(1 of 12) 1600045 Anand Barapatre Keshaw Ram Aadil Harit Jha Department of Biotechnology, Guru Ghasidas Vishwavidyalaya (A Central University), Bilaspur, Chhattisgarh, India Research Article Biodegradation of Malachite Green by the Ligninolytic Fungus Aspergillus flavus The azo class of synthetic dyes represents one of the most industrially used dyes as well as a major class of environmental contaminants, which possess one or more azo bonds ( N¼N ) along with aromatic rings and sulfonic groups. Due to its recalcitrant nature and toxicity for animals and human beings, the elimination of these dyes from the environment is essential. The present study focuses on the biodegradation of such azo dye, malachite green (MG), through a potent ligninolytic fungus, Aspergillus flavus (F10). A. flavus (F10) completely decolorized MG (150 mg L 1) within 6–8 days in optimized Kirk’s basal medium under static aerobic conditions at pH 5.8. Sucrose and sodium nitrate were efficient carbon and nitrogen sources, respectively. The products obtained after degradation were examined using UV-vis spectrophotometry, Fourier transform IR spectroscopy, and liquid chromatography-mass spectroscopy. The metabolic intermediate products were identified as N-demethylated and N-oxidized metabolites, including primary and secondary arylamines, which confirms the involvement of laccase and manganese peroxidase in decolorization and degradation of MG. The end products of MG degradation were nontoxic. A. flavus (F10), immobilized by entrapment on natural and synthetic polymeric matrices was found to be a more efficient degrader of MG as compared to free cells. Keywords: Azo dyes; Degradation pathway; Immobilization; Metabolism; Wastewater treatment Received: January 19, 2016; revised: August 4, 2016; accepted: January 16, 2017 DOI: 10.1002/clen.201600045 supporting information may be found in the online version of this article at the : Additional publisher’s web-site. 1 Introduction At present, around 100 000 different commercial dyes are available on the industrial scale and more than 7  105 metric tons of dyestuffs are produced annually worldwide. It is estimated that 10–50% of these reactive dyes used in textile processing are released as a wastewater discharge [1]. Azo dyes represent a major group of dyes utilized at the industrial level. The induced or self-reductive cleavage of the azo bond is responsible in the production of toxic amines, which create serious environmental concern because of their color, bio-recalcitrant nature and potential toxicity toward animals and human beings [2, 3]. Malachite green (MG), the oldest man-made azo dye, is structurally related to triphenylmethane and is extensively used as ecto-parasiticide, fungicide, food coloring agent, food additive, medical disinfectant, and industrial dye. Correspondence: Dr. Herit Jha, Department of Biotechnology, Guru Ghasidas Vishwavidyalaya (A Central University), Bilaspur, Chhattisgarh 495009, India E-mail: harit74@yahoo.co.in Abbreviations: BPD, biphenyl derivative; 2,6-DMP, 2,6-dimethoxy phenol; FTIR, fourier transform IR spectroscopy; KBNM, Kirk’s basal nutrient medium; LC-MS, liquid chromatography-mass spectroscopy; LMG, leucomalachite green; MG, malachite green; MGC, malachite green carbinol; MnP, manganese peroxidase. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim However, it is also reported for its multi-organ toxicity, DNAdamage, carcinogenic, and mutagenic behavior toward mammalian cells, aquatic life, and many other organisms [4–6]. Several physical, chemical, and physico-chemical methods have been applied with limited success to remove MG from wastewater, including adsorption, chemical precipitation, photodegradation, osmosis, and membrane filtration [7]. These methods are not only costly and inefficient, but also produce large amounts of sludge. Therefore, biological degradation of MG is receiving considerable attention as an eco-friendly, efficient and low-cost alternative method [6, 8]. In the last few years, a wide range of microbes, including bacteria, fungi, algae, and yeast have been reported for decolorization, transformation and complete mineralization of azo dyes [7, 9–12]. Sometimes, the degradation products of the dye such as aromatic amine and phenolics have higher toxicity and lower biodegradability as compared to the dye itself. In such cases, fungi show strong adaptability, efficient removal, and mineralization of these aromatic compounds due to their enzymatic machinery [12]. Lignin-degrading enzymes such as peroxidase and laccase have excellent ability to cleave the aromatic and fused aromatic phenolic structures through their oxido-reductive enzymatic system. Thus, these ligninolytic fungi are suitable for degradation and mineralization of a wide range of synthetic aromatic dyes [3, 13]. Several researchers claim the involvement of ligninolytic enzymes: laccase, manganese peroxidase www.clean-journal.com Clean – Soil, Air, Water 2017, 45 (4) 1600045 1600045 (2 of 12) A. Barapatre et al. and lignin peroxidase in azo dye degradation through hydroxylation and ring cleavage [14–17]. Immobilization of the microorganisms improves their activity. Attachment is one of the most common methods to immobilize microorganisms, whereby microorganisms adhere to the surface of the material by self-adhesion or chemical bonding [13]. The