Chapter II. Study on the Decolorization of Commonly Used Disperse Dyes in the Textile Industry

Transcription

1 Chapter II Study on the Decolorization of Commonly Used Disperse Dyes in the Textile Industry

2 Disperse Dyes D isperse dyes are colorants with low water solubility that, in their disperse colloidal form, are suitable for dyeing and printing hydrophobic fibers and fabrics. Forerunners of the disperse dyes were the ionamine dyes of British Dyestuffs Corp.; these were N-methanesulfonic acids of aminoazo or aminoanthraquinone dyes that release the N-methanesulfonic acid group in the dyeing process and, thereby, precipitated as disperse dyes on the acetate fibers. The understanding of this mechanism in 1923 initiated the development of genuine disperse dyes. British Celanese and British Dyestuffs Corp. were the first companies to introduce these dyes into the market for coloring acetate fibers. The dyes were dispersed with sulforicinoleic acid, soap, or Turkey red oil (Fourness, 1956). From 1924 to 1930, products of other companies appeared on the market, initially as pastes; later, when the materials could be dried successfully without interfering with their dispersibility, they were also marketed as powders. Since 1950, the production of disperse dyes has increased sharply, closely following the growth in worldwide production of synthetic fibers, especially polyester [poly(ethylene terephthalate)] fibers, production of which has grown steadily from in 1970 to about 16x10 6 t/a in 1998 (Acordis, 1998). Furthermore, new dyeing processes necessitated the development of special disperse dyes. For instance, dyes characterized by special ease of sublimation are preferred for transfer printing (Moore, 1974). The demand for new fastness properties such as thermo-migration fastness and automotive light fastness (Hihara, 1997; Lampe et al., 1992; Ulrich 1992;

3 Vonhone and Stuck, 1992), also led to new dyes, as has the ongoing pressure on market prices. Models for the dyeing of polyester fibers with disperse dyes have been developed (McDowell, 1980). When the dye is applied from aqueous medium, it is adsorbed from the molecularly dispersed aqueous solution onto the fiber surface and then diffuses into the interior of the fiber. The following parameters determine the rate of dyeing and, to some extent, the leveling properties; (1) the dissolution rate during the transition from the dispersed crystalline state of the dye into the molecularly dispersed phase, and (2) the diffusion rate at the fiber surface and, especially, in the interior of the fiber. The rates of both processes vary with temperature. Differences in geometry and polarity of the dye molecules can lead to wide variations in these finishing or dye-specific properties and can have a marked effect on the absorption characteristics of all dyes, irrespective of whether single component or combination dyeing processes are used. For instance, uneven dyeing may occur when an unequal distribution of particle size results in insufficient dispersion stability and hence crystal growth and precipitation at the substrate surface. Industrially applied disperse dyes are based on numerous chromophore systems. Approximately 60 % of all products are azo dyes, and 25 % are anthraquinone dyes, with the remainder distributed among quinophthalone, methine, naphthalimide, naphthoquinone, and nitro dyes (Muller, 1968). Azo dyes are currently employed to create almost the entire range of shades; anthraquinone derivatives are used for red, violet, blue, and turquoise. The remaining dye classes are used mainly to produce yellow shades. Fastness properties and often also the structures are disclosed in the Color Index (Color Index International, 1992). For this study, four disperse dyes were chosen based on the

4 frequency of application in an industry called United Bleachers (P) Limited at Mettupalayam, Tamil Nadu, India. Chosen group of disperse dyes were Disperse Red F3BS, Disperse Yellow F3B, Disperse Yellow GR and Disperse T Blue. Soil samples were also therefore chosen from the same site of the industrial ground. The objective was to isolate and identify the best decolorizing bacteria.

