Production of cellulases and hemicellulases by the straw mushroom, Volvariella volvacea

Mycal. Res. 98 (9), 1019-1024 (1994)

1019

Printed in Great Britain

Production of cellulases and hemicellulases by the straw mushroom, Volvariella volvacea

Y. J. CAl, J. A. BUSWELL AND S. T. CHANG Department of Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong

Carboxymethylcellulase (CMCase), Avicelase and [3-glucosidase production was monitored in submerged cultures of two strains (V 14 and V 34) of Volvariella volvacea grown on Avice!. All three cellulolytic enzymes were detectable in culture supernatants, although levels of CMCase and Avicelase were conSiderably higher in cultures of V. volvacea V 14. [3-Glucosidase activity was also observed in mycelial extracts of both fungal strains. Addition of 0'2 % Tween 80 to cultures markedly enhanced extracellular cellulase activity, especially in V. volvacea V 34. Cellulases, together with xylanase and both intra- and extracellular [3-xylosidase, were also recorded in cultures of V. volvacea V 34 grown on paddy straw.

The edible straw mushroom, Volvariella volvacea (Bull. ex Fr.) Singer, is a fungus indigenous to the tropics and sub-tropics. It has been cultivated for many years in China and other Asian countries and, in more recent years, straw mushroom cultivation has developed in both North America and Europe. In contrast to other important cultivated mushroom species, e.g. Lentinus edodes and Pleurotus sajor-caju, V. volvacea is unable to grow well on 'woody' materials with a substantial lignin content. Rice straw is the natural substrate for growth and fruiting, but the introduction of cotton waste' composts' has resulted in earlier fructification and in higher and more stable yields (Chang, 1974). Mushrooms produce a wide range of extracellular enzymes that enable them to degrade complex lignocellulosic substrates into soluble substances which can then be absorbed by the mushroom for nutrition (Wood & Fermor, 1982). Consequently, growth and fruiting of V. volvacea will depend largely upon the ability of the fungus to utilize the cellulose and hemicellulose components of rice straw and other lignocellulosic materials as a nutritional source. This, in turn, will be determined by the mushroom's capacity to synthesize the hydrolytic enzymes necessary to degrade the polysaccharides into low-molecular-weight sugars which can be readily assimilated. In 1989/90, world production of V. volvacea amounted to 207000 tonnes (fresh weight), thereby rating the mushroom as the fifth most important among industrially cultivated varieties (Chang & Miles, 1991). However, despite the commercial importance of the straw mushroom, little is known about the enzyme systems used by the fungus to degrade the composted substrates adopted for cultivation. The present study was undertaken to determine the ability of V. volvacea to produce cellulolytic and xylanolytic enzymes, and to

explain the preference of the fungus for high-cellulose, lowlignin-containing growth substrates.

MA TERIALS AND METHODS Fungus

V. volvacea, strains V 14 and V 34, were taken from the Department's stock culture collection. The fungi were maintained on Potato Dextrose Agar (Difco) slants by periodic transfer.

Growth media DMS medium (BuswelL Mollet & Odier, 1984) containing the following (per litre) of distilled water was used in experiments to monitor cellulase and hemicellulase production: KH 2 P0 4, 0-2 g; MgS0 4 _ 7H 2 0, 0-05 g; CaCI 2 _2H 20, 0-013 g; 2,2dimethylsuccinic acid, 1-46 g; L-asparagine, 1-0 g; NH4 NO a, 0-5 g; yeast extract (Difco), 0-1 g; trace element solution (Kirk et al., 1978), 1-0 ml; vitamin solution (Kirk et al., 1978),0-5 ml. When Tween 80 was included in the medium, it was added to 0-2 % (v Iv). The medium was adjusted to pH 6-0 with 2M KOH and filter-sterilized (Millipore). Cellulose (Sigmacell, Type 20; Sigma), 10 g, or paddy straw, 10 g, served as the major carbon source and was sterilized by autoclaving in the growth flasks before the DMS medium was added. The lignocellulosic substrate was prepared by grinding paddy straw to pass a 0-9 mm mesh size and drying overnight at 60°C before use. In experiments designed to detect ligninolytic enzymes, the growth medium contained glucose (I %, w Iv) as the carbon source, and the levels of asparagine and NH 4 NO a were reduced to 0-1 g and 0-05 g, respectively.

