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The effect of junlon PW110 and tween 80 on the production of cellulolytic enzymes by Coprinus cinereus
Mycol. Res.
9S (9): 1077-1081 (1991)
1077
Printed in Great Britain
The effect of Junlon PWII0 and Tween 80 on the production of cellulolytic enzymes by Coprinus cinereus
KAMARIAH LONG AND JEREMY S. KNAPP Department of Microbiology, University of Leeds, Leeds, L52 9fT. UK
The effect of Junlon PWllO (an acrylic acid polymer) and of Tween 80 (a nonionic surfactant) on the production of cellulolytic and xylanolytic enzymes by the basidiomycete fungus Coprinus cinereus was investigated. The inclusion of each material in culture media resulted in significant increases in the production of extracellular cellulases and a mixture of the two was even more effective. Junlon PWllO also stimulated growth of C. cinereus and the production of xylanase and 13-xylosidase. Tween 80 had no effect on xylanase production although it did increase 13-xylosidase production. Junlon PWllO and Tween 80 in combination increased the rate of production of cellulase by cultures as well as its final yield. The cultivation of filamentous fungi in agitated, submerged culture can be problematical due to heterogeneity of morphology. Most fungi, and especially basidiomycetes, tend to produce morphologically and physiologically heterogeneous pellets of variable and often large size (Burkholder & Sinnot, 1945; Jones et at 1988 b; Pirt, 1966; Trinci & Thurston, 1976), making representative sampling difficult. Dispersed filamentous growth, on the other hand, is desirable because of the increased homogeneity and ease of sampling. The addition of polymeric additives such as Carbopol (carboxypolymethylene) (Elmayergi, Scharer & Moo.Young, 1973) or Junlon PWllO (an anionic acrylic polymer) Oones et al., 1988 b} to fungal cultures encourages dispersed mycelial growth, leading to increases in the rate and extent of growth. Despite investigations of the effects of polymer addition on fungal growth and morphology (e.g. Trinci, 1983; Byrne & Ward, 1987; Jones, Moore & Trinci, 1988a; Morrin & Ward, 1989) little is known of the effects of these materials on the production of fungal extracellular enzymes (Elmayergi et al., 1973; Moo-Young, 1976). During an investigation into the production of cellulolytic enzymes by the basidiomycete fungus Coprinus cinereus (Schaeff. ex Fr.) S. F. Gray problems were encountered in taking representative samples due to the pelleted growth of the organism in submerged, shaken cultures on media containing cellulose. Mild ultrasonic treatment of suspensions of oidia prior to inoculation and an increase in the rate of shaking led to a decrease in pellet size. Nevertheless the effects of Junlon PWllO (and the related polymer Hostacerin) on the growth and cellulase and xylanase production were investigated. The nonionic surfactant Tween 80 is well known to improve cellulase yie!ds from fungal cultures (Reese & Maguire, 1969) and its effects, alone and in combination with Junlon PWllO, on cellulase and xylanase production by C. cinereus were also studied.
MA TERIALS AND METHODS General chemicals were obtained from BDH Ltd (Poole, Dorset) and were of Analar grade where commercially available and otherwise of GPR grade. Biochemicals were from Sigma, Avice! from Fluka and Solka Floc from James Rivers Corp. (Hackensack, NJ, U.s.A.), Junlon PWllO and Montanox 20 were gifts from Honeywill and Stein Ltd (Sutton, Surrey). Hostacerin PN73 was a gift from Hoechst Ltd.
Organisms and cultural conditions Coprinus cinereus IMI 140506 (monokaryon) was obtained from the Commonwealth Mycological Institute (Kew, London). Stock cultures were maintained on slopes of malt extract agar (MEA) (Oxoid Ltd, Basingstoke, Hants, UK.). Oidia of C. cinereus were harvested in sterile distilled water from 7-day-old cultures on MEA slopes incubated at 37°C. Spore suspensions were pooled and subjected to gentle ultrasonic treatment (ultrasonic water bath, Sonicor Instrument Corp., Copiague, NY) for 1'75 min. Experiments were conducted in 500 ml Erlenmeyer flasks containing 200 ml of sterile mineral salts medium and 1 % w Iv Avicel inoculated with a spore suspension (5 ml) containing ca 1'0--1'3 x 10 7 spores ml- 1 . The composition of the mineral salts medium was as follows (g 1- 1): urea, 0'3; proteose peptone, 0'5; KH 2 P0 4, 2; (NH4}2S04' 1'4; MgS0 4. 7H 2 0, 0'3; CaCl 2, 0'3; FeS0 4, 0'005; ZnS04' 7H 20, 0'0014; CoCl 2, 0'002; MnS0 4 . H 20, 0'0016; thiamin, 0'001; Tween 80, 0'6. Urea and thiamin were sterilised separately by membrane filtration (pore size 0'2 J.,lm) and added to the medium aseptically. Cultures were incubated at 37° on an orbital shaker at 210 rev. min- 1 (25 mm displacement). For experiments with polymeric additives the cellulose mineral salts medium
Effect of Junlon on cellulase production and polymer solutions were autoclaved together at 1'034 x 10 5 Nm -2 for 20 min. The pH of the medium was adjusted to 7'0 with sterile 2 M NaOH.
Biomass estimation
Mycelium was harvested by centrifugation at 2750 g for 15 min and washed twice with distilled water before estimation of biomass of the final pellet as cell protein by the method of Pavlostathis, Miller and Wolin (1988). Protein was assayed by the method of Bradford (1976), with bovine serum albumen in 1 MNaOH as the protein standard. Junlon PWllO and Tween 80 were shown not to affect the cell protein assay in any way. The supernatant (culture filtrate) was retained for enzyme assays and measurement of pH. Mycelial samples were taken in duplicate and each of these was assayed for protein in duplicate.