5 Materials and Methods The microorganisms present in the soil from the effluent disposal site of a textile dyeing industry named United Bleachers Pvt. Ltd., Mettupalayam, Tamil Nadu, India, were aseptically transported to the lab bacterial cultures were raised in nutrient agar. Based on morphological difference, bacterial isolates were identified and pure cultures were raised. From these pure cultures, decolorization studies were carried out on the chosen group of disperse dyes such as Disperse Red F3BS, Disperse T Blue, Disperse Yellow F3B, Disperse Yellow GR after prior spectrophotometric studies as explained in Materials and Methods of Chapter I. Decolorization studies were performed in nutrient broth initially to test the decolorization potential of the bacterial isolates with 100 mg/l dye concentration set at 7 ph. The decolorization was followed by maintaining for 24 hours at 37 0 C and 150 rpm in a Shaker-Incubator. The decolorization experiment was also repeated in minimal media following similar protocol which is described elsewhere in thesis. Best decolorizing bacteria for the individual dyes were mixed as consortium and tested for decolorizing abilities of the respective dyes in nutrient broth and minimal media. All the experiments were conducted in triplicate and the average was calculated to represent the decolorization activity. Strain Identification and Sequencing Bacterial cells from best decolorizing culture strains were collected by centrifugation (5000 g for 10 min) and subjected to sequential digestion by lysozyme (2.5

6 mg/ml, 37 0 C for 1 h) and proteinase K (200 mg/ml in 1% SDS, 55 0 C for 1 h), followed by incubation in 1% CTAB and 0.7 M NaCl at 65 0 C for 15 min. After extraction with phenol/chloroform, DNA was recovered by ethanol precipitation and then dissolved in double distilled water. The 16S rrna gene was amplified by PCR in a 50 ml reaction system using universal primer mentioned in the Materials and Methods of Chapter I (Jing et al., 2004) under the following conditions: 1 Taq Buffer, 0.2 mm of each dntp, 0.2 mm of each primer and 1 U Taq polymerase (Eppendorf, Germany). An initial denaturing period of 5 min was followed by 30 cycles at 94 0 C for 1min, 55ºC for 1 min, 72 0 C for 2 min, and the final extension (72 0 C) time was 10 min (Su et al., 2007). Then the PCR products were sent for sequencing (Chromous Biotech Pvt. Ltd., Bangalore, India). The sequence was deposited at NCBI to identify it by BLAST search.

7 Results Spectrum Study Absorbance measurements were performed using UV/VIS spectrophotometer. Wavelength in maximum absorbance ( max) were used for respective dyes. Disperse red F3Bs, Disperse T Blue, Disperse Yellow F3B and Disperse Yellow GR had max of 546 nm, 600 nm, 430 nm and 410 nm, respectively. Isolation of Bacterial Cultures From the soil sample taken from the United Bleacher s Pvt. Limited, Mettuppalayam, Tamil Nadu, bacterial cultures were raised in the laboratory and 46 bacterial isolates were identified based on the colony morphology and named as UBL-JMC01 to UBL-JMC46 due to obvious reasons of the contribution of respective institutions. Decolorization in Nutrient Broth All the 46 isolates were individually screened for their ability to decolorize the four disperse dye models chosen such as Disperse Red F3BS, Disperse T-Blue, Disperse Yellow F3B and Disperse Yellow GR (Table 2.1; Fig. 2.4 a,b & d)). Among these, Disperse Yellow GR was decolorized to a maximum of 92.2±0.24 % by JMC-UBL02 strain. Eleven strains of the 46, decolorized Yellow GR to an average of 64 %. These eleven strains happened to be JMC-

8 UBL - 05, 08, 10, 12, 22, 29, 33, 39, 40, 43 and 45. All the other strains of these 46 did not decolorize the Disperse Yellow GR to any significant level. JMC-UBL02 and JMC-UBL35 demonstrated a significant decolorization of Disperse Yellow F3B with 91.8 ± 0.14 % and 84.5 ± 0.09 % respectively. JMC-UBL25 and JMC-UBL21 revealed a decolorization ability of about 71 % approximately. Other than these four strains, none of the isolate could decolorize Disperse Yellow F3B to a significant level. Disperse Red F3BS was decolorized to a maximum of ± 0.27 % by JMC-UBL02 followed by JMC-UBL45 to about 53.0±0.17 %. In contrast, Disperse T-Blue was not decolorized by any of the 46 isolates beyond 31.8±0.25 % by JMC-UBL-03. Decolorization in Minimal Media Decolorization of the four dyes in Minimal media was performed with all the 46 isolates. In this, JMC-UBL02 alone demonstrated a moderate level of decolorization of three dyes namely, Disperse red F3BS (62.31 ± 0.24 %), Disperse Yellow F3B (72.75 ± 0.27 %) and Disperse Yellow GR (76.52 ± 0.41 %) after 48 hours of incubation, whereas, rest of isolates demonstrated low/negligible decolorization. There was no significant decolorization in Disperse T-Blue by any of these isolates (Table 2.2).

