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Abstract The production of ligninolytic enzymes by the fungus Phanerochaete chrysosporium BKM-F-1767 (ATCC. 24725) in laboratory-scale bioreactors was ...
Bioprocess Engineering 20 (1999) 531±535 Ó Springer-Verlag 1999

Production of manganese peroxidase and laccase in laboratory-scale bioreactors by Phanerochaete chrysosporium S. RodrõÂguez, M.A. Longo, C. Cameselle, A. SanromaÂn

genetics and molecular biology are well-documented [2, 3]. Nutrient nitrogen limitation has been thought to be the major factor triggering activation of the lignin degrading system in P. chrysosporium, although limitation of carbon, sulphur and manganese have also been shown to stimulate it [4]. Moreover, lignin degradation does not occur without the presence of a readily metabolizable cosubstrate such as glucose [2]. On the contrary, the increased rates of lignin metabolism are connected to increased cosubstrate consumption rates [5]. In 1983 and 1984, two extracellular enzymes, lignin peroxidase (LiP) and manganese peroxidase (MnP), were discovered in P. chrysosporium [6, 7, 8]. These enzymes have been demonstrated to be the major components of the lignin degradation system of this microorganism [9]. The production of enzymes using solid-substrate fermentation has been developed from the ``koji'' process. List of symbols Economically, this type of process presents many advanSSF Solid-state fermentation tages [10] such as superior volumetric productivity, reMnP Manganese-dependent peroxidases duced energy requirements, simple handling and improved product recovery. However, many problems are encountered in control of different parameters and scale1 up, especially due to the choice of reactor design [11]. One Introduction of the great advantages of solid-state processes could be White-rot fungi are able to degrade all the three major components in wood at similar rates [1]. To understand the possible reduction of the production costs, by using high amounts of solid substrate, although many problems the mechanism of wood degradation by these microormust be solved in the ®eld of monitoring and control of ganisms, and especially the action on the recalcitrant parameters in these conditions [12]. polymer lignin, their natural habitat needs to be consiIn the present work, two laboratory-scale bioreactors dered. The natural growth environment of these fungi, wood, means low moisture content, reduced oxygen partial ®lled with inert carriers (polyurethane foam in one case and nylon sponge in the other) were studied, in order to pressure and low nitrogen concentration. The most thoroughly studied white-rot fungus is Pha- determine the more suitable support for ligninolytic enzyme production by P. chrysosporium. Moreover, the renerochaete chrysosporium (asexual stage known as Sposults obtained were compared to those obtained in rotrichum pulverulentum). Its lignin-degrading ability, growth requirements, culture conditions, mode of attack previous experiments in static ¯ask cultures. on lignin, enzymes secreted during lignin degradation,

Abstract The production of ligninolytic enzymes by the fungus Phanerochaete chrysosporium BKM-F-1767 (ATCC 24725) in laboratory-scale bioreactors was studied. One bioreactor was ®lled with cubes of polyurethane foam and the other with cubes of nylon sponge, in order to determine the more suitable carrier to produce high ligninolytic enzyme activities by this fungus. Both cultivations were carried out in batch. Manganese-dependent peroxidase activities about 600 U l)1 were achieved in the bioreactor ®lled with cubes of nylon sponge, while up to 500 U l)1 were detected in that ®lled with cubes of polyurethane foam. Furthermore, quite high levels of laccase appeared in both cultures: maximum activities of 114 U l)1 and 62 U l)1 were obtained on nylon and polyurethane supports, respectively.

2 Materials and methods Received: 25 June 1998

S. RodrõÂguez, M.A. Longo, C. Cameselle, A. SanromaÂn Department of Chemical Engineering, University of Vigo, E-36200 Vigo, Spain Correspondence to: S. RodrõÂguez This research was ®nanced by Xunta de Galicia (Project XUGA30113A96).