1078 reducing sugars liberated from xylan (from oat spelt, Sigma). The reaction mixture contained 0'5 ml diluted culture filtrate, 1 ml xylan solution 1 % (wIv) and 0'5 ml 0-05 M phosphate buffer (pH 6-4). Controls were included in which culture filtrate or substrate was omitted. Assays were incubated at 50° for 30 min. Reducing sugar levels were determined by the dinitrosalicylic acid method (Miller, 1959) and enzyme activities expressed as ~mol reducing sugar released. l3-xylosidase activity was determined by the method used for l3-glucosidase, but with p-nitrophenyl I3-D-xylopyranoside as substrate and 0-05 M phosphate, pH 6'4, as buffer. The results given are the means of assays carried out on duplicate cultures, all enzyme assays were performed, at least, in duplicate and in the case of filter paper cellulase in triplicate. All experiments were repeated. The statistical significance of some of the differences observed was calculated using the one-way analysis of variance and least significant difference tests.
Enzyme assays
Junlon PWllO and Tween 80 where shown to have no effect on assays for enzyme activity or on the measurement of reducing sugars. Total cellulase activity was measured by incubation of 50 mg of filter paper (Whatman no. 1) and 1'5 m10-05 Mcitrate buffer (pH 6-0) with 0-5 ml culture filtrate (Mandels & Weber, 1969). Activity is quoted as ~mol redUcing sugar produced by 0'5 ml of enzyme solution during incubation at 50° for 1 h. The concentration of reducing sugars, expressed as glucose, was measured by the dinitrosalicylate method of Miller (1959). I3-Glucosidase was determined by measuring the release of p-nitrophenol from p-nitrophenyl I3-D-glucopyranoside. The assay mixture contained 1 ml 5 mM, p-nitrophenyl I3-Dglucopyranoside, 1'6 ml of 0-05 M citrate buffer (pH 6-0) and 0-4 ml diluted culture filtrate (1/20 dilution). The mixture was incubated at 50° for 30 min and glycine buffer, pH 10-8, added to stop the reaction. The concentration of p-nitrophenol was determined spectrophotometrically at 430 nm. Carboxymethyl cellulase (CMCase) activity was measured by the method of Wood & Bhat (1988). The assay mixture contained 1-0 ml carboxymethylcellulose sodium salt 1 % (wIv) (degree of substitution ca 0'4, BDH) in 0-05 M citrate buffer (pH 6-0) and 0'6 ml of the same buffer. Diluted culture filtrate (0-4 ml of a 1/20 dilution) was added to the reaction mixture and incubated at 50° for 15 min. This method is based on the initial rate of reaction and activity is quoted as ~mol of reducing sugar expressed as glucose. Glucose was measured using the Somogyi-Nelson method as described by Wood & Bhat (1988). Avicelase activity was assayed by the method of Wood & Bhat (1988) using Avice!, pH 101, as a substrate. The reaction mixture consisted of a 1 % (wIv) suspension of Avicel in 1'8 ml 0-05 Mcitrate buffer (pH 6'0) and 0-2 ml culture filtrate. The mixture was incubated at 50° for 2 h, centrifuged (2750 g for 15 min) and 1 ml of the supernatant was removed and analysed for soluble reducing sugar (expressed as glucose) using the Somogyi-Nelson method (Wood & Bhat, 1988). Xylanase activity was determined by measuring the
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23456789 Time (days)
Fig.!. The effect of Junlon PWIOO and Tween 80 on (A) pH, (B) cell protein concentration and (C) total cellulase production by Coprinus cinereus growing on Avice! as the major carbon source. (e) Control cultures with no additions; (0) Tween 80 only; (.) Junlon PWIOO only; (..J Junlon PWIOO and Tween 80. Bars represent standard errors, where no bars are shown the error was smaller than the symbol. Maximum cell protein values occurred on day 3 and for controls and cultures containing only Tween 80 these values were not significantly different. However both of these were significantly different (P = 0'05) from the other two cultures which also differed significantly from each other. For cellulase, the maximum activities of all cultures were significantly different from each other (P ~ 0'05), as were the activities of all cultures on day 7 (P = 0.01).
K. Long and J. S. Knapp
1079 0·8
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RESULTS During growth on Avicel as the principle source of carbon and energy, Coprinus cinereus produced extracellular cellulases. In control cultures, biomass and total cellulase (measured as Filter Paper Activity) generally declined after 3 and 4 d growth respectively (Fig. 1). The presence of Junlon PWll0 at 0'1 % (w Iv) was always associated with an increase in cell protein (Fig. 1) and led to dispersed filamentous growth rather than
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234567
Time (days) Fig. 2. The effect of Junlon PWIOO and Tween 80 on the production of (A) Avicelase, (B) carboxymethylcellulase (CMCase) and (C) ~ glucosidase during the growth of Coprinus cinereus on Avice! as the major carbon source. (.) Control cultures with no additions; (0) only Tween 80; (.) only Junlon PWIOO; (.6.) Junlon PWIOO and Tween 80. Bars represent standard errors, where no bars are shown the error was smaller than the symbol. Avicelase activities of all cultures were significantly different (P :::; 0'05) on day 3. When the maximum activities occurring in the different cultures were compared, the control was not significantly different from the cultures containing only Junlon PWllO but all other differences between cultures were significant (P = 0·0l). CMCase activities in all cultures on day 3 were significantly different (P = 0·0l). When maximum CMCase activities were compared the control did not differ significantly from the cultures containing only Junlon PWllO but all other differences were significant (P = 0·0l). ~-glucosidase activities of all the different treatments were compared on day 7 and all differences were significant (P = 0·0l).
