Ultrastructural Aspects of Wood Delignification by Phlebia (Mearulius) Tremellosust

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.

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