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Cellulases and hemicellulases of Volvariella volvacea

Culture conditions Cultures for assaying polysaccharidase activities were grown up in 40 ml DMS medium in ISO ml Erlenmeyer flasks after inoculation with I ml fragmented mycelial suspension. The fungal inoculum was prepared by suspending the mycelium from a 4-d-old culture of V. volvacea grown on Potato Dextrose Broth (Difco) in 50 ml sterile distilled water followed by 45 s treatment in a Waring blender. Flasks were incubated at 32 0 with gyratory shaking at ISO rpm. Culture supernatants and fungal mycelium were assayed daily for carboxymethylcellulase (CMCase), Avicelase, l3-glucosidase, xylanase and 13xylosidase as shown. Growth conditions adopted for monitoring ligninolytic enzyme production were the same except that cultures were grown in stoppered flasks, flushed with 100% 02' and kept stationary. Fungal biomass was estimated on the basis of wet-weight determinations following initial washing of the mycelium on a sieve to remove unchanged Avicel substrate and filtration using a 2'5 cm glass microfibre filter (GF I A; Whatman).

method of Leatham & Stahmann (1981), using ABTS (2,2'azino-bis-3-benthiazoline-6-sulphonate according to Bourbonnais & Paice (1988) and by the syringaldazine method described by Leonowicz & Grzywnowicz (1981). Lignin peroxidase was assayed by monitoring the oxidation of veratryl alcohol to veratraldehyde spectrophotometrically at 310 nm according to Tien & Kirk (1984), and manganesedependent peroxidase was assayed by the method described by Glenn et al. (1983). All assays were performed in duplicate using three culture replicates. Enzyme activities are expressed in units defined as the quantity of enzyme required to form I ~mol product min- 1 under the conditions of assay.

Protein determination Protein in culture supernatants and fungal extracts was determined by the Lowry method (Lowry et aI., 1951) using bovine serum albumin as standard.

RESULTS Cellulolytic activities in cultures grown on cellulose

Fungal extracts Harvested mycelium, maintained below 10 0, was homogenized in KOH-KH 2P0 4 buffer (50 mM, pH 7'2) using a glass homogenizer. After centrifugation at 1000 g for IS min at 4 0, the supernatant was retained for assay of intracellular 13glucosidase and l3-xylosidase.

Enzyme assays CMCase activity was determined by measuring the amount of reducing sugar released in reaction mixtures containing: 1'7 ml 50 mM KOH-KH 2P0 4 (pH 6'2), 0'8 ml 2% (w/v) carboxymethylcellulose (CMC) (Sigma) solution and 0'5 ml culture supernatant. Mixtures were maintained at 500 for 30 min and the reducing sugar determined by the SomogyiNelson method (Somogyi, 1952) using 520 nm wavelength and glucose as a standard. CMC is normally adopted as the substrate in the assay of endoglucanases (endo- 1,4-13glucanases, EC 3 . 2 . I . 4). Avicelase and xylanase were measured using the same procedure except that 1% (w Iv) Avicel (Sigma) or 1% (w Iv) oat-spelt xylan (Sigma), respectively, replaced CMC as the substrate. Avice! is normally adopted as a substrate in the assay of cellobiohydrolases (exo1,4-I3-glucanases, EC 3 . 2 . I . 91). Intra- and extracellular 13glucosidase and j3-xylosidase were determined by measuring the amount of p-nitrophenol released in reaction mixtures containing: 0'9 ml 50 mM KOH-KH 2P0 4 (pH 6'2), 0'05 ml 40 mM p-nitrophenyl-j3-D-glucopyranoside or 25 mM pnitrophenyl-j3-D-xylopyranoside, and 0'05 ml fungal extract or 0'05 ml culture supernatant, respectively. Mixtures were maintained at 45 0 for 30 min and, after addition of 3 ml 1M sodium carbonate, the p-nitrophenol released measured spectrophotometrically at 400 nm. Laccase activity in culture supernatants was determined using three different methods previously described in the literature: with o-tolidine using the

