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Enzyme and Microbial Technology 49 (2011) 478– 484

Contents lists available at SciVerse ScienceDirect

Enzyme and Microbial Technology

j our na l ho me p age: www.elsev ier .com/ locate /emt

zo dye decolorization by a laccase/mediator system in a membrane reactor:nzyme and mediator reusability

aura Mendozaa,b, Maria Jonstrupa, Rajni Hatti-Kaula, Bo Mattiassona,∗

Department of Biotechnology, Lund University, P.O. Box 124, Getingevägen 60, SE-221 00 Lund, SwedenInstituto de Investigaciones Fármaco – Bioquímicas, Facultad de Ciencias Farmacéuticas y Bioquímicas, Universidad Mayor de San Andrés, Casilla Postal 3239, La Paz, Bolivia

r t i c l e i n f o

rticle history:eceived 21 June 2011eceived in revised form 18 August 2011ccepted 18 August 2011

eywords:zo dyes

a b s t r a c t

This paper presents the use of a membrane-integrated reactor system with recycling of laccase andmediator for azo dye decolorization. From initial screening of different laccases and mediators, Trametesversicolor laccase and syringaldehyde provided the best system for decolorization. Decolorization yieldsof 98, 88, 80 and 78% were obtained for Red FN-2BL, Red BWS, Remazol Blue RR and Blue 4BL, respectively.The reaction parameters were optimized and a membrane reactor was set up for dye decolorization inbatch mode with reuse of the enzyme. Between 10 and 20 batches could be run with decolorization yields

accaseediatorembrane reactor

EG-TEMPOyringaldehyde

from 95 to 52% depending on the dye type. To study the possibility of reusing both enzyme and mediator,the reactor was run using 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) coupled to polyethylene glycol(PEG). Nine batches were run for the treatment of Remazol Blue RR, providing decolorization yields of96–78%. Cost analysis of the processes showed that the costs of laccase/syringaldehyde or laccase/TEMPOwere almost equal when running 20 batches, but the cost for the PEG-TEMPO was higher. However, theadvantages associated with reuse of the mediator should motivate further development of the concept.

. Introduction

Laccases are enzymes, produced by white rot fungi and alsother organisms, which catalyze the oxidation of phenolic com-ounds using molecular oxygen as electron acceptor [1]. The rangef substrates can be broadened to include also non-phenolic com-ounds using low molecular weight mediators. The mediator actss electron acceptor during the oxido-reduction reaction, and par-icipates in the oxidation of substrates that due to their high redoxotential or size cannot be oxidized by laccase alone [2]. Laccasesrom white rot basidiomycetes are increasingly being used in aariety of industrial and environmental applications such as in theood, paper and textile industries [3]. Laccase mediated degrada-ion can be achieved either using whole fungi or with isolatednzymes. When using whole fungal cells, it has been shown dif-cult to maintain a stable process under non-sterile conditions [4].he use of enzymes involves higher costs associated with enzymesolation; this might however be overcome by the advantages ofess sensitivity to the wastewater composition, e.g., high salinity

nd faster reactions [5]. The main limitation for wider applicationf laccase/mediator systems is the cost of the enzyme and media-or and in some cases mediator toxicity [6]. Enzymes are used in an

∗ Corresponding author. Tel.: +46 46 222 8264; fax: +46 46 222 4713.E-mail address: Bo.Mattiasson@biotek.lu.se (B. Mattiasson).

141-0229/$ – see front matter © 2011 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2011.08.006

© 2011 Elsevier Inc. All rights reserved.

immobilized or entrapped form in several large-scale applicationsto enable their reuse and to lower the process cost. An interestingapproach would be to reuse also the mediator to provide furthercost reduction as well as to avoid process stream contamination.

We have chosen azo dyes as model compounds to study laccaseand mediator reuse. Azo dyes are the most widely used colorants intextiles industries [7,8] and they are very recalcitrant as they havebeen developed to withstand bleaching by UV-light and detergents.Conventional methods for treatment of wastewaters from textileindustries have limitations such as incomplete dye degradation orsludge formation leading to disposal problems [8,9]. Laccases orlaccase/mediator systems have been used for decolorization of awide variety of dyes and the results are highly promising [10–12].Reports on sequential dye decolorization by laccase/mediator sys-tems in reactors are however few [13–15] and studies regarding thepossibility of recycling the mediator are lacking. A way of reusingthe mediator could be to couple it to a large polymer such aspolyethylene glycol (PEG) to increase its molecular size, and thenuse it in a membrane reactor where both enzyme and mediatorcould be retained. PEG-coupled mediators have previously beensuccessfully used for oxidation of alcohols to carbonyl compounds[16,17] and it would thereby be interesting to study if the sameapproach could be used for dye decolorization.