materials commonly used in the attachment procedure are synthetic foams like polyurethane foam [13], nylon sponge [13], natural material like Luffa cylindrica sponge [18], sand particles [3], and stainless steel sponge [13]. The immobilized material provides an advantage to the fungal cells and offers the organism a natural habitat as well as additional nutrients needed to stimulate the production of ligninolytic enzymes involved in dye degradation. Different dye biodegradation studies revealed that the immobilized fungi have a number of advantages over free fungal cells, such as easy separation of cells from liquid medium, protection from shear damage and reduction in protease activity [19]. Furthermore, cell immobilization lowers the apparent broth viscosity and makes the rheological features more favorable for oxygen and mass transfer [20]. In the present study, a ligninolytic fungus Aspergillus flavus (strain F10) was used for the degradation of the azo dye MG. The medium was optimized for efficient degradation of MG by using free and immobilized fungus and MG degradation was analyzed by various spectroscopic techniques. The degradation pathway based on Liquid chromatography-mass spectroscopy (LC-MS) detection of intermediates is proposed. Phytotoxicity and microbial toxicity of MG degradation products was also assessed. 2 Material and methods 2.1 Chemical and reagents Malachite green, guaiacol, 2,6-dimethoxy phenol (2,6-DMP) and ethyl acetate were purchased from Hi-Media (Mumbai, India). All other chemicals and reagents used were of high-purity analytical grade and purchased from Merck (India). Ultra-pure Millipore water (ELIX, Merck Millipore, India) was used for enzyme assay. media. MG dye was added to each flask at the concentration of 150 mg L 1 in sterile condition. The inoculated flasks were placed under static and shaking conditions for 8–10 days at 37°C [15, 23]. Samples withdrawn after every 48 h were used to determine the percent of dye decolorization. The percentage decolorization of MG was determined by monitoring the change in absorbance at A618, that is, lmax of MG using UV-vis spectrophotometry. The decolorization percentage was calculated as follows: Decolorizationð%Þ ¼  AI AF AI   100 ð1Þ where AI is the initial absorbance and AF is the final absorbance. The experiments were performed in triplicate. 2.4 Optimization of media components and conditions for degradation of malachite green One-step optimization method for process optimization was adopted, in which a single factor was varied for each experiment, while keeping the previously optimized variable constant. For the optimization of efficient MG degradation, the medium components (carbon, Section 2.4.1, and nitrogen sources, Section 2.4.2) and medium pH, Section 2.4.3, were optimized. The KBNM medium with 150 mg L 1 MG was used for the optimization process. Assay condition and preparation were according to Section 2.3. 2.4.1 Effect of different carbon sources To enhance MG decolorization and degradation by A. flavus (F10) the carbon source of the medium (KBNM) was optimized. A total of six different carbon sources as glucose, sucrose, maltose, mannitol, lactose, and starch were used at the concentration of 1% w/v. The inoculated medium was incubated under shaking conditions (120 rpm) for 8 days at 37°C and pH 5.8 (after optimization of initial decolorization step). In subsequent experiments, the effect of concentrations of the optimum carbon source (sucrose) was monitored. The percentage decolorization of MG was determined intermittently. 2.4.2 Effect of nitrogen sources 2.2 Organism, growth medium, and dye stock preparation A. flavus strain F10 (KC911631.1), a ligninolytic fungus, was used for the degradation and decolorization of MG. The isolation and identification method is described elsewhere [21]. A. flavus (F10) was maintained in potato dextrose agar at 4°C. Kirk’s Basal Nutrient Medium (KBNM) was used for the degradation study [22]. KBNM was composed of w/v 1% D-glucose, 0.3% NaNO3, 0.05% MgSO4, 0.02% KH2PO4, 0.002% CaCl2
2H2O, and showed a pH of 5.8. The MG stock was prepared at a concentration of 1 mg mL 1 in sterile distilled water, filter sterilized and stored for further use at 4°C. In the MG degradation study, the initial concentration of dye was kept at 150 mg L 1. A total of seven different nitrogen sources like ammonium sulfate, ammonium nitrate, sodium nitrate, beef extract, peptone, malt extract, and yeast extract (0.02% w/v) were used in the presence of 1% w/v sucrose (optimum carbon source), and the flasks were kept in shaking condition for 8 days under optimum conditions of pH and temperature. The percent decolorization of MG was determined intermittently. 2.4.3 Effect of different initial pH To check the effect of initial pH on the decolorization and degradation efficiency, the pH of the medium was varied (3.8, 4.8, 5.8, 6.8, and 7.8). The percent decolorization of MG was determined intermittently. The experiment was performed in triplicate. 