Bioresource Technology 99 (2008) 8999–9003
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Decolorization and degradation of Disperse Blue 79 and Acid Orange 10, by Bacillus fusiformis KMK5 isolated from the textile dye contaminated soil Yogesh M. Kolekar a, Shrikant P. Pawar b, Kachru R. Gawai a, Pradeep D. Lokhande a, Yogesh S. Shouche b, Kisan M. Kodam a,* a b
Biochemistry Division, Department of Chemistry, University of Pune, Pune, Maharashtra 411007, India National Centre for Cell Science, Pune University Campus, Pune, Maharashtra 411007, India
a r t i c l e
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Article history: Received 9 October 2007 Received in revised form 30 April 2008 Accepted 30 April 2008 Available online 17 June 2008 Keywords: Acid Orange 10 Bacillus fusiformis Biodegradation Disperse Blue 79
a b s t r a c t The release of azo dyes into the environment is a concern due to coloration of natural waters and due to the toxicity, mutagenicity and carcinogenicity of the dyes and their biotransformation products. The dye degrading bacterial strain KMK 5 was isolated from the textile dyes contaminated soil of Ichalkaranji, Maharashtra, India. It was identiﬁed as Bacillus fusiformis based on the biochemical and morphological characterization as well as 16S rDNA sequencing. KMK 5 could tolerate and degrade azo dyes, Disperse Blue 79 (DB79) and Acid Orange 10 (AO10) under anoxic conditions. Complete mineralization of DB79 and AO10 at the concentration of 1.5 g/l was observed within 48 h. This degradation potential increased the applicability of this microorganism for the dye removal. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Dyes and dyestuffs are widely used within the food, pharmaceutical, cosmetic, textile and leather industries. Over 10,000 commercially available dyes exist and more than 7 105 tons of dyestuffs are produced annually. Among these dyes, azo dyes are the most widely used; these account for over 60% of the total number of dye structures known to be manufactured (Meyer, 1981; Zollinger, 1987). Because color in wastewater is highly visible and affects esthetics, water transparency and gas solubility in water bodies, and especially because many dyes are made from known carcinogens, such as benzidine and other aromatic compounds, dye wastewaters have to be treated (Banat et al., 1996). Azo dyes can be distributed in monoazo, diazo, and triazo classes, are available in six application categories: acid, basic, direct, disperse, azoic, and pigments. Since 1970s, there is signiﬁcant increase in the use of disperse dyes (Weber and Adams 1995). The largest amount of azo dyes are used for the textile dyeing, and it has been estimated that about 10% of the dyestuff used during the dyeing process does not bind to the ﬁbers and therefore released into sewage treatment system or the environment (Clarke and Anliker, 1980; Chudgar, 1985). Since some of the dyes are harmful, dye containing waste pose an important environmental problem (Verma and Madamwar, 2003). Decolorization of azo dyes
* Corresponding author. Tel./fax: +91 20 25691728. E-mail address: [email protected]
(K.M. Kodam). 0960-8524/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2008.04.073
by bacterial strains typically initiated by azoreductase involving anaerobic reduction or cleavage of azo bond (Zimmermann et al., 1982). Reductive cleavage of azo bond leading to the formation of aromatic amines is the initial reaction during the bacterial metabolism of azo dyes. The resulting metabolites could further be degraded under aerobic or anaerobic conditions (Seshadri et al., 1994) by a mixed bacterial community (Chung and Stevens, 1993). Therefore, an aerobic–anaerobic sequential environment was proposed for bacterial degradation or mineralization of azo dyes (Banat et al., 1996). In recent years, many studies have been focused on various microorganisms, which are able to biodegrade and biosorb the dyes in wastewater. These include bacteria, fungi and algae, capable of decolorizing a wide range of dyes with high efﬁciency (Fu and Viraraghavan, 2002). The decolorization of sulfonated azo dyes by different Pseudomonas species under static conditions (anaerobic incubation of azo dyes) has been reported (Banat et al., 1996; Puvaneshwari et al., 2002). But most of these studies have emphasized only on the decolorization/degradation of dye wastewater, not discussing much about the product released by the cleavage of azo group. Unfortunately most azo dyes are recalcitrant to aerobic degradation by bacterial cells (Bras et al., 2001). Dispersed blue 79 (DB79) is one of the colorants with more application in the textile industry. It is used in the dyeing of polyester, nylon, diacetate and triacetate of cellulose as well as in acrylic ﬁbers. The reductive cleavage of the azo linkage of the DB79 results in the formation of 2-bromo-4,6-dinitroaniline, which is toxic and mutagenic (Weber and Adams 1995).
