Metabolites characterisation of laccase mediated Reactive Black 5 biodegradation by fast growing ascomycete fungus Trichoderma atroviride F03

International Biodeterioration & Biodegradation 104 (2015) 274e282

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Metabolites characterisation of laccase mediated Reactive Black 5 biodegradation by fast growing ascomycete fungus Trichoderma atroviride F03 Liyana Amalina Adnan a, b, Palanivel Sathishkumar a, Abdull Rahim Mohd Yusoff a, b, *, Tony Hadibarata a, c a

Centre for Environmental Sustainability and Water Security (iPASA), Research Institute for Sustainable Environment, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysia Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysia c Department of Environmental Engineering, Faculty of Civil Engineering, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysia b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 April 2015 Received in revised form 21 May 2015 Accepted 24 May 2015 Available online xxx

In this study, fast growing ascomycete fungus Trichoderma atroviride F03 was explored to biodegrade bisazo dye, Reactive Black 5 (RB5). The maximum RB5 biodegradation (91.1%) was achieved in the culture medium supplemented with an appropriate carbon source (glucose, 20 g l
1), and nitrogen source (yeast extract, 20 g l
1) at pH 5 and 27  C. The laccase produced by T. atroviride F03 was involved in the RB5 biodegradation processes. The metabolites such as (I) 1,2,4-trimethylbenzene, (II) 2,4-ditertbutylphenol, and (III) benzoic acid-TMS) were identified as the biodegradation products of RB5 using gas chromatography-mass spectrometry (GCeMS). The presence of these metabolites suggested that RB5 biodegradation was initiated by the cleavage of azo bond forming naphthalene-1,2,8-triol and sulphuric acid mono-[2-(toluene-4-sulfonyl)-ethyl] ester. The sulphuric acid mono-[2-(toluene-4-sulfonyl)-ethyl] ester was further desulphonated to 1,2,4-trimethylbenzene. Then, the oxygenated ring of C1 and C2 naphthalene-1,2,8-triol was cleaved to 2-(2-carboxy-ethyl)-6-hydroxy-benzoic acid. The degradation of 2-(2-carboxy-ethyl)-6-hydroxy-benzoic acid could be proceeded with two pathways: (i) decarboxylation and methylation to form 2,4-ditertbutylphenol and (ii) decarboxylation mechanism that induced the formation of benzoic acid-TMS. Finally, this study proved that T. atroviride F03 might be a good candidate in treating textile effluent containing azo dye as this treatment does not generating aromatic amines. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Azo dye Biodegradation Laccase Metabolic pathway Reactive black 5 Trichoderma atroviride F03

1. Introduction Demand for the utilization of synthetic dyes has increased worldwide, as more than 3000 azo dyes are used by various industries (Jadhav et al., 2013). The higher utilization of azo dye, Reactive Black 5 (RB5) by these industries is due to its excellent properties in limiting energy consumption, remaining economical, producing brilliant colours, and containing reactive groups þ (-SO
4 Na ) that bond covalently to cellulose, which reduces the amount of colour lost during textile dyeing (Chen et al., 2011). However, the desperation of industrial companies to gain profit has

* Corresponding author. Centre for Sustainable Environment and Water Security (IPASA), Research Institute for Sustainable Environment, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysia. E-mail address: [email protected] (A.R. Mohd Yusoff). http://dx.doi.org/10.1016/j.ibiod.2015.05.019 0964-8305/© 2015 Elsevier Ltd. All rights reserved.

diverted their concerns for the environment and human being, thus causing catastrophic deterioration by their actions. For example, the ingestion of RB5 through food and water intake increases the probability of having intestinal cancer, impedes the cerebral growth of a fetus and cause allergic reactions to the respiratory tract (Usha et al., 2011; Husain and Husain, 2012). In addition, the elution of RB5 wastes from textile industries into river will affect the photosynthesis process therefore, suppressing the concentration of oxygen needed by aquatic life. This is due to the presence of chromophoric bis-azo group (eN]Ne) which gives the dye its colour thus, reduces the penetration of sunlight into the water (Wang et al., 2013). Furthermore, RB5 contains of stringent aromatic molecules and bis-azo bonds, which are difficult to be degraded by any mechanism, even by conventional wastewater treatment such as photo-oxidation and flocculation (Hussain et al., 2013).

