Process Biochemistry 37 (2001) 549–554

Design of a new rotating drum bioreactor for ligninolytic enzymeproduction by Phanerochaete chrysosporium grown on an inert

support

Alberto Domınguez, Isabel Rivela, Susana Rodrıguez Couto, Ma Angeles Sanroman *Department of Chemical Engineering, Uni�ersity 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 newbioreactor 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-foldhigher than the former, achieving maximum manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP) activities of 1350and 364 U/l, respectively. Moreover, laccase activity was also detected, showing a highest activity of 56 U/l. In addition, the invitro decolorisation of a model dye (Poly R-478) by the extracellular liquid obtained in the bioreactor was monitored in order toassess its ligninolytic ability. A percentage of Poly R-478 decolorisation of about 19% was achieved, after 15 min of dyeincubation. © 2001 Elsevier Science Ltd. All rights reserved.

Keywords: Aeration rate; Ligninolytic enzymes; Nylon sponge; Phanerochaete chrysosporium ; Rotating drum bioreactor; Solid-state fermentation

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1. Introduction

The white-rot fungus Phanerochaete chrysosporiumsecretes, during its secondary metabolism, severallignin-degrading enzymes including lignin peroxidase(LiP), manganese-dependent peroxidase (MnP) and lac-case. The secondary metabolism in this fungus is trig-gered by nitrogen [1], carbon or sulphur [2] deprivation.In addition to lignin, the above-mentioned enzymes arealso able to degrade a wide range of hazardous environ-mental pollutants [3,4]. Their application to industrialprocesses (biobleaching, biopulping, decolorisation,etc.) on a large scale requires the production of highamount of enzyme at low cost. Thus, the design of asystem permitting the continuous production of ligni-nolytic enzymes efficiently is required.

Solid-state fermentation (SSF) involves the growth ofmicroorganisms on moist solid substrates in the absenceor near absence of free liquid [5]. The physical nature of

the medium is quite different from that in submergedfermentation. For example, a solid bed is more difficultto mix effectively than a liquid broth, and as a conse-quence, O2 supply and heat removal can be restricted inSSF processes [6]. The kind of solid materials that canbe employed in this type of cultivation are classified intwo main categories: inert support and non-inert sup-port. The former acts as an attachment place (e.g.plastic foams) whereas the latter also functions as asource of nutrients (e.g. crop wastes).

SSF has gained importance in recent years due toseveral advantages over submerged fermentation suchas superior productivity, simpler techniques, reducedenergy requirements, low wastewater output, and im-proved product recovery [7–9]. Nevertheless, bioreactordesign aspects have not been received enough attentionby researchers of SSF and the present state of the artdoes not indicate an ideal type of bioreactor for solidstate processes.

In earlier reports, a high production of extracellularligninolytic enzymes in semi-solid-state cultures hasbeen achieved and the utility of such cultures has beendemonstrated [10–12].

* Corresponding author. Tel.: +34-986-812-304; fax: +34-986-812-382.

E-mail address: sanroman@uvigo.es (M.A. Sanroman).

0032-9592/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.

PII: S 0 0 3 2 -9592 (01 )00233 -3

A. Domınguez et al. / Process Biochemistry 37 (2001) 549–554550

Among the diverse types of support tested for semi-solid-state processes in previous papers [11–13], nylonsponge was preferred in the present work, mainly dueto its inert nature, which allowed study the efficiency ofthe bioreactor without interactions of a series of vari-ables related to the composition of a non-inert support.Moreover, its physical features (high roughness, hydro-phobic nature and high porosity), permit good attach-ment of the fungus to the carrier as well as efficientoxygen and nutrients diffusion into the reactor bed.

In previous work [14,15], different bioreactorconfigurations were assayed to produce ligninolytic en-zymes in semi-solid-state conditions. Those resultsshowed that the choice of an adequate reactor configu-ration is essential operating in such conditions. Most ofthese configurations are a modification of conventionalbioreactors. Hence, this paper focuses on the develop-ment of a new bioreactor configuration, which pro-duces high ligninolytic activities, functioning insemi-solid-state conditions, for a long time period with-out operational problems.

The bioreactor configuration employed in the presentwork is very appropriate to operate with immobilisedbiomass. In order to apply this bioreactor to semi-solid-state processes the volume of the culture medium incontact with the microorganism has been reduced toconditions near to semi-solid-state.

2. Materials and methods

2.1. Microorganism and growth medium

P. chrysosporium BKM-F-1767 (ATCC 24725) wasgrown on a medium prepared according to Tien andKirk [16] with 10 g glucose/l as carbon source, andreplacement of dimethylsuccinate with 20 mM acetatebuffer (pH 4.5) [17]. The fungus was grown in 90 ml ofthis medium at 37 °C in complete darkness for 48 h.After this, the whole culture was homogenised in ablender for 1 min. This homogenate suspension wasused to inoculate (10% v/v) the production medium forthe preinoculum.