In the present paper, we have examined the possibility of recy-cling both laccase and mediator for dye decolorization. Four azodye preparations used in Bolivian and Indian textile industries wereused as model compounds. Initial studies involved screening of lac-

icrobial Technology 49 (2011) 478– 484 479

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L. Mendoza et al. / Enzyme and M

ases and mediators for efficient dye decolorization, after which therocess parameters were optimized in batch studies. A membraneeactor was set up to first study the repeated use of the laccasend after that the repeated use of the mediator TEMPO coupled to

PEG molecule. Finally a cost analysis was conducted to evaluatehe competitiveness of the developed enzyme/mediator process.

. Materials and methods

.1. Materials

Remazol Blue RR and Cibacron Red FN-2BL were supplied by a textile industryn Tirupur, India. Blue 4BL and Red BWS were purchased from Química AlemanaLa Paz, Bolivia). Laccase from Trametes versicolor, N-hydroxyphthalimide (HPT),-hydroxyanthranilic acid (HAA), syringaldehyde (SA), acetosyringone (AS), aceto-anillone (AV), syringic acid (Sac), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfoniccid) diammonium salt (ABTS), p-coumaric acid (C), 2,2,6,6-tetramethylpiperidine-oxyl (TEMPO), vanillin (V), viorulic acid (Vac) and poly(ethylene glycol)-bis-EMPO (PEG-TEMPO) were purchased from Sigma–Aldrich (Schnelldorf, Germany).yceliophthora thermophila laccase was obtained from Novozymes (Denmark) andalerina sp. strain HC1 laccase was produced in our laboratory. EDTA was obtained

rom Merck (Darmstadt, Germany). The ultrafiltration polysulfonate membrane0 kDa cut off was purchased from Sartorius AG (Goettingen, Germany).

.2. Screening of optimal conditions for dye decolorization by theaccase-mediator system

Stock solutions of Remazol Blue RR (�max 613 nm), Red FN-2BL (�max 526 nm),lue 4BL (�max 604 nm) and Red BWS (�max 513 nm) were prepared in water atoncentrations of 1 g/l.

To select an efficient laccase-mediator system, laccase from three differentources (T. versicolor, M. thermophila and Galerina sp. strain HC1) and several medi-tors (HPT, HAA, SA, AS, AV, Sac, ABTS, C, TEMPO, V and Vac) were compared forecolorization of the dyes. The experimental conditions were 50 mg dye/l, 100 U/l

accase and 50 �M mediator in 50 mM sodium acetate buffer, pH 5. The experimentsere performed in 1 ml cuvettes at room temperature. Controls without mediator

ddition were also prepared to study the decolorization efficiency by laccase alone.ecolorization was determined spectrohotometrically by monitoring the decrease

n absorbance at �max of each dye using an Ultrospec 1000 UV–vis spectrophotome-er (Pharmacia Biotech, Sweden).

The reaction conditions were optimized using the most efficient mediator (SA)nd laccase (T. versicolor). The effect of enzyme concentration (100, 500 and 1000 U/l)nd mediator concentration (50, 100 and 500 �M) was studied and the efficiencyas evaluated on several dye concentrations (25, 50 and 100 mg/l) in 50 mM sodium

cetate buffer at pH 5. The influence of pH (pH 3–5, 50 mM sodium acetate buffer;H 6–7, 50 mM sodium phosphate buffer), ionic strength (0–50 mM sodium acetateuffer at pH 5) and salt concentration (0–100 mM NaCl at pH 5) were evaluatedsing 50 mg dye/l, 100 U/l laccase and 50 �M mediator.

PEG-TEMPO was used as mediator in the experiments on mediator reuse. Toptimize the PEG-TEMPO concentration, each dye (50 mg/l) was incubated with lac-ase (100 U/l) and varying concentrations of PEG-TEMPO (50, 100, 500 and 750 mg/lorresponding to 25, 50, 250 and 375 �M TEMPO) at pH 5. The optimal enzyme con-entration (100, 500 and 1000 U/l) was then determined using the optimal mediatoroncentration.

