APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1986, p. 239-245 Vol. 52, No. 2 0099-2240/86/080239-07$02.00/0 Copyright © 1986, American Society for Microbiology Ultrastructural Aspects of Wood Delignification by Phlebia (MeArulius) tremellosust

R. A. BLANCHETTE'* AND I. D. REID2t Department of Plant Pathology, University of Minnesota, St. Pauil, Minnesota 55108,1 a(id Plant Bioteclhnology Research Institute, National Research Council of Canada, Saskatoon, Saskatchewan, Canada S7N OW92 Received 3 March 1986/Accepted 30 April 1986

Wood from aspen and birch that had been decayed for 12 weeks by Phlebia tremellosus had averages of 30 and 31% weight loss, respectively, and 70% lignin loss. Digestibility increased from averages of 21 and 13% for sound aspen and birch to 54 and 51% for decayed aspen and birch. Individual wood sugar analyses of decayed birch blocks indicated an average loss of 10% glucose, 45% xylose, and 19% mannose. Micromorphological studies demonstrated the removal of middle lamellae and separation of cells. Vessels also separated at perforation plates. Electron microscopy with Os04-glutaraldehyde-fixed and KMnO4-fixed wood showed that lignin was progressively removed first from the secondary cell wall layers, beginning at the lumen surface, and later from the compound middle lamella. Extensive degradation of lignin was found throughout the secondary wall and middle lamella region between cells. In cells with advanced decay, the middle lamella between cells was completely degraded, but cell corner regions remained.

Basidiomycetes that cause white rots can degrade wood understood. This investigation was done to examine the by simultaneously attacking lignin, cellulose, and hemicel- ultrastructural changes that occur during preferential lulose or by specifically removing individual cell wall com- delignification in vitro of aspen (P. tremiuloides) and birch ponents (3, 5, 21). Preferential lignin degradation has been (Betiila papyrifera) by P. tremellosius. documented for several white-rot fungi (1, 4, 10, 20, 25, 32, 34, 36) and for mutants that lack cellulase activity (9). Fungi that selectively remove lignin generally also degrade a MATERIALS AND METHODS substantial amount of hemicellulose but only small amounts of cellulose (3, 5, 11). Distinct micromorphological differ- Sapwood blocks with greater radial surface (0.7 by 2.0 by ences between simultaneously white-rotted and selectively 1.5 cm; height by width by length) were cut from freshly delignified wood have been demonstrated (3, 5). Ultrastruc- harvested birch (B. papyrifera Marsh.) and aspen (P. tural observations of advanced stages of white rot, decayed tremiuloides Michx.). Additional aspen blocks with greater in the forest under natural conditions, have been published tangential surface (2.0 by 0.7 by 0.7 cm) and birchwood (3-5). The selectively delignified wood contained cells with- wafers (0.7 by 0.1 by 2.0 cm) were also used. Wood was out middle lamellae; the cellulose-rich S2 layer of the sec- dried to a constant weight at 60°C. The wood was then ondary wall was the primary tissue remaining. Controlled prepared for decay by P. tremellosius (Schrad.:Fr.) Nakas. laboratory studies have demonstrated that selective lignin and Burds. (Syn. Meriuliius tremellosius Schrad.:Fr.) with degradation can also be obtained in chambers designed to isolate PRL 2845 by previously described techniques (25). accelerate decay of wood blocks (25). Recently, delignifica- After 12 weeks, wood blocks and wafers were dried at tion by Phlebia tremellosus, used in solid-state fermentation loss or for electron ofPopulus tremuloides, was shown to be technically feasible 60°C to determine weight prepared (31). This fungus removed 52% of the lignin with only a 12% microscopy. Dried samples were used to determine lignin, weight loss of wood. The in vitro digestibility of the wood, wood sugar content, and cellulase digestibility by previously with a cellulase preparation, increased from 18 to 53%. described techniques (8, 30-32). The highest concentration of lignin within the woody cell Wood blocks used for transmission electron microscopy wall is in the middle lamella region and cell corners (15, 29, were either (i) fixed in 2.5% glutaraldehyde in 0.05 M 35). However, in birch this area may contain less than 19% phosphate buffer (pH 7.2) for 24 h at 4°C followed by a of the total cell wall lignin (14). Fungi that preferentially phosphate buffer wash and fixation in 2.0% OS04 in 0.05 M remove large amounts of lignin from wood must have the phosphate buffer for an additional 24 h at 4°C, or (ii) fixed in capability of degrading lignified areas throughout the cell 2.0% KMnO4 in distilled water for 3 h at 4°C. Samples were wall without extensive loss of cellulose. How a fungus can placed under low vacuum during fixation. After fixation, cause such a specific attack on wood is not completely samples were dehydrated with a graded acetone series and embedded in Spurr (hard consistency formulation) embed- ding medium (37). Polymerization was done at 70°C. Sec- * Corresponding author. tions were cut with a diamond knife, and sections from t Paper 14,803 of the scientific journal series of the Minnesota glutaraldehyde-Os04-fixed wood were stained with 0.5% Agricultural Experiment Station. for t Present address: Biotechnology Research Institute, A/S Hospi- uranyl acetate. Sections of wood were also prepared tal, Royal Victoria, Pavillon Hersey, Montreal, Quebec, Canada scanning electron microscopy by methods previously de- H3A lAl. scribed (3). 239 240 BLANCHETTE AND REID APPL. ENVIRON. MICROBIOL.