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Fig. 3. The effect of Junlon PWllO and Tween 80 on the production of (A) ~-xylosidase and (B) xylanase by Coprinus cinereus growing on Solka Floc as the major carbon source. (.) Control cultures with no additions; (0) only Tween 80; (.) only Junlon PWllO; (.6.) Junlon PWllO and Tween 80. Bars represent the standard errors, where no bar is shown the error was smaller than the symbol. ~-xylosidase activities were compared on day 7 and activities of all cultures were significantly different (P = 0.01). Xylanase activities were compared on days 5 and 7. The control and the cultures containing only Tween 80 did not differ significantly from each other and cultures containing only Junlon PWllO did not differ from those containing Junlon PWllO plus Tween 80. However the cultures without Junlon PWllO differed significantly (P = 0'01) from those containing Junlon PWIIO.
the pelleted form found in control cultures. Junlon PWll0 increased maximum cellulase activity by ca 30 % but the general pattern of production was similar to controls. pH profiles in cultures containing Junlon PWll0 differed only slightly from those of controls (Fig. 1), and it is unlikely that pH differences significantly affected growth or enzyme production. The inclusion of Tween 80 (0'6 g-1 1) in the culture medium also markedly increased cellulase activity, and a combination of Tween 80 and Junlon PWll0 resulted in even higher cellulase activity. The production of cellulase during growth on Avicel appeared to be largely growth-associated in the presence and absence of Junlon PWll0 but in the presence of Tween 80 (with or without Junlon PWll0) cellulase activity continued to increase for, at least, a further 3 days (generally peaking on the 7th day of growth) while biomass concentration decreased. In the presence of Tween 80 (0'6 g-1 I) a Junlon PWll0 concentration of 0'1 to 0'15% (w/v) was optimal for production of cellulase but as little as 0'025 % (w Iv) Junlon PWIlO increased the rate of cellulase accumulation compared with controls and with cultures containing only Tween 80.
Effect of Junlon on cellulase production Hostacerin PN73 (an anionic co-polymer of acrylic acid and acrylamide) had a similar effect to Junlon PW1l0 on both growth of, and cellulase production by, C. cinereus. Combinations of Hostacerin PN73 (0·1-0·2% w/v) and Tween 80 were as effective as Junlon PW1l0 and Tween 80, but the nonionic surfactant Montanox 20 (polyoxyethylene sorbitan monolaurate ester) at 0·1 % w Iv strongly inhibited growth and cellulase production. The effect of Junlon PW1l0 and Tween 80 on the production of individual enzyme activities of the cellulase complex was also determined (Fig. 2). The production of Avicelase was only very slightly increased by Junlon PW1l0 alone (ca 6% increase in maximum yield) but was greatly improved by Tween 80 (ca 167%) and even more so by a combination of the two additives (ca 235 %). Addition of Junlon PW1l0 also improved the production of CMCase (ca 42 %) and l3-glucosidase (ca 52·5%) but to a lesser degree than Tween 80, which increased maximum yield by 229% and 195 % respectively. Again the inclusion in the growth medium of both Junlon PW1l0 and Tween 80 was most effective resulting in increases of ca 400% in CMCase and 265 % in 13glucosidase yield. The effects of}unlon PW1l0 and Tween 80 (alone or in combination) on the production of cellulolytic enzymes by C. cinereus when grown on Solka Floc SW40 as the major carbon source were similar to those noted during growth on Avice!. Junlon PW1l0 generally increased the initial rate of enzyme production as well as the final yield (Figs 1, 2).
The presence of ]unlon PW1l0 also stimulated production of xylanase (by ca 20%) and l3-xylosidase (by ca 40%) by C. cinereus growing on Solka Floc SW40 (Fig. 3). Tween 80 had negligible effect on xylanase activity although it did stimulate l3-xylosidase activity (by 17 to 25 %).
DISCUSSION Both Tween 80 and the acrylic acid polymers stimulated the production of cellulolytic enzymes by C. cinereus. Although Tween 80 is well known to improve cellulase production by a range of fungi (e.g. Reese & Maguire, 1969; Schewale & Sadana, 1978; Hung et al., 1988), its effects on C. cinereus have not previously been reported. Tween 80 did not appear to affect the growth of C. cinereus in this study and prolonged production of cellulases long after the cessation of growth. The ability of Junlon PW1l0 and Hostacerin PN73 to increase cellulase production has not previously been reported, although the anionic polymer Carbopol has been shown to increase the rate of cellulase production by Trichoderma viride (Moo-Young, 1976) and of amylase production by Aspergillus niger (Elmayergi et al., 1973). Most previous studies do not show the effect of additives on the timing of exoenzyme production. The effects of Junlon PW1l0 and Tween 80 on cellulase production appear to be additive and may even be synergistic. ThiS, together with the temporal differences in their effects, suggests that, at least for C. cinereus, Junlon PW1l0 and Tween 80 affect cellulase production by different mechanisms. Junlon PW1l0 increased the growth of C. cinereus, in agreement with the findings of Jones et al. (1988b). A similar
1080 effect has been noted for Carbopol (Elmayegi et aI., 1973) and it seems reasonable to propose that these polymers increase cellulase production simply by increasing the extent of growth. This may be due to encouragement of mycelial rather than pelleted growth. Jones et al. (1988a) have suggested that the coating of spores and hyphae with Junlon PW1l0 reduces their tendency to aggregate, thus giving rise to dispersed filamentous rather than pelleted growth. The related polymer Carbopol has also been shown to coat fungal mycelia (Morrin & Ward, 1989) and it has been proposed that the presence of polymers may stimulate mass transfer of nutrients to the biomass (Byrne & Ward, 1987). Somewhat contradictory results have been reported by Mukhopadhyay & Chose (1977), who found that pelleted cultures of T. viride gave higher cellulase production than dispersed filamentous cultures. However, their results were obtained using different cultivation systems (shake flasks giving pelleted growth and stirred tank fermenters giving filamentous growth) and factors other than growth form (e.g. different rates of enzyme inactivation) may have been important. It is difficult to be certain whether the elevated levels of cellulolytic enzymes observed are due to de novo synthesis. 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 activity of cellulase in cultures. Although increased levels of cellulolytic enzymes in the presence of ]unlon PW1l0 may be due to increased growth, this is unlikely to explain the effect of Tween 80. As a surfactant, Tween 80 may interact with cell membranes and other surfaces, altering membrane permeability and encouraging release or solubilisation of exoenzymes (Reese, 1972). On the other hand, Castanon & Wilke (1981) showed that Tween 80 increased the rate of cellulose hydrolysis in cell-free systems and improved recovery of cellulolytic activity after saccharification. It was proposed that Tween 80 hindered immobilisation of cellulase on the insoluble substrate. Cellulases are subject to inactivation at air-liquid interfaces which has been shown to be preventable by the addition of the non-ionic detergent Triton X100, the fluorinated surfactant Zonyl FSN or even proteins (Reese, 1980; Kim et al., 1982). Incidentally, Triton XI00 has also been shown to enhance heat stability in some microbial extracellular Iipases (Bucky, Robinson & Hayes, 1987). The effect of Tween 80 may be due to one or a combination of these mechanisms. It is interesting that Tween 80 has negligible effect on xylanase production by C. cinereus as it has been shown to increase xylanase production in a range of fungi (Reese & Maguire, 1969; Reese, 1972). The toxicity of the surfactant Montanox 20 to C. cinereus is also surprising in view of its structural similarity to Tween 80. From a practical point of view the combination of Tween 80 and ]unlon PW1l0 offers the possibility of further significant increases in the rate of cellulase production and the final yield of cellulase from cultures of C. cinereus. As both Tween 80 and anionic polymers have been shown separately to improve significantly the production of cellulase and other exoenzymes in a range of fungi, it is likely that the
K. Long and
J. S. Knapp
combination will also have beneficial effects on other fungi which are perhaps more likely than C. cinereus to be commercially exploited in the near future. The authors gratefully acknowledge the financial support of the Malaysian Agricultural Research and Development Institute, which provided a scholarship to K. L.