All the tests were run in duplicates.

.3. Laccase stability in presence of mediator

To investigate the effect of the mediator on the enzyme stability, 5 ml of 1000 U/l. versicolor laccase was incubated in water, with 25 �M SA, 25 �M TEMPO orEG-TEMPO amount giving equivalent mediator concentration. PEG-TEMPO wasuantified by its UV absorption at �max, 249 nm. The samples were incubated atH 5 in room temperature during 40 days. Samples of 50 �L were taken at regularime intervals for determination of residual laccase activity. The experiment waserformed in triplicate.

Laccase activity was measured according to Childs and Bardsley [18], and wasased on the spectrophotometric determination of the enzyme-catalyzed oxidationf ABTS at 420 nm.

.4. Scale up of the process and reusability of the laccase/mediator system

.4.1. Reusability of the laccaseA 500 ml reactor with 300 ml working volume was set up for batch wise

ecolorization of the dyes (Fig. 1). The reactor was coupled to a polysulfonate ultra-

ltration membrane with a nominal molecular weight cut-off of 10 kDa, whichermitted retention of the soluble enzyme in the reactor for use in a subse-uent batch. Dye adsorption to the membrane was determined before startinghe experiments by passing 300 ml of dye solution (50 mg/l) over the membraneepeatedly until no more adsorption was observed. The reactor was operated for

Fig. 1. Schematic diagram of reactor set-up for dye decolorization.

decolorization of 50 mg/l dye using 100 U/l laccase from T. versicolor and 25 �M SAat room temperature. The treatment of Red FN-2BL and Remazol Blue RR was runin water adjusted to pH 5, while the treatment of Red BWS and Blue 4BL was run insodium acetate buffer at pH 5. The enzyme was added at the start of the experimentand the reaction was run until no further decolorization was achieved, after whichthe reactor was emptied using a peristaltic pump set at 50 ml/min, until approxi-mately 10 ml of retentate remained in the reactor. A new batch was started by addingdye solution (50 mg/l) and syringaldehyde (25 �M) and filling up to 300 ml withwater or water/buffer. At the start of each batch, an aliquot of 50 �l was collectedfor measuring residual laccase activity in the reactor.

The possibility to stabilize the enzyme during the reaction with one of the dyes,Red FN2BL, was also investigated by addition of 1 mM EDTA at the start of eachbatch.

2.4.2. Reusability of the laccase and mediatorThe recyclability of the mediator, PEG-TEMPO (25 �M) was investigated using

100 U/l T. versicolor laccase to decolorize Remazol Blue RR (50 mg/l) at pH 5. Thereactor was set up as described in Section 2.4.1 with the exception that only dyeand water was added when starting a new batch. Retention of PEG-TEMPO in thereactor was confirmed by measuring the absorbance of the permeate and retentateat 249 nm

3. Results and discussion

3.1. Screening of laccases and mediators

Preliminary screening of decolorization of four azo dyes(50 mg/l) was done using laccase (100 U/l) from different sources– T. versicolor, M. thermophila and Galerina sp. HC1 (isolated in ourlaboratory from a soil sample in Bolivian Amazon region) – in thepresence of different mediators (HPT, HAA, SA, AS, AV, Sac, ABTS, C,TEMPO, V and Vac). The enzyme from T. versicolor was shown to bethe most efficient enzyme (data not shown). Red FN-2BL was com-pletely decolorized in 1 min and 5 min with SA and AS, respectively,as mediators. About 95–98% decolorization was achieved in 60 minin the presence of TEMPO, V and ABTS. Decolorization of Red BWSwas less efficient, 85–88% in 60 min, with SA, AS, or ABTS as media-tor, while maximum decolorization of about 80% was achieved forRemazol Blue RR in 5 min, and 73–78% for Blue 4BL in 10 min usingSA or AS as mediators.

Using M. thermophila and Galerina sp. strain HC1 laccases decol-orization yields of 55–80% were obtained for Remazol Blue RR,Blue 4BL and Red FN-2BL in combination with SA, AS or ABTS after60 min. Only 30–45% decolorization of Red BWS was reached in60 min using these enzymes and mediators. Without any mediatorthe highest decolorization efficiencies were 2, 20, 43 and 47% for

Red FN-2BL, Red BWS, Blue 4BL and Remazol Blue RR, respectively.

