Effects of Incubation Time and Temperature on In Vitro Selective Delignification of Silver Leaf Oak by Ganoderma colossum

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1995, p Vol. 61, No /95/$ Copyright 1995, American Society for Microbiology Effects of Incubation Time and Temperature on In Vitro Selective Delignification of Silver Leaf Oak by Ganoderma colossum J. E. ADASKAVEG, 1 * R. L. GILBERTSON, 2 AND M. R. DUNLAP 3 Department of Plant Pathology 1 and Facility of Advanced Instrumentation, 3 University of California, Davis, California 95616, and Department of Plant Pathology, University of Arizona, Tucson, Arizona Received 1 August 1994/Accepted 15 October 1994 The effects of incubation time and temperature on the ability of isolates of the chlamydosporic and thermophilic fungus Ganoderma colossum (Fr.) C. F. Baker to cause selective delignification of Quercus hypoleucoides A. Camus were evaluated by standard in vitro agar block tests. Chemical and scanning electron microscopy studies of decayed wood were used to determine the extent of selective delignification or simultaneous decay caused by each fungal isolate. At 35 C, the percent weight loss increased from 6.1% after 4 weeks to a maximum of 32.5 to 33.0% after 16 and 20 weeks of incubation. The average percent Klason lignin-chlorite holocellulose ratios (PKL/CHC) decreased from 0.35 in the control wood block to 0.22 in wood blocks incubated for 12 weeks; this indicated selective delignification. The average PKL/CHC increased for the 16- and 20-week incubation periods, indicating greater removal of polysaccharides during longer incubation periods. In temperature studies, the percent weight loss after 12 weeks was 26 to 27% between 30 and 40 C and less than 16% for the 25 and 45 C treatments. The average PKL/CHC ranged from 0.18 to 0.16 between 35 and 40 C, whereas they were 0.23 and 0.31 for the 25 and 45 C treatments, respectively. Scanning electron microscopy confirmed an optimum temperature range near 35 to 40 C and incubation times of 8 to 12 weeks for selective delignification. Under these conditions, ray parenchyma, fiber tracheids, and vessels were devoid of middle lamella; pit regions of cells were visible with significantly enlarged apertures; and individual cells were separated and clearly delimited. Extensive delignification of wood occurred throughout the wood blocks evaluated. Incubation times longer than 12 weeks resulted in greater degradation of wood cell walls and thus in greater removal of the polysaccharide component of the wood. For incubation times of 4 weeks or a temperature of 25 C, limited to no degradation of cells was observed. At 45 C, walls of fiber tracheids were eroded and ray parenchymal cells were extensively degraded, indicating that simultaneous degradation of cell walls occurred. Thus, the incubation temperature influenced the type of decay by G. colossum observed on oak wood blocks: extensive selective delignification at 35 to 40 C after more than 8 weeks of incubation or simultaneous decay at 45 C with 14% weight loss after 12 weeks of incubation. Isolates of G. colossum may prove useful in studies on mechanisms of delignification and biotechnological applications (e.g., biopulping) of lignin-degrading fungi. The largest group of fungi capable of extensive wood degradation is classified in the Basidiomycota. Within this phylum, two ecological groups of wood decay fungi are generally recognized: brown rot and white rot fungi. Brown rot fungi cause extensive degradation of cellulose and hemicellulose with limited modification of lignin. Fungi that cause white rots, however, degrade all major structural components of wood cell walls, including cellulose, hemicellulose, and lignin. The rate and extent of polysaccharide and lignin removal can differ among different white rot fungi. Some white rot fungi [e.g., Trametes versicolor (L.: Fr.) Pilát] remove all cell wall components simultaneously, whereas others (e.g., Phanerochaete chrysosporium Burds. in Burds. & Eslyn) can preferentially degrade lignin or selectively delignify wood prior to polysaccharide