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After a primary growth phase, at the onset of sec- ondary metabolism, the filamentous white-rot ba- sidiomycete, Phanerochaete chrysosporium, excretes.

Biotechnology Letters 22: 1443–1447, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.

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Extracellular ligninolytic enzyme production by Phanerochaete chrysosporium in a new solid-state bioreactor I. Rivela, S. Rodr´ıguez Couto & A. Sanrom´an∗ Department of Chemical Engineering, University of Vigo, 36200 Vigo, Spain Author for correspondence (Fax: +34 986 812382; E-mail: [email protected]) Received 1 June 2000; Revisions requested 28 June 2000; Revisions received 17 July 2000; Accepted 18 July 2000

Key words: immersion bioreactor, LiP, MnP, Phanerochaete chrysosporium, solid state fermentation

Abstract The production of manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP) by the fungus Phanerochaete chrysosporium (ATCC 24725) in a new bioreactor, the Immersion Bioreactor, which grows cells under solid-state conditions, was studied. Maximum MnP and LiP activities were 987 U l−1 and 356 U l−1 , respectively. The polymeric dye, Poly R-478, was degraded at 2.4 mg l−1 min−1 using the extracellular culture filtrate.

Introduction After a primary growth phase, at the onset of secondary metabolism, the filamentous white-rot basidiomycete, Phanerochaete chrysosporium, excretes two families of peroxidases into the culture medium: lignin peroxidase (LiP) and manganese peroxidase (Tien & Kirk 1983, Gold et al. 1984). These peroxidases have catalysed the initial oxidation of a wide variety of chemicals including structurally diverse (Banat et al. 1996, Buckley & Dobson 1998). The culture conditions affect fungal physiology as well as the expression and activity of the ligninolytic enzymes (Dosoretz et al. 1990). To increase the production of both MnP and LiP, optimisation through nutritional supplementation, addition of detergents and inducers, and variation of culture conditions has been carried out. In solid-state fermentation (SSF) the moist water insoluble solid substrate is consumed by microorganisms in the absence of free liquid (Marsh et al. 1998). The main advantage of SSF systems is that they simulate the fermentation reactions occurring in nature (Lonsane et al. 1985). The increasing interest around SSF in the last years has focused on its application in food fermentation and enzyme production. SSF is a technique, whose advantages and inconveniences have already been indicated

on repeated occasions (Hesseltine 1977, Lonsane et al. 1985). The factors in favour of this type of cultivation are the increased productivity and product concentration, comparable or superior to that of submerged fermentation, and a more economical cost because it is possible the use of agro-industry residues as support (Murthy et al. 1993). Nevertheless, there is very little information regarding engineering aspects of SSF and few designs of bioreactors are available in the literature (Lonsane et al. 1985, Pandey 1992, Durand et al. 1993, Marsh et al. 2000). Their application to industrial processes on a large scale requires the use of an efficient production system. In previous work, we achieved a high production of extracellular ligninolytic enzymes in semi-solid-state cultures and have demonstrated the utility of such cultures (Rodríguez et al. 1997, Rodríguez Couto et al. 1998a,b, 1999). The objective of this paper is to develop and establish the conditions allowing the enzymatic production in a bioreactor for a long time with no operational problems. Thus, a new bioreactor design, operating in solid-state conditions with cubes of nylon sponge as support, was developed. Furthermore, the degradation capability of the ligninolytic complex produced has been determined.

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Fig. 1. Schematic diagram of an Immersion Bioreactor, 225 mm height and 232 mm internal diameter (working volume of 2.5 l). (1) Medium, (2) wire mesh baskets 30 mm height and 165 mm internal diameter and (3) pneumatic system.

Materials and methods Microorganism and growth medium Phanerochaete chrysosporium BKM-F-1767 (ATCC 24725) was grown on a medium prepared according to Tien & Kirk (1988) with 10 g glucose l−1 as carbon source, except dimethylsuccinate was replaced by 20 mM acetate buffer (pH 4.5) (Dosoretz et al. 1990). The fungus was grown in 90 ml of this medium at 37 ◦ C in complete darkness for 48 h. After this, the entire culture was homogenised in a blender for 1 min. This homogenate suspension was used to inoculate (10% v/v) the production medium.

