Sequential treatment via Trametes versicolor and UV/TiO2/RuxSey to reduce contaminants in waste water resulting from the bleaching process during paper production

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Chemosphere 67 (2007) 793–801

Sequential treatment via Trametes versicolor and UV/TiO2/RuxSey

to reduce contaminants in waste water resulting from thebleaching process during paper production

Aura M. Pedroza a,b, Rodolfo Mosqueda c, Nicolas Alonso-Vante d,Refugio Rodrıguez-Vazquez a,*

a Department of Biotechnology and Bioengineering, CINVESTAV-IPN, Avenue I.P.N. 2508, Mexico D.F. 07360, Mexicob Department of Microbiology, University Javeriana, Avenue 7 No 43-82, Colombia

c National School of Biological Sciences-IPN, Mexicod Laboratory of electrocatalysis UMR-CNRS 6503, University of Poitiers, F-86022 Poitiers, France

Received 26 April 2006; received in revised form 3 October 2006; accepted 5 October 2006Available online 22 November 2006

Abstract

An efficient sequential, biological and photocatalytic treatment to reduce the pollutant levels in wastewater due to the bleaching pro-cess during paper production is reported. For a biological pre-treatment, 800 ml of non-sterilized effluent was inoculated with Trametes

versicolor immobilized in polyurethane foam, with 25 g l�1 glucose, 6.75 mM CuSO4, and 0.22 mM MnSO4 added, and cultured at 25 �Cwith an air flow of 800 ml min�1 for 8 d. The fungus did not inhibit growth of the heterotropic populations of the effluent. After 4 d ofculture, the chemical oxygen demand (COD) reduction and colour removal (CR) were 82% and 80%, respectively, with laccase (LAC)and manganese peroxidase (MnP) activities of 345 U l�1 and 78 U l�1, respectively. The COD reduction and CR correlated positively(p < 0.0001) with LAC and MnP activities. Chlorophenol removal was 99% of pentachlorophenol, 99% of 2,3,4,6-tetrachlorophenol(2,3,4,6-TCP), 98% of 3,4-dichlorophenol (3,4-DCP) and 77% of 4-chlorophenol (4-CP), while 2,4,5-trichlorophenol (2,4,5-TCP)increased to 0.2 mg l�1. The pre-treated effluent was then exposed to a photocatalytic treatment. The treatment with photolysis resultedin 9% CR and 46% COD reduction, 42% CR and 60% COD reduction by photocatalysis, and 62% CR and 85% COD reduction byheterogeneous photocatalysis with the system TiO2/RuxSey (Fig. 4). With this treatment the bacterial and fungal populations alsodecreased by 5 logarithmic units with respect to the biological treatment alone (Fig. 5). The total sequential treatment resulted in a92% CR (from 5800 UC), 97% COD reduction (from 59 g l�1) and 99% chlorophenol removal at 96 h and 20 min.� 2006 Elsevier Ltd. All rights reserved.

Keywords: Trametes versicolor; Effluent bleaching; Chlorophenols; Photocatalysis; Titanium dioxide; Ruthenium–selenium chalcogenide

1. Introduction

Different raw materials such as pine wood and sugar-cane bagasse are used for paper production. These materi-als are treated by physical–chemical methods to eliminatethe lignin and obtain white paper. Residues generated by

0045-6535/$ - see front matter � 2006 Elsevier Ltd. All rights reserved.

doi:10.1016/j.chemosphere.2006.10.015

* Corresponding author. Tel.: +52 55 50613316; fax: +52 55 50613313.E-mail addresses: [email protected] (A.M. Pedroza), rero-

[email protected], [email protected] (R. Rodrıguez-Vazquez).

the pulp and paper industry are characterized by havinghigh levels of chemical oxygen demand (COD), colourand more than 500 different adsorbable organic halidecompounds (AOX) (Savant et al., 2005). The low molecu-lar weight chloro-organics are the major contributors tomutagenicity and bioaccumulation, due to their hydropho-bicity and ability to penetrate cell membranes. Anotherproblem with this waste water is its dark brown colour,which is unacceptable because it inhibits the natural pro-cess of photosynthesis in streams, due to the poor penetra-tion of sunlight (Sahoo and Gupta, 2005).

