Use of Fungi in Pulping Wood: An Overview of Biopulping Research

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1 7 Use of Fungi in Pulping Wood: An Overview of Biopulping Research T. Kent Kirk, Richard R. Burgess, and John W. Koning, Jr. Introduction Fresh wood chips destined and stored for pulp production are rapidly colonized by a variety of microorganisms, including many species of fungi. These organisms compete vigorously while easily assimilable foodstuffs last, and then their populations decrease. They are replaced by fungi that are able to degrade and gain nourishment from the cell wall structural polymers: cellulose, hemicelluloses, and lignin. Left unchecked, these last colonizers. mostly white-rot fungi," eventually decompose the wood to carbon dioxide and water. Some of them selectively degrade the lignin component, which is what chemical pulping processes accomplish. Biopulping is the concept of deliberately harnessing whiterot fungi for pulping. Pulp is produced from wood by either chemical delignification, mechanical separation of the cells (fibers), or combinations of chemical and mechanical methods. Mechanical pulping methods are used increasingly because they give much higher yields (80% to 90% based on the wood) than chemcial methods (40% to 50% yields). They also are less polluting than chemical methods. and mills using these methods are much less expensive to buiid. Currently, about 25% of world pulp production is by mechanical means. The main disadvantages of mechanical pulping methods are the production of lower quality pulps, which are unsuitable for fiber products that need high strength properties, and the amount of energy required for production (and consequent cost). Chemical pretreatment of wood chips are used to enhance the strength properties of mechanical pulps. Making such chemimechanical pulps, however, generates chemical waste streams that mush be treated, and it lowers the pulp yield by removing wood substance (mainly hemicelluloses and lignin). Biopulping as studied to date is actually biomechanical pulping, the use of fungi to replace chemicals in pretreating wood for mechanical pulping. 99

2 100 / Kirk, Burgess, and Koning Past Work on Biopulping Early chemical analyses of wood partly decayed by certain white-rot fungi revealed that lignin had often been removed selectively; that is, the cellulose content had increased. Actually, some naturally white-rotted woods are so heavily delignified that they resemble chemical pulps and can be made into paper with excellent properties. Thus, the concept of biopulping was probably obvious to early investigators. Perhaps the first serious consideration of fungal delignification for pulping was by researchers at the West Virginia Pulp and Paper Company (now Westvaco Corporation) research laboratory in the United States in the 1950s. Their investigation resulted in a published article (Lawson and Still, 1957) that surveyed 72 lignin-degrading fungi and summarized what was known about how the fungi degrade lignin. At that time, very little was known. At about the same time, a study of the effect of natural decay of pine by white-rot fungi on chemical pulping showed that most paper strength properties increased as the extent of decay increased (Reis and Libby, 1960: Kawase, 1962). Any research on biopulping per se that might have been done by various companies from the 1950s to the present has not been published. Also unpublished, except as an internal report at the Forest Products Laboratory, was a 1972 study of biomechanical pulping by T. K. Kirk and Prof. Knut P. Kringstad, then at North Carolina State University in Raleigh (USA). Aspen wood chips were partly decayed by Rigidoporus ulmarius (Sow.: Fr. ) Imaz. and mechanically fiberized to a pulp, and the pulp was made into paper. Pulping these chips required fewer revolutions in the pulping apparatus than did the pulping of untreated control wood, suggesting lowered energy consumption; also, the paper from the biomechanical pulp was stronger. Similar work was done shortly thereafter at the Swedish Forest Products Laboratory (STFI) in Stockholm, and the first published paper on biopulping per se (Ander and Eriksson, 1975) described results very similar to those of Kirk and Kringstad. In 1976, the Swedish researchers patented a method for producing cellulose pulp (Eriksson et al., 1976). After the initial study, this group worked on various aspects of biopulping, primarily with the white-rot fungus Sporotrichum pulverulentum Novobranova. Mean while, our work at the Forest Products Laboratory of the USDA Forest Service in Madison focused on the mechanism of lignin degradation by a white-rot fungus that was tentatively referred to as Peniophora G. Both fungi were chosen because they grew and degraded lignin quite rapidly in comparison to other fungi; they also produced copious conidia and thus were easy to manipulate. It was a surprise to both laboratories when the two fungi were found to be synonymous, and they are now classified as Phanerochaete chrysosporium Burds. (Burdsall and Eslyn. 1974). The Swedish researchers made a number of contributions to biopulping (review: Eriksson and Kirk, 1985). They described the growth rates of P. chrysosporium

