Eco-friendly biodegradation of a reactive textile dye Golden Yellow HER by Brevibacillus laterosporus MTCC 2298

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<ul><li><p>e22</p><p>N</p><p>BiodegradationPhytotoxicity</p><p>TChebseC. Tas4 (</p><p>rent alfonaters. Des canto as hal., 200</p><p>decolorize awide range of commercially available textile dyes (Jinqiand Houtian, 1992; Kim et al., 1995; Parikh and Madamwar, 2005;Parshetti et al., 2007). The features like omnipotence, faster growth,facultative nature and high adaptability are the desirable qualitiesof bacterial community for the bioremediation. Brevibacillus</p><p>from NCIM, National Chemical Laboratory, Pune, India. All thebacterial strains were maintained routinely on the nutrient slantscontaining (g l1): NaCl, 5.0; bacteriological peptone, 5.0; yeastextract, 2.0; beef extract, 1.0 and agaragar 15.0 at 4 C.</p><p>2.2. Dyes and chemicals</p><p>A textile dye Golden Yellow HER was obtained from YashwantTextile Processing, Ichalkaranji, Kolhapur, India. Remaining all</p><p>* Corresponding author. Tel.: 91 231 2609152; fax: 91 231 2691533.</p><p>Contents lists availab</p><p>International Biodeterior</p><p>.e</p><p>International Biodeterioration &amp; Biodegradation 63 (2009) 582586E-mail address: (S.S. Gomare).Various physical (Astrid et al., 2004; Anastasios et al., 2005;Theodora et al., 2006), chemical (Kabita et al., 2001) and biolog-ical treatments (Bustard et al., 1998; Hao et al., 2000; Robinsonet al., 2001) have been reported to remove colors from dye con-taining wastewater. Environmental biotechnology relies upon thepollutant degrading capacities of naturally occurring microbialconsortium in which bacteria play central role (Liu and Sufta,1993).</p><p>Number of microorganisms has been studied for their ability to</p><p>2. Materials and methods</p><p>2.1. Microorganisms and culture conditions</p><p>B. laterosporus MTCC 2298 was obtained from Microbial TypeCulture Collection, Chandigarh, India. Pseudomonas aeruginosaNCIM 2036 and Azotobacter vinelandii NCIM 2821 were obtained1. Introduction</p><p>More than 2000 structurally diffeuse (Vijaykumar et al., 2007). Sua large group that gives diverse colothe dyes, their loss in wastewateroriginal concentration for basic dyesdyes (ONeill et al., 1999; Tan et0964-8305/$ see front matter 2009 Elsevier Ltd.doi:10.1016/j.ibiod.2009.03.005are non-toxic to the common crops such as Sorghum vulgare and Phaseolus mungo. Also, degradationproducts are non-toxic to B. laterosporus as well as ecologically important bacteria like Pseudomonasaeruginosa and Azotobacter vinelandii.</p><p> 2009 Elsevier Ltd. All rights reserved.</p><p>zo dyes are currently ind azo dyes representpending on the class ofrange from 2% of theigh as 50% for reactive0; Boer et al., 2004).</p><p>laterosporus strains are eco-friendly, since they have been studiedfor biological control (Edmar et al., 2004). B. laterosporus MTCC2298 showed the potential for the degradation of various azo dyesincluding Golden Yellow HER (Gomare and Govindwar, 2009).Present study was undertaken to conrm the biodegradation ofa commonly used reactive textile dye Golden Yellow HER by B.laterosporus. Also, analytical techniques such as TLC and HPLC, FTIRand GCMS have been performed for the concrete conclusion.Golden Yellow HERBrevibacillus laterosporus4-(3-amino-2-hydroxy-cyclopentylamino)-benzene-sulfonic acid and 5-amino-cyclohex-3-ene-sulfonicacid (peak 3,m/z 183). Phytotoxicity results suggested that degradation products of Golden Yellow HERKeywords:1, m/z 526), 4-(3-hydrazino-2-hydroxy cyclopentylamino)-benzene-sulfonic acid (peak 2, m/z 455),Eco-friendly biodegradation of a reactivHER by Brevibacillus laterosporus MTCC</p><p>Sushama S. Gomare*, Dhawal P. Tamboli, AnuradhaDepartment of Biochemistry, Shivaji University, Vidhyanagar, Kolhapur 416004, India</p><p>a r t i c l e i n f o</p><p>Article