2.3 Decolorization protocol A 100 mL KBNM was prepared in 250 mL Erlenmeyer flasks and the pH was adjusted to 5.8 with 0.1 N HCl or 0.1 N NaOH. After sterilization by autoclaving at 121°C, 20 min at 15 psi pressure, three disks (10 mm) of 7 days old culture of A. flavus were inoculated in the © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 2.5 Dye adsorption study To detect any adsorption of dye on fungal mycelia, sterilized distilled water was used without any addition of KBSM or additional nutrients. The dye (0.001–0.02%, w/v) was added to sterilized distilled water, pH www.clean-journal.com Clean – Soil, Air, Water 2017, 45 (4) 1600045 General 5.8. The pH was adjusted with 0.1 N HCl. Three culture discs (10 mm diameter, 7 days old culture) were inoculated and incubated under shaking conditions (120 rpm) at 37°C. Due to the absence of any nutrient, the growth of A. flavus (F10) was negligible and dye removal observed in the absence of mycelial growth and enzyme activity was assumed to be due to the absorption on fungal mycelia. (3 of 12) 1600045 2.8.2 Manganese peroxidase 2.6 Determination of MG degradation efficiency of flavus (F10) The manganese peroxidase (MnP) activity was determined spectrophotometrically at 469 nm, based on the oxidation of 2,6-DMP (e469 ¼ 49 600 M 1 cm 1) by the MnP system which forms a quinone dimer. Assay mixtures (3 mL) were composed of 100 mM sodium tartrate buffer (pH 4.5), 1 mM 2,6-DMP, 1 mM MnSO4, 0.1 mM H2O2 and 100 mL of the sample. One unit (U) was defined as the amount of enzyme that releases 1 mmole of the dimeric product of 2,6-DMP oxidation per min measured at 469 nm [26]. To investigate the degradation efficiency and tolerance of the fungus against MG, degradation was carried out with varying concentrations (100–1000 mg L 1) of dye. The optimized medium (KBNM; carbon source—sucrose, nitrogen source—sodium nitrate and pH 5.8) was used to determine MG degradation efficiency. The fungal inoculated flasks were incubated by shaking (120 rpm) at 37°C for 8 days. 2.9 Analysis of MG degradation by UV-vis spectrophotometry, Fourier transform IR spectroscopy, and liquid chromatography-mass spectroscopy (LC-MS) 2.7 Decolorization of malachite green dye by immobilized flavus (F10) To assess the degradation efficiency under immobilized conditions, inert support material (polyurethane foam, vegetable foam, stainless steel sponge, clay, and ash brick) was used for the immobilization of A. flavus (F10). A 250 mL flask containing about 50 mL of optimized medium (KBNM; carbon source—sucrose, nitrogen source—sodium nitrate and pH 5.8) and four to five pieces of the carrier material (100  150 mm) were inoculated with A. flavus (F10) (1% spore inoculum, concentration of 1  109 spores) [13, 18, 24]. The flasks were incubated at 37°C at stationary condition with occasional shaking for 7–10 days. About three pieces of each immobilized material were used in the MG degradation study. 100 mL of optimized KBNM containing 150 mg L 1 MG was inoculated with three pieces each of immobilized A. flavus (F10) and incubated at 37°C for 8 days under shaking conditions (120 rpm). The inert materials and their preparation are as follows: Polyurethane foam cubes (Scotch Brite, India, density, 20 kg m 3) were pretreated by washing once in methanol and twice in distilled water and then autoclaved at 121°C and 15 psi for 20 min. Thereafter, the cubes were dried at room temperature overnight [18]. Vegetable foam cubes (Scotch Brite, India) were pretreated by boiling for 10 min and autoclaved at 121°C and 15 psi for 20 min. Thereafter, the cubes were dried overnight at room temperature [18]. Pieces of stainless steel sponge (Scotch Brite) were pretreated as described above for polyurethane foam [13]. Clay and ash bricks pieces were obtained from a local supplier at Bilaspur, CG and were autoclaved at 121°C and 15 psi for 20 min and dried at 60°C in the oven prior to use. 2.8 Enzyme assays UV-vis spectrophotometry was used to assess the degradation of MG. A fixed volume (3 mL) of the medium was periodically withdrawn from the incubation flask, and the UV-vis spectrum recorded from 200 to 700 nm. The MG degradation intermediates were recovered as ethyl acetate fractions by liquid-liquid extraction for further analysis. The procedure adopted for the recovery of MG degradation intermediates was according to Parshetti et al. [27]. In brief, the decolorized medium (days 4 and 8) was withdrawn, centrifuged at 10 000 rpm for 20 min and the supernatant used to extract degraded metabolites with equal volumes of ethyl acetate. After proper shaking (about 10 min), the ethyl acetate fraction was separated by a separatory funnel. The ethyl acetate fractions were dried under vacuum using a vacuum evaporator in the presence of anhydrous Na2SO4. The crystals obtained were dissolved in a small volume of methanol and used for further analysis [27]. Fourier transform IR spectroscopy (FTIR) analysis of biodegraded MG (products of days 4 and 8) was carried out using an IR affinity-1 spectrometer (Shimadzu, Japan) with MG dye as a control. The FTIR analysis was performed in the mid IR region of 400–4000 cm 1 with 64 scans. The samples were mixed with the spectroscopic grade KBr in the ratio of 1:100 (w/w). The LC-MS analysis was performed according to Du et al. [7]. The degradation products were analyzed using HPLC (Agilent 1200 series, equipped with a reversed-phase C-18 analytical column of 100 mm length  3 mm and 2.6 mm particle size, ACCUCORE-C18) coupled with MS (UPLC-TQD mass-spectrometer, Waters). The analysis was carried out using electro-spray ionization in positive ion mode. The flow rate was kept at 1 mL min 1. The compounds were resolved using solvent