Investigation of dyes degradation intermediates with ... - Springer Link

Eur Food Res Technol (2011) 233:751–758 DOI 10.1007/s00217-011-1569-7

ORIGINAL PAPER

Investigation of dyes degradation intermediates with Scytalidium thermophilum laccase Sonia Ben Younes • Zouhaier Bouallagui Adel Gargoubi • Sami Sayadi

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Received: 5 July 2011 / Revised: 15 August 2011 / Accepted: 22 August 2011 / Published online: 4 September 2011 Ó Springer-Verlag 2011

Abstract Scytalidium thermophilum laccase was able to successfully decolourise Congo Red, Bromo-Cresol Green, Malachite Green, Phenol Red and Indigo Carmine under optimised conditions. The cited dyes belonging to three different classes were named azo, triarylmethane and indigoid. The decolourisation rates were 100, 95, 76, 57 and 22 mg h-1 U-1 for Indigo Carmine, Malachite Green, Bromo-Cresol Green, Congo Red and Phenol Red, respectively. The degradation products were characterised by UV–vis and FT-IR techniques, and their cytotoxicity was monitored. UV–visible absorption spectra and FT-IR analysis showed a complete degradation of Congo Red, Bromo-Cresol Green and Malachite Green, a partial degradation of Phenol Red and a transformation of Indigo Carmine. Toxicity study revealed that most of the treated dyes were less toxic than those before treatment, especially for Malachite Green. In fact, Scytalidium thermophilum laccase degraded Malachite Green into non-toxic products. Scytalidium thermophilum laccase constitutes a powerful tool for effective bioremediation of rich-dye textile effluents and was, therefore, found worthy of investigation for potential applications in restoration work and other biotechnological uses. Keywords Triarylmethane dyes Azo dyes Indigoid dyes Laccase FT-IR Detoxification

S. B. Younes (&) Z. Bouallagui A. Gargoubi S. Sayadi (&) Laboratoire Des Bioproce´de´s Environnementaux, Poˆle d’Excellence Re´gional AUF, (PER-LBP), Centre de Biotechnologie de Sfax, Universite´ de Sfax, Route de Sidi Mansour Km 6, BP ‘‘1177’’, 3018 Sfax, Tunisie e-mail: [email protected]

Introduction Textile industry wastewaters are one of the most polluting worldwide. In particular, the release of coloured effluents into the environment is undesirable not only due to their colour but also because many synthetic dyes, and their breakdown products are toxic and/or mutagenic [1, 2]. Generally, dyes are considered to be stable towards light and temperature and resist to the microbial attack, making them recalcitrant compounds [3]. The textile industry accounts for two-thirds of the total dyestuff market. During the dyeing process, approximately 10–15% of the dyes used are released into the wastewater [2]. The presence of these dyes in the aqueous ecosystem is the cause of serious environmental and health concerns [4, 5]. The structural diversity of dyes is attributed to the presence of different chromophoric groups [6]. These groups will contribute to the classification of dyes into azo, anthraquinone, nitro, nitroso, triphenylmethane, xanthene, acridine, thiazole, sulphur, indigoid, phthalocyanine dyes, etc. Amongst these dyes, Azo dyes, which are aromatic compounds with one or more (–N=N–) groups, are the most important and largest class of synthetic dyes used in commercial applications [7]. Several processes are used to treat textile effluents to achieve decolourisation. These include physicochemical methods including filtration, coagulation, activated carbon and chemical flocculation [2, 5]. Such methods are effective but expensive and involve the formation of a concentrated sludge that creates a secondary disposal problem [5]. However, all of them have shortcomings, and an effective and inexpensive alternative would be very useful. In recent years, new biological processes, including aerobic and anaerobic bacteria and fungi, have been developed for dyes degradation and wastewater reuse [3]. For fungi,