filled with cubes of fibrous nylon sponge and placed into the bioreactor vessel. They were moving upwards and downwards by means of a pneumatic system, remaining 90 s outside and 10 s inside the medium. Temperature was maintained at 37 ◦ C by circulation of temperature controlled water. Air was supplied in a continuous way at 1 vvm. The production medium composition was the same as the growth medium. Moreover, sorbitan polyoxyethylene monooleate (Tween 80, 0.5% v/v) and veratryl alcohol (3,4-dimethoxybenzyl alcohol; 2 mM final concentration) were added at the beginning of the cultivation in order to stimulate the ligninolytic enzyme production (Rodriguez & Rättö 1998b). This medium was inoculated with 10% (v/v) homogenised mycelium. In vitro Poly R-478 decolourisation The in vitro Poly R-478 decolourisation was monitored at 520 nm, which is the maximum visible absorbance of the polymeric dye, according to Kim et al. (1995). The reaction mixture contained sodium malonate (6 mM; pH 4.5), manganese sulphate (0.1 mM), hydrogen peroxide (0.4 mM), crude enzyme and dye (Poly R-478: 0.03 g l−1 ) in a total volume of 1 ml. The MnP activity of crude enzyme employed was 200 U l−1 . Analytical determinations Reducing sugars They were measured by the dinitrosalicylic acid method using D-glucose as standard, according to Ghose (1987).

Carrier The bioreactor was filled with 5 mm cubes of fibrous nylon sponge (Scotch Brite, 3M Company, Spain), which will act as a supporting matrix on which the mycelium can be bound. The nylon sponge was pre-treated according to Linko (1991) by boiling for 10 min and washing thoroughly three times with distilled water. Then, the carriers were dried at room temperature overnight and autoclaved at 121 ◦ C for 20 min until used. Design of an Immersion Bioreactor The bioreactor used was designed by our group. It consists of a jacketed cylindrical glass vessel with a round bottom (Figure 1). Two wire mesh baskets, were

Nitrogen ammonium content It was assayed by the phenol/hypochlorite method described by Weatherburn (1967), using NH4 Cl as standard. Mn (II)-dependent peroxidase activity It was assayed spectrophotometrically by the method of Kuwahara et al. (1984). One activity unit was defined as the amount of enzyme that oxidised 1 µmol of dimethoxyphenol per minute and the activities were expressed in U l−1 . Lignin peroxidase activity It was analysed spectrophotometrically according to Tien & Kirk (1984). One unit (U) was defined as the

1445 amount of enzyme that oxidised 1 µmol veratryl alcohol in 1 min, and the activities were reported as U l−1 . Laccase activity It was determined spectrophotometrically as described by Niku-Paavola et al. (1990) with ABTS (2,20 -azinodi-[3-ethyl-benzo-thiazolin-sulphonate]) as substrate. One activity unit was defined as the amount of enzyme that oxidised 1 µmol ABTS per min. The activities were expressed in U l−1 .

Results and discussion Enzymatic production in the Immersion Bioreactor Several bioreactor types have been employed in submerged or immobilised conditions with the aim of obtaining high ligninolytic enzyme activities (Moreira et al. 1995, 1997, Bosco et al. 1996, Laugero et al. 1996). Nevertheless, there are only a few studies on the production of such enzymes in solid-state bioreactors. When the cultivation is carried out in solid-state conditions, the selection of an adequate bioreactor design as well as studies on engineering and scale-up are absolutely necessary. Among the diverse types of supports tested for this fermentation process, the most interesting ones were nylon sponge and corncob. The former, in particular, is an inert material, which allowed us to study the efficiency of the biorreactor without interactions of a series of variables related to the composition of natural support. Pre-cultures of the fungus must be made in order to inoculate the bioreactor with enough inoculum, otherwise fungus growth would be delayed and the product formation rate could be unsatisfactory. This pre-cultivation was realised in a tray bioreactor, operating with cubes of nylon sponge as support, for six days. In order to determine the suitability of the bioreactor developed in the present paper to produce ligninolytic enzymes, a culture in batch process was performed, operating with aeration and an immersion frequency of 0.01 s−1 . Glucose consumption (see Figure 2), measured as reducing sugars, was very low and nitrogen was not consumed until the third day, which caused a delay in the appearance of the ligninolytic activities, since the depletion of this nutrient activates the ligninolytic system of this fungus. MnP activity first