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The toxicity and dangerousness of polluted water containing azo dye alerted the researchers to propose any possible methods to combat this problem. Unfortunately, most of the existing methods, such as activated carbon, coagulation, membrane filtration and irradiation, have suffered from a lot of disadvantages, such as high costs and being non-environmentally friendly due to the use of toxic oxidizing chemical reagents (Wang et al., 2008). Furthermore, they tend to produce a high amount of chemical sludge that requires complicated and costly secondary treatment (Kalpana et al., 2012). Although the bacterial treatment is economical: however, there is a high tendency for formation of toxic aromatic amine (Vyrides et al., 2014). Fortunately, treatment with ascomycota fungi has been promising in cutting costs and providing an environmentally friendly procedure. This is due to the utilization of natural redox mediators in catalysing the enzymatic mechanism, which are produced by the fungus itself (Rodriguez-Couto, 2012). The non-specificity of lignin degrading enzymes by fungus, which are lignin peroxidase (LiP; EC 1.11.1.14), manganese peroxidase (MnP; EC 1.11.1.13) and laccase (p-diphenol: dioxygen oxidoreductases; EC 1.10.3.2), enable them to degrade a wide range of xenobiotic compounds regardless of the spatial arrangements of their atoms or their structural isomers (Sathishkumar et al., 2010; Perlatti et al., 2012). In this study, ascomycete fungus Trichoderma atroviride F03 has been chosen due to its remarkable properties of growing fast and degrading high percentage of RB5. The preferable ligninolytic enzyme mechanism by T. atroviride F03 in aerobic condition promotes the oxidation reaction, thus reduces the tendency of aromatic amines production (Leung and Pointing, 2002). Thus, this research focused on monitoring the optimum conditions for the growth of T. atroviride F03 and RB5 biodegradation. Further, the ligninolytic enzymes involved in the RB5 biodegradation and metabolites formation were identified. This is the first report that demonstrates the new biodegradation pathway of RB5 by T. atroviride without generating aromatic amines.

275

2013). The sequence was compared with other 18S rDNA and internal transcribed spacer (ITS) region sequences that were obtained from Basic Local Alignment Search Tool (Blast) on the National Center for Biotechnology (NCBI) server (http://www.ncbi. nlm.nih.gov/BLAST). The phylogenetic tree was constructed based on the neighbour-joining method using Molecular Evolutionary Genetics Analysis 5 (MEGA5) (Saroj et al., 2014). Finally, the sequence was submitted to NCBI. 2.3. Seed and growth medium For the inoculum preparation, mycelium discs (5 mm diameter) of isolated fungus from a well grown plate culture were transferred into seed medium consisted of 2 g malt extract powder in 100 ml of distilled water, and was grown at 25  C on a shaker incubator for 48 h. Then, 5% (v/v) seed culture was used as inoculum for further experiment. The growth medium was prepared according to the method of Adnan et al. (2014) with slight modification associate with the growth of isolated fungus. The growth medium (100 ml) containing yeast extract (20 g l
1), glucose (20 g l
1), and RB5 (50 mg l
1) were sterilized by autoclaving for 15 min at 121  C. 2.4. Effect of parameters on RB5 biodegradation The effect of pH and temperature on the RB5 biodegradation by isolated fungus was monitored from pH 3 to 8 and 20e35  C with the above mentioned growth medium. Then, in order to know the effect of nutrient factors, the growth medium was prepared based on the different combinations of 2% of carbon sources (glucose, fructose and galactose) and 2% nitrogen sources (yeast extract, ammonium nitrate and ammonium chloride) under optimized pH and temperature on RB5 biodegradation. All experiments were performed in duplicate and reported values are the average of at least two experiments. 2.5. RB5 biodegradation