2.2. Carrier

The bioreactor was filled with 5-mm cubes of fibrousnylon sponge (Scotch Brite, 3M Company, Spain),which acted as a supporting matrix on which themycelium can be bound. The nylon sponge was pre-treated according to Linko [18] by boiling for 10 minand washing thoroughly three times with distilled wa-ter. Then, the carriers were dried at room temperatureovernight and autoclaved at 121 °C for 20 min untilused.

2.3. Bioreactor configuration

A new bioreactor configuration, based on conven-tional rotating drum bioreactors, was designed by ourresearch group. It consists of a wire mesh cylinder,which rotates slowly (3 rpm), inside a cylindrical glassvessel containing the culture medium at its lower part.The wire mesh cylinder contains the carrier (cubes offibrous nylon sponge) together with the fungus. Whenthe wire mesh cylinder rotates, the carrier and thefungus are impregnated with the culture medium and,at the same time, they are in contact with the air of theupper part of the vessel, permitting a suitable oxygentransfer (Fig. 1).

2.4. Production medium and operation conditions

The production medium composition was the sameas the growth medium. Moreover, sorbitan poly-oxyethylene monooleate (Tween 80, 0.05% v/v) andveratryl alcohol (3,4-dimethoxybenzyl alcohol; 2 mMfinal concentration) were added at the beginning of thecultivation in order to stimulate ligninolytic enzymeproduction [12].

Pre-cultures of the fungus were made in order toinoculate the bioreactor with enough inoculum, other-wise fungus growth would be delayed and the productformation rate could be inadequate. These pre-cultureswere performed in a tray bioreactor, operating withcubes of nylon sponge as a support. It was maintained

Fig. 1. Scheme of the rotating drum bioreactor employed: 1, medium level; 2, line with air diffusers; 3, wire mesh cylinder; and 4, rotating device.

A. Domınguez et al. / Process Biochemistry 37 (2001) 549–554 551

in a chamber at 37 °C and 90% humidity, to avoidevaporation, in complete darkness. After 6 days, thecolonised sponge was transferred to the bioreactor, thisbeing the starting time for the experiments.

The rotating bioreactor was kept in an incubator at37 °C, and humidified air was supplied to the bioreac-tor in a continuous way at two different aeration levels(0.5 vvm and 1 vvm) in order to study the effect of thisparameter on the production of ligninolytic enzymes.

The bioreactor operated in batch and in continuousmode. Samples were collected once a day from thecentral part of the bioreactor by means of a sterilisedpipette, centrifuged at 400 rpm for 10 min andanalysed.

The experiments were realised in duplicate and tripli-cate samples of each one were analysed. The values inthe figures correspond to mean values, being the stan-dard deviation less than 15%.

2.5. Analytical determinations

2.5.1. Reducing sugarsWere measured by a dinitrosalicylic acid method

using D-glucose as standard, according to Ghose [19].

2.5.2. Ammonium nitrogen contentWas assayed by a phenol-hypochlorite method de-

scribed by Weatherburn [20], using NH4Cl as astandard.

2.5.3. Mn (II)-dependent peroxidase acti�ity (MnP)Was assayed spectrophotometrically by the method

of Kuwahara et al. [21]. One activity unit was definedas the amount of enzyme that oxidised 1 �mol ofdimethoxyphenol per minute and the activities wereexpressed in U/l.

2.5.4. Lignin peroxidase acti�ity (LiP)Was analysed spectrophotometrically according to

Tien and Kirk [22]. One unit (U) was defined as theamount of enzyme that oxidised 1 �mol veratryl alco-hol in 1 min, and the activities were reported as U/l.

2.5.5. Laccase acti�ityWas determined spectrophotometrically as described

by Niku-Paavola et al. [23] with ABTS (2,2�-azino-di-[3-ethyl-benzo-thiazolin-sulphonate]) as substrate. One ac-tivity unit was defined as the amount of enzyme thatoxidised 1 �mol ABTS per min. The activities wereexpressed in U/l.

2.5.6. In �itro Poly R-478 decolorisationThis was monitored at 520 nm, which is the maxi-

mum visible absorbance of the polymeric dye PolyR-478 [24]. The reaction was carried out directly in thespectrophotometer cubette, and the reaction mixture

Fig. 2. Glucose and ammonium consumption and ligninolytic activi-ties obtained by P. chrysosporium, grown on nylon sponge, in arotatory drum bioreactor operating in batch at an aeration rate of 0.5vvm.

contained sodium malonate (6 mM; pH 4.5), man-ganese sulphate (0.1 mM), hydrogen peroxide (0.4mM), extracellular liquid (containing mainly MnP) andPoly R-478 (0.003%) in a total volume of 1 ml. TheMnP activity of the extracellular liquid employed was100 U/l (final concentration in the cubette). The reac-tion was initiated by the addition of H2O2 and theabsorbance was measured immediately after adding theH2O2 and 15 min later.