2.1 Microorganisms and medium P. chrysosporium BKM-F-1767 (ATCC 24725) was maintained at 37 °C on 2% malt agar slants and plates. Spores were harvested, ®ltered through glass wool, and kept at )20 °C [13]. The growth medium was prepared as described by Tien & Kirk [14] with 10 g glucose per litre as carbon source, and 20 mM acetate buffer (pH 4.5) instead of dimethyl-

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(SDS-PAGE), using a 10% polyacrylamide gel in a Bio-Rad Ready Gel vertical electrophoresis unit. Isoelectric points were evaluated by isoelectric focussing (IEF), using a wide range (pH 3±10) gel from Bio-Rad. Protein bands were detected by silver staining and their properties were determined by comparison with a suitable standard (Molecular Weight Standards, Low Range, for 2.2 SDS-PAGE and IEF Standards, pI range 4.45±9.6 for isoCulture conditions The culture medium was identical to the growth medium, electric focussing, both from Bio-Rad). except that sorbitan polyoxyethylene monooleate (Tween 80, 0.5% v/v) and veratryl alcohol (2 mM ®nal concen3 tration) were added at the beginning of the cultivations. Results and discussion Cultivations in 1.0-l (working volume) bioreactors were Solid-state cultures are characterized by the development carried out at 37 °C with aeration. The reactors were ®lled of microorganisms in an environment of low water actiwith 5-mm cubes of polyurethane foam in one case and vity, upon a damp insoluble material, which functions as a with 5-mm cubes of ®brous nylon sponge (Scotch BriteTB, physical support. Fungal mycelia penetrate into the par3M Company, Spain) in the other. These carriers will act as ticles of the substrate [19]. The utilization of solid subsa supporting matrix on which the mycelium can be bound. trates by the microorganisms is affected by several The free culture liquid was gently agitated by means of the physical and chemical factors. Among the physical factors, accessibility of substrate to microbes, ®lm effects, mass air bubbles. Before use, the cubes of nylon sponge were boiled for effects and the physical morphology (i.e. porosity and 10 min and thoroughly washed three times with distilled particle size) of the substrate, are outstanding [20]. Among the chemical factors, the chemical nature of the substrate water. Polyurethane foam cubes were washed once in methanol, then three times with distilled water. Both car- is a remarkable feature to have in account [19]. In the present work, P. chrysosporium has been cultiriers were dried at 60 °C and autoclaved until use. vated in a 1-l laboratory-scale bioreactor, using two different inert materials as supports. When polyurethane 2.3 foam was employed as a carrier, glucose consumption was Analytical determinations very slow; its concentration decreased at an approximately Reducing sugars: they were measured by the dinitrosalicylic acid method using D-glucose as standard, according constant rate of 0.55 g l)1 day)1. On the contrary, nitrogen to Ghose [16]. was totally consumed in two days. Manganese peroxidase activity ®rst appeared on day 3 (17 U l)1), then increased Nitrogen ammonium content: it was assayed by the phe- progressively up to a maximum value on days 7 and 8 nol-hypochlorite method described by Weatherburn [17], (478 U l)1 and 477 U l)1, respectively) (Fig. 1). After that, MnP activity decreased sharply, although some new enusing NH4Cl as standard. zyme seemed to be produced from day 12 onward. Laccase activity was also assayed throughout the culLaccase activity: it was determined spectrophotometrically as described by Niku-Paavola et al. [18] with ABTS (2,2¢- tures. This enzyme ®rst appeared on day 6 (24 U l)1) and azino-di-[3-ethyl-benzothiazolinsulphonate]) as substrate. attained a maximum level (62 U l)1) on day 8 (Fig. 1). A decrease in activity after day 11, similar to that mentioned The laccase reaction mixture contained 2.3 ml enzyme diluted to buffer (0.025 M succinic acid, pH 4.5) and 0.7 ml above for MnP, was observed. Nevertheless, laccase pro0.02 M ABTS. The reaction was monitored by measuring duction was not recovering during the following days. In the change in A436 for 2 min. One activity unit was de®ned spite of the generally held belief that this enzyme was absent in P. chrysosporium [21], the results demonstrated as the amount of enzyme that oxidized 1 lmol of ABTS the ability of this fungus as laccase producer, and agreed per minute. The activities were expressed in U l)1. with a recent report by Dittmer et al. [22] and with a previous work [23]. Mn(II)-dependent peroxidase activity: it was determined Comparison of the results with those previously respectrophotometrically by the method of Kuwahara et al. [7]. The reaction mixture contains 200 ll of 250 mM so- ported [24] for stationary ¯ask cultures in the same condium malonate (pH 4.5), 50 ll of 20 mM 2,6 dimethoxy- ditions (Table 1), indicated that higher MnP production levels were attained in bioreactors, whereas slightly lower phenol, 50 ll 20 mM MnSO4áH2O and 600 ll of diluted laccase activities were detected. culture ¯uid (200 ll of enzyme sample plus water). The An attempt of preliminary characterization of the proreaction was started by adding 100 ll of 4 mM H2O2. One teins produced during the culture was undertaken. Samactivity unit was de®ned as the amount of enzyme that ples collected from the bioreactor ®lled with polyurethane oxidized 1 lmol of dimethoxyphenol per minute. The foam at days 7, 8 and 10, in which high enzymatic activactivities were expressed in U l)1. ities (»500 MnP U l)1, 30±60 laccase U l)1) had been detected, were studied by polyacrylamide gel electrophoresis 2.4 in denaturing conditions. A major protein band at moElectrophoresis lecular weight around 40 kDa, and some minor bands at Molecular weights of the proteins produced were deterslightly lower molecular weights (between 30 and 35 kDa) mined by gel electrophoresis in denaturing conditions succinate [15]. The fungus was grown in 90 ml of this medium at 37 °C in complete darkness for 48 hours. After this, fungus and medium were homogenized for 1 min. This homogenate was used to inoculate (10% v/v) the fermentation medium.

