Process Biochemistry 37 (2001) 549– 554 www.elsevier.

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Design of a new rotating drum bioreactor for ligninolytic enzyme production by Phanerochaete chrysosporium grown on an inert support
´ ngeles Sanroma Alberto Domı ´nguez, Isabel Rivela, Susana Rodrı ´guez Couto, Ma A ´n *
Department of Chemical Engineering, Uni6ersity of Vigo, Lagoas -Marcosende s /n, E -36200 Vigo, Spain Received 23 April 2001; received in revised form 28 May 2001; accepted 13 June 2001

Abstract The production of ligninolytic enzymes by the white-rot fungus Phanerochaete chrysosporium BKM-F-1767 was studied in a new bioreactor configuration based on a standard rotating drum bioreactor. P. chrysosporium was grown on cubes of nylon sponge, and cultivation was carried out in batch. Two aeration levels: 0.5 and 1 vvm were tested. The latter led to activities about 3-fold higher than the former, achieving maximum manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP) activities of 1350 and 364 U/l, respectively. Moreover, laccase activity was also detected, showing a highest activity of 56 U/l. In addition, the in vitro decolorisation of a model dye (Poly R-478) by the extracellular liquid obtained in the bioreactor was monitored in order to assess its ligninolytic ability. A percentage of Poly R-478 decolorisation of about 19% was achieved, after 15 min of dye incubation. © 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Aeration rate; Ligninolytic enzymes; Nylon sponge; Phanerochaete chrysosporium ; Rotating drum bioreactor; Solid-state fermentation

1. Introduction The white-rot fungus Phanerochaete chrysosporium secretes, during its secondary metabolism, several lignin-degrading enzymes including lignin peroxidase (LiP), manganese-dependent peroxidase (MnP) and laccase. The secondary metabolism in this fungus is triggered by nitrogen [1], carbon or sulphur [2] deprivation. In addition to lignin, the above-mentioned enzymes are also able to degrade a wide range of hazardous environmental pollutants [3,4]. Their application to industrial processes (biobleaching, biopulping, decolorisation, etc.) on a large scale requires the production of high amount of enzyme at low cost. Thus, the design of a system permitting the continuous production of ligninolytic enzymes efficiently is required. Solid-state fermentation (SSF) involves the growth of microorganisms on moist solid substrates in the absence or near absence of free liquid [5]. The physical nature of
* Corresponding author. Tel.: + 34-986-812-304; fax: + 34-986812-382. ´ . Sanroma E -mail address: sanroman@uvigo.es (M.A ´ n).

the medium is quite different from that in submerged fermentation. For example, a solid bed is more difficult to mix effectively than a liquid broth, and as a consequence, O2 supply and heat removal can be restricted in SSF processes [6]. The kind of solid materials that can be employed in this type of cultivation are classified in two main categories: inert support and non-inert support. The former acts as an attachment place (e.g. plastic foams) whereas the latter also functions as a source of nutrients (e.g. crop wastes). SSF has gained importance in recent years due to several advantages over submerged fermentation such as superior productivity, simpler techniques, reduced energy requirements, low wastewater output, and improved product recovery [7–9]. Nevertheless, bioreactor design aspects have not been received enough attention by researchers of SSF and the present state of the art does not indicate an ideal type of bioreactor for solid state processes. In earlier reports, a high production of extracellular ligninolytic enzymes in semi-solid-state cultures has been achieved and the utility of such cultures has been demonstrated [10 –12].