3 Use of Fungi in Pulping Wood / 101 through wood, finding that colonization of pulpwood chips is unlikely to be ratedetermining. Scanning and transmission electron microscopy were used to show the growth patterns in wood and the degradation patterns of the cell walls. The group conducted studies on biomechanical pulping, showing energy savings and paper strength improvements. A considerable effort went into developing cellulase-less mutants of selected white-rot fungi for biopulping (Johnsrud and Eriksson, 1985). Attempts by the group to scale up the biopulping process were not notably successful (Samuelsson et al., 1980). That work, however, was undoubtedly premature because insufficient information was available on how to scale up the fungal treatment. Subsequent work on a large scale with bagasse, done in cooperation with Cuban scientists, gave more promising results (Johnsrud et al., 1987). Biopulping received little attention outside of Sweden until our recent investigations. In one small study, Bar-Lev et al. (1982) reported that treatment of a coarse mechanical pulp with P. chrysosporium decreased the energy required for further fiberization and increased paper strength properties. Akamatsu et al. (1984) found that treatment of wood chips with any of 10 white-rot fungi decreased mechanical pulping energy; with three of the fungi (Trametes sanguinea, T. coccinea, and Coriolus hirsutus), treatment increased paper strength. Biopulping Consortium Research Taken together, the results of these various studies suggested to us in 1986 that biomechanical pulping merited a comprehensive investigation. Consequently, in April 1987 a cooperative research program on biopulping was established. involving the Forest Products Laboratory, the University of Wisconsin Biotechnology Center, and nine pulp and paper and related companies. The number of companies in Biopulping Consortium had grown to 20 by April The overall objective of the 5-year consortium research effort is to evaluate the scientific and technical feasibility of using a fungal pretreatment with mechanical pulping to save energy and/or improve pulp and paper properties. In addition. we have assumed that the fungal pretreatment will have less environmental impact than have chemical pretreatment, a significant factor in its own right. The Biopulping Consortium research group is divided into six closely coordinated teams. The fungal research team screens species and strains of white-rot fungi from culture collections, as well as new isolates. Screening is based on growth and wood decay rates and on selectivity for lignin degradation in wood. The team also works to optimize the fungal pretreatment and, importantly, produces fungal-treated chips for evaluation by the pulp and paper research team. The pulp and paper team determines energy consumption required for pulp production and measures pulp and paper properties. The enzyme team seeks to determine which of the extracellular enzymes secreted during the fungal

