Biodegradation of malachite green by Micrococcus sp ...

International Biodeterioration & Biodegradation 78 (2013) 108e116

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Biodegradation of malachite green by Micrococcus sp. strain BD15: Biodegradation pathway and enzyme analysis Lin-Na Du a, b,1, Ming Zhao a,1, Gang Li b, Fang-Cheng Xu b, Wen-Hua Chen b, Yu-Hua Zhao a, * a b

College of Life Science, Zhejiang University, 310058 Hangzhou, Zhejiang, PR China Department of Agriculture and Biotechnology, Wenzhou Vocational College of Science and Technology, 325006 Wenzhou, Zhejiang, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 July 2012 Received in revised form 20 November 2012 Accepted 14 December 2012 Available online 10 February 2013

Malachite green (MG) is extensively used, although it is carcinogenic and mutagenic. In our previous study, the novel Micrococcus sp. strain BD15 was observed to efficiently decolorize MG. The aims of this study were to identify the metabolites after degradation by this strain and to identify the enzymes involved in degradation. UVeVisible, FTIR, GCeMS and LCeMS analyses were performed to determine the degradation products, and our results indicate that the intermediates of MG degradation include 4-(Dimethylamino) benzophenone, Michler’s ketone, 4-(methylamino)benzophenone, 4-aminobenzophenone, 4methylaminobenzoic acid, 4-hydroxyl-N,N-dimethylaniline, N,N-dimethylaniline, hydroxyl-4-(dimethylamino)benzophenone and 4-hydroxyl-aniline. In addition, enzyme analysis revealed that laccase and NADH-DCIP reductase are involved in the degradation of MG. To our knowledge, this is the first study of the detailed biodegradation pathway of MG by Micrococcus sp. strains. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Biodegradation Micrococcus sp. Malachite green Enzyme analysis Pathway

1. Introduction Malachite green (MG) (Basic Green 4; CI 42000) is extensively used in the aquaculture industry worldwide as a biocide to control external fungal and protozoan infections of fish and as a dye in the silk, wood, cotton, leather, paper, and acrylic industries (Saha et al., 2012; Lee and Kim, 2012; Moumeni and Hamdaoui, 2012). In recent years, reports of the enduring toxic, carcinogenic and mutagenic characteristics of this compound have increased (Yang et al., 2011; Chen et al., 2012). Moreover, MG has a complex aromatic molecular structure recalcitrant to degradation and can reduce the transmission of sunlight in bodies of water, thereby affecting the aquatic biota of the habitat. Hence, MG will produce irreversible damage to the environment if discharged into water. Recently, MG has been listed as a priority chemical for carcinogenicity assessment by the U.S. Food and Drug Administration (Srivastava et al., 2004; Shedbalkar and Jadhav, 2011). Nonetheless, MG is still utilized in many regions worldwide due to its low cost, good efficacy, and a lack of suitable alternatives (Srivastava et al., 2004; Shedbalkar and Jadhav, 2011). Therefore, it is urgent to explore effective methods to solve this problem (Daneshvar et al., 2003). * Corresponding author. Tel.: þ86 571 88208557. E-mail address: [email protected] (Y.-H. Zhao). 1 These authors contributed equally to this work. 0964-8305/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ibiod.2012.12.011

Several physicochemical methods have been utilized for the removal of MG from wastewater, such as photo-Fenton reagent (Chen et al., 2002), ozonation (Kusvran et al., 2011), and microwave-assisted catalysts (Ju et al., 2008). However, each of these methods has limitations, such as high cost, secondary pollution and low efficiency (Patidar et al., 2012). Biological treatment has already drawn considerable attention due to its eco-friendly, efficient and low-cost characteristics (Deng et al., 2008). In our previous work, a novel Micrococcus sp. strain (BD15) that outperformed most of the strains previously reported in MG degradation was isolated, which provided a solid foundation for our present study (Du et al., 2012). The mechanisms of MG degradation by several physicochemical methods have been proposed in the literature (Cha et al., 2001; Chen et al., 2002; Chen et al. 2007; Berberidou et al., 2007; Gao et al., 2008; Ju et al., 2008; Ju et al. 2009; Chen et al., 2010; Kusvran et al., 2011; Patidar et al., 2012), generally including reaction disposition, sequence and velocity of the N-demethylation step, destruction of the conjugated structure and further ring opening reactions. Nonetheless, there are few reports of the sufficient mechanisms of MG biodegradation. Chen et al. (2010) proposed a partial degradation mechanism by Shewanella decolorationis NTOU1 under anaerobic conditions because only 4(Dimethylamino)benzophenone, N,N,N0 -trimethyl-4,40 -benzylidenediamine and N,N-dimethylaminophenol were detected.

