Biomass 15(1988) 93-101 Selection of White-rot Fungi for Biopulping* Robert A. Blanchette, Todd A. Burnes Department of Plant Pathology, University of Minnesota. St Paul, Minnesota 55108, USA Gary F. Leatham & Marilyn J. Effland USDA Forest Products Laboratory, Madison. Wisconsin 53705, USA (Received 17 November 1987; accepted 29 November 1987) ABSTRACT Different rates of wood decay and ligninolytic activity were found in wood decayed by various white-rot fimgi. Chemical and ultrastructural analyses showed wood decayed by Coriolus versicolor consisted of a nonselective attack on all cell wall components. Lignin degradation was restricted to the cell wall adjacent to hyphae or around the circumference of cell lumina. Decay by Phellinus pini, Phlebia tremellosus, Poria medullapanis and Scytinostroma galactinum was selective for lignin degradation. Secondary walls were void of lignin and middle lamelae were extensively degraded. A diffuse attack on lignin occurred throughout all cell wall layers. Variation in ligninolytic activity was found among strains of Phanerochaete chrysosporium. Differences in weight loss as well as lignin and polysaccharide degradation were also found when wood of different coniferous and deciduous tree species was decayed by various white-rot fungi. Key words: White-rot fungi, wood decay, Coriolus versicolor, Phellinus pini, Phlebia tremellosus, Poria medullapanis, Scytinostroma galactinum. INTRODUCTION White-rot fungi are a diverse group of organisms that are able to degrade lignin. There are many morphologically different patterns of white rot *Paper No. 15,534, Scientific Journal Series. Minnesota Agriculture Experiment Station, St Paul, Minnesota 55108. 93 Biomass 1988 Elsevier Applied Science Publishers Ltd. England. Printed in Great Britain
94 R. A. Blanchette, T. A. Burnes, G. F. Leatham, M. J. Effland that occur in wood due to the variation in the way lignin and polysaccharides are removed. Differences in the amount of lignin, cellulose and hemicellulose degraded from wood and the sequence in which these cell wall components are attacked have been reported. 1-3 Some white-rot fungi have the ability to selectively remove extensive amounts of lignin with only slight losses of cellulose and moderate to low losses of hemicellulose. 1,12 Lignin degradation maybe restricted to isolated areas of the wood and occur in pockets of decay 5 or may be extensive throughout large areas of the substrate. 6-8 Decay resulting from these wild type organisms is chemically and morphologically similar to decay by cellulase-less mutants previously reportedly 9,10 There are many white-rot fungi that are not selective for lignin degradation and large losses of polysaccharides also are removed. Coriolus versicolor is an example of a white-rot fungus that causes a simultaneous degradation of all cell wall components. It is a species that has been repeatedly used in assays as a representative of all white-rot fungi. However, there is a great deal of variation among the white rots. 4 In addition to those that are either selective or nonselective for lignin degradation, it is possible to find fungi that cause both types of white-rot attack within one substrate. 1,6,11 This results in a mottled pattern of delignified wood and simultaneous white-rotted wood. Other types of white-rot attack also have been reported. 4 The degradation of lignin by white-rot fungi, especially those that selectively degrade lignin from wood, is a characteristic that makes them ideally suited for industrial applications where lignin or various phenolic compounds must be altered or removed. The potential use of white-rot fungi for biopulping, biobleaching and treatment of pulp mill waste effluents has been reviewed 13,14 and is also discussed by others in these proceedings. To obtain the best possible species for use in new biotechnological processes, numerous species of white-rot fungi have been screened for rapid growth and superior rates of lignin degradation. 15,16 This paper describes the results from selection studies where several white-rot fungi were Screened for potential industrial use, examines the diversity among different strains of fungi and evaluates several whiterotters on different deciduous and coniferous woods. MATERIALS AND METHODS Laboratory decay studies were completed with wood blocks of Abies balsamea, Acer saccharum, Alnus rubra, Betula papyrifera, Picea mariana, Pinus resinosa, Pinus strobus, Populus tremeloides and Tilia