4 102 / Kirk, Burgess, and Koning pretreatment are beneficial for pulping and which are not beneficial. Emphasis is on the components of the lignin- and cellulose-degrading systems. The molecular genetics team has focused on P. chrysosporium and ultimately seeks to engineer improved strains for biopulping. Lignin- and cellulase-degrading systems again are the focus. An engineering and scale-up team is looking at the fungal pretreatment as an engineered solid substrate fermentation. and it is working with the fungal research team to determine critical parameters. Supporting the other teams is an information group. Using sophisticated computer search strategies, the information team screens the scientific literature and specializes in retrieving information from particular sources, such as Japanese patent applications. The industrial partners partially fund the project and provide input during semiannual meetings with the researchers. The industrial partners are provided with both research results and synopses of the expanding world literature of biotechnology as it affects or might affect the pulp and paper industry. Participation in the consortium also provides the industrial partners with ready personal access to biotechnology researchers (most of the companies do not have them in house) and acquaintance with students, postdoctoral associates, and technicians, who constitute a potential employee pool. The consortium has made good research progress. Some of the key published findings are summarized in the following paragraphs. Details are given in the cited papers. Research was initiated by screening species and strains of white-rot fungi for selective removal of lignin from wood blocks (Otjen et al., 1987: Blanchette et al., 1988). Wide variation was found among species and among strains within certain species. For example. in 12 weeks Peniophora hydnoides (Cke. and Mass. ) M.P. Chris. [ =Phanerochaete rimosa (Cke. ) Burds. ] removed 26% of the lignin and 24% of the glucan (cellulose) from birch wood. whereas P. chrysosporium Burds. (strain BKM F-1767) was highly selective and removed 73% of the lignin and only 15% of the glucan. Similarly, in 12 weeks, Heterobasidion annosum (Fr. ) Bref. removed nearly equal proportions of lignin and glucan (26% and 23%) from pine, whereas Ceriporiopsis subvermispora (Pil. ) Gilbn. et Ryv. removed 50% and 3% of lignin and glucan, respectively. Within the species P. chrysosporium, strain HHB removed 51% and 48% of the lignin and glucan from birch wood, wherease strain BKM F-1767, as noted, removed 73% and 15% of lignin and glucan. respectively, pointing to substantial intraspecies variation. Based on these initial screenings. several species-and in some cases, specific strains were chosen for biopulping studies. Screening continues. however, and some interesting new fungi have recently been selected for further study. A total of over 200 strains have been screened. Although selective removal of lignin does not correlate strictly with efficacy of biopulping pretreatment, the fungi selected by this method have proved to be effective for biopulping. Better screening methods are needed. A somewhat faster method for screening for selective lignin removal was described recently by Nishida et al. ( 1988); that

5 Use of Fungi in Pulping Wood / 103 method is based on the formation of color during growth of test strains of guaiacol-wood meal agar plates. The Biopulping Consortium reported recently on a more targeted biopulping screening procedure based on the effect of fungal treatment of coarse pulp on pulp trainability (Leatham and Myers, 1990). The method could be used to predict fungal efficacy insofar as improved paper strength properties were concerned, but it did not predict energy savings. An introductory study with Dichomitus squalens (Karst. ) Reid and P. chrysosporium B KM F-1767 with aspen wood chips showed large improvements in the paper strength properties of biomechanical pulps in comparison to the properties of controls (Myers et al., 1988). The chips in that study and in other studies described here-were initial] y supplemented with glucose, glutamate, and other nutrients prior to introducing the fungi. Dichometus squalens was allowed to decay the wood for 7 weeks, and P. chrysosporium for 4 weeks. Even so, total loss in wood weight was less than 270. The fungal pretreatment decreased the brightness (whiteness) of the pulps in this and in all studies to date, which is somewhat surprising because white-rot fungi are so named because they eventually bleach wood. Although the pulps are not difficult to bleach, the necessity of bleaching is a negative aspect of biopulping. Subsequent studies with additional fungi and aspen wood chips confirmed the enhancement of paper strength properties and also demonstrated that large energy savings for the pulping are possible (Leatham et al., 1990a, b. C). The fungi varied greatly in their effectiveness with aspen. Trametes versicolor had essentially no effect, despite good lignin degradation, whereas C. subvermispora, Phlebia tremellosa (Schrad.: Fr. ) Nakas. et Burds., and Phlebia brevispora Nakas. were quite effective. The fungi also varied greatly in their effectiveness for pretreating aspen compared to pine. Interesting] y, there was little correlation between removal of specific components of the wood by the fungi and efficacy of the fungal pretreatment for either energy savings or paper strength property improvement. This is unfortunate because such a correlation could have pointed to more rapid screening methods. There was also little correlation between energy savings and paper improvement. indicating that the changes in the wood cell walls that provide the beneficial effects are different for energy savings and for paper strength property improvement. Fortunately, pretreatment with some fungi, including P. chrysosporium, Phlebia subserialis (Bourd. et Galz. ) Donk, and P. brevispora, resulted in both energy savings and paper improvement. Properties of paper from aspen wood pulped by six commercial pulping processes and by biomechanical pulping were recently compared. Results showed that the biomechanical process produced a pulp that is comparable to a chemithermomechanical pulp in overall properties (Wegner et al., in press). Over 100 biopulping runs have now been completed on a 2 5-kg scale. Most of the work has been with P. chrysosporium on aspen and C. subvermispora on southern pine. Some of the most promising data obtained thus far are given in Table 7.1. These data are not atypical, but such results are not always obtained,