2Materials and methods 2.1Chemicals Reactive Black 5 (RB5), Trimethylchlorosilane (TMCS), 1,2,4trimethylbenzene, 2,4-ditertbutylphenol, and benzoic acid were purchased from SigmaeAldrich (St. Louis, Missouri, USA). Malt extract agar (MEA), glucose and yeast extract were obtained from Scharlau (Scharlab S.L., Sentmenat, Spain). 2.2. Isolation, screening, and identification of RB5 biodegrading fungal strain The fungal strains isolated from the bark of well grown trees in Recreational Forest, UTM, Johor, Malaysia were cultured in MEA plate and incubated at 25  C for a week. Further, the fast growing isolates were selected from MEA plate and inoculated into RB5 (50 mg l
1) amended MEA plates. The agar medium was regularly monitored for fungal growth and biodegradation activities for every 24 h. Finally, the fast growing and RB5 rapid biodegrading efficient fungal strain was selected for further identification and RB5 biodegradation studies. For identification, the DNA of the fungal tissue body was extracted using a Wizard® Genomic DNA Purification Kit (Promega, USA, Cat No: A1120). The polymeraseechain reaction (PCR) reaction was performed as follows: 1 cycle at 94  C for 2 min, 25 cycles at 94  C for 35 s, 50  C for 30 s and 72  C for 3 min and ended with 1 cycle at 72  C for 10 min (Hadibarata et al.,

The RB5 biodegradation by isolated fungus was carried out in liquid medium under optimized culture conditions. The culture supernatant harvested from the medium at every 24 h interval was centrifuged at 10 000  g for 20 min and used for further analysis. The RB5 biodegradation analysis was divided into three experimental parts: (i) measurement of RB5 biodegradation percentage by UVeVisible spectrophotometer and electrochemical analysis, (ii) assay of ligninolytic enzyme activities, and (iii) characterisation of RB5 biodegradation metabolites using thin-layer chromatography (TLC), UV-Visible spectrophotometer, gas chromatography-mass spectrometry (GCeMS) analysis and attenuated total reflectancefourier transform infrared spectroscopy (ATR-FTIR). All the experiments were performed in duplicate and reported values are the average of least two experiments as to obtain valid results. Data were analysed by one-way analysis of variance (ANOVA) with the TukeyeKramer multiple comparison test. 2.6. UV-visible spectrophotometer and electrochemical analysis The residual RB5 concentration was measured from 200 to 750 nm in UVVisible spectrophotometer (PerkineElmer, Germany) and calculated the area under the plot. Since, this approach takes into account the conversion of RB5 into new compounds absorbing at different wavelengths, the ratio of the area under the visible spectrum is almost equal to or lower than the ratio of the initial absorbance. A control test (un-inoculated medium) was also performed in parallel. RB5 biodegradation was calculated as follows (eq. (1)):

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A
At D¼ i  100 Ai

(1)

where D is the percentage of RB5 biodegradation, Ai is the area under the absorption spectrum curve from 200 to 750 nm at zero time and At is the area under the absorption spectrum curve at determined time point. The electrochemical analysis for biodegradation of RB5 was performed by Autolab Potentiostat/Galvanostat 30 (PGSTAT 30) coupled with a Voltammetric Analytical Stand (VA 663) Metrohm (Zurich, Switzerland). The standard procedure for voltammetry analysis was as follows: a series volume of 50 mg l
1 RB5 (20, 60 and 100 ml) was added into Britton Robinson buffer (BRB), which was prepared by dissolving boric acid (2.47 g), glacial acetic acid (2.3 ml) and orthophosphoric acid (2.7 ml) in distilled deionized water (1000 ml) (Yusoff et al., 1998). The percentage of RB5 biodegradation was calculated as follows (eq. (2)):

D¼

Ii
If  100 Ii

50e500. The initial temperature was programmed at 80  C for 2 min, raised from 80  C to 140  C at 15  C min
1, then to 290  C at 25  C min
1 and held for 6 min. The flow rate, interface temperature and injection volume were regulated at 1.5 ml min
1, 275  C and 1 ml, respectively, while the Helium pressure was maintained at 1 ml min
1. The identification of the individual total ion metabolite's peaks was done by comparison with the Wiley7 mass spectra database (Adnan et al., 2015). Furthermore, to validate the metabolites characterisation by GCeMS, their absorption spectra (UVVis) and retention time (GC) were compared with standard compounds: 2,4-ditertbutylphenol, 1,2,4-trimethylbenzene and benzoic acid. The biodegradation of metabolites were detected by GCeMS with and without trimethylchlorosilane (TMCS) derivatization. Also, the possible biodegradation and biosorption of the RB5 metabolites was further analysed based on infrared (IR) spectroscopic studies. Approximately 0.05 g of fungal biomass in RB5 treated culture medium was dried and ground before being directly analysed by ATR-FTIR in the mid-infrared region of 650e4000 cm-1 with 16-scan speed (Santo et al., 2013).