A: acetonitrile (60%, v/v) and solvent B: 20 mM ammonium acetate (40%, v/ v). The operating conditions of the mass spectrometer were as follows: sheath gas flow rate of 55 arb, awx/sweep gas flow rate of 5 arb, spray voltage of 4.5 kV, capillary temperature of 300°C, capillary voltage of 46 V, and tube lens offset of 10 V. 2.8.1 Laccase Laccase activity was measured as described by Arora et al. [25] with slight modification. The 3 mL reaction mixture containing guaiacol (2 mM, e450 ¼ 12 100 M 1 cm 1) in acetate buffer (10 mM, pH 5) and 0.5 mL of enzyme extract was incubated at 25°C for 2 h. The absorbance change at 450 nm was measured using UV-vis spectrophotometry (UV-1800, Shimadzu, Japan). Laccase activity was expressed in international unit mL 1 (IU mL 1). One activity unit (U) was defined as the amount of enzyme required to oxidize 1 mmole guaiacol per minute at 450 nm. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 2.10 Phytotoxicity and microbial toxicity study of MG degradation products The phytotoxicity study was carried out to assess the toxicity of MG degradation products using the plant-based bioassay. The ethyl acetate extracted products (days 4 and 8) were dissolved in water to obtain a final concentration of 500 ppm. The study was carried out at room temperature. 10 mL MG (500 ppm) and its degradation products (500 ppm) were added per day to each of ten seeds of www.clean-journal.com Clean – Soil, Air, Water 2017, 45 (4) 1600045 1600045 (4 of 12) A. Barapatre et al. Vigna radiata. Water was used as control [27]. All results were recorded in terms of percent germination, length of the plumule (shoot), and radicle (root) was recorded after seven days and subsequently germination index (GI) was calculated as follows: GI ¼   GL  100 GC  LC ð2Þ where G and L are germination and radicle growth of the seeds germinated in the test solution, and GC and LC are the germination and radicle growth of seeds in the control (distilled water), respectively. According to Zucconi et al. [28], GI values <50% are considered to be indicative of high toxicity, values between 50 and 80% represent low toxicity, and GI values >80% are considered to represent non-phytotoxicity. The toxicity of the MG degradation products was assessed against microbes. The toxicity was tested on Staphylococcus aureus and Pseudomonas aeruginosa by the well diffusion method. 100 mL of MG degradation products (days 4 and 8) and MG at 500 ppm were used. Water was used as a blank. 2.11 Statistical analysis An analysis of variance (ANOVA) was carried out to test for the differences between the control and test samples in the statistical program Minitab 17.0. Significance of difference was defined at the 0.01 (p < 0.001;  ), 0.1 (p < 0.01;  ) and 5% level (p < 0.05;  ). 3 Results and discussion 3.1 Optimization of different parameters for MG degradation For MG degradation, KBNM containing 150 mg L 1 of dye was used for the optimization of nutritional and pH conditions during degradation. It was found that MG degradation reached 70% after 8 days of incubation under shaking condition (120 rpm). Without shaking, MG degradation was 30% on day 8. Under shaking conditions, the high decolorization observed might be due to better oxygen transfer and nutrient distribution as compared to static condition [29]. All the further optimization experiments were performed under shaking condition (120 rpm) at 37°C. demonstrate that some fungi, including A. flavus, prefer monosaccharaides like glucose and fructose as the carbon supplement during degradation of triphenylmethane dye [23, 29, 32]. Laccase and manganese peroxidase activity was periodically assayed during carbon source optimization (Fig. 1b and c). Laccase activity was observed in the initial stage of degradation, where manganese peroxidase was active during the later stage of degradation. Liu et al. [33] had also studied MG degradation by Trametes versicolor, T. hispida, Fome lignosus, Coriolus hirsutus and found that the laccase activity was high at the initial stage of degradation, while MnP was active during the later stage. 3.1.2 Effect of nitrogen sources A total of seven different nitrogen sources were used to optimize the nitrogen source for MG degradation (Fig. 1d). High rate of degradation was observed for five nitrogen sources—ammonium nitrate, ammonium sulfate, sodium nitrate, beef extract, and yeast extract after eight days of incubation. The least decolorization (22.5%) was observed in the medium containing malt extract. Sodium nitrate was found to be the most preferred source of nitrogen and in sodium nitrate containing medium, the decolorization of MG reached 79.3% on day 4 and up to 95% after 6 days of incubation. The laccase and MnP activity was also assessed during the degradation process, and it was observed that the activity trend of both enzymes was almost same as the activity found during carbon source optimization (Fig. 1e and f). The laccase and MnP activity was highly influenced by different nitrogen sources. In yeast extract and sodium nitrate containing medium, laccase was active throughout the 8 days of incubation, while with other nitrogen sources the activity was high in the middle part of incubation time (i.e., on day 4). A significant level of MnP activity was found in the medium containing sodium nitrate, peptone and beef extract on days 6 and 