The higher efficiency of T. versicolor laccase may be attributedto its higher redox potential, 0.78 V [19], in comparison to theother laccases (laccase from M. thermophila has a redox poten-

480 L. Mendoza et al. / Enzyme and Microbial Technology 49 (2011) 478– 484

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ig. 2. UV–visible scan of (a) Red FN-2BL, (b) Red BWS, (c) Remazol Blue RR and

ersicolor laccase and 50 �M syringaldehyde at pH 5 in 50 mM sodium acetate buffe

ial of 0.46 V [20] while that of Galerina sp. laccase has not yeteen determined). The high efficiency of SA and AS as mediatorsbserved in this study corresponds well with the reports fromamarero et al. [21] and Murugesan et al. [22]. The high oxida-ive capacity of these mediators is related to the electron donorransfer of the methoxy substituents at the benzenic ring [21].urthermore, these mediators are derived from lignin, an abun-ant renewable resource, and can potentially be produced in a costfficient way [21].

UV–visible spectra of the dye solutions before and after treat-ent with the T. versicolor laccase-syringaldehyde system are

hown in Fig. 2 and it can be seen that only Red FN2BL wasompletely decolorized. The rate and extent of decolorization isependent on dye structure and in particular on the functionalroups in the aromatic region and their interactions with the azoond [23]. Incomplete decolorization could also be attributed tohe heterogeneous quality of the dye formulations, since interme-iates are usually not removed during production, and moreoverome dyestuffs can be mixtures of several different dye types, lead-ng to differences in recalcitrance of the molecules in the finalroduct. It is also clear that in spite of the decolorization of theyes, the UV absorbance is still predominant suggesting that theromatic structures were not completely degraded. Determinationf the degradation products were not in the scope of this study,

ut it has previously been shown that laccase mediated degra-ation of azo dyes can result in detoxification of the dyes sincehe nitrogen in the azo bond is released as molecular nitrogen,

ue BWS before (continuous line) and after (dotted line) treatment with 100 U/l T.

thereby preventing formation of aromatic amines [24]. Further-more, when polymerization of reactants takes place, then toxicityis reduced

3.2. Optimization of parameters using T. versicolor laccase and SA

Based on the above results, laccase decolorization conditionsusing T. versicolor laccase and SA were optimized. Optimal con-ditions with respect to laccase and SA concentrations variedfor achieving maximal decolorization of the four dyes (data notshown). Increasing the enzyme concentration from 100 to 500 U/lled to a slight decrease in decolorization efficiency, and thenremained steady at higher enzyme concentration. SA concentra-tion of 50–100 �M provided the highest decolorization efficiency,and further increase in concentration to 500 �M had a negativeimpact probably due to loss of laccase activity. At optimal enzymeand mediator concentrations, 98–100% decolorization of Red FN-2BL was reached at the tested dye concentrations up to 100 mg/l.For Remazol Blue and Blue 4BL 87–85% decolorization was reachedat 25–50 mg/l dye and approximately 80% at 100 mg/l. Decoloriza-tion of Red BWS decreased from 78 to 69% with the increase in dyeconcentration from 25 to 100 mg/l.

Further experiments were done using 100 U/l laccase and 50 �MSA to achieve high decolorization yields at minimal costs and activ-

ity loss. Decolorization was highest at pH 3–5 (Fig. 3a), attributed tothe low pH-optimum for laccase activity [15] and possibly the lowsyringaldehyde stability at higher pH [21]. At pH 6 the decoloriza-

L. Mendoza et al. / Enzyme and Microbial Technology 49 (2011) 478– 484 481

Fig. 3. Effect of (a) pH, (b) buffer strength and (c) NaCl concentration on the decol-o4o

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rization of Red FN-2BL (white), Red BWS (stripes), Remazol Blue RR (grey) and BlueBL (black) using 100 U/l laccase and 50 �M syringaldehyde. Figures show resultsbtained after 5 min treatment.

ion yields reached 73, 20, 56 and 34%, for the dyes Red FN-2BL, RedWS, Remazol Blue RR and Blue 4BL, respectively, and at pH 7, theecolorization yields were only 2–4% (Fig. 3a). The ionic strengthf the buffer had no effect on the decolorization of Red FN-2BLnd Remazol Blue RR; the reaction was equally efficient in waterdjusted to pH 5 as in 10–50 mM acetate buffer at the same pHFig. 3b). On the other hand, the decolorization of Red BWS andlue 4BL was considerably lower in water, but was significantly

mproved in buffer already at low ionic strength (10 mM).Textile wastewaters can contain NaCl at concentrations over