794 A.M. Pedroza et al. / Chemosphere 67 (2007) 793–801

Physical, chemical, electrochemical and biological meth-ods have been developed to remove these pollutants fromwaste water (Thompson et al., 2001). Conventional aerobicand anaerobic treatments are the most widely used meth-ods to remove COD, biological oxygen demand (BOD),chlorophenols and colour (Zourari et al., 2002; Ramsayet al., 2005; Wu et al., 2005). Because these individual pro-cesses are generally limited, combinations of biological andphysical–chemical treatments, which can be complemen-tary, have also been tested to explore their technical feasi-bility, mainly when there are recalcitrant compoundspresent in the effluent. Li et al. (2005) reported that theuse of the combination of ozone and biologically activatedcarbon to treat effluents enhanced the efficiency of contam-inant removal. Other authors have treated textile effluents,using the sequence Phanerochaete chrysosporium/ozone,and found that this combination increased the efficiencyof removal of colour and total organic carbon (TOC)and reduced toxicity (Kunz et al., 2001).

The biological process using Basidiomycetes involvesmultiple biochemical reactions that have to take placesimultaneously: cleavage of intermonomeric linkages,demethylation, hydroxylation, side chain modificationsand aromatic ring fission, followed by dissimilation of thealiphatic metabolites produced (Sahoo and Gupta, 2005).The common feature of the pathways for degradation ofchlorophenols involves the production of ligninolyticenzymes like laccase (LAC) (EC 1.10.3.2), lignin peroxi-dase (LiP) (EC 1.11.1.14) and manganese peroxidase(MnP) (EC 1.11.1.13). The action of these enzymes resultsin phenoxy radical formation followed by nucleophilic sub-stitution to release Cl� (Limura et al., 1996). Other mech-anisms present among the bioremediation processes arecolour and COD biosorption, which involve physical–chemical interactions with the fungal biomass (Fu andViraraghavan, 2001).

On the other hand, heterogeneous photocatalysis withtitanium dioxide has been shown to be potentially advanta-geous and useful for the treatment of waste water from thepaper industry (Gouvea et al., 2000; Yeber et al., 2000;Chang et al., 2004). This process offers other advantages,such as complete mineralization and the lack of a waste-solids disposal problem, and is also environmentallyfriendly and relatively cheap (Krysa et al., 2005). The pho-tocatalytic mechanism starts when a photon with energy hv

matches or exceeds the band gap energy, Eg, of the semi-conductor. Conduction electrons, e�cb, are promoted fromthe valence band into the conduction band (CB), leavinga hole, hþvb behind. The electron–hole pair can react withelectron donors and electron acceptors (Hoffmann et al.,1995). The hole is a strong oxidant that can either oxidizea compound directly or react with electron donors likewater to form OH� radicals, which in turn react with pollu-tants such as chlorophenols, dyes, and organic compounds(Peiro et al., 2001; Wang et al., 2001; Aguedach et al.,2005). One efficient electron acceptor is molecular oxygen(O2), which forms a superoxide anion (O2) after capturing

the electron. The photocatalytic degradation process canbe accelerated by this process (Gerischer and Heller,1992). Hydrogen peroxide can be produced when a surfaceOH radical reacts with O�2 or by direct photolysis. It can actas a CB band electron acceptor, like O2, and subsequentlyform OH radicals (OH). These radicals react with organicpollutants leading to the total mineralization of most ofthem (Kusvuran et al., 2005).