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especially white-rot micro-organisms, the biodegradation potential has been attributed to ligninolytic enzymes, which are capable of non-specifically breaking down heterogeneous macromolecular lignin structures [5]. White biotechnology seems to be the magic solution using in particular white-rot fungi producing enzymes like lignin peroxidase (LiP), manganese-peroxidase (MnP) and laccase that degrade many aromatic compounds [8]. The potential of these fungi to oxidise phenolic, non-phenolic, soluble and non-soluble dyes was largely discussed [5]. Many studies demonstrated that white-rot fungi can degrade a wide variety of structurally diverse dyes such as azo, anthraquinone, heterocyclic, triphenylmethane and polymeric dyes due to the lignin-degrading enzymes system [3]. Laccase preparations obtained from white-rot fungi increased up to 25% of the decolourisation rate of individual commercial triarylmethane, anthraquinonic and indigoid textile dyes [9]. In contrast, MnP and LiP were reported as the main enzymes involved in dyes decolourisation by Phanerochaete chrysosporium and Bjerkandera adusta [5]. However, the long fungal growth cycle and the complexity of the textile effluents, which are extremely variable in composition, limit the performance of these fungi. Furthermore, and despite the successful achievement of the stable operation of continuous fungal bioreactors for the treatment of synthetic dye solutions, application of white-rot fungi for the removal of dyes from textile wastewaters faces many problems such as large volumes produced, the synthetic dyes class and control of biomass [5]. Thus, enzyme decolourisation and degradation has appeared as an environmentally friendly and cost-competitive alternative [5]. Thus, the main purpose of this study was to determine the dye degradation intermediates of three classes of dyes using exclusively a Scytalidium thermophilum laccase under optimised conditions. The degradation products were also characterised using UV–vis and FT-IR analyses. The toxicity of treated and untreated dyes was monitored.

Materials and methods Chemicals All the dyes—Phenol Red (PR), Malachite Green (MG), Bromo-Cresol Green (BCG), Congo Red (CR) and Indigo Carmine (IC)—were purchased from Sigma-Aldrich and Fluka and used without further purification. Table 1 shows their chemical structure and their main characteristics. Fungal strain and culture conditions Scytalidium thermophilum strain (accession number: FJ560721) was isolated from a locally prepared compost in

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Eur Food Res Technol (2011) 233:751–758

the north of Tunisia [10]. Strain ST26 was identified using the universal ITS1 and ITS4 primers according to White et al. [10, 11]. Scytalidium thermophilum produces laccase and protease activities under optimised conditions [10]. The isolate was maintained on 2% (w/v) malt extract agar at 4 °C. The fungal inoculum was cultivated on the same media at 45 °C. For laccase production and induction studies, the optimised medium was used as described by Ben Younes and Sayadi [12]. The pH of the solution was adjusted to 5.5. The mycelia were homogenised using a Waring blender for 5 min at 20,000 rpm. The homogenized culture was used to inoculate 250-ml Erlenmeyer flasks containing 100 ml of medium and induced by 250 lM of copper [10, 12]. Cultures were incubated at 45 °C in a rotary shaker at 200 rpm, and samples were taken periodically. After 5 days of fungal growth, laccase activity reached its maximum and the cultures were harvested and filtered through filter paper [12]. The clear filtrate was concentrated 5 times at 4 °C and used as crude enzyme preparation for in vitro decolourisation experiments [10, 12]. Enzyme and protein assays Laccase activity was assayed using 10 mM DMP (2,6Dimethoxyphenol) in 100 mM sodium citrate buffer, pH 5 (e469 = 27,500 M-1 cm-1), as described by Ben Younes [12, 13]. Enzymatic reactions were carried out at room temperature (22–25 °C). One unit of enzyme activity was defined as the amount of enzyme oxidising 1 lmol of substrate per min. Extracellular proteins were determined by the Bradford method [14], using Bio-Rad protein assay and bovine serum albumin as standard [12, 13]. UV–vis analysis UV–visible spectral analysis was carried out using UV-spectrophotometer (UV-1800 Shimadzu), and changes in the absorption spectrum of the dye degradation products, produced during biodegradation using Scytalidium thermophilum laccase under optimised conditions (400–800 nm), were recorded and compared with the control [15]. Fourier Transform Infrared (FT-IR) analysis After complete decolourisation, the reaction solution was used to extract metabolites with equal volume of ethyl acetate. The extracts were dried over anhydrous Na2SO4 and evaporated to dryness in rotary evaporator. The crystals obtained were dissolved in small volume of methanol and used for analysis [2]. The Fourier Transform Infrared Spectroscopy (FT-IR) analysis of controls and extracted metabolites was done on Thermo Scientific Nicolet 380