6 104 / Kirk, Burgess, and Koning for reasons that are under investigation. Factors influencing fun gal treatment time and overall efficacy are becoming clearer but much remains to be learned. Preliminary engineering calculations have also been completed on the fungal treatment (Wail et al., 1990). Several potential problems that generally occur in solid substrate fermentations ( Hesseltine, 1972) must be considered. These include system heterogeneity, difficulty of process monitoring and control. and large temperature and concentration gradients within the fermenter. In the case of biopulping, other aspects must also be recognized: (1) white-rot fungi grow more slowly than do most industrial fungi (although some. including P. chrysosporium, grow relatively rapidly); (2) pulp is a low-value, high-volume product: and (3) wood chips are large particles with significant intraparticle diffusion resistance. It is not yet clear whether asepsis will be required in largescale fungal pretreatment of wood chips. Calculations suggest that aeration will be a significant factor. An economics calculation based on energy savings alone indicated that in constructing a new mill (cost about $60 million), an investment of $6 million or $9 million for the fungal treatment facility could be justified if the fungal treatment resulted in 25% or 40% energy savings, respectively (Harpole, 1989). Simultaneous investigations have been aimed at understanding the basic mechanism of the beneficial effects of fungal pretreatment. Electron microscopy has been used to observe the growth and degradation patterns of P. chrysosporium in aspen wood chips (Sachs et al., 1989). The fungus grew rapidly over the chip surfaces, forming a mycelial network (Fig. 7. i); within the cells, erosion troughs could be seen on the lumen surfaces (Fig. 7.2). Three-week treatment with the fungus caused the normally rigid wood cell wall structure to swell and fragment (Fig. 3). Earlier work by Ruel et al. (i 98 i. i 984) showed that attack of spruce wood by P. chrysosporium resulted in a swelling and disruption of the lignin in the ceil walls. The lignin later appeared to aggregate into granules. The lignin-

7 Figure 7.1 Mycelial network of Phanerochaete chrysosporium on surface of aspen wood chip after 3 weeks of growth ( x 35) (Sachs et al ). H. hypha. 105

8 Figure 7.2 Erosion troughs (E) produced by enzymes secreted by Collapsed) Hyphae (H) of Phanerochaete chrysosporium lying on the lumen wall of aspen wood (Sachs et al., 1989). 106

9 Figure 7.3 The normally rigid wood cell wall structure of aspen wood (A) was modified by 3-week treatment with Phanerochaete chrysosporium (B) (Sachs et. al ).Modifications included cell will swelling (a). cnzymatic softening or relaxing, resulting in partial collapse of cell structure (b), localized areas of wall thinning (c). and fragmentatlon (d). 107

10 108 I Kirk, Burgess. and Koning rich middle lamella between the cells was resistant to attack. A cellulase-negative mutant caused only lignin swelling. Research on the enzymes of biopulping has focused mainly on the lignindegrading system of P. chrysosporium, with the assumption that lignin degradation is important to effective fungal pretreatment. Figure 7.4 illustrates schematically the probable makeup of the ligninolytic system in this organism. The key extracellular enzymes are thought to be lignin peroxidase and glyoxal oxidase. The latter oxidizes the metabolites glyoxal and methyl glyoxal and coupled reduction of molecular oxygen to hydrogen peroxide, which activates lignin peroxidase. Lignin peroxidase oxidizes nonphenolic aromatic nuclei in lignin by one electron, generating aryl cation radicals: these degrade nonenzymatically via many reactions. Most of those reactions result in polymer cleavages, generating both aromatic and aliphatic products. These are taken up by the hyphae and mineralized. A second kind of peroxidase, manganese peroxidase. oxidizes Mn 2+ to Mn 3+, which in turn can oxidize phenolic units in lignin. The role of such oxidation, if any, and that of manganese peroxidase are not yet clear. The aromatic metabolize veratryl alcohol seems to play multiple roles, including stimulation of biosynthesis of the enzymes and electron transfer reactions during substrate degradation. Biopulping Consortium enzyme research has helped characterize glyoxal oxidase (Kersten, 1990), and the possible roles of Mn 3+ (Popp et al.,