Selection of white-rot fungi for biopulping 95 americana. Blocks of wood were prepared, inoculated and incubated as previously described. 1 Selection of several white-rot fungi for use in these studies was made from screening large numbers of different white rot fungi. 15,16 Sample preparations for ultrastructural investigations were those of Blanchette and Reid 7 and Blanchette et al. 17 Lignin and wood sugar analyses were done using the methods of Effland 18 and Pettersen et al., 19 respectively. RESULTS Large differences in amounts of lignin removed from birch wood were found among the white-rot fungi tested (Table 1). The percent of glucose and xylose lost during degradation, representing the cellulose and xylan content of the wood, was also different among the different fungi. C. versicolor was least selective for lignin degradation whereas extensive preferential lignin removal was found in wood decayed by Scytinostroma galactinum. Lignin loss by all white-rot fungi tested was always accompanied by a loss of some polysaccharide (Table 1). Ultrastructural observations of transverse sections from decayed birch wood fixed in KMnO 4 showed different patterns of lignin degradation from the wood. C. versicolor removed all cell wall components of the secondary wall from the cell lumen toward the middle lamella (Figs 1(A) and (B)). Cell wall degradation and lignin removal occurred directly adjacent to fungal hypha and within a shallow zone of the secondary wall located next to the cell lumen. The loss of lignin never extended very deep within the secondary wall (Figs 1 (A) and (B)). In parts of the secon-
96 R. A. Blanchette, T. A. Burnes, G. F. Leatham, M. J. Effland Figs 1 (A) to (D). Transmission electron micrographs of transverse sections from birch wood fixed with KMnO 4 ((A) and (B)). Decay by Coriolus versicolor showing an erosion of the cell wall. The secondary wall was degraded from the lumen toward the middle lamella. In some areas, decay advanced through the secondary walls and middle lamella of adjacent cells resulting in holes forming within the cell walls (arrowheads). Lignin degradation was restricted to the exposed surface of the cell wall around the circumference of the lumina ((C) and (D)). Decay by Phellinus pin showing extensive delignification of the cells. Secondary walls were void of lignin and middle lamella was degraded (arrows). Lignin degradation was evident throughout all cells. The delignified secondary walls appeared slightly swollen but no erosion troughs or holes were evident. dary wall where portions of the wall had been totally degraded, the exposed middle lamella was also attacked and holes developed between cells (Figs 1(A) and (B)). In wood degraded by Phellinus pini, Poria medulla-panis and Scytinostroma galactinum cells were void of lignin. The secondary wall layers were free of lignin and middle lamella had been completely degraded. A type of diffuse attack on lignin is shown in
Selection of white-fungi for biopulping 97 birch wood decayed by P. pini (Figs 1(C) and (D)). Lignin was removed throughout the cells but no erosion of the secondary wall layers was evident. The secondary walls were slightly altered, however, after delignification and appeared to be swollen when compared to cells of sound wood. In a few delignified cells a thinning of the secondary wall was also apparent. In these cells the cellulose-rich secondary wall was partially degraded after lignin had been removed. This situation was more common in cells of wood degraded by Dichomitus squalens. Delignified fibers were eroded from the lumen causing an overall thinning of remaining secondary walls. Cellulose degradation by D. squalens was also evident in the wood sugar analyses of decayed wood (Table 1). Although some white-rot fungi, such as D. squalens, preferentially degrade lignin during early stages of degradation, cellulose utilization may be initiated after delignification. Differences in lignin and polysaccharide losses were also evident in wood decayed by different strains of the white-rot fungus. Phanerochaete chrysosporium. Five different isolates differed in the extent of weight loss resulting in the wood as well as lignin and polysaccharide degradation (Table 2). Strain BKM-F-1767 caused the greatest amount of lignin loss (73%) with only slight loss of glucose and moderate loss of xylose (15 and 55%, respectively). The effect of different wood substrates on decay by white rot fungi was also determined (Table 3). Large differences in the percent weight loss and degradation of lignin, glucose and xylose were evident. In general, C. vericolor caused a nonselective degradation of all cell wall components. Greater amounts of decay were found in the deciduous wood species and in balsam fir than in spruce and pine wood. P. pini and Phlebia tremellosus caused a preferential degradation of lignin in several deciduous and coniferous woods. Some substrates, such as P. resinosa,
