1996 Microbial Decolorization Of Textile Dye Containing Effluents A Review

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Bioresource Technology 58 (1996) 217-227 Copyright © 1997 Elsevier Science Limitcd Printed in Great Britain. All rights rescrvcd 0960-8524/96 $15.00 ELSEVIER PII:S0960-8524(96)O0113-7 MICROBIAL DECOLORIZATION OF TEXTILE-DYECONTAINING EFFLUENTS: A REVIEW Ibrahim M. Banat, a* Poonam Nigam, h Datel Singh " & Roger Marchant h "Department of Biology University of the UAE, P.O. Box 17551, Al-Ain, UnitedArab Emirates hBiotechnology Research Group, School of Applied Biological and Chemical Sciences, University of Ulster, Coleraine BT52 1SA, UK 'Department of Microbiology, CCS Haryana Agricultural University. Hisar 125004, India (Received 5 June 1996; revised version received 9 August 1996; accepted 13 August 1996) mers and organic products. There are more than 8000 chemical products associated with the dyeing process listed in the Colour Index (Society of Dyers and Colourists, 1976) while over 100000 commercially available dyes exist with over 7 x 105 metric tons of dyestuff produced annually (Meyer, 1981; Zollinger, 1987). These dyes include several structural varieties of dyes, such as acidic, reactive, basic, disperse, azo, diazo, anthraquinone-based and metal-complex dyes. The only thing in common is their ability to absorb light in the visible region. Colour is the first contaminant to be recognized in wastewater and has to be removed before discharging into waterbodies or on land. The presence of very small amounts of dyes in water (less than 1 ppm for some dyes) is highly visible and affects the aesthetic merit, water transparency and gas solubility in lakes, rivers and other waterbodies. The removal of colour from wastewaters is often more important than the removal of the soluble colourless organic substances, which usually contribute the major fraction of the biochemical oxygen demand (BOD). Methods for the removal of BOD from most effluents are fairly well established; dyes, however, are more difficult to treat because of their synthetic origin and mainly complex aromatic molecular structures. Such structures are often constructed to resist fading on exposure to sweat, soap, water, light or oxidizing agents (Poots et al., 1976; McKay, 1979) and this renders them more stable and less amenable to biodegradation (Fewson, 1988; Seshadri et al., 1994). Government legislation is becoming more stringent in most developed countries regarding the removal of dyes from industrial effluents, which is in turn becoming an increasing problem for the textile industries. Environmental-protection agencies in Europe are promoting prevention of transferral of pollution problems from one part of the environment to another. This means that for most textile industries, developing on-site or in-plant facilities to Abstract Water-pollution control is presently one of the major areas of scientific activity. While coloured organic" compounds generally impart only a minor fraction of the organic load to wastewater, their colour renders them aesthetically unacceptable. Effluent discharge from textile and dyestuff industries to neighbouring water bodies and wastewater treatment systems is currently causing significant health concerns to environmental regulatory agencies. Colour removal, in particular, has recently become of major scientific interest, as indicated by the multitude of related research reports. During the past two decades, several physico-chemical decolorization techniques have been reported, few, however, have been accepted by the textile industries. Their lack of implementation has been largely due to high cost, low efficiency and inapplicability to a wide variety of dyes. The ability of microorganisms to carry out dye decolorization has received much attention. Microbial decolorization and degradation of dyes is seen as a cost-effective method for removing these pollutants from the environment. Recent fundamental work has revealed the existence of a wide variety of microorganisms capable of decolorizing an equally wide range of dyes. In this review we have examined biological decolorization of dyes used in textile industries and report on progress and limitations. Copyright © 1997 Elsevier Science Ltd. Key words: Microbial decolorization, colour removal, dyes, textile effluent, azo dyes, wastewater treatment. INTRODUCTION Textile industries consume substantial volumes of water and chemicals for wet processing of textiles. These chemicals are used for desizing, scouring, bleaching, dyeing, printing and finishing. They range from inorganic compounds and elements to poly*Author to whom correspondence should be addressed. 217 218 L M. Banat, P. Nigam, D. Singh, R. Marchant treat their own effluents before discharge is fast approaching actuality. Recently, state and federal agencies in the USA have been requiring lower effluent colour limits ( < 200 units of American Dye Manufacturers Institute, ADMI) (McCurdy et al., 1992). TEXTILE DYES: ENVIRONMENTAL CONCERNS Interest in the pollution potential of textile dyes has been primarily prompted by concern over their possible toxicity and carcinogenicity. This is mainly due to the fact that many dyes are made from known carcinogens, such as benzidine and other aromatic compounds, all of which might be reformed as a result of microbial metabolism (Clarke & Anliker, 1980). It has been shown that azo- and nitro-compounds are reduced in sediments (Weber & Wolfe, 1987) and in the intestinal environment (Chung et al., 1978), resulting in the regeneration of the parent toxic amines. Anthraquinone-based dyes are the most resistant to degradation due to their fused aromatic structures, which remain coloured for long periods