(2)

Where D is the percentage of RB5 biodegradation, Ii and If referred to initial and final peak current at the reduction potentials of
0.38 V and
0.55 V for RB5, respectively. All the experiments were subjected with triplicate as to obtain valid results and the average values were inserted into the data. 2.7. Ligninolytic enzyme activity assay Laccase activity was determined according to the method of Wolfenden and Wilson (1982) using ABTS as substrate. LiP activity was determined using the veratryl alcohol oxidation assay as described by Tien and Kirk (1984). MnP activity was assayed according to the method of Paszcynski et al. (1985) using vanillylacetone as substrate. The activity was expressed as units per volume and one unit of activity was defined as the amount of enzymes that oxidized 1 mmol of substrates per min. Furthermore, in order to confirm the laccase involvement in the RB5 biodegradation, the crude supernatant from culture medium (4th day) grown without RB5 was incubated with sodium azide (0.1 M) for 1 h at room temperature. After that, 10 ml of sodium azide treated culture supernatant was added into 50 mg l
1 of RB5 solution (50 ml) to monitor the biodegradation process. The residual concentration of RB5 and laccase activity was further analysed as per standard assay conditions. 2.8. TLC, GCeMS, and FTIR analysis The characterisation of metabolites was carried out to study the intermediate products from the biodegradation of RB5 under optimized conditions up to 8 days. To check the RB5 biodegradation products, the samples were acidified with 1 M of H2SO4 and extracted with 20 ml ethyl acetate (three repetitions) (Hadibarata et al., 2007). Each extracted samples were combined and concentrated with a rotary evaporator. The TLC technique was applied for preliminary detection of metabolites where the spots were visualized under UV-light (short and long wavelength) and sprayed with a bromocresol green solution as to indicate the presence of carboxylic acid compound. Hexane and ethyl acetate with a ratio of 1:1 (v/v) was chosen as the best solvent system for column chromatography (silica gel 60, 0.2e0.5 mm) as it successfully separated the TLC spots with the retention factor (Rf) between 0.2 and 0.8 (Hadibarata et al., 2012). After that, the characterisation of metabolites using GCeMS (Agilent Technologies) was performed with an HP5 column (25 m  0.2 mm; ID 0.3 mM) that was equipped with a detector at 1.3 eV, scan intervals of 1s and a mass range of

3. Results and discussion 3.1. Isolation, screening, and identification of RB5 biodegrading fungal strain Six different fungal strains (F01 to F06) isolated from the Recreational Forest in Universiti Teknologi Malaysia were screened for their growth and RB5 biodegradation efficiency on RB5 amended MEA plates. Among the fungal isolates, strain F03 showed rapid growth and maximum RB5 biodegradation efficiency. Therefore, in this study, fungal strain F03 was selected for further identification and RB5 biodegradation. The growth of fungal strain F03 on MEA plate was rapid and appeared as cream-coloured puffs, and the edge of the colony soon became green due to the increase in fungal sporulation which occurs mainly in the Trichoderma species (Monkemann et al., 1997). Then, to confirm the species of fungal strain F03, molecular characterisation was performed and compared with the NCBI Nucleotide Collection database, thus resulting in a phylogenetic tree (Fig. 1). Sequence analysis of the ITS regions of the nuclear encoded rDNA showed significant alignment for T. atroviride with 99% identities. 3.2. Optimization of culture conditions for RB5 biodegradation by T. atroviride F03 The pH and temperature of the growth medium are the most important factors for the growth of fungus and RB5 biodegradation. The changes of solution pH altered the adsorption of fungal surface electrical charge (cell wall) that consists of several functional groups (thiol and phosphate) towards the ionic dye (Arica and Bayramonglu, 2007). Hence, it must be regulated as to enhance RB5 biodegradation. Table 1 illustrates the effect of pH and temperature on the biodegradation of RB5 by T. atroviride F03. The pH of the liquid growth media was monitored at different pH ranging from 3 to 8, and the maximum percentage (72.4%) of RB5 biodegradation by T. atroviride F03 was achieved at pH 5. This is due to the fact that pH 5 was the optimum point for the secretion and mechanisms of ligninolytic enzymes (Marlida et al., 2000). The effect of temperature on biodegradation results clearly shows that the maximum percentage of RB5 biodegradation was found to be 87.2% at 27  C. The biodegradation of RB5 increased proportionally up to a certain point of temperature (27  C), beyond that the biodegradation was considerably decreased. This might be due to the denaturation of the enzyme active sites, which reduced