REFERENCES Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 12, 248-254. Bucky, A R., Robinson, D. S. &< Hayes, P. R. (1987). Factors affecting the heat stability of lipase produced by a strain of Pseudomonas f/uorescens. Food Chemistry 23. 159--173. Burkholder, P. R. &< Sinnott, E. W. (1945). Morphogenesis of fungus colonies in submerged shaken cultures. American Journal of Botany 32, 424-431. Byrne, G. S. &< Ward, O. P. (1987). Effect of polymers on pelleting of Rhiwpus arrhizus. Transactions of British Mycological Society 89, 367-371. Castanon, M. &< Wilke, C. R. (1981). Effects of the surfactant Tween 80 on enzymatic hydrolysis of newspaper. Biotechnology and Bioengineering 23, 1365-1372. Elmayergi, H., Scharer, j. M. &< Moo-Young, M. (1973). Effects of polymer additives on fermentation parameters in a culture of Aspergillus niger. Biotechnology and Bioengineering 15, 845-859. 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. jones, P., Moore, D. &< Trinci, A P. j. (1988a). Effects of junlon and Hostacerin on the electrokinetic properties of spores of Aspergillus niger, Phanaerochaete chrysosporium and Geotrichum candidum. Journal of General Microbiology 134, 225-240. jones, P., Shahab, B. A, Trinci, A P. j. &< Moore, D. (1988 b). Effect of polymeric additives, especially junlon and Hostacerin, on growth of some basidiomycetes in submerged culture. Transactions of British Mycological Society 90, 577-583.
(Received for publication 17 Mny 1990 and in revised form 26 March 1991)
1081 Kim, M. H., Lee, S. B., Ryu, D. D. Y. &< Reese, E. T. (1982). Surface deactivation of cellulase and its prevention. Enzyme and Microbial Technology 4, 99--103. Mandels, M. &< Weber, j. (1969). The production of cellulases.1n Cellulases and their Applications, Advances in Chemistry Series 95 (ed. G. j. Hajny &< E. T. Reese), pp. 391-413. Washington DC: American Chemical Society Publications. Miller, G. L. (1959). Use of dinitrosalicylic acid for determination of reducing sugars. Analytical Chemistry 31, 426-428. Moo-Young, M. (1976). U.S. Patent 3975236; August 17, 1976. Described in Industrial Enzymes from Microbial Sources, Recent Advances. (ed. M. G. Halpern), pp. 246--247. Park Ridge, New jersey: Noyes Data Corporation. Morrin, M. &< Ward, O. P. (1989). Studies on interaction of Carbopol-934 with hyphae of Rhizopus arrhizus. Mycological Research 92, 265-272. Mukhopadhyay, S. N. &< Ghose, T. K. (1977). Studies on cellulase production in relation to morphological variations of Trichoderma viride QM 9414. In Proceedings of Bioconversion Symposium (ed. T. K. Ghose), pp. 97-109. New Delhi, India: Indian Institute of Technology. Pavlostathis, S., Miller, T. L. &< Wolin, M. j. (1988). Fermentations of insoluble cellulose by continuous cultures of Ruminococcus albus. Applied and Environmental Microbiology 54, 2655-2659. Pirt, S. j. (1966). A theory of the mode of growth of fungi in the form of pellets in submerged culture. Proceedings of the Royal Society, London, Series B 166, 369-373. Reese, E. T. (1972). Enzyme production from insoluble substrates. Biotechnology & Bioengineering Symposium 3, 43---62. Reese, E. T. (1980). Inactivation of cellulase by shaking and its prevention by surfactants. Journal of Applied Biochemistry 2, 36--39. Reese, E. T. &< McGuire, A (1969). Surfactants as stimulants of enzyme production by microorganisms. Applied Microbiology 17, 242-245. Shewale, j. G. &< Sadana, j. C. (1978). Cellulase and ~-glucosidase production by a basidiomycete species. Canadian Journal of Microbiology 24, 1204-1216. Trinci, A P. j. (1983). Effect of junlon on morphology of Aspergillus niger and its use in making turbidity measurements of fungal growth. Transactions of British Mycological Society 81, 408-412. Trinci, A P. j. &< Thurston, C. F. (1976). Transition to the non-growing state in eukaryotic micro-organisms. In The Survival of Vegetative Microbes (ed. T. R. G. Gray &< j. R. Postgate), pp. 55-80. Cambridge, U.K.: Cambridge University Press. Wood, T. M. &< Bhat, K. M. (1988). Method for measuring cellulase activities. In Methods in Enzymology 160 A. (ed. W. A Wood &< S. T. Kellogg), pp. 87-112. London: Academic Press.
