Bioresource Technology 102 (2011) 10359–10362

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Bioresource Technology

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Biodegradation pathway and detoxification of the diazo dye Reactive Black 5by Phanerochaete chrysosporium

Naeimeh Enayatizamir a, Fatemeh Tabandeh b,⇑,1, Susana Rodríguez-Couto c,d,⇑,1, Bagher Yakhchali b,Hossein A. Alikhani e, Leila Mohammadi e

a Department of Soil Science, Faculty of Agriculture, Shahid Chamran University, Ahvaz, Iranb Industrial and Environmental Biotechnology Department, National Institute of Genetic Engineering and Biotechnology (NIGEB), P.O. Box 14155-6343, Tehran, Iranc CEIT, Unit of Environmental Engineering, Paseo Manuel de Lardizábal 15, 20018 San Sebastian, Spaind IKERBASQUE, Basque Foundation for Science, Alameda de Urquijo 36, 48011 Bilbao, Spaine Department of Soil Science Engineering, Faculty of Soil and Water Engineering, University College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran

a r t i c l e i n f o

Article history:Received 24 June 2011Received in revised form 26 August 2011Accepted 31 August 2011Available online 8 September 2011

Keywords:BiodegradationFixed-bed bioreactorLigninolytic enzymesPhanerochaete chrysosporiumReactive Black 5

0960-8524/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.biortech.2011.08.130

⇑ Corresponding authors at: CEIT, Unit of EnviroManuel de Lardizábal 15, 20018 San Sebastian, Spain+34 943 213076 (S. R.-Couto), tel./fax: +98 21 445803

E-mail addresses: taban_f@nigeb.ac.ir (F. Ta(S. Rodríguez-Couto).

1 These authors contributed equally to this work.

a b s t r a c t

The in vivo biodegradation of the diazo dye Reactive Black 5 (RB5) by Phanerochaete chrysosporium immo-bilised on cubes of nylon sponge and on sunflower-seed shells (SS) in laboratory-scale bioreactors wasinvestigated. The SS cultivation led to the best results with a decolouration percentage of 90.3% in 72 hfor an initial RB5 concentration of 100 mg/L. It was found that the addition of 0.4 mM veratryl alcohol(VA) into the medium considerably increased the decolouration rate in SS cultivation. However, the addi-tion of VA had no effect in the nylon cultivation. Thin layer chromatography (TLC) revealed that RB5 wastransformed into one metabolite after 24 h. UV–vis spectroscopy and Fourier Transform Infrared (FT-IR)also confirmed the biodegradation of RB5. Toxicity of RB5 solutions before and after fungal treatment wasassayed using Sinorhizobium meliloti as a sensitive soil microorganism. P. chrysosporium transformed thetoxic dye RB5 into a non-toxic product.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Azo dyes, widely used in the textile industry, are considered re-calcitrant xenobiotic compounds due to the presence of a nitrogendouble bond (–N@N–) and other groups (i.e. sulphonic group) thatare not easily biodegraded. In addition, most azo dyes are mutagenicand carcinogenic to living organisms (Nilsson et al., 1993). The cur-rent physical and/or chemical techniques to remove these dyes fromeffluents present the following drawbacks: high cost, low efficiency,potential production of highly toxic by-products and in-applicabil-ity to a wide variety of dyes (Novotny et al., 2004). Thus, biodegrada-tion is seen as an environmental/eco-friendly and less expensivealternative.

By far white-rot fungi are the most efficient micro-organisms inbreaking down synthetic dyes because of the non-specific nature oftheir lignin-degrading enzymes (Wesenberg et al., 2003). Moststudies dealing with wastewater treatment have been performed

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nmental Engineering, Paseo. Tel.: +34 943 212800; fax:99 (F. Tabandeh).

bandeh), srodriguez@ceit.es

under sterile conditions which are unpractical at large scale dueto operational costs. Therefore, search for methods with white-rot fungi operating under non-sterile conditions are the subjectof future research.

The aim of the present paper was to study the degradation anddetoxification of the recalcitrant diazo dye Reactive Black 5 (RB5)by the white-rot basidiomycetePhanerochaete chrysosporium immo-bilised on two kinds of supports (inert and non-inert) in fixed-bedlaboratory-scale bioreactors operating under non-sterile conditions.In addition, the resulting biodegradation intermediates and metab-olites and their toxicity were investigated.

