Decolourization and Biodegradation of Reactive Azo Dyes by Mixed Culture

Transcription

1 Indian Journal of Biotechnology Vol 2, April 23, pp Decolourization and Biodegradation of Reactive Azo Dyes by Mixed Culture P P Vijaya, P Padmavathy and S Sandhya* National Environmental Engineering Research Institute, CSIR Complex, Chennai 6 113, India Received 3 October 21; accepted 6 July 22 Synthetic dyes, azo dye in particular, are widely found in the effluents from textile industries. The persistence and toxicity of these compounds cause adverse impact in the receiving streams. A mixture of isolated cultures, from domestic sewage treatment plant was found to decolourize reactive azo dyes, Red RB, Blue M2B and Yellow, efficiently in the absence of added external nitrogen source. After shaking incubation for 48 hrs, mixture of cultures removed these dyes and decolourization was 95% for Red RB and Blue M2B and 5% for Yellow. The mixed culture could degrade 5 mgll Red RB efficiently with the release of 84.9 mgll of ammonia. There was also degradation of 1 mgll of Blue M2B and 5 mgll of Yellow dyes but the release of ammonia was not observed in Blue M2B and Yellow. Decolourization was remarkably enhanced when peptone was used in the medium. Azoreductase activity was high in the presence of reactive azo dyes and the enzyme could decolourize the reactive dyes much faster when decolourization was studied as cell free extract in the presence of individual dyes. Keywords: azoreductase, azo dyes, decolourization, biodegradation Introduction Reactive dyes are becoming more popular in the textile industry. There are more than 8 chemical products associated with the dyeing process listed in the Colour Index (1976). While over 1,, commercially available dyes exist, over 7x1 5 metric tons of the dyes stuff are produced annually (Zdlinger, 1987). These dyes include several structural varieties of dyes, such as acidic, reactive, basic, disperse, azo, diazo, anthaquinone based and metal complex dyes. The only thing in common is their ability to absorb light in the visible region. Reactive dyes are the colour molecules capable of forming covalent bond between the dye molecules and the fibres, which are usually cellulose fibres. However, reactive dyes hydrolyze easily resulting in a high portion of unfixed reactive dyes, which have to be washed off during the dyeing process. As much as 5% of the initial dye load is present in the dye bath effluent (Shore, 1995). Colour is the first contaminant to be recognized in textile wastewaters and has to be removed before discharging into the receiving water body. Many different methods have been used to purify these effluents, but none of them can be described as perfect (Idaka et al, 1987). Biological treatment systems may have * Author for correspondence: Tel: 23125, ; Fax: sswami_in@yahoo.com promising application for the removal of azo compounds since it is widely reported that the azo dyes are reduced by anaerobic sludges/sediments and or bacterial cultures (Brown & Hamburger, 1987; Chung & Stevens, 1992). Under anaerobic conditions, azo dyes can be reduced to form aromatic amines, which are considered to be toxic, mutagenic and carcinogenic to animals (Brown & Hamburger, 1987). Toxicity, mutagenicity and carcinogenicity of azo dyes are, therefore, of major concern from human health point, particularly with occupational groups such as dyestuff and textile dyers. These workers have been reported to have incidences of bladder cancer (Chung & Stevens, 1992). Although azo dyes were long considered nearly non-degradable or non-transformable by bacteria under aerobic condition (Wuhrmann et al, 198), efforts to isolate microorganisms capable of transforming azo compounds have continued. The total mineralisation of azo dyes to carbon-dioxide, ammonia and water by aerobic sludge has been documented (Hu, 1994; Ogawa & Yatome, 199). On an average, more than 5% of the dye remained unchanged or was slightly modified (Chung, 2). Kulla (1981) described degradation pathways for sulphonated azo dyes by Pseudomonas strains previously adopted to grow on the corresponding carboxylated azo dyes. Idaka et al (1987) described the reductive fission of azo bonds by P. cepacia. In the present work, the aim was to isolate bacteria that were capa-

2 26 INDIAN J BIOTECHNOL, APRIL 23 ble of decolourizing reactive azo dyes Red RB, Blue M5B and Yellow. The rate of decolourization and specific growth rate of consortia with respect to dye decolourization as well as azoreductase activity of these cultures, when grown on respective dyes were also studied. Materials and Methods Isolation of Reactive Azo dyes Degrading/ Decolourizing Bacteria Bacterial culture was isolated from an aerated lagoon system treating the city sewage. Sewage sample, 1 ml along with 25 mg/l of dye solution and 2 mg/l of peptone and yeast extract, respectively was incubated on shaker. After 24 hrs, 1% inoculate from the flask was inoculated in fresh sewage along with dye. Five such transfers were made. After the fifth enrichment, 1% inoculate was transferred to basal salt medium (BSM) of composition as described earlier (Vijaya & Sandhya, 22). The mixture of cultures was incubated at 3 C. Periodically the sample was withdrawn and cells were palleted by centrifugation at 1, rpm for 1 min in a R-24 research centrifuge (Remi Instruments, India). The supernatant was scanned from 2-5 nm in UV spectrophotometer (Shimadzu UVI6A, Japan). Reactive dyes, commercial grade dyes, Red RB, Blue M2B and Yellow were supplied by Dye Star India Ltd, Mumbai, India. All the three dyes belong to reactive azo group with absorbance maxima of 52 nm for Red RB, 59 for Blue M5B and 42 for Yellow. The structure of the dyes is given in Fig. 1. Enzyme Assay The biomass from the culture broth (1L) was collected by centrifugation and washed with phosphate buffer (.1M) several times. Cells were disrupted in portions by ultrasonic treatment for 15 min in an ice bath. Particle-free cell extracts were made by centrifugation at 5, rpm for 2 min. The protein content of the cell extract and azoreductase activity was measured according to Lowery et al (1951) and Wuhrmann et al (198). Evaluation of Bio-kinetics Constants Batch experiments have been conducted by using mix culture and all the three reactive azo dyes to determine specific growth rate (u), maximum specific growth rate ().!max), and half velocity constant (K), ).! is NapS@"~H an~~~3na Blue M5B CI 3414 Yellow CI 1914 ~=..oo~ S3Na~S3Na Red RB CI 1855 Fig. I-Structure of azo dyes. related to ).!max and K, as described by Monad equation (Gaudy & Gaudy, 198). Results and Discussion Isolation of Dyes Decolourizing Bacteria The mixture of cultures degrading and decolourizing Red RB, Blue M2B and Yellow dyes were isolated from sewage treatment plant by enrichment culture technique. The consortia was grown in a basal salt medium (BSM) along with Red RB dyes 25-5 mg/l, concentrations during which the decolourization and biodegradation achieved was about 9-95%. The consortia grown on Blue M5B could biodegrade 25-2 mg/l only as further increase in concentration was not degraded. In the case of Yellow dye, the mixture of culture could biodegrade only 25-5 mg/l of dye within 96 hrs. In case of Red RB decolourization occurred within 48 hrs, whereas for blue M5B it was 72 hrs and for Yellow 96 hrs. The decolourization of dyes against the time is shown in Figs 2, 3.and 4 for Red RB, Blue M5B, and Yellow, respectively. However, increase in the initial dye concentration resulted in decreasing reaction rates of decolourization of dyes. Final ph of the culture broth was increased from 7. to 7.6. Generally biological decolourization is due to bio-adsorption (Hu, 1994) or biodegradation (Ogawa & Yatome, 199; Kulla, 1981; Zimmermann et al, 1984; Paszczynski et al, 1992). To ensure that the decojourization was not simply a function of ph variation, the visible adsorption was assayed after adjusting ph to 7. of decojourized broth and shows

3 VIJA YA et al: DECOLOURIZATION AND BIODEGRADATION OF REACTIVE AZO DYES , ~. 1.8 o:;:,.6. o.4.,.8 U.6 U.4.2 o j.~ 25 mgll - 5 mgll -- 1 mg/l -- 2 mg/l j i ~---=-~O_O_mg/l ~ 4 mg/l - 5 mg/l -.-J Time, hrs mgll mgll Oomglll Fig. 2-Biodegradation of Red RB by mixture of culture as measured by reduction in dye concentration , ,.8 o :;:,.6 o Time, hrs j--- 25mg/l mg/ mg/l -- 2~J Fig. 3-Biodegradation of Blue M5B by mixture of culture as measured by reduction in dye concentration. Fig. 4--Biodegradation of Yellow by mixture of culture as measured by reduction in dye concentration. no colour, indicating the total decolourization of dyes. This was further confirmed by monitoring the reduction in chemical oxygen demand (COD) of the decolourized broth and ammonia generation during the growth (Table 1). About % of COD reduction was observed for all the three dyes with 84.9 mg/l of ammonia generation from Red RB, 21.4 mg/l from Blue M5B and 2.8 mg/l from Yellow, respectively. Initial step on the biodegradation of azo compounds is a reductive cleavage of the azo bridge, by azoreductase (Chung, 2). The enzyme azoreductase is considered to be sensitive to oxygen (Wuhrmann et al, 198). However, azo dyes could be reduced under aerobic or microaerophillic conditions. Idaka et al (1987) reported the apparent aerobic reduction of simple azo compounds by Aeromonas hydrophilia. An experiment was performed to test whether the dye decolourizing activity was constitutive or inducible. Cultures grown in the presence of reactive dyes only Table I-COD removal and ammonia formation during the biodegradation of azo dyesby consortia of culture Dyes Conc COD* mg/l COD Removal Final ph Ammonia formation mg/l mg/l Initial Final % Initial Final Red RB BlueM5B Yellow Initial ph 7. COD of medium in absence of dye was 81 mg/l Soluble COD was monitored for a medium containing 2 mg/l of peptone and dye concentration, respectively

4 262 INDIAN J BIOTECHNOL, APRIL 23 show azoreductase activity (data not shown) indicating inducible nature of the enzyme. The azoreductase activity was estimated for the consortia growing on Red RB, Blue M5B and Yellow, respectively and given in Table 2. Higher azoreductase activity was obtained on Blue M5B than the other two. Higher the azoreductase acivity, higher the decolourization. The exponential curves suggest a first order relationship of decolourization reactive dyes vs time (hr) for initial dye concentration. The values obtained for bio-kinetics constants of azo dyes Red RB, Blue M5B and Yellow have been presented in Table 3 which indicate that the average value of Il at various concentrations of Red RB dye increased with corresponding increase in dye concentration up to 2 mg/l and remains constant till 4 mg/i. But further increase in dye concentration declines the growth. Whereas in Blue M5B and Yellow Il decreases with the concentration. Ilmax and K, have been evaluated by plotting Lineweaver-Burk Plot (I/Il versus 1/s) for the Red RB and results are given in Table 3. Ilmax and K, Table 2-Decolourization of azo dyes by azoreductase from consortia Dyes Cone Protein Azo- Decolourimg/l mg/l reductase zation U/ml % Red RB Blue M5B Yellow Table 3-Kinetics constants determination for reactive azo dyes under batch conditions Dyes Cone )l mg/l mg/hr Red RB Blue M5B Yellow )lmax mg/hr.799 Ks mg/l could not be obtained for Blue M5B and Yellow as these dyes are not easily biodegradable. Coloured-dye-wastewater treatment is an arduous task. Wide ranges of ph, salt concentrations and chemical structures often add to the complication. Although decolourization is a challenging process to the textile industry, II great potential for microbial decolourizing system exists for achieving total colour removal with only few hours of exposure. Acknowledgement The authors are thankful to Dr S N Kaul, Director grade scientist and Dr R N Singh, Director, NEERI, Nagpur, for their permission to publish this manuscript. They are thankful to Council of Scientific and Industrial Research, New Delhi for funding the research. References Brown D & Hamburger B, The degradation of dyestuffs Part Ill-Investigation of their ultimate degradibility. Chemosphere, 16, Chung K T, 2. Mutagenicity and carcinogenicity of aromatic amines metabolites produced from azo dyes. Environ Carcino & Ecotox Rev, 18, Chung K T & Stevens S E, The reduction of azo dyes by the intestinal microflora. Crit Rev Microbial, 18, Colour Index, Society of Dyers and Colourists, Colour Index. 3 rd edn, Yorkshire, UK. Gaudy A F Jr & Gaudy E T, 198. Microbiology for Environmental Scientists and Engineers. McGraw-Hili Publishing Co, New York. Hu T L, Decolourization of reactive azo dyes by transformation with Pseudomonas luteola. Bioresource Technol, 49, Idaka E et al, Some properties of azoreductase produced by Pseudomonas. Bull Environ Contam Toxicol, 39, Kulla H G, Aerobic bacterial degradation of azo dyes. in Microbial Degradation of Xenobiotics Recalcitrant Compounds, edited by T Leisinger, A M Cook, J Nuesh & R Hutter. Academic Press, London. Pp with the folin phe- Lowery H et al, Protein measurement nol reagent. J Bioi Chern, 193, Ogawa T & Yatome C, 199. Biodegradation of azo dyes in multistage rotating biological contractor immobilised by assimilating bacteria. Bull Environ Contam Toxicol, 44, Paszczynski A et al, Mineralization of sulphonated azo dyes and sulfanilic acid by Phenerochaete chrysosporium and Streptomyces chromofuscus. Appl Environ Microbial, 58, Shore J, Dyeing with Reactive Dyes. Adden Press, UK.

5 VIlA YA et al: DECOLOURIZA TION AND BIODEGRADATION OF REACTIVE AZO DYES 263 Vijaya P P & Sandhya S, 22. Decolourization and biodegradation of methyl red by mixed culture. Environmentalist (In Press). Wuhrmann K et al, 198. Investigation of rate determining factors in the microbial reduction of azo dyes. Eur J Appl Microbiol Biotechnol, 9, Zdlinger H, Colour Chemistry-Synthesis, Properties and Application of Organic Dyes and Pigments. VCH Publication, New York. Zimmermann T et al, Comparison of two bacterial azoreductases acquired during adaptation to growth on azo dyes. Arch Microbiol, 138,