of time. Basic dyes have high brilliance and therefore higher colour intensity, making them more difficult to decolorize, while metal-based complex dyes, such as chromium-based dyes, can lead to the release of chromium, which is carcinogenic in nature, into water supplies. Some disperse dyes have also been shown to have a tendency to bio-accumulate (Anliker et al., 1981; Baughman & Perenich, 1988) and heavy-metal ions from textile effluents have also been reported at high concentrations in both algae and higher plants exposed to such effluents (Srivastava & Prakash, 1991). The lack of data on the properties of many dyes has been the main problem in assessing broad classes of dyes to identify common characteristics. The above-mentioned classification as acid, direct, dispersant etc. is based on their dyeing mode or main structural moieties, both of which are inadequate for environmental evaluation purposes. Although dyes constitute only a small portion of the total volume of waste discharge in textile processing, these compounds are not readily removed by typical microbial-based waste-treatment processes (Brown et al., 1981). Furthermore, dyes can be detrimental to the microbial population present in such treatment works and may lead to decreased efficiency or treatment failure in such plants (Ogawa et al., 1988). Similar adverse effects have also been detected for aquatic microbial populations and the aquatic environment in general (Richardson, 1983; Michaels & Lewis, 1985), or for laboratory cultures (Ogawa et al., 1989) exposed to such dyes. The economic removal of polluting dyes is gaining great importance, particularly as new European Community (EC) regulations on industrial effluent discharge are at present being enforced. Since many textile plants have rural locations and municipal treatment costs are increasing, both industries and scientists are becoming compelled to search for innovative novel treatments and technologies directed particularly towards the decolorization of dyes in effluents. Dyes usually have a very low rate of removal ratio for BOD to COD (BOD/COD less than 0.1) (De Angelis & Rodrigues, 1987). Biological methods, being cheap and simple to use, have been the main focus of recent studies on dye degradation and decolorization. This review therefore aims at evaluating the state of the art on the subject. DECOLORIZATION TECHNIQUES In the past, municipal treatment systems were mainly used for the purification of textile mill wastewaters. These systems, however, depended mainly on biological activity and were mostly found inefficient in the removal of the more resistant synthetic dyes; less sensitive, yet more effective methods, therefore, were developed and tested for dye removal. Primarily they depended on using physicalor chemical-treatment processes, occasionally in conjunction with biological treatment (Groff, 1993; Wilking & Frahne, 1993; Mishra & Tripathy, 1993). The physical and chemical techniques were numerous and included physico-chemical flocculation combined with flotation, electroflotation, flocculation with Fe(II)/Ca(OH)2, membrane-filtration, electrokinetics coagulation, electrochemical destruction, ion-exchange, irradiation, precipitation, ozonation, adsorption and the Katox treatment method involving the use of activated carbon and air mixtures (Lin & Lin, 1993; Hosono et al., 1993; Ogfitveren & Koparal, 1994; Ulker & Savas, 1994; Lin & Liu, 1994; Chang & Lin, 1994; Shu et al., 1994; Huang et al., 1994; Spadaro & Tenganathan, 1994; Adams et al., 1995; Mao & Smith, 1995). Some of these techniques have been shown to be effective, although they have shortcomings. Among these are: excess amount of chemical usage or sludge generation with obvious disposal problems; costly plant requirements or operating expenses; lack of effective colour reduction, particularly for sulfonated azo dyes; and sensitivity to a variable wastewater input. Certain treatment schemes may be applicable for some textile mills using one or two types of dyes, but not for other mills or dye mixtures. Other techniques involve chemical oxidation using sodium hypochlorite to remove the colour. They, however, release a lot of aromatic amines which are carcinogenic, or otherwise toxic compounds; these subsequently aggravate the problem (Anliker, 1979). Utilizing the recalcitrance of dyes and affinity to adhere to surfaces as a means of removing them through adsorption, which would not involve biodegradation and release of intermediate products, has also been suggested by several authors (De Microb&l decolorization of textile dyes: a review Angelis & Rodrigues, 1987; Nawar & Doma, 1989). These above-mentioned technologies have been the subjects of several fairly recent reviews (Groff, 1991; 1993; Wilking & Frahne, 1993; Mishra & Tripathy, 1993). No one specific treatment process seems to be able to handle decolorization of all textile wastewaters and, generally, a customized process, probably involving a combination of methods, could be more applicable. BIOLOGICAL DECOLORIZATION PROCESSES The treatment of textile wastewater by purely biological processes may be possible even without the inclusion of other carbon sources, e.g. municipal wastewater. This has been the subject of intensive research in recent years. Such a situation was predicted earlier by McKay (1979) who concluded that "decolorization through biological systems would receive increased attention in the future". Among the many biological systems available are: Aerobic activated-sludge or rotating biofilm reactors (Shaul et al., 1991; Jiang & Bishop, 1994). Aerobic-anaerobic packed-bed reactors (Schliephake et al., 1993; Lin & Liu, 1994). Aerobic-anaerobic fluidized-bed reactors (Seshadri et al., 1994). Aerobic-anaerobic