8. Levin et al. [34] had found that different nitrogen sources significantly affect the production (onset time and amount) of ligninolytic enzymes, which ultimately affects the degradation of azo dye. Although A. flavus (F10) mediated degradation of MG was found to be similar in sodium nitrate and yeast extract containing medium, sodium nitrate, being inorganic and cheap, was chosen as an optimized nitrogen source. Kumar et al. [29, 32] reported sodium nitrate as an efficient nitrogen supplement for triphenylmethane dye (methylene blue and brilliant blue) degradation by Aspergillus sp. while Yang et al. [30] reported sodium nitrate for MG degradation by Penicillium sp. 3.1.1 Effect of different carbon sources In an attempt to enhance degradation efficiency, a study was carried out to identify the most preferred carbon source. The maximum 99% decolorization was observed with sucrose (1%, w/v), whereas 98% decolorization was observed in a medium containing glucose, lactose, and mannitol, while 87, 20, and 4.6% decolorization was observed with maltose, starch, and in control, respectively at eight days of incubation (Fig. 1a). Sucrose was the most preferable source of carbon, whereas starch was the least preferable source. It was found that the decolorization of MG reached 85% at day 4 and 98% at day 6 in a medium containing sucrose as the carbon source. The maximum decolorization of MG by A. flavus (F10) was achieved in a medium containing sucrose and glucose. Sucrose was selected as the optimal carbon source. Yang et al. [30] and Jin et al. [31] also had found sucrose to be an efficient carbon source during MG degradation for two other Ascomycetes, Penicillium, and A. fumigatus, respectively. Previous studies © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 3.1.3 Effect of initial pH The effect of initial pH (3.8–8.8) of degradation medium on the biodegradation efficiency of MG was analyzed (Fig. 2a), and it was found that the MG decolorization efficiency of A. flavus (F10) differs with change in the initial pH of the medium. Maximum decolorization of 98.3% was observed at pH 5.8. The decolorization efficiency decreased at higher pH, that is, 6.8–8.8, whereas at lower pH, that is, 3.8 and 4.8 no decolorization was observed. It was found that in fungus mediated textile dye removal system, the optimum growth, decolorization and biodegradation varied for the pH range of 4–6, depending on the fungal species, medium composition, and types of dye [15]. Fungi show better decolorization and biodegradation activities at acidic or neutral pH [1, 35]. Yang et al. [30] reported the maximum decolorization of MG by Penicillium sp. at pH  6 (5.8). The activities of laccase and manganese www.clean-journal.com Clean – Soil, Air, Water 2017, 45 (4) 1600045 General (5 of 12) 1600045 Figure 1. Percentage decolorization of MG, manganese peroxidase, and laccase activity during MG degradation media optimization. (a–c) MG decolorization under the influence of different carbon sources, laccase activity, and manganese peroxidase activity, respectively. (d–f) MG decolorization under the influence of different nitrogen sources, laccase activity, and manganese peroxidase activity, respectively. peroxidase (Fig. 2b and c) were found to be similar to the carbon optimization step, taht is, laccase activity was present at the initial stage of MG degradation while manganese peroxidase was active during the second half period of incubation. 3.2 Dye adsorption Dye absorption was also tested along with degradation to omit any possibility of reduction in color due to absorption of dye. It was found that, only 2–3% of decolorization occurs despite the absence of enzymatic activity, probably due to absorption. This confirms that the decolorization was mainly due to the fungal metabolic mediated degradation of MG and not due to the absorption. Similar results were reported by Lalitha et al. [23] for A. flavus. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 3.3 MG degradation efficiency of A. flavus (F10) The degradation efficiency of MG was determined at increasing concentrations of MG dye (100, 250, 500, 750, and 1000 mg L 1). It was found that the degradation efficiency decreased with increasing concentration of dye (Fig. 3). The complete 100% decolorization of MG was observed at 100, 250, and 500 mg L 1 within 8 days. The rate of decolorization decreased beyond 500 mg L 1 dye concentration, indicating a reduction in decolorization efficiency with an increase in dye concentration. Only 42 and 10% decolorization was observed for 750 and 1000 mg L 1 dye concentration, respectively, after 8 days of incubation. During MG degradation, a rapid increase in biomass concentration was observed in the first 2 days of incubation followed by a slow increase and maximum growth up to 3 days, beyond which the concentration started decreasing. The concentration of fungal www.clean-journal.com Clean – Soil, Air, Water 2017, 45 (4) 1600045 1600045 (6 of 12) A. Barapatre et al. Figure 2. (a) MG degradation, (b) laccase activity, and (c) manganese peroxidase activity, under the influence of initial pH of decolorization media. biomass decreased with the increase in the initial concentration of dye. It was assumed that after three days, A. flavus (F10) consumed most of the supplemented carbon, but this consumption decreased with an increase in the initial concentration of dye, thus indicating that the higher concentration of dye inhibits the growth of fungal biomass. Papinutti et al. [36] found that during MG degradation by Phanerochaete chrysosporium and F. sclerodermeus, MG stops the growth of both fungi at 64 mM (¼ 23.35 mg L 1) and 128 mM (¼ 46.7 mg L 1), respectively, due to its toxicity. These results indicate the toxicity of MG at higher dye concentration. However, A. flavus (F10) may degrade high MG concentration if the incubation time is increased. 