Various methods are known for the fixation of nano-scale TiO2 onto a support matrix. The thin film can, there-after, be modified with different elements in order toincrease its efficiency and diminish the electron–hole sur-face recombination process. Thus, the surface of TiO2 ana-tase was modified by RuxSey nanoparticles (/ � 2 nm) toproduce TiO2/RuxSey interfaces (Villarreal et al., 2004).The cluster-like material (RuxSey), synthesized in colloidalform, is efficient for the reduction of molecular oxygen inacid medium (Alonso-Vante, 2003). The accumulated elec-trons on the surface of the semiconductor particles, photo-generated via low UV illumination, are transferred throughthe catalyst (RuxSey) to enhance the reduction of themolecular oxygen present in the electrolyte. In this way,the holes are available for the photo-oxidation of organicmolecules, thus favoring the photocatalytic process with ashorter timescale.

The objective of this study was to investigate the effi-ciency of the treatment of bleaching effluents with sequen-tial processes, i.e., biological pre-treatment with Trametes

versicolor immobilized on polyurethane foam followed byphotocatalysis using RuxSey surface-modified nanostruc-tured TiO2 thin films. The most important parameters wereevaluated, namely decolorization, COD reduction and chlo-rophenol removal. Both processes were conducted undernon-sterile conditions at room temperature (25 ± 3 �C).

2. Materials and methods

2.1. Biological pre-treatment with T. versicolor

2.1.1. Microorganisms and production of the immobilized

biomass

The fungus T. versicolor PUJ3 (Microbial Collection,University Javariana, Colombia) was cultured in a 1-l Erlen-meyer flask with 300 ml of wheat bran broth and 100polyurethane foam (PUF) cubes of dimensions 1 cm ·1 cm · 1 cm. The PUF was standardized for the fungalimmobilization in submerged cultivation (Pedroza et al.,2005). Under these conditions, the cubes have a high amountof fungal biomass that produces LAC and MnP. Ten agarplugs of 5 mm diameter that were cut from active fungus(8 d old) and grown on extract wheat bran plates were usedas inoculum. The culture was incubated in an orbital shakerat 120 rpm and 25 �C, for 9 d (Rosas et al., 2005).

2.1.2. Characterization of the bleaching effluentThe chemical, physical and microbiological characteris-

tics of the effluent before and after treatment are presented

Table 1Chemical, physical and microbiological characterizations of effluent before and after the sequential treatment with T. versicolor/TiO2/RuxSey

Parameter Effluent withouttreatment

Pre-treatment withT. versicolor immobilized

Post-treatment withUV/TiO2/RuxSey

Total sequential efficiency T. versicolor

TiO2/RuxSey (%)

Time 0 4 d 20 minpH 5.5 4.1 4.8Unit color 5800 1147 435 92Chemical oxygen

damand (g l�1)59 10.4 1.6 97

Pentachlorophenol(mg l�1)

13 0.1 – 99

2,3,4,6-Tetrachlorophenol(mg l�1)

12.1 0.1 – 99

2,4,5-Trichlorophenol(mg l�1)

8.4 8.6 0.1 99

3,4-Dichlorophenol(mg l�1)

11.3 0.12 <0.1 99

4-Chlorophenol(mg l�1)

15 3.4 <0.1 99

Bacteria (CFU ml�1) 12 · 102 50 · 107 85 · 102 0Yeast (CFU ml�1) 23 · 102 34 · 106 92 · 102 0

The data correspond to the mean values of three replicates. The total efficiency was calculated taking as a basis the initial concentrations of the effluentwithout biological treatment.

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in Table 1. The pH was adjusted to 5.5 for experiments inthe bioreactor. The colour of the effluent was measuredaccording to the APHA (1999). The COD was determinedby the open reflux titration method. The chlorophenolswere analyzed by HPLC, according to the methodologyof Rios and Calva (1999). The changes in the microbialpopulations in the effluent without treatment and the efflu-ent treated with the sequence T. versicolor/TiO2/RuxSey

and photocatalytic controls were investigated. Colonyforming units per milliliter (CFU ml�1) was the measure-ment used for these determinations (Doyle, 1997). Theeffluents were stored at 4 �C in the dark until further use.