TABLE 1. Selective loss of lignin and increased accessibility of (Fig. 2d). This electron-dense region of the secondary wall wood carbohydrates after decay by P. trernellosius became more extensive in areas of advanced decomposition (Fig. 2e and f). In contrast, the middle lamella region became No. Lignin Cellulase Wood replicatesof Wt( losss loss digestibility less electron dense and less visible as compared with sound wood. The first observable loss of middle lamella was in the Birch, expt 1 region between cells (Fig. 2e, arrowheads). The cell corners Undecayed 5 0± O O ± 3 15 ± 1 persisted and were still evident in advanced stages of decay. Decayed blocks 5 23 + 3 59 ± 6 51 ± 2 In most cells, the secondary wall did not change significantly Decayed wafers 5 35 3 73 ± 1 51 ± 1 in size or shape. The only difference was a tendency to Birch, expt 2 become more intensively stained. However, in some cells Undecayed 5 1±0 0 ± 0 12 0 from areas with advanced stages of decay, the secondary Decayed blocks 5 39 2 81 ± 1 50 1 wall appeared thin, and small depressions were seen (Fig. 2f, Aspen arrows). Undecayed 5 0±0 0 1 21 0 Sections from decayed aspen wood had a similar pattern Decayed blocks of degradation. The middle lamella region between cells was Radial 10 31 1 71 ± 1 54 1 degraded, and secondary wall layers became more electron Tangential 10 28 2 68 ± 4 54 1 dense (Fig. 2g). Cells from wood with advanced decay had extensively degraded middle lamellae and cells separated aMean ± standard error. All values are percentage of original weight. (Fig. 2h and i). The cell corners remained least affected. The secondary wall layers were densely stained but otherwise RESULTS remained relatively unaltered. Lignin within the woody cell wall becomes electron dense At 3 months after inoculation, aspen and birch blocks had after KMnO4 fixation (6). The micrographs of sound wood lost an average of 30 and 31%, respectively, of their dry presented here (Fig. 3a to c) showed good definition of the weight and 70% of their lignin. Birch wafers had an average lignified areas of the cell wall. The most intensely stained weight loss of 35% and an average lignin loss of 73% (Table zone was the middle lamella and cell corner region. The SI, 1). The selective degradation of lignin increased the acces- S2, and S3 layers of the secondary wall have a less electron- sibility of wood polysaccharides to enzymatic hydrolysis, as dense appearance and a diffuse lignin distribution (Fig. 3a to shown by their increased solubilization by a crude cellulase c). Thin sections of birch (Fig. 3d to f) and aspen (Fig. 3g to preparation. i) that were decayed for 12 weeks had little contrast, The extent of decay varied substantially between repli- resulting from appreciably less staining than sound wood cates and between experiments (Table 1). However, all (Fig. 3d to i). Hyphae were observed in the lumina of cells, samples appeared to follow the same trend of lignin loss but the surrounding hyphal sheath or slime layer (Fig. 3d, e, versus weight loss. Therefore, we can assume that all the and g to i) was not as evident as in sections fixed with samples represent different stages in a common decay proc- Os04-glutaraldehyde. Secondary wall layers were not as ess. There were no clear differences between aspen and easily distinguished as in sound wood. The middle lamella birch, between wafers and blocks, or between radial and was extensively degraded throughout the wood (Fig. 3d and tangential blocks. g). In cells less severely degraded, the middle lamella Wood sugar analyses of decayed birch blocks indicated contained small individual granules between cells instead of that only small amounts of glucose (4 to 17%) were lost when a continuous electron-dense region as observed in sound 74 to 83% of the lignin was degraded (Table 2). Substantially wood (Fig. 3e, h, and i). The cell corners remained and were more xylose was lost than any other wood sugar. Only trace the most intensively stained zones within the sections (Fig. amounts of arabinose and galactose were present in sound 3d to i). Remnants of the middle lamella and cell corner and decayed wood. region can be seen at higher magnification in Fig. 3f, h, and i. Noninoculated wood blocks were examined with a scan- ning electron microscope and compared with decayed sam- ples (Fig. 1). Cells of decayed wood separated at the middle DISCUSSION lamella region when sectioned. The loosely arranged paren- chyma cells, fiber cells, and vessel elements readily de- P. taemellosuis causes a specific attack on lignin within tached from adjacent cells (Fig. la to d). Vessel elements wood. After 12 weeks of degradation, 60 to 85% of the lignin also separated at perforation plates (Fig. le and f). The is degraded with only a small loss of cellulose. The enzymes morphology of the defibrated cell walls can be seen in Fig. responsible for this selective removal of lignin appear to lf. No fungal hyphae were found among the exposed cells begin action on the cell wall immediately surrounding the where they had been separated from one another. Erosion hypha. The dark staining that occurred in the secondary wall troughs or lysed zones were not apparent. of Os04-glutaraldehyde-fixed wood indicates that the area Micrographs of noninoculated birch and aspen fixed with was more reactive to poststaining with uranyl acetate. Since Os04-glutaraldehyde and poststained with uranyl acetate are presented in Fig. 2a to c. Decayed wood blocks contained areas with apparently different stages of decay. Some parts TABLE 2. Weight, lignin, and wood sugars lost from birchwood of each wood block contained incipient stages of decompo- decayed by P. trernellosius for 12 weeks sition with little alteration of the cell wall, while other areas % lost consisted of advanced stages of decay. Mycelia of P. tremel- Wood block no. losus were evident in cell lumina. The hyphae, seen in Wt loss Lignin Glucose Xylose Mannose transverse section in Fig. 2d to i, had hyphal sheaths or slime 1 39.2 74.5 17.5 45.1 14.1 layers surrounding them. During incipient stages of decay in 2 34.3 75.2 4.1 39.4 28.5 birch, the cell wall beneath the hyphae was electron dense 3 40.5 83.2 7.6 50.6 14.8 VOL. 52, 1986 DELIGNIFICATION BY P. TREMELLOSUS 241

