ACCELERATION OF AZO DYE DECOLORIZATION USING REDOX MEDIATORS AND COMPLETE BIODEGRADATION OF THE AROMATIC AMINES IN A SEQUENTIAL BIOREACTOR

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ABSTRACT Azo dye degradation takes place in two steps i.e. aromatic amine formation from azo dyes in anaerobic conditions and completes mineralization of the resultant aromatic amines in aerobic conditions. It is reported in literature that azoreductases are responsible for the azo dye bond reduction and protocatechuates in oxidation of the aromatic amines into simple products. It is also reported that the addition of redox mediators to anaerobic culture of bacteria resulted in significantly increased reduction rates of azo dye. In this study, decolorization was achieved in anaerobic Reactor-1 with facultative anaerobic strains, TJ-1, and the oxidation of resultant aromatic amines was achieved by aerobic bacterial strains, TJ-2. Significant decolorization rates were achieved using redox mediators. NADH and FADH (or FAD and NAD) were used as natural redox mediators whereas Quinones-AQS (anthraquinone-2-sulfonate) was used as a synthetic redox mediator. Quinones showed the highest rate of decolorization. The possible mechanisms are proposed. The results show that bacterial oxidation of redox mediators transfers the electrons to azo dyes, accelerating the reduction of azo bond in dye. The above experiments were done with 1 mM of azo dye Methyl Orange (MO) and varying concentration of redox mediators from 0.5 mM 2.0 mM. Keywords: Methyl Orange (MO), FADH, NADH, TJ-1, TJ-2, Quinones-AQS

(anthraquinone-2-sulfonate), Redox Mediator

Introduction: Removal of dyes is a major concern when treating textile-processing wastewater. The vast majority (60% to 70%) of the dyes applied in textile-processing industries are azo compounds, characterized by azo (-N = N-) bridges linking substituted aromatic structures [1]. It is estimated that 10% to 40% of the dyes used for textile dyeing end up in the wastewater. This fraction has increased over the last decades because of the increasing use of reactive dyes, a class of water soluble dyes with a relatively low degree of fixation [2, 3]. Discharge of dyes into the environment is avoided, not only for aesthetic reasons, but also because many azo dyes and their breakdown products are toxic to aquatic life [4] and mutagenic to humans [5]. Azo dyes are generally persistent under aerobic conditions [6, 7].

Figure 1: Steps in azo dye degradation (adapted from PhD thesis, Taruna Joshi, IIT Kanpur) However, under anaerobic conditions, they undergo relatively easy reductive fission, yielding colorless aromatic amines. The reduction of azo dyes is therefore closely

associated with their decolorization. The aromatic amines released from azo dye reduction, generally require aerobic conditions for their degradation [8]. The most logical treatment strategy for complete degradation of azo dyes is therefore a sequentialanaerobic-aerobic approach with, for instance, an upflow anaerobic sludge blanket (UASB) reactor as the first stage. In a previous study, screening of the anaerobic decolorization of 20 widely varying types of azo dyes revealed that all azo dyes studied were reduced in the presence of the electron-donating primary substrate [9]. The reaction followed first-order kinetics, with half-life times varying greatly between dyes. The reactive azo dyes with triazyl reactive groups were slowly reduced. For these commonly occurring reactive dyes, long contact times might be necessary to reach an extent considered satisfactory (>90%) for decolorization. Consequently, they pose a serious problem for applying high-rate anaerobic treatment as the first stage in the biological degradation of azo dyes. Therefore, methods to improve the rate of azo reduction are clearly needed. To overcome the problem of slow azo dye reduction rates, redox mediators, that is, compounds that speed up reaction rates by shuttling reducing equivalents between (terminal) electron donors and electron acceptors may be helpful. Enzyme cofactors like FAD are known as effective redox mediators for azo dye reduction [10, 11, 12] and, in addition, artificial quinones can act as redox mediators: In abiotic systems, quinones have been shown to accelerate chemical azo dye reduction by sulfide [13] as well as electrochemical azo dye reduction [14] and, in biological systems, they were shown to accelerate azo dye reduction by anaerobically incubated aerobic biomass [15, 16] as well as azo dye reduction by anaerobic granular sludge [9]. For the sequential treatment, anaerobic and aerobic environments can be provided either in a single reactor for different periods or in two separate reactors. In a simultaneous treatment system, decolorization takes place in anaerobic zones of the biofilm or in immobilized microbes entrapped in a gel matrix [17, 18]. Furthermore, decrease in toxicity between the effluents of the anaerobic stage and anaerobicaerobic stage seems to provide indirect evidence for the removal of aromatic amines. As reviewed and reported earlier [15], various substituted aminobenzene and aminonaphthalene and aminobenzidine compounds have been found to be aerobically biodegradable. However, they generally require the enrichment of specialized cultures.

