BIOLOGICAL DECOLORIZATION OF CARCINOGENIC AZO DYE: AN ECOFRIENDLY APPROACH

Transcription

1 Original Research Article Biotechnology International Journal of Pharma and Bio Sciences ISSN BIOLOGICAL DECOLORIZATION OF CARCINOGENIC AZO DYE: AN ECOFRIENDLY APPROACH MERINA PAUL DAS* 1, MANAS BHOWMICK 1 AND MAGGIDI REYNOLDS 1 *1Department of Industrial Biotechnology, Bharath University, Chennai, India ABSTRACT Dyes are extremely used in textile, leather, paper industries for colorations. But unsafe disposal of these dyes into the environment causes serious damage and negative effect to the human, plant and animal health. Microbial decolorization is a low cost and is frequently applied process for the decolorization of textile dyes. Methyl red (MR) is a carcinogenic textile dye which is used for the coloration of various products. The objective of the study was to isolate the active bacterial strains which can decolorize the azo dye Methyl red. Among thirteen isolates, four potential bacteria (MR-1, MR-2, MR-3, MR-4) with methyl red decolorizing ability were selected for identification and used for the decolorization process. All the four microorganisms showed maximum dye degradation after 48 h of incubation. The improvement of the decolorization of methyl red was obtained by optimization of various nutritional and environmental factors for each isolate. The results from this study show the potential and practical applications of these bacterial isolates to treat the textile effluent containing MR dye. KEYWORDS: Azo Dye, Methyl red, Decolorization, Optimization. MERINA PAUL DAS Department of Industrial Biotechnology, BharathUniversity,Chennai,India. B

2 INTRODUCTION Textile effluent released from industries is a complex mixture of many polluting substances such as organo chlorine based pesticides, heavy metals, pigments and dyes 1 and must be treated before discharged into environment because of their recalcitrant nature and potential toxicity to animals and human 2-4. Azo dyes account for about one-half of all dyes produced and are the most commonly used synthetic dyes in the textile, food, paper making, color paper printing, leather and cosmetic industries 5. The release of high colored compounds into water bodies is undesirable not only because of their impact on photosynthesis of aquatic plants, algae by absorption of light but also due to the carcinogenic nature of many of these dyes and their breakdown products 6. However, beyond color, the presence of these dyes in aqueous ecosystems presents serious environmental and health concerns as a result of the toxicity of the free dyes themselves and their transformation into toxic, mutagenic and carcinogenic amines, primarily as result of anaerobic microbial reductive cleavage of the azo bond 6-8, as a result dye decolorization has been a primary goal of dye wastewater treatment processes.there were many reports about the use of physiochemical methods for dye removal from colored effluents Currently used physiochemical techniques for the treatment of colored wastewaters such as: adsorption, precipitation, chemical oxidation, photodegradation or membrane filtration have serious restrictions because high costs caused by intensive energy requirements, formation of hazardous by-products 12, generation of significant amount of sludge, which must 13 be handled and can cause secondary pollution problems Microbial decolorization is an ecofriendly and cost competitive alternative to chemical decomposition process 17 and effective in removing dyes from large volumes of effluents 15. A number of microorganisms have been found to be able to decolorize textile dyes including bacteria, fungi, and yeasts 18.Methyl red (MR) or C.I. Acid Red is a synthetic azo dye, and is a dark red crystalline powder. It is a commonly used monoazo dye in laboratory assays, textiles and other commercial products; however, it may cause eye and skin sensitization 19 and pharyngeal or digestive tract irritation if inhaled or swallowed 20. Azo dyes like methyl red are known to be major human carcinogens besides being environmental damage. The present study was focused on screening of indigenous bacterial strains, isolated from dye contaminated sites, which had the potential to decolorize the textile dye Methyl red in vitro conditions and optimization of the factors influencing the process. MATERIALS AND METHODS Dyes and chemicals The diphenyl azo dye used was Methyl red (MR) and the chemical structure of Methyl red shown in Figure 1. The textile dye Methyl red (2-(N,N-Dimethyl-4- aminophenyl)azobenzenecarboxylic acid) was obtained from Sigma-Aldrich, (USA). The MR dye (Molecular formula: C 15 H 15 N 3 O 2 ) has a molar mass of g mol 1. The absorbance was measured with a UV-vis spectrophotometer at the maximum absorption wavelengths (λ max = 410 nm). All the media and chemicals were of analytical grade with highest purity and procured from Hi-Media, Mumbai (India). Figure 1 Chemical structure of Methyl red Sample collection Samples were collected from the zone of industry, Nagalkeni (Latitude 12 57'39" N; Longitude 80 8'13" E), Chennai, Tamil Nadu; from different places such as drainage canal at 2-3 cm depth of soil surface those carry dye effluent which is 2 km far from the industries. Samples were in the form of untreated soil from which dye degrading bacteria were isolated. All the samples were collected in sterile polythene bag and preserved at 4 C in refrigerator and samples were tested within 24 h of collection. Isolation and screening of dye-decolorizing bacteria The soil samples were serially diluted up to 10-8 dilutions, in distilled water and 1 ml sample from 10-3 to 10-7 were pour plated in Nutrient Agar (NA) (Hi-Media, Mumbai, India) plates. The plates were kept for incubation at 37 C for 24 h in an inverted position. Well grown bacterial colonies were picked and further purified by streaking. Isolated morphologically distinct bacterial isolate were tested for their ability to decolorize textile azo dye, Methyl red by streaking the isolate on dye contained agar test tubes separately which were kept at 37 C for 48 h. The concentration for azo dye was 1.0 mg/ml. The isolates B