2.1.3. Biological reactor and operational conditions

The biological treatment was performed in reactorsmade up of glass tubing (6 cm inner diameter, 50 cm long)packed with 160 polyurethane foam cubes colonized withT. versicolor and 800 ml of non-sterilized bleaching efflu-ent, supplemented with glucose and ammonium sulfateup to a C/N ratio of 405/1 (wt/wt) and induction forLAC and MnP activity (6.75 mM CuSO4 and 0.22 mMMnSO4, respectively). The reactors were maintained at25 �C, with an air flow rate of 800 ml min�1. The processwas evaluated for 8 d. The values in the figures correspondto the mean values of two replicates with a standard devi-ation of less than 15%. The effluent treated by the biologi-cal system was stored under refrigeration at 4 �C untilfurther use in the photocatalytic reactor. The immobilizedbiomass was observed by scanning electron microscopy(SEM).

2.1.4. Scanning electron microscopyThin-sliced sections of the matrix were obtained in order

to observe the mycelial growth in the polyurethane foam.

The thin sections were then prepared and fixed for 1 h withglutaraldehyde at 2.5% v/v, and were then washed with0.1 M phosphate buffer (pH 7.0) and fixed with osmiumtetraoxide at 1% v/v for 2 h. The fixed particles were dehy-drated using a series of ethanol washings with increasingethanol concentration (70, 85, 95 and 100%) for 5 min.After 24 h, the specimens were mounted on stainless steelstubs and coated immediately with gold in an ion coaterand examined using a JEOL-JSM-6300.

2.1.5. Enzymatic activity

The enzymatic activity of MnP was determined by theoxidation of 0.01% p/v Phenol Red (Merck 70156870) in20 mM succinic buffer (Sigma S-5047) at pH 4.5 (Michelet al., 1991). LAC activity was determined through the oxi-dation of 0.5 mM ABTS (Sigma A1888) in 100 mM sodiumacetate buffer at pH 4.5 (Tinoco et al., 2001). The enzy-matic unit was defined as the formation of 1 lmol of prod-uct per min, under the evaluated conditions.

2.2. Post-treatment with photocatalytic TiO2 modified by

colloidal RuxSey

2.2.1. Electrophoretically produced TiO2 thin filmsmodified with a RuxSey colloidal solution

The TiO2 thin films were deposited onto aluminum foilsof 2 mm thickness at room temperature by an electrochem-ical process according to the methodology reported byPeiro et al. (2002). In our experiments, the TiO2 (DegussaP-25) concentration was increased to 1% w/v. The electro-phoretic and cathodic deposition was carried out by apply-ing 3 V for 90 min between the substrate and an electrodeof platinum of similar dimensions. The thin film was driedat 450 �C for 60 min. The modifications of the thin films

Fig. 1. Photocatalytic reactor of 1 l.

796 A.M. Pedroza et al. / Chemosphere 67 (2007) 793–801

were done by dipping them in the colloidal RuxSey at50% v/v for 5 min. Thereafter, the modified films weredried and annealed at 230 �C for 2 h under a nitrogenatmosphere to eliminate the organic stabilizer (Villarrealet al., 2004). The thin films were observed by SEM (Floridoet al., 2005).

The electrolytic suspension contained TiO2 nanocrys-tals and peroxotitanium at pH 1.3, which is lower thanthe pH of the point of zero charge of TiO2 (pH 6.2).This fact determines that both compounds were posi-tively charged. Consequently, when a constant voltagewas applied between the anode and the cathode, the peroxycomplex migrated toward the aluminum substrate (cath-ode). The complex reacted with electrogenerated OH–and precipitated as a hydrate of insoluble peroxytitaniumTiO3(H2O)x. The positively charged TiO2 nanoparticlesalso moved toward the aluminum cathode and depositedwith the peroxytitanium hydrate.