In shaken cultures containing DMS medium supplemented with I % crystalline cellulose (Avicel) as carbon source, both strains of V. volvacea grew as a single large mycelial pellet (Fig. I). Although the medium was clear of cellulose particles within 2 d, most of the cellulose remained unchanged and enclosed within the mycelial pellet. Avicelase, CMCase and 13glucosidase activity was detectable in culture supernatants after 4 d incubation although the levels of Avicelase and CMCase produced by V. volvacea V 34 were appreciably lower compared to strain V 14 (Figs 2 and 3). The growth rates of the two fungi were comparable under the conditions used. However, growth and enzyme production patterns were markedly affected by the addition of 0-2 % (v Iv) Tween 80 to the growth medium. Under these conditions the fungi grew as numerous discrete pellets, and enzyme levels were significantly higher (Figs 4 and 5). This effect on enzyme production was

Fig. 1. V. volvacea, strain V 34, grown on cellulose in agitated submerged culture. The flask on the left shows the less heterogeneous growth obtained in medium supplemented with 0'2% (v/v) Tween 80.

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Figs 4 and 5. Production of extracellular protein (0), CMCase (D), Avicelase (L,,) and I'-glucosidase ('\7) by V. volvacea grown in

much more pronounced with strain V 34 where, at peak production times, CMCase, Avicelase and ~-glucosidase levels were approximately 16-, 5- and 4-fold higher, respectively (Figs 4 and 5). In the case of A vicelase and ~-glucosidase, this increase in enzyme production was largely reflected in parallel increases in extracellular protein levels. However, the specific activity of CMCase also increased when the fungus was grown in the presence of Tween 80. ~-Glucosidase were also detected in mycelial extracts of both V. volvacea strains grown on A vice!. Intracellular enzyme levels recorded over the first 6-8 days of fungal growth were approximately 2- to 3-fold higher (based on specific activity) compared to extracellular ~­ glucosidase although, in older cultures (10 d), no significant difference between intra- and extracellular enzyme levels was evident. Addition of Tween 80 to the culture medium caused a drop in the intracellular levels of ~-glucosidase in V. volvacea V 14, but intracellular enzyme levels in strain V 34 were unaffected (data not shown).

submerged agitated culture on crystalline cellulose in the presence of Tween 80 (0'2%, v/v). Fig. 4 (top panel), strain V 14; Fig. 5 (bottom panel). strain V 34. Other conditions as in Fig. 2.

lower and 50% higher, respectively, compared to cultures containing crystalline cellulose as growth substrate, whereas the amounts of CMCase produced were broadly similar in both cases. However, total extracellular protein levels were about three times higher when paddy straw served as the growth substrate. In paddy straw cultures supplemented with Tween 80, enzyme activities were again detectable after 48 h (Fig. 7). Under these conditions, ~-glucosidase levels were approximately 2-fold higher compared to Tween-supplemented cellulose cultures. Peak production of CMCase, measured after 4 d, was almost double that observed in cellulose cultures before falling to similar levels. Total extracellular protein levels were comparable in both sets of Tween-supplemented cultures.

Cellulolytic activities in cultures grown on paddy straw CMCase, Avicelase and ~-glucosidase production was also observed in submerged cultures of V. volvacea V 34 in which the natural substrate, paddy straw, replaced cellulose as the growth substrate. Under these conditions, all three enzyme activities were detectable after only 48 h and levels remained roughly constant over a 10-d experimental period (Fig. 6). Avicelase and ~-glucosidase titres were approximately 50%

Xylanases of V. voIvacea V 34

V. volvacea V 34 grew very poorly in shaken cultures on extracted oat-spelt or birchwood xylan, and no xylanase was detectable in the growth medium. However, when xylan was replaced by paddy straw the fungus produced low but measurable amounts of extracellular xylanase and ~-xylosidase (Fig. 8). Both enzymes were detectable after 48 h incubation;

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Figs () and 7, Production of extracellular protein (0), CMCase (0), Avicelase (D.) and [3-glucosidase ('\7) by V. volvacea (V 34) grown in submerged agitated culture using paddy straw as substrate. Fig. 6 (top panel) in the absence of Tween 80; Fig. 7 (bottom panel) in the presence of 0'2% (vjv) Tween 80. Other conditions as in Fig. 2.

xylanase activity peaked after 4 d, whereas j3-xylosidase levels remained relatively constant over a 10-d experimental period (Fig. 8). Intracellular l3-xylosidase was also readily detectable in extracts of fungal mycelium grown on paddy straw. Peak activity observed after 4 d was 0-01 IV mg protein-I. Higher levels of xylanase were recorded in Tweensupplemented cultures of V. volvacea V 34, although the enhancement effect was not as evident as observed with enzymes of the cellulolytic complex (Fig. 8). Supplementation of culture medium with 0'2 % Tween 80 had relatively little effect on intracellular j3-xylosidase; specific activity peaked after 6 d at 0'12 IV mg protein-I.