9S (9): 1077-1081 (1991)
1077
Printed in Great Britain
The effect of Junlon PWII0 and Tween 80 on the production of cellulolytic enzymes by Coprinus cinereus
KAMARIAH LONG AND JEREMY S. KNAPP Department of Microbiology, University of Leeds, Leeds, L52 9fT. UK
The effect of Junlon PWllO (an acrylic acid polymer) and of Tween 80 (a nonionic surfactant) on the production of cellulolytic and xylanolytic enzymes by the basidiomycete fungus Coprinus cinereus was investigated. The inclusion of each material in culture media resulted in significant increases in the production of extracellular cellulases and a mixture of the two was even more effective. Junlon PWllO also stimulated growth of C. cinereus and the production of xylanase and 13-xylosidase. Tween 80 had no effect on xylanase production although it did increase 13-xylosidase production. Junlon PWllO and Tween 80 in combination increased the rate of production of cellulase by cultures as well as its final yield. The cultivation of filamentous fungi in agitated, submerged culture can be problematical due to heterogeneity of morphology. Most fungi, and especially basidiomycetes, tend to produce morphologically and physiologically heterogeneous pellets of variable and often large size (Burkholder & Sinnot, 1945; Jones et at 1988 b; Pirt, 1966; Trinci & Thurston, 1976), making representative sampling difficult. Dispersed filamentous growth, on the other hand, is desirable because of the increased homogeneity and ease of sampling. The addition of polymeric additives such as Carbopol (carboxypolymethylene) (Elmayergi, Scharer & Moo.Young, 1973) or Junlon PWllO (an anionic acrylic polymer) Oones et al., 1988 b} to fungal cultures encourages dispersed mycelial growth, leading to increases in the rate and extent of growth. Despite investigations of the effects of polymer addition on fungal growth and morphology (e.g. Trinci, 1983; Byrne & Ward, 1987; Jones, Moore & Trinci, 1988a; Morrin & Ward, 1989) little is known of the effects of these materials on the production of fungal extracellular enzymes (Elmayergi et al., 1973; Moo-Young, 1976). During an investigation into the production of cellulolytic enzymes by the basidiomycete fungus Coprinus cinereus (Schaeff. ex Fr.) S. F. Gray problems were encountered in taking representative samples due to the pelleted growth of the organism in submerged, shaken cultures on media containing cellulose. Mild ultrasonic treatment of suspensions of oidia prior to inoculation and an increase in the rate of shaking led to a decrease in pellet size. Nevertheless the effects of Junlon PWllO (and the related polymer Hostacerin) on the growth and cellulase and xylanase production were investigated. The nonionic surfactant Tween 80 is well known to improve cellulase yie!ds from fungal cultures (Reese & Maguire, 1969) and its effects, alone and in combination with Junlon PWllO, on cellulase and xylanase production by C. cinereus were also studied.
MA TERIALS AND METHODS General chemicals were obtained from BDH Ltd (Poole, Dorset) and were of Analar grade where commercially available and otherwise of GPR grade. Biochemicals were from Sigma, Avice! from Fluka and Solka Floc from James Rivers Corp. (Hackensack, NJ, U.s.A.), Junlon PWllO and Montanox 20 were gifts from Honeywill and Stein Ltd (Sutton, Surrey). Hostacerin PN73 was a gift from Hoechst Ltd.
Organisms and cultural conditions Coprinus cinereus IMI 140506 (monokaryon) was obtained from the Commonwealth Mycological Institute (Kew, London). Stock cultures were maintained on slopes of malt extract agar (MEA) (Oxoid Ltd, Basingstoke, Hants, UK.). Oidia of C. cinereus were harvested in sterile distilled water from 7-day-old cultures on MEA slopes incubated at 37°C. Spore suspensions were pooled and subjected to gentle ultrasonic treatment (ultrasonic water bath, Sonicor Instrument Corp., Copiague, NY) for 1'75 min. Experiments were conducted in 500 ml Erlenmeyer flasks containing 200 ml of sterile mineral salts medium and 1 % w Iv Avicel inoculated with a spore suspension (5 ml) containing ca 1'0--1'3 x 10 7 spores ml- 1 . The composition of the mineral salts medium was as follows (g 1- 1): urea, 0'3; proteose peptone, 0'5; KH 2 P0 4, 2; (NH4}2S04' 1'4; MgS0 4. 7H 2 0, 0'3; CaCl 2, 0'3; FeS0 4, 0'005; ZnS04' 7H 20, 0'0014; CoCl 2, 0'002; MnS0 4 . H 20, 0'0016; thiamin, 0'001; Tween 80, 0'6. Urea and thiamin were sterilised separately by membrane filtration (pore size 0'2 J.,lm) and added to the medium aseptically. Cultures were incubated at 37° on an orbital shaker at 210 rev. min- 1 (25 mm displacement). For experiments with polymeric additives the cellulose mineral salts medium
Effect of Junlon on cellulase production and polymer solutions were autoclaved together at 1'034 x 10 5 Nm -2 for 20 min. The pH of the medium was adjusted to 7'0 with sterile 2 M NaOH.
Biomass estimation
Mycelium was harvested by centrifugation at 2750 g for 15 min and washed twice with distilled water before estimation of biomass of the final pellet as cell protein by the method of Pavlostathis, Miller and Wolin (1988). Protein was assayed by the method of Bradford (1976), with bovine serum albumen in 1 MNaOH as the protein standard. Junlon PWllO and Tween 80 were shown not to affect the cell protein assay in any way. The supernatant (culture filtrate) was retained for enzyme assays and measurement of pH. Mycelial samples were taken in duplicate and each of these was assayed for protein in duplicate.