0 mM [25]. Decolorization of Red FN-2BL was not affected by NaCloncentration up to 100 mM, while the extent of decolorizationas reduced with increasing NaCl concentration for the other dyes

Fig. 3c). As reported earlier [26], NaCl could have an inhibitory

Fig. 4. Decolorization of Remazol Blue RR (50 mg/l) using 100 U/l of T. versicolorlaccase and 25 �M syringaldehyde (�), 25 �M TEMPO (�) or 25 �M PEG-TEMPO(�) at pH 5.

effect on the laccase activity; nevertheless, over 50% decoloriza-tion of all the dyes was still achieved up to 100 mM NaCl. The highefficiency observed for Red FN-2BL also at high NaCl concentrationsmight be due to the short time required for decolorization of thisdye (<1 min).

3.3. Optimization of parameters using T. versicolor laccase andPEG-TEMPO

TEMPO coupled to PEG with an average molecular weight ofapproximately 5 kDa is commercially available, and was evalu-ated as a mediator for dye decolorization even though TEMPOwas not the optimal mediator in the present study. When com-paring the decolorization of Remazol Blue RR using PEG-TEMPOwith free mediators used at a concentration of 25 �M each (Fig. 4),it can be seen that although the rate of decolorization was lowerwith PEG-TEMPO (0.0003 Abs min−1) than with both free TEMPO(0.0016 Abs min−1) or syringaldehyde (0.006 Abs min−1), ratherefficient decolorization could still be achieved.

The decolorization efficiency with 0.1 U/ml laccase increasedwith increasing PEG-TEMPO concentration to 250 �M for treatmentof Red FN-2BL, Blue 4BL and Red BWS (data not shown). Decoloriza-tion of Blue 4BL and Red BWS increased from 81 to 92% and from 55to 89%, respectively, with the increasing mediator concentration.Although Red FN2BL was completely decolorized at all mediatorconcentrations tested, the reaction time was decreased from 96 to23 h with increase in PEG-TEMPO concentration from 25 to 250 �M.In case of Remazol Blue RR, complete decolorization was achievedirrespective of the mediator concentrations used, with only minordifference in decolorization rate.

The reaction rate could also be improved significantly byincreasing the enzyme concentration from 0.1 to 1 U/ml, resultingin reduction of the time required for complete decolorization from23–96 h to 4–8 h for the different dyes.

3.4. Effect of mediators on laccase stability

Laccase stability during the process is an important parame-ter for long term operation and low process costs. The activity ofT. versicolor laccase was followed during 40 days in the absenceand presence of mediators. In the absence of the mediator, a lineardecrease in activity from 1000 U to 290 U/l was observed during

the first 15 days followed by a gradual decrease to yield an almostinactive enzyme. In the presence of 50 �M syringaldehyde, thelaccase was deactivated already within 30 min (data not shown).Reducing the mediator concentration to 25 �M resulted in nearly a

482 L. Mendoza et al. / Enzyme and Microbial Technology 49 (2011) 478– 484

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ig. 5. Residual laccase activity during incubation in water (�), 25 �M syringalde-yde (�), 25 �M TEMPO (�) or 25 �M PEG-TEMPO (�) at pH 5. Initial laccase activitysed was 1000 U/l.

imilar deactivation profile as without the mediator, yielding resid-al activity of 130 and 180 U/l in the presence of TEMPO and SA,espectively, in 15 days (Fig. 5). Mediator-induced loss of enzymectivity has been attributed to the degradation of essential aminocid residues or the glycosyl moieties at the surface of the enzyme6,27,28]. In contrast, the residual laccase activity was 274 U/l in theample with PEG-TEMPO, suggesting that the deactivation effect ofhe mediator is reduced by coupling to the polymer.

.5. Dye decolorization using T. versicolor laccase andyringaldehyde in a membrane reactor

The optimized conditions were applied for dye decolorizationn a reactor with an integrated ultrafiltration membrane to enableetention and recycling of the laccase. The enzyme (100 U/l) coulde recycled for 10–20 batches, the reaction time required to obtainaximal decolorization being dependent on the dye (Table 1). Dye

ecolorization due to adsorption to the membrane was less than% for all dyes and no enzyme activity was detected in the effluent,hich confirmed retention of the enzyme in the rector.