sequential batch or continuous-flow reactors (Loyd et al., 1992; Ganesh et al., 1994; Oxspring et al., 1996). Anaerobic batch reactors (Carliell et al., 1995). Several microbial cultures have been tested or are implicated in textile dye decolorization as follows. DECOLORIZATION CAPABILITY OF THE FUNGUS P H A N E R O C H A E T E CHRYSOSPORIUM Bio-decolorization of lignin-containing pulp and paper wastewater, as measured by the decrease in 219 colour absorption, using two white-rot Basidiomycete fungi: Phanerochaete chrysosporium and Tinctoporia sp., was reported as early as 1980 (Eaton et al., 1980; Fukuzumi, 1980). Both were clear examples of colour removal through microbial degradation of polymeric lignin molecules. Since then, the wood-rotting P. chrysosporium in particular has been the subject of intensive research related to the degradation of a wide range of recalcitrant xenobiotic compounds, including azo dyes. Table 1 shows recent reports of various percentage decolorizations achieved by this fungus. The mechanism of colour removal involves a lignin peroxidase and Mndependent peroxidase or laccase enzymes (Michel et al., 1991). The decolorization of three polymeric dyes Polymeric B-411, Polymeric R-481 and Polymeric Y-606 (Sigma) by P. chrysosporium was confirmed by Glenn and Gold (1983). Their results suggested that the decolorization was a secondary metabolic activity linked to the fungus' ligninolytic-degradation activity. The process, however, was slow and optimum decolorization needed up to 8 days. Phanerochaete chrysosporium was also shown to biodegrade the azo- and heterocyclic-dyes Orange II, Tropaeofin O, Congo Red and Azure B (Cripps et al., 1990). The extent of colour removal varied depending on the dye complexity, nitrogen availability in the media and ligninolytic activity in the culture. At low nitrogen concentrations 90% of the colour was removed within the initial 6 h, while when excess nitrogen was provided, up to 5 days were required to achieve 63-93% decolorization of the above-mentioned dyes (Cripps et al., 1990). A series of other dyes were also tested and were decolorized by P. chrysosporium at various concentrations, particularly when veratryl alcohol was present in the medium. Veratryl alcohol is believed to stimulate the ligninase activity, which seems to be linked to decoiorization (Paszczynski & Crawford, 1991; Table 1. Recent reports on strains of Phanerochaete chrysosporium capable of dye decolorizations Dye and concentration Percent removal/time" Amaranth (67 mg/l) Orange G (67 mg/l) Several azo dyes separately (12-20 pmol) Sulfonated azo dyes and sulfanilic acid (200 mg/l) 18 commercial azo dyes (unknown concentration) 10 polyaromatic azo dyes (200 mg/1) Orange II (57 /tm) Tropaeolin (7(} pro) Azure B (100 pm) Congo Red (76 pro) Polymeric B-411 (200 mg/l) Polymeric R-481 (200 mg/1) Polymeric Y-606 (200 rag/l) 90% (3 days) 90% (3 days) 23-48% (15 days) Reference Chao and Lee (1994) Spadaro et al. (1992) Up to 40% (2l days) Paszczynski et al. (1992) 40-73% (5 days) (8 dyes); 0.0% (5 days) for (10 dyes) 45-98% (10 days) Capalash and Sharma (1992) 100% (24 h) 95% (24 h) 91% (24 h) 93% (24 h) unknown (8 days) unknown (6 days) unknown (14 days) Cripps et al. (1990) Paszczynski and Crawford (1991) Glenn and Gold (1983) "In all cases the removal mechanism was a lignin peroxidase and Mn-dependent peroxidase system. 220 L M. Banat, P. Nigam, D. Singh, R. Marchant Ollikka et al., 1993; Sayadi & EUouz, 1995). Capalash and Sharma (1992) tested the biodegradation of 18 azo dyes using P. chrysosporium and only eight were degraded, with 40-70% colour removal. This degradation was mainly through the lignin-degrading enzyme system or adsorption to cell mass. Substitution with sulfo groups on the aromatic component of some azo dyes did not seem to significantly affect the biodegradability of the azo dyes (Paszczynski et al., 1992; Pasti-Grigsby et al., 1992). Spadaro et al. (1992), in contrast, showed that when aromatic rings of dyes had substituted hydroxyl, amino, acetamido or nitro functions, mineralization was greater than for those with unsubstituted rings. The use of high lignin-peroxidase-producing medium for growing P. chrysosporium immobilized on polyurethane foam significantly improved the decolorization achieved in olive-mill wastewater (Sayadi et al., 1995; 1996). The role of lignin peroxidase in a strain of P. chrysosporium has been doubted, however, in a recent report (Kirby et al., 1995) as only extremely low enzyme activities (<100 U 1-~) were detectable upon the decoiorization of several synthetic textile dyes. In the majority of the above cases it was generally observed that nitrogen-limitation increased ligninolytic activities through increased lignin peroxidase and Mn-dependent peroxidase and therefore enhanced decolorization (Perie & Gold, 1991). Con- trary to that, however, Chao and Lee (1994) reported higher decolorization rates when their strains were pre-grown in nitrogen-rich media, while slower or no decolorization occurred with strains pre-cultured in low-nitrogen media. The degradation of some xenobiotics by other white-rot fungi is known to occur under non-ligninolytic conditions and would mainly be through the laccase enzyme activity (Dhawale et al., 1992). OTHER DECOLORIZING FUNGI Several other fungi have been shown to be capable of decolorization (Table 2). Neurospora crassa was reported to decolorize diazo dyes by Corso and coworkers (1981). They achieved 89-91% colour removal within 24 h incubation when the concentration of the dye Vermelho Reanil P8B ranged between 16.0 and 20.0 mg 1-1, which resembles the range of concentrations found in industrial dye effluents. Schizophyllum c o m m u n e also decolorized