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Department of Biotechnology, B.V. Bhoomaraddi College of Engineering and Technology, Hubli,. Karnataka, 580031, India; E-mail: [email protected].
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Biodegradation of industrially important textile dyes by actinomycetes isolated from activated sludge Zabin K. Bagewadi, Amitkumar G. Vernekar, Aishwarya Y. Patil, Abhijit A. Limaye, Vandana M. Jain Department of Biotechnology, B.V. Bhoomaraddi College of Engineering and Technology, Hubli, Karnataka, 580031, India; E-mail: [email protected]

ABSTRACT Azo dyes, which are widely used in textile industries when left in water bodies without any treatment cause environmental pollution and in turn are toxic,carcinogenic and mutagenic. The efficient treatment of the sludge from the industries is not economical and a challenging task. Biodegradation of an Azo dye Reactive Yellow was carried out by microorganisms isolated from the activated sludge by enrichment culture techniques. The isolates were maintained on PDA slants with 0.005% (5mg/100ml) of the dye. The isolated organisms were acclimatized to different concentrations of dye in VMM initially from 0.005-0.200% (mg/100ml). Based on the cultural and morphological characteristics the isolates were identified as actinomycetes. The consortium was developed by mixing five actinomycetes and the degradation pattern for 0.01% (mg/100ml) of dye for actinomycetes A, B and Consortium was found to be 97.44%, 97.45% and 94% respectively in 15 days. The results obtained from the experiments revealed that the degradation of dye depends on the concentration of dye as well as the growth of the actinomycetes. The media containing 0.02% (mg/100ml) dye was degraded in 3 days, 0.05% (mg/100ml) in 8 days and 0.1% (mg/100ml) in 15 days by actinomycete B, with adsorption process taking place simultaneously. The growth pattern of the actinomycetes was studied and the concentration of dye adsorbed by the actinomycetes A, B and Consortium was found to be 85mg, 80mg and 44mg in 100ml respectively. The enzymes responsible for degradation such as lignin peroxidase, laccase and tyrosinase showed steady activities during the 8 days of incubation. Biosorption of Reactive Yellow dye was carried out using dead biomass for different concentration of dye and 90% of dye was adsorbed in 1 hour effectively. Keywords: Azo dyes, biodegradation, Reactive Yellow dye, consortium, adsorption, biosorption Abbreviations: PDA - Potato Dextrose Agar, VMM - Vogel’s Mineral Media, MSM - Mineral Salt Media, LiP - Lignin peroxidase

INTRODUCTION Biodegradation of azo dyes is being considered as an environment friendly and cost effective option which also offers environmental control. Environmental pollution due to urbanization and rapid growth of industries has an adverse effect on human health and ecology [1]. Azo dyes constitute the largest and most versatile class of synthetic dyes used in the textile, pharmaceutical, paper, food and cosmetic industry due to their ease in production and variety in colour compared to natural dyes [2,3]. These are the main class of dyes used in textile industries which are one of the reasons of environmental pollution. These are compounds containing one or more R1 = N= R2 groups where R1 and R2 are substituted rings responsible for the intense colour [4,5]. These dyes are produced from known carcinogens and mutagens like benzidene. The annual production of dyes worldwide is about 8×105 metric tons and about 50% of these dyes are azo dyes [6]. The unadhered dye from the textile industries is left into the water bodies without any treatment which significantly affect the Research Article, Biotechnol. Bioinf. Bioeng. 2011, 1(3):351-360 © 2011 Society for Applied Biotechnology. Printed in India; ISSN 2249-9075

