Detoxification of malachite green by Pleurotus florida ...

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Detoxification of malachite green by Pleurotus florida laccase produced under solid-state fermentation using agricultural residues Palanivel Sathishkumar

a c

a

b

, Thayumanavan Palvannan , Kumarasamy Murugesan &

Seralathan Kamala-Kannan

c

a

Laboratory of Bioprocess and Engineering, Department of Biochemistry, Periyar University, Salem, Tamil Nadu, 636 011, India b

School of Environmental Science and Engineering, Pohang University of Science and Technology, San 31, Hyojadong, Namgu, Pohang, 790 784, South Korea c

Bioremediation Laboratory, Division of Biotechnology, Advanced Institute of Environment and Bioscience, College of Environmental and Bioresource Sciences, Chonbuk National University, Iksan, 570 752, South Korea Accepted author version posted online: 01 May 2012.Version of record first published: 25 May 2012.

To cite this article: Palanivel Sathishkumar , Thayumanavan Palvannan , Kumarasamy Murugesan & Seralathan KamalaKannan (2013): Detoxification of malachite green by Pleurotus florida laccase produced under solid-state fermentation using agricultural residues, Environmental Technology, 34:2, 139-147 To link to this article: http://dx.doi.org/10.1080/09593330.2012.689359

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Environmental Technology Vol. 34, No. 2, January 2013, 139–147

Detoxification of malachite green by Pleurotus florida laccase produced under solid-state fermentation using agricultural residues Palanivel Sathishkumara,c , Thayumanavan Palvannana∗ , Kumarasamy Murugesanb and Seralathan Kamala-Kannanc∗ a Laboratory

of Bioprocess and Engineering, Department of Biochemistry, Periyar University, Salem, Tamil Nadu 636 011, India; of Environmental Science and Engineering, Pohang University of Science and Technology, San 31, Hyojadong, Namgu, Pohang 790 784, South Korea; c Bioremediation Laboratory, Division of Biotechnology, Advanced Institute of Environment and Bioscience, College of Environmental and Bioresource Sciences, Chonbuk National University, Iksan 570 752, South Korea

b School

Downloaded by [Chonbuk National University] at 23:28 09 April 2013

(Received 1 August 2011; final version received 12 April 2012 ) Laccase was produced from Pleurotus florida under solid-state fermentation, and the production was optimized by response surface methodology. The predicted maximum laccase production of 8.81 U g−1 was obtained by the optimum concentration of malt extract, banana peel, wheat bran and CuSO4 , which was found to be 0.69 g, 10.61 g, 10.68 g and 77.15 ppm, respectively. The validation results suggested that the laccase production was 7.96 U g−1 in the optimized medium, which was close to the predicted value. Decolorization efficiency of P. florida laccase was evaluated against malachite green (MG). Rapid decolorization of MG dye was observed, and a dark-coloured precipitate was formed in the reaction mixture. HPLC analysis indicated that the laccase enzyme degraded MG by the demethylation process. The toxicity of MG was reduced to 67% after the treatment with laccase, which was confirmed by a phytotoxicity study. Keywords: decolorization; laccase; malachite green; N -demethylation; Pleurotus florida; response surface methodology

1. Introduction Laccases (benzenediol:oxygen oxidoreductases, EC 1.10.3.2) are oxidase enzymes belonging to the multicopper oxidase family, and catalyse the oxidation of an array of aromatic substrates concomitantly with the reduction of molecular oxygen to water [1,2]. Several studies have demonstrated that the laccases are widely distributed in fungi, plants, insects and also some bacterial species, but the efficient producers are white-rot fungi [3–7]. These oxidative enzymes are being increasingly evaluated for a variety of biotechnological applications owing to their broad substrate range [7–18]. However, the application of these enzymes in processes requires a high amount of production at low cost. Therefore, research in this area is oriented toward the search for novel, efficient production systems. Submerged fermentation (SF) does not mimic the natural living conditions for white-rot fungi. Solid-state fermentation (SSF), defined as the fermentation of solids in the absence of free water, has the advantage of supporting the growth and metabolism of microorganisms under moisture conditions [19]. Production of enzymes by SSF on agricultural residues has gained much attention because of its higher productivity and low production cost [20]. The use of such wastes, besides providing alternative substrates, ∗ Corresponding