wastewater from a bagasse-pulping plant (Belsare & Prasad, 1988). A strain of a Trichoderma sp., belonging to the fungi imperfecti, was also shown to decolorize lignin-containing hardwood-extraction-stage leachplant effluent (Prasad & Joyce, 1991). Up to 85% colour removal was achieved after 3 days cultiva- Table 2. Recent reports on various fungi capable of dye decolorizations Culture Aspergillus sojae Dye and concentration Amaranth (10 mg/1) B-10 Sudan III (10 mg/l) Congo Red (10 mg/l) Myrothecum verrucaria Orange II (200 mg/1) 10B (Blue) (200 mg/1) RS (Red) (200 mg/l) Myrothecum sp. Neurospora crassa Pycnoporus cinnabarinus Trichoderma sp. Candida sp. NR = Not reported. Orange II (100 mg~) 10B (Blue) RS (Red) (100 mg/1) Vermelho Reanil P8B (16-32 mg/1) Pigment plant effluent (unknown concentration) Hardwood extraction effluent (unknown concentration) Procyon Black SPL (100 mg/1) Procyon Blue MX2G (100 mg/i) Procyon Red HE7B (100 rag/l) Procyon Orange HER (100 mg/I) Percent removal/time 97.8% (5 days) 97.4% (5 days) 93.0% (5 days) 70.0% (5 h) Mechanism NR 86.0% (5 h) 95.0% adsorption 25-91% (24 h) 58-98% (24 h) 81-98% (24 h) 89-91% (24 h) 90% (3 days) 85% (3 days) adsorption 93.8% (2 h) 96.8% (2 h) 98.9% (2 h) 96.8% (2 h) adsorption (5 h) Reference Ryu & Weon (1992) NR NR adsorption Brahimi-Horn et al. (1992) adsorption Mou et al. (1991) adsorption adsorption adsorption Corso et al. (1981) extracellular Schliephake et al. (1993) oxidases ligninolytic Prasad & Joyce (1991) enzymes adsorption adsorption adsorption De Angelis & Rodrigues (1987) Microbial decolorization of textile dyes: a review tion. Another white-rot fungus, Pycnoporus cinnabarinus, was also found to tolerate and decolorize high concentrations of dyes in a packed-bed reactor within 48-72 h incubation. An extracellular oxidase activity similar to the veratryl alcohol, which acts as an inducer for ligninolytic activity in P. chrysosporium, was observed in P cinnabarinus, which confirmed its suitability for the degradation of dye effluent (Schliephake et al., 1993). Enrichment procedures designed to obtain microbial agents suitable for decolorizing dye-containing wastewater, by Mou and co-workers (1991), resulted in the isolation of several strains of fungi capable of decolorization. These included strains of Myrotheciurn verrucaria and of Ganoderma sp. Up to 99% decolorization was observed after 48 h incubation, which was mainly through adsorption to the fungus mycelium and was effective for a wide range of dyes. Myrothecium verrucaria was shown to have a very strong binding affinity to some azo dyes, which were recoverable by extraction with methanol, suggesting a hydrophobic-hydrophillic interaction in the dyebinding mechanism (Brahimi-Horn et al., 1992). De Angelis and Rodrigues (1987) tested colour removal of textile dyes using a Candida sp. yeast biomass (produced by the vast ethanol production industries in Brazil) and demonstrated 93-98% decolorization for several Procyon dyes at 100 mg/l. A strain of Aspergillus sojae B-10 was also shown to be able to decolorize the azo dyes Amaranth, Congo Red and Sudan III in nitrogen-poor media after 3-5 days incubation (Ryu & Weon, 1992). The decolorization of molasses wastewater has also been investigated and several fungal cultures capable of decolorization have been isolated. One culture possessing this capability was found to belong to the Basidiomycete group (Hongo, 1973). Further screening, utilizing melanoidin, the major colouring substance in molasses, resulted in isolation of Coriolus sp. 20. Its decolorizing ability was associated with the enzyme sorbose oxidase (Watanabe et al., 1982). Subsequent investigation by Ohmomo et al. (1985) yielded Coriolus versicolor Ps4a, Mycelia sterilia D90, Aspe.'gillus fumigatus G-2-6 and A. oryzae. Several other wood-rotting fungi capable of decolorizing a wide range of structurally different dyes were also isolated and found more effective than P. chrysosporium (Knapp et al., 1995) Recently, other facultative anaerobic fungi capable of growth on dyes as sole carbon sources have been reported. They, however, do not seem to be able to carry out decolorization (Marchant et al., 1994; Nigam et al., 1995a; b). They appear to cleave some of the bonds in these dyes to use as carbon sources, yet do not affect the chromophore centre of the dyes. This capability might be of significance when a consortium of microorganisms is employed in degrading dye-containing effluents when other decolorizers are present. Both types, the degrading and the decolorizing microorganisms, would ultima- 221 tely benefit from each other's activities to achieve more complete or faster biodegradation. Success in the decolorization of paper-mill bleach-plant effluent has also been recently reported using unidentified marine fungi which produced the enzymes laccase, manganese peroxidase and lignin peroxidase (Raghukumar et al., 1996). DECOLORIZING WITH BACTERIAL CULTURES Numerous bacteria capable of dye decolorization have been reported (Table 3). Efforts to isolate bacterial cultures capable of degrading azo dyes started in the 1970s with reports of a Bacillus subtilis (Horitsu et al., 1977), then Aeromonas hydrophila (Idaka & Ogawa, 1978), followed by a Bacillus cereus (Wuhrmann et al., 1980). Isolating such microorganisms proved to be a difficult task. Extended periods of adaptation in chemostat conditions were needed to isolate the first two Pseudomonas strains capable of dye decoiorization (Kulla, 1981). An azoreductase enzyme was responsible for the initiation of the degradation of the Orange II dye by these strains and substituting any of the groups near the azo group's chemical structure hindered the degradation (Zimmermann et al., 