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Biodegradation and Detoxification of Malachite Green by a Newly Isolated

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Biodegradation and Detoxification of Malachite Green by a Newly Isolated Biocatalysis and Agricultural Biotechnology 20 (2019) 101183 Contents lists available at ScienceDirect Biocatalysis and Agricultural Biotechnology journal homepage: www.elsevier.com/locate/bab Biodegradation and detoxification of malachite green by a newly isolated bioluminescent bacterium Photobacterium leiognathi strain MS under RSM T optimized culture conditions Shubham S. Sutara,1, Prasanna J. Patila,1, Asif S. Tambolib, Devashree N. Patila, Onkar A. Apinea, ∗ Jyoti P. Jadhava, a Department of Biotechnology, Shivaji University, Vidyanagar, Kolhapur, Maharashtra, 416004, India b Department of Biochemistry, Shivaji University, Vidyanagar, Kolhapur, Maharashtra, 416004, India ARTICLE INFO ABSTRACT Keywords: The isolated bioluminescent bacterium was identified as Photobacterium leiognathi strain MS using 16 S rRNA Bioluminescence sequence analysis and subjected to malachite green (MG) dye degradation. The Box-Behnken based response Response surface methodology surface methodology (RSM) was employed in optimizing medium conditions exhibiting maximum growth at pH Malachite green 8.0, temperature 30 °C, NaCl 3.5%, and peptone 6.5%. Photobacterium leiognathi strain MS was capable of tol- Biodegradation erating high concentration of MG (1.0 gL-1) with 92.50% decolorization potency within 24 h. UV-Vis, FTIR, and Phytotoxicity LC-MS QTOF analyses confirmed biodegradation of MG into several metabolites as well as its catabolism Cytotoxicity pathway. A significant increase in the activity of laccase was obtained which revealed its major involvement in the degradation of MG dye. Moreover, phytotoxicity and cytotoxicity analyses illustrated that the metabolites generated were less toxic than the parental compound. 1. Introduction Previous studies have been reported on different groups of MG de- colorizing microorganisms including yeast (Jadhav and Govindwar, Bioluminescent bacteria are the foremost copious and widely dis- 2006), fungi (Jasinska et al., 2012; Shedbalkar and Jadhav, 2011), tributed light-emitting organisms. They hold numerous remarkable microalgae (Daneshvar et al., 2007) and bacteria which embrace Ci- flamboyant properties, including ability to grow at low temperature trobacter sp. (An et al., 2002), Mycobacteria sp. (Jones and Falkinham, (psychrophiles), high salinity (halophiles) and pressure tolerance 2003), Aeromonas sp. (Ren et al., 2006), Pseudomonas sp. (Wu et al., (barophiles). Malachite green (MG) is a compound belonging to a group 2009), Achromobacter sp. (Wang et al., 2011), Exiguobacterium sp. of triphenylmethane dyes (Yang et al., 2015), usually used in the textile (Wang et al., 2012) and Micrococcus sp. (Du et al., 2013). industries as coloring agents for papers, toys and plastic varieties The present study aimed to determine the use of P. leiognathi strain (Cheriaa and Bakhrouf, 2009). It is also used as a potential agent for MS for the biodegradation and detoxification of MG dye. As per the treating fungal and protozoal infections in aquaculture and fisheries literature review, the bioluminescent microorganisms and members of (Shedbalkar and Jadhav, 2011). In the medical field, it has been utilized genus Photobacterium have not yet been reported to degrade and de- as a disinfectant and anthelmintic (Singh and Nakate, 2013). Potential toxify the MG dye. The experimental data from LC-MS QTOF analysis exposure to MG occurs to the people working in dye and aquaculture led us to propose a potential possible pathway for degradation of MG industries (Culp and Beland, 1996). Its toxicological effects include dye. carcinogenesis, mutagenesis, teratogenesis, chromosomal aberrations, and respiratory toxicity (Srivastava et al., 2004). Hence, it is not ap- 2. Materials and methods proved by the United States Food and Drug Administration and is listed as a priority chemical for carcinogenicity. But, due to its low cost, good 2.1. Dye and chemicals efficacy and lack of appropriate alternatives, it is still used in many areas throughout the world. Malachite green hydrochloride (Basic green 4), practical grade, CAS ∗ Corresponding author. Department of Biotechnology, Shivaji University, Kolhapur, 416004, India. E-mail address: [email protected] (J.P. Jadhav). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.bcab.2019.101183 Received 3 March 2019; Received in revised form 24 May 2019; Accepted 28 May 2019 Available online 28 May 2019 1878-8181/ © 2019 Published by Elsevier Ltd. S.S. Sutar, et al. Biocatalysis and Agricultural Biotechnology 20 (2019) 101183 No.: 123333-61-9 was purchased from Himedia Co. Ltd. Stock solution (Table 1). They were designated as A, B, C, and D and boundary con- of the dye was prepared in distilled water (D/W) and sterilization was ditions for each factor with actual design are depicted in Table 1. These performed using 0.22 μm syringe filter (Axiva). All other chemicals four factors were prescribed into three coded levels, +1, 0, −1 for were of analytical grade and highest purity. high, intermediate, and low value, i.e., pH (6, 8 and 10), temperature (25 °C, 30 °C and 35 °C), NaCl (0.5%, 3.5% and 6.5%) and peptone 2.2. Isolation and identification of bioluminescent bacteria, bioluminescence (0.5%, 6.5% and 12.5%). measurements, and acclimatization The relationship between dependent and independent variables was explained by a second order polynomial equation in RSM (Eq.). A marine fish, saundali (Lactarius lactarius) was used as a source to n n n n 2 isolate bioluminescent bacteria. For 16S rRNA sequence analysis, DNA Y=+ααAαAαAA0 ∑∑ii +ii i + ∑∑ij i j sequencing was accomplished from Agile life science technologies, i1==i1 i1 ==j1 (1) India Pvt. Ltd. Pune. The phylogenetic tree was constructed using α MEGA 7 software and the evolutionary history was inferred using the where, Y is the predicted response; 0 is model intercept; Ai and Aj are α α α ffi maximum likelihood method. For bioluminescence measurements, the independent variables; i, ii and ij are regression coe cients for ff ‘ ’ bioluminescence intensities were measured at room temperature using linear, square and interaction e ects respectively. n indicates the a Varian Cary Eclipse Fluorescence Spectrophotometer (Agilent number of independent variables and (n = 4) in this study. All ex- Technologies, USA, Instrument Serial Number: MY14410002) (Watkins periments were carried out in triplicate. Bioluminescent bacterial cul- et al., 2018). tures were grown in a shaking condition (120 rpm) and their lumines- To increase the dye decolorizing potency, acclimatization was per- cence output in a.u. (arbitrary unit) and corresponding optical densities formed by exposing isolated strain (P. leiognathi strain MS) to various (ODs) were estimated after an incubation period of 18 h. Absorbance MG dye concentrations (Wang et al., 2011). It was performed up to 1.0 measurement was performed using a UV-Visible spectrophotometer gL-1 concentration of MG dye. All assays were performed at pH 7.0 and (UV-1800, Shimadzu, Japan). Design expert software (Version 11.0, 27 ± 2 °C room temperature under static condition. The acclimatized Stat-Ease Inc., Minneapolis, USA) was used for statistical analysis and strain was consistently maintained on dye agar plates and used for experimental design. further experiments. 2.4. Decolorization assay 2.3. Statistical media optimization by experimental design Decolorization of MG was expressed in terms of percentage (%) by Based on the preliminary experiments, variables such as pH, tem- few modifications in previously described method (Deng et al., 2008). A perature, NaCl, and peptone were identified to have strong effects on UV-Visible spectrophotometer (UV-1800, Shimadzu, Japan) was used the responses. Therefore, these factors were elected as the variables for absorbance measurement. Decolorization experiments were per- tested in the 29 run experiment of the Box-Behnken design experiment formed in triplicate and its activity was calculated as follows: Table 1 Box-Behnken design matrix with variables along with actual and predicted responses. Run Independent variables Responses Coded variables OD (Y1) Light intensity (Y2) (a.u.) A:pH B: C: NaCl D: Peptone Actual Predicted Externally Studentized Actual Predicted Externally Studentized Temperature (˚C) (%) (%) response Response Residuals Response Response Residuals 1 0 −1 −1 0 0.6020 0.5812 1.205 455.66 445.02 1.643 2 0 0 +1 0 1.24 1.28 −1.524 770.66 775.82 −0.528 3 0 −1 +1 +1 0.5330 0.5428 −0.547 390.32 399.30 −1.345 4 0 0 +1 0 1.22 1.28 −2.617 768.88 775.82 −0.717 5 0 0 +1 0 1.28 1.28 0.063 775.77 775.82 −0.005 6 −1 0 +1 +1 0.5540 0.5370 0.965 401.69 403.62 −0.271 7 0 +1 +1 +1 0.6780 0.6768 0.064 544.56 536.33 1.222 8 +1 0 −1 0 0.4850 0.4810 0.220 355.68 369.01 −2.191 9 +1 0 +1 −1 0.3450 0.3562 −0.625 255.33 250.57 0.681 10 +1 −1 +1 0 0.6350 0.6458 −0.601 511.73 513.30 −0.220 11 +1 0 0 0 0.5890 0.5783 0.594 431.22 429.37 0.261 12 00 −1 −1 0.3940 0.3916 0.131 282.33 269.92 1.994 13 0 0 +1 0 1.33 1.28 2.558 783.44 775.82 0.792 14 0 −1 0 0 0.6120 0.6065 0.301 489.55 478.92 1.642 15 −1 +1 +1 0 0.8650 0.8560 0.502 685.55 682.13 0.485 16 00 0 −1 0.4800 0.4690 0.616 352.66 355.42 −0.389 17 0 0 0 +1 0.6070 0.6111 −0.227 462.66 473.22 −1.628 18 0+1 +1 −1 0.5780 0.5722 0.322 419.35 415.09 0.608 19 0 −1+1−1 0.4580 0.4632 −0.285 342.55 355.49 −2.105 20 −1 −1 +1 0 0.5550 0.5555 −0.025 404.72 402.53 0.309 21 00 −1 +1 0.4210 0.4338 −0.717 321.77 317.17 0.659 22 +1 +1 +1 0 0.5870 0.5883 −0.071 429.99 430.33 −0.047 23 +1 0 +1 +1 0.5300 0.5214 0.478 387.25 378.65 1.283 24 −10 −1 0 0.5250 0.5397 −0.827 372.55 379.11 −0.952 25 −1 0 0 0 0.6890 0.6970 −0.443 568.93 560.31 1.287 26 −10 +1 −1 0.5150 0.5179 −0.158 360.88 366.65 −0.831 27 0 0 +1 0 1.31 1.28 1.484 780.33 775.82 0.461 28 0+1 −1 0 0.6010 0.6007 0.016 448.66 456.46 −1.149 29 0 +1 0 0 0.8150 0.8300 −0.849 656.32 664.12 −1.150 2 S.S.
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