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photosynthetic activity of the species present due to reduced light penetration [7]. The different physico-chemical methods used for colour removal of waste are membrane flocculation, filtration, precipitation, adsorption by activated carbon, photodegradation and ozonation [8]. These methods are expensive and less effective as they merely concentrate the dyes and do not degrade them. Hence, secondary treatment such as incineration has to be performed [9]. Microorganisms which are as known nature’s recyclers, convert toxic compounds to harmless products such as carbon dioxide and water [10]. The various organisms which degrade dyes are fungi, bacteria and actinomycetes [1,11]. The dyes are completely decolorized by these organisms in 8 to 10 days [1]. It is also known that a mixture of organisms degrade a dye better as compared to individual organisms as their complexity enables them to act on a variety of pollutants [12]. They are like black boxes whose constituent populations are not known and are used for environmental remediation. The decolourisation takes place in anoxic conditions that is in aerobic conditions the organism grows and in the anaerobic condition they degrade the dye [2]. But the intermediates formed in aerobic conditions are more toxic and difficult to degrade. The degradation rate also depends on the different conditions such as high salinity of the medium, temperature, pH and also static or shaking conditions [13]. The degradation is found to be most effective under static conditions. The best media for degradation was Vogel’s Mineral media as compared to mineral salt media and basal mineral media [4,14]. The media is also supplemented with different carbon and nitrogen sources for better degradation of dye. The carbon source generally used are glucose, sucrose, starch and nitrogen sources used are yeast extract, beef extract and tryptone [4,15]. The organisms are adapted to different concentrations of dyes from 0-250mg/100ml of dye concentration [13]. The mechanism of microbial decolorization of azo dyes involves the reductive cleavage of azo linkages under anaerobic conditions resulting in the formation of colorless aromatic amines. The biodegradation of relatively simple sulfonated amino-benzene and amino-naphthalene compounds under aerobic conditions is simple and effective [16]. White rot fungi such as Phanerochaete chrysosporium, Pleurotus ostreatus and Irpex lacteus have gained importance in the biodegradation of dye for their ability to produce extracellular lignolytic enzymes such as lignin peroxidase (LiP), Mn-dependent peroxidase (MnP), laccase and Mn-independent enzymes which are non-specific and can attack a wide variety of complex aromatic dyestuff [17-20]. Azo reductase activity has been detected in many bacteria, such as Sphingomonas xenophaga BN6, Pigmentiphaga kullae K24 and Caulobacter subvibrioides C7-D and so on [15]. These organisms are efficient in degradation of recalcitrant and xenobiotic compounds. The degradation ability of the organisms also depends on the oxidation potential of the dye [21]. Biosorption of the dyes is also a promising technique which will enhance the color removal of the effluent stream containing a mixture of dye. Biosorption can be defined as a process in which solids of natural origin are employed for sequestration or separation of solids from the effluent streams. The interaction between the cell wall ligands and adsorbates can be explained by ion exchange, complexation, coordination and microprecipitation which are responsible for color removal. For this process living or dead biomass can be used as biosorbent [22]. Living cells require constant nutrient supply and the toxicity of dyes which will harm them. On the other hand dead biomass can avoid such problems and can be regenerated for use [23]. However much less studies have been carried out on degradation of reactive yellow dye by actinomycetes; hence, the present study was carried out to investigate the ability of some actinomycetes to degrade the reactive yellow dye. The present study also investigates the enzymes involved in the degradation and biosorption of dye on dead biomass [24].

MATERIALS AND METHODS Reactive yellow dye was collected from textile industry (Gadag, India). All chemicals used were of the highest purity and of the analytical grade. The organisms were isolated from microbial