2. Methods

2.1. Micro-organisms

P. chrysospoium RP 78 was a kind gift from Dr. D. Cullen (ForestProducts Laboratory, Madison, WI, USA). It was maintained onpotato dextrose agar (PDA) as previously described (Ghasemi etal., 2010). Sinorhizobium meliloti (168) was provided from themicrobial collection of the Department of Soil Science Engineering,University of Tehran, Iran, and then re-cultured in yeast extractmannitol agar (YMA) with Congo Red (Vincent, 1970).

Table 1RB5 decolouration (%) obtained along time for different RB5 concentrations by P.chrysosporium immobilised on sunflower-seed shells in a fixed-bed bioreactor (eachdye concentration corresponds to a different batch).

Time (h) Decolouration (%)

[RB5] (mg/L) 15 40 60 80* 100*

24 80 ± 7.3 56 ± 5.2 7 ± 0.5 29 ± 2.8 52 ± 5.048 – 68 ± 6.3 28 ± 2.6 80 ± 7.7 77 ± 7.472 – 73 ± 6.5 52 ± 4.9 87 ± 8.4 90 ± 8.796 – 81 ± 7.9 83 ± 7.8 89 ± 8.8 92 ± 8.9

* Addition of 0.4 mM veratryl alcohol.

10360 N. Enayatizamir et al. / Bioresource Technology 102 (2011) 10359–10362

2.2. Carriers

Inert supports: 0.5 � 0.5 � 0.7 cm cubes of nylon sponge(Scotch Brite™, 3M Company, Spain). The cubes were pre-treatedas indicated in Linko (1991).

Non-inert supports: Sunflower (Helianthus annuus) seeds shells(SS) were obtained and pre-treated as indicated in Rodríguez-Coutoet al. (2009). The chemical composition of the SS according toDemirbas and Akdeniz (2002) is 17% lignin, 48.4% cellulose and34.6% hemicellulose.

2.3. Culture media and operation conditions

The growth medium was prepared according to Tien and Kirk(1988) with 10 g/L glucose as a carbon source, except dimethylsuc-cinate was replaced by 20 mM acetate buffer (pH 4.4). The culturesconsisted of 0.95 g of nylon cubes or 1.5 g of SS, according to theexperiment, and 20 mL of growth medium. Three agar plugs (diam-eter, 7 mm), taken from a 7-day-old PDA culture, were used asinoculum. The flasks were statically incubated at 37 �C in darknessfor 7 days when manganese-dependent peroxidase (MnP) activitywas maximised. Then, 8 g of nylon or 8 g of SS, according to theexperiment, colonised by the fungus were transferred to thebioreactor.

A fixed-bed tubular bioreactor with dimensions of 20 cm heightand 4.5 cm in internal diameter (working volume of 200 mL) wasfilled with 8 g of nylon cubes or 8 g of SS colonised by the fungusand kept at room temperature (25 �C) and aeration rate of0.5 vvm. The culture medium composition was: 20 mM acetate buf-fer (pH 4.4) and 2 g/L glucose, in the case of nylon cultivation andwithout glucose in the SS one. Dye was added under non-sterileconditions in successive batches, increasing the dye concentrationin each batch (15, 40, 60, 80 and 100 mg/L). At the beginning of eachbatch the bioreactor was poured out and filled with fresh mediumsterilised beforehand and after 2–3 h the dye was added under non-sterile conditions. Samples from the middle part of the bioreactorwere collected, centrifuged (8000g, 10 min) and analysed.

2.4. Enzyme assays and decolouration studies

Lignin peroxidase (LiP) and MnP activities were determinedaccording to Tien and Kirk (1984) and Kuwahara et al. (1984),respectively. Analyses were done in triplicate and the mean andstandard deviations were calculated with Microsoft Excel Pro-gramme. The decolouration of the diazo dye Reactive Black 5(RB5) (C26H21N5Na4O19S6; CI 20505; synonym: Remazol Black B;kmax 597 nm) by P. chrysosporium immobilised either on nylon orSS in a fixed-bed bioreactor was performed. Stock solutions (2 g/Lin water) were stored in the dark at room temperature. The resid-ual dye concentration was spectrophotometrically measured from450 to 700 nm and calculated measuring the area under the plot.Dye decolouration was expressed in terms of percentage.