Red FN-2BL underwent the fastest decolorization and althoughigh decolorization efficiency could be obtained during 10 batches,he reaction rate decreased with the number of batches from only

min for 95% decolorization in the first batch to 1 h for 91% decol-rization in the last batch. The decreased reaction rate was due tohe successive decrease in enzyme activity with every batch untilt was nearly completely lost after the 10th batch (Table 1).

The decolorization rate of Red BWS was slower, requiring–1.6 h for maximal decolorization. Relatively high enzyme activity>25%) could be maintained during 15 batches, although the extentf decolorization decreased from 84 to 52%.

High decolorization yields (94–85%) were obtained for Rema-ol Blue RR during 20 batches, even though only 4% of the laccasectivity remained during the last batch. Decolorization of this dyeequired longer reaction time, 2–3 h for the initial batches andlmost 7 h for the last batches.

Ten batches could be run for treatment of Blue 4BL, resultingn 72–61% decolorization in 1–2 h. The enzyme activity decreasedo 49% of the initial activity after the 10th batch. Although theesidual enzyme activity and the decolorization yield were satisfac-ory, the experiment was terminated due to accumulation of darkroducts retained by the membrane. The lignin oxidizing enzymes

ncluding peroxidases and laccase catalyze the oxidation of phe-olic compounds by generation of free radicals, which can further

nitiate polymerization or degradation. It has been proposed thatzo compounds are “degraded” asymmetrically and symmetrically

laccase and 25 �M PEG-TEMPO at pH 5. Absorbance at 613 nm (♦); laccase activ-ity (�). The laccase and mediator were maintained in the reactor using a 10 kDamembrane.

by ligninases [29,24]; the latter can lead to formation of prod-ucts prone to polymerization and precipitation [22]. The precipitatecould also arise from dye degradation products reacting with pro-tein resulting in colored conjugates.

The possibility to stabilize the enzyme during the reaction wasinvestigated by including EDTA, which plays a protecting role forproteins especially under oxidative stress [30]. In the presence of1 mM EDTA, the number of batches for decolorization of Red FN-2BLcould be increased from 10 to 19 batches and the reaction rate wasincreased as well. Even though 69% decolorization was achievedin the 19th batch with 20% residual activity, the experiment wasterminated due to accumulation of dark polymerization products,as described above (data not shown).

3.6. Dye decolorization using T. versicolor laccase andPEG-TEMPO in a membrane reactor

The possibility of reusing both laccase and PEG-TEMPO in themembrane reactor was evaluated using Remazol Blue RR, since ahigh degree of decolorization could be achieved with a low con-centration of the mediator. The retention of the mediator wasconfirmed by scanning the permeate and retentate after passinga 25 �M PEG-TEMPO solution over the membrane and revealedinsignificant mediator loss. The membrane reactor was operatedusing 1000 U/l laccase and 25 �M PEG-TEMPO. Even though PEG-TEMPO showed lower decolorization rate than syringaldehyde at25 mM, it was possible to run 9 batches without adding extraenzyme or mediator (Fig. 6). Almost complete decolorization (96%)was obtained in the first batch in 4.5 h, but decreased to 85–78%in the following batches and the time required increased to 27 hin the final batch. According to an earlier study, PEG-TEMPO couldbe recycled 6 times for the synthesis of carbonyl compounds withconversion yields ranging from 99 to 74% [16].

There have so far been no other reports on the repeateduse of PEG-TEMPO (or any other mediator) for dye decoloriza-tion. Chhabra et al. [14] reported the recovery of 70% of ABTSthrough ammonium sulphate precipitation after use with laccasefor dye decolorization in a membrane reactor. The mediator washowever not reused in the continuous reactor. Even though thedecolorization rate is slower with PEG-TEMPO than with the free

mediator, the high molecular weight mediator has the advantageof being retained in the system. The results open the possibilityfor further development of the concept and optimization of thesystem, e.g. by using different polymers with varying molecular

L. Mendoza et al. / Enzyme and Microbial Technology 49 (2011) 478– 484 483

Table 1Decolorization and enzyme activity during batch-wise enzymatic treatment in a membrane reactor using laccase from T. versicolor. Syringaldehyde (25 �M) was used asmediator.