degradation (9, 15). Fungi capable of selective delignification may have diverse uses, e.g., biopulping of wood in the paper industry, bioremediation of chemical wastes in soil and water, and improving the digestibility of lignocellulosic food sources for animal consumption (7, 16, 18, 21). Desirable characteristics of white rot fungi capable of delignifying wood include rapid growth, tolerance of high temperatures, asexual sporulation, and the ability to cause extensive selective delignification of substrates with minimal growth and loss of carbohydrates (5, 11, 22). One * Corresponding author. fungus with these characteristics, Phanerochaete chrysosporium, has been used extensively in research on regulation mechanisms (12, 17, 19, 23) and applications of lignin degradation (18). More recently, other fungi such as Ceriporiopsis subvermispora (Pil). Gilbn. & Ryv. (10, 18) and Phlebia tremellosa (Schrad.: Fr.) Nakas. & Burds. (24) have been evaluated for their usefulness in biopulping processes. Taxonomic investigations on basidiomes and cultures of fungi in the Ganoderma lucidum (W. Curt.: Fr.) Karst. complex have identified several species that occur in North America (3, 4). Studies of culture characteristics and temperature relationships of these fungi have indicated that two species, G. colossum (Fr.) C. F. Baker and Ganoderma lucidum, can reproduce asexually with the production of chlamydospores in culture and have optimum temperatures above 30 C for growth in culture. In studies evaluating Ganoderma species for their potential to cause selective delignification of silver leaf oak (Quercus hypoleucoides A. Camus), white fir [Abies concolor (Gord. & Glend.) Lindl. ex Hildebr.], and other species of wood (2, 5), G. colossum, G. lucidum, G. oregonense Murr., and G. tsugae Murr. were identified as species capable of causing selective delignification of oak wood. In white fir wood, however, most species evaluated caused a simultaneous decay (2, 5). Of the Ganoderma species capable of causing selective delignification of oak, G. colossum is the only species that produces abundant chlamydospores, has an optimum temperature of 40 C for growth in culture, and is capable of growth at 45 C and higher 138

2 VOL. 61, 1995 DELIGNIFICATION OF SILVER LEAF OAK BY G. COLOSSUM 139 FIG. 1. PWL (A) and PKL/CHC (B) of oak after 4, 8, 12, 16, or 20 weeks of incubation for decay caused by G. colossum in agar block decay chambers incubated at 35 C. Data are means and standard deviations where n 10 for PWL and n 6 for PKL/CHC. (4, 5). In these initial studies, however, experiments were conducted at only one temperature (35 C) and incubation time (12 weeks). The purpose of this study was to determine the effects of time and temperature of incubation on selective delignification of silver leaf oak caused by G. colossum, using Klason lignin and chlorite holocellulose analyses and scanning electron microscopy to determine biochemical and morphological changes in wood structure after decay. MATERIALS AND METHODS Sources of fungi and wood. Basidiomes of G. colossum were collected in Florida. Collection RLG15829 was obtained from Celtis laevigata Willd., University of Florida, Gainesville, and collection JEA529 was collected by J. Gibson from Phoenix canariensis Hort. ex. Chabaud, University of Florida, Gainesville. These isolates were obtained by plating context tissue of basidiomes on 2% malt extract agar. Sound wood for test blocks was cut from silver leaf oak obtained from Bear Canyon, Santa Catalina Mountains, Coronado National Forest, Ariz. Decay tests. Agar block decay chambers were prepared by a modification of the method described by Adaskaveg and Gilbertson (2). Fungi were grown on 2% malt extract agar for 1 week prior to inoculation of decay chambers. Wood blocks (2 by 2 by 4 cm) were cut from freshly cut trees, dried at 100 C for 16 h in a recirculating oven, and weighed. The blocks were soaked in distilled water for 10 min, blotted dry, and sterilized at 120 C and kpa (16 lb/in 2 )for1h in an autoclave. They were then placed on autoclaved, 3-mm, V-shaped glass rods in 