2.2.2. Experimental set-up and photocatalytic reactions

The photocatalytic experiments were carried out in abeaker with capacity of 1 l , a double cylindrical jacket ofquartz for the refrigeration system to maintain the temper-ature at 25 �C. A light source of Hg vapour UV lamp(254 nm), which was placed at a distance of 5.5 cm and isparallel to a 15 · 30 Teflon racket, supported the thin filmsof TiO2 and/or TiO2/RuxSey (2.5 cm · 2.5 cm) (Fig. 1).Prior to illumination, and for every treatment, the systemwas bubbled with air in darkness for 60 min to establishan adsorption–desorption equilibrium. During the experi-ments, air flowing at a rate of 150 ml min�1 was continu-ously bubbled into the effluent. Four treatments wereevaluated: direct photolysis (UV), adsorption in darknessusing the semiconducting substrate (TiO2), photocatalysiswith the non-modified surface of TiO2 (TiO2/UV), andphotocatalysis with the modified surface with chalcogenide

(TiO2/RuxSey/UV). The UV, TiO2 in darkness and non-modified thin film (TiO2/UV) experiments were used ascontrols. The analytical determinations evaluated for thephotocatalysis experiments were the same as those usedduring biological pre-treatment. The values in the figurescorrespond to the mean values of three replicates with astandard deviation of less than 15%.

3. Results and discussion

3.1. Biological pre-treatment with T. versicolor

Colour is one of the main concerns in waste water(Thompson et al., 2001). Colour removal (CR) is carriedout by various eukaryote organisms; however, T. versicolor

is one of the most efficient organisms. In this study T. ver-

sicolor, under non-sterile conditions, decreased the colourduring the first 24 h, and a level of 80% decolorizationwas attained by day 4 (Fig. 2). The mechanisms involvedin the decolorization could be associated with physicaladsorption followed by a biochemical mechanism. The firstmechanism is not associated with metabolism but implies abiosorption through the formation of hydrophilic–hydro-phobic interactions between the compound and the cellwall (Fu and Viraraghavan, 2001). The second mechanismis associated with primary and secondary metabolism, inwhich the fungus uses a co-substrate, like glucose, and thento degrade the most toxic compounds and to remove col-our, during the secondary metabolism. This process is med-iated by MnP and LAC enzymes (Kasikara et al., 2005;Ramsay et al., 2005). The decolorization could be carriedout by oxidation (via removal of one or two electrons) ofthe recalcitrant chromophoric groups of the lignin to formradical cationic species that can follow different degrada-tion routes (Bergbauer et al., 1991). During this studyenzymatic activity was observed to increase progressively

Fig. 2. Biological pre-treatment with immobilized Trametes versicolor: (d)% decolorization, (m) COD removal, (j) laccase activity, (�) MnPactivity, 8 days, 25 �C, flow air: 800 ml min�1, non-sterilized conditions.

A.M. Pedroza et al. / Chemosphere 67 (2007) 793–801 797

until it reached maximum values between day 4 and 5 of345 U l�1 LAC and 78 U l�1 MnP (Fig. 2). Statistically apositive linear correlation (p < 0.0001) exists between thepercentage of decolorization, COD reduction and enzy-matic activity.

In a previous report Pedroza et al. (2005), using thepath of steepest ascent methodology, demonstrated thatthe addition of 6.75 mM CuSO4, 0.22 mM MnSO4 and25 g l�1 glucose, as a carbon source, had a significant effect(p < 0.0001) on the enzymatic activity (MnP and LAC) anddecolorization. They determined that these agents act aspossible metallic-type inducers (Cu and Mn) and substrate(glucose) for the fungus. We used these concentrations inthe biological treatment of the effluent in the bioreactorand found that the fungus grew inside the foam, attaininga maximum biomass per cube of 193 mg cm�3 in 4 d. Onthe other hand, we observed by SEM that the colonization

Fig. 3. Biological system: (A) polyurethane foam 18·, (B) Trametes versicolor ifilms of TiO2 600·, (D) characteristics of mesoscopic surface of TiO2/RuxSey

of the fungal biomass was inside the foam and an extensivemycelium network was recovered completely from thesurface, generating biosorption of coloured organic com-pounds in some areas (Fig. 3A and B). Under the evaluatedconditions, both mechanisms could have contributed toremoval of the pollutants by the fungus (biosorption 12%and biological removal 80%).