9 Effect of Consortium Based on the decolorization performance of the 46 bacterial isolates, four different consortiums were developed using the best decolorizing bacteria for each dye. Decolorization experiment was carried out in both Nutrient broth and minimal media using these consortia. Thus, JMC-UBL02 and JMC-UBL45 were mixed together for developing consortium (named as C-DR) to decolorize Disperse Red F3BS. This resulted in a decolorization up to ± 0.48 % in nutrient broth (Table 2.3) after 24 hours of incubation, whereas that of Minimal media produced only ± 0.43 %. The latter produced ± 0.19 % of decolorization at 48 hours (Table 2.4). Further incubation of the nutrient broth beyond 24 hours and minimal media beyond 48 hours did not result in any further decolorization from their earlier levels. For the decolorization of Disperse Yellow F3B, five isolates namely, JMC-UBL01, 02, 25, 35 and 45 were employed in preparing the consortia (named as C-DYF). This consortium could efficiently decolorize the dye to a maximum of ± 0.47 % in nutrient broth at 24 hours of time interval (Table 2.3). Whereas, in minimal media, there was only ± 0.32 % of decolorization at 48 hours (Table 2.4). Interestingly, the decolorizing performance of the consortia is comparable to that of the individual isolatejmc-ubl-02 which demonstrated 92 % approximately in 24 hours.

10 Twelve bacterial isolates such as JMC-UBL02, 05, 08, 10, 12, 22, 29, 33, 39, 40, 43 and 45 were employed for the development of consortia (named as C-DYGR) to decolorize Disperse Yellow GR. There was decolorization of the dye upto ± 0.28 % in nutrient broth at 24 hours (Table 2.3). However, this is lower by 10% when compared to the decolorization of the same dye using JMC-UBL-02 (92.2 % at 24 hours in nutrient broth). In case of minimal media, the decolorization reached a maximum of ± 0.27 % at 48 hours (Table 2.4). As none of the 46 isolates could individually decolorize Disperse T-Blue (named as C-DTB) in nutrient broth, a consortium was developed with all the 46 bacterial strains. This consortium was tested for its decolorizing ability in nutrient broth and minimal media. Results revealed very low and negligible levels of decolorization when used as consortia (Table 2.3, 2.4). Of all the studies carried out in nutrient broth and minimal media, JMC- UBL01, JMC-UBL02, JMC-UBL03, JMC-UBL-43 and JMC-UBL-45 revealed potential decolorizing abilities and therefore subjected to molecular identification procedures. Subsequent blasting of the resulting sequence revealed the identification of isolates and are tabulated (Table 2.5).

11 Table 2.1 Decolorization of four disperse dyes in nutrient media by bacterial isolates (JMC-UBL01- JMC-UBL46). All the experiments were performed in triplicates and the average was calculated to represent the decolorization activity in percentage (%). ND- No Decolorization S. No. Isolates D Red F3BS D T Blue D Yellow F3B D. Yellow GR 1 UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± 0.45 ND 8.8 ± UBL ± ± 0.35 ND 4.6 ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± 0.52 ND 22 UBL ± ± ± ± UBL ± ± ± ± 0.09

12 24 UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL 42 ND 24.5 ± 0.21 ND ND 43 UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± 0.45 Table 2.1(Cont.); Decolorization of four disperse dyes in nutrient media by bacterial isolates (JMC-UBL01- JMC-UBL46). All the experiments were performed in triplicates and the average was calculated to represent the decolorization activity in percentage (%). ND- No Decolorization

13 S. No. Isolates D.Red F3B D T Blue D. Yellow F3B Yellow GR 1 UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± 0.45 ND ± UBL ± ± 0.27 ND ± UBL ± ± 0.37 ND ND 7 UBL ± ± 0.22 ND ND 8 UBL ± ± 0.19 ND ± UBL ± ± ± ± UBL ± 0.53 ND ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± o ± ± UBL ± ± 0.27 ND ± UBL ± ± ± ± UBL ± ± ± 0.38 ND 18 UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± 0.22 ND 22 UBL ± ± ± ± 0.11 Table 2.2 Decolorization of four disperse dyes in minimal media by bacterial isolates (JMC-UBL01- JMC- UBL46). All the experiments were performed in triplicates and the average was calculated to represent the decolorization activity in percentage (%). ND- No Decolorization.