1078 reducing sugars liberated from xylan (from oat spelt, Sigma). The reaction mixture contained 0'5 ml diluted culture filtrate, 1 ml xylan solution 1 % (wIv) and 0'5 ml 0-05 M phosphate buffer (pH 6-4). Controls were included in which culture filtrate or substrate was omitted. Assays were incubated at 50° for 30 min. Reducing sugar levels were determined by the dinitrosalicylic acid method (Miller, 1959) and enzyme activities expressed as ~mol reducing sugar released. l3-xylosidase activity was determined by the method used for l3-glucosidase, but with p-nitrophenyl I3-D-xylopyranoside as substrate and 0-05 M phosphate, pH 6'4, as buffer. The results given are the means of assays carried out on duplicate cultures, all enzyme assays were performed, at least, in duplicate and in the case of filter paper cellulase in triplicate. All experiments were repeated. The statistical significance of some of the differences observed was calculated using the one-way analysis of variance and least significant difference tests.
Enzyme assays
Junlon PWllO and Tween 80 where shown to have no effect on assays for enzyme activity or on the measurement of reducing sugars. Total cellulase activity was measured by incubation of 50 mg of filter paper (Whatman no. 1) and 1'5 m10-05 Mcitrate buffer (pH 6-0) with 0-5 ml culture filtrate (Mandels & Weber, 1969). Activity is quoted as ~mol redUcing sugar produced by 0'5 ml of enzyme solution during incubation at 50° for 1 h. The concentration of reducing sugars, expressed as glucose, was measured by the dinitrosalicylate method of Miller (1959). I3-Glucosidase was determined by measuring the release of p-nitrophenol from p-nitrophenyl I3-D-glucopyranoside. The assay mixture contained 1 ml 5 mM, p-nitrophenyl I3-Dglucopyranoside, 1'6 ml of 0-05 M citrate buffer (pH 6-0) and 0-4 ml diluted culture filtrate (1/20 dilution). The mixture was incubated at 50° for 30 min and glycine buffer, pH 10-8, added to stop the reaction. The concentration of p-nitrophenol was determined spectrophotometrically at 430 nm. Carboxymethyl cellulase (CMCase) activity was measured by the method of Wood & Bhat (1988). The assay mixture contained 1-0 ml carboxymethylcellulose sodium salt 1 % (wIv) (degree of substitution ca 0'4, BDH) in 0-05 M citrate buffer (pH 6-0) and 0'6 ml of the same buffer. Diluted culture filtrate (0-4 ml of a 1/20 dilution) was added to the reaction mixture and incubated at 50° for 15 min. This method is based on the initial rate of reaction and activity is quoted as ~mol of reducing sugar expressed as glucose. Glucose was measured using the Somogyi-Nelson method as described by Wood & Bhat (1988). Avicelase activity was assayed by the method of Wood & Bhat (1988) using Avice!, pH 101, as a substrate. The reaction mixture consisted of a 1 % (wIv) suspension of Avicel in 1'8 ml 0-05 Mcitrate buffer (pH 6'0) and 0-2 ml culture filtrate. The mixture was incubated at 50° for 2 h, centrifuged (2750 g for 15 min) and 1 ml of the supernatant was removed and analysed for soluble reducing sugar (expressed as glucose) using the Somogyi-Nelson method (Wood & Bhat, 1988). Xylanase activity was determined by measuring the
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Fig.!. The effect of Junlon PWIOO and Tween 80 on (A) pH, (B) cell protein concentration and (C) total cellulase production by Coprinus cinereus growing on Avice! as the major carbon source. (e) Control cultures with no additions; (0) Tween 80 only; (.) Junlon PWIOO only; (..J Junlon PWIOO and Tween 80. Bars represent standard errors, where no bars are shown the error was smaller than the symbol. Maximum cell protein values occurred on day 3 and for controls and cultures containing only Tween 80 these values were not significantly different. However both of these were significantly different (P = 0'05) from the other two cultures which also differed significantly from each other. For cellulase, the maximum activities of all cultures were significantly different from each other (P ~ 0'05), as were the activities of all cultures on day 7 (P = 0.01).
K. Long and J. S. Knapp
1079 0·8
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RESULTS During growth on Avicel as the principle source of carbon and energy, Coprinus cinereus produced extracellular cellulases. In control cultures, biomass and total cellulase (measured as Filter Paper Activity) generally declined after 3 and 4 d growth respectively (Fig. 1). The presence of Junlon PWll0 at 0'1 % (w Iv) was always associated with an increase in cell protein (Fig. 1) and led to dispersed filamentous growth rather than
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234567
Time (days) Fig. 2. The effect of Junlon PWIOO and Tween 80 on the production of (A) Avicelase, (B) carboxymethylcellulase (CMCase) and (C) ~ glucosidase during the growth of Coprinus cinereus on Avice! as the major carbon source. (.) Control cultures with no additions; (0) only Tween 80; (.) only Junlon PWIOO; (.6.) Junlon PWIOO and Tween 80. Bars represent standard errors, where no bars are shown the error was smaller than the symbol. Avicelase activities of all cultures were significantly different (P :::; 0'05) on day 3. When the maximum activities occurring in the different cultures were compared, the control was not significantly different from the cultures containing only Junlon PWllO but all other differences between cultures were significant (P = 0·0l). CMCase activities in all cultures on day 3 were significantly different (P = 0·0l). When maximum CMCase activities were compared the control did not differ significantly from the cultures containing only Junlon PWllO but all other differences were significant (P = 0·0l). ~-glucosidase activities of all the different treatments were compared on day 7 and all differences were significant (P = 0·0l).
