuously in advance of cellulose degradation.


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Selective White-Rot Selective white-rot fungi have the ability to remove large quantities of lignin from the cell wall without destruction of cellulose. Lignin is preferentially removed from the cell wall causing a loosening of cells. Hemicellulose is degraded concomitantly with lignin and cellulose is left unmodified to a large extent (3,13,14). After incubation of wood chips for 6 weeks with Ceriporiopsis subvermispora, or Dichomitus squalens, T E M observations demonstrated disintegration of the wood tissue by complete dissolution of the middle lamella. Nevertheless, the wood fibres showed no sign of visible damage (19). By employing selective staining of lignin and cellulose with safranin and astra blue, it was obvious that the middle lamellae and the wood cell walls had both been delignified (20). Greater birefringence was observed when wood was viewed in polarized light (21). The crystalline nature of cellulose was not destroyed which is in contrast to the removal of cellulose by simultaneous white-rot. Blanchette and Reid (8) demonstrated progressive stages of selective delignification by Phlebia tremellosa by fixation of wood with O s 0 glutaraldehyde and post-staining with uranyl acetate. Delignification of the cell wall started directly adjacent to the hyphae and progressed through the S layer to the middle lamella, which was gradually lost. This occurred around the entire circumference of the cell wall. Remnants of cell corners persisted. The ability to degrade hardwood vessels varies among selective white-rot fungi: e.g. Dichomitus squalens does not delignify the vessels while Phlebia tremellosa removes lignin selectively from vessels (8). This might be related to the ability to cope with the higher guaiacyl content of vessels (18,22). Syringyl lignin is degraded more rapidly than guaiacyl lignin (23-26). Both types of attack, simultaneous as well as selective decay, can be produced by the same white-rot fungus and even in the same piece of wood. Factors that determine different modes of fungal degradation remain obscure. Unpublished results of the authors have lead us to the conclusion that the moisture content of wood is one of the determining factors. 4

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Penetration of Enzymes Selective white-rot fungi are used in so called "biopulping processes" to treat wood chips prior to mechanical or chemical pulping. After 2 - 3 weeks of incubation, weight loss of the wood chips is only about 2%, but cell wall components are already modified, leading to energy savings of approximately 30% in mechanical pulping (27), or lower kappa numbers in sulphite (19) or Kraft pulping (28). The chemical nature of these effects was found recently to be depolymerization of cell wall lignin (28) by fungal metabolites. This



79 demonstrates that lignin is attacked by fungal metabolites in the cell wall and middle lamella in the early phases of colonisation. Based on immunogold labelling experiments carried out by Srebotnik etal. (29) and confirmed later by Daniel (30) and Blanchette (31), it is now generally accepted that the enzymes secreted by fungi into the lumen - their molecular weights are usually above 20 k D - are too large to penetrate the cell wall until relatively late stages of decay. Even when insulin (5.7 kD) was used as a size marker molecule after 2 weeks of colonisation of the wood samples by Ceriporiopsis subvermispora, it could be identified only in the inner area of the wood cell wall, close to the lumen (30). Consequently, the depolymerization of lignin leading to biopulping effects must be caused by molecules smaller than 5.7 kD which clearly excludes the involvement of any direct enzymatic activity in the cell wall. These low molecular weight agents involved in selective whiterot have become one of the main scientific targets in white-rot research.

L o w Molecular Weight Agents Various reactive low molecular weight agents may be formed directly or indirectly by oxidative fungal enzymes and have been proposed to participate in delignification starting at the cell lumen surface and penetrating deeper into the wood cell wall. Several of the systems proposed to generate low molecular weight agents are reviewed below:

Manganese Peroxidase /Mn(II) / Oxalate Manganese peroxidase (MnP) is a very common extracellular enzyme produced by simultaneous as well as selective white-rot fungi. Its catalytic cycle includes two one-electron reducing stepsby Mn(II) (32, 33). The Mn(III) formed is then chelated and released from the enzyme by the fungal metabolite oxalate in vivo. The relatively stable Mn(III) oxalate is able to oxidize phenolic lignin compounds and has been discussed as a diffusible agent acting in the wood cell wall, distant from MnP located in the fibre lumen. Since phenolic lignin makes up only a small portion of the cell wall lignin, total delignification as observed in selective white-rot is considered unlikely to be caused by Mn(III) alone. However, Hofrichter et al. (34) have recently succeeded in creating conditions favourable for the efficient depolymerisation of native lignin by the M n P / Mn(II) couple. Furthermore, Mn0 -deposits accumulate during white-rot decay, and are probably formed by disproportionate of MnP-generated Mn(III). This may then be "reactivated" by reduction to Mn(II) (35) or the formation of reactive oxalate complexes, which may contribute to the selective delignification of the wood cell wall (36). 2


80 Manganese Peroxidase /Mn(II) / Oxalate / Cellobiose Dehydrogenase (CDH) C D H is oxidized by molecular oxygen and metal ions such as Fe(III) and Cu(II) to form hydrogen peroxide and reduced metal ions. Fe(II) and Cu(I) react with hydrogen peroxide to generate hydroxyl radicals which in turn are proposed to demethoxylate and hydroxylate non-phenolic lignin. Thus, nonphenolic lignin is converted to phenolic lignin which can then be attacked by MnP-generated Mn(III) (37). The hydroxyl radical may also be formed by other pathways, e.g. via hydroquinone redox cycling involving semiquinones produced by peroxidase or laccase, which reduce both Fe(III) and 0 to provide the ingredients for Fenton-type hydroxyl radical formation (38). Downloaded by UNIV LAVAL on September 16, 2015 | http://pubs.acs.org Publication Date: March 31, 2003 | doi: 10.1021/bk-2003-0845.ch005

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Manganese Peroxidase /Mn(II) / Oxalate/Lipids The oxidative potential of M n P can be extended by the presence of lipids. Peroxidation of unsaturated fatty acids is promoted by Mn(III) resulting in the formation of peroxyl radicals which are diffusible, potentially ligninolytic agents (39). Watanabe et al. (40) suggested that Mn(III) directly abstracts hydrogen from fatty acids to form acyl radicals. This system has been shown to depolymerise both phenolic as well as non-phenolic lignin (41). Substantial amounts of fatty acids and hydroperoxides have recently been detected in cultures of Ceriporiopsis subvermispora, providing further support that lipidderived radicals may in fact be involved in selective white-rot degradation (42).

Lignin Peroxidase / Veratryl Alcohol Lignin peroxidase (LiP) has the highest redox potential of all enzymes believed to be involved in lignin degradation, and in principle is able to oxidise phenolic as well as non-phenolic lignin. However, for steric reasons discussed above its action on lignin is restricted to surface areas of the wood fibre. The veratryl alcohol radical, generated during turnover of L i P when compound II is reduced to the resting state by veratryl alcohol, was proposed by Harvey et al. (43) to act as a charge transfer system in wood. However, due to its short lifetime (44) this radical is not expected to diffuse into deeper areas of the cell wall. A very recent study proposes self-propagation of chemical reactions initiated by lignin peroxidase, involving lignin-derived peroxyl radicals (45). However, it is not known whether such radicals would be able to depolymerize lignin inside the wood cell wall.


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Laccase / Mediators Laccase, a phenol oxidase which is produced by most white-rot fungi, oxidizes the phenolic moieties in lignin which primarily leads to the polymerisation of lignin. However, when laccases are combined with low molecular weight charge transfer agents, so-called mediators, they can delignify wood pulps. A significant kappa reduction can be achieved in pulp bleaching when using synthetic mediators carrying N - O H groups (46). The effect of the laccase/mediator-system is probably based on ketone formation by the mediator radical, which makes the lignin molecule more susceptible to alkaline hydrolysis during extraction. In addition to pulp bleaching, the laccase/mediator system is also able to depolymerise non-phenolic guaiacyl lignin (47). However, the occurrence of natural laccase mediators during white-rot decay has not been demonstrated so far.