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0 0

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8 10 12 Time (days)

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Activity (U l-1)

Glucose (g l-1), Nitrogen (mg l-1)

S. RodrõÂguez et al.: Production of manganese peroxidase and laccase in laboratory-scale bioreactors

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Fig. 1. Ligninolytic activities by P. chrysosporium in a laboratory Fig. 2. Ligninolytic activities by P. chrysosporium in a laboratory bioreactor ®lled with polyurethane foam. Symbols: j MnP activity bioreactor ®lled with nylon sponge. Symbols: j MnP activity )1 )1 )1 (U l ) d Laccase activity (U l ) e Glucose (g l ) and n Nitrogen (U l)1) d Laccase activity (U l)1) e Glucose (g l)1) and n Nitrogen ammonium (mg l)1) concentrations during the cultivation ammonium (mg l)1) concentrations during the cultivation Table 1. Ligninolytic activities obtained in stationary ¯asks cul- although in that case, no laccase activity had been detected tures and in bioreactors by P. chrysosporium employing poly(Table 1). urethane foam and nylon sponge as carriers

Stationary ¯asks Bioreactor

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were identi®ed. Then, the selected samples were studied by isoelectric focussing, in order to investigate the isoelectric points of the produced enzymes. The major band was detected at pI 4.0. The values obtained for molecular weight and isoelectric point of the proteins agreed with previous results reported for peroxidases from P. chrysosporium. It was not possible to distinguish the different isozymes composing the ligninolytic system in the assayed conditions, since all of them have very similar properties [25, 26]. The behaviour of P. chrysosporium was also studied using a bioreactor ®lled with cubes of nylon sponge. Glucose consumption was also very slow, decreasing at an average rate of 0.47 g l)1 day)1. On the other hand, nitrogen consumption was slower than with the polyurethane carrier and it was not totally consumed until day 5. Manganese peroxidase activity ®rst appeared on day 3 (21 U l)1) and then increased gradually up to a maximum value of 571 U l)1 on day 9 (Fig. 2). Afterwards, the activity levels remained nearly constant (around 500 U l)1) from days 9 to 14 and ®nally they decreased abruptly during the late stages of the experiment. On the other hand, laccase was also produced in nylon sponge cultures. This enzyme ®rst appeared on day 1 (75 U l)1) and attained a maximum level (114 U l)1) on day 19 (Fig. 2). In this case, laccase activity was detected throughout the whole culture time. Cultivation of P. chrysosporium in ¯ask cultures, using nylon sponge as support, had given place to higher MnP production levels than the experiments presented here,

A preliminary protein characterization study was also carried out on samples from the bioreactor ®lled with nylon sponge at days 9, 11 and 14. Similar protein bands than for the polyurethane supported cultures were detected. Nevertheless, an additional weak band at molecular weight 65 kDa was observed in the sample taken at day 14. This could be due to the appearance in the cultures of other enzymes (proteases, glyoxal oxidase) or some protein from cell degradation [27, 28]. The study of MnP production pro®les showed that the high activity levels were maintained longer in the cultures on nylon sponge than in those on polyurethane foam (6 and 3 days, respectively). This could indicate either that the peroxidases produced on the former carrier are more stable, or that a smaller amount of potential enzyme deactivators (i.e. hydrogen peroxide, proteases) [29, 30] are produced during the culture in these conditions. A similar phenomenon was observed on laccase activity: it decreased strictly to zero from day 11 onward in the bioreactor ®lled with cubes of polyurethane foam whereas a more or less continuous activity level was obtained in the bioreactor ®lled with cubes of nylon sponge from day 1 onward. In addition to this, laccase activities obtained with nylon sponge were also higher than those achieved with polyurethane foam. Therefore, the use of nylon sponge as a carrier allowed maintenance of both MnP and laccase activities for longer times. Thus, it can be asserted that nylon sponge is a more suitable carrier for ligninolytic enzyme production than polyurethane foam. This can be due to the higher porosity of the nylon carrier, which allows a better oxygen transfer into the mycelium mats, as well as a better diffusion of the nutrients. Besides, this porosity could facilitate the access of the added veratryl alcohol and Tween 80 to the supportbound microorganism, and increase their effect as production inducers and/or enzyme activity protection agents [2]. Moreover, the fungus grew better and faster in nylon sponge than in polyurethane foam. Biomass estimation was almost three times higher in the bioreactor ®lled with nylon sponge than in the one ®lled with polyurethane