helps to solve environmental problems that are caused by their disposal. In addition, most of these wastes contain lignin and/or cellulose and hemicellulose, which act as mediators of the ligninolytic activities. Furthermore, most of them are rich in sugars, which makes the whole process much more economical. All these factors make such wastes very suitable as raw materials for the production of secondary metabolites by microorganisms of industrial significance [21]. In recent years, laccase produced under the SSF system using agricultural residues has proved to be an effective bioremediation agent for synthetic dyes [9,22–24]. Synthetic dyes are common contaminants of water, the production of these compounds was estimated to be around 7 × 105 tons per year, and the quantity of dyes discharged into the environment was assumed to be about 40% [25–27]. Malachite green (MG) is a triphenylmethane dye used as a direct dye for the dyeing of silk, wool, jute and leather [28], and it is also widely used as fungicidal agent to prevent fungal infection in fish hatcheries [29,30]. Consequently, the public may be exposed to this dye and its metabolites through the consumption of MG-treated fish [29]. Malachite green is considered as a potential carcinogen [31], and it is also highly toxic to mammalian cells and aquatic organisms even at trace concentrations [32,33]. Because of the toxicity to biotic communities, studies that can detoxify

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these toxic dyes and its metabolites are of great interest. Synthetic dye decolorization has been reported by many researchers. Sometimes, the degradation products are more highly toxic than the parent compounds. Thus, it is essential to evaluate the toxicity after the decolorization process. Pleurotus florida is a white-rot fungus, which has great biotechnological importance, and its application in bioremediation of industrial effluents and decolorization of synthetic dyes through their ligninolytic enzyme system is well known [14,34]. In this study, agricultural residues were used as the substrate to produce extracellular laccase from P. florida NCIM 1243 under the SSF process, and the production was optimized by response surface methodology (RSM). Further, the laccase enzyme was used to evaluate its efficiency for the decolorization and detoxification of MG dye. The decolorization mechanism was elucidated using UV-visible spectrophotometry, HPLC and LC-ESI-MS analysis. 2. Materials and methods 2.1. Chemicals and dye 2,2 -Azinobis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and MG were purchased from Sigma-Aldrich, USA, and all other chemicals were of analytical grade. 2.2. Fungal strain Pleurotus florida NCIM 1243 was purchased from the National Collection of Industrial Microorganisms (NCIM), National Chemical Laboratory (NCL), Pune, India, and was grown on potato dextrose agar (PDA) plates incubated at 28 ◦ C for about seven days. Thereafter, the plates were maintained at 4 ◦ C until used. The fungus was subcultured every three months. 2.3. Agricultural residues and inert material Agricultural residues (banana peel and wheat bran) were procured from a local market and were used as a substrate for the laccase production from P. florida under the SSF condition. Crab shells were collected from a fish market, washed with distilled water and dried at 50 ◦ C. The dried shells were powdered and used as an inert material to make up the total weight in the optimization studies under SSF.

enzyme activities were expressed in U g−1 or U mL−1 . Protein content was estimated by the method of Lowry et al. [36] using bovine serum albumin (BSA) as a standard. 2.5. Culture conditions Fermentation was performed in two stages: seed growth and laccase production. For the seed growth, the mycelium from a plate culture was inoculated into the seed medium (2% malt extract broth) and grown at 28 ◦ C on a shaking incubator for 48 h. Five per cent (v/w) of seed culture was used as inocula for the laccase production optimization experiments. 2.6.