Materials and methods: Chemicals All chemicals used were of analytical grade. The common names of all chemicals have been used for convenience. Methyl Orange of The British Drug House Limited (BDH), England purchased from M/s Pragati Chemicals, Kanpur was used in this study. Medium The growth medium (GM) for anaerobic decolorisation consisted of yeast extract (YE) 3 g, (NH4)2SO4 0.5 g, KH2PO4 2.66 g, Na2HPO4 4.32 g per liter of distilled water. GM for aerobic degradation of aromatic amine formed in anaerobic reactor consisted of Na2HPO4 2g, KH2PO4 1g, NH4Cl .5g, MgCl2 .1g, CaCl2 0.05g, ABS .4-1.6 g per liter of distilled water. The pHs of both mediums was adjusted to 7.0, autoclaved at 121 oC for 15 min. For developing MO decolorizing culture, GM was amended with 50 mg/l MO and inoculated with mixed culture TJ1. Aerobic culture was developed with TJ2 mixed culture. Experimental Setup The reactor consisted of cylindrical perspex column (dia 13cm, length 19cm). The reactor was filled with ceramic rings (inner dia. 3mm) up to 14cm. Contents of 2L reactor were transferred to the SBR. Total reactor volume to the exit point was 1.7L and working volume was 1.28L. Feed solution (influent) entered vertically at the bottom through the sludge bed. (Fig.2). Feed solution consisted of glucose (600mg/L); pH of the feed was around 7.5 + 0.3. Reactor was maintained at ambient temperature of 30 - 35C. MO dye concentration was kept constant viz. 1mM in all experiments conducted in this study.

Figure 2: Schematic diagram Anaerobic Aerobic sequential batch reactor (adapted from PhD thesis, Taruna Joshi, IIT Kanpur) (1) Feed tank (2) Peristaltic pump (3) Anaerobic fixed film sequential batch rector (4) Aerobic reactor (5) Sparger (6) Air pump Analytical methods Dye decolorization was estimated by measuring the absorbance in a UV-vis spectrophotometer at 472nm. Samples were centrifuged at 1100g (3400 rpm) for 10 min. Supernatant was used for analysis. Uninoculated controls were always included in experiments. All the experiments were performed in triplicate. The standard variation was observed to be below 10%. Gram staining and biochemical tests were performed as per standard procedures (Cappuccino and Sherman, 1999). Determination of Total Amines using p- dimethylaminobenzaldehyde: 0.8 ml of distilled water and 0.05 ml of 1 M HCl were added to 0.2 ml of culture supernatant, and then 3ml of ethanol, 0.5ml of 5% p-dimethylaminobenzaldehyde in ethanol were further added. After 10 minutes, the absorbance was measured at 444nm in a UV-vis spectrophotometer [19]. Calibration Figure was prepared with required amines in the concentration range of 0 - 100mg/l. Results and Discussion:

Azo dye decolorisation in anaerobic reactor: Azo dyes become colorless as a result of reductive splitting of azo group with formation of aromatic amines: Ar1-N=N=Ar2 Ar1-NH2 + Ar2-NH2. MO produces N, N-dimethyl1,4-phenylenediamine and sulphanilic acid after reductive cleavage of azo bond. Experiment were done with 1 mM of MO and varying concentration of redox mediator NADH, FAD and AQS viz. 0.5mM, 1.0mM and 2 mM. The decolorization of the MO in these batch experiment show the increment in decolorization rates with increasing molar concentration of redox mediators. Within these redox mediators synthetic redox mediator AQS has shown significantly high decolorisation rates than natural redox mediators NADH, FAD. Between FAD and NADH, FAD has shown high decolorisation rate. Application of redox mediators: The results clearly establish that with redox mediators it is feasible to obtain high decolorization rates. AQS increases the reaction rate by acting as a redox mediator that shifts electrons between its oxidized, quinone forms (AQS) and its reduced hydroquinone form (AH2QS). The mechanism of redox mediation by quinones comprises two reactions: the oxidation of the hydroquinone by a terminal electron acceptor and the reduction of quinone by an electron donor.
2.0 m Redox m M ediator

5 Control abs at 472 nm 4 NA DH FAD 3

A QS

0 0 5 10 15 time(hrs) 20 25 30 35

Figure 3: Effect of different redox mediator on dye decolorization. Redox Mediators 2mM

AQS
7

abs at 472 nm

Control 0.5 mM

1.0 mM

2.0 mM

0 0 5 10 15 20 time(hrs) 25 30 35 40

Figure 4: Effect of different molar concentration of redox mediator (AQS) Oxidation of aromatic amines in aerobic reactor: Amines produced N,N-Dimethyl-P-Phenylenediamine (C8H12N2), Sulfanilic acid (4aminobenzene sulfonic acid, C6H7NO3S) in anaerobic reactor were autoclaved and media of aerobic reactor with TJ2 bacterial consortia was then added. Amine studies revealed that amines were slowly degrading in the aerobic reactor. A decline in amine concentrations was observed. Amines were degraded within 12 hours. Amines were mineralized into simpler products. N,N-dimethyl-1,4-phenylenediamine, after exposure to aerobic conditions, underwent autoxidation and polymerization yielding colored products which was evident from a largely altered UV-vis spectrum.

amine degradation
0.4

0.35

0.3

0.25 abs at 440nm

0.2

0.15

0.1

0.05

0 -1 4 9 time(hrs) 14 19 24

Figure 5: Amine degradation in aerobic reactor Conclusions: Synthetic redox mediators can be used effectively to carry out decolorisation. Quinones are the most effective than natural redox mediators. Higher the molar concentration of redox mediators higher the decolorisation rate. Azo dye can be completely mineralized in sequential bioreactor system viz. aerobic and anaerobic. Acknowledgements: Financial aid from the Department of Science and Technology (DST), Government of India is gratefully acknowledged. We also thank Arti Tiwari, Research Associate, IIT Kanpur for valuable help extended in carrying out the experiments. References: 1. Carliell, C.M., Barclay, S.J., Naidoo, N., Buckley, C.A., Mulholland, D.A., Senior, E., Microbial decolorization of a reactive azo dye under anaerobic conditions, Water SA, 21, pp. 61-69 (1995).

