biotreatment of synthetic textile wastewater using ...

BIOTREATMENT OF SYNTHETIC TEXTILE WASTEWATER USING ANOXIC-AEROBIC SEQUENTIAL BIOREACTOR Manjinder Singh Khehra, Harvinder Singh Saini, Bhupinder Singh Chadha and Swapandeep Singh Chimni* Department of Microbiology, *Department of chemistry, Guru Nanak Dev University, Amritsar, Punjab, India. KEYWORDS: Azo dye, decolorization, Acid Red 97, bacterial consortium, anoxic-aerobic sequential bioreactor.

ABSTRACT: Aim: The aim of this study was to develop a sequential anoxic-aerobic process for the treatment of textile wastewater. Material and methods: The degradation of textile azo dye Acid Red-97 was evaluated using laboratory scale anoxic up flow fixed-film column reactor (UFCR) and a continuously stirred aerobic reactor (CSAR) operated in sequence. Results: The anoxic-aerobic treatment process resulted in the 98% color and 95% COD removal. Conclusion: Such a treatment process can find application in pilot scale treatment of actual textile wastewater after appropriate scaling up.

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1. INTRODUCTION Colored wastewater is a consequence of continuous processes both in the dye manufacturing industries and dye consuming industries. The textile processing industries (TPI) produce high quantities of effluents with varying composition depending on the wet processes employed (EPA 1978). It had been estimated that about 10-15% of the dyestuffs used during dyeing processes does not bind to the fibers and is released into the environment (Tan et al. 2000). The scale of problem is enormous as the annual market for dyes is more than 7 x 105 tonnes per year (Robinson et al.2001). About 60-70% of all known dyes produced are azo dyes, making them the largest group of synthetic colorants (Carliell et al. 1995). Their chemical structure is characterized by one or more azo groups (-N=N-) substituted with benzene or naphthalene groups, which may contain many different substituents. There are rare examples for the presence of azo group in natural products and thus industrially produced azo dyes are xenobiotic compounds. Due to their xenobiotic nature, azo dyes are not completely degraded by conventional wastewater treatment processes. (Pagga and Brown 1986). This can lead to acute effects on flora and fauna of the ecosystem due to the toxicity of the dyes, abnormal coloration and reduction in photosynthesis because of the absorbance of light that enters the water (Slokar and Le Marechal 1998). The presence of unnatural color is aesthetically unpleasant and tends to be associated with contamination (Waters 1995). Moreover, some of the azo dyes as well as their breakdown products are cytotoxic (Bhaskar et al., 2003) or carcinogenic (Pinheiro et al., 2004). The new environment regulations concerning textile products have banned the discharge of colored waste in natural water bodies (Maximo et al., 2003). Therefore, an effective and economic treatment of effluents of textile industries has become a necessity for clean production technology for textile industries. The treatment systems based on physical and chemical methods for removal of dyes from effluents are being widely used (Robinson et al.2001). However, these procedures have inherent drawbacks as they generate a significant amount of sludge or causes secondary pollution due to formation of hazardous by-products (Zhang et al., 2004). The new guidelines for effluent and sludge disposal have generated interest in use of biological treatment of TPI wastewaters, as they are known to achieve complete mineralization without producing any toxic sludge. Most of the azo dyes are not aerobically decolorized in activated sludge system, as the azo bond is susceptible to reduction under low redox potential (Carliell et al. 1995). The sequential anaerobic-aerobic reactor configurations have been proposed for decolorization of textile dyestuffs (Rajaguru et al. 2000; Kapdan et al. 2003; Sponza and Isik 2002; Tan et al. 2000). In light of these facts, a sequential anoxic-aerobic bioreactor was designed for the decolorization and degradation of C.I. Acid Red 97, a sulfonated azo group textile dye commonly used in textile dyeing industries established in and around the city of Amritsar. 2. MATERIALS AND METHODS 2.1. Chemicals The azo dye Acid Red 97 commonly used in TPI of this region (Fig. 1) was obtained from Punjab Rang Udyog, Amritsar, Punjab (India) a dye-manufacturing unit. The media components and chemicals were purchased from Himedia Labs. Bombay (India). All chemicals used were of analytical grade. Polyurethane foam (PUF), used as immobilization support, was purchased from local department store (Amritsar, Punjab, India). Heleco ’05, ΤΕΕ, Αθήνα, 3-6 Φεβρουαρίου 2005