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Fig. 1. Phylogenetic tree of ascomycete fungus T. atroviride F03.

the amount of cell viability for the ligninolytic enzymatic activities in degrading the structure of targeted dye molecules (Khan et al., 2013). Enayatzamir et al. (2009) observed the maximum rate of RB5 biodegradation exhibited at room temperature, which is similar to this result. The effect of different carbon and nitrogen sources on the RB5 biodegradation by T. atroviride F03 are shown in Table 1. The obtained results confirm that glucose was the best carbon source for T. atroviride F03 because it provided the highest percentage of RB5 biodegradation (91.1%), while fructose and galactose exhibited only 37.3% and 57.2% of biodegradation. The simplicity and less hindered structure of glucose compared to the complex cyclic structures of fructose and galactose enhanced the degradation of glucose (Bankar et al., 2009). In the case of nitrogen source, yeast extract was the most excellent nitrogen source for the growth of T. atroviride F03 as it provided the highest percentage of RB5 biodegradation up to 91.1%,

Table 1 The effect of different parameters on the RB5 biodegradation by T. atroviride F03. Parameters

RB5 biodegradation (%) Day 4

Effect of pH 3 4 5 6 7 8 Effect of temperature 20  C 27  C 37  C Effect of nitrogen sources Yeast extract Ammonium nitrate Ammonium chloride Effect of carbon sources Glucose Fructose Galactose

43.8 48.1 59.4 52.2 45.4 42.0

± ± ± ± ± ±

Day 8 0.53 0.65 0.85 0.67 0.35 0.37

52.1 62.2 72.4 64.5 52.1 46.8

± ± ± ± ± ±

0.70 0.67 0.38 1.14* 0.54 0.85

65.4 ± 0.40 73.9 ± 0.78 58.1 ± 0.53

72.4 ± 0.46 87.2 ± 0.24 63.3 ± 0.43

85.5 ± 1.13* 24.1 ± 0.75 31.9 ± 0.65

91.1 ± 0.56 29.1 ± 0.53 71.7 ± 0.90

85.5 ± 1.13* 29.9 ± 0.95 46.6 ± 0.57

91.1 ± 0.56 37.3 ± 0.60 57.2 ± 0.49

compared to the use of ammonium nitrate and ammonium chloride, which exhibited only 29.1% and 71.7%, respectively. This might be due to the preference of enzyme secreted by T. atroviride F03 in biodegrading RB5 under organic nitrogen containing medium. This deduction was supported by Saravanakumar and Kathiresan (2014) that the highest percentage decolourisation of malachite green by Trichoderma sp. was reported in yeast extract supplemented medium (5.8 mg l
1). 3.3. RB5 biodegradation by T. atroviride F03 3.3.1. UV-vis spectrophotometer and electrochemical analysis The maximum absorbance of RB5 that occurred at 597 nm was indicated by the blue-black colour arising from the long conjugated p-system that linked the chromophoric azo double bond group. The spectrum of RB5 showed a decrease in absorbance at 597 nm and shifted to 423, 312, and 358 nm after biodegradation by T. atroviride F03 (Fig. S1). These shifts were due to the degraded chromophoric azo compound of the dye, colour change from blue-black to yellow, and the presence of benzene ring with the substitution group or the conjugated carbonyl group in the molecule, respectively (Enayatizamir et al., 2011). The biodegradation of RB5 was further analysed using differential pulse cathodic stripping voltammetry (DPCSV), due to its sensitivity in detecting compounds down to the sub-ppb level. The voltammogram peaks at
0.38 V and
0.55 V which were indicated by the detection of each electro-active RB5 azo compound were decreased to 78.06% and 88.46% respectively, after being treated with T. atroviride F03 (Fig. S2). The different reduction potentials of RB5 voltammogram peaks were due to the electron density alteration that was affected by eOH and eNH2 groups in which ortho-position with bis-azo bond (Guaratini et al., 2001). The voltammogram peak of RB5 at
0.55 V was shifted to a lower negative potential (
0.49 V) after treated with T. atroviride F03. This might be due to a decrease in the pH of the treated medium that resulted from the formation of a carboxylic acid compound (a metabolic product). The TLC analysis further verified the detection of carboxylic acid as it formed yellow spot when sprayed using bromocresol green (data not shown).