2. Cooper, P., Removing color from dye house waste waters-a critical review of technology available, J. Soc. Dyers Col, 109, pp. 97-100 (1993). 3. ONeill, C., Hawkes, F.R., Hawkes, D.L., Lourenco, N.D., Pinheiro, H.M., Delee, W., Color in textile effluents sources, measurement, discharge consents and simulation: A review, J. Chem. Technol. Biotechnol., 74, pp. 1009-1018 (1999b). 4. Chung, K.T., Stevens, S.E.J., Degradation of azo dyes by environmental microorganisms and helminthes, Environ. Toxicol. Chem., 12, pp. 2121-2132 (1993). 5. Chung, K.T., Cerniglia, C.E., Mutagenicity of azo dyes: Structure activity relationships, Mutat. Res., 277, pp. 201-220 (1992). 6. Pagga, U., Brown, D., The degradation of dyestuffs: Part II. Behavior of dyestuffs in aerobic biodegradation tests, Chemosphere, 15, pp. 479-491 (1986). 7. Shaul, G.M., Holdsworth, T.J., Dempsey, C.R., Dostal, K.A., Fate of water soluble azo dyes in the activated sludge process, Chemo sphere, 22, pp. 107-119 (1991). 8. Brown, D., Laboureur, P, The aerobic biodegradability of primary aromatic amines, Chemosphere, 12, pp. 405-414 (1983). 9. van der Zee, F.P., Lettinga, G., Field, J.A., Azo dye decolorisation by anaerobic granular sludge, Chemosphere, 44, pp. 1169-1176 (2001). 10. Fujita, S., Peisach, J., The stimulation of microsomal azoreduction by flavins, Biochim. Biophys. Acta., 719, pp. 178-189 (1982). 11. Gingell, R., Walker, R., Mechanism of azo reduction by Streptococcus faecalis II: The role of soluble flavins, Xenobiotica, 1, pp. 231-239 (1971). 12. Russ, R., Rau, J., Stolz, A., The function of cytoplasmic flavin reductases in the reduction of azo dyes by bacteria, Appl. Environ. Microbiol., 66, pp. 1429-1434 (2000). 13. van der Zee, F.P., Lettinga, G., Field, J.A., The role of (auto) catalysis in the mechanism of anaerobic azo reduction, Water. (2000). 14. Bechtold, T., Burtscher, E., Turcanu, A., Anthraquinones as mediators 465, pp. 80-87 (1999). for the indirect cathodic reduction of dispersed organic dyestuffs, J. Electroanal. Chem., Sci. Technol., 42, pp. 301-308

15. Keck, A., Klein, J., Kudlich, M., Stolz, A., Knackmuss, H.J., Mattes, R., Reduction of azo dyes by redox mediators originating in the naphthalenesulfonic acid degradation pathway of Sphingomonas sp. strain BN6, Appl. Environ. Microbiol., 63, pp. 3684-3690 (1997). 16. Kudlich, M., Keck, A., Klein, J., Stolz, A., Localization of the enzyme system involves in anaerobic reduction of azo dyes by Sphingomonas sp. strain BN6 and effect of artificial redox mediators on the rate Environ. Microbiol., 63, pp. 3691-3694 (1997). 17. Field , J.A., Cervantes, F.J., van der Zee, F.P., Lettinga, G., Role of quinones in the biodegradation of priority pollutants: A review, Water Sci. Technol ., 42, pp. 215-222(2000). 18. Kudlich, M., Bishop, P.L., Knackmuss, H.J., Stolz, A., Simultaneous anaerobic and aerobic degradation of the sulfonated azo dye Mordant Yellow-3 by immobilized cells from a naphthalenesulfonate-degrading mixed culture, Appl. Microbiol. Biotechnol., 46, pp. 597603 (1996). 19. Oren, A.S., Gurevich, P., and Henis, Y., Reduction of nitrosubstituuted aromatic compounds by the halophilic anaerobic bacteria eubacteria Haloanaerobium praevalens and Sporohalobacter marismortui, Appl. Environ. Microbiol., 57, pp. 3367-3370 (1991). of azo dye reduction, Appl.

List of Figures: Figure 1: Steps in azo dye degradation Figure 2: Schematic diagram Anaerobic Aerobic sequential batch reactor (1) Feed tank (2) Peristaltic pump (3) Anaerobic fixed film sequential batch rector (4) Aerobic reactor (5) Sparger (6) Air pump Figure 3: Effect of different redox mediator on dye decolorization. Redox Mediators Figure 4: Effect of different molar concentration of redox mediator (AQS) Figure 5: Amine degradation in aerobic reactor

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