3.5 Analysis of MG degradation 3.4 Degradation of malachite green dye by immobilized fungal strain 3.5.1 UV-vis spectroscopy Five different inert materials viz. vegetable foam, polyurethane foam, stainless steel gauge, clay brick, and ash brick were used to immobilize Figure 3. Efficiency of A. flavus (F10) toward MG decolorization at different MG concentration (100–1000 mg L 1). © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim A. flavus (F10) by the attachment method to check the efficiency of MG degradation (Fig. 4a). Studies showed that, in contrast to MG decolorization, the efficiency of immobilized A. flavus (F10) was higher compared to free fungus. It was found that the fungus immobilized on polyurethane foam and clay brick decolorized MG up to 99 and 98.9% within 6 days of incubation (Fig. 4b). Stainless steel sponge also showed efficient decolorization, which was 83% on day 4 and 98.7% on day 6. At the end of 8 days of incubation, about 97–99% of decolorization was achieved by all five immobilized fungi. An overall study showed that decolorization efficiency of the A. flavus (F10) was improved significantly by immobilization. UV-vis spectroscopy is the preliminary technique to determine the degradation of MG, because in biodegradation, either the major absorbance peak disappears completely, or a new peak appears [11]. Figure 5 depicts the day-wise UV-vis spectral analysis of MG decolorization. The characteristic MG peak I at 618 nm (lmax for MG) decreased completely in 8 days when incubated with A. flavus (F10). The other two characteristic peaks of MG at 315 and 420 nm decreased during the days of treatment, which is the evidence of degradation [37]. In contrast, peak III at 254 nm initially decreased up to day 4, then it subsequently increased and gradually hypochromically shifted toward higher wavelength. The peak obtained at 254 nm corresponds to single-benzene vibrations, possibly produced by the degradation of triphenylmethane ring structure. Simultaneously, a significant small spectral absorption band with lmax at 370 nm emerged at the end stage of degradation which possibly represented the formation of a new metabolite. The emergence of the peak at 370 nm could be attributed to the vibration www.clean-journal.com Clean – Soil, Air, Water 2017, 45 (4) 1600045 General (7 of 12) 1600045 Figure 4. (a) Immobilization of A. flavus (F10) on different inert material, (b) decolorization efficiency of A. flavus (F10) immobilized on different inert materials. AB, ash brick; CB, clay brick; PF, polyurethane foam; SS, stainless steel sponge; VF, vegetable foam. Figure 5. UV-vis spectrum of MG decolorization by A. flavus (F10). © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim of a conjugated polycyclic aromatic structure. The peak at 220 nm is also attributed to the production of single-benzene derivatives, which is also the proof of the enzymatic destruction of a triphenylmethane moiety of MG. In A. flavus (F10) mediated MG degradation, the absorbance of the characteristic peak of MG (618 nm) decreased and new peaks were observed (at 220, 315, and 420 nm), suggesting degradation and decolorization [37]. These peaks correspond to mono, di-, and tri-benzene rings containing compounds. According to previous reports, it was observed that if the specific absorption peaks decreased proportionally with incubation time, the decolorization might be due to the biosorption phenomenon, whereas in biodegradation, the major absorbance peak disappears or an entirely new peak appears [11]. Therefore, in the present study, the decolorization of MG is attributed to degradation rather than to biosorption by A. flavus (F10). www.clean-journal.com Clean – Soil, Air, Water 2017, 45 (4) 1600045 1600045 (8 of 12) A. Barapatre et al. 3.5.2 Characterization of MG degraded products by FTIR Figure 6. FTIR spectra of MG and A. flavus (F10) mediated MG degradation product; (a) MG (control), (b) MG degradation product day 4, (c) MG degradation product day 8. A significant variation was observed in the FTIR spectra of MG, and its degradation products by A. flavus (F10) are shown in Fig. 6. A new peak arises in the FTIR spectra of biodegraded products (days 4 and 8) at 3784 and 3698 cm 1, representing OH stretching vibrations due to the formation of hydroxylated metabolites. In addition, sharp peaks at 2925 and 2855 cm 1 correspond to CH stretching by asymmetric CH2 groups, indicating the methylene-substituted metabolites obtained at increased frequency in degradation products [27]. The peaks at 1635, 1588, and 1380 cm 1, formed by NH or CN stretching vibrations in amine I, II, and III groups totally disappeared in the degradation products providing evidence of the MG degradation. The peak at 1585 cm 1, corresponding to C¼C stretching of the mono-substituted and para-disubstituted benzene rings, were prominent in control MG and day 4 MG degradation products, while it disappeared in the day 8 degradation products [38]. The FTIR analysis of MG degradation compounds confirms the structural and functional changes in MG during degradation. The FTIR analysis also confirms the formation of hydroxylated and methylene substituted benzene (mono-substituted and para-disubstituted) related metabolites during degradation. The intensity of signal peaks Figure 7. Total ion count chromatogram of the MG degradation products analyzed by LC-MS. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.clean-journal.com Clean – Soil, Air, Water 2017, 45 (4) 1600045 General (9 of 12) 1600045 Figure 8. Proposed pathway for degradation of MG adopted by A. flavus (F10), based on the identified degradation product through LC-MS analysis. of these products completely disappeared in the final degraded product, confirming the complete degradation of MG [9, 37]. 3.5.3 LC-MS analysis of MG degradation products LC-MS analysis is one of the most reliable techniques combining both sensitivity and selectivity to identify and quantify MG degradation © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim metabolites in complex matrices. LC-MS analysis of MG degradation products was performed after 4 days (Fig. 7a) and 8 days of incubation (Fig. 7b). The total ion chromatogram revealed four new peaks that persisted for 24 h, but with varying intensities. The retention time (Rt) of day 4 products peaks were 0.72, 0.9, 1.14, 1.53, 1.71, 10.73, and 13.01 min, and correspondingly, the protonated molecular ion peaks www.clean-journal.com Clean – Soil, Air, Water 2017, 45 (4) 1600045 1600045 (10 of 12) A. Barapatre et al. were at 367, 317, 363, 361, 413, 279, and 224 m/z, respectively. On the other hand, the retention times of day 8 products peaks were 0.73, 1.12, 1.51, 1.71, 2.91, 4.91, 8.86, 10.94, and 13.13 min and correspondingly, the protonated molecular ion peaks were at 203, 212, 355, 371, 226, 100, 88, 279, and 224 m/z, respectively. It was found that the degradation product obtained in the day 8 sample was different and with lower molecular mass than that from day 4 degradation products, which supports A. flavus (F10) decolorization and biodegradation ability of the MG. LC-MS analysis identified many degradation products, and based on these products the proposed MG degradation pathway is presented in Fig. 8 (see also Supporting Information Tab. S1). Bacterial and fungal mediated degradation of MG occur either through a direct step-wise demethylation and hydroxylation process or through an oxidative breakdown reaction followed by a stepwise demethylation process [7, 8, 38]. The presence of MG and leucomalachite green (LMG) in the medium was confirmed by LC-MS analysis. The A. flavus (F10) mediated degradation of MG is initiated by hydroxylation of LMG, consequently forming leucomalachite green dehydrate (MW 367), which is successively demethylated and dehydroxylated by the action of A. flavus laccase and form malachite green carbinol (MGC, MW 347). MGC is also formed by another route in which MG is demethylated by the action of laccase. This carbinol form of MG is further attacked by manganese peroxidase and laccase and two electrons are abstracted from the phenolic ring of the MGC to form corresponding carbonium ion. This carbonium ion is further hydrated by a nucleophilic attack of water, converting it into the biphenyl derivative (4-(dimethylamino) benzophenone) [39, 40]. These biphenyl derivatives undergo demethylation and benzene ring removal reaction to form dibenzyl methane (MW 167). In another degradation route, MG is demethylated thrice by the action of laccase and MnP and successively forms three metabolic products, desmethyl malachite green (MW 315), didesmethyl malachite green (MW 302), and tridesmethyl malachite green (MW 287), respectively, without any ring cleavage reaction [7, 30, 39]. Compounds with MW 106 and 88 were detected as final products, and assumed that they might be oxo-(phenyl) methylium and oxalic acid, respectively. The biodegradation pathway of MG by fungi in literature also supports the proposed pathway of degradation [8, 38–41]. Several researchers claim the direct involvement of ligninolytic enzymes (laccase and MnP) in azo dye degradation [6, 14–17, 41]. In this study, the proposed pathway of MG degradation also confirms the activity of laccase and MnP. 3.6 Phytotoxicity and microbial toxicity study of MG degradation products 3.6.1 Phytotoxicity Untreated dyeing effluents may cause serious environmental and health hazards, despite being disposed off in water bodies, assuming that this water is used for irrigation purpose, thereby affecting the growth of plants. Thus, it is important to assess the phytotoxicity of the dye before and after degradation. V. radiata was thus used as a model to assess the toxic effect of MG degradation products. The percentage germination, root length, shoot length and GI index are presented in Tab. 1. It was found that the control seeds had 100% germination (ten seeds) with a mean shoot length and root length of 20.06  3.77 and 8.37  2.19 cm, respectively. Seed germination was reduced to 66.67% when seeds were treated with MG (100 ppm). The shoot and root lengths were reduced by 27.5 and 73.4% in comparison to control. Whereas in the seeds treated