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Table 1 Characteristics of the used dyes Name, colour index (C.I.)

Chemical structure

kmax (nm)

Phenol Red

410

C.I. Basic green 4 (Malachite Green) C.I. 42000

610

Bromo-Cresol Green

617

C.I. Direct red 28 (Congo Red) C.I. 22120

499

Indigo Carmine C.I. 73015

608

FT-IR. The spectra were collected within a scanning range of 400–4,000 cm-1. Toxicity studies Cytotoxicity was studied for all of the treated and untreated dyes. Cell viability of human cervix HeLa cells was assessed using the MTT assay [16]. Cells harvested by trypsinisation at 60–80% confluence were seeded in 96-well plates at 2,000 cells per well in 100 ll RPMI 1640 medium. All plates were incubated at 37 °C in the presence of 5% CO2 for 24 h to allow cells to attach. Cytotoxicity was evaluated at 5 and 50% (V/V) final concentrations starting from a concentration dye working solution prepared at 200 mg l-1, and for a treating period of 48 h. Subsequent to this incubation, the

medium was removed, and 100 ll new medium and 10 ll of MTT solution (5 mg ml-1 in PBS) were added. Four hours later, 100 ll of SDS solution (10%) was added to each well. Following to formazan dissolution, optical density was read at 570 nm using a microplate reader ELX 800. The growth rate was expressed as the percentage of control cells treated with 5% or 50% PBS. Statistical analysis All the results, related to the toxicity determination, were the average of three replicates of two separate experiments. They were statistically analysed by SAS software (Version 8) using Duncan test performed after analysis of variance (ANOVA).

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Results and discussion Decolourisation study In this study, Scytalidium thermophilum laccase was chosen to separately decolourise the five dyes: Bromo-Cresol Green, Malachite Green, Phenol Red, Congo Red and Indigo Carmine. In previous studies, aiming the physicochemical influencing decolourisation parameters, conditions were optimised to achieve higher decolourisation rates (data in publication). In fact, high decolourisation rates of the cited dyes were obtained operating under acidic pH conditions (6 for CR and MG and 5 for PR, BCG and IC), high temperatures (65 °C for PR, BCG and IC, 60 °C for CR and 50 °C for MG) and low ionic strengths (0.3 mol/l for IC, 0.2 mol/l for CR, 0.05 mol/l for PR, 0.1 mol/l for BCG and 0.75 mol/l for MG). The use of a small amount of crude S. thermophilum laccase degraded MG, CR and BCG totally, PR partially and transformed IC (data in publication). Using these conditions, S. thermophilum laccase could achieve effectively the complete decolourisation for BCG, CR, PR and MG and the transformation for IC (Table 2). The decolourisation rate showed no relationship between the chemical structure of the dyes and the reaction kinetic. The decolourisation rates were 22, 57, 76, 95 and 100 mg h-1 U-1 for PR, CR, MG, BCG and IC, respectively. These results are in agreement with the findings reported by Franciscon [5], demonstrating that the chemical structures of the dyes deeply influence their decolourisation rates. Dyes with simple structures and low molecular weights usually exhibit higher rates of colour removal, whereas colour removal is more difficult with highly substituted and high molecular weight dyes [2, 17, 18]. It has been reported that the turnover rate of monoazo dyes increased with increasing dye concentration, whereas the turnover rate of the diazo and triazo dyes remained constant when the dye concentration increased [5, 19] as observed for CR. Usually, the presence of sulphonates in reactive dye structures results in low levels of colour removal that is the case of PR. However, this is not applicable to BCG, usually exhibiting high levels of colour removal independently from the number of sulphonate