235-ml French square glass decay chambers containing 50 ml of malt extract agar (1 block per chamber). Six decay chambers were used for each isolate and temperature treatment. Six noninoculated wood blocks served as controls. Assembled decay chambers were incubated in the dark for 12 weeks at 25, 30, 35, 40, or 45 C or, in separate experiments, for 0 (control wood block), 4, 8, 12, 16, and 20 weeks at 35 C. Five blocks for each isolate were brushed clean FIG. 2. PWL (A) and PKL/CHC (B) of oak after 12 weeks of incubation for decay caused by G. colossum in agar block decay chambers incubated at 25, 30, 35, 40, or 45 C. Data are means and standard deviations where n 10 for PWL and n 6 for PKL/CHC. of superficial mycelium, oven dried with control wood blocks, and weighed as described above. The sixth block was used for scanning electron microscopy. Percent weight loss (PWL) was determined as follows: (weight loss of oven-dried wood/weight of oven-dried original wood) 100. For each treatment, one wood block with a weight loss approximately equal to the mean of the five replications was chosen for chemical analysis. Klason lignin and chlorite holocellulose analyses were performed by techniques described previously (2, 14). These analyses were conducted on three different samples for the median wood block for each incubation period or temperature treatment and fungal isolate. Data were expressed as a ratio of the percent Klason lignin to percent chlorite holocellulose (PKL/CHC). Percentages were based on the weight of each component compared with the weight of the wood sample analyzed. Values of PKL/CHC that were lower than the PKL/CHC value of the control wood block indicated preferential degradation of lignin. Data on percent weight loss and data from the different chemical analyses for wood samples decayed by each isolate were analyzed by analysis of variance and least significant difference mean separation procedures of SAS version 6.04 (SAS Institute Inc., Cary, N.C.). Electron microscopy. For scanning electron microscopy (SEM), one block of each of the six replications was cut into 100- to 200-mm 3 pieces and placed in 4% formaldehyde 1% glutaraldehyde (Ted Pella Inc., Redding, Calif.) in 0.10 M phosphate buffer (ph 7.2) for 72 h at 25 C. Samples were then placed in an alcohol dehydration series of 20, 50, 80, 95, 95, 100, and 100% ethanol for 15 to 30 min per treatment and either stored in alcohol or critical point dried with carbon dioxide. Dried wood samples were then sectioned with a razor blade or broken to an approximate size of 5 by 5 by 3 mm, exposing one of three sections of wood: transverse, radial, or tangential. Sections were then mounted on SEM stubs, sputter coated to a thickness of 30 nm with gold, and observed in an ISI DS-130 scanning electron microscope. Pits of fiber tracheid and ray parenchyma cells were compared for nondecayed and decayed wood. For this, 25 pits of each cell type were randomly selected on scanning electron photomicrographs for different temperature and incubation period treatments and the length and width of pit apertures were measured. Data were analyzed by analysis of variance and least significant difference mean separation procedures of SAS.

3 140 ADASKAVEG ET AL. APPL. ENVIRON. MICROBIOL.

4 VOL. 61, 1995 DELIGNIFICATION OF SILVER LEAF OAK BY G. COLOSSUM 141 FIG. 3. Scanning electron micrographs of radial and tangential faces of oak nondecayed or decayed by G. colossum RLG after 0 (control), 4, 8, 12, 16, or 20 weeks at 35 C. Bars, 25 m. (A) Radial section of nondecayed (control) wood showing uniseriate ray cells (r) and densely packed fiber tracheids (t). (B) Radial section of decay after 4 weeks. Individual ray cells (r) and bordered pits of fiber tracheids (t) were observed in delignified wood. (C and D) Tangential sections showing delignified rays (r) and fiber tracheids (t) after 8 weeks. Overlapping tapered ends of fiber tracheids and gaps (arrowheads) between wood cells were observed where middle lamella was removed. (E and F) Tangential sections showing delignified rays (r) and fiber tracheids (t) after 