Ligninolytic enzymes No lignin peroxidase, Mn-dependent peroxidase or laccase activity was detected in cultures of V. volvacea V 14 and V 34 grown under a range of culture conditions (see Materials and Methods) which support the production of these enzymes in other fungi.

DISCUSSION The two strains of V. volvacea used in this investigation (V 14 and V 34) both grew well on crystalline cellulose and produced significant levels of the major enzymes of the cellulase complex. Xylanolytic enzymes were also detected in culture supernatants of V. volvacea V 34 grown on paddy straw. However, V. volvacea grew as a large globose pellet in submerged culture when using the type of inoculum employed and cellulose particles as the sole carbon source. The heterogeneity of the culture, which affects the morphological and physiological development of fungal hyphae in many basidiomycetes, can be reduced dramatically when polymeric additives (e.g. lunlon PW 110) are included in the fungal growth medium (Jones et aI., 1988). This same response has now been observed in cultures of V. volvacea supplemented with Tween 80. Addition of Tween 80 to submerged cultures of V. volvacea also enhanced cellulase and, to a lesser extent, xylanase production. Tween-mediated stimulation of cellulase (Reese & Maguire, 1969; Hung et al., 1988; Long & Knapp, 1991), xylanase (Reese & Maguire, 1969) and lignin peroxidase production (Asther et al., 1987) by a range of fungi are already documented, although we are unaware of any previous reports of similar effects in V. volvacea. Recently, vegetable oils, various fatty acids including palmitic, stearic and oleic acids, and Tween-type surfactants, have been reported to promote the growth of V. volvacea (Li et al., 1992). Tween 80 increased mycelial growth by 156% (Li et al., 1992) and it seems reasonable to assume that increased enzyme levels in V. volvacea were due simply to the stimulatory effect of Tween 80 on biomass production. Mycelial yields in Tweensupplemented cultures were certainly higher compared to those with no supplementation. However, other factors such as enhanced release of the enzymes from cells, increased release of enzymes from binding to the residual insoluble substrates or decreased inactivation of enzymes might account for the greater activities observed (Long & Knapp, 1991). As a surfactant, Tween 80 may interact with cell membranes, altering membrane permeability and encouraging release or solubilization of exoenzymes (Reese, 1972). Supplementation of the growth medium of V. volvacea with sunflower oil, which is rich in oleic acid, altered membrane permeability and resulted in increased glucose uptake (Li et al., 1992). The stimulatory effect of Tween 80 on cellulase secretion by Trichoderma reesei was linked to enhanced formation and!or activity of dolichol phosphate mannose synthase, thereby elevating the level of O-glycosylation which appears to be obligatory for protein secretion (Kruszewska, Palamarczyk & Kubicek, 1990). Possible involvement of one or more of these mechanisms in Tween-mediated enhanced cellulase activity in V. volvacea is currently under investigation. Unlike several other cultivated mushroom species (e.g. P. sajor-caju, L. edodes), V. volvacea is unable to grow well on substrates which contain high amounts of lignin. The natural substrate for V. volvacea is paddy straw which has a relatively low lignin content (Dale, 1987), and the preference of this mushroom for 'less-lignified', high-cellulose substrates is further exemplified by the increased production yields

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Fig. 8. Xylanase and extracellular f3-xylosidase production in shake cultures of V. volvacea (V 34) grown on paddy straw. e, 0, Xylanase; _, D, f3-xylosidase. Cultures were unsupplemented (open symbols) or supplemented (closed symbols) with 0'2% (v/v) Tween 80. Other conditions as in Fig. 2.