2.5. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis(SDS–PAGE)

Extracellular fluid was filtered (0.4 lm filter paper, Whatman)and precipitated with trichloroacetic acid (TCA). Precipitated pro-teins were solubilised in sample buffer (90 mM tris–HCl, pH 6.8).SDS–PAGE (13% v/v) was performed according to the method ofLaemmli (1970). Protein bands were visualised by silver staining.

2.6. Analysis of dye degradation metabolites

Analysis of dye degradation was performed by using filteredsamples (0.45 lm). UV–vis region spectra (200–700 nm) were

done with a double-beam spectrophotometer Helios alfa (ThermoSpectronic, Cambridge, UK). The decolourised RB5 solutions, col-lected at 4, 10, 24, and 48 h and the original RB5 solution(15 mg/L; absorbance at 597 nm: 0.5) were spotted on commercialthin layer chromatography (TLC) plates (Silica gel, 10 � 20 cm,fluorescent indicator at 254 nm, Fluka). The plates were developedby using the solvent system: methanol/ethylacetate (1:1 v/v) andsome drops of ammonium solution. The chromatograms wereobserved under UV (254 nm) and by iodine vapours. For FourierTransform Infrared (FT-IR) analysis, lyophilised samples wereground with KBr powder and pressed between two metal platesat 40 MPa.

2.7. Toxicity studies

A colony of freshly isolated S. meliloti was cultured in yeastmannitol broth (YMB) overnight. Toxicity of RB5 and its biotrans-formation products were assessed by inoculating 5% (v/v) froman overnight culture into test tubes containing YMB and dye at dif-ferent concentrations or the decolourised medium previouslyfiltered (0.2 lm) in a final volume of 3 mL. The tubes were incu-bated on a rotatory shaker at 28 �C and 130 rpm. After 24 h,serial dilutions on physiological serum (NaCl 0.9% v/v) were pre-pared and 0.1 mL was transferred to YMA and after 48 h viable col-onies were counted.

3. Results and discussion

3.1. Decolouration of RB5

For RB5 concentrations ranging from 15 to 60 mg/L higherdecolouration rate was achieved in the bioreactor with P. chrysos-porium immobilised on SS but similar decolouration percentageswere attained in both bioreactors at the end of each batch (Tables1 and 2). In addition, it was shown that for RB5 concentrationshigher than 40 mg/L the decolouration rate diminished consider-ably. This likely indicates substrate inhibition at high dye concen-trations (Balu and Radha, 2009). The addition of VA (0.4 mM) to theculture medium had only a positive effect on the SS cultivation andincreased both the decolouration rate and the decolouration per-centage. By operating with SS as a support, a decolouration per-centage of about 90% in 72 h at initial dye concentrations of 80and 100 mg/L was obtained (Table 1). However, operating withnylon sponge a decolouration percentage of 45% after 72 h for aninitial dye concentration of 80 mg/L was attained (Table 2). Thismight indicate a different composition in the enzymatic complexsecreted by each culture. It has been shown that the addition ofVA into fungal cultures leads to higher LiP activities and dye decol-ouration percentages but has no significant effect on MnP produc-tion (Bibi et al., 2010). Also, the lignin content of the SS could haveinduced higher LiP activities. Decolouration was likely due to theMnP and LiP enzymes secreted by the fungus since physical

Table 2RB5 decolouration (%) obtained along time for different RB5 concentrations by P.chrysosporium immobilised on nylon sponge in a fixed-bed bioreactor (each dyeconcentration corresponds to a different batch).

Time (h) Decolouration (%)

[RB5] (mg/L) 15 40 60 80*

24 48 ± 4.3 – 34 ± 3.1 30 ± 2.848 83 ± 8.1 – – 34 ± 3.272 – 84 ± 8.2 47 ± 4.5 45 ± 4.396 – – 80 ± 7.9 47 ± 4.6

* Addition of 0.4 mM veratryl alcohol.

Fig. 1. Thin layer chromatography (TLC) analysis of the parent (line 1) anddecolourised dye at different stages (4, 10, 24 and 48 h, lines 2–5, respectively).

y = -2,3352x + 754,43R² = 0,9876

0

100

200

300

400

500

600

700

800

0 50 100 150 200 250 300 350

CFU

×10^

6/m

L

RB5 (mg/L)

Fig. 2. Number of viable cells of S. meliloti when exposed at different concentrationsof Reactive Black 5 (RB5) for 48 h.