12 weeks. Bordered pits and gaps between fiber tracheids were observed. (G and H) Radial sections of decayed wood after 16 and 20 weeks, respectively. Extensive selective delignification of fiber tracheids (t) and severe degradation of rays (r) were observed. RESULTS Weight loss and degradation of structural components. The average PWL and chemical analyses of decayed wood blocks for both isolates of G. colossum studied are shown in Fig. 1 and 2. Values for PWL and PKL/CHC for each isolate were not corrected for the PWL of the controls ( 3%) that occurred during sterilization and preparation procedures. Temperature and incubation time significantly (P 0.01) influenced weight loss and changes in PKL/CHC caused by G. colossum. No significant differences (P 0.10) in weight loss or PKL/CHC were observed between isolates of G. colossum used in this study. Furthermore, there was no significant relationship between isolates and temperature or isolates and incubation time (P 0.10). PWL increased from 6.1 to 32.5% from 4 to 16 weeks at 35 C (Fig. 1A). After 20 weeks, PWL remained at 33.0%. Chemical analyses of these wood blocks indicated that both Klason lignin and chlorite holocellulose were removed from the oak wood blocks exposed to G. colossum when compared with noninoculated wood blocks. Values of PKL/CHC averaged from 0.35 for the control wood block to a low of 0.22 for the 12-week incubation period (Fig. 1B). In studies comparing temperature effects after a 12-week incubation for decay, PWL averaged 26 to 27% between 30 and 40 C for the isolates evaluated (Fig. 2A). Minimum decay levels of 15.2 and 14.5 PWL occurred at 25 and 45 C, respectively. Average values of PKL/CHC decreased incrementally from 0.25 at 25 C to 0.17 at 40 C, compared with 0.33 in the noninoculated control wood blocks (Fig. 2B). At 45 C, PKL/ CHC was Morphological evidence of decay. Scanning electron microscopy indicated that in radial and tangential sections of nondecayed oak, fiber tracheids and ray parenchyma cells were densely packed and adhered to each other (Fig. 3A). Bordered pits had thickened walls and were not clearly distinguishable in most fiber tracheid cells observed (Fig. 3A). At 35 C, decay was observed in wood tissues for all incubation times evaluated (4 to 20 weeks) and ranged from minimal changes to extensive degradation. After 4 weeks, minimal decay was observed in ray parenchyma and fiber tracheids (Fig. 3B). Individual ray parenchyma cells and bordered pits of fiber tracheids were readily discernible. Extensive selective delignification was observed after 8 weeks (Fig. 3C and D). In these wood samples, fiber tracheids were observed with overlapping tapered ends, whereas ray parenchyma cells commonly had exposed pits (Fig. 3C). Additionally, fiber tracheids were often loosely arranged (Fig. 3D). After 12 weeks, individual fiber tracheids and ray parenchyma cells were distinctly separated and pits of both cell types were enlarged (Fig. 3E and F). Analyses of pit size in the 12-week treatment indicated that pit aperatures of decayed fiber tracheids were significantly different in size from (P 0.05) and nearly twice as large as (average, 29.7 by 45.8 m) those in nondecayed wood (11.3 by 26.5 m). Pit apertures of ray parenchyma were also significantly (P 0.05) enlarged compared with the control (Fig. 3E and F). As in the 8-week treatment, ray parenchyma and fiber tracheids were loosely arranged within the wood tissues. After 16 and 20 weeks, fiber tracheids were similar in appearance to those in the 12-week treatment (Fig. 3G); however, ray parenchyma cells were severely degraded (Fig. 3H). Evidence of minimal decay, selective delignification, and simultaneous decay was observed in the inoculated wood exposed for 12 weeks to the different temperatures evaluated (Fig. 4). Minimal decay was observed at 25 C (Fig. 4A); individual fiber tracheids and pits of ray parenchyma and fiber tracheids were not clearly discernible. Initial stages of delignification were evident at 30 C (Fig. 4B), and extensive delignification was observed at 35 C (Fig. 4C and D). Vessels had exposed