3 showed significant decolorization of dyes, were used for further work. Biochemical identification of dye degrading isolates Selected isolates were grown on nutrient agar (Hi-Media, India) plates. Based upon the staining reactions, physiological and biochemical characteristics 21 of the isolates were identified according to Bergey's Manual of Determinative bacteriology 22 and the organism was identified upto genus level. Textile dye decolorization assay Decolorization activity was expressed in terms of percentage decolorization and was determined by monitoring the decrease in absorbance of the dye. The culture media used for decolorization study contained (g/l); Peptone 5; Yeast extract 3; Beef extract 2; NaCl 5; K 2 HPO 4 5; KH 2 PO 4 1; MgSO 4.7H 2 O 0.1. The ph of the medium was adjusted to ml of sterile media amended with textile dye (1.0 %) and subsequently inoculate with potential dye decolorizing strains. The flasks were kept in mechanical shaker (200 rpm) and incubated at 37±1 C for 48 h. After 2 d, aliquots of the culture media were withdrawn, centrifuged at 10,000 rpm for 10 min at room temperature to separate the bacterial cell mass. The supernatant was used for analysis of decolorization and the uninoculated isolate media supplemented with dye was used as control. The percentage of decolorization of the tested dye was determined by measuring the absorbance of the supernatant at λmax of the dye i.e., 410 nm 23. The percentage decolorization was calculated according to the following formula Optimization of parameters Optimization of dye decolorization was carried in 250 ml Erlenmeyer flasks with 100 ml of medium and incubated at orbital shaker (200 rpm) for 48 h at 35 C. In all optimization study, 1.0 % of dye was used. This batch experiment was run under various culture conditions varying one at a time while keeping others constant. All decolorization experiments were performed in three sets. Effect of temperature on dye decolorization In order to optimize the decolorization process of Methyl red, the decolorization was carried out at wide range of temperature. 250 ml of decolorization media supplemented with inoculums and dye, and incubated at temperatures 25, 30, 35, 40 and 45 C for 48 h while kept other parameters constant. The % dye decolorization was measured. Effect of ph on dye decolorization After optimization of incubating temperature, media ph was adjusted into 4, 5, 6, 8 and 9 by addition of either 1N NaOH or 1N HCl in order to study its effect on decolorization of Methyl red by isolated strain at respective optimized temperature. The % dye decolorization was measured of each ph separately for dye decolorizing bacteria while other parameters were same. Effect of carbon source on dye decolorization To study the effect of carbon on decolorization of Methyl red, monosaccharide, disaccharide and polysaccharides were used. For efficient decolorization, different carbon sources such as fructose, glucose, lactose, sucrose and starch (0.1 % each) were added individually to culture media with Methyl red (1 mg/ml) for inoculums and kept for 48 h in shaking incubator at respective optimized temperature, and ph. The % dye decolorization was measured of each carbon sources separately for dye decolorizing bacteria. Effect of nitrogen source on dye decolorization To study the effect of nitrogen on maximum decolorization of Methyl red, different nitrogen sources such as, urea, beef extract, peptone, ammonium sulfate and sodium nitrate (0.1 % each) were added individually to culture media and inoculated with isolate keeping other factors constant. All the flasks were kept for 48 h in shaking conditions at respective optimized temperature, ph, and carbon sources and they were analyzed for percent decolorization. RESULTS AND DISCUSSION Screening and identification of potential strain A total of thirteen morphologically different colonies of bacteria were isolated, purified and screened for biodecolorization study of Methyl red. Of the cultures were tested, finally four bacterial isolates were selected on the basis of dye decolorization from moderate to intense. The isolation of different bacteria from the sample indicates the natural adaptation of isolates to survive in the presence of toxic dye. The potential strains were designated as MR-1, MR-2, MR-3 and MR-4 which were identified by the phenotypic characteristics and other biochemical tests. Based on the morphological and cultural characteristics of MR-1, MR-2, and MR-4 were identified as Pseudomonas sp. and MR-3 was identified as Staphylococcus sp. comparing with Bergey s manual of determinative bacteriology (Table 1). B