Y.M. Kolekar et al. / Bioresource Technology 99 (2008) 8999–9003
The main objective of the study was to observe the decolorization and degradation potential of the isolated bacterium from the textile contaminated site and propose a plausible mechanism of degradation.
with the dye AO10 and DB79 at a concentration of 0.25, 0.5, 0.75, 1.0, 1.25 and 1.5 g/l to study the time duration required for the dye decolorization. 2.7. Extraction and analysis of biotransformed products
2. Methods 2.1. Microorganism and culture medium The microorganism present in the soil from the efﬂuent disposal site of a textile-dyeing industry located in Ichalkaranji (Maharashtra, India) was enriched in nutrient broth medium containing 50 mg of the dye/l and later on increasing the dye concentration. The visibly morphological different isolates were isolated and the one with the highest dye degradation potential was used for the further study. The bacterial isolate (KMK 5) was cultured at 37 °C in nutrient medium (pH 9.0) (Kodam et al., 2005). The culture was maintained on nutrient agar (Hi Media, Mumbai, India) slants at 4 °C as well as glycerol stocks at 20 °C. 2.2. PCR ampliﬁcation and DNA sequencing of the 16S rDNA gene The PCR ampliﬁcation and DNA sequencing of the 16S rDNA of the strain KMK5 was carried out as described earlier (Vijaykumar et al., 2007). 2.3. Dyestuff and chemicals The textile dyes, DB79 and AO10 were a generous gift from local textile industry, Ichalkaranji, Maharashtra, India. All the chemicals used were of the highest purity available and of analytical grade. 2.4. Analytical methods Absorbance of the supernatant withdrawn at different time intervals were measured at the maximum absorption wavelength for the dye DB79 (kmax = 547 nm) and AO10 (kmax = 480 nm) in the visible region on a Shimadzu double beam spectrophotometer (UV 1601). The percentage of decolorization was calculated from the difference between initial and ﬁnal absorbance values. 2.5. Optimization of parameters To study the effect of static anoxic and shaking condition (100 rpm) on the decolorization efﬁciency of KMK5, the isolate was cultivated for 24 h in conical ﬂasks containing 100 ml nutrient broth and was amended separately with 0.5 g/l of AO10 and DB79. To determine the effect of pH on decolorization the isolate KMK5 was cultivated for 24 h in conical ﬂasks containing 100 ml nutrient broth of varying pH (6–11) and was amended with 0.5 g/l of AO10 and DB79 separately. Similarly, the optimum temperature of KMK5-mediated dye decolorization was determined by evaluating the dye decolorization at 20, 25, 30, 37 and 45 °C. The effect of salt concentration on the dye decolorization efﬁciency of KMK5 was determined in the presence of NaCl (1–4%). After different time intervals aliquot (5 ml) of the culture media was withdrawn, centrifuged at 10,000g for 10 min in a refrigerated centrifuge (Dupont Sorvall RC-5B) to separate the bacterial cell mass. The supernatant was used for analysis of decolorization and all the experiments were repeated in triplicates. 2.6. Concentration studies The culture KMK5 was cultured for 24 h in conical ﬂasks containing 100 ml nutrient broth. After 24 h, the media was amended
The supernatants after decolorization were extracted with dichloromethane and dried over anhydrous sodium sulfate. The solvent was evaporated and the residue was ﬁrst examined by thin layer chromatography. It was further puriﬁed by column chromatography using silica and the fractions were collected and subjected to GC–MS and IR spectroscopy. GC–Mass Spectra were recorded on Shimadzu Gas Chromatograph Mass Spectrometer (GCMS-QP 5050) and infrared spectra were determined on Shimadzu FT-IR Spectrophotometer (FT-IR-8400).