The growth media containing 50 mg l
1 RB5 were agitated at 120 rpm to assay for effect of growth's condition. All experiments were performed in duplicate and reported values are the average of at least two experiments and SD (±) is significantly different from the control at, *P <0.001, by one-way analysis of variance (ANOVA) with TukeyeKramer comparison test.

3.3.2. Ligninolytic enzyme activities In order to know the role of ligninolytic enzymes such as such as laccase, LiP, and MnP on RB5 biodegradation by T. atroviride F03, the

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Fig. 2. (a) Laccase production profile and (b) confirmation of laccase mediated RB5 biodegradation by T. atroviride F03 crude culture (0.1 M of sodium azide used as laccase inhibitor).

biodegradation experiment was performed under optimized conditions up to 9 days and the enzyme production was monitored every 24 h interval. Fig. 2a demonstrates the profile of enzyme production by T. atroviride F03 during RB5 biodegradation. The

results show that the laccase production was started on the 2nd day and the maximum activity was found to be 5.8 U ml
1 at 4th day; whereas, LiP and MnP were not detected in the biodegradation medium. In addition, the rate of RB5 biodegradation was almost

Table 2 Mass spectral and UV-vis analysis of the principal metabolites detected during RB5 biodegradation by T. atroviride F03. Metabolites

Wavelength (nm)

Rt (min)

I

262

5.04

120, 105, 77

II

271

9.97

206, 191, 163, 91

III

276

7.18

194, 179, 135, 105, 77

m/z

Possible structure

Abbreviations: m/z: mass to charge ratio, t: retention time, UV-Vis: Ultraviolet Visible . R The red line indicated UV-vis spectrum of standard compound.

1,2,4-trimethylbenzene (confirmed with a standard)

2,4-ditertbutylphenol (confirmed with a standard)

Benzoic acid-TMS (confirmed with a standard)

UV-vis spectrum

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84% decreased after laccase inhibited by sodium azide (0.1 M) in the crude culture of T. atroviride F03 (Fig. 2b). The remaining 16% of the degradation may be due to some other enzyme activity, which were not detected in this study. This result concludes that the laccase was involved in RB5 biodegradation process, which was predominantly produced by T. atroviride F03. Previous report of Chakroun et al. (2010) proved that T. atroviride produced extracellular laccase. Furthermore, Dhouib et al. (2005) noticed that T. atroviride produced laccase alone, but not LiP and MnP, which is similar to this study. Several studies were reported on the biodegradation of RB5 by laccase produced from Trametes pubescens (Roriz et al., 2009), and Pleurotus florida (Sathishkumar 2014); however, there was no detailed degradation mechanism on this dye by T. atroviride.

279

3.3.3. Metabolites analysis The decrease at the wavelength of 597 nm and increase in the absorbance at the UV region indicated the cleavage of the chromophoric azo bond and parent dye biodegradation into aromatic compounds. This was further proven by the detection of three metabolites from the biodegradation of RB5 by T. atroviride F03 using UV-Vis spectrophotometric and GCeMS (Table 2). The characterisations of these metabolites were confirmed using authentic standards. The incubation of RB5 on the 3rd day revealed one metabolite namely 1,2,4-trimethylbenzene (I) (8.32  10
4 M) which showed lmax of 262 nm and retention time (tR) of 5.04 min which was similar to the standard compound of 1,2,4trimethylbenzene. The GCeMS spectrum of metabolite I in Fig. 3a

Fig. 3. The mass spectra profile for metabolites of RB5: (a) 1,2,4-trimethylbenzene, (b) 2,4-ditertbutylphenol, and (c) benzoic acid-TMS derivatives.

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revealed a molecular ion [Mþ] at m/z 120 which strongly correlated with the MS properties of authentic standard 1,2,4-trimethylbenzene. Prolonged incubation for the RB5 biodegradation by T. atroviride F03 led to the formation of 2,4ditertbutylphenol (II) (4.36  10
4 M) and benzoic acid (III) (2.57  10
4 M). Metabolite II had a retention time of 9.97 min and MS properties of Mþ at m/z 206 (Fig. 3b). The retention time and the MS properties of this compound was identical with the authentic standard of 2,4-ditertbutylphenol. Then, metabolite (III) exhibited similar UV-Vis properties (lmax: 276 nm) with the standard compound benzoic acid. Benzoic acid was subjected with derivatization as to enhance its detection by GCeMS and it revealed the retention