Table 2 Decolourisation rate of the used dyes

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Dyes

Decolourisation rate (mg/h/U)

IC

100

MG

96

BCG CR

76 57

PR

22

groups in the dye structure, reinforcing the hypothesis that steric hindrance and dyes molecular weight may be responsible for the difference in decolourisation times. It has also been reported that a correlation between the enzyme redox potential and its activity towards the substrates could influence the decolourisation rate [5, 20]. Thus, the ability of the biocatalysts to degrade dyes depends on the structural characteristics of the dye, the temperature and the pH of the medium, the presence of intermediates and the difference between the redox potentials of the biocatalyst and the dye [5]. UV–Vis characterisation Laccase-catalysed oxidation reactions are attracting increasing interest because of the synthetic potential of the mild oxidative reagent. Dyes transformations have some importance, because they are used as analytical reagents and may even be used in diagnostic methods in medicine [21]. Additionally, they have significant industrial importance in the enzyme-assisted decolourisation of denim dyestuff. UV–visible spectroscopy is much more sensitive in aqueous solutions, because water is practically transparent in the spectral region between 200 and 800 nm. The absorption coefficients in the UV–visible range can be very high, and thus the sensitivity is also excellent. The biodegradation of the chosen dyes was monitored by UV–vis analysis (Fig. 1). Figure 1 shows a plot of the UV–visible spectra during the laccase-catalysed dyes oxidation, without redox mediator. For untreated dyes, as shown in Fig. 1, PR, CR, IC, MG and BCG presented a major absorbance peak in the visible at 410, 499, 608, 610 and 617 nm, respectively, which are responsible for the intense colours of the used dyes. The wide band absorption between 200 and 300 nm was observed for all the treated and untreated dyes. For laccase treated dyes, the characteristic absorbance peak in the visible region has disappeared, indicating complete decolourisation except for Indigo Carmine (Fig. 1). The decrease of the visible peak intensity at 608 nm of Indigo carmine, resulting in a new peak emerged at 550 nm, was probably due to the apparition of a red coloured product. It is important to note a residual component with a red colour is still present (peak around 550 nm) when the reaction depletes. The spectrum of an unidentified final product that is labelled ‘‘red’’ because of its colour is seen in Fig. 1e. The presence of the typical absorption peak of the hydrogenated azo bond structure (–NH–NH–) at 245 nm in the spectra of CR seems to indicate only partial azo bond disruption after laccase biodegradation. The absorbance peaks in the visible region disappear indicating CR complete decolourisation [5]. To confirm whether it was a complete degradation or a partial transformation, FT-IR analyses were carried out.


b

4

3

Absorbance

a Absorbance

Fig. 1 UV–vis spectra of the treated (light dotted line) and untreated dyes (dark dotted line) (a Malachite Green, b BromoCresol Green, c Phenol Red, d Congo Red, e Indigo Carmine) under optimised conditions

755

2

1

0 200

300

400

500

600

4

3

2

1

0 200

700

300

Wavelength (nm)

d

4

3

2

300

400

500

600

700

Wavelength (nm)

Absorbance

e

600

700

800

4

3

2 1

1

0 200

500

Wavelength (nm)

Absorbance

Absorbance

c

400

0 200

300

400

500

600

700

Wavelength (nm)

4

3

2

1

0 200

300

400

500

600

700

800

Wavelength (nm)