history:Received 22 January 2009Received in revised form14 March 2009Accepted 25 March 2009Available online 26 April 2009</p><p>a b s t r a c t</p><p>Brevibacillus laterosporus Munder static condition at tization performance was o(74%) at the pH 7.0 and 30 </p><p>Biodegradation pathway wolites are the 2,5-Dichloro-</p><p>journal homepage: wwwAll rights reserved.C 2298 showed 87% decolorization of Golden Yellow HER within 48 hconcentration 50 mg l1; however no signicant change in the decolor-rved under shaking condition. Decolorization performance was maximumLC and HPLC analysis conrmed the biodegradation of Golden Yellow HER.proposed using GCMS and FTIR spectral analysis. Mainly elected metab-3-hydrazino-2-hydroxy cyclopentylamino-) dibenzene-sulfonic acid (peaktextile dye Golden Yellow98</p><p>. Kagalkar, Sanjay P. Govindwar</p><p>lsevier .com/locate/ ib iodle at ScienceDirect</p><p>ation &amp; Biodegradation</p></li><li><p>decolorization performance was studied by using an orbital shaker</p><p>tion performance was studied by the repeated addition of dye</p><p>mid IR region of 4004000 cm1 with 16-scan speed (Perkins</p><p>2.6. Toxicity studies</p><p>Toxicity effects of control dye and its degradation products werestudied for two important crops such as Sorghum vulgare andPhaseolus mungo. Toxicity of control dye studied at differentconcentrations and a concentration (0.1 g l1) showing inhibitoryeffect on the growth of seeds was selected for further studies. Tenseeds of each crop allowed germinating in a lter paper beddedpetriplate with daily watering of 5.0 ml solutions (i.e. control dye/its degradation product). Simultaneously, a control set with theplain water supply was carried out. Toxicity effect was measured interms of percent germination and lengths of plumule and radicalafter 6 days. Relative seed germination, relative root elongation andgermination index (GI) were calculated by the formulae (Gomareet al., in press) as given below:</p><p>Relative seed germination %</p><p>Germination index GI</p><p>oration &amp; Biodegradation 63 (2009) 582586 583Elmer 783 Spectrophotometer). GCMS analysis was carried outusing a Hewlett Packard 989 B MS Engine, equipped withintegrated gas chromatograph and an HP1 column (30 m long and(50 mg l1) into a batch culture (100 ml) after every 48 h andremoval of dye during each cycle was monitored. All the decolor-ization experiments were carried out in triplicate. Lignin peroxi-dase, laccase, tyrosinase, aminopyrine N-demethylase, MGreductase and DCIP reductase activities were determined usingprocedures reported from our laboratory (Gomare and Govindwar,2009). Lignin peroxidase, laccase and tyrosinase activities wereassayed by monitoring the formation of propanaldehyde fromn-propanol, oxidized ABTS from ABTS and catechol quinone fromcatechol respectively. NADHDCIP reductase and MG reductaseactivities were determined by monitoring reduction of DCIP andmalachite green respectively. Aminopyrine N-demethylase activitywas determined by measuring formaldehyde liberated by usingNash reagent (Gomare and Govindwar, 2009).</p><p>2.4. Extraction of degradation products</p><p>Biomass was removed after decolorization (10 000 g at 4 Cfor 20 min) and supernatant was extracted for degradation prod-ucts with an equal volume of ethyl acetate. The ethyl acetate extractwas evaporated in vacuum over anhydrous Na2SO4 and dried. Thedried sample was dissolved in 2 ml HPLC grade methanol and usedfor analytical studies.