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Fig. 3. The effect of Junlon PWllO and Tween 80 on the production of (A) ~-xylosidase and (B) xylanase by Coprinus cinereus growing on Solka Floc as the major carbon source. (.) Control cultures with no additions; (0) only Tween 80; (.) only Junlon PWllO; (.6.) Junlon PWllO and Tween 80. Bars represent the standard errors, where no bar is shown the error was smaller than the symbol. ~-xylosidase activities were compared on day 7 and activities of all cultures were significantly different (P = 0.01). Xylanase activities were compared on days 5 and 7. The control and the cultures containing only Tween 80 did not differ significantly from each other and cultures containing only Junlon PWllO did not differ from those containing Junlon PWllO plus Tween 80. However the cultures without Junlon PWllO differed significantly (P = 0'01) from those containing Junlon PWIIO.
the pelleted form found in control cultures. Junlon PWll0 increased maximum cellulase activity by ca 30 % but the general pattern of production was similar to controls. pH profiles in cultures containing Junlon PWll0 differed only slightly from those of controls (Fig. 1), and it is unlikely that pH differences significantly affected growth or enzyme production. The inclusion of Tween 80 (0'6 g-1 1) in the culture medium also markedly increased cellulase activity, and a combination of Tween 80 and Junlon PWll0 resulted in even higher cellulase activity. The production of cellulase during growth on Avicel appeared to be largely growth-associated in the presence and absence of Junlon PWll0 but in the presence of Tween 80 (with or without Junlon PWll0) cellulase activity continued to increase for, at least, a further 3 days (generally peaking on the 7th day of growth) while biomass concentration decreased. In the presence of Tween 80 (0'6 g-1 I) a Junlon PWll0 concentration of 0'1 to 0'15% (w/v) was optimal for production of cellulase but as little as 0'025 % (w Iv) Junlon PWIlO increased the rate of cellulase accumulation compared with controls and with cultures containing only Tween 80.
Effect of Junlon on cellulase production Hostacerin PN73 (an anionic co-polymer of acrylic acid and acrylamide) had a similar effect to Junlon PW1l0 on both growth of, and cellulase production by, C. cinereus. Combinations of Hostacerin PN73 (0·1-0·2% w/v) and Tween 80 were as effective as Junlon PW1l0 and Tween 80, but the nonionic surfactant Montanox 20 (polyoxyethylene sorbitan monolaurate ester) at 0·1 % w Iv strongly inhibited growth and cellulase production. The effect of Junlon PW1l0 and Tween 80 on the production of individual enzyme activities of the cellulase complex was also determined (Fig. 2). The production of Avicelase was only very slightly increased by Junlon PW1l0 alone (ca 6% increase in maximum yield) but was greatly improved by Tween 80 (ca 167%) and even more so by a combination of the two additives (ca 235 %). Addition of Junlon PW1l0 also improved the production of CMCase (ca 42 %) and l3-glucosidase (ca 52·5%) but to a lesser degree than Tween 80, which increased maximum yield by 229% and 195 % respectively. Again the inclusion in the growth medium of both Junlon PW1l0 and Tween 80 was most effective resulting in increases of ca 400% in CMCase and 265 % in 13glucosidase yield. The effects of}unlon PW1l0 and Tween 80 (alone or in combination) on the production of cellulolytic enzymes by C. cinereus when grown on Solka Floc SW40 as the major carbon source were similar to those noted during growth on Avice!. Junlon PW1l0 generally increased the initial rate of enzyme production as well as the final yield (Figs 1, 2).
The presence of ]unlon PW1l0 also stimulated production of xylanase (by ca 20%) and l3-xylosidase (by ca 40%) by C. cinereus growing on Solka Floc SW40 (Fig. 3). Tween 80 had negligible effect on xylanase activity although it did stimulate l3-xylosidase activity (by 17 to 25 %).
DISCUSSION Both Tween 80 and the acrylic acid polymers stimulated the production of cellulolytic enzymes by C. cinereus. Although Tween 80 is well known to improve cellulase production by a range of fungi (e.g. Reese & Maguire, 1969; Schewale & Sadana, 1978; Hung et al., 1988), its effects on C. cinereus have not previously been reported. Tween 80 did not appear to affect the growth of C. cinereus in this study and prolonged production of cellulases long after the cessation of growth. The ability of Junlon PW1l0 and Hostacerin PN73 to increase cellulase production has not previously been reported, although the anionic polymer Carbopol has been shown to increase the rate of cellulase production by Trichoderma viride (Moo-Young, 1976) and of amylase production by Aspergillus niger (Elmayergi et al., 1973). Most previous studies do not show the effect of additives on the timing of exoenzyme production. The effects of Junlon PW1l0 and Tween 80 on cellulase production appear to be additive and may even be synergistic. ThiS, together with the temporal differences in their effects, suggests that, at least for C. cinereus, Junlon PW1l0 and Tween 80 affect cellulase production by different mechanisms. Junlon PW1l0 increased the growth of C. cinereus, in agreement with the findings of Jones et al. (1988b). A similar
1080 effect has been noted for Carbopol (Elmayegi et aI., 1973) and it seems reasonable to propose that these polymers increase cellulase production simply by increasing the extent of growth. This may be due to encouragement of mycelial rather than pelleted growth. Jones et al. (1988a) have suggested that the coating of spores and hyphae with Junlon PW1l0 reduces their tendency to aggregate, thus giving rise to dispersed filamentous rather than pelleted growth. The related polymer Carbopol has also been shown to coat fungal mycelia (Morrin & Ward, 1989) and it has been proposed that the presence of polymers may stimulate mass transfer of nutrients to the biomass (Byrne & Ward, 1987). Somewhat contradictory results have been reported by Mukhopadhyay & Chose (1977), who found that pelleted cultures of T. viride gave higher cellulase production than dispersed filamentous cultures. However, their results were obtained using different cultivation systems (shake flasks giving pelleted growth and stirred tank fermenters giving filamentous growth) and factors other than growth form (e.g. different rates of enzyme inactivation) may have been important. It is difficult to be certain whether the elevated levels of cellulolytic enzymes observed are due to de novo synthesis. 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 activity of cellulase in cultures. Although increased levels of cellulolytic enzymes in the presence of ]unlon PW1l0 may be due to increased growth, this is unlikely to explain the effect of Tween 80. As a surfactant, Tween 80 may interact with cell membranes and other surfaces, altering membrane permeability and encouraging release or solubilisation of exoenzymes (Reese, 1972). On the other hand, Castanon & Wilke (1981) showed that Tween 80 increased the rate of cellulose hydrolysis in cell-free systems and improved recovery of cellulolytic activity after saccharification. It was proposed that Tween 80 hindered immobilisation of cellulase on the insoluble substrate. Cellulases are subject to inactivation at air-liquid interfaces which has been shown to be preventable by the addition of the non-ionic detergent Triton X100, the fluorinated surfactant Zonyl FSN or even proteins (Reese, 1980; Kim et al., 1982). Incidentally, Triton XI00 has also been shown to enhance heat stability in some microbial extracellular Iipases (Bucky, Robinson & Hayes, 1987). The effect of Tween 80 may be due to one or a combination of these mechanisms. It is interesting that Tween 80 has negligible effect on xylanase production by C. cinereus as it has been shown to increase xylanase production in a range of fungi (Reese & Maguire, 1969; Reese, 1972). The toxicity of the surfactant Montanox 20 to C. cinereus is also surprising in view of its structural similarity to Tween 80. From a practical point of view the combination of Tween 80 and ]unlon PW1l0 offers the possibility of further significant increases in the rate of cellulase production and the final yield of cellulase from cultures of C. cinereus. As both Tween 80 and anionic polymers have been shown separately to improve significantly the production of cellulase and other exoenzymes in a range of fungi, it is likely that the
K. Long and
J. S. Knapp
combination will also have beneficial effects on other fungi which are perhaps more likely than C. cinereus to be commercially exploited in the near future. The authors gratefully acknowledge the financial support of the Malaysian Agricultural Research and Development Institute, which provided a scholarship to K. L.