L.-N. Du et al. / International Biodeterioration & Biodegradation 78 (2013) 108e116

Oxidative enzymes and NADH-DCIP reductase have been claimed to be responsible for dye decolorization by bacteria, and laccase and NADH-DCIP reductase have been verified to be involved in the degradation of MG by strain BD15. In this study, we report the detailed biodegradation mechanisms of MG based on the successful detection of intermediates using advanced methods. 2. Materials and methods 2.1. Dye and chemicals 4-methylaminobenzoic acid, 4-dimethylaminophenol, and 4(Dimethylamino)benzophenone were purchased from Sigma Co. Ltd. All other chemicals were of analytical grade and purchased from Sinopharm Chemical Reagent Company, China. For MG, the dye content was S85%. 2.2. Organism and media Micrococcus sp. strain BD15 was isolated from sewage and preserved at College of Life Science, Zhejiang University. Luria-Bertani medium (LB) contained 10.0 g l1 Tryptone, 5.0 g l1 Yeast Extract, and 10.0 g l1 NaCl, pH 7.0e7.2. The MG solution contained 100 mg MG in 1 l distilled water. 2.3. Determination of biodegradation products of MG by strain BD15 All experiments were performed in triplicate with an initial concentration of MG of 100 mg l1. The strain BD15 was cultured in LB medium shaken at 200 rpm at 30 C overnight. The cultures were centrifuged at 8000 rpm for 5 min, and the cells were collected and washed three times with distilled water. The cells were inoculated into the MG solution at an initial cell density of 0.1e 0.3 g l1 (dry weight) and cultured at static conditions at 30 C (Deng et al., 2008). At a specified interval, the cultures were collected and tested by different analytical methods. 2.3.1. UVevisible and LCeMS analysis For UVevisible and LCeMS analysis, the cultures were collected at specific time points and centrifuged at 12,000 rpm for 10 min. The supernatants were analyzed with a UV-3100PC spectrophotometer (Shanghai MAPADA Instruments Co., Ltd.) and by liquid chromatography-mass spectrometry (LCeMS, Agilent Series 1200 coupled with a LCQ DECA XP MAX mass spectrometer, Thermo). LCeESIeMS analysis was performed with an HPLC system (Agilent 1200 Series equipped with a reverse-phase C-18 analytical column of 150 mm length 2.1 mm and 3.5 mm particle size, ZORBAX SBC18) coupled with the mass spectrometer (Thermo Finnigan, LCQ DECA XP MAX). The HPLC measurement was performed with 50:50 (v/v) acetonitrile: 20 mM ammonium acetate as the mobile phase at a flow rate of 1.0 ml min1. The detection wavelength was 370 nm. The mass spectrometer settings were as reported in our previous study (Du et al., 2011). 2.3.2. FTIR and GCeMS measurement To prepare the samples for FTIR and GCeESIeMS measurement, 10 ml of culture was extracted with trichloromethane (3 2 ml) and concentrated to 1 ml. The concentrated extraction was spread over potassium bromide (KBr) powder and dried under light for 10 min. The dried samples were then analyzed on a Bruker Veefor 22 FTIR spectrometer to collect the FTIR spectra at 400e4000 cm1. Detailed information regarding GCeESIeMS measurement is described in our previous study (Du et al., 2011).

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2.4. Enzyme assays The activities of tyrosinase, laccase, Mn-peroxidase (MnP), lignin peroxidase (LiP), NADH-DCIP reductase and MG reductase, which may relate to decolorization, were also tested in this study using methods described in our previous studies (Du et al., 2011). One unit of tyrosinase, laccase, MnP and LiP activity was defined as a change in absorbance units min1 mg protein1. The extinction coefficients of 90 mM1cm1 and 1.47 105 mM1cm1 were used to calculate the NADH-DCIP reductase and MG reductase activities, respectively.