1982). Several other decolorizing Pseudomonas and Aeromonas species were then reported by a Japanese group (Ogawa et al., 1986; Yatome et al., 1987; Ogawa & Yatome, 1990; Yatome et al., 1990). A considerable period passed before an upsurge in interest in bacterial cultures took place in the western world. Haug et al. (1991) described a bacterial consortium capable of mineralizing the sulfonated azo dye Mordant Yellow. An alternation, however, from anaerobic to aerobic conditions was required to achieve complete degradation. This was necessary as different members of the consortium needed different conditions for optimum reaction and the main azo-bond cleavage needed the reductase enzymes, which are mainly functional under anaerobic conditions (Haug et al., 1991). In a review, Groff and Kim (1989) described a host of bacterial cultures with capabilities to carry out decolorization, including a Rhodococcus sp., Bacillus cereus, a Plesiomonas sp. and Achromobacter sp. Paszczynski and co-workers (1992) compared the efficiency of a soil actinomycete culture, Streptomyces chromofuscus, to that of the previously described fungus P. chrysosporium and concluded that the soil bacterium could carry out decolorization but to a lesser extent than the white-rot fungus (Paszczynski et al., 1992). The presence of sulfo groups on the aromatic component of some azo dyes seemed to significantly inhibit the biodegradability of the sulfonated azo dyes by bacteria (Kulla et al., 1983). This is contrary to the results reported for these dyes degraded with the fungal cultures described earlier. A Rhodococcus sp. capable of effectively decoloriz- 222 I.M. Banat, P. Nigam, D. Singh, R. Marchant ing two sulfonated azo dyes, Orange II and Amido Black, was used to clone DNA fragments coding for azoreductase into a mutant which had lost decolorization capability, thereby conferring sulfonated azo-dye decolorization ability (Heiss et al., 1992). Several other actinomycete stains have been reported with a capability to decolorize reactive dyes, including anthraquinone, phthalocyanine and azo, through adsorption of dyes to the cellular bidmass without any degradation (Zhou & Zimmermann, 1993). Other Cu-based azo dyes, such as formazan-copper complex dyes, were completely decolorized through degradation by the same actinomycete strains (Zhou & Zimmermann, 1993). Recently, a bacterium, Pseudomonas luteola, with the ability to remove the colour of reactive azo dyes, such as Red G, RBB, RP2B and V2RP, has been isolated from dyeing-wastewater-treatment sludge. Complete degradation was observed for the latter three dyes, while only azo-bond cleavage was Table 3. Recent reports on bacterial cultures capable of dye decolorizations Culture Aeromonas hydrophila var 24B Aeromonas hydrophila var 24B Bacillus subtilis IFO 1 3 7 1 9 Bacillus subtilis IFO 3002 Klebsiella pneumoniae RS-13 Pseudomonas cepacia IaNA Pseudomonas cepacia 13NA Pseudomonas cepacia 13NA (immobilized system) Dye and concentration various azo dyes (0.2 mmol/l) various azo dyes (10-100 mg/l) 2-carboxy 4' dimethyleamino benzene (0.045 mmole) p-aminoazobenzene (30 mg/1) Methyl Red (MR) (100 mg/1) C.I. Acid Orange 12 (10 mg/l) C.I. Acid Orange 20 (10 rag/l) C.I. Acid Red 88 (37 mg/l) Orange I (0.045 mmole) Percent removal/time 40-100% (24 h) 50-90% (24 h) 100% (20 rain) Mechanism azoreductase (cell free extract) azoreductase (cell free extract) azoreductase (in growing cells) 80-90% (30 h) 100% (24 h) 65% (8 h) 87% (8 h) 94% (8 h) = 90% (10 h) azoreductase Horitsu et al. (1977) azoreductase Wong & Yuen (1996) azoreductase Ogawa et al. (1986) p-aminoazobenzene (10 rag/l) 60-80% (10 h RT) Red G (100 rag/i) RBB (100 mg/l) Pseudomonas stutzeri various azo dyes (0.1 retool/l) 37.4% (2 days) 93.2% (2 days) 92.4% (2 days) 88.0% (2 days) 90% (8-20 min) Pseudomonas stutzeri Orange I (0.045 mmole) = 80% (20 h) Orange II (0.045 mmole) = 80% (20 h) Azo-reactive Red 147 (150 mg/l) Azo-copper Red 171 (180 rag/i) Anthraquinone Blue 114 (280 mg/l) Formazan Blue 209 (80 mg/l) Phthalocyanine Blue 116 (200 mg/l) Mordant Yellow (unknown) 29.0% (14 days) Pseudomonas luteola RP2B (100 mg/1), VERP (100 rag/I), IAM 12097 Streptomycetes BWI30 Mixed bacterial culture RT = Retention time. 73.0% (14 days) 27.0% (14 days) 70.0% (14 days) 39.0% (14 days) 50% (5 days) Reference Yatome et al. (1987) Idaka & Ogawa (1978) Yatome et al. (1991) azoreductase azoreductasee azoreductase (cell free extract and growing cells) azoreductase Yatome et al. (1991) azoreductase Hu (1994) Ogawa & Yatome (1990) azoreductase azoreductase azoreductase azoreductase (cellular extract) Yatome et al. (1990) azoreductase (cell free extract) azoreductase (cell free extract) adsorption Yatome et al. (1991) Zhou & Zimmermann (1993) adsorption adsorption adsorption adsorption azoreductase Haug et al. (1991) Microbial decolofization of textile dyes: a review observed for Red G dye in this culture (Hu, 1994). Other bacterial strains of Klebsiella pneumoniae RS13 and Acetobacter liquefaciens S-1 capable of decolorizing Methyl Red (MR) have also been reported as suitable for future applications in azodyes-containing industrial effluents (Wong & Yuen, 1996). DECOLORIZING WITH ALGAL CULTURES To our knowledge there has been only one report of algae capable of degrading azo dyes through an induced form of an azoreductase (Jinqi & Houtian, 1992). Several species of Chlorella and Oscillatoria were capable of degrading azo dyes to their aromatic amines and to further metabolize the aromatic amines to simpler organic compounds or CO2. Some were even capable of utilizing a few azo dyes as their sole source of carbon and nitrogen. Using such algae was proposed by the authors for use in stabilization ponds, as they can play a