The optimization of laccase production was carried out using the central composite design of RSM (CCD-RSM). Four independent variables, malt extract, banana peel, wheat bran and CuSO4 , at five levels (−2, −1, 0, +1, and +2) were used in this study. The range and levels of these variables are given in Table 1. According to the design, 30 runs, replicated six times at central points, were performed, and the laccase production was considered as the response (dependent) variable [37]. The significant interaction between and optimum level of the variables were tested for the maximum laccase production. 2.7. Statistical analysis Statistical calculations were performed using Design Expert software 7.0 (Stat Ease, Minneapolis, USA). Quadratic and interaction effects of the independent variables were evaluated for the RSM. The Fisher’s F-test for analysis of variance (ANOVA) was performed on the experimental data to evaluate the statistical significance of the model. The statistical significance of regression coefficients was evaluated using Student’s t-test. Three-dimensional surface plots were drawn to illustrate the main and interactive effects of the independent variables on laccase production. The optimum values of the selected variables were obtained, and a validation of the predicted model was also undertaken. All experiments were performed in triplicate, and the data were expressed as the mean values of experiments. 2.8.

2.4. Laccase assay and protein estimation Laccase activity was measured using ABTS as the substrate at 30 ◦ C [35]. The assay mixture contained 50 mM sodium acetate buffer (pH 5.6), 1 mM ABTS and the laccase source. One unit of activity was defined as the amount of enzyme that oxidized 1 μmol of ABTS per minute. The absorbance increase of the assay mixture was monitored at 420 nm (ε420 = 36.0 mM−1 cm−1 ) in a UV-visible spectrophotometer (Perkin-Elmer Lambda 25, Germany). The

Optimization of laccase production by RSM

Enzyme extraction and partial purification

Extracellular enzyme extraction and purification were performed according to the protocol described previously [24]. The enzyme from the SSF was extracted by soaking the culture overnight with 100 mM sodium acetate buffer, pH 5.6, at 4 ◦ C. The culture supernatant was filtered and centrifuged at 12, 000 × g for 15 min to remove the fine particles. The supernatant was concentrated by a Millipore Amicon ultrafiltration stirred cell (Millipore Corporation, USA) through filter of 10 kDa, until a 20-fold concentration was

Environmental Technology Table 1. Full factorial CCD matrix and observed laccase production.


S. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Malt Banana extract (g) peel (g) A

B

0.4 [−1] 0.8 [+1] 0.4 [−1] 0.8 [+1] 0.4 [−1] 0.8 [+1] 0.4 [−1] 0.8 [+1] 0.4 [−1] 0.8 [+1] 0.4 [−1] 0.8 [+1] 0.4 [−1] 0.8 [+1] 0.4 [−1] 0.8 [+1] 0.2 [−1] 1.0 [+2] 0.6 [0] 0.6 [0] 0.6 [0] 0.6 [0] 0.6 [0] 0.6 [0] 0.6 [0] 0.6 [0] 0.6 [0] 0.6 [0] 0.6 [0] 0.6 [0]

5.0 [−1] 5.0 [−1] 15 [+1] 15 [+1] 5.0 [−1] 5.0 [−1] 15 [+1] 15 [+1] 5.0 [−1] 5.0 [−1] 15 [+1] 15 [+1] 5.0 [−1] 5.0 [−1] 15 [+1] 15 [+1] 10 [0] 10 [0] 2.0 [−2] 18 [+2] 10 [0] 10 [0] 10 [0] 10 [0] 10 [0] 10 [0] 10 [0] 10 [0] 10 [0] 10 [0]

Wheat bran (g)

CuSO4 (mM)