3. Results and discussion 3.1. Possible intermediates of MG biodegradation by strain BD15 3.1.1. UVevisible spectra Fig. 1 depicts the UVeVis spectral analysis of MG before and after decolorization. The characteristic MG peak 4 at 620 nm (lmax for MG) decreased completely within 30 min when incubated with Micrococcus sp. strain BD15 as did the two peaks at 315 nm and 420 nm. In contrast, the slight peak 2 at 254 nm increased and was gradually hypsochromically shifted, whereas peak 1 was slightly red shifted over time. Simultaneously, a significant spectral band that likely represented a new metabolite with an absorption maximum at 370 nm (peak 3) emerged. According to previous reports, the absorption peaks would decrease proportionally if the decolorization were caused by biosorption, whereas with biodegradation, either the major visible light absorbance peak would disappear or a new peak would appear (Asad et al., 2007). Therefore, the decolorization of MG in the present study is attributed to degradation rather than biosorption. In addition, the inset figure (Fig. 1) shows that with increased incubation time, the intensity of peaks 2 and 3 increased, whereas the intensity of peak 1 initially decreased for 1 h and subsequently increased. The decreased intensity of peak 1 was an indication of diminished conjugated structure, and thereafter, the continuous incremental red shift could have been caused by sequential reactions that accumulated metabolites that resonate at this wavelength. It is known that conjugated polycyclic aromatic compounds have strong auxochromic moieties that appear at a wavelength longer than the band position of single-benzene derivatives. Therefore, it can be surmised that peaks 1 and 2 were

Fig. 1. UVevisible analysis of an MG solution before and after inoculation with strain BD15. The inset figure is the absorbance of peaks 1, 2, and 3.

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produced by single-benzene vibrations, and peak 3 could be attributed to the vibration of a conjugated polycyclic aromatic structure. Furthermore, 4-dimethylaminobenzophone (DLBP) was reported by Ju et al. (2009) to be one of the main products yielded via attack on a central carbon of MG, with an obvious absorbance at 360 nm and a corresponding blue shift. Therefore, peak 3 could be deduced to represent DLBP. 3.1.2. FTIR spectra Remarkable variations in the fingerprint region (4000e 400 cm1) of the FTIR spectra of MG and its metabolites by strain BD15 are shown in Fig. 2. The FTIR spectra of biodegraded products with new peaks at 3784 cm1 and 3698 cm1 for eOH stretching vibrations represent the formation of hydroxylated metabolites, and the new peaks at 3451 cm1 and 1174 cm1 corresponding to stretching vibrations are caused by eNH2 or eNR2 groups. In addition, the sharp peaks at 2925 cm1 and 2855 cm1 for eCH stretching by asymmetric CH2 groups indicate the yield of methylene-substituted metabolites, whereas the slight peak at 1726 cm1 corresponds to the C]O stretch of ketones. The peaks at 1635 cm1, 1588 cm1 and 1380 cm1 are caused by eNH or eCN stretching vibrations in amine I, II, and III groups. Moreover, new peaks at 1112 cm1 (CeC) and 759 cm1 (PeOeP or PeOeC) can be inferred to be bacterial secretions. Therefore, the results of FTIR analysis indicate that the conspicuous exposed chemical groups in the metabolites of MG include eOH, eC]O and eNH2, which substantiate the degradation of MG by strain BD15. 3.1.3. Intermediates of MG identified by GCeMS measurement GCeMS-based metabolite analysis has profound implications for discovering the mode of action of drugs or herbicides and helps unravel the effect of altered gene expression on metabolism and organism performance in biotechnological applications (Schauer et al., 2005). Though only 20% of the total compounds present can be detected using this technique in scientific research because internal factors such as the volatility, melting temperature, and structure of the compounds and various external GC conditions are important for a successful final elution, this technique is irreplaceable because of its unique ability to detect compounds with high volatilities, low boiling temperatures and low molecular weights with high sensitivity (Jonsson et al., 2004; Schauer et al., 2005). As shown in Fig. 3, GCeMS analysis of MG and its products after complete degradation identified three possible metabolites using

Fig. 2. FTIR analysis of MG before (B) and after (A) degradation by strain BD15 for 24 h.

Fig. 3. GCeMS analysis of MG and its degradation products.