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basidiomycete from Phanerochaete chrysosporium. Biochem. foam. The higher microorganism concentration could exBiophys. Res. Commun. 114 (1983) 1077±1083 plain the higher activity levels detected in nylon cultures. 7. Kuwahara, M.; Glenn, J.K.; Morgan, M.A.; Gold, M.H.: SeThe better growth can be due to the higher roughness and paration and characterization of two extracellular H2O2-deporosity of the nylon carrier, which offers a larger surface pendent oxidases from ligninolytic cultures of Phanerochaete area to the microorganism and makes easier its anchorage. chrysosporium. FEBS Lett. 169 (1984) 247±250 Besides, the different chemical composition of these car8. Tien, M.; Kirk, T.K.: Lignin-degrading enzyme from the hyriers could also in¯uence the growth of the fungus. Despite menomycete Phanerochaete chrysosporium Burds. Science. 221 (1983) 661±663 having been traditionally regarded as inert supports, some 9. Gold, M.H.; Alic, M.: Molecular biology of the lignin-deresearchers have reported degradation of this kind of grading basidiomycete Phanerochaete chrysosporium. Micromaterials during microorganism cultivation, which could biological Reviews. 57(3) (1993) 605±622 provoke the release of potentially toxic compounds in the 10. Cannel, E.; Moo-Young, M.: Solid-state fermentations sysculture media [31]. tems. Process Biochem. 6 (1980) 2±7

4 Conclusions From the obtained results, it can be concluded that nylon sponge appears to be a very suitable carrier for MnP production and, above all for laccase production in a laboratory-scale bioreactor. This is probably due to the higher roughness and porosity of this carrier, which makes easier the anchorage of the fungus to the carrier and permits a better oxygenation of the fungus and diffusion of medium components. On the other hand, when nylon sponge was used as a carrier laccase activity appeared earlier than by employing polyurethane foam. This can also be related to a better and faster growth of the fungus in this carrier. It may be that the fungus needs to attain a certain degree of growth in order to produce this enzyme. Thus, by employing a bioreactor ®lled with cubes of nylon sponge in a medium supplemented both with Tween 80 (0.5% v/v) and veratryl alcohol (®nal concentration 2 mM) in semi-solid-state conditions high MnP and laccase activities were achieved. It suggests the possibility of applying this system to large-scale processes. Anyway, studies of cultivations in larger bioreactors (`scaling up') are underway. References

1. Kirk, T.K.; Fenn, P.: Formation and action of the Ligninolytic system in Basidiomycetes. In: Hollaender, A (Ed.): Trends in the Biology of Fermentations for Fuels and Chemicals, p. 131. New York: Plenum 1981 2. Kirk, T.K.; Farrell, R.L.: Enzymatic ``combustion'': The microbial degradation of lignin. Annu. Rev. Microbiol. 41 (1987) 465±505 3. Alic, M.; Gold, M.H.: Genetics and molecular biology of the lignin-degrading basidiomycete Phanerochaete chrysosporium. In: More genes manipulations in fungi, pp. 1459±1464. Academic Press 1991 4. Bonnarme, P.; Jeffries, W.: Mn(II) regulation of lignin peroxidase and manganese-dependent peroxidases from lignin degrading white rot fungi. Appl. Environm. Microbiol. 56 (1990) 210±217 5. Leisola, M.; Ulmer, D.; Haltmeier, T.; Fiechter, A.: Rapid solubilization and depolymerization of puri®ed kraft lignin by thin layers of Phanerochaete chrysosporium. Eur. J. Appl. Microbiol. Biotechnol. 17 (1983) 117 6. Glenn, J.K.; Morgan, M.A.; May®eld, M.B.; Kuwahara, M.; Gold, M.H.: An extracellular H2O2-requiring enzyme preparation involved in lignin biodegradation by the white-rot