time (tR) of 7.18 min with an MS spectrum of Mþ at m/z 194 (Fig. 3c). Some of the metabolites which were naphthalene-1,2,8-triol, sulphuric acid mono-[2-(toluene-4-sulfonyl)-ethyl] ester and 8-hydroxy-[1,2]-naphthoquinone were not detected in the culture extract; thus, the presence of these compounds in fungal mycelium were further verified using ATR-FTIR analysis. The FTIR spectrum of the treated sample shows significant peaks at 1347.15 cm-1, 1033.21 cm-1, and 812.82 cm-1 which were equivalent to asymmetric SeO stretching vibration, S]O group and CeS stretching, respectively (Fig. S3). This result proved that the sulphonic compound was present in the fungal body. Plus, the specific peaks at 2923.68 cm-1, 1520.67 cm-1 and 1054.90 cm-1 were in correlation

Fig. 4. The proposed pathway of RB5 biodegradation by ascomycete fungus T. atroviride F03. Compounds in the bracket were not identified in the culture extract.

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with CeH (alkane stretching), C]C stretching vibration in the benzene ring and CeO stretching, respectively. C]O bond (ketone) and OeH deformations were reported by the peaks at 1682.79 cm-1, and 1347.15 cm-1, respectively. These peaks indicated the quinone and phenolic derivatives nature of metabolites were formed after RB5 biodegradation. The proposed biodegradation pathway of RB5 by T. atroviride F03 is shown in Fig. 4. The RB5 biodegradation was initiated by the cleavage of the bis-azo bond and followed by deamination and hydroxylation which were mediated by laccase to produce naphthalene-1,2,8-triol and sulphuric acid mono-[2-(toluene-4sulfonyl)-ethyl] ester. These findings contradict with the previous report of Wang et al. (2008), that the degradation of RB5 azo bond by Rhodopseudomonas palustris led to the formation of toxic aromatic compound 7-amino-8-hydroxy-1,3-naphthoquinone-3,6disulfonate-1,2-diimime. The loss of amino compound (-NH2) that bearing by RB5 was likely mediated by deamination and hydroxylation as these mechanisms were main role of laccase in degrading substrate (Patel et al., 2013). Then, the desulphonation of sulphuric acid mono-[2-(toluene-4-sulfonyl)-ethyl] ester led to the formation of 1,2,4-trimethylbenzene (I) which was detected in the culture extract at earlier incubation. The present study proved that RB5 biodegradation mechanism proceeded with the aromatic ring fission of naphthalene-1,2,8-triol, where its oxygenated ring at C1 and C2 position was cleaved to 2-(2-carboxy-ethyl)-6-hydroxy-benzoic acid via 8-hydroxy-[1,2]naphthoquinone. After that, the biodegradation of 2-(2-carboxyethyl)-6-hydroxy-benzoic acid could be further degraded via two possible pathways: (i) it undergone decarboxylation and methylation to form 2,4-ditertbutylphenol (detected at 7th day) and (ii) it transformed to benzoic acid by decarboxylation mechanism. The synthetic cofactor such as H2O2 which bearing high oxidizing agent was not added into the culture medium due to the dependency of laccase to only molecular oxygen thus, indicated that this treatment is environmentally safe. The production of phenolic and carboxylic acid metabolites after the maximum activity of laccase at 4th day of incubation, deduced that the oxidative mechanism for RB5 biodegradation occurred after the secretion of laccase from T. atroviride F03. 4. Conclusions The present study suggests that T. atroviride F03 was able to biodegrade RB5 efficiently by the secretion of extracellular laccase. The dependency of laccase to only molecular oxygen, without the need for cofactors and oxidizing agent deduced that it is a simple process. Further, the characterisation of metabolites proved that this biodegradation process did not generate toxic aromatic amines, thus it is an eco-friendly nature treatment. Acknowledgement A part of this research was financially supported by Fundamental Research Grant Scheme from Ministry of Education, Malaysia (Vote R.J130000.7809.4F312), which is gratefully acknowledged. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ibiod.2015.05.019. References Adnan, L.A., Sathishkumar, P., Hadibarata, T., Yusoff, A.R.M., 2015. Biodegradation pathway of acid red 27 by white-rot fungus Armillaria sp. F022 and

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