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In order to apply this bioreactor to semi-solidstate processes the volume of the culture medium in contact with the microorganism has been reduced to conditions near to semi-solid-state. 2 mM final concentration) were added at the beginning of the cultivation in order to stimulate ligninolytic enzyme production [12]. Pre-cultures of the fungus were made in order to inoculate the bioreactor with enough inoculum. rotating device. nylon sponge was preferred in the present work. the carriers were dried at room temperature overnight and autoclaved at 121 °C for 20 min until used. The fungus was grown in 90 ml of this medium at 37 °C in complete darkness for 48 h. the whole culture was homogenised in a blender for 1 min. This homogenate suspension was used to inoculate (10% v/v) the production medium for the preinoculum. which produces high ligninolytic activities. medium level. permitting a suitable oxygen transfer (Fig. 2. Carrier The bioreactor was filled with 5-mm cubes of fibrous nylon sponge (Scotch Brite. permit good attachment of the fungus to the carrier as well as efficient oxygen and nutrients diffusion into the reactor bed. Most of these configurations are a modification of conventional bioreactors.4.1. Microorganism and growth medium P. 3M Company. Moreover. 2. functioning in semi-solid-state conditions.5) [17]. hydrophobic nature and high porosity). 2. It was maintained 2. different bioreactor configurations were assayed to produce ligninolytic enzymes in semi-solid-state conditions. Domı ´nguez et al. based on conventional rotating drum bioreactors. at the same time. Hence.05% v/v) and veratryl alcohol (3. Those results showed that the choice of an adequate reactor configuration is essential operating in such conditions. operating with cubes of nylon sponge as a support. These pre-cultures were performed in a tray bioreactor. 1. and replacement of dimethylsuccinate with 20 mM acetate buffer (pH 4. which acted as a supporting matrix on which the mycelium can be bound. line with air diffusers. Bioreactor configuration A new bioreactor configuration. / Process Biochemistry 37 (2001) 549 – 554 Among the diverse types of support tested for semisolid-state processes in previous papers [11 – 13]. It consists of a wire mesh cylinder. Spain).2. they are in contact with the air of the upper part of the vessel. Scheme of the rotating drum bioreactor employed: 1. Production medium and operation conditions The production medium composition was the same as the growth medium. In previous work [14.550 A. 1). was designed by our research group. Materials and methods 2. which rotates slowly (3 rpm). The nylon sponge was pretreated according to Linko [18] by boiling for 10 min and washing thoroughly three times with distilled water. this paper focuses on the development of a new bioreactor configuration. the carrier and the fungus are impregnated with the culture medium and. When the wire mesh cylinder rotates. otherwise fungus growth would be delayed and the product formation rate could be inadequate. Moreover. The bioreactor configuration employed in the present work is very appropriate to operate with immobilised biomass. and 4.3.15]. wire mesh cylinder. sorbitan polyoxyethylene monooleate (Tween 80. which allowed study the efficiency of the bioreactor without interactions of a series of variables related to the composition of a non-inert support. The wire mesh cylinder contains the carrier (cubes of fibrous nylon sponge) together with the fungus. 3. mainly due to its inert nature. chrysosporium BKM-F-1767 (ATCC 24725) was grown on a medium prepared according to Tien and Kirk [16] with 10 g glucose/l as carbon source. After this. 0. its physical features (high roughness. . Then. for a long time period without operational problems.4-dimethoxybenzyl alcohol. Fig. inside a cylindrical glass vessel containing the culture medium at its lower part. 2.

[23] with ABTS (2. 2.5. 2.5. The reaction was initiated by the addition of H2O2 and the absorbance was measured immediately after adding the H2O2 and 15 min later. Ammonium nitrogen was totally depleted in 2 days (Fig. and the activities were reported as U/l. Analytical determinations 2. based on conventional rotating drum bioreactors. extracellular liquid (containing mainly MnP) and Poly R-478 (0. 3.5 66m Glucose. and humidified air was supplied to the bioreactor in a continuous way at two different aeration levels (0. One activity unit was defined as the amount of enzyme that oxidised 1 mmol of dimethoxyphenol per minute and the activities were expressed in U/l. Dom´ ınguez et al. to avoid evaporation. After 6 days.4. Reducing sugars Were measured by a dinitrosalicylic acid method using D-glucose as standard. was tested for the production of ligninolytic enzymes in semi-solid-state conditions. Results and discussion Several bioreactor configurations have been employed to obtain ligninolytic enzymes not only in submerged but also in immobilised conditions [25 – 28]. The MnP activity of the extracellular liquid employed was 100 U/l (final concentration in the cubette). oscillated until the 5th day.5. according to Ghose [19]. Ammonium nitrogen content Was assayed by a phenol-hypochlorite method described by Weatherburn [20]. One unit (U) was defined as the amount of enzyme that oxidised 1 mmol veratryl alcohol in 1 min. pH 4. in complete darkness. Mn (II) -dependent peroxidase acti6ity (MnP) Was assayed spectrophotometrically by the method of Kuwahara et al. The reaction was carried out directly in the spectrophotometer cubette. a new bioreactor design.6.2. The bioreactor operated in batch and in continuous mode.29]. centrifuged at 400 rpm for 10 min and analysed. this being the starting time for the experiments. and the reaction mixture Fig.4 mM). being the standard deviation less than 15%.3.A. The activities were expressed in U/l. In the present report. Lignin peroxidase acti6ity (LiP) Was analysed spectrophotometrically according to Tien and Kirk [22]. in a rotatory drum bioreactor operating in batch at an aeration rate of 0.5 vvm and 1 vvm) in order to study the effect of this parameter on the production of ligninolytic enzymes. using NH4Cl as a standard. chrysosporium. 3.5. the colonised sponge was transferred to the bioreactor. In contrast there are few studies on the production of such enzymes in bioreactors operating in semi-solid-state conditions [6. [21]. The values in the figures correspond to mean values. 2.5. 2. and from there onwards. hydrogen peroxide (0.5.2%-azino-di-[3ethyl-benzo-thiazolin-sulphonate]) as substrate. 2. / Process Biochemistry 37 (2001) 549 – 554 551 in a chamber at 37 °C and 90% humidity. grown on nylon sponge. contained sodium malonate (6 mM.5 vvm. The experiments were realised in duplicate and triplicate samples of each one were analysed. The rotating bioreactor was kept in an incubator at 37 °C. . measured as reducing sugars. it was progressively consumed.5). 2). Glucose and ammonium consumption and ligninolytic activities obtained by P. In 6itro Poly R -478 decolorisation This was monitored at 520 nm. Rotatory drum bioreactor at 0.5. which is the maximum visible absorbance of the polymeric dye Poly R-478 [24].1. manganese sulphate (0.5. 2.1.003%) in a total volume of 1 ml. One activity unit was defined as the amount of enzyme that oxidised 1 mmol ABTS per min. 2. Samples were collected once a day from the central part of the bioreactor by means of a sterilised pipette.1 mM). Laccase acti6ity Was determined spectrophotometrically as described by Niku-Paavola et al.