an identification program from the NIST library. MG, which eluted with a retention time of 27.46 min, gradually degraded into infinitesimal quantities. After 1 h, a possible new intermediate peak that eluted at 6.69 min was captured and identified as silylated 4aminophenol, which could not be tested at later time points, indicating that silylated 4-aminophenol formed during the initial phase of degradation and was then transformed into other compounds for further degradation. Another apparent metabolite that eluted at 20.40 min increased gradually with incubation time (0e 10 h) and was confirmed to be 4-(Dimethylamino)benzophenone (abbreviation: 4-DLBP) according to the NIST library. In sum, the two intermediates detected by GCeMS could be the result of cleavage of the entire conjugated chromophore structure, leading to aminobenzene derivatives. By contrast, an earlier report by Saquib and Muneer (2003) described the detection of 4aminobenzoic acid and N-methylaniline from the photocatalytic degradation of crystal violet through GCeMS analysis, signifying that different chemical bond-breaking dispositions due to different strategies can lead to the differential accumulation of compounds. 3.1.4. Products of MG identified by LCeMS measurement LCeMS analysis is a versatile system, combining both sensitivity and selectivity, and it is deemed the most reliable technique to identify and quantify chemical compounds in complex matrices (Zille et al., 2005). HPLC analysis of MG and its degradation products taken at 2 h (Fig. 4B) and 24 h (Fig. 4C) and the total ion chromatogram (TIC) of LCeMS are shown in Figs. 4 and 5, respectively. In the HPLC plot, four new peaks emerged and persisted for 24 h, with variations in intensity. The retention times (tR) of these peaks were 1.32 min, 1.55 min, 1.87 min and 7.86 min, and correspondingly, their protonated molecular ion peaks were at 118, 152, 138, and 226 m/z, respectively. MG, with a retention time of 17.40 min (Fig. 4A), could no longer be detected after incubation for 2 h, which further supports the conclusion that highly efficient decolorization of MG by strain BD15 is caused by biodegradation. To preclude the interference of bacterially secreted organic compounds, verification was performed without any dyestuff, and the supernatant was withdrawn for LCeMS analysis. In comparison with the results in Supplementary Fig. 1, no peak was detected at 1.32 min for the MG solution alone (Supplementary Fig. 1a). However, in the presence of strain BD15, a new peak at 1.32 min emerged, which implies that it consisted of a bacterial secretion rather than a degraded metabolite. Therefore, combined with data from FTIR analysis and GCeMS, the three other new peaks in the LC were identified as degraded metabolites. The degradation products were verified by HPLC with standards purchased from Sigma Co. Ltd (data not shown) as 4methylaminobenzoic acid (m/z 152, abbreviation: 4-MBAc), 4-


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Fig. 4. LCeMS analysis of MG and its degradation products. (A) MG; (B) degradation for 2 h; (C) degradation for 24 h.

dimethylaminophenol (m/z 138, 4-DMAP) and 4-(Dimethylamino) benzophenone (m/z 226, 4-DLBP). Furthermore, by contrasting the plots in Fig. 4, we also discovered that 4-MBAc sharply increased initially (2 h) and decreased noticeably over the next 22 h, whereas both 4-DMAP and 4-DLBP accumulated consistently. Other small, possibly degraded organics were not apparent as peaks in the HPLC scheme but could still be detected by mass spectrometry, including Michler’s ketone (m/z 269, retention time, 1.37 min), 4-(methylamino)benzophenone (m/z 212, 4-MLBP, retention time, 4.16 min), 4-aminobenzophenone (m/z 198, 4-BP, retention time, 1.76 min), N,N-dimethylaniline (m/z 122, retention time, 0.64 min), 4hydroxyl-N,N-dimethylaniline (m/z 138, retention time, 1.84 min), and hydroxyl-4-(dimethylamino)benzophenone (m/z 243, OH-4DLBP, retention time, 1.81 min). Identification of all of the intermediates by solitary mass spectrometry was not possible because the concentration of intermediates present in the analysis solution and the amount

obtained after separation on the solid phase column, HPLC separation protocols, the types of molecules and other factors could affect analysis by mass spectrometry (Gokulakrishnan et al., 2012). Therefore, it is imperative to include several advanced testing methods to guarantee that few of the metabolites are missed. Moreover, it is well known that a smaller proportion of the total compounds can be identified by GCeMS compared with HPLC, which was also observed in our work. It is likely that the volatilities of several intermediates are too low to be eluted under the gas chromatographic conditions used and the polarities were high enough that these compounds eluted with the solvent phase during HPLC analysis (Kusvran et al., 2011). 3.2. Proposed MG degradation pathway by strain BD15 From the large number of aromatics identified by the detection methods discussed above, a possible degradation pathway is

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Fig. 5. The mass spectra of MG and its degradation products identified by LCeMS analysis. (A) m/z 330, malachite green (retention time, 17.40 min); (B) m/z 226, 4-(Dimethylamino) benzophenone (retention time, 7.84 min); (C) m/z 269, Michler’s ketone (retention time, 1.37 min); (D) m/z 212, 4-(methylamino)benzophenone (retention time, 4.61 min); (E) m/z 198, 4-aminobenzophenone (retention time, 1.76 min); (F) m/z 152, 4-methylaminobenzoic acid (retention time, 1.55 min); (G) m/z 138, 4-hydroxyl-N,N-dimethylaniline (retention time, 1.84 min); (H) m/z 122, N,N-dimethylaniline (retention time, 0.64 min); (I) m/z 243, hydroxyl-4-(dimethylamino)benzophenone (retention time, 1.81 min).