11. Lonsane, B.K.; Ghildyal, N.P.; Budiatman, S.; Ramakrishna, S.V.: Engineering aspects of solid-state fermentation. Enzyme Microb. Technol. 7 (1985) 258±265 12. Durand, A.; de la Broise, D.; BlacheÁre, H.: Laboratory scale bioreactor for solid state processes. J. Biotechnol. 8 (1988) 59±66 13. JaÈger, A.; Croan, C.; Kirk, T.K.: Production of ligninases and degradation of lignin in agitated submerged cultures of Phanerochaete chrysosporium. Appl. Environ. Microbiol. 50 (1985) 1274±1278 14. Tien, M.; Kirk, T.K.: Lignin peroxidase of Phanerochaete chrysosporium. Meth. Enzymol. 161 (1988) 238±249 15. Dosoretz, C.G.; Chen, H.C.; Grethlein, M.E.: Effect of oxygenation conditions on submerged cultures of Phanerochaete chrysosporium. Appl. Microb. Biotech. 34 (1990) 131±137 16. Ghose, T.K.: Measurement of cellulase activities. Pure Appl. Chem. 59 (1987) 257±268 17. Weatherburn, M.W.: Phenol-hypochlorite reaction for determination of ammonia. Anal. Chem. 28 (1967) 971±974 18. Niku-Paavola, M.L.; Raaska, L.; ItaÈvaara, M.: Detection of white-rot fungi by a non-toxic stain. Mycological Research. 94(1) (1990) 27±31 19. Pandey, A.: Recent process developments in solid-state fermentation. Process Biochem. 27 (1992) 109±117 20. Knapp, J.S.; Howell, J.A.: Topics in Enzymes and Fermentation Biotechnology. In: Wiseman, A. and Howard, E. (Eds.), vol. 4, pp. 85±143 1985 21. Thurston, C.F.: The structure and function of fungal laccases. Microbiology. 140 (1994) 19±21 22. Dittmer, J.K.; Patel, N.J.; Dhawale, S.W.; Dhawale, S.S.: Production of multiple laccase isoforms by Phanerochaete chrysosporium grown under nutrient suf®ciency. FEMS Microbiol. Lett. 149 (1997) 65±70 23. RodrõÂguez Couto, S.; Santoro, R.; Cameselle, C.; SanromaÂn, A.: Laccase production in semi solid cultures of Phanerochaete chrysosporium. Biotechnol. Lett. 19 (1997) 995±998 24. RodrõÂguez Couto, S.; Santoro, R.; Cameselle, C.; SanromaÂn, A.: ComparacioÂn de dos tipos de soportes inertes en la produccioÂn de MnP por Phanerochaete chrysosporium durante cultivos en estado semi-soÂlido. XI Encontro GalegoPortugueÂs de QuõÂmica, Ferrol, Nov. 1997 25. Linko, S.: Production of lignin peroxidase by immobilized Phanerochaete chrysosporium. PhD thesis, Department of Food Science, Wageningen Agricultural University, Wageningen, The Netherlands (1993) 26. De Jong, E.: Physiological roles and metabolism of fungal aryl alcohols. PhD thesis, Helsinki University of Technology (1991) 27. Dass, S.B.; Dosoretz, C.G.; Reddy, C.A.; Grethlein, H.E.: Extracellular proteases produced by the wood-degrading fungus Phanerochaete chrysosporium under ligninolytic and non-ligninlolytic conditions. Arch. Microbiol. 163 (1995) 254±258

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30. Bonnarme, P.; Asther, M.: In¯uence of primary and se28. Kersten, P.J.; Kirk, T.K.: Involvement of a new enzyme, condary proteases produced by free or immobilized cells of glyoxal oxidase, in extracellular H2O2 production by Phathe white-rot fungus Phanerochaete chrysosporium on lignin nerochaete chrysosporium. J. Bacteriol. 169 (1987) 2195±2201 peroxidase activity. J. Biotechnol. 30 (1993) 271±282 29. Tonon, F.; Odier, E.: In¯uence of veratryl alcohol and hydrogen peroxide on ligninase activity and ligninase produc- 31. Deguchi, T.; Kakezawa, M.; Nishida, T.: Nylon biodegradation by lignin-degrading fungi. Appl. Environm. Microbiol. 63 tion by Phanerochaete chrysosporium. Appl. Environm. (1997) 329±331 Microbio. 54 (1988) 466±472

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