11 Use of Fungi in Pulping Wood / 109 unpublished). For recent reviews of lignin degradation by P. chrysosporium, see Kirk (1988) and Shoemaker and Leisola (1990). Molecular genetics research in connection with the Biopulping Consortium has been directed at elucidating the organization, structure, and function of the lignin peroxidase and cellulase (cellobiohydrolase) genes. That research has shown that multiple genes encode lignin peroxidases and cellulases (Schalch et al., 1989; and unpublished results). DNA hybridization of chromosomes separated by electrophoresis showed that the lignin peroxidase genes are clustered on a single chromosome. However, the cellulase genes are on at least three different chromosomes (unpublished results). In summary, our results to date and those of other investigations indicate that biopulping is a promising concept that deserves to be evaluated more completely. The key question, of course, is whether the pretreatment can be done economically; that is, whether the value of the beneficial effects exceeds the costs. Finding a good answer to that question requires more complete investigations of the many facets of biopulping. Acknowledgments The authors thank the following companies for support of the Biopulping Consortium research effort: Boise Cascade; Celulosa Arauco y Constitution S.A.; Champion International Corp.: Chimica del Friuli; Consolidated Papers, Inc.; Dow Chemical Co.; Great Northern Nekoosa Corp.: James River Corp.; Leykam A/S; Potlatch Corp.: Procter & Gamble; Sandoz Chemicals Corp.: Scott Paper Co.; Sproat-Bauer. Inc.; Union Camp Corp.; and Weyerhaeuser Paper Co. We also thank the Consortium researchers for their hard work. References Akamatsu. 1., Yoshihara, K., Kamishima. H., and Fujii. T Influence of white-rot fungi on poplar chips and thermo-mechanical pulping of fungi-treated chips. Mokuzai Gakkaishi 30: Bar-Lev, S. S. Kirk, T. K., and Chang, H.-m Fungal treatment can reduce energy requirements for secondary refining of TMP. Tappi Journal 65: Blanchette, R. A., Burnes, T. A.. Leatham, G. F., and Effland, M. J Selection of white-rot fungi for biopulping. Biomass 15: Burdsall, H. H.. Jr.. and Eslyn. W. E A new Phanerochaete with a chrysosporium imperfect state. Mycotaxon 1:

12 110 / Kirk, Burgess, and Koning Eriksson, K.-E., Ander. P.. Henningsson, B., Nilsson, T.. and Goodell, B Method for producing pulp. June , U. S., Patent No Eriksson, K.-E.. and Kirk. T. K Biopulping, biobleaching and treatment of kraft bleaching effluents with white-rot fungi. Pp. 27 I -294 in C. L. Cooney, and A. E. Humphrey (eds.), The Principles of Biotechnology: Engineering Considerations. In M. Moo-Young, (ed.), Comprehensive Biotechnology: The Principles, Applications and Regulations of Biotechnology in Industry, Agriculture and Medicine. Pergamon Press, New York. Has-pole, G. B., Leatham, G. F., and Myers, G. C Economic assessment of biomechanical pulping. In Proceedings of the International mechanical pulping conference 1989 Mechanical pulp-responding to the end product demands; 1989 June 6-8; Helsinki. 2: Hesseltine, C. W Solid state fermentations. Biotechnology and Bioengineering 14: Johnsrud, S. C., and Eriksson, K.-E Cross-breeding of selected and mutated homokaryotic strains of Phanerochaete chrysosporium K-3: New cellulase deficient strains with increased ability to degrade lignin. Applied Microbiology and Biotechnology 21: Johnsrud, S. C.. Fernandez, N., Lopez, P.. Guitierrez, I., Saez, A., and Eriksson, K.-E Properties of fungal pretreated high yield bagasse pulps. Nordic Pulp & Paper Research Journal, Special Issue 2: Kawase, K Chemical components of wood decayed under natural conditions and their properties. Journal of Faculty of Agriculture, Hokkaido University 52: Kersten, P. J Glyoxal oxidase of Phanerochaete chrysosporium: Its characterization and activation by lignin peroxidase. Proceedings of the National Academy of Sciences USA 87: Kirk, T. K Lignin degradation by Phanerochaete chrysosporium. ISI Atlas of Science: Biochemistry I: Lawson, L. R., Jr. and Still, C. N The biological decomposition of lignin Literature survey. Tappi Journal 40:56A-80A. Leatham, G. F.. and Myers, G. C A PFI mill can be used to predict biomechanical pulp strength properties. Tappi Journal 73: Leatham, G. F., Myers, G. C.. and Wegner, T. H. 1990a. Biomechanical pulping of aspen chips: energy savings resulting from different fungal treatments. Tappi Journal 73: Leatham, G. F., Myers. G. C., Wegner, T. H., and Blanchette, R. A. 1990b. Biomechanical pulping of aspen chips: paper strength and optical properties resulting from different fungal treatments. Tappi Journal 73: Leatham. G. F., Myers, G. C., Wegner, T. H., and Blanchette, R. A. 1990c. Energy savings in biomechanical pulping. Pp in T. K. Kirk and H.-m. Chang (eds.), Biotechnology in Pulp and Paper Manufacture. Butterworth Publishers, Stoneham, MA. Myers, G. C., Leatham, G. F., Wegner, T. H., and Blanchette, R. A Fungal