3. Results and discussion

Several bioreactor configurations have been em-ployed to obtain ligninolytic enzymes not only in sub-merged but also in immobilised conditions [25–28]. Incontrast there are few studies on the production of suchenzymes in bioreactors operating in semi-solid-stateconditions [6,29]. In the present report, a new bioreac-tor design, based on conventional rotating drum biore-actors, was tested for the production of ligninolyticenzymes in semi-solid-state conditions.

3.1. Rotatory drum bioreactor at 0.5 ��m

Glucose, measured as reducing sugars, oscillated un-til the 5th day, and from there onwards, it was progres-sively consumed. Ammonium nitrogen was totallydepleted in 2 days (Fig. 2).

A. Domınguez et al. / Process Biochemistry 37 (2001) 549–554552

MnP activity first appeared on the 7th day (179 U/l),then increased reaching its maximum value on the 10thday (570 U/l), after which it gradually decreased. As forLiP, activity appeared on the 4th day (12 U/l), thenincreased peaking on the 7th day (144 U/l), and fromthere onwards it diminished, although two peaks withactivity around 80 U/l appeared on 12th and 15th day.

3.2. Rotatory drum bioreactor at 1 ��m

As it can be observed in Fig. 3, ammonium nitrogenwas totally consumed on the 3rd day. Glucose, mea-sured as reducing sugars, was depleted at an averagerate of 0.65 g/l day.

MnP activity was present on the 5th day (70 U/l) andthen increased up to a maximum value of 1350 U/l onthe 11th day. These activities are about 3-fold higherthan those attained operating at an aeration level of 0.5vvm. This indicates that the aeration rate is a keyparameter in this type of processes.

On the other hand, LiP activity was present on the1st day (76 U/l), peaking on the 6th day (364 U/l).These values are also about 3-fold higher than thoseobtained at an aeration level of 0.5 vvm. Moreover,laccase was produced, starting on the 1st day (12 U/l)and peaking on the 3rd day (56 U/l). This enzyme wasnot detected in the bioreactor operated at an aerationlevel of 0.5 vvm.

The results obtained clearly show that operating atan aeration rate of 1 vvm is more suitable than at one

of 0.5 vvm, to produce ligninolytic enzymes under theconditions assayed in the present report. This might bedue to a higher aeration rate that would produce abetter mix in the culture medium, making the oxygenand nutrients more accessible for the microorganism,and avoiding support clogging, as well.

In addition, these activities are superior to thoseattained employing other bioreactor configurations(Table 1), showing the efficiency of the design tested.This could be due to the rotating bioreactor producingbetter aeration and nutrients diffusion, since the wiremesh cylinder that contains the carrier (cubes of nylonsponge) and the fungus is gently agitated by the contin-uous rotation. Moreover, this movement also avoidssupport clogging.

Subsequently, continuous bioreactor operation wasattempted, obtaining MnP and LiP activities around100 and 300 U/l, respectively without operational prob-lems. This is a very interesting finding, since accordingto the literature, attempts to produce LiP continuouslyhave been unsuccessful [30]. To our knowledge, thelevels of LiP achieved are higher than those reported todate. Therefore, it can be asserted that the configura-tion developed in the present work is probably suitablefor the continuous production of LiP.

3.3. pH e�olution

An evaluation of pH during the process at an aera-tion rate of 1 vvm was carried out. The results obtained

Fig. 3. Glucose and ammonium consumption and ligninolytic activities obtained by P. chrysosporium, grown on nylon sponge, in a rotatory drumbioreactor operating in batch at an aeration rate of 1 vvm.

A. Domınguez et al. / Process Biochemistry 37 (2001) 549–554 553

Table 1Maximum ligninolytic enzyme activities obtained employing different bioreactor configurations, operating with nylon sponge as support, in batchprocess

MnP (U) LiP (U/l)Type of bioreactor LiP (U)MnP (U/l) Reference

398 229Fixed bed (250 ml) 571593 Rodrıguez Couto et al. [31]780 400780 400Tray (1 l) Rodrıguez Couto et al. [32]

2467 356Immersion at 1 vvm (2.5 l) 890987 Rivela et al. [33]1140 144570 288Rotating drum at 0.5 vvm (2 l) This work2700 364 728 This workRotating drum at 1 vvm (2 l) 1350

Fig. 4. pH evolution in a rotatory drum bioreactor operating inbatch, at an aeration rate of 1 vvm.

4. Conclusions

In view of the results attained, it can be concludedthat the bioreactor configuration developed in thepresent paper is appropriate for the production ofligninolytic enzymes in semi-solid-state conditions.Moreover, it allowed the continuous production of LiPat high activity levels for long times without opera-tional problems.

Acknowledgements

This research was financed by Xunta(PGIDT00PXI30118PR).

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3.4. Poly R-478 decolorisation

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The percentage of dye decolorisation obtained after15 min of dye incubation was 19%, which shows thedegrading potential of the ligninolytic fluid produced.

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