</p><p>2.5. Analytical techniques</p><p>Thin layer chromatogram was obtained on the pre-coatedsilica gel plates with mobile phase containing methanol and spotswere visualized in an iodine chamber. HPLC analysis was carriedout at 30 C by using Water Model 2487 equipped with dual lUVVis detector (set at 379 nm) and C18 column (symmetry,4.6 250 mm). Samples (10 ml) injected to separate dye productsat the ow rate 0.5 ml min1 using an isocratic mobile system i.e.methanol for 10 min. FTIR spectral analysis was carried out in theadjusted at 30 C and 150 rpm. Pre-grown (under static condition)culture was kept on the shaker after dye addition. Effect of pH onthe decolorization performance was studied by adjusting differentpH (pH 3.0, 5.0, 7.0, 9.0 and 11.0) of the pre-grown culture beforedye addition. Effect of temperature on the decolorization perfor-mance was studied by incubating pre-grown (at 30 C) culture atdifferent temperatures (5, 15, 30 and 45 C) after the dye addition.Flasks were incubated at respective temperatures for 30 min beforeaddition of dye. Effect of repeated dye addition on the decoloriza-chemicals were obtained from Hi-media Laboratories Pvt. Ltd.,Mumbai, India.</p><p>2.3. Decolorization experiments, physicochemical parameters andenzyme assays</p><p>B. laterosporus culture grown for 36 h (an exponential phase)was used for the dye decolorization. Decolorizationwas carried outunder static condition at 30 C using 250 ml Erlenmeyer asks.Aliquot (5 ml) withdrawn after decolorization was centrifuged(4000 g for 20 min) and residual dye content in the supernatantwas measured according to the procedure reported earlier byGomare and Govindwar (2009). Effect of shaking condition on the</p><p>S.S. Gomare et al. / International Biodeteri0.25 mm id). % Seed germination % Root elongation100</p><p>Microbial toxicity studies carried out for the bacterial strainexploited for the decolorization i.e. B. laterosporus as well asa phosphate solubilizing bacterium P. aeruginosa and a nitrogenxing bacterium A. vinelandii. The nutrient medium containing 1.5%agar was used for plating. Toxicity of control dye studied atdifferent concentrations and the concentration (1.0 g l1) showingdistinct zone of inhibition was selected for further studies. Toxicityeffect was measured in terms of zone of inhibition (diameter in cm)after 24 h incubation at 30 C.</p><p>3. Results and discussion</p><p>3.1. Decolorization of Golden Yellow HER and physicochemicalparameters</p><p>Literature survey revealed that there are no reports on thebacterial decolorization of Golden Yellow HER or the biological No: of seeds germinated in the extractNo: of seeds germinated in the dye control</p><p> 100</p><p>Relative root elongation %</p><p> Mean root elongation in the extractMean root elongation in the dye control</p><p> 100Fig. 1. Comparison of the FTIR spectra of control dye Golden Yellow HER and itsdegradation products extracted after 48 h.</p></li><li><p>of the Golden Yellow HER up to repeated V cycles of the dyeadditions and then declined up to 75% in the cycle VI. Moosvi et al.(2005) found that 93, 94 and 68% decolorization of Reactive Violet 5in the cycle I (40 h), II (24 h) and III (24 h) respectively by thebacterial consortium RVM-11.1.</p><p>3.2. Analysis of Golden Yellow HER degradation products</p><p>Thin layer chromatogram of control dye and its degradationproducts extracted after 48 h showed different spots having Rfvalue 0.98 and 0.91 respectively (data not shown). The HPLC of</p><p>S.S. Gomare et al. / International Biodeterioration &amp; Biodegradation 63 (2009) 582586584treatment of textile efuents containing this widely used textiledye. B. laterosporus decolorized 87% of Golden Yellow HER within48 h under static condition; however decolorization performanceremained unchanged under shaking condition. Recently, acceler-ated decolorization of structurally different azo dyes by newlyisolated bacterial strains has reported, where, shaking increasedthe time required for complete decolorization of dye compared tothat which was required under static conditions (Khalid et al.,2008). The pH of culture medium of B. laterosporus at the time ofdye addition was 8.0. B. laterosporus showed maximum decolor-ization of Golden Yellow HER by the bacterial culture withoutadjusting the