3. Results and discussion 3.1. Identiﬁcation of the isolated strain This isolate KMK5 was identiﬁed as Bacillus fusiformis on the basis of its 16S rDNA sequence. The 16S rDNA sequence KMK5 was a continuous stretch of 1403 bp. The strain KMK5 is deposited in National Collection of Industrial Microorganisms (NCIM), Pune, India, with the accession number NCIM 5294. The sequence of the 16S rDNA gene of the strain KMK5 is available under the GenBank accession number EU090727. 3.2. Evaluation of optimum conditions The dye decolorization of azo dyes AO10 and DB79 were studied under static and agitation conditions with an initial dye concentration of 0.5 g/l. It was observed that under static anoxic conditions, the dye decolorization of DB79 and AO10 was 75% and 90% within 24 h as compared to 37% and 30% decolorization under agitation, respectively (Fig. 1a). Therefore, static conditions were adopted to investigate bacterial dye decolorization in further experiments. The results were similar to those of studies on Escherichia coli NO3 (Chang and Kuo, 2000), Pseudomonas luteola (Chang et al. 2001) and Rhodopseudomonas palustris (Liu et al., 2006). It was speculated that under agitation conditions, the aerobic respiration of the strain might dominate the utilization of NADH and deprive the azoreductase from obtaining electrons from NADH to decolorize azo dyes (Stolz, 2001). The optimum pH for KMK5-mediated dye decolorization was pH 9.0. Nonetheless, KMK5 was also capable of decolorizing the dyes at a wider pH range of 6–11 with an appreciable efﬁciency on either side of the pH optima (Fig. 1b). Majority of the azo dye reducing species of Bacillus and Pseudomonas reported (Kalme et al. 2007; Chang et al., 2001; Suzuki et al., 2001) so far were able to reduce the dye at pH near neutrality. The decolorization of the dyes AO10 and DB79 were studied within a temperature range of 20 to 45 °C. The optimum temperature for the KMK5-mediated dye decolorization was 37 °C (Fig. 1c). The bacterial isolate KMK5 was also tested for its ability to decolorize the dyes DB79 and AO10 in the presence of increasing salt concentration (0.5–4%). It was observed that 85–95% decolorization of AO10 was observed within 48 h for 0.5–3% salt concentration, However, signiﬁcant dye decolorization of AO10 (78%) was observed in the presence of 4% salt concentration (Fig. 1d). The ability of KMK5 to reduce the dye AO10 even at high salt concentration (up to 4%) suggested that KMK5 is a halo-tolerant culture and can be used to treat the efﬂuents containing high amounts of salt. The increase in salt concentration (above 1%) signiﬁcantly affects the decolorization of DB79.
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Fig. 1. Effect of (a) agitation, (b) pH, (c) temperature and (d) salinity, and on the dye decolorization efﬁciency of KMK5 was studied for 24 h in nutrient medium containing 0.5 g/l dye, 1% NaCl, at 37 °C, pH 9.0 and under static anoxic conditions, unless speciﬁed otherwise.
3.3. Effect of different concentration of dye on decolorization Percent decolorization of DB79 and AO10 by B. fusiformis KMK5 was varied with initial concentrations (0.25–1.5 g/l) when studied up to 48 h. The 90% decolorization of DB79 was observed within 16, 28 and 32 h for the dye concentration of 0.25, 0.5 and 0.75 g/ l, respectively. While the time required for the higher concentrations of DB79 (1, 1.25 and 1.5 g/l) was 36 h (Fig. 2a). At 0.25, 0.5 and 0.75 g/l of AO10, 95% dye decolorization was observed within 16, 24 and 28 h, respectively. However, for the dye AO10 concentration above 1 g/l, the decolorization time was 36 h (Fig. 2b). The potential of the KMK5 was such a high that it decolorizes the dye with the same efﬁciency even at high concentrations of DB79 and AO10. Many bacteria capable of reducing azo dye reported were isolated from dye contaminated site (Coughlin et al., 1999; Asad et al., 2007). This is an indication of adaptation of the microorganisms to the toxic levels of azo dye in the environment. The isolate KMK5 could tolerate the dye AO10 and DB79 up to 1.5 g/l (3.32 mM) which is in contrast to the toxic effect reported for the azo dye AO10 in concentrations within 0.037–0.051 mM (Seshadri et al., 1994). This observation is of signiﬁcance for bioremediation since it indicates the ability of the isolate KMK5 to withstand high concentration of azo dye during the bioremediation process. The strain KMK5 was able to decolorize the dye at a concentration much higher than other bacterial strains isolated from differ-