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populations present in activated sludge, samples collected from waste disposal sites of textile processing and dye manufacturing units in and around Gadag, India. Inoculum was prepared by dissolving 10% (w/v) of sludge sample in distilled water and filtered using filter paper. The organisms were isolated from the activated sludge containing active microbes by enrichment culture techniques. The sample was serially diluted and organism were isolated by spread plate and pour plate method using MSM (g/l) K2HPO4 (6.3), KH2PO4 (1.82), NH4NO3 (1), MgSO4.7H2O (0.2), CaCl2.2H2O (0.1), FeSO4.7H2O (0.1), Na2MoO42H2O (0.06), MnSO4 (0.06). The pH of medium was adjusted to 7 before autoclaving at 121°C for 20 min. After sterilization the medium was supplemented with 0.02% reactive yellow dye (20mg/100ml) as a sole source of carbon and nitrogen and 500µl of culture inoculums was poured into plates [7]. The consortium was developed by mixing the five actinomycetes and some of the isolates that are capable of degrading the dye (0.05%) were selected as potent isolates in the consortium [1]. The actinomycetes were capable of utilizing dye as the sole source of carbon and nitrogen. Enrichment of isolates was carried out by supplementing 2% glucose (2g/100ml) to the media with dye. The medium used for degradation is VMM 50X (g/L), prepared in water (750ml), with media components of trisodiumcitrate (130), (NH4)2H2PO4 (144), KH2PO4 (80), MgSO4.7H2O (10), CaCl2.2H2O (5), Biotin 10mg/100ml (2.5), trace elements (5ml) [solution (g/l) C6H8O7.H2O (50), Fe(NH4)2(SO4)2.6H2O (10), ZnSO4.7H2O (50), Na2MoO4.2H2O (0.5), MnSO4.H2O (0.5), H3BO3 (0.5), CuSO4.5H2O (2.5)]; make up the final volume to 1litre. The pH of medium was adjusted to 7 before autoclaving at 121°C for 20 min. After sterilization 5% of inoculum was added and adapted to different concentration of dye, ranging from 0.005% to 0.25% in 1X VMM media in 250 ml Erlenmeyer flasks, under static conditions [25,26]. Enrichment of isolates was carried out by supplementing additional carbon source as glucose 2% (2g/100ml) to the media containing reactive yellow dye, for enhanced growth in biomass. Degradation was studied under enriched conditions [4]. The identification of the potential dye degrading organism was done on the basis of its morphological, cultural, and physiological characteristics [27]. Microscopic analysis by Lacto-phenol cotton blue staining was carried out. The organism used for degradation studies were maintained on PDA slants with 0.005% of reactive yellow dye. Samples (1.5 ml) were taken at different intervals under sterile conditions for measurements. The samples were centrifuged at 10,000 rpm for 15 min. The supernatant and cell pellet was evaluated using UV-Visible spectrophotometer (Labomed) method. To determine percentage reduction, the supernatant was read at the λ max = 410nm of the dye and biomass was read at 600 nm [1]. The decolorization percentage was calculated as I-F/I×100, where, I is the initial absorbance and F is the absorbance of decolorized medium. The cell pellet was resuspended in equal volume of methanol to extract the dye adsorbed by the cells. The methanol samples were centrifuged at 10,000 rpm for 15 min and supernatant was read at λ max= 410nm of dye used and the concentration of dye absorbed was calculated using the standard plot [9,28]. The cells were harvested and centrifuged at 10,000 rpm for 10 min at 4°C. The cell pellet was suspended in 50 mM phosphate buffer (pH 7.4) for sonication (Ultrasonic homogenizer, Sartorius) with sonicator out put 80 amplitude maintaining temperature at 4°C and giving 5 strokes each of 10 s with 10 min interval. This extract was centrifuged and the supernatant was used as a source of crude intracellular enzyme [10]. Lignin peroxidase (LiP), laccase and tyrosinase activities were assayed in cell homogenate. LiP activity was determined in reaction mixture of 5ml containing 100mM n-propanol, 250M tartaric acid, 10mM H2O2 by monitoring the formation of propanaldehyde at 300nm [17]. Laccase was determined in a reaction mixture of 2ml containing 5mM O-tolidine, 20mM acetate buffer, 0.2ml of enzyme solution at 366nm. Tyrosinase activity was determined in reaction mixture of 2ml containing 0.01% catechol in 0.1 M phosphate buffer (pH 7.4) at 495 nm [18]. One unit of the enzyme activity was defined as a change in absorbance U/mg