pit regions, and individual cells were identified, whereas fiber tracheids were separated from each other and had exposed bordered pits (Fig. 4C). Gaps occurred between these cells where the middle lamella was degraded (Fig. 4D). Rays were also delignified with exposed pit regions. At 40 C, evidence of selective delignification and extensive decay of wood tissues was observed (Figs. 4E and F). Fibers and vessels had exposed pits and significantly (P 0.05) enlarged pit apertures. Ray parenchyma cells were extensively decayed, with coalescing and enlarged pit apertures (mean diameter, 25.8 m) that were significantly greater (P 0.05) than those in wood decayed at 30 C (7.5 m). In some regions of wood, ray parenchyma cells were completely degraded (Fig. 4F). Simultaneous decay was observed at 45 C (Fig. 4G). The middle lamella was not degraded, and bordered pits of fiber tracheids and pit regions of vessels were not clearly discernible. Rays were nearly completely degraded, and gaps in walls of fiber tracheids were observed (Fig. 4G). Additionally, pits of rays adjacent to vessels and fiber tracheids were enlarged and coalescing (Fig. 4G). DISCUSSION Decay caused by G. colossum was dependent on time of incubation and temperature as indicated by PWL and PKL/ CHC. Both isolates used in this study responded similarly for the incubation time and temperature ranges evaluated. Maximum selective delignification occurred after 12 weeks of incubation at 35 C, as indicated by the lowest PKL/CHC ratio (0.22), compared with the control wood blocks (0.35). An average PWL of only 17.4% occurred at 8 weeks, whereas after 16 and 20 weeks PWL was greater than 30%. In our previous study evaluating decays caused by Ganoderma species, one incubation time of 20 weeks at 35 C was used to evaluate decay caused by G. colossum (5). Therefore, the selective delignification potential of G. colossum was underestimated in the earlier study because longer incubation times did not increase selective delignification. Instead, PKL/CHC increased from 0.22 to 0.30 for longer incubation times (Fig. 1). The increase in these values indicated a greater removal of wood polysaccharides. Thus, delignification of oak caused by G. colossum is time dependent, with preferential removal of lignin in the first 8 to 12 weeks of decay, whereas greater degradation of cell wall polysaccharides occurred during longer incubation periods. Previous studies with other decay fungi have also shown that longer incubation times did not increase selective delignification (20, 25), and other in vitro evaluations screening fungi for

5 142 ADASKAVEG ET AL. APPL. ENVIRON. MICROBIOL.

6 VOL. 61, 1995 DELIGNIFICATION OF SILVER LEAF OAK BY G. COLOSSUM 143 FIG. 4. Scanning electron micrographs of radial and transverse sections of oak decayed by G. colossum RLG after 12 weeks at 25, 30, 35, 40, or 45 C. Bars, 25 m. (A) Tangential section of wood decayed at 25 C showing little degradation of fiber tracheids (t) and rays (r). (B) Radial section of decay at 30 C showing partial selective delignification of wood tissues as indicated by exposed bordered pits of fiber tracheids (t) and rays (r). (C and D) Tangential sections of wood after decay at 35 C. Delignified vessels (v) and fiber tracheids with exposed pits and gaps (arrowheads) appear between cells where middle lamella is absent. (E and F) Radial sections of wood after decay at 40 C, showing extensive selective delignification of vessels (v) and fiber tracheids (t). Parenchyma cells of rays (r) were extensively degraded. Boreholes (arrowhead) and coalescing bordered bits (double arrowhead) were also commonly observed. (G) Radial section of wood decayed at 45 C. Fiber tracheids (t) had gaps in their cell walls (arrowheads), whereas the middle lamella remained mostly intact. Rays were extensively decayed with enlarged, coalescing pits (double arrowhead). selective delignification have been conducted for 12 weeks (1, 11, 22). In our 12-week decay studies evaluating the effects of temperature, lower PKL/CHC occurred in wood blocks exposed to temperatures from 25 to 40 C with an