appeared on the fourth day (100 U l−1 ) and peaked on the tenth day (1293 U l−1 ). The profile of LiP activity produced was rather irregular, starting on the second day (100 U l−1 ) and reaching a maximum value of 350 U l−1 on the thirteenth day. It is worth drawing attention to the fact that with the bioreactor employed, a decrease in the restrictions to gaseous inter-particular diffusion was observed, which furnishes a more appropriate environment for the production of ligninolytic enzymes. Finally, one should note that, in this bioreactor no operation problems have been detected in all fermentation days. In a previous work (Rodriguez Couto 2000) the production of ligninolytic enzymes by the fungus Phanerochaete chrysosporium in packed-bed tubular bioreactors, operating in semi-solid-state conditions, was studied. In both bioreactors, immersion and packed-bed, the cultivation was carried out in discontinuous mode. In Table 1, the maximum MnP and LiP activity and the constancy index are summarised. The constancy index was calculated with the activities obtained three days before and three days after the maximum value (admax ), and the equation employed to calculate it, is the following: MnP or LiPconstancy index = (admax−3 + admax−2 + admax−1 + admax + admax+1 + admax+2 + admax+3 )/7(admax). When the enzymatic production achieved in a packedbed bioreactor filled with nylon sponge was analysed, it was observed that MnP activity started on the sixth day (145 U l−1 ) and it increased, reaching a maximum value of 1593 U l−1 on the tenth day. On the other hand, LiP activity appeared on the first day (23 U l−1 ) and the highest value was achieved on the sixth day (229 U l−1 ). In this case, an irregular profile for both ligninolytic enzymes was observed, and this behaviour is determined by the low constancy index for the MnP and LiP obtained (Table 1), which indicates that the enzymes obtained are easily deactivated. On the other hand, using the packed-bed reactor in these conditions numerous operational problems were encountered, which did not appear operating with the immersion one. The results obtained clearly show that operating with the bioreactor developed in this work, named Immersion Bioreactor, is possible to obtain high ligninolytic enzyme activities without operational problems. Moreover, the enzymes produced were not de-

1446 Table 1. Maximum MnP and LiP activities (a) and the C.I.: constancy index (calculated as: admax−3 + admax−2 + admax−1 + admax + admax+1 + admax+2 + admax+3 )/7(admax )) operating with a Packed-bed and an Immersion Bioreactor. MnP Max (U l−1 ) Packed-bed (200 ml) Immersion (2500 ml)

Max (U)

C.I.

LiP Max (U l−1 )

Max (U)

C.I.

1593

319

0.284

229

46

0.354

987

2468

0.72

356

890

0.578

activated earlier. This is due to this design permiting a good attachment of the fungus to the carrier as well as a proper oxygen and nutrients diffusion into the bioreactor. Decolourisation of Poly R-478 Some studies demonstrated a good correlation between biodegradation of aromatic pollutants and decolourisation of polymeric dyes by ligninolytic fungi. Therefore, the decolourisation of Poly R-478 is a simple method to assess the degrading capability of ligninolytic enzymes. The aim of this paper is to examine the potential and the ability of the ligninolytic complex secreted by the white-rot fungus P. chrysosporium, operating in an immersion bioreactor in solid-state conditions, to decolourise in vitro the polymeric dye Poly R-478. The decolourisation was carried out directly in the spectrophotometer cubette and Poly R-478 degradation was monitored at 520 nm, which is its maximum visible absorbance according to Kim et al. (1995). The evolution of the decolourisation is shown in Figure 3, and with these data the initial degradation rate of the dye was determined. The value obtained was 2.4 mg l−1 min−1 , which suggests that the extracellular ligninolytic enzymes secreted in the immersion bioreactor are efficient in dye degradation.

Fig. 2. Glucose and ammonium consumption and MnP and LiP activities obtained in an Immersion solid-state Bioreactor.

Conclusions The bioreactor configuration designed and employed in the present report has shown to be very suitable to produce ligninolytic enzymes in solid-state conditions, since it permitted to operate for a long time without operational problems. Moreover, the ligninolytic complex secreted degraded efficiently the polymeric dye Poly R-478, which indicates that the enzymes

Fig. 3. Profile of maximum absorbance of PolyR-478 at 520 nm, using the extracellular culture filtrate with MnP activity of crude enzyme of 200 U l−1 .

1447 obtained are adequate for their application to several biotechnology processes. Therefore, in view of these encouraging results studies on a large scale are underway.

Acknowledgement This research was financed by DGES (Spanish government) PB97-0668.

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