The results obtained in this study are interesting, sincethe fungus carries out the bioremediation process of thewaste water from the pulping industry without requiringsterile conditions, reaching a high (80%) colour removalin 96 h with 25 g l�1 glucose as co-substrate. In compari-son, Sahoo and Gupta (2005) reported 60% decolorizationof effluent in 72 h with a supplement of 20 g l�1 glucoseusing free cells of T. versicolor but under sterile conditions.On the other hand, Satwinder et al. (1998) demonstratedthat P. chrysosporium, in a non-sterile effluent of anaerobi-cally digested black liquor, biobleached up to 85% in 7 dwith the addition of 10 g l�1 glucose at pH 5.5. The effi-ciency of decolorization was similar to our results, but intheir study the original residues had lower colour units.

The COD removal showed similar behavior as colourunits, attaining 82% of up-to-date removal with a positivecorrelation (Fig. 1) between percentage of COD reductionand chlorophenols (p < 0.0001) (Fig. 2). Wu et al. (2005)demonstrated that by immobilizing the biomass of severalwhite rot-fungi, the concentration of carbon, nitrogen,and inducers, as well as pH, had a significant effect onthe black liquor treatment. We found similar results withglucose as a source of carbon, low nitrogen concentration,copper and manganese addition and acidic pH.

In relation to the quantified chlorophenols (Table 1), weobserved during the biological treatment that removalstarted in the first 24 h with the biosorption processesand further degradation of the organic compounds. Maxi-mum removal was attained after 4 d with 99% forpentachlorophenol (PCP) and 2,3,4,6-tetrachlorophenol(2,3,4,6-TCP) and 77% for 4-chlorophenol (4-CP) and aconcentration increase of 8.6 mg l�1 for 2,4,5-tricholoro-phenol (2,4,5-TCP). The latter was possibly generated bythe dechlorination of highly chlorinated compounds like

mmobilized into support 430·. Photocatalytic system: (C) thickness of thin1000·.

798 A.M. Pedroza et al. / Chemosphere 67 (2007) 793–801

pentachlorophenol and tetrachlorophenol. A positive cor-relation (p < 0.0001) between COD reduction, decoloriza-tion, chlorophenol removal and LAC activity wasobtained.

The removal of chlorophenols may be associated with aco-metabolism process, and it becomes more efficient in thepresence of a simple co-substrate. The use of fermentablesugar allows a decrease in pH up to 4.1. A similar behaviorwas observed by Ullah et al. (2000), who used T. versicolor

for PCP removal and found that pH 5.0 was associatedwith glucose consumption and a greater removal (90%)was obtained, showing a strong correlation with thelevels of LAC expression. The enzymatic degradation ofaromatic chlorinated compounds starts with an oxidativedechlorination due to the LACs, resulting in the forma-tion of p-quinone (2,6-dichloro-1,4-quinone or tetrachlo-robenzoquinone) or cationic radicals that result in afurther dechlorination, which could occur via a nucleo-philic attack on a molecule of water, producing chloroqui-nones (Kadhim et al., 1999). These compounds are strongoxidizers and can be transformed into the correspondinghydroxyquinones, eliminating the atoms of chlorine com-pletely so that the ring opens upon the action of MnPand LAC to form sour beta intermediary ketoadipic acidthat enters into the Krebs cycle (Limura et al., 1996; Reddyet al., 1998).

When using the biological reactor without sterilizing thewaste water, the bacteria (12 · 102 CFU ml�1) and fungi(23 · 102 CFU ml�1) present in them increased progres-sively until we obtained recounts of 50 · 107 CFU ml�1

for heterotrophic bacteria and 34 · 106 CFU ml�1 for het-erotrophic fungi (Fig. 5). Cooperation was possiblebetween T. versicolor and the other microorganisms. Thissituation is very favourable since the system could be usedin reactors of higher capacity without the need to controlthe sterility. This diminishes the risk of losing efficiencydue to the inhibition or death of the Basidiomycete.