FIG. 1. Scanning electron micrographs of aspen and birch decayed for 12 weeks by P. tremellosus. (a to c) Radial (a) and tangential (b and c) sections of aspen wood showing a separation of fibers, ray parenchyma cells, and vessels. The sectioned wood separated between cells, and individual cells were apparent. (d) Cells were loosely arranged but no erosion troughs or lysed zones were evident in the cell walls. (e) Vessel elements separated at perforation plates (arrowheads). (f) Radial section of birchwood with exposed scalariform perforation plate where vessel element had been pulled away (arrowheads). this stain causes cellulose within the plant cells to become tacked the cell wall from the lumen inward toward the more electron dense (7), these dark zones beneath the middle lamella region. In advanced stages of decay, the hyphae could indicate the early removal of lignin. The entire secondary wall was similarly affected. Apparently, the unbound cellulose fibrils would then have a greater affinity cell wall was attacked from the entire surface of the lumen. for the uranyl acetate. Further evidence is provided by Action of the lignin-degrading enzymes was not restricted to observations of KMnO4-fixed specimens. The distribution of direct contact with hyphae, and degradation occurred at lignin, identified by deposits of electron-dense granules, considerable distances from the fungus. became less conspicuous in areas of the cell wall beneath Once alterations of the secondary wall layers had oc- hyphae. As degradation within the cell wall continued, a curred, the middle lamella was destroyed between cells. The diffuse, dark electron-dense stain in the Os04-glutaralde- KMnO4-fixed wood clearly showed the loss of a continuous hyde-fixed wood and a lack of contrast in the KMnO4-fixed electron-dense region at the middle lamella. The degradation wood were apparent. This indicates that the enzymes at- of the middle lamella continued until it was no longer 242 BLANCHETTE AND REID APPL. ENVIRON. MICROBIOL.

FIG. 2. Transmission electron micrographs of transverse sections from OsO4-glutaraldehyde-fixed birch and aspen. Bar, 5 ,um. (a) Sound birchwood showing a group of fiber cells. (b) Sound aspen with electron-dense middle lamella and cell corner regions and less electron-dense secondary wall layers. (c) Cell walls of two adjacent birchwood cells showing the cell corner (cc), middle lamella region (ml), and secondary wall (Sl and S2). The SI and S2 layers of the secondary wall were not clearly defined. (d to f) Birchwood degraded by P. tremellosus for 12 weeks. Hyphae (hy) were located in the lumina of cells and were surrounded by a hyphal sheath. Cells from a less severely decayed area of a wood block showed incipient stages of decay. (d) A dense staining of the secondary wall was evident beneath the hyphae (arrowheads). Cells from areas with more advanced decay were without middle lamellae between cells (e, arrowheads). Secondary walls contained a diffuse electron dense stain. Cell corners persisted (f, circled areas). In some cells, the secondary wall appeared thin, and small depressions were seen (f, arrows). (g to i) Aspen wood decayed for 12 weeks by P. tremellosus. Incipient stages of decay showed hyphae (hy) in cell lumina and dark secondary walls. Middle lamellae were partially degraded (g). Cells from wood with more advanced decay showed a complete degradation of middle lamella between cells (h) and a separation of cells (i), but remnants of the cell corner regions remained.