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adsorption on the supports was negligible. SDS–PAGE of the cul-ture extract produced showed two main bands with molecularweights of about 45 and 38 kDa as visualised by silver staining(data not shown), which likely corresponds to MnP and LiPenzymes, respectively.

3.2. Analysis of the extracted metabolites

The UV–vis absorption spectra of RB5 decolouration showed aband with maximum absorption at 597 nm (visible region) andtwo bands with maximum absorption at 313 and 203 nm (UVregion). The former is responsible for the dark blue colour arisingfrom aromatic rings connected by azo groups and the latter areassociated with ‘‘benzene-like’’ structures in the molecule. After10 and 24 h two visible bands at 560 and 525 nm and three bandsat 277, 230 and 212 nm for 10 h and 206.5 nm for 24 h in the UVregion were detected. The absorption increase at 24 h may beattributed to the formation of coupling products. This change inthe maximum absorption indicated partial degradation of thedye. The bands at UV region related to 24 and 10 h probably corre-sponded to quinone and benzene rings with substitution groups.After 48 h the visible bands disappeared whereas two bands (212and 277 nm) at UV region remained. These changes indicated azogroup cleavage and dye transformation.

TLC results are shown in Fig. 1. The parent dye RB5 resolved inTLC into a single spot of Rf value of 0.78. The 10 and 24-h fungal-treated samples also showed a spot at this Rf but much lessintense, which could correspond to the remaining dye, togetherwith a spot at an Rf of 0.36. In addition, the 24-h fungal-treatedsample also showed a spot at an Rf of 0.25, only visible underiodine vapours which indicates its non-aromatic nature. In the48-h fungal-treated sample the spots at Rf of 0.78 and 0.25 disap-peared whereas only appeared a spot at 0.38. This indicated thatthe dye was totally converted into one metabolite after 48 h of fun-gal treatment.

The FT-IR spectra of RB5 showed vibrations at 1600–1450 cm�1

(aromatic C@C stretching), 640–900 cm�1 (aromatic @C–H bend-ing) and the left part of 3000 cm�1 (aromatic @C–H stretching).10 h after the reaction started the aromatic part vibrations startedbranching and after 24 h a new vibration at 3321–3395 cm�1

appeared, implying destruction of N@N and formation of NH2.After 48 h of reaction under oxidative conditions, almost all thecharacteristic absorption bands of RB5 disappeared.

The decolouration occurred in two stages. In the first stage thedye concentration decreased rapidly within 10 h and the colour ofthe solution remained purple. In the second stage, dye concentra-tion decreased slowly within 48 h and the solution became colour-less. This two-step decolouration likely occurred due to thedifferent reactivity of the two azo-bonds in RB5 is attributed tothe reactivity difference between the hydroxyl (–OH) and the ami-no (–NH2) groups, which are the activating groups of aromaticelectrophilic substitution at the ortho position related to the azobonds.

3.3. Toxicity studies

Fig. 2 indicates the changes in the viability of cells in responseto the treatment with different concentrations of dye. It can beclearly seen that the viability of cells dropped sharply by increasingthe dye concentration and EC50 was 164 mg/L. The toxicity of sam-ples at different biodegradation stages (10 and 24 h) indicated thatP. chrysosporium converted the toxic dye RB5 into a non-toxicmetabolite (p < 0.05).

4. Conclusions

The above results showed that the decolouration ability ofP. chrysosporium was greatly influenced by the support used. Thisis likely due to the support influences both the type and proportionof the enzymes produced, which are responsible for dye decolour-ation. This underlines the importance of selecting a suitable sup-port in this type of process. The ability of P. chrysosporium todegrade RB5 was mainly due to the secretion of the extracellularenzyme MnP. The effectiveness of degradation was confirmed byTLC, FT-IR and toxicity studies, which indicated that P. chrysospori-um transformed the diazo dye RB5 into a non-toxic metabolite.

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Acknowledgements

This research was financed by the National Institute of GeneticEngineering and Biotechnology (NIGEB project 329) and the Span-ish Ministry of Education and Science (Project CTQ2007-66541).Some part of the work was performed at the Department of Chem-ical Engineering from Rovira i Virgili University (Tarragona, Spain).

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