obtained when V. volvacea is cultivated on cotton wastes (Chang, 1974). This aversion of V. volvacea for highly lignified substrates might be due in part to a higher sensitivity to lignin-derived phenolic monomers and tannin substances, and to a more pronounced effect of these compounds on the production and/or activity of the straw mushroom's cellulases and hemicellulases. Several monomeric phenols have been reported to inhibit fungal growth (Akin & Rigsby, 1985; Shea & Buswell, 1992; Buswell & Eriksson, 1994), and the hydrolytic enzymes which catalyse the breakdown of the cellulolytic and hemicellulolytic components of plant cell walls (Martin & Akin, 1988; Shi, Y. L., Buswell, J. A. & Yu, H. S., unpublished results). Moreover, inhibition of extracellular endoglucanases of the rumen bacterium, Fibrobacter succinogenes, by condensed tannins from birdsfoot trefoil was evident at tannin concentrations as low as 25 I-lg ml~l (Bae et al., 1993). In terms of mycelial growth at least, V. volvacea exhibits different sensitivity profiles to lignin-related phenols and tannin derivatives compared to P. sajor-caju and L. edodes (Buswell, Cai & Chang, 1993; Cai, Buswell & Chang, 1993). However, the inhibition patterns, and the observed qualitative and quantitative differences compared to L. edodes and P. sajor-caju, were not explicit enough to conclude that the distribution of these compounds in lignocellulosic wastes alone is responsible for the poor growth of V. volvacea on lignified substrates (BusweII et al., 1993; Cai et al., 1993). On the basis of the results presented here, the mushroom's poor growth on substrates with higher lignin content is undoubtedly reflected in the apparent inability of the fungus to synthesize any of the recognized lignin-transforming enzymes. No lignin peroxidase, manganese peroxidase or laccase production was detected in the two strains of V. volvacea used in this study, even though they were grown under a range of culture conditions which support the production of these enzymes by known ligninolytic fungi such as Phanerochaete chrysosporium, Le. static culture and increased oxygen tension (Buswell, 1991). The cellulose and hemicellulose components of the plant cell wall are intimately

associated with the lignin moiety, which presents a barrier to the hydrolytic enzymes catalysing the degradation of the polysaccharides (Buswell, 1991). Thus, since V. volvacea apparently lacks a ligninolytic or lignin-transforming system, fungal access to the polysaccharide components will be restricted, thereby markedly reducing the capacity of the fungus to grow and fruit on lignified substrates. This work was supported by grants from the Sir Haddon Cave Fund and the Hong Kong Research Grants Council (grant no. CUHK 18/92 M).

REFERENCES Akin. D. E. &< Rigsby, L. L. (1985). Influence of phenolic acids on rumen fungi. Agronomy Journal 77. 180-182. Asther, M., Corrieu. G .• Drapon, R. &< Odier. E. (1987). Effect of Tween 80 and oleic acid on ligninase production by Phanerochaete chrysosporium INA-l2. Enzyme and Microbial Technology 9. 245-249. Bae. H. D .. McAllister. T. A., Yanke. J.. Cheng. K.-I. &< Muir. A. D. (1993). Effect of condensed tannins on endoglucanase actiVity and filter paper digestion by Fibrobaeter succinogenes S85. Applied and Environmental Microbiology 59. 2132-2138. Bourbonnais. R. &< Paice. M. G. (1988). Veratryl alcohol oxidases from the lignin-degrading basidiomycete Pleurotus sajor-caju. Biochemical Journal 255. 445-450. Buswell. j. A. (1991). Fungal degradation of lignin. In Handbook of Applied Mycology. Vol. 1. Soil and Plants (ed. A. K. Arora. B. Rai, G. Mukerji &< G. Knudsen). pp. 425-480. Marcel Dekker: New York. Buswell. I. A.. Cai. Y. I. &< Chang. S. T. (1993). Fungal- and substrateassociated factors affecting the growth of individual mushroom species on different lignocellulosic substrates. In Mushroom Biology and Mushroom Products (ed. S. T. Chang. j. A. Buswell &< S. W. Chiu). pp. 141-150. Chinese University Press: Hong Kong. BuswelL I. A. &< Eriksson. K.-E. L. (1994). Effect of lignin-related phenols and their methylated derivatives on the growth of eight white-rot fungi. World Journal of Microbiology and Biotechnology 10. 169-174. Buswell. I. A.. Mollet. B. &< Odier. E. (1984). Ligninolytic enzyme production by Phanerochaete chrysosporium under conditions of nitrogen sufficiency. FEMS Microbiology Letters 25. 295-299. Cai. Y. I.• Buswell. I. A. &< Chang, S. T. (1993). Effect of lignin-related phenolic monomers on the growth of the edible mushrooms Lentinus edodes, Pleurotus sajor-caju and Volvariella volvacea. World Journal of Microbiology and Biotechnology 9. 503-507. Chang. S. T. (1974). Production of the straw mushroom (Volvariella volvacea) from cotton wastes. Mushroom Journal 21. 348-354. Chang. S. T. &< Miles. P. G. (1991). Recent trends in world production of cultivated mushrooms. Mushroom Journal 503. 15-18. Dale. B. E. (1987). Lignocellulose conversion and future fermentation technology. Trends in Biotechnology 5. 287-291. Glenn. I. K.. Morgan, M. A.. Mayfield. M. B.• Kuwuhara, M. &< Gold. M. H. (1983). An extracellular H.O.-requiring enzyme preparation involved in lignin degradation by the white-rot basidiomycete Phanerochaete chrysosporium. Biochemical and Biophysical Research Communications 114. 1077-1083. Hung. B. R.• Lara. L.. Patron. M. A.. Ugarova. N. N.. Bechstedt, W. &< Clappes. S. (1988). Tween 80 and proteose peptone effect on cellulase production. Acta Biotechnologica 8. 461-464. lones. P.• Shahab. B. A.. Trinci. A. I. P. &< Moore. D. (1988). Effect of polymeric additives. especially Junlon and Hostacerin, on growth of some basidiomycetes in submerged culture. Transactions of the British Mycological Society 90, 577-583. Kirk. T. K.• Schultze, E., Connors. W. I.• Lorenz. L. F. &< Zeikus, I. G. (1978). Influence of culture parameters on lignin metabolism by Phanerochaete chrysosporium. Archives of Microbiology 117. 277-285.