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protein/ min of the enzyme. The protein content was assayed by Bradford method taking Bovine Serum Albumin (BSA) as standard. The cells were autoclaved and were separated through filtration and the biomass was kept for drying in hot air oven at 37°C for 2days. Motor and pestle was used to grind the biomass into powdered form. For adsorption equilibrium experiments, the biosorbent (0-1 g) and the VMM (50 ml) containing dye (0.02%) were placed in a 250ml flask and then shaken for 2 days in a shaking incubator. The dye concentration of the solutions was analyzed using UV-VIS spectrophotometer (Labomed) at a wavelength of 410 nm [29]. The amount of dye adsorption at equilibrium, q (mg/g), was obtained as q = (Ci-Ce) V/W, where q is the equilibrium amount adsorbed on the biomass(mg/ml), Ci is the initial solution concentrations(mg/ml), Ce is the equilibrium solution concentrations (mg/ml),V is the volume of the solutions (ml) and W is the weight of the biomass used (g) [30,31].

RESULTS AND DISCUSSION The organisms were isolated from activated sludge using VMM with reactive yellow dye as sole source of carbon and nitrogen. Six different isolates were obtained. The isolates were round and had pits, elevated and had hard and leathery morphological characteristics. The colonies were adhered to the media tightly and some colored pigments were also produced on the surface of the colony. Microscopic observation of the isolates showed spore chain arrangement. Similar results were obtained for actinomycete strain Streptomyces olivochromogenes which showed both aerial and substrate mycelium and the microscopic view showed the spore chain arranged in spirals with smooth surfaces. Nearly 30-50 spores were present in the spore chain [27]. Thus the morphology was similar to that of actinomycetes. The reductive cleavage of the –N=N– bond is the initial step in the degradation of azo dyes. Decolorizing of azo dyes occurs under aerobic, anaerobic and microaerobic conditions. There are few organisms that are able to grow on azo compounds as the sole carbon source. These reductively cleave the azo bonds and use the amines as their source of carbon and energy [31]. The reactive yellow dye was scanned using UV-VIS spectrophotometer and the maximum absorbance was found at 410 nm and is shown in figure 1. Decolorisation of reactive yellow dye was varied at initial concentration (20-200mg /100ml) under static condition. The growth and degradation pattern of actinomycete A and B was studied as shown in figure 2 and figure 3 respectively. The consortium was used for degradation of 0.1% dye concentration and the growth curve was studied as shown in figure 4. At 20, 50 and 100 mg/100ml reactive yellow dye concentration, 100%, 99%, 98% dye degradation was observed respectively by actinomycete B. The degradation pattern of 0.05% of Reactive yellow dye by actinomycete B is shown in figure 7 and its respective adsorption pattern is shown in figure 8. The time required for respective decolourization was 3, 8, 15 days. As the concentration of dye was increased the time required for decolorisation varied from 15 to 40 days (100-200mg/100ml). Similarly, the time required for degradation increased with increasing concentrations of the Direct Red 5B from 50-1000mg/l by Sphingobacterium sp. [26]. But the decolorisation was not observed at 250mg/100ml, instead the high dye concentration was toxic for the cell growth. The depression of dye decolorization at higher dye concentration is due to higher inhibition at high dyestuff concentration [31]. It is usually noticed that consortium can better degrade the dye which was not true for our consortium, this may be due to the co-metabolism of isolates to degrade dye which inhibited the process. This was also found as the organism EAP3 could not survive in the consortium in spite of showing a good degradation of 82% when inoculated alone at a dye concentration of 50% of Sirius Yellow dye [1]. Another phenomena observed was as the concentration of dye increased the degradation rate of dye decreased and in turn the rate of adsorption of dye on cell surface increased [28]. At 100% reactive yellow dye concentration the

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decolorization of dye was 97%, 98%, 94% as shown in figure 5 and the adsorption of dye on cell was 87.27, 80, 44.45mg/100ml for actinomycetes A, B and consortium respectively as shown in figure 6.