14 23 UBL ± ± ± ± UBL 24 ND ± ± ± UBL ± ± ± ± UBL 26 ND ND ± ± UBL ± ± ± 0.51 ND 28 UBL ± ± ± 0.34 ND 29 UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL 32 ND ± ± ± UBL ± ± ± ± UBL ± 0.18 ND ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL 39 ND ± ± ± UBL ± ± ± ± UBL 41 ND ± ± ± UBL 42 ND 14.2 ± 0.15 ND ND 43 UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± UBL ± ± ± ± 0.27 Table 2.2 (Cont.) Decolorization of four disperse dyes in minimal media by bacterial isolates (JMC-UBL01- JMC-UBL46) ND- No Decolorization.

15 Dye(s) D. R F3BS D. T Blue D. Y F3B D.Y GR Consortium C-DR C-DTB C-DYF C-DYGR 24 Broth hrs ± ± ± ± 0.28 Table 2.3 Decolorization of disperse dyes in nutrient media by four different consortiums (C-DR; C-DTB; C-DYF and C-DYGR) Dye(s) D. R F3BS D. T Blue D. Y F3B D.Y GR Consortium C-DR C-DTB C-DYF 24 Minimal hrs ± ± ± ± hrs ± ± ± ±0.27 Table 2.4 Decolorization of disperse dyes in minimal media by four different consortiums (C-DR; C-DTB; C-DYF and C-DYGR). All the experiments were performed in triplicates and the average was calculated to represent the decolorization activity in percentage (%). S.No. Isolate Identified as GenBank Accession No. 1 JMC-UBL01 Pseudomonas sp. HM JMC-UBL02 Enterococcus faecalis HM JMC-UBL03 B. thurunginesis HM JMC-UBL43 Azoarcus sp. HM JMC-UBL45 Lysinibacillus sp. HM Table 2.5 Isolates identified by 16s rdna sequencing method with GenBank Acc. No.

16 Figure 2.1: Absorption spectrum of the Disperse Red F3BS dye in visible region ( nm) following optimal conditions; Solid line and dotted line represent the spectrum of the dye before and after decolorization respectively.

17 Figure 2.2: Absorption spectrum of the Disperse Yellow F3B dye in visible region ( nm) following optimal conditions; Solid line and dotted line represent the spectrum of the dye before and after decolorization respectively.

18 Figure 2.3: Absorption spectrum of the Disperse Yellow GR dye in visible region ( nm) following optimal conditions; Solid line and dotted line represent the spectrum of the dye before and after decolorization respectively.

19 A B C D Figure 2.4: Flasks showing the decolorization of dyes in comparison with respective control in Nutrient broth. A Disperse Red F3BS; B Disperse T Blue; C Disperse Yellow F3B and D Disperse Yellow GR.

20 Discussion F our structurally different disperse dyes with the same dye concentration of 100mg/l was used in this research for decolorization. From this study, a potential bacterial isolate- JMC-UBL02 was identified that could decolorize a maximum of 92 % of decolorization in Disperse Yellow F3B and Disperse Yellow GR followed by 83 % approximately in Disperse Red F3B. This variation in the decolorization of different dyes might be attributed to the structural diversity of the dyes. (Kalyani et al., 2008). It should be noted that although the percentages did not reach 100%, the liquid appeared colorless indicating efficient decolorization process to have involved and also the potent strain that could possibly be investigated to apply in this regard (Khadijah et al., 2009). This JMC-UBL02 which was identified as a potent decolorizing agent was later found to be Enterococcus faecalis based on sequencing methods and Biochemical profiling. The decolorizing ability is attributed to the presence of its azo-reductase enzyme. Azoreductase activity has been identified in several species of bacteria recently, such as Caulobacter subvibrioides strain C7-D, Xenophilus azovorans KF46F, Pigmentiphaga kullae K24, Enterobacter agglomerans, Enterococcus faecalis, and Staphylococcus aureus (Mazumder et al., 1999; Blu mel et al., 2002; Blu mel and Stolz 2003; Moutaouakkil et al., 2003; Chen et al., 2004, 2005). This azo-reductase is reported to be the key enzyme expressed in azo-dyedegrading bacteria and catalyses the reductive cleavage of the azo bond. All the three model dyes in this chapter happens to be azo-group of dyes, except the Disperse T-Blue