discernible except at the cell corners. These cell corner White-rot fungi that attack various hardwoods appear to regions persisted without noticeable alterations. The type of preferentially degrade the syringyl lignin component (12, 17, lignin found in the cell corners may influence degradation. 19). Decay by P. tremelloslus may also be influenced by the Vessels and cell corners in hardwoods have high guaiacyl type of lignin found within the aspen and birch. The guaiacyl lignin contents; other cells and morphological regions of lignin content of the cell corners could be responsible for the each cell wall have a syringyl-rich lignin fraction (16, 24). lack of degradation in these areas. The vessels of both aspen VOL. 52, 1986 DELIGNIFICATION BY P. TREMELLOSUS 243

I

*i

FIG. 3. Transmission electron micrographs of KMnO4-fixed birch and aspen. Bar, 2 p.m. (a) Sound wood with all cell wall layers clearly discernable: ml, middle lamella; cc, cell corner; SI, S2, and S3, secondary cell wall layers. (b) Cell walls of two adjacent aspen wood cells. (c) Cell corner region of sound birchwood. (d to f) Birchwood decayed by P. tremellosus for 12 weeks showing extensive degradation of middle lamellae between cells. Hyphae (hy) were located in cell lumina. Secondary walls had little electron density and contrast as compared with sound cell walls (a to c). SI to S2 layers were still apparent (f). Cell corners were least affected and remained electron dense when most of the middle lamella had been removed (d, circled areas). (g to i) Aspen wood decayed for 12 weeks by P. tremellosus. Cells were free of a continuous middle lamella region between cells, and secondary walls were poorly stained. Cell corners (g, circled areas) were not removed. Cells from a wood block with less advanced decay showed the middle lamella region partially degraded (h and i). This region contained a discontinuous line of electron-dense granules. Cell corners were only partially degraded. and birch in the present study separated from surrounding taneous white rot that decayed all cell wall components. cells but were not degraded. The separation of vessels at Erosion troughs and electron-dense granules are usually perforation plates, however, could indicate that the ratio of observed around the hyphae of white-rotters that cause a guaiacyl to syringyl lignin within vessel elements is not the simultaneous rot (2, 22, 33). The degradation of the cell wall same as in the perforation plate zone. is localized to the immediate area around the fungus. As The ultrastructure of selective lignin removal demon- decay progresses, the secondary wall is destroyed, and the strated that a type of degradation and enzymatic attack had middle lamella is degraded in the lysed zone. The secondary taken place that was different from one caused by a simul- wall may also become progressively thinned from the lumen 244 BLANCHETTE AND REID APPL. ENVIRON. MICROBIOL. toward the middle lamella (21, 38). Messner and creases substantially and reaffirm that lignin is removed Stachelberger (22) have provided a schematic drawing of the throughout the cell wall layers. decay process caused by Trametes hirsuta (Wulf.:Fr.) Pilat. Our results indicate that selective lignin removal by P. ACKNOWLEDGMENTS tremellosus does not follow this type of attack. sheaths were around of P. tremel- We thank M. J. Effland of the Forest Products Laboratory, Hyphal present hyphae Madison, Wis., for analyses of individual wood sugars, Michael losus within the lumina of wood cells. Os04-glutaraldehyde Carlson for his technical assistance with the transmission electron fixation resulted in better visualization of the sheath than did microscopy, and C. S. Mallard for lignin and digestibility analyses. KMnO4 fixation. Hyphal sheaths appear to be a morpholog- ical feature of many white- and brown-rot fungi (27, 28). LITERATURE CITED Sheaths around wood decay fungal hyphae may serve as 1. Ander, P., and E.-K. Eriksson. 1977. Selective degradation of media for housing and transporting lignocellulose wood components by white-rot fungi. Physiol. Plant. depolymerizing agents and also may serve as sources of 41:239-248. support and nutrition. The extensive degradation of lignin by 2. Blanchette, R. A. 1980. Wood decomposition by Phellinus P. tremellosus indicates that the sheath may act as an (Fomes) pini: a scanning electron microscopy study. Can. J. interface between the fungus and cell wall. de- Bot. 58:1496-1503. However, 3. Blanchette, R. A. 1984. 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