Figure 1. λmax for reactive yellow dye was found to be 410nm.

Figure 2. Time course evolution of isolate A growth by taking OD at 600 nm and concentration of reactive yellow dye remaining in supernatant at initial concentration of 100%.

Dye biosorption indicated that the dominant mechanism of dye removal by the fungus was probably bioaccumulation [24] and extraction with methanol recovered the major part of the decolorized dye under static conditions of Acid Black 210 by Vibrio harveyi and is mainly due to adsorption [32]. The enrichment studies was done by adding glucose as carbon source along with reactive yellow dye which showed better growth of the isolates and hence the dye degradation was more effective. The enrichment studies of actinomycete B resulted in 97.11% dye degradation and

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0.375 mg/100 ml of dye adsorbed on the cell surface on day 9. Large portion of the dye was degraded and very little portion of dye was adsorbed on the cells due to better growth. The effect of different carbon and nitrogen sources was also carried out in this work [18] and molasses was found to be the best carbon source, although by addition of glucose degradation of dye was found to be 80%.

Figure 3. Time course evolution of isolate B growth by taking OD at 600 nm and concentration of reactive yellow dye remaining in supernatant at initial concentration of 100%.

Figure 4. Time course evolution of Consortium growth by taking OD at 600 nm and concentration of reactive yellow dye remaining in supernatant at initial concentration of 100%.

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Figure 5. Reactive yellow dye degradation at initial dye concentration of 100% under static conditions by different isolates with respect to time.

Figure 6. Adsorption profile of reactive yellow dye by different isolates with respect to time.

The intracellular enzymes were extracted from the isolate B at 0.05% (mg/100ml) dye concentration with enrichments was subjected to enzyme assay. LiP, tyrosinase, laccase assay showed that the degradation of dye was mainly due to mixture of these enzymes. The time course of laccase, tyrosinase and LiP is shown in figure 9. The enzyme activity decreased with decrease in dye concentration and hence the total protein content also decreased. The highest activity of laccase, tyrosinase, LiP was found to be 0.223, 0.104, 0.309 (unit/mg/min) on day 9, day 6 and day 3 respectively which was also stated in saying that after complete decolorization of dye enzymes like tyrosinase, laccase, manganese peroxidase activity were decreased in 96 h in the batch culture [18]. The biosorption technique showed 90% dye adsorption by dead biomass of actinomycete B in 1 hour for 0.02% and 0.05% of reactive yellow dye concentration. The maximum uptake of reactive

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yellow dye was found to be 13.19mg/g and 22.31mg/g respectively of dead biomass which was also

investigated by others [30]. In this article, the dead biomass of Aspergillus niger was taken and biosorption was observed to be complete in 10 minutes. Reports [33] showed that adsorption by Corynebacterium glutamicum resulted in the maximum uptake of dye reactive yellow 2 at pH of 2 to be 155 mg/g of dead biomass.

Figure 7. Degradation studies of reactive yellow dye by isolate B (50mg/100ml) with enrichment.

Figure 8. Adsorption of reactive yellow dye by isolate B on its cell surface at initial concentration of 50mg/100ml with enrichment.

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Figure 9. Time course of laccase, tyrosinase, and LiP activity in Units/mg/min. in isolate B during reactive yellow dye degradation at initial concentration of 50mg/100ml with enrichment.

The present research work reports a novel decolorization process by actinomycetes A and B, which were able to degrade reactive yellow dye with the help of lignolytic enzymes such as lignin peroxidase, laccase and tyrosinase activity produced at static condition. The addition of glucose as additional carbon source has a significant effect on the degradation of dye and growth of organism. Also the complete degradation was carried out in aerobic condition hence these actinomycetes can be employed for degradation of reactive yellow dye coupled with biosorption for effective removal of colour from the effluent stream.