FT-IR characterisation Since the signals obtained from IR spectroscopy are caused by substance-specific vibration modes of the molecules, they can readily be attributed to functional groups. Thus, infrared spectroscopy is a widely used tool for qualitative characterisation of materials and, to a lesser extent, for quantitative analysis also. FT-IR spectra of treated and untreated dyes are shown in Fig. 2. Structure of triarylmethane, azo and indigoid dyes has many common characteristics. The FT-IR spectra of untreated dyes showed the specific peaks in fingerprint region (1,500–500 cm-1) for the mono-substituted and para-disubstituted benzene rings, which is supporting to the peak at 1,585 cm-1 for the C=C stretching of the benzene ring [22]. The observed peaks in the region (3,300–3,500 cm-1) are attributed to the O–H stretching. Absorption at 2,900 cm-1 of control dyes was C–H stretch band of CH2 and CH3 groups. All tested dyes, except MG, present a characteristic peak in the range 1,100–1,000 cm-1 related to the S=O stretch band with the most significant

absorbance peak being at 1,084 cm-1 and another one in the range 740–690 cm-1 for the C–S stretching vibrations due to sulphur compound bonded to the activated carbons. The presence of these functionalities is consistent with the reported functionalities of fixed sulphur in activated carbons prepared from sulphur and sulphur-based compounds [23]. Furthermore, the peaks at 1,170 cm-1 for the C–N stretching vibrations and at 2,923 cm-1 for C–H stretching of asymmetric –CH3 group give the perception of MG structure (Fig. 2c) [23]. If is noticed the chemical structure of CR (Table 1), it can be seen that the bands located within the range 1,610–1,630 cm-1 and at 1,402 cm-1 were due to azo linkages –N=N– on aromatic structures and –N=N– stretching on CR (Fig. 2a), respectively [5]. The bands at 1,737 assigned to the C=O stretching of PR (Fig. 2d) and IC (Fig. 2b) as well as a one characteristic of BCG (Fig. 2a) due to the band located at the region 510–590 cm-1 assigned to the Br group. Comparison of FTIR spectra of control dye with metabolites extracted after 100% decolourisation clearly

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Fig. 2 FT-IR spectra of BromoCresol Green (a), Indigo Carmine (b), Malachite Green (c), Phenol Red (d) and Congo Red (e) before and after (red dotted line) laccase treatment

indicated the biodegradation of the initial dye compound by Scytalidium thermophilum laccase. The characteristic band at 1,585 cm-1 of C=C of the benzene ring of treated IC persisted even after the increase of the exposure time with laccase. Furthermore, the bands at 1,585 cm-1 assigned to aromatic skeletal vibrations [24] have been shifted, broadened and reduced after biocatalyst treatment confirming the complete degradation of PR, CR, MG and BCG (Fig. 2). By comparing the spectral characteristics of both treated MG and CR, a peak at 1,261 cm-1 for –C–N– stretch with supporting peak at 1,098 cm-1 might be attributed to the

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formation of primary and secondary amines [2, 22]. The sharp peak at 800 cm-1 for disubstituted benzene derivatives indicates aromatic nature of amines in the case of MG [22]. For CR, the strong bands at 1,062.7, 1,178.4 and 1,266.6 cm-1 attributed to S=O stretching and at the same time, the band at 1,610.5 cm-1 attributed to –N=N– stretching disappeared after laccase treatment, confirming the previous UV–vis results about azo linkage disruption [25]. Once again for the CR spectra (Fig. 2e), the reduction of the azo linkage peak followed by the formation of two bands in the carbonyl region at around 1,680–1,600 cm-1 was consistent with an amide derived from ammonia or a

Eur Food Res Technol (2011) 233:751–758 Table 3 Cell viability of HeLa cells towards treated and untreated dyes