role in aromatic amine removal. MOST RECENT ADVANCES Studies carried out at the authors' laboratories have resulted in the isolation of various fungi and mixed bacterial cultures capable of growth on several kinds of azo, diazo and reactive dyes, both under aerobic and anaerobic conditions. Obtaining these cultures proved to be a time-consuming and demanding task (Nigam et al., 1995a; b; 1996a; b). Two mixed bacterial cultures, namely PDW and PDC, capable of decolorizing textile dyes, were isolated from enrichment cultures that were kept growing in minimal media containing dyes as sole carbon sources 223 and anaerobic conditions for over a year (Nigam et al., 1996a). An investigation into the efficiency of growth and decolorization for these cultures, PDW and PDC, concluded they were facultative, with an ability to grow under both aerobic and anaerobic conditions, but with highest growth rate and decolorization ability under anaerobic conditions. Both growth and decolorization in the two mixed cultures were enhanced in rich media supplemented with yeast extract. A distinct advantage was noticed for culture PDW which, upon purification, was found to be composed of at least two bacterial strains, Alcaligenes faecalis and Comomonas acidovorans (Nigam et al., 1996a). The results of a typical decolorization experiment with various individual types of textile dyes using the PDW-mixed culture are shown in Table 4. Percentage colour removal as measured by colour absorbency varied between 67-88% for eight out of a total of nine dyes tested. Only one of the dyes (Remazol Turquoise Blue G133) was not decolorized (Nigam et al., 1996b). It is of interest to note that this dye was the only one that had a phthalocyanine chemical composition. Whether this structural difference was the reason for its resilience, or whether there were other factors, remains to be seen. Figure 1 shows a colour absorbance scan for an effluent sample containing a mixture of all the previously described dyes in the presence of the PDW-mixed-bacterial culture after 1, 3 and 4 days. Although significant decolorization of the effluent mixture did occur ( = 80% after 4 days) its extent was less than that observed for most of the individual dye components (Table 1). This might be due to the presence of dye #8 (Remazol Turquoise Blue) which remained in the effluent and imparted the residual colour, while most other component dyes were completely decolorized within 4 days. Table 4. Percentage colour removal for nine textile dyes by a PDW-mixed-bacterial culture under laboratory-culture conditions Textile dye Cibacron Red C-2G Remazole Navy Blue GG Remazole Red RB Cibacron Orange CG Remazol Golden Yellow RNL Hisperse Navy D2GR Remazol Blue B Remazol Turquoise Blue G133 Remazol Black B Absorbancer,ax (nm) Dye type Decolorization (%) 24 h 4 days 515 620 Reactive Diazo 88 80 100 100 525 Diazo 89 100 489 Reactive 79 100 410 Azo 78 100 543 Disperse 68 80 590 660 Diazo Phthalocyanine 76 08 100 < 20 600 Diazo 67 100 Decolorization expressed as percent decrease of absorbancem~x for each dye after 24 h and 4 days incubation at 26°C under anaerobic conditions. Dye concentration was 500 mg/l. 224 L M. Banat, P. Nigam, D. Singh, R. Marchant It is also of interest to note that the two components obtained in pure culture upon the purification of PDW described above did not have the independent capability to decolorize any of the dyes. The advantages of mixed cultures are apparent as some strains can collectively carry out complex biodegradation tasks that no individual strain can achieve independently. Similar other mixed bacterial cultures have also been reported recently (Knapp & Newby, 1995) Further testing involving a selection of cheap support media for biofilm development suitable for active textile dye decolorization using the PDW-mixed-bacterial culture was pursued. Several support materials were investigated, including gravel, calcium alginate beads, polystyrene chips, polyurethane foam chips, nylon-web cubes, inert polyethylene chips, sea-shell powder and highly porous volcanic rocks composed of silicon and aluminium oxides (Nigam & Marchant, 1995). The results of a typical experiment showing decoloriza- 0.800 0.600 ~,,~ Mixture of dyes (Effluent) day 0 < 0.400 0000 300 400 500 600 Wavelength (nm.) 700 Fig. 1. Decolorization of textile-plant effluent containing a mixture of dyes by PDW-mixed-bacterial culture under laboratory-scale culture conditions. tion of three commercially utilized dyes and a highly coloured textile effluent, all of which were obtained from a local textile factory, are shown in Table 5. Such results demonstrate the ease with which biofilms were obtained using PDW on different media, many of which are cheap and readily available. It also highlighted the effectiveness of colour removal for a representative collection of azo, diazo and reactive dyes. Total decolorization was also achieved for the textile effluent which contained a mixture of dyes of unknown composition. Developing such bacterial systems should make it possible to apply them to large-scale effluent-treatment plants. CONCLUSION Coloured-dye-wastewater treatment and decolorization presents an arduous task. Wide ranges of pH, salt concentrations and chemical structures often add to the complication. Among the most economically viable choices available for effluent treatment/decolorization, and the most practical in terms of manpower requirements and running expenses to adopt and develop, appear to be the biological systems. At present biological systems are known to be capable of dealing with BOD and COD reduction or removal through conventional aerobic biodegradation. They have, however, an inherent problem in their inability to remove