4 Table 1 Morphological, physiological and biochemical characteristics of the dye decolorizing strains Characters Isolate MR-1 Isolate MR-2 Isolate MR-3 Isolate MR-4 Morphology Rod shaped, gram - Rod shaped, gram -ve, irregular Cocci shaped, gram +ve, rough Rod shaped, gram -ve, raised ve, smooth green fluroscent yellow colour raised yellow colony, green colour colony, obligate colour colony, colony,obligate aerobic and without facultative anaerobic and aerobic and without obligate aerobic and endospore without endospore endospore without endospore Motility +ve +ve -ve +ve Catalase +ve +ve +ve +ve Oxidase +ve +ve -ve +ve Methy red -ve -ve -ve -ve Voges- Proskauer -ve -ve +ve -ve Indole production -ve +ve -ve -ve Citrate utilization +ve +ve +ve +ve Nitrate reduction +ve +ve +ve -ve Hydrolysis of starch -ve -ve -ve +ve Fermentation Glucose -ve +ve +ve -ve Lactose -ve -ve +ve -ve Sucrose -ve -ve +ve -ve Mannitol +ve -ve +ve +ve Optimization of growth conditions for maximum decolorization All the four bacterial cultures was incubated in nutrient media of varying cultural conditions with 0.1 % of Methyl red dye for 48 h under shaking conditions separately. The optimization study was done using different physicchemical parameters, such as, temperature, ph and nutritional factors, such as, carbon and nitrogen sources. After the incubation period, the cell free extract were used to determine the optimum decolorization ability of the isolates of added dye. Percentage of decolorization was calculated with respect to control. Effect of temperature on decolorization of dye Incubation temperature is known to influence directly the overall growth and development of any organism, hence the temperature effects significantly on the decolorization study. The temperature effect was investigated in a wide range from C. As evident from the data presented in Figure 2, all the isolates showed maximum % decolorization of Methyl red at 35 C. The percentage of decolorization decreased with temperature either increased or decreased. Much lower as well as higher temperature failed to support any growth or biodecolorization process. Among the Methyl red by dye decolorizing strains, Staphylococcus sp. MR-3 produced significant activity. This optimum temperature was used for each isolate in further optimization process. Figure 2 Effect of temperature on decolorization of Methyl red by dye decolorizing strains B