Low Molecular Weight Peptides Tanaka et al. (48) discovered peptides, produced by white-rot fungi, which are of the molecular weight range of 1000-5000 D. These peptides have been proposed to catalyse a redox reaction between molecular oxygen and an electron donor to produce hydroxyl radicals via reaction with hydrogen peroxide. This may lead to ligninolytic reactions similar to those described above for C D H .

Conclusion Current research suggests that lignin depolymerization is a highly complex process. Numerous pathways have been suggested and it is difficult to assess their individual contributions to ligninolysis. However, it can be assumed that radical reactions involving Mn(III) and reduced oxygen species play a key role in selective lignin degradation by white-rot fungi. In this regard, we propose another mechanism, the coordinated Cu-system, which may also play a key role in white-rot decay and which is discussed in the second section of this chapter.

The Coordinated Copper System Introduction Hypothetical Pathway for the Generation of Active Oxidants from Hydrogen Peroxide by Coordinated Copper The transition metals Fe and Cu are the active centres of all the enzymes assumed to be involved in lignin degradation. These metals are coordinated by



82 amino acids and/or N-atoms of protoporphyrins. The concentrations measured for these metal ions in wood are 9.2 ppm for Fe(III) and 0.7 ppm for Cu(II) (49). There is strong evidence that Fe is involved in the mechanism of brown-rot (50) resulting mainly in the degradation of cellulose and hemicellulose. Recently, siderophores containing catechol groups that are able to reduce and solubilise the Fe attached to cellulose have been isolated (57, 52). In Fenton chemistry reactions, Fe(II) generates hydroxyl radicals from hydrogen peroxide produced by both brown-rot and white-rot fungi (50, 38). These radicals are strong oxidants and degrade cellulose and hemicellulose and can alter lignin chemistry. Besides its function as the active centre in laccases, almost no attention has been paid in the past to the possible involvement of Cu in cell wall degrading reactions; although Cu(I) would also lead to Fenton reactions. One of the reasons might be that the main focus has been on Fenton chemistry and Fe is the more likely reaction partner as its concentration in wood is higher than that of Cu. It was demonstrated by Watanabe et al. (53) that C u coordinated by low molecular weight organic compounds like pyridine together with peroxides either hydrogen peroxide or lipid hydroperoxides - leads to very strong oxidative reactions. These reactions are able to oxidize various dyes, pulp lignin and even a non-phenolic synthetic lignin model compound (Figure 1). Compared to uncoordinated Cu, the coordinated reaction was much more effective. The products obtained after the reaction of the non-phenolic lignin dimer with Cu/ pyridine/ H 0 were similar to those after oxidation of similar substrates with L i P / H 0 (54). The Cu/ pyridine/ H 0 products were not consistent with oxidation by free hydroxyl radicals, which would instead lead to aromatic hydroxylation or hydrogen abstraction (55). The nature of the active oxidant is not known, but since the substrate was oxidized by a single electron transfer reaction it may be speculated that a Cu-centered oxo or peroxo complex (56) is the one-electron oxidant produced by the system (Figure 2). Like the other models for lignin degradation, one can only conclude the existence of the coordinated-Cu system in nature from the detection of the reaction components in treated wood and comparing the results achieved to wood degraded naturally by selective white-rot fungi. As discussed earlier, Cu and hydrogen peroxide are present in white-rotted wood. Evidence for the production of compounds containing a pyridine nucleus by fungi is provided in the literature (57) and includes, homarine, pycolic acid, dipycolic acid, fusaric acid, dehydrofusaric acid, nicotinamide, P Q Q etc. In the first step of the reaction Cu(II) is reduced to Cu(I) by either H 0 or reducing groups contained in wood or pulp lignin. Cu(I) together with H 0 2

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Figure 1. HPLC analysis and radioactive products obtained after incubation of a [ C]-labelled non-phenolic lignin dimer (I) with Cu/H 0 in the presence and absence of pyridine. Veratraldehyde (II) was the only main oxidation product in the presence of the coordinator pyridine.

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Wood Deterioration and Preservation - American Chemical Society

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