Cellulases and hemicellulases of Volvariella volvacea Kruszewska, J., Palamarczyk, G. & Kubicek, C. P. (1990). Stimulation of exoprotein secretion by choline and Tween 80 in Trichoderma reesei QM 9414 correlates with increased activities of dolichol phosphate mannose synthase. journal of General Microbiology 136, 1293-1298. Leatham, G. F. & Stahmann, M. A. (1981). Studies on lacease of Lentinus edodes: Specificity, localization and association with the development of fruiting bodies. journal of General Microbiology 125, 147-157. Leonowicz, A. & Gryzywnowicz, K. (1981). Quantitative estimation of laccase forms in some white-rot fungi using syringaldazine as substrate. Enzyme and Microbial Technology 3, 55-58. Li, Y, Cho, K. Y" Wu, Y. Z. & Nair, N. G. (1992). The effect of lipids and temperature on the physiology and growth of Volvariella volvacea. World journal of Microbiology and Biotechnology 8, 621-626. Long, K. & Knapp, J. S. (1991). The effect of Junlon PWUO and Tween 80 on the production of cellulolytic enzymes by Coprinus cinereus. Mycological Research 95, 1077-1081. Lowry, O. H., Rosebrough, N.)., Farr, A. L. & Randall, R. J. (1951). Protein measurements with the Folin phenol reagent. journal of Biological Chemistry 193, 265-275.

(Accepted 9 February 1994)

1024 Martin, S. A. & Akin, D. E. (1988). Effect of phenolic monomers on the growth and "'-glucosidase of Bacteroides ruminicola and on the carboxymethylcellulase. "'-glucosidase and xylanase from Bacteroides succinogenes. Applied and Environmental Microbiology 54, 3019-3022. Reese. E. T. (1972). Enzyme production from insoluble substrates. Biotechnology & Bioengineering Symposium 3, 43-62. Reese, E. T. & McGuire, A. (1969). Surfactants as stimulants of enzyme production by microorganisms. Applied Microbiology 17, 242-245. Somogyi, M. (1952). Notes on sugar determination. journal of Biological Chemistry 195, 19-23. Shea, S. K. & BuswelL J. A. (1992). Effect of lignin-derived phenols and their methylated derivatives on the growth of Lentinus spp. Letters in Applied Microbiology 15, 12-14. Tien, M. & Kirk, T. K. (1984). Lignin-degrading enzyme from Phanerochaete chrysosporium: purification, characterization and catalytic properties of a unique H2 0 2-requiring oxygenase. Proceedings of the National Academy of Sciences of the U.S.A. 81, 2280--2284. Wood, D. A. & Fermor, T. R. (1982). Nutrition of Agaricus bisporus in compost. Mushroom journal 114, 194-197.