colour. Although decolorization is a challenging process to both the textile industry and the wastewater-treatment facilities that must treat them, the literature suggests a great potential for microbial decolorizing systems for achieving total colour removal and (occasionally) with only hours of exposure. Such biological processes could be adopted as a pre-treatment decolorization step, combined with the conventional treatment system (e.g. activated sludge) to reduce the BOD and COD, as an effect- Table 5. The use of different laboratory-scale biofilms systems for textile dyes and effluent decolorization (Nigam & Marchant, 1995) Biofilm support matrix Volcanic rock (Kissirs) Sea-shell powder Inert polyethylene chips Nylon-web cubes Polyurethane foam chips Vermiculite (SiO2) Polystyrene chips Gravel Calcium alginate beads Time required for complete decolorization (h) R. Golden Yellow (azo dye) R. Navy Blue (diazo dye) Cibacron Red (reactive dye) Textile effluent 11 13 10 10 12 11 14 14 11 10 12 13 8 7 9 9 9 7 9 8 9 10 10 10 16 12 34 18 15 38 16 13 35 15 13 32 Microbial decolorization of textile dyes: a review ive alternative for use by the textile-dyeing industries. Concerted efforts are still required to establish biological decolorization systems. The techniques by which decolorization occurs vary and among them adsorption seems of great significance for future development in bio-removal or bio-recovery of dye substances. One of the routes still to be explored is the use of thermotolerant or therrnophilic microorganisms in decolorization systems. This would be of advantage as many textile and other dye effluents are produced at relatively high temperatures (50-60°C), even after a cooling or heat-exchange step. The availability of thermotolerant organisms may consequently reduce cost significantly, through removing the need for further removal of low-grade heat and through allowing more immediate treatment. The role of molecular biology has yet to feature prominently in this vital area of environmental protection. REFERENCES Adams, S. D., Fusco, W. & Kanzelmeyer, T. (1995). Ozone, hydrogen peroxide/ozone and UV/ozone treatment of chromium- and copper-complex dyes: decolorization and metal release. Ozone Sci. Engng, 17, 149-162. Anliker, R. (1979). Ecotoxicology of dyestuffs--a joint effort by industry. J. Ecotoxicol. Environ. Safety, 3, 59-74. Anliker, R., Clarke, E. A. & Moser, P. (1981). Use of the partition coefficient as an indicator of bio-accumulation tendency of dyestuffs in fish. Chemosphere, 10, 263-274. Baughman, G. L. & Perenich, T. A. (1988). Fate of dyes in aquatic systems: I. Solubility and partitioning of some hydrophobic dyes and related compounds. Environ. Toxicol. Chem., 7, 183-199. Belsare, D. K. & Prasad, D. Y. (1988). Decolorization of effluent from the bagasse-based pulp mills by the whiterot fungus Schikophyllum commune. Appl. Microbiol. Biotechnol., 28, 301-306. Brahimi-Horn, M. C., Lim, K. K., Liany, S. L. & Mou, D. G. (1992). Binding of textile azo dyes by Mirothecium verrucaria--Orange I1, 10B (blue) and RS (red) azo dye uptake for textile wastewater decolorization. J. Ind. Microbiol., 10, 31-36. Brown, D., Hitz, H. R. & Schefer, L. (1981). The assessment of the possible inhibitory effect of dyestuffs on aerobic wastewater bacteria. Experience with a screening test. Chemosphere, 10, 245-261. Capalash, N. & Sharma, P. (1992). Biodegradation of textile azo dyes by Phanerochaete chrysosporium for potential application in azo dye degradation and decolorization in wastewater. World J. Microbiol. Biotechnol., 8, 309-312. Carliell, C. M., Barclay, S. J., Naidoo, N., Buckley, C. A., Mulholland, D. A. & Senior, E. (1995). Microbial decolorisation of a reactive azo dye under anaerobic conditions. Water SA (Pretoria), 21, 61-69. Chang, C. J. & Lin, J. C. (1994). Elimination of dyestuffs slurry pollution using liquid phase oxidation. J. Chinese Inst. Chem. Engng, 25, 215-220. Chao, W. L. & Lee, S. L. (1994). Decoloration of azo dyes by three white rot fungi: influence of carbon source. World J. Microbiol. Biotechnol., 10, 556-559. 225 Chung, K. T., Fulk, G. E. & Egan, M. (1978). Reduction of azo dyes by intestinal anaerobes. Appl. Environ. Microbiol., 35, 5588-5620. Clarke, E. A. & Anliker, R. (1980). Organic dyes and pigments. In The Handbook of Environmental Chemistry, Vol. 3, Part A. Anthropogenic Compounds, ed. O. Hutzinger. Springer, Heidelberg, pp. 181-215. Corso, C. R., De Angelis, D. F., De Oliveira, J. E. & Kiyan, C. (1981). Interaction between the diazo dye, 'Vermelho Reanil' P8B. and Neurospora crassa strain 74A. Eur. J. Appl. Microbiol. Biotechnol., 13, 64-66. Cripps, C., Bumpus, J. A. & Aust, S. D. (1990). Biodegradation of azo and heterocyclic dyes by Phanerochaete chrysosporium. Appl. Environ. Microbiol., 56, 1114-1118. Dhawale, S. W., Dhawale, S. S. & Dean-Ross, D. (1992). Degradation of phenanthrene by Phanerochaete chrysosporium occurs under ligninolytic as well as nonligninolytic conditions. Appl. Environ. Microbiol., 58, 3000-3006. DeAngelis, F. E. & Rodrigues, G. S. (1987). Azo dyes removal from industrial effluents using yeast biomass. Arquiros De Biologia E. Technologia (Curitiba), 30, 301-309. Eaton, D., Chang, H. M. & Kirk, T. K. (1980). Fungal decolorization of Kraft bleach plant effluent. Tappi J., 63, 103-109. Fewson, C. A. (1988). Biodegradation of xenobiotic and other persistent compounds: the causes of recalcitrance. Trends Biotechnol., 6, 148-153. Fukuzumi, T. (1980). Microbial decolorization and defoaming of pulping waste liquors in lignin biodegradation. In Microbiology, Chemistry and Potential Applications, Voi. t, ed. T. K. Jurj, T. Higuchi & H. Chang. CRC Press, Boca Raton, FL, pp. 215-230. Ganesh, R., Boardman, G. D. & Michelsen, D. (1994). Fate