ent contaminated sites (Nachiyar and Rajkumar, 2003). The time taken by KMK5 to decolorize the dye compares favorably with the reports by Pseudomonas sp., which required periods of more than 7 days (50 mg/l) to achieve 80% decolorization for synthetic dyes (Senan and Abraham, 2004). P. ﬂuorescens was able to decolorize 20 mg/l at an efﬁciency of 40–80% (Khehra et al. 2005). The isolate KMK5 could decolorize AO10 and DB79 at much above the reported dye concentration in the wastewaters and thus could be used successfully in treatment of textile wastewaters. 3.4. Identiﬁcation of metabolic intermediates Incubation of the azo dyes, with B. fusiformis KMK5 resulted in the decolorization of the dyes AO10 and DB79 and the biotransformed metabolites were characterized by GC–MS and FT-IR. The results of FT-IR analysis of the dye DB79 parent and sample obtained after decolorization showed various peaks. The FT-IR spectra of DB79 parent dye displays peaks at 3452, 2924, 1740 and 1440 cm 1, for –OH and –NH stretching vibration, aromatic –CH stretching vibration, >[email protected]
stretching and –[email protected]
– stretching vibration, respectively. While peaks at 1520 and 1329 cm 1 are for –NO2 group and a peak at 619 shows the presence of –Br on the dye. The FT-IR spectra of degradation product displays peak at 3263 cm 1 for –OH stretching indicating hydroxylation of the product, a peak at 2924 and 1740 cm 1 for –CH and >[email protected]
stretching, for the formation of an intermediate with carbonyl group. The
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During the degradation there is asymmetric cleavage of azo bond in AO10 resulting in formation of phenyl hydrazine, which was conﬁrmed by the standard GC–MS library data, this is further converted to aniline. While the naphthalene part of the dye was further biodegraded with opening of one ring, the formation of aldehyde as one of the intermediate is conﬁrmed from the IR data.
60 0.25 g/l 0.50 g/l 0.75 g/l 1.0 g/l 1.25 g/l 1.50 g/l
Time in h 100
4. Conclusion The results, thus, obtained have characterized and identiﬁed a new dye-degrading strain, B. fusiformis KMK5, from an efﬂuent contaminated site of textile dyeing industry. This observation has established that the bacteria are adaptive in nature and can degrade the contaminants. The ability of the strain to tolerate, decolorize and degrade azo dyes at high concentration gives it an advantage for treatment of textile industry wastewaters. However, potential of culture needs to be demonstrated for its application in treatment of real dye bearing wastewaters using appropriate bioreactors. Acknowledgements
0.25 g/l 0.5 g/l 0.75 g/l 1.0 g/l 1.25 g/l 1.5 g/l
Time in h Fig. 2. Effect of initial dye concentration (g/l) on the decolorization efﬁciency of (a) Disperse Blue 79 (DB79) and (b) Acid Orange 10 (AO10) by the bacterial isolate KMK5.
peak of –Br is disappeared in the product indicating the debromination or dehalogenation reaction. The analysis of biodegradation products of DB79 by GC–MS also showed that there is asymmetric cleavage of the azo bond in DB79 leading to the formation of hydrazine molecule, while another product was immediately debrominated to form dinitrobenzene and the nitro groups were further reduced. Our report does not ﬁnd the formation of 2-bromo-4,6-dinitroaniline, which is in contrast to that reported earlier by chemical and sediment mediated reduction of DB79 (Weber and Adams, 1995). Based on the analysis of the intermediates, the degradation pathway of DB79 in B. fusiformis KMK5 is different from those of chemical and sediment mediated reduction of DB79 (Weber and Adams, 1995) and sequential batch reactor (Melgoza et al., 2004). The results of FT-IR analysis of AO10 parent dye and sample obtained after decolorization showed various peaks. The FT-IR spectra of AO10 control dye displays peaks at 3483, 2929, 1660 and 1440 cm 1, for –OH stretching vibration, aromatic –CH stretching vibration, –[email protected]
– stretching and –[email protected]
– stretching vibration, respectively. While peak near 1065 cm 1 is for –[email protected]
, indicates sulphoxide nature of the dye. The IR spectra of degradation product displays peak at 3263 cm 1 for –OH stretching. During the degradation of aromatic amines of AO10 there is formation of aromatic aldehyde as an intermediate which was conﬁrmed by the spot test using 2,4-dinitrophenyl hydrazine reagent which indicated color test due to presence of aldehyde.
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