Number ofbatches

Red FN-2BL Red BWS Remazol Blue RR Blue 4BL

Decolorization(%)

Activity(%)

Time(h)

Decolorization(%)

Activity(%)

Time(h)

Decolorization(%)

Activity(%)

Time(h)

Decolorization(%)

Activity(%)

Time(h)

1 95 100 0.1 84 100 1.1 94 100 2.7 71 100 1.62 94 72 0.15 74 82 1.2 94 73 2.3 72 80 1.83 94 33 0.17 72 88 0.8 94 64 2.8 61 83 1.64 94 24 0.22 69 75 1.0 94 63 3.3 68 80 1.85 93 16 0.3 67 73 1.0 94 44 2.8 66 64 1.36 92 13 0.4 67 67 1.2 94 38 2.8 64 66 1.57 93 8 0.5 62 70 1.4 94 37 3.2 63 65 1.88 94 6 0.7 62 52 1.2 94 34 3.0 62 56 1.29 92 5 0.9 59 59 1.2 94 21 3.0 63 56 1.610 91 5 1.0 60 53 1.2 93 21 3.0 61 49 1.611 55 30 1.3 94 14 3.212 55 37 1.5 93 16 3.713 56 31 1.3 93 12 3.714 55 29 1.5 94 11 4.515 52 26 1.6 94 8.0 4.316 93 7.0 5.017 93 7.0 4.718 93 6.0 6.719 93 5.0 7.020 85 4.0 6.3

Table 2Cost analysis for dye decolorization using the laccase/mediator systems. The cost is expressed in Euros per gram of treated dye.

Number of batches aSyringaldehyde + Laccase aTEMPO + Laccase bPEG-TEMPO + Laccase

1 0.86 0.85 9.45 0.20 0.19 1.9

10 0.12 0.10 0.9520 0.07 0.06 0.47

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eights, and other mediator compounds. Other supports that haveeen used for immobilization of TEMPO include silica, polystyrenend sol–gel [31]. Cofactor-polymer conjugates such as NAD+-olyethyleneimine, NAD+-polylysine, NAD+-PEG and NADP+-PEGave been successfully used in continuous enzyme reactors [32–34]nd the modification with a polymer has been shown not to haveny notable effect on the kinetic constants of the reactions [34].igh product yields have been reported with low leakage of theofactor through the membrane during long periods of reaction33,35], which can reduce the cost of the process significantly. Yetnother advantage was the reduced cofactor inactivation by dimer-zation [36].

.7. Cost analysis

A cost analysis for dye treatment using the different lac-ase/mediator systems is shown in Table 2. The process costs areimilar when syringaldehyde and free TEMPO are used as medi-tors; the costs for running 20 batches would be 0.07 vs. 0.06uros per gram of dye for syringaldehyde and TEMPO, respec-ively. Syringaldehyde would however be preferred since it leadso faster decolorization. The application of PEG-TEMPO would cost

ore than the other mediators (0.47 Euros per gram of decolorizedye) based on current prices and the process cost is comparableo the free mediators first after reusing PEG-TEMPO for 200–300

atches. PEG-TEMPO can be considered as a model compound toemonstrate the possibility of mediator reuse. Other macromolec-lar mediators should also be screened to improve the cost and thefficiency. Moreover large scale application of a reusable mediator

would probably lower the production cost of the polymer boundform.

4. Conclusions

This study has shown that the efficiency of laccase mediator sys-tems for decolorization of azo dyes varies with the nature of the dye.It has also shown that running the process in a membrane reactorallows the recycling of both the enzyme and the mediator. Althoughthe kinetics of Red FN-2BL decolorization was the fastest, the lossof enzyme activity occurred much more rapidly than during decol-orization of Remazol Blue RR, permitting recycling of the enzymefor double number of batches for the latter dye. It is encouragingto realize that coupling to a soluble polymer does not prevent themediator from functioning as mediator, although the kinetic con-stants were affected. The possibility of reusing the enzyme and themediator is important in order to promote their use in large-scalereactions. Moreover, environmental problems associated with theloss of the mediator in the wastewater will be reduced.

Acknowledgement

The financial support of Swedish Agency for Research Develop-ment Cooperation (SIDA-SAREC) is gratefully acknowledged.

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