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Fig. 5. (continued).

proposed in Fig. 6. Some undetected intermediates are shown in brackets in Fig. 6, such as phenol, 4-Dimethylaminobenzoic acid, and the N-demethylated MG products (p-dimethylaminophenyl)(p-methylaminophenyl) phenylmethylium (DM-PM) and (pdimethylaminophenyl)(p-methylaminophenyl) phenylmethylium (D-PM). It is supposed that these compounds may have accumulated to trace quantities and were spontaneously biotransformed and whose existence can be inferred from some of the compounds detected. The complexity of the degradation process is caused by the multiple sites prone to OH radical attack on MG and its byproducts in aqueous solution (Oturan et al., 2008), and generally,

the generation of a carbon-centered radical is succeeded by the destruction of dye chromophore structures (Chen et al., 2007). Given the identification of N-dimethylated aromatic amines such as 4-DLBP (m/z 226), Michler’s ketone (m/z 269) and 4-DMAP (m/z 138) and based on previous reports of photo-Fenton treatment of MG (Chen et al., 2002; Gao et al., 2008) and microwave-enhanced H2O2-based treatment (Ju et al., 2009), it is clear that the first step of biodegradation in the present study is initiated by hydroxylation of the central carbon of MG. In essence, decolorization did take place and involved the cleavage of different CeC bonds of the central carbon to generate either 4-DMAP (m/z 138) and 4-DLBP (m/z 226) through one pathway or Michler’s ketone (m/z 269)

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Fig. 6. The proposed MG degradation pathway by strain BD15.

and phenol (m/z 95) through another. Due to the large quantities of 4-DLBP (m/z 226) and 4-DMAP (m/z 138), it can be inferred that the former pathway is dominant during the first stage of degradation, which is consistent with a previous report by Chen et al. (2010). Nonetheless, mass studies report that attack by a hydroxyl radical could also result in N-demethylation during the initial degradation of MG. Chen et al. (2007) and Saha et al. (2012) proposed that the N-demethylation degradation of MG takes place in a stepwise manner to yield N-demethylated MG species under basic conditions, whereas Ju et al. (2008) contend that no stepwise photochemical process was observed for the N-demethylation reactions. Berberidou et al. (2007) proposed parallel and competing pathways between N-demethylation reactions and destruction of the conjugated structure. Likewise, in biodegradation research, Chen et al. (2010) discovered that only one methyl group was removed from the parent structure before reductive splitting of the triphenylene rings, and the N-demethylation process may influence the velocity of the subsequent reactions. Cha et al. (2001) suggested that N-demethylation is mediated by the cytochrome P450 system in their study. Despite the disagreements detailed above, most investigators have reached a consensus in their UVevisible analysis that an apparent hypsochromic spectral shift slightly deflected at 620 nm was observed, implying that a basic structure similar to MG existed, as did the N-demethylation products detected by LCeMS, such as DM-PM and D-PM. However, in this study, a spectral hypsochromic shift between 595 nm and 620 nm, DM-PM and D-PM were not detected. We suppose that our failure to capture DM-PM and D-PM may be due to their immediate biotransformation, which is characterized by a reaction velocity several orders of magnitude faster than conventional physicochemical-catalyzed reactions. This is likely because, in one respect, enzymes can engulf their specific substrates within the active site to minimize the radius of