grown on nylon sponge. LiP activity was present on the 1st day (76 U/l). This enzyme was not detected in the bioreactor operated at an aeration level of 0. 3. ammonium nitrogen was totally consumed on the 3rd day. was depleted at an average rate of 0.5 vvm. then increased peaking on the 7th day (144 U/l).2. then increased reaching its maximum value on the 10th day (570 U/l). these activities are superior to those attained employing other bioreactor configurations (Table 1). These activities are about 3-fold higher than those attained operating at an aeration level of 0. Dom´ ınguez et al. 3. This could be due to the rotating bioreactor producing better aeration and nutrients diffusion. continuous bioreactor operation was attempted. Therefore. this movement also avoids support clogging. it can be asserted that the configuration developed in the present work is probably suitable for the continuous production of LiP. to produce ligninolytic enzymes under the conditions assayed in the present report. Glucose and ammonium consumption and ligninolytic activities obtained by P. Subsequently. These values are also about 3-fold higher than those obtained at an aeration level of 0. chrysosporium.5 vvm. although two peaks with activity around 80 U/l appeared on 12th and 15th day.5 vvm. This might be due to a higher aeration rate that would produce a better mix in the culture medium.3. / Process Biochemistry 37 (2001) 549 – 554 MnP activity first appeared on the 7th day (179 U/l). obtaining MnP and LiP activities around 100 and 300 U/l.65 g/l day. 3. as well. and avoiding support clogging.552 A. As for LiP. since the wire mesh cylinder that contains the carrier (cubes of nylon sponge) and the fungus is gently agitated by the continuous rotation. after which it gradually decreased. and from there onwards it diminished. 3. starting on the 1st day (12 U/l) and peaking on the 3rd day (56 U/l). measured as reducing sugars. since according to the literature. This is a very interesting finding. MnP activity was present on the 5th day (70 U/l) and then increased up to a maximum value of 1350 U/l on the 11th day. In addition. To our knowledge. Moreover. attempts to produce LiP continuously have been unsuccessful [30]. activity appeared on the 4th day (12 U/l). showing the efficiency of the design tested. Rotatory drum bioreactor at 1 66m As it can be observed in Fig. This indicates that the aeration rate is a key parameter in this type of processes. The results obtained Fig.5 vvm. laccase was produced. . pH e6olution An evaluation of pH during the process at an aeration rate of 1 vvm was carried out. On the other hand. in a rotatory drum bioreactor operating in batch at an aeration rate of 1 vvm. Glucose. making the oxygen and nutrients more accessible for the microorganism. The results obtained clearly show that operating at an aeration rate of 1 vvm is more suitable than at one of 0. respectively without operational problems. Moreover. the levels of LiP achieved are higher than those reported to date. peaking on the 6th day (364 U/l).

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