C

D

5.0 [−1] 40 [−1] 5.0 [−1] 40 [−1] 5.0 [−1] 40 [−1] 5.0 [−1] 40 [−1] 15 [+1] 40 [−1] 15 [+1] 40 [−1] 15 [+1] 40 [−1] 15 [+1] 40 [−1] 5.0 [−1] 80 [+1] 5.0 [−1] 80 [+1] 5.0 [−1] 80 [+1] 5.0 [−1] 80 [+1] 15 [+1] 80 [+1] 15 [+1] 80 [+1] 15 [+1] 80 [+1] 15 [+1] 80 [+1] 10 [0] 60 [0] 10 [0] 60 [0] 10 [0] 60 [0] 10 [0] 60 [0] 2.0 [−2] 60 [0] 18 [+2] 60 [0] 10 [0] 20 [−2] 10 [0] 100 [+2] 10 [0] 60 [0] 10 [0] 60 [0] 10 [0] 60 [0] 10 [0] 60 [0] 10 [0] 60 [0] 10 [0] 60 [0]

Laccase productiona (U g−1 ) 0.73 ± 0.04 0.62 ± 0.06 0.66 ± 0.09 0.69 ± 0.04 0.72 ± 0.05 0.59 ± 0.02 0.47 ± 0.02 0.37 ± 0.01 1.36 ± 0.10 5.84 ± 0.36 2.39 ± 0.18 7.18 ± 0.43 2.10 ± 0.05 5.62 ± 0.11 2.81 ± 0.21 6.34 ± 0.24 0.21 ± 0.08 5.29 ± 0.36 0.85 ± 0.01 4.14 ± 0.18 6.46 ± 0.11 5.41 ± 0.30 0.16 ± 0.02 6.81 ± 0.03 7.34 ± 0.27 7.35 ± 0.31 7.29 ± 0.46 7.30 ± 0.18 7.33 ± 0.23 7.28 ± 0.30

Note: Crab shells were used as an inert material for making up the total weight (35 g). a Laccase production activities are means (n = 3) with standard deviations.

achieved. The enzyme was precipitated with ammonium sulphate to a final concentration of 80% (w/v). After standing in the ammonium sulphate solution for 5 h at 4 ◦ C, the precipitate was collected by centrifugation at 21, 000 × g for 15 min, resuspended with 100 mM sodium acetate buffer (pH 5.6) and dialyzed against the same buffer for 12 h. The resulting solution was used as the enzyme source.

2.9. Electrophoresis analysis Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to Laemmli [38] using a 4% stacking gel and a 12% separating gel. Protein bands were stained with Coomassie brilliant blue R-250, and the approximate molecular weight of the purified laccase was determined along with the standard protein markers (Fermentas, Canada).

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2.10. MG dye decolorization experiment Decolorization efficiency of P. florida laccase was evaluated against MG dye. The decolorization reaction was conducted in citrate–phosphate buffer (50 mM, pH 4.0) with dye (50 ppm) and laccase (2.5 U mL−1 ) at 40 ◦ C. After the incubation, the control and the laccase-treated reaction mixtures were filtered through 0.45 μm hydrophilic PTFE filters (Millipore) and subjected to UV-visible spectrophotometry, HPLC and LC-ESI-MS analyses. The residual dye concentration (%) in the reaction mixture was measured in a UV-visible spectrophotometer (Perkin–Elmer Lambda 25, Germany). Dye decolorization was calculated by means of the following formula: D(%) =

(Aini − Aobs ) × 100 Aini

(1)

where D is the decolorization of dye (%), Aini is the initial area and Aobs is the final area. The HPLC analysis was performed in an RPHPLC (Agilent1100 series, Agilent, Waldbronn, Germany) equipped with a Zorbax SB C-18 (Applied Biosystems) column and autosampler injection. The solvent system was 70% acetonitrile in 0.1% orthophosphirc acid (v/v) at a flow rate of 1 mL min−1 . Using the autosampler injection mode, 10 μL of sample was injected into the C-18 column. LC-ESI-MS (Agilent 1100 HPLC gradient system with API 2000, Applied Biosystems) was used to identify the fragmentation ions of MG in positive mode. Acetonitrile was used as the solvent system, with gradient elution at a constant temperature of 25 ◦ C.