molecular interactions and to isolate substrates from unfavorable solvents. Another reason is that the active site is a small compact region containing polar-enhanced radicals such as eOH, eCOOH, e SH and others that are inclined to donate electrons in the nonpolar surroundings. Therefore, to be consistent with above statement and the GCeMS inspection of 4-aminophenol and 4-DLBP, the degradation of MG could still occur via two consecutive N-demethylation steps and further into 4-aminophenol and 4-DLBP (Fig. 6) in addition to the destruction of the chromophore structure. In fact, 4aminophenol can also be generated through rapid N-demethylation from 4-DMAP (Oturan et al., 2008). Subsequently, one major intermediate, 4-DLBP (m/z 226), may undergo further ring opening reactions preceded by central carbon hydroxylation to produce OH-4-DLBP (m/z 243), though its ring-open intermediates were not studied in this work. Moreover, previous reports have established that dimethylamino substitutes are liable to be dealkylated, with the formation of an iminium transition ion, prior to amine formation (Gao et al., 2008; Oturan et al., 2008; Gokulakrishnan et al., 2012), and it has been confirmed by mass spectra that 4-DLBP can alternatively undergo two successive Ndemethylation steps into N,N-dimethylaniline (m/z, 122) and 4aminobenzophenone (m/z 198) without initial hydroxylation. In contrast, Ju et al. (2009) reported that 4-DLBP initially hydroxylated and successively demethylated in a single step. Furthermore, significant quantities of 4-MBAc (m/z 152) were detected by HPLC analysis and small quantities of N-dimethylaniline (m/z 122) by MS and in previous reports by Saquib and Muneer (2003) and Chen et al. (2010). We propose that Michler’s ketone could be further degraded into Ndimethylaniline and 4-Dimethylaminobenzoic acid, which spontaneously demethylates one methyl group into the relevant stable 4MBAc compound. It has been supposed that N-dimethylaniline should degrade further via N-methylation into aniline and then into nitrobenzene for further degradation (Berberidou et al., 2007).


Fig. 7. The activity of laccase in strain BD15 at different dye concentrations.

3.3. Enzyme analysis Recently, oxidative enzymes and NADH-DCIP reductase have been reported to be responsible for dye decolorization in bacteria (Wu et al., 2009). In the present study, tyrosinase, laccase, lignin peroxidase, Mn-peroxidase, NADH-DCIP reductase and MG reductase were studied to ascertain whether these enzymes are responsible for the decolorization of MG. Our results confirm that laccase and NADH-DCIP reductase are involved in this process. Our results show that there was no significant activity by tyrosinase, lignin peroxidase, Mn-peroxidase, and MG reductase, whereas the activity of laccase and NADH-DCIP reductase was significant. A schematic of the results is shown in Figs. 7 and 8. Our results demonstrate that significant enzymatic activity by laccase and NADH-DCIP reductase occurs in MG-containing solutions. With increasing time (1 he12 h) and dye concentrations (100 mg l1e900 mg l1), the enzymatic activity of laccase increased (from 0.242 U mg of protein1 min1 to 21.512 U mg of protein1 min1), whereas the reverse trend was observed for

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NADH-DCIP reductase, i.e., the enzymatic activity gradually decreased with increasing MG concentration and time (from 21.511 U mg of protein1 min1 to 0.539 U mg of protein1 min1). Kumar et al. (2012) investigated the degradation of synthetic dyes by the temperature-stable laccase and found that purified laccase could efficiently decolorize various dyes, especially MG, without the addition of redox mediators. These authors also proposed that laccase was capable of modifying the chemical structure of MG, resulting in its complete degradation, which was confirmed by HPLC (Kumar et al., 2012). The variation in laccase activity in the present study can be explained as follows. First, laccase is an inducible enzyme and requires time to be fully expressed. In addition, higher MG concentrations require more enzymes to degrade the dye, which triggers further enzyme expression. Both Shedbalkar and Jadhav (2011) and Yang et al. (2011) reported decreased NADH-DCIP activity in MG treatment using Penicillium sp., which is consistent with the results of this study. However, many studies utilizing bacteria and our previous work on MG degradation by Pseudomonas sp. strain DY1 described amplification of NADH-DCIP activity (Du et al., 2011). Nevertheless, both laccase and NADH-DCIP reductase act as highly efficient biocatalysts of MG biodegradation. 4. Conclusion The present study described the degradation products of MG by strain BD15, and based on our results, a possible degradation pathway is proposed. Enzyme analysis indicates that laccase and NADH-DCIP reductase may be involved in the degradation of MG by this strain. To our knowledge, this is the first report of the detailed biodegradation mechanism of MG by bacteria. Moreover, further study will emphasize the identification of the principal genes involved in biodegradation and thereby further the practical application of this strain. Acknowledgments This study was supported by the National Natural Science Foundation of China (31070079), the National Key Technology Research and Development Program of the Ministry of Science and Technology of China (2012BAC17B04), the Science and Technology Project of Zhejiang Province (2008C13014-3), and the Science and Technology Project of Zhejiang Province (2010C 13G 2010074). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ibiod.2012.12.011. References

Fig. 8. The activity of NADH-DCIP reductase in strain BD15 at different dye concentrations.

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