with 100 ppm of days 4 and 8 degradation metabolites, 38.9 and 19.9% decrease in shoot length, and 57.7 and 25.4% decrease in root length was observed, in comparison to control. In the present study, it was found that the end products of MG degradation (day 8 product) were non-toxic to V. radiata, while the metabolites produced after day 4 of incubation caused toxicity. The toxicity of the day 4 metabolites was due to partially degraded aromatic products and MG. MG and partially degraded metabolites cause adverse effect on V. radiata, which was assessed in terms of germination index. The overall results demonstrated that the lignin degrading fungus, A. flavus (F10), has the capacity to completely mineralize MG. 3.6.2 Microbial toxicity The study of toxic effect of MG degradation products on microbial growth was performed against P. aeruginosa and S. aureus. Table 2 shows the growth inhibition zone of the extracted degradation metabolites. The microbial toxicity and phytotoxicity confirm the low toxicity of degradation metabolites. It is reported that some of the degradation products can be equal to or more toxic than MG [8, 27]. In microbial toxicity studies the end product did not cause any growth inhibition of tested microbes, confirming the formation of non-toxic end products. The discharge of treated dyeing effluents is mostly in water bodies, and this water may be used for agriculture purposes. Thus, it is a necessary concern to evaluate the toxicity of the MG degradation metabolites [11]. It was found that MG and day 4 degradation products create the zone of inhibition, while degradation metabolites of day 8 did not Table 1. Phytotoxicity of malachite green and A. flavus (F10) mediated MG degradation products Parameter Germination (%) Germination index (GI%) Shoot (cm) Root (cm) Root/shoot Control (water) MG (100 ppm) MG degraded product (day 4; 100 ppm) MG degraded product (day 8; 100 ppm) 100 – 20.06  3.77 8.37  2.19 1 : 2.4 66.67 16.44 14.56  1.29 2.06  0.76 1 : 7.03 77.78 24.93 11.54  1.69 2.69  0.41 1 : 4.31 100 60.48 16.07  1.95 5.02  1.57 1 : 3.2 Values represent mean  standard deviation, (n ¼ 10). Values within in a row are significantly different according to one way ANOVA.  p < 0.001.  p < 0.05. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.clean-journal.com Clean – Soil, Air, Water 2017, 45 (4) 1600045 General References Table 2. Microbial toxicity of MG and its degradation products Diameter of inhibition zone (cm) Pseudomonas aeruginosa Sample Water (control) MG MG degraded product (day 4) MG degraded product (day 8) Staphylococcus aureus 500 mg L 1 1000 mg L 1 500 mg L 1 1000 mg L 1 ND 1.8 1.2 ND 2.2 1.6 ND 0.8 ND ND 1.0 ND ND ND ND ND ND, not detected. create any inhibition zone. These results also confirm that the products/metabolites formed at the end of MG degradation by A. flavus (F10) were nontoxic. 4 Concluding remarks This work evaluated A. flavus strain F10 ability to decolorize the common industrially used azo dye MG. The results of liquid-phase batch decolorization experiments showed that extracellular enzymes of A. flavus could efficiently decolorize and degrade MG. Decolorization up to 98–99% was achieved for 150 mg L 1 MG after 6 days under optimum conditions. The MG decolorization mechanisms adopted by A. flavus involve enzymatic reactions like ring cleavage, demethylation, and hydroxylation. UV-vis, FTIR, and LC-MS analysis of MG degradation products further confirm the proposed degradation pathway. The toxicity of the A. flavus mediated MG degradation products was found to be significantly lower toward the plants and microbes showing its high detoxification capability. The use of immobilized growing cells for decolorization and degradation of dye seems to be more promising than the use of free cells, due to reusability and stability. It is reported for the first time that A. flavus immobilized on different materials degraded MG. This study also suggests that A. flavus mediated dye degradation can be applied as an alternate environment friendly decolorization/degradation system for high concentrations of commercial azo dyes, and can be integrated with existing wastewater treatment systems. Acknowledgement The authors gratefully acknowledge the funding by University Grant Commission (UGC), New Delhi, India (vide nos. F.41-543/2012 (SR)) and DBT BUILDER (BT/PR 7020/INF/22/172/2012). The authors also acknowledge support of SAIF-CDRI (Lucknow, India) for LC-MS analysis, Department of Pharmacy, Guru Ghasidas Vishwavidyalaya, Bilaspur, Chhatisgarh, India, for FTIR analysis. The authors are thankful to the Dr. Anurag Chauhan, Assistant Professor, Department of English, Guru Ghasidas Vishwavidyalaya, Bilaspur, India for editing the manuscript. The authors have declared no conflict of interest. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim (11 of 12) 1600045 [1] R. Khan, P. Bhawana, M. H. Fulekar, Microbial Decolorization and Degradation of Synthetic Dyes: A Review, Rev. Environ. Sci. Biotechnol. 2013, 12, 75–97. [2] Y. Fu, T. Viraraghavan, Fungal Decolorization of Dye Wastewater: A Review, Bioresour. Technol. 2001, 79, 251–262. [3] S. Andleeb, N. Atiq, G. D. Robson, S. Ahmed, An Investigation of Anthraquinone Dye Biodegradation by Immobilized Aspergillus flavus in Fluidized Bed Bioreactor, Environ. 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