optimum range near 35 to 40 C (Fig. 2). Little to no selective delignification occurred at 45 C (Fig. 2B), although the average PWL was Thus, these studies show that the conditions for selective delignification of silver leaf oak are near 35 to 40 C after 8 to 12 weeks of incubation. The temperatures of 25 and 45 C represent the lower and upper limits for growth of G. colossum in culture (4), and therefore these temperatures were not conducive to selective delignification. In a study on decay tests for screening white rot fungi for their potential to delignify wood, Setliff and Eudy (25) concluded that most wood decay fungi, with the possible exception of tropical wood decay fungi, grow better at 30 C than at higher temperatures. Subsequently, most screening studies have used one temperature between 27 and 30 C (5, 11, 22). Our study with G. colossum, however, shows that higher temperatures will probably be required to determine the full potential of subtropical and tropical fungi to delignify wood. Scanning electron micrographs of decayed wood correlated with chemical data on decay and confirmed the optimal conditions for selective delignification. This is the first study to use SEM to illustrate the effects of time and temperature of incubation on decay by a wood-rotting fungus. Morphologically, selective delignification was observed as removed middle lamella, clearly defined fiber tracheids with overlapping tapered ends, fiber tracheids that had exposed bordered pits with enlarged apertures, and ray parenchyma and vessels with exposed pit regions (Fig. 3C to G and 4C to F). Simultaneous decay was observed as degradation of entire cell walls (e.g., ray parenchyma), coalescing pits of ray parenchyma and vessels, and localized degradation of middle lamella and the S 2 wall layer of fiber tracheids (Fig. 3H and 4G). Spatially, selective delignification was uniformly distributed throughout the wood blocks that were exposed to decay from 8 to 16 weeks at 35 C or after 12 weeks at 35 to 40 C. Selective delignification was less evident morphologically in the 4-week incubation at 35 C and after 12 weeks at 25 and 30 C. Although some selective delignification occurred at these times and temperatures, as shown by chemical analyses, longer incubations ( 8 weeks) and higher temperatures were needed before selective delignification by G. colossum could be readily detected by SEM. As indicated in the chemical data (Fig. 2B) and as observed by SEM (Fig. 4G), simultaneous decay was observed at 45 C. Simultaneous decay was reported for G. colossum on white fir and date palm wood but not oak wood (1, 5). Thus, this is the first report of a wood decay fungus that caused a temperaturedependent selective delignification of oak wood involving greater than 14% weight loss after 12 weeks of incubation. Research on industrial applications during the last several years has led to evaluation of the usefulness of several white rot fungi for biopulping of wood and for bioremediation of toxic wastes that contaminate soil and water (10, 16, 18). For example, several fungi, including P. chrysosporium, P. tremellosa, and Phlebia brevispora Nakas. of aspen and P. chrysosporium and Ceriporiopsis subvermispora of pine wood chips have been successfully used in biopulping (6, 10, 18, 24). Use of these fungi generally resulted in improvement of paper quality and in reduction of energy costs in paper production (18, 24). Recently, selected Ganoderma species have been shown to cause extensive selective delignification of hardwoods (2, 5, 9, 13) and conifers (5, 8). In this study, we determined that G. colossum can cause different degrees of selective delignification of wood blocks (decay times of 4 to 12 weeks) at temperatures of 30 to 40 C. Thus, from this and a previous study (5), these data suggest that G. colossum may be an excellent white rot fungus for additional studies on industrial applications of biopulping and bioremediation, as well as biochemical mechanisms of selective delignification. ACKNOWLEDGMENTS We thank Peggy A. Mauk for technical assistance throughout this study and