REFERENCES Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 12, 248-254. Bucky, A R., Robinson, D. S. &< Hayes, P. R. (1987). Factors affecting the heat stability of lipase produced by a strain of Pseudomonas f/uorescens. Food Chemistry 23. 159--173. Burkholder, P. R. &< Sinnott, E. W. (1945). Morphogenesis of fungus colonies in submerged shaken cultures. American Journal of Botany 32, 424-431. Byrne, G. S. &< Ward, O. P. (1987). Effect of polymers on pelleting of Rhiwpus arrhizus. Transactions of British Mycological Society 89, 367-371. Castanon, M. &< Wilke, C. R. (1981). Effects of the surfactant Tween 80 on enzymatic hydrolysis of newspaper. Biotechnology and Bioengineering 23, 1365-1372. Elmayergi, H., Scharer, j. M. &< Moo-Young, M. (1973). Effects of polymer additives on fermentation parameters in a culture of Aspergillus niger. Biotechnology and Bioengineering 15, 845-859. 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. jones, P., Moore, D. &< Trinci, A P. j. (1988a). Effects of junlon and Hostacerin on the electrokinetic properties of spores of Aspergillus niger, Phanaerochaete chrysosporium and Geotrichum candidum. Journal of General Microbiology 134, 225-240. jones, P., Shahab, B. A, Trinci, A P. j. &< Moore, D. (1988 b). Effect of polymeric additives, especially junlon and Hostacerin, on growth of some basidiomycetes in submerged culture. Transactions of British Mycological Society 90, 577-583.
(Received for publication 17 Mny 1990 and in revised form 26 March 1991)
1081 Kim, M. H., Lee, S. B., Ryu, D. D. Y. &< Reese, E. T. (1982). Surface deactivation of cellulase and its prevention. Enzyme and Microbial Technology 4, 99--103. Mandels, M. &< Weber, j. (1969). The production of cellulases.1n Cellulases and their Applications, Advances in Chemistry Series 95 (ed. G. j. Hajny &< E. T. Reese), pp. 391-413. Washington DC: American Chemical Society Publications. Miller, G. L. (1959). Use of dinitrosalicylic acid for determination of reducing sugars. Analytical Chemistry 31, 426-428. Moo-Young, M. (1976). U.S. Patent 3975236; August 17, 1976. Described in Industrial Enzymes from Microbial Sources, Recent Advances. (ed. M. G. Halpern), pp. 246--247. Park Ridge, New jersey: Noyes Data Corporation. Morrin, M. &< Ward, O. P. (1989). Studies on interaction of Carbopol-934 with hyphae of Rhizopus arrhizus. Mycological Research 92, 265-272. Mukhopadhyay, S. N. &< Ghose, T. K. (1977). Studies on cellulase production in relation to morphological variations of Trichoderma viride QM 9414. In Proceedings of Bioconversion Symposium (ed. T. K. Ghose), pp. 97-109. New Delhi, India: Indian Institute of Technology. Pavlostathis, S., Miller, T. L. &< Wolin, M. j. (1988). Fermentations of insoluble cellulose by continuous cultures of Ruminococcus albus. Applied and Environmental Microbiology 54, 2655-2659. Pirt, S. j. (1966). A theory of the mode of growth of fungi in the form of pellets in submerged culture. Proceedings of the Royal Society, London, Series B 166, 369-373. Reese, E. T. (1972). Enzyme production from insoluble substrates. Biotechnology & Bioengineering Symposium 3, 43---62. Reese, E. T. (1980). Inactivation of cellulase by shaking and its prevention by surfactants. Journal of Applied Biochemistry 2, 36--39. Reese, E. T. &< McGuire, A (1969). Surfactants as stimulants of enzyme production by microorganisms. Applied Microbiology 17, 242-245. Shewale, j. G. &< Sadana, j. C. (1978). Cellulase and ~-glucosidase production by a basidiomycete species. Canadian Journal of Microbiology 24, 1204-1216. Trinci, A P. j. (1983). Effect of junlon on morphology of Aspergillus niger and its use in making turbidity measurements of fungal growth. Transactions of British Mycological Society 81, 408-412. Trinci, A P. j. &< Thurston, C. F. (1976). Transition to the non-growing state in eukaryotic micro-organisms. In The Survival of Vegetative Microbes (ed. T. R. G. Gray &< j. R. Postgate), pp. 55-80. Cambridge, U.K.: Cambridge University Press. Wood, T. M. &< Bhat, K. M. (1988). Method for measuring cellulase activities. In Methods in Enzymology 160 A. (ed. W. A Wood &< S. T. Kellogg), pp. 87-112. London: Academic Press.