2.11. Detoxification experiment The phytotoxicity of MG and laccase-treated MG dye on ryegrass (Lolium perenne) seeds was assessed by the method of Zucconi et al. [39] with minor modification. In brief, dye samples were used at 1:6 dilutions. Ten seeds were immersed in each test sample and incubated in the dark. After 120 h of incubation, the percentage of seed germination and the root length of seeds immersed in the MG and laccase-treated MG dye solutions and in deionized water were also determined. The values obtained for the deionized water were considered as the control. The germination index (GI) was calculated as follows: GI =

GP × La Lc

(2)

where GP is the number of germinated seeds expressed as a percentage of control values, La is the average value of root length in the dye solutions, and Lc is the average value of root length in the control. All experiments were performed in triplicate, and the data were expressed as the mean values of experiments.


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3. Results and discussion 3.1. Laccase production optimization It has been well documented that agricultural residues can be used to produce extracellular laccase enzyme because they may act as a source of carbon and nitrogen and also as an inducer for the laccase production [4,24,40,41]. Therefore, in the present study, agricultural residues were used to produce the laccase from P. florida under the SSF process. For the optimization study, primarily significant variables for the laccase production were screened, and highly positive influencing factors such as malt extract, banana peel, wheat bran and CuSO4 were selected for the CCD-RSM to evaluate the interactive effect and optimum level of the variables for the maximum laccase production. Banana peel was selected as a substrate for laccase production by P. florida as it has a high content of carbohydrates and minerals. It can be easily metabolized by microorganisms, and it has the physical integrity to serve as a supporting material. Also, its ascorbic acid content exerts an inhibitory effect against bacteria [42]. Furthermore, the banana-processing industry generates a huge amount of solid wastes, which are dumped in landfills, rivers, oceans and unregulated dumping grounds. Therefore, their reutilization would help to diminish the pollution problems caused by their disposal. Wheat bran is commonly being used for the cultivation of microorganisms in SSF processes [43]. It is also the best substrate for laccase production by white-rot fungi [44] because of its lignin and aromatic compounds [45–47]. Malt extract involved in the effective stimulation of the growth of fungal mycelia, which contain the aromatic amino acids tryptophan and tyrosine, might be responsible for better laccase enzyme yields [48]. Copper sulphate has been selected in this study because laccase mRNA levels in several white-rot species were significantly increased in media containing high concentrations of cupric ions. Multiple putative cis-acting elements, termed metal responsive elements (MREs), were identified in the promoter region of several laccase genes that are transcriptionally activated by copper [49–51]. RSM is widely used to study the effect of several variables and to seek the optimum conditions for a multivariable system [37]. A summary of the variables and their variation levels are given in Table 1. SSF was carried out according to the CCD design matrix for 120 h, and laccase production activity was assayed from extracellular enzymes obtained from the SSF. The results were analysed using Design Expert 7.0 statistical software and the response surface was generated. The ANOVA was used for the determination of the significant parameters. The ANOVA of the quadratic regression model demonstrates that it was a highly significant model, as was evident from the Fisher’s F-test with a very low probability value (F-value = 61.43) (Table 2). Values of ‘Prob > F’ less than 0 indicate that the model term was

Table 2. Analysis of variance for the response surface quadratic model. Source

Sum of Degree of Mean squares freedom square F-value P-value

Model 244.48 A 28.54 B 3.50 C 0.21 D 73.82 A2 40.69 B2 56.91 C2 6.24 D2 29.17 0.015 A∗ B A∗ C 0.35 A∗ D 17.28 B∗ C 0.12 B∗ D 1.14 C∗ D 0.026 Residual 4.26 Lack of Fit 4.26 Cor Total 248.74

14 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 10 29

17.46 28.54 3.50 0.21 73.82 40.69 56.91 6.24 29.17 0.015 0.35 17.28 0.12 1.14 0.03 0.28 0.43

61.43 100.39 12.30 0.76 259.68 143.13 200.20 21.96 102.61 0.053 1.24 60.81 0.44 4.01 0.09