David Thompson for assistance in the chemical analyses. REFERENCES 1. Adaskaveg, J. E., R. A. Blanchette, and R. L. Gilbertson Decay of date palm wood by white-rot and brown-rot fungi. Can. J. Bot. 69: Adaskaveg, J. E., and R. L. Gilbertson In vitro decay studies of selective delignification and simultaneous decay by the white rot fungi Ganoderma lucidum and G. tsugae. Can. J. Bot. 64: Adaskaveg, J. E., and R. L. Gilbertson Basidiospores, pilocystidia, and other basidiocarp characters in several species of the G. lucidum complex. Mycologia 80: Adaskaveg, J. E., and R. L. Gilbertson Cultural studies of four North American species in the Ganoderma lucidum complex with comparisons to G. lucidum and G. tsugae. Mycol. Res. 92: Adaskaveg, J. E., R. L. Gilbertson, and R. A. Blanchette Comparative studies of delignification caused by Ganoderma species. Appl. Environ. Microbiol. 56: Akhtar, M., M. C. Attridge, G. C. Myers, T. K. Kirk, and R. A. Blanchette Biomechanical pulping of loblolly pine with different strains of the white-rot fungus Ceriporiopsis subvermispora. Tappi J. 75: Akin, D. E., Sethuraman, W. H. Morrison III, S. A. Martin, and K.-E. Eriksson Microbial delignification with white rot fungi improves forage digestibility. Appl. Environ. Microbiol. 59: Blanchette, R. A Selective delignification of eastern hemlock by Ganoderma tsugae. Phytopathology 74: Blanchette, R. A Delignification by wood-decay fungi. Annu. Rev. Phytopathol. 29: Blanchette, R. A., T. A. Burnes, M. M. Eerdmans, and M. Akhtar Evaluating isolates of Phanerochaete chrysosporium and Ceriporiopsis subvermispora for use in biological pulping processes. Holzforschung 46: Blanchette, R. A., T. A. Burnes, G. F. Leatham, and M. Effland Selection of white-rot fungi for biopulping. Biomass 15: Bonnarme, P., M. Delattre, G. Corrieu, and M. Asther Activation of lignin and manganese peroxidase excretion in Phanerochaete chrysosporium by fungal extracts. Appl. Microbiol. Biotechnol. 37: Dill, I., and G. Kraepelin Palo podrido: model for extensive delignification of wood by Ganoderma applanatum. Appl. Environ. Microbiol. 52: Effland, M Modified procedure to determine acid-insoluble lignin in wood and pulp. Tappi 60: Eriksson, K. E., R. A. Blanchette, and P. Ander Microbial and enzymatic degradation of wood and wood components. Springer-Verlag, New York. 16. Eriksson, K. E., and T. K. Kirk Biopulping, biobleaching, and treatment of kraft bleaching effluents with white rot fungi, p In C. L.

7 144 ADASKAVEG ET AL. APPL. ENVIRON. MICROBIOL. Cooney and A. E. Humphrey (ed.), Comprehensive biotechnology. Pergamon Press, Toronto. 17. Faison, B. D., and T. K. Kirk Factors involved in the regulation of ligninase activity in Phanerochaete chrysosporium. Appl. Environ. Microbiol. 49: Kirk, T. K., R. P. Burgess, and J. W. Koning, Jr Use of fungi in pulping wood: an overview of biopulping research, p In G. F. Leatham (ed.), Frontiers in industrial mycology. Proceedings of Industrial Mycology Symposium. Routledge, Chapman & Hall, New York. 19. Kirk, T. K., and R. L. Farrell Enzymatic combustion : the microbial degradation of lignin. Annu. Rev. Microbiol. 41: Kirk, T. K., and W. E. Moore Removing lignin from wood with white-rot fungi and digestibility of resulting wood. Wood Fiber 4: Myers, G. C., G. F. Leatham, T. H. Wegner, and R. A. Blanchette Fungal pretreatment of aspen chips improves strength of refiner mechanical pulp. Tappi J. 71: Otjen, L., R. A. Blanchette, M. Effland, and G. Leatham Assessment of 30 white rot basidiomycetes for selective lignin degradation. Holzforschung 41: Perez, J., and T. W. Jeffries Roles of manganese and organic acid chelators in regulating lignin degradation and biosynthesis of peroxidases by Phanerochaete chrysosporium. Appl. Environ. Microbiol. 58: Reid, I. D Biological pulping in paper manufacture. Trends Biotechnol. 9: Setliff, E. C., and W. W. Eudy Screening white-rot fungi for their capacity to delignify wood, p In T. K. Kirk, T. Higuchi, and H.-M. Chang (ed.), Lignin biodegradation: microbiology, chemistry, and potential applications, vol. 1. CRC Press, Inc., Boca Raton, Fla.