5 Effect of ph on decolorization of dye In order to determine the optimum ph for high level of biodecolorization, the test strains were grown in nutrient medium with different hydrogen ion concentrations at optimized temperature. Figure 3 shows the decolorization efficiency of Methyl red by dye decolorizing strains. The effect of ph on biodecolorization efficiency of MR was examined in the range of 4 to 9. As bacterial growth is ph dependent, each isolate showed maximum dye decolorization at specific ph. Based on the results, the optimum ph for MR decolorization was for MR-1 at 4, for MR-2, 3 at 6, and for MR-4 at 9 which were used for the rest of the study. Among these isolates, MR-3 produced a maximum decolorization of 86 % at ph 6. Figure 3 Effect of ph on decolorization of Methyl red by dye decolorizing strains Effect of carbon sources on decolorization of dye A variety of carbon sources such as, monosaccharides, disaccharides, polysaccharides are known to be utilized by bacteria, are known to influence its growth and therefore decolorization activity. To investigate the role of nutritional factors on biodecolorization, the percent of decolorization was measured using different carbon sources for each Methyl red dye decolorizing bacteria with optimized temperature and ph. Similarly like ph, here also each isolate showed maximum dye decolorization at specific carbon sources. The optimum carbon source for Methyl red was found as fructose for MR-1 and MR-2 and lactose for MR-3 and MR-4 (Figure 4). Among these isolates, MR-3 produced a high level of decolorization of 88 %. Figure 4 Effect of carbon sources on decolorization of Methyl red by potential strains B

6 Effect of nitrogen sources on decolorization of dye Bacterial growth is largely affected by nitrogen sources utilized by them, which reflects on its decolorization activity. Five different nitrogen sources at a concentration of 0.1 % were used in the present study to find out the ideal nitrogen sources having enhanced MR biodecolorization. The test was performed for each potential strain with their respective optimum temperature, ph and carbon sources. The optimum nitrogen source was found as peptone for MR-1 and MR-3, ammonium sulphate for MR-2, and urea for MR-4 (Figure 5). Maximum MR decolorization was exhibited by MR-3 and MR-4 than MR-1 and MR-2, but appreciable result was showed by Staphylococcus sp. MR-3 of 93 % with all other optimized parameters compared to MR-4 (90 %). Figure 5 Effect of nitrogen sources on decolorization of Methyl red by potential strains The isolated strain Staphylococcus sp. MR-3 showed significant MR decolorization (dye concentration of 1.0 %) result which is higher than Bacillus subtilis (89 %) used for decolorization of Methyl red with concentration of % 24. Therefore, this bioremediation process can be scaled up with the optimized conditions and the potential strain Staphylococcus sp. MR-3 can be used as useful ecofriendly tool to treat the waste water containing Methyl red dye. CONCLUSION the presence of dyes with complex aromatic structure. These azo dyes are generally recalcitrant to biodecolorization due to their xenobiotic nature. In this study, Methyl red decolorizing strains were screened and various physicochemical and nutritional parameters were optimized in order to get maximum decolorization. Under optimal conditions, Staphylococcus sp. MR-3 completely decolorized 1mg/ml of MR within 48 h of incubation. The high MR decolorization ability enables this bacterium to be used in the biological treatment of industrial effluent containing azo dyes. Dye industry waste water is difficult to treat because of REFERENCES 1. Saraswathy K, Balakumar S, Biodecolorization of Azo Dye (Pigmented red 208) Using Bacillus firmus and Bacillus laterosporus, Journal of Biosciences Technology, 1: 1 7, (2009). 2. Levine WG, Metabolism of Azo Dyes: Implication for Detoxification and Activation, Drug and Metabolic Research, 23: , (1991). 3. Hildenbrand S, Schmahl FW, Wodarz R, Kimmel R, Dartsch PC, Azo Dyes and Carcinogenic Aromatic Amines in Cell Cultures, International Archives of Occupational Environment and Health, 72: 52 56, (1999). 4. Martins MAM, Queiroz MJ, Silvestre AJD, Lima N, Relationship of Chemical Structure of Textile Dye on the Preadaptation Medium and the Potentialities of their Biodegradation by Phanerochaete chrysosporium, Research Microbiology, 153: , (2012). 5. Chang SJ, Lin YC, Decolorization kinetics of recombinant Escherichia coli strain harboring azo dye decolorization determinants for Rhodococcus sp., Biotechnology Letters, 23: , (2001). 6. Weisburger JH, Comments on the history and importance of aromatic and heterocyclic amines in public health, Mutation Research, 506/507: 9 20, (2002). B

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