REFERENCES [1] Senan RC, Abraham TE. Biodegrad. 2004, 15:275-280. [2] Pandey A, Singh P, Iyengar L. Int J Biodeter. Biodegrad. 2007, 59:73-84. [3] Isika M, Teresa SD. Enzyme Microb Technol. 2007, 40:934-939. [4] Barraga BE, Costa C, Marquez MC. Dyes and Pigments 2007, 75:73-81. [5] Jirasripongpun K, Nasanit R, Niruntasook J, et al. Thammasat Int. J. Sc. Technol. 2007, 12:6-11. [6] Zhao X, Hardin IR. Dyes and Pigments 2007, 72:322-332. [7] Deivea FJ, Domingueza A, Barrioa T, et al. J. Hazard Material 2010, 182:735-742. [8] Junnarka N, Srinivas Murty D, Bhatt NS, et al. World J Microbiol. Biotechnol. 2006, 22:163-168. [9] Modi HA, Rajput G, Ambasana C. Biores. Technol. 2010, 101:6580-6583. [10] Joshi SM, Inamdar SA, Telke AA, et al. Int. J. Biodeter. Biodegrad. 2010, 64:622-628. [11] Asgher M, Shah SAH, Ali M, Legge RL. World J Microbiol. Biotechnol. 2006, 22:89-93. [12] Dafale N, Nageswara Rao N, Sudhir U, et al. Biores. Technol. 2008, 99:2552-2558. [13] Mona HM, Mabrouk EM, Yusef HH. J. Appl. Sci. Res. 2008, 4:262-269. [14] Asgher M, Shah SAH, Ali M, et al. World J Microbiol. Biotechnol. 2006, 22:89-93. [15] Gou M, Qu Y, Zhou J, et al. J. Hazardous Materials 2009, 170:314-319. [16] Pinheiro HM, Touraud E, Thomas O. Dyes and Pigments 2004, 61:121-139. [17] Chaube P, Indurkar H, Moghe S. Asiatic J. Biotechnol. Res. 2010, 3:220-226. [18] Kamle SD, Parshetti GD, Jadhav SU, et al. Biores. Technol. 2006, 07:1405-1410. [19] Saratale RG, Saratale GD, Kalyani DC, et al. Biores. Technol. 2009, 100:2493-2500. [20] Mane UV, Gurav PN, Deshmukh AM, et al. Malaysian J. Microbiol. 2008, 4:1-5. [21] Tauber MM, Gubitz GM, Rehorek A. Biores. Technol. 2008, 99:4213-4220.

360 [22] Bhole BD, Ganguly B, Madhuram A. Curr. Sci. 2004, 86:12-25. [23] Malarvizhi R, Wang MH, Ho YS. World Appl. Sc. J. 2010, 8:930-942. [24] Sivarajasekar N, Baskar R, Balakrishnan V. J. Chem. Technol. Metall. 2009, 44:157-164. [25] Azeem Khalid, Muhammad Arshad , David E.Crowley. Appl Microbiol Biotechnol. 2008, 78:361-369. [26] Tamboli DP, Kagalkar AN, Jadhav MU, et al. Biores. Technol. 2010, 101:2421-2427. [27] Balagurunathan R, Radhakrishnan M, Somasundaram ST. Aust. J. Basic Appl. Sci. 2010, 4:698-705. [28] Khehra MS, Saini HS, Sharma DK, et al. Water Research 2005, 39:5135-5141. [29] Wu J, Kyoung-Sook KIM, Sung NC, et al. J. Gen. Appl. Microbiol. 2009, 55:51-55. [30] Franciscona E, Piubelia F, Garbogginic FF, et al. Enzyme and Microbial Technology 2010, 46:360-365. [31] Junnarkar N, Srinivas Murty D, Bhatt NS, et al. World J Microbiol. Biotechnol. 2006, 22:163-168. [32] Ozdemir G, Pazarbasi B, Kocyigit A, et al. World J Microbiol Biotechnol. 2008, 24:1375-1381. [33] Won SW, Han MH, Yun YS. Water Research 2008, 42:4847-4855.