98 R. A. Blanchette, T. A. Burnes, G. F. Leatham, M. J. Effland had very high losses of lignin when degraded by P. pini, whereas decayed P. strobus wood lost less lignin. P. tremellosus appeared to cause large losses of lignin with relatively low amounts of weight loss in the deciduous woods as well as in balsam fir. DISCUSSION These results indicate that there are many wild type white-rot fungi that selectively degrade large amounts of lignin from wood. The strain of the
Selection of white-rot fungi for biopulping 99 fungus and type of wood substrate may significantly influence the rate of degradation and selectivity of lignin removal. Differences in ligninolytic activity among strains of P. chrysosporium demonstrate the diversity that exists within a species. The results presented here show that strain BKM-F-1767 caused greater losses of lignin than the other strains tested. Differences in ligninolytic activity among strains of P. chrysosporium have recently been reported. 20 In this study, differences in ligninase activity and titer were noted. The greatest activity was from strain BKM-F-1767. The results from these studies demonstrate that once suitable species of white-rot fungi have been obtained, increases in ligninolytic activity, growth rate, or other desirable characteristics may be obtained by strain selection. Selection of the best strain of S. galactinum was found to be successful and an isolate with very high ligninolytic activity was identified (author, unpublished data). The results obtained for the best strain tested are presented in Table 1. The degradation of different wood substrates by C. versicolor, P. pini, and P. tremellosus showed considerable variation in overall degradation and ligninolytic activity (Table 3). The type of wood used in various pulping processes may require a specific white-rot fungus that is best suited for the particular substrate. In general, white-rot fungi such as C. versicolor cause greater losses in deciduous woods than coniferous woods. This has been previously reported by many investigators. 21,22 The results presented here, however, indicate that other white-rot fungi, such as P. pini and P. tremellosus, can degrade substantial amounts of lignin at relatively low weight losses in wood from many different species of trees. The selection of the best species, or strain of a particular species will ultimately depend on the substrate to be degraded. Ultrastructural observations confirmed the lignin and wood sugar analyses and demonstrated that there are different patterns of cell wall attack by white-rot fungi. The removal of lignin from the cell wall by C. versicolor was restricted to the wall layers near the hypha or cell lumen. Lignin removal did not extend deeply into the adjacent cell wall layers. Recently, white-rotted wood treated with bromine and analyzed with energy dispersive X-ray microanalysis showed similar results. 17 In contrast, decay by P. pini and other selective lignin degraders removed extensive amounts of lignin from all cell wall layers. Although during early stages of attack the secondary wall was delignified from the lumen toward the middle lamella, 17 lignin degradation ultimately occurred throughout the cell wall. In later stages of decay, cell walls may be completely delignified leaving the cellulose-rich secondary wall relatively unaltered. The results presented here indicate that there are many wild type species of white-rot fungi with great potential for use in biopulping.
100 R. A. Blanchette, T. A. Burnes, G. F. Leatham, M. J. Effland Selection of strains with superior ligninolytic activity for use on a particular wood substrate is possible and should provide an array of excellent isolates. ACKNOWLEDGEMENTS The author thanks L. Otjen for data in Table 2 and A. Abad for technical assistance. Cultures of Scytinostroma galactinum were provided by Drs J. Worrall and T. Harrington. REFERENCES
Selection of white-rot fungi for biopulping 101