Fig. 4. Photocatalytic treatment of effluent, previously treated with Trametes

RuxSey, (�) TiO2 in dark. Air flow: 120 ml min�1, pH: acid, temperature: 25 �

3.2. Post-treatment with photocatalytic TiO2 modified by

colloidal RuxSey

3.2.1. Characterization of the thin films of TiO2/RuxSey

The thin films of TiO2 obtained by electrophoresiscontained approximately 1.56 mg cm�2 of TiO2, and thethickness of the film was approximately 19.5 lm (seeFig. 3C and D). The films were characterized by X-ray dif-fraction, indicating the anatase phase [with (101), (004),(20 0) orientation] and rutile phase [with (10 1), (110),(21 1) orientation]. The mesoscopic surface of the thin filmof TiO2 was successfully modified with the colloidal Rux-Sey50% v/v according to Villarreal et al. (2004) (data notshown).

3.2.2. Efficiency of photocatalytic activityThe photocatalytic post-treatment yielded colour

removal and COD reduction. The results obtainedunder four different experimental conditions are shown inFig. 4. The treatments with UV, UV/TiO2, and TiO2, inthe dark were used as controls for the photocatalysis withRuxSey surface-modified TiO2. The best results, with sig-nificant differences among treatments, were obtainedwith UV/TiO2/RuxSey (p < 0.0001). Villarreal et al. (2004)working with this chalcogenide material (RuxSey) in colloi-dal form found that when modifying the surface of theTiO2 with RuxSey, the transfer of photogenerated electronsto adsorbed molecular oxygen was facilitated via the chal-cogenide catalyst, leaving a hole available for the oxidationof organic compounds. In their study, formic acid was usedas a model molecule. The modification led to an increase ofup to 650-fold the oxidation of the acid with respect to anon-modified TiO2. In our study, the efficiency may besmaller due to the complex composition of the residue. Inthe controls, with non-modified TiO2, a 42% CR and60% reduction in COD were recorded. On the other hand,the sole use of ultraviolet light gave 9% CR and 46% reduc-

versicolor: 60 min of irradiation, (m) UV, (d) UV/TiO2, (j) UV/TiO2/C.

Fig. 5. Changes in the microbial populations as a function of the type ofeffluent treatment. (A) none, (B) biological treatment alone, (C) UV/TiO2/RuxSey after biological pretreatment, (D) photolysis control, (E) photo-catalytic control on UV/TiO2 (hashed bars) heterotrophic bacteria (whitebars) heterotrophic fungus.

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tion in COD during the same test period. Further, TiO2 inthe dark removed 13% of the colour and reduced COD by31%, probably due to the adsorption of some compoundsonto the surface of the TiO2. The decolorization achievedby treatment with UV/TiO2 alone and TiO2/RuxSey waslow in comparison with that obtained in other studies (Yeberet al., 1999, 2000). This behavior could be attributed to theoxidation of the aluminum foil when it was exposed to ultra-violet light. On the other hand, the effluent used in our studywas more concentrated in colour and COD than the effluentused in the studies by Yeber et al. (2000).

To evaluate the photocatalytic activity of TiO2/RuxSey

under conditions of high COD, we carried out a comple-mentary experiment using an effluent without biologicaltreatment (59 g l�1 COD and 5800 colour units). We foundthat during 60 min of evaluation, there was no decrease inthe colour or reduction in COD. We found significant dif-ferences that demonstrate that it is necessary to first initiatebiological treatment to diminish COD and colour, in orderto avoid inhibition of the TiO2 activity. One possible rea-son is the excess of COD that can be adsorbed onto the sur-face of the TiO2 catalyst. This phenomenon can lead to adecrease in the number of reactive sites of TiO2. Anotherexplanation may be related to the low capacity of the UVradiation to penetrate an effluent that contains a solid sus-pension and dark colour. This fact was reported by Amatet al. (2005), who found that high COD (11 g l�1) affectedthe photocatalytic activity of TiO2, provoking a 20% lowerCOD reduction.