13 Use of Fungi in Pulping Wood / 111 pretreatment of aspen chips improves strength of refiner mechanical pulp. Tappi Journal 71: Nishida, T., Kashino, Y.. Mimura. A., and Takahara, Y Lignin biodegradation by wood-rotting fungi. I. Screening of lignin-degrading fungi. Mokuzai Gakkaishi 34: Otjen, L., Blanchette, R., Effland, M., and Leatham, G Assessment of 30 white rot basidiomycetes for selective lignin degradation. Holzforschung 41: Reis, C. J. and Libby, C. E An experimental study of the effect of Fomes pini (There) Lloyd on the pulping qualities of pond pine Pinus serotina (Michx) cooked by the sulfate process. Tappi Journal 43: Rue]. K.. Barnoud, F., and Eriksson, K.-E Micromorphological and ultrastructural aspects of spruce wood degradation by wild-type Sporotrichum pulverulentum and its cellulase-less mutant Cel 44. Holzforschung 35: Ruel, K.. Barnoud, F., and Eriksson, K.-E Ultrastructural aspects of wood degradation by Sporotrichum pulverulentum Observations on spruce, wood impregnated with glucose. Holzforschung 38: Sachs, I. B., Leatham, G. F., and Myers, G. C Biomechanical pulping of aspen chips by Phanerochaete chrysosporium: Fungal growth pattern and effects on wood cell walls. Wood and Fiber Science 21: Samuelsson, L. Mjoberg, P. J., Harder, N.. Vallander, L.. and Eriksson. K.-E Influence of fungal treatment of the strength versus energy relationship in mechanical pulping. Svensk Papperstidning 8: Schalch, H., Gaskell, J., Smith. T. L., and Cullen. D Molecular cloning and sequences of lignin peroxidase genes of Phanerochaete chrysosporium. Molecular and Cellular Biology 9: Shoemaker. H. E., and Leisola, M. S. A Degradation of lignin by Phanerochaete chrysosporium. Journal of Biotechnology 13:10 I Wall, M. B., Lightfoot, E. N.. Cameron, D. C.. Cockrem, M. C. M., and Leatham, G. F Design of a biopulping reactor. Transactions of the Mycological Society of the Republic of China. In press. Wegner, T. H.. Leatham, G. F.. Myers. G. C., and Kirk. T. K Biological treatments as an alternative to chemical pretreatments in high-yield wood pulping. Tappi Journal. In press. In: Leatham, Gary F., ed. Frontiers in industrial mycology. Proceedings of Industrial Mycology symposium; 1990 June 25 26; Madison, WI. New York: Routledge, Chapman & Hall; Chapter 7. Printed on Recycled Paper