21 which is an anthraquinone based one. This is probably the reason that JMC-UBL02 could very well decolorize the three disperse dyes (Disperse Red F3BS, Yellow F3B and Yellow GR) while not the Disperse T-blue. It is believed that the anthraquinone dyes are more recalcitrant than the azo dyes such as the Disperse Red F3BS and Disperse Yellow F3B (Zhang et al., 2007). The poor decolorization might be probably due to the toxic effects of the dye and/or inadequate biomass concentrations for the uptake of higher concentrations of dye as well as blockage of active sites of azoreductase by dye molecules with different structure (Tony et al., 2009). JMC-UBL02 is a promising isolate for further investigation into its optimization of the decolorizing conditions and application for effluent treatment which is discussed in later chapters. Decolorization of dyes may take place by adsorption (Aravindhan et al., 2007) or degradation (Kumar et al., 2007). In the case of adsorption, dyes are only adsorbed on the surface of bacterial cells whereas; new compounds come into being when dyes are degraded by bacterial enzymes during the degradation process. In adsorption, examination of the absorption spectrum reveals that all peaks decrease approximately in proportion to each other. If dye removal is attributed to biodegradation or biotransformation, either the major visible light absorbance peak completely disappears or a new peak appears (Yu and Wen, 2005). Dye adsorption can also be easily judged by an evidently colored cell pellet, whereas, those retaining their original colors are accompanied by the occurrence of biodegradation (Chen et al., 2003). Simple agitation of the broth also would reveal the re-

22 appearance of the original color if adsorption had been taking place. Irrespective of the percentage of decolorization of these dyes by all or any of the 46 isolates, the spectral analysis of the degraded dyes reveal that biotransformation has occurred and not bioadsorption. The efficiency of decolorization process depends on the survival, adaptability and activities of the enzymes produced by microorganism present in the mixed culture (Senan and Abraham, 2004). As reports reveal, the development of dye decolorizing consortia (such as C-DR, C-DYF and C-DYGR) from the isolates collected from the waste disposal site of the textile industry indicates the natural adaptation of these organisms survive in the presence of toxic dyes (Chen et al., 2003; Adedayo et al., 2004; Khehara et al., 2005; Ren et al., 2006). In the present study of decolorization of disperse and reactive dyes (discussed in Chapter I), shaking condition or aerated condition was followed through a handful of isolates that showed significant decolorization. There are contradictory reports about the effect of shaking/agitation on microbial decolorization of synthetic dyes. According to some authors, decolorization is enhanced by shaking while according to others by static conditions, because of better oxygen transfer and nutrient distribution as compared to the stationary cultures. On the contrary, according to Kalyani et al. (2009), agitated culture of Pseudomonas sp. SUK1 showed almost no decolorization in 24 h, while the static culture decolorized more than 96% of the initial dye concentration (300 mg.l 1 ) of Reactive Red 2 in 6 h. Similarly, Husseiny (2008), while studying the biodegradation of Reactive Red 120

23 and Direct Red 81 by Aspergillus niger, found that the static conditions were more efficient than the shaking. Higher enzymatic activities are observed in static conditions (Kaushik and Malik 2009; Hazrat, 2010: Novotny et al., 2004). This could also be a reason for the failure of decolorization by most of the isolates and especially with Disperse T-blue by the isolates. Therefore, evaluation of the static condition and other optimization conditions remain as significant experiments that are discussed in the chapters to be followed. The growth quality of the non-decolorizing bacterial isolates, was better in the control than in disperse dye-containing media, indicates the adverse effect of dyes to bacterial growth. This result was similar to that of (Chen et al., 2004; Widhi et al., 2007). It was also reported that dye toxicity to microorganisms inhibits metabolic activities; decreasing the growth rate and / or biomass needed for decolorization which leads to the decrease of decolorization activity (Sumathi and Manju, 2000). The results, thus, obtained have characterized and identified few novel dye-degrading bacterial strains from an effluent contaminated site of textile dying industry. This observation has established that the bacteria are adaptive in nature and can degrade the pollutants. The ability of the strain to tolerate, decolorize and degrade disperse group of dyes 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 culture conditions/bioreactor systems.