757

IC

PR

CR

MG

BCG

Bf

Af

Bf

Af

Bf

Af

Bf

Af

Bf

Af

5%

100

98

94

101

94

86

33

96

85

25%

94

94

83

94

77

73

10

82

73

85

50%

92

93

65

94

76

63

4

57

61

75

5%

90

88

87

97

75

87

12

93

87

102

25%

70

74

63

105

77

80

3

29

81

91

50%

60

63

57

108

66

67

2

10

63

75

50 mg/l 98

200 mg/l

primary amine. These two bands disappeared, and a new peak around 1,680 cm-1 appeared. The presence of this additional group, due to the conjugation of C=C and C=O groups, suggested that this peak could belong to a carbonyl group of carboxylic acid [26]. The fact that no new peaks appeared between 3,300 and 3,500 cm-1 (attributed to azo bonds and OH groups in a position relative to the azo linkage) and in the region between 1,340 and 1,250 cm-1 (–NH2) after the laccase treatment suggested that the azo linkage could be transformed into N2 or NH3 [5]. Moreover, the presence of new peaks at 850 and 950 cm-1 and the peak at 1,140 cm-1 that could belong to acetate, formates, propionates, benzoates, etc. suggested that the products were undergoing irreversible chemical changes, probably due to concomitant biodegradation and autoxidation reactions of the products formed during the reductive dye degradation [5]. In addition, the persistence of the sulphur groups of sulphonated dyes (PR, BCG and CR) even after laccase treatment reflects their general stability. These results suggest that Scytalidium thermophilum laccase was characterised by a non-specific action on aromatic and phenyl groups in dyes, as substrates, and it attacks actively these xenobiotics and recalcitrant compounds. Cytotoxicity determination The cytotoxicity test demonstrated that dyes degradation using S. thermophilum laccase and under optimised conditions was not sufficient to remove the toxicity of Indigo Carmine (*60%). Conversely, a significant decrease in toxicity after laccase treatment of MG (*80%) was observed when the initial concentration was 5%. At high concentration of MG, we can attribute the increase of toxicity due to the amine group spread in the solution [5]. It was concluded that even if the laccase were able to decolourise the dyes under optimised conditions, the toxicity cannot be removed completely but also we can decrease the percentage of toxicity (Table 3).

Cytotoxic effects of untreated dyes at both concentrations 50 and 200 mg l-1, introduced to cell culture medium at different percentages, showed a total cell growth that was slightly higher than 60% except for the Malachite Green, presenting a sharp dropping of cell growth that has not exceeded 40% (Table 3). Subsequent to the laccase catalysed decolourisation, a significant cytotoxicity decrease of MG was observed. Similarly, a slight increase of cell growth in presence of treated BCG and PR was shown. CR cytotoxicity profile was not affected by the enzymatic decolourisation, and the same cell behaviour was seen when cells were incubated with native and biodegraded dye. Malachite Green, an N-methylated diaminotriarylmethane dye, has previously been reported to be highly cytotoxic to mammalian cells. The present work shows that the enzyme catalysed decolourisation was associated with the detoxification of MG. Comparable statements could be reported for untreated PR, BCG and CR, but to a less extent as they did not reveal high cytotoxicity under our experimental conditions. Fisher’s test for the variance analysis, performed with untreated and laccase treated dyes, indicated that these later resulted in a statistically significant cell growth improvement with Pr [ F-value less than 0.001. The laccase catalysed dyes degradation, dyes chemical structure and the interaction between dyes and enzyme treatment exhibited a confidence level above 99%, especially for MG. A low value of the variation coefficient (7.52) indicated a great reliability of the trials. In conclusion, cytotoxicity method can be usefully applied for toxicity detection in dyes contaminated wastewaters.

Conclusion Using Scytalidium thermophilum laccase, all the dyes tested were totally and rapidly decolourised under optimised conditions. Some differences in decolourisation kinetics depending on the dye structure and class were confirmed by the UV–vis analysis. Decolourisation was strongly

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dependent on the enzyme required amount. After dye decolourisation, degradation intermediates products were analysed by FT-IR analysis. Cytotoxicity assays were evaluated and showed a complete detoxification of MG. Dyes decolourisation and detoxification with S. thermophilum crude laccase, shown to be effective even without the presence of any mediators, may have great advantages for the development of treatment methods based on laccase considered to be a cost-benefit in biotechnological application perspective.

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