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OH

OH

SO3Na N

N

N

N

NaO3S

Figure 1: Chemical structure of dye Acid Red 97 2.2. Composition of synthetic dye wastewater (SDW) Mineral salts medium (MSM) of the composition as reported previously (Sharma et al. 2004) was used. The MSM was supplemented with 0.1% (w/v) of yeast extract, 2.8mM glucose and dye AR-97 (100mgl-1) from their respective filter sterilized stock solutions to prepare synthetic dye wastewater (SDW) feed for the bioreactor. 2.3. Inoculum for bioreactor A consortium designated HM-4, based on four bacterial strains viz. Bacillus cereus, Pseudomonas putida, Pseudomonas fluorescence and Stenotrophomonas acidaminiphila was used as inoculum for the UFCR. The bacterial strains were isolated from soil and sludge samples collected from waste disposal sites of local TPI and dye manufacturing units. 2.4. Experimental lab-scale sequential bioreactor The laboratory scale sequential bioreactor (Fig. 2) consisted of an upflow fixed-film column reactor (UFCR), a sedimentation tank and a continuously stirred aerobic reactor (CSAR).

Figure 2: Schematic diagram of anoxic aerobic sequential bioreactor; 1 feed tank, 2 peristaltic pump, 3 UFCR, 4 sedimentation tank, 5 CSAR, 6 air from compressor, 7 gases out, 8 magnetic stirrer, 9 sludge settling tank, 10 sludge reloading and 11 effluent/sludge out. UFCR: The UFCR reactor was built from borosilicate glass column of 28 cm height and 3.4 cm internal diameter. The PUF pieces of 5-8mm size were first washed with methanol and then thoroughly rinsed with distilled water prior to their use as immobilization support. The glass column was filled with PUF to a bed height of 26cm. The total volume of the reactor was 235ml and the working volume of the reactor filled with PUF was 87ml (37% of the total volume). The UFCR was fed in upflow mode by a peristaltic pump (Miclins, India) at an average flow rate of 7.0mlh-1 with an average hydraulic retention time (HRT) of 12.4h. The activated cell suspension of consortium HM-4 was fed to UFCR in a loop for three days for entrapment of microbial cells Heleco ’05, ΤΕΕ, Αθήνα, 3-6 Φεβρουαρίου 2005

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on the support particles. This was followed by a feed of MSM broth supplemented with 0.1% (w/v) yeast extract and 2.8mM glucose for the development of biofilm on support particles. The reactor was then fed with SDW containing 20mgl-1 of AR-97. The dye concentration in feed was progressively increased upto 100mgl-1 over a stabilization period of three months. CSAR: The CSAR was designed using a reagent bottle having inner dimensions of 13.3cm diameter and height of 19cm with working volume of 2.0L. The contents of the reactor were agitated with a magnetic stirrer and were aerated continuously so as to maintain the dissolved oxygen (DO) level at 5.0±1mgl-1. The CSAR was fed in continuous mode with the output of UFCR during stabilization so as to enrich the cultures capable of degrading the intermediates formed during UFCR treatment. After the stabilization period, the CSAR was operated in batches of 22 days, including 20 days of continuous stirring of contents and two days for settling of sludge. The treated sample was removed by aspiration and replaced with effluent from UFCR collected in sedimentation tank to start the next batch. The system was operated at ambient temperature that varied from 20°- 45° C and pH was between 6 and 7. 2.4. Analytical methods Samples collected from UFCR and CSAR at regular time intervals were centrifuged at 10,000rpm for 15min and the supernatant was used for further analysis. 2.4.1. Spectrophotometric analysis: Cell-free supernatant of UFCR reactor was read at 495nm (λmax of AR-97) using Shimadzu UV-1601 (Kyoto, Japan) spectrophotometer. The dye free medium was used as blank and SDW containing 100mg of AR-97 per liter was used as reference for calculating percentage decolorization as per following equation: Decolorization (%) = (F- O) X100 F Where F = Absorbance of feed and O = Absorbance of UFCR output Aliquots (5ml) of the cell free supernatant of feed, UFCR output and CSAR output samples was scanned in the range of 200-800nm to observe shifting of peaks due to transformations of dye after UFCR and CSAR treatment. 2.4.2. Thin layer chromatography (TLC): The cell-free supernatant of output of the UFCR and CSAR were extracted twice with an equal volume of n-butanol to extract the residual dye and biotransformed products of AR-97. The SDW was also extracted the same way. The pooled extracts were concentrated on a rotary vacuum evaporator (Buchii R-114, Switzerland). The concentrated samples were resolved on Silica gel HF254 TLC plates using hexane/ethyl acetate/methanol (5:3:2 v/v) as developing solvent. The resolved chromatograms were observed under UV light (254nm) and by exposure to iodine vapors. 2.4.3. Nuclear Magnetic Resonance Spectroscopy (NMR): The n-butanol extracted samples of SDW and output of UFCR and CSAR were completely dried using rotary evaporator. The dried samples were dissolved in CD3OD (Aldrich, USA) and transferred to NMR tubes (Wilmad, USA). The 1H NMR spectra of the dried samples were recorded using a 200-MHz, Brucker AMX 300 NMR spectrometer to observe the structural transformations in dye molecule during bioreactor treatment. 2.4.4. Estimation of chemical oxygen demand (COD): COD of SDW, UFCR and CSAR output was determined according to standard methods (APHA, 1989). 3. RESULTS The consortium HM-4, capable of complete decolorization of different azo dyes viz. Acid Red 97, Acid Red 88, Acid Red 119, Reactive Red 120, Acid Blue 113 and Acid Brown 100 at flask level under anoxic conditions, was used to develop biofilm on PUF support pieces in UFCR. Heleco ’05, ΤΕΕ, Αθήνα, 3-6 Φεβρουαρίου 2005