pH; however, decolorization was more than 60% inthe broader range of pH (i.e. 5.09.0). No change in pH of themedium observed during dye decolorization. Decolorization ofGolden Yellow HER by B. laterosporus was 62, 72, 74, 67, and 57%within 48 h at the pH 3.0, 5.0, 7.0, 9.0, and 11.0 respectively.Maximum decolorization of azo dyes by Pseudomonas sp. has beenreported between the pH 6.0 and 9.0 (Mali et al., 2000). Maximumrate of decolorizationwas between pH 7.0 and 8.5; however furtherincrease in pH caused decrease in the decolorization rate (Moosviet al., 2005). B. laterosporus exhibited maximum decolorization at30 C whereas, about 60% dye removal observed even at 45 C.Decolorization of Golden Yellow HER by B. laterosporus was 22, 43,86, and 64% within 48 h at 5, 15, 30, and 45 C respectively. Nodecolorization was observed by the autoclaved cells or abioticcontrol. The dye decolorization activity of consortium NBNJ6 wasincreased with the increase in incubation temperature from 25 to37 C and further increase in temperature resulted in the marginalreduction in decolorization activity (Junnarkar et al., 2006). Declinein decolorization activity at higher temperature can be attributed tothe loss of cell viability or to the denaturation of the enzymes(Pearce et al., 2003). Klebsiella pneumoniae RS-13 exhibited decol-orization of methyl red at temperatures varying from 23 to 37 C,whereas decolorization was completely inhibited at 45 C (Wong</p><p>Fig. 2. Gas chromatogram of degradation products of the Golden Yellow HER extractedafter 48 h.and Yuen, 1996). Degradation products formed and saturated in theculture medium may affect the cell viability and decolorizationactivity. B. laterosporus exhibited the ability to decolorize about 80%</p><p>Table 1GCMS data of the Golden Yellow HER degradation products extracted after 48 h.</p><p>PeakNo.</p><p>RT m/z (percent relative intensity of the predominant ions agged in the</p><p>1. 21.042 43 (73), 57 (27), 73 (27), 83 (47), 107 (7), 113 (100), 139 (10), 141 (33), 16341 (20), 353 (7), 377 (7), 399 (7), 411 (7), 427 (10), 441 (7), 479 (7), 4</p><p>2. 21.733 43 (64), 57 (64), 82 (7), 85 (93), 98 (7), 113 (100), 131 (93), 141 (43), 156(25), 327 (7), 343 (14), 356 (14), 369 (14), 384 (7), 399 (7), 412 (7), 42</p><p>3. 23.433 55 (13), 68 (19), 70 (6), 86 (75), 98 (6), 112 (6), 126 (6), 140 (13), 155 (4. 25.067 53 (56), 69 (50), 70 (56), 85 (50), 100 (56), 121 (38), 134 (50), 141 (100),1</p><p>281 (63), 300 (31), 313 (63), 331 (56), 345 (31), 355 (75), 371 (56), 387(38), 538 (25).control dye showed a single peak at retention time 1.593, whereastwo peaks of the degradation products extracted after 48 h at theretention time 2.299 and 2.453 (data not shown). Overall ndingssuggested biotransformation of the dye. The degradation productsof Golden Yellow HER are analyzed by FTIR analysis. FTIR spectrumof control dye displayed a peak at 3431 cm1 for NeH stretch,a peak at 2924 cm1 for CH3 stretch, a peak at 1574 cm1 foreN]Ne stretch, a peak at 1487 cm1 for ring vibrations, a peak at671 cm1 for CeH bend, a peak at 1188 cm1 for SO2 stretch whereas peaks at 1082, and 1039 cm1 for CeN stretch as well as a peak at615 cm1 for CeCl stretch suggested aromatic azo nature of the dyeand conrmed its chemical structure (Fig. 1). In recent years,vibrational spectroscopies such as Fourier transform infrared (FTIR)(Goodacre et al., 2000; Oberreuter et al., 2002) have been devel-oped for analyzing characteristics of the different samples. FTIRspectrum of degradation products displayed the peaks at 3241,1330, and 1106 cm1 suggested the formation of NeNe disubsti-tuted sulfonamides from the parent dye molecules. Disappearanceof a peak at 1574 cm1 for an azo stretch clearly indicated thebreaking of azo bond by B. laterosporus that would be an essentialand foremost step for the col...</p></li></ul>


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