of azo dyes in sludges. Water Res., 28, 1367-1376. Glenn, J. K~ & Gold, M. H. (1983). Decolorization of several polymeric dyes by the lignin-degrading Basidiomycete Phanerochaete chrysosporium. Appl. Environ. Microbiol., 45, 1741- 1747. Groff, K. A. (1991). Textile wastes. J. Water Pollut. Control Fed., 63,459-462. Groff, K. A. (1993). Textile waste--textile industry wastewater waste disposal; a review. Water Environ. Res., 65, 421-423. Groff, K. A. & Kim, B. R. (1989). Textile wastes. J. Water Pollut. Control Fed., 63, 872-876. Haug, W., Schmidt, A., Nortemann, B., Hempel, D. C., Stolz, A. & Knackmuss, H. J. (1991). Mineralization of the sulphonated azo dye mordant yellow 3 by a 6-aminonaphthatene-2-sulphonate-degrading bacterium consortium. AppL Environ. Microbiol., 57, 3144-3149. Heiss, G. S., Gowan, B. & Dabbs, E. R. (1992). Cloning of DNA from a Rhodococcus strain conferring the ability to decolorize sulfonated azo dyes. FEMS Microbiol. Lett., 99, 221-226. Hongo, M. (1973). Japanese Patent 870421, issued 1973. Horitsu, H., Takada, M., ldaka, E., Tomoyeda, M. & Ogawa, T. (1977). Degradation of p-aminoazobenzene by Bacillus subtilis. Eur. J. Appl. Microbiol., 4, 217-224. Hosono, M., Arai, H., Aizawa, M., Yamamoto, I., Shimizu, K. & Augiyama, M. (1993). Decoioration and degradation of azo dye in aqueous solution supersaturated with oxygen by irradiation of high-energy electron beams. Appl. Radiat. Iso., 44. 1199-1203. Hu, T. L. (1994). Decolorization of reactive azo dyes by transformation with Pseudomonas luteola. Biores. Technol., 49, 47-51. Huang, C. R., Lin, Y. K. & Shu, H. Y. (1994). Wastewater decolorization and TOC-reduction by sequential treatment. Am. Dyestuff Reporter, 83, 15-18. 226 I.M. Banat, P Nigam, D. Singh, R. Marchant Idaka, E. & Ogawa, Y. (1978). Degradation of azo compounds by Aeromonas hydrophila var. 2413. J. Soc. Dyers and Colourists, 94, 91-94. Jiang, H. & Bishop, P. L. (1994). Aerobic biodegradation of azo dyes in biofilms. Water Sci. Technol., 29, 525-530. Jinqi, L. & Houtian, L. (1992). Degradation of azo dyes by algae. Environ. Poll., 75, 273-278. Kirby, N., McMullan, G. & Marchant, R. (1995). Decolorization of an artificial textile effluent by Phanerochaete chrysosporium. Biotechnol. Lett., 17, 761-764. Knapp, J. S. & Newby, P. S. (1995). The microbiological decolorization of an industrial effluent containing a diazo-linked chromophore. Water Res., 29, 1807-1809. Knapp, J. S., Newby, P. S. & Reece, L. P. (1995). Decolorization of dyes by wood-rotting Basidiomycete fungi. Enzyme Microbial Biotechnol., 17, 664-668. Kulla, H. G. (1981). Aerobic bacterial degradation of azo dyes. In Microbial Degradation of Xenobiotics and Recalcitrant Compounds, FEMS Symposium 12, ed. T. Leisinger, A. W. Cook, R. Hutter & J. Nuesch. Academic Press, London, pp. 387-399. Kulla, H. G., Klausener, F., Meyer, U., Liideke, B. & Leisinger, T. (1983). Interference of aromatic sulfo groups in the microbial degradation of the azo dyes Orange I and Orange II. Arch. Microbiol., 135, 1-7. Lin, S. H. & Lin, C. M. (1993). Treatment of textile waste effluent by ozonation and chemical coagulation. Water Res., 27, 1743-1748. Lin, S. H. & Liu, W. Y. (1994). Continuous treatment of textile water by ozonation and coagulation. J. Environ. Engng (N. Y.), 120, 437-446. Loyd, C. K., Goardman, G. D. & Michelsen, D. L. (1992). Anaerobic/aerobic treatment of a textile dye wastewater. Hazard. Ind. Wastes, 24, 593-601. Mao, H. Z. & Smith, D. W. (1995). Toward elucidating mechanism and kinetics of ozone decolorization and dechlorination of pulp-mill effluents. Ozone Sci. Engng, 17, 419-448. Marchant, R., Nigam, P. & Banat, I. M. (1994). An unusual facultatively anaerobic fungus isolated from prolonged enrichment culture conditions. Mycol. Res., 98, 757-760. McCurdy, M. W., Boardman, G. D., Michelsen, D. L. & Woodby, B. M. (1992). Chemical reduction and oxidation combined with biodegradation for the treatment of a textile dye. In 46th Proc. Purdue Industrial Waste Conf. Lewis Publishers, MI, pp. 229-234. McKay, G. (1979). Waste colour removal from textile effluents. Am. Dyestuff. Reporter, 68, 29-36. Meyer, U. (1981). Biodegradation of synthetic organic colorants. In Microbial Degradation of Xenobiotic and Recalcitrant Compounds, FEMS Symposium 12, ed. T. Leisinger, A. M. Cook, R. Hutter & J. Nuesch. Academic Press, London, pp. 371-385. Michaels, G. B. & Lewis, D. L. (1985). Sorption and toxicity of azo and triphenyl methane dyes to aquatic microbial populations. Environ. Toxicol. Chem., 4, 45 -50. Michel Jr, F. C., Dass, S. B., Grulke, E. A. & Reddy, C. A. (1991). Role of manganese peroxidases and lignin peroxidases of Phanerochaete chrysosporium in the decolorization of kraft bleach plant effluent. Appl. Environ. Microbiol., 57, 2368-2375. Mishra, G. & Tripathy, M. (1993). A critical review of the treatments of decolorization of textile effluent. Colourage, 40, 35-38. Mou, D. G., Lim, K. K. & Shen, H, P. (1991). Microbial agents for decolorization of dye wastewater. BiotechnoL Adv., 9, 613-622. Nawar, S. S. & Doma, H. S. (1989). Removal of dyes from effluents using low-cost agricultural byproducts. Sci. Total Environ., 79, 271-279. Nigam, P., Banat, I. M., Singh, D. & Marchant, R. (1996a). Microbial process of fast decolorization of textile effluent containing azo, diazo and reactive dyes. Proc. Biochem., 31, 435-442. Nigam, P., McMullan, G., Banat, I. M. & Marchant, R. (1996b). Decolorization of effluent from the textile industry by a microbial consortium. Biotechnol. Lett., 18, 117-120. Nigam, P., Banat, I. M., Oxspring, D., Marchant, R., Singh, D. & Smyth, W. F. (1995a). A new facultative anaerobic filamentous fungus capable of growth on recalcitrant textile