For chlorophenols (Table 1), the greatest removal wasobserved for 2,4,5-trichlorophenol 2,4,5-TCP, (3,4-DCP)and 4-chlorophenols (4-CP) with TiO2/RuxSey. These aro-matic compounds had strong electronegativity and exhib-ited low affinity for adsorption onto TiO2 at acidic pH(Krysa et al., 2005). As a result, an important number ofreactive sites on TiO2 were available to enhance removal,as observed. Degradation of chlorophenolic compoundscould take place mainly in a homogeneous phase, as shownby Peiro et al. (2001), via reaction with photogeneratedOH� radicals in the solution. The second step could corre-spond to the formation of a r-complex between the carbonatoms of the ring, where the substitution will take place,and the OH� radical.

The microbial populations were diminished by 1, 2 and 5logarithmic units (Fig. 5) upon treatment with UV, UV/TiO2 and UV/TiO2/RuxSey, respectively. Coleman et al.(2005) reported that TiO2 has a strong effect on bacterialpopulations because the ultraviolet light causes irreversibledamage to the DNA and the photocatalytic process pro-duces species that are strong oxidizers. Thus, this interactionmay have caused cell wall damage followed by cytoplasmicmembrane damage and the loss of the capacity to divide(Rincon and Pulgarın, 2004; Robertson et al., 2005).

Several other investigations that included combinedtreatments increased the removal of pollutants from paperand textile effluents (Gomes et al., 2000; Kunz et al., 2001;Li et al., 2005). However, none of them reported a

sequence similar to ours, except Kunz et al. (2001), whoused P. chrysosporium and ozone for treatment of textileeffluents.

This is the first study of a sequential treatment of aneffluent from the paper industry using T. versicolor and athin film of TiO2 modified by colloidal RuxSey, whichcould be used for other types of effluents due to the highremoval efficiency of chlorophenolic compounds, microbialcounts, and COD. TiO2 modified with the colloid of Rux-Sey had previously been used only for the degradation offormic acid electrolytes (Alonso-Vante, 2003).

In conclusion, a sequential set of processes for CODreduction, colour removal, and chlorophenolic compounddegradation of a bleaching effluent were studied. Eachprocess showed specific merit. The fungus T. versicolor

interacted with bacteria and yeasts (50 · 107 CFU ml�1

heterotrophic bacteria, 34 · 106 CFU ml�1 heterotrophicfungi), diminishing the colour in 80% (from 1147 UC),COD in 82% (from 10.4 g l�1) and chlorophenol removalwas 99% of pentachlorophenol (PCP), 99% of 2,3,4,6-tetra-chlorophenol (2,3,4,6-TCP), 98% of 3,4-dichlorophenol(3,4-DCP) and 77% of 4-chlorophenol (4-CP), while 2,4,5-trichlorophenol (2,4,5-TCP) increased to 0.2 mg l�1 in4 d. The photocatalytic process with thin films of TiO2

modified by colloidal RuxSey demonstrated increased effi-ciency of CR and COD removal with respect to thecontrols. The result obtained was 62% CR (from 435),85% COD (1.6 g l�1) reduction and 99% chlorophenolremoval after 20 min of irradiation. Table 1 shows theindividual and total efficiencies for the sequential treat-ment (biological-UV/TiO2/RuxSey), where 92% of colourremoval (from 5800 UC), 97% for COD reduction (from59 g l�1) and 99% for chlorophenol removal were obtainedafter 96 h and 20 min total time. Bacterial and fungal

800 A.M. Pedroza et al. / Chemosphere 67 (2007) 793–801

populations diminished by 5 logarithmic units comparedwith the biological treatment alone.

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