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The microbial populations immobilized on support pieces were progressively exposed to increasing concentrations of AR-97 from 20mgl-1 to final concentration of 100mgl-1 over a period of three months to avoid toxic shock to microorganisms. The UFCR showed 98% decolorization of SDW supplemented with 100mgl-1 of AR-97. The dye was not adsorbed on support material as evident from the extraction of support particles with n-butanol. The microbial populations comprising of rods and cocci formed biofilm on the surface of support material bound by matrix of extracellular polysaccharide (EPS) fibers as observed from scanning electron micrographs (data not shown). 3.1. Analytical methods 3.1.1. Spectrophotometric analysis: The UV-Visible spectra of bioreactor feed containing azo dye AR-97 showed maximum absorption at 495nm and also showed peaks at 308nm and 236nm (Fig. 3). The treatment of this dye containing feed by UFCR resulted in the shifting of absorption peaks at 293nm and 236nm and complete disappearance of peak at 495nm rendering the feed solution colorless.

Figure 3: UV-Visible scans of SDW feed (

), UFCR output (-----), CSAR output (..….)

The shifting of peaks indicated the biotransformation of dye molecule to some other metabolites. The treatment of output of UFCR by CSAR caused further degradation of these metabolites as evident from the scan of CSAR output sample with disappearance of peaks at 293nm and 238nm. Two new peaks were observed at 306nm and 234nm with comparatively low concentration. 3.1.2. TLC analysis: The parent dye AR-97 resolved in TLC into a single spot of Rf value 0.61 (Fig. 4). The solvent extracted samples of UFCR output showed complete disappearance of spot corresponding to dye with concomitant appearance on two new spots with Rf values 0.52 and 0.78. The CSAR treated sample resolved into spots with Rf values 0.06, 0.37, 0.48 and 0.87 indicating to the conversion of dye molecule into some other metabolites during treatment by sequential bioreactors. Heleco ’05, ΤΕΕ, Αθήνα, 3-6 Φεβρουαρίου 2005 5

Figure 4: Thin layer chromatogram of AR-97 (A) dye feed, UFCR output (B) and CSAR output (C). 3.1.3. 1H NMR analysis: A comparison of the 1H NMR spectrum of the SDW with that of the output of UFCR and CSAR indicated the biotransformation of AR-97 dye molecule as evident by the loss of signals characteristics of the dye AR-97 after treatment in sequential bioreactor. The 1H NMR spectrum of CSAR output showed complete loss of signals in aromatic region (δ 7.0-9.0) indicating the conversion of naphthalene and benzene moieties of the parent dye molecule to some unrelated metabolic intermediates (Fig. 5). 3.1.4. Estimation of COD: The COD load of SDW supplemented with 100mgl-1 of AR-97 was 1648mgl-1. The COD of 100mg of AR-97 dye was 588mgl-1. After UFCR treatment, COD was reduced to 316mgl-1 resulting in 64% reduction in COD. It was further decreased to 85mgl-1 after CSAR treatment. Thus, the overall reduction of COD load by 95% was achieved after the sequential treatment of SDW. 4. DISCUSSION In this study, biodegradation of an azo dye AR-97 was studied using anoxic-aerobic sequential bioreactor. AR-97 is an intensely colored diazo dye having a biphenyl moiety and two naphthalene rings with sulfonate functional groups and is recalcitrant to biological degradation (Fig. l). The sequential bioreactor constituted an up flow fixed-film column reactor (UFCR) having polyurethane foam (PUF) as immobilization support and a continuously stirred aerobic reactor (CSAR). A consortium of four acclimatized bacterial strains belonging to Stenotrophomonas sp., Pseudomonas sp. and Bacillus sp., isolated from waste disposal sites of Heleco ’05, ΤΕΕ, Αθήνα, 3-6 Φεβρουαρίου 2005

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textile processing industries (TPI) was used as inoculum for developing the sequential bioreactor.

A

B

C Figure 5: 1H NMR spectra of (A) AR-97 in bioreactor feed (B) output of UFCR and (C) output of CSAR The development of contiguous biofilm on the support after immobilization of the consortium on the PUF support in UFCR was evident from scanning electron micrographs (data not shown). The anoxic conditions in the column provided suitable environment for decolorization of dye, as the chromophore of azo dyes is susceptible to reduction under anaerobic conditions (Donlon et al. 1997). The concerted metabolic activity of the immobilized bacterial populations resulted in almost complete decolorization (98%) and up to 82% COD removal during anoxic treatment of synthetic dye wastewater. The analytical studies based on UV-Visible, TLC and NMR spectroscopy indicated accumulation of amionnaphthlene derivative in the output of UFCR. Similar observations have been reported by Hayase et al. 2000 regarding accumulation of 4amiono-1-naphthalene sulfonic acid after decolorization of azo dye Bordeaux S. These amino Heleco ’05, ΤΕΕ, Αθήνα, 3-6 Φεβρουαρίου 2005

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derivatives/intermediates are reported to be degraded/transformed under aerobic conditions (Kapdan et al. 2003). Thus, output of UFCR was treated in CSAR and resulted in 90% decrease in COD load. The treatment of SDW by sequential treatment system thus resulted in overall 98% removal of COD load. The 1H NMR spectrum of the solvent extracted samples of CSAR output showed disappearance of signals in aromatic region (δ 7.0-9.0). The thin layer chromatogram of CSAR output resolved into spots of unrelated Rf values that were not visible in UV light but could only be visualized after exposure to iodine vapors indicating to the conversion of dye molecule into some other metabolites during treatment by sequential bioreactor (Fig. 4C). These observations indicated the conversion of aromatic metabolites produced after UFCR treatment into nonaromatic intermediates (Fig. 4 and Fig. 5). Complete mineralization of azo dyes based on substituted benzene rings by anaerobic/aerobic sequential has been reported in literature (Tan et al. 2000). However, there are very few reports on degradation of sulfonated naphthalene rings based azo dyes. Haug et al. (1991) reported complete degradation of Mordant Yellow 3, a naphthalene and benzene rings based azo dye, using co-culture of Sphingomonas sp. BN6 and Pseudomonas sp. BN9 at flask level. The aminonaphthalene intermediates formed after anoxic treatment of AR-97 dye, was degraded by the consortium during aerobic treatment in CSAR. Thus, the consortium has capability of working under both anoxic as well as aerobic conditions. Biodegradation of some other textile dyes viz. Acid Red 88, Acid Red 119, Reactive Red 120, Acid Blue 113 and Acid Brown 100 using sequential bioreactor in being carried out. Further work on improving the activity of the microbial populations in CSAR to achieve faster aerobic degradation rate and toxicity assessment of the effluent treated at different stages is being carried out. The sequential treatment system will be scaled upto pilot scale for treatment of effluents from local TPI. 5. ACKNOWLEDGEMENT Harvinder Singh Saini gratefully acknowledges Council of Scientific and Industrial Research (CSIR), New Delhi (India) for funding this study. Manjinder Singh Khehra thanks Guru Nanak Dev University for financial assistance provided in the form of Senior Research Fellowship. 6. REFERENCES 1. APHA 1989 ‘Standard methods for the examination of water and wastewater’, Washington DC: APHA. 2. Bhaskar M., A. Gnanamani, R.J. Ganeshjeevan, R. Chandrasekar, S. Sadulla and G. Radhakrishnan (2003) ‘Analyses of carcinogenic aromatic amines released from harmful azo colorants by Streptomyces sp. SS07’ Journal of Chromatography A, Vol. 1018, pp. 117-123. 3. Carliell C.M., S.J. Barclay and C.A. Buckley (1995) ‘Microbial decolourization of a reactive azo dye under anaerobic conditions’ Water SA, Vol. 21(1), pp. 61–69. 4. Donlon B.A., E. Razo-Flores, M. Luijten, H. Swarts, G. Lettinga and J.A. Field (1997) ‘Detoxification and partial mineralization of the azo dye mordant orange I in a continuous upflow anaerobic sludge-blanket reactor’ Applied Microbiology and Biotechnology, Vol. 47(1), pp. 83-90. 5. EPA, Textile processing industry EPA-625/778-002. U.S. Environmental Protection Agency, Washington, 1978. 6. Hayase N., K. Kouno and K. Ushio 2000 ‘Isolation and characterization of Aeromonas sp. B-5 capable of decolorizing various dyes’ Journal of Bioscience and Bioengineering, Vol. 90(5), pp. 570-573. Heleco ’05, ΤΕΕ, Αθήνα, 3-6 Φεβρουαρίου 2005

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7. Kapdan I.K., M.Tekol and F. Sengul (2003) ‘Decolorization of simulated textile wastewater in an anaerobic-aerobic sequential treatment system’ Process Biochemistry, Vol. 38, pp. 1031-1037. 8. Maximo C., M.T.P. Amorim and M. Costa-Ferreira (2003) ‘Biotransformation of industrial reactive azo dyes by Geotricum sp. CCMI 1019’ Enzyme Microbial Technology, Vol. 32, pp. 145-151. 9. Pagga U. and D. Brown (1986) ‘The degradation of dyestuffs. Part II. Behavior of dyestuffs in aerobic biodegradation tests’ Chemosphere, Vol. 15, pp. 479-491. 10. Pinheiro H.M., O. Thomas and E. Touraud (2004) ‘Aromatic amines from azo dye reduction: status review with emphasis on direct UV spectrophotometric detection in textile industry wastewaters’ Dyes and Pigments, Vol. 61, pp. 121-139. 11. Rajaguru P., K. Kalaiselvi, M. Palanivel and V. Subburam (2000) ‘Biodegradation of azo dyes in a sequential anaerobic-aerobic system’ Applied Microbiology and Biotechnology, Vol. 54(2), pp. 268-273. 12. Robinson T., G. McMullan, R. Marchant and P. Nigam (2001) ‘Remediation of dyes in textile effluent: a critical review on current treatment technologies with a proposed alternative’ Bioresource Technology, Vol. 77, pp. 247–55. 13. Sharma D.K,, H.S. Saini, M. Singh, S.S. Chimni, B.S. Chadha (2004) ‘Biological treatment of textile dye Acid violet-17 by bacterial consortium in an up-flow immobilized cell bioreactor’ Letters in Applied Microbiology, Vol. 38, pp. 345-350. 14. Slokar Y.M. and A.M. Le Marechal (1998) ‘Methods of decoloration of textile wastewater’ Dyes and Pigments, Vol. 37(4), pp. 335–56. 15. Sponza D.T. and M. Isik (2002) ‘Decolorization and azo dye degradation by anaerobic/aerobic sequential process’ Enzyme and Microbial Technology, Vol. 31, pp. 102-110. 16. Tan N.C.G., A. Borger, P. Slender, A.V. Svitelskaya, G. Lettinga and J.A. Field (2000) ‘Degradation of azo dye Mordant Yellow 10 in a sequential anaerobic and bioaugmented aerobic bioreactor’ Water Science and Technology, Vol. 42(5-6), pp. 337-344. 17. Waters B.D (1995) ‘The dye regulator’s view. In: Cooper P, editor. Colour in dyehouse effluent’ Bradford, UK: Society of Dyers and Colourists, pp. 23–6. 18. Zhang F., A. Yediler, X. Liang and A. Kettrup (2004) ‘Effects of dye additives on the ozonation process and oxidation by-products: a comparative study using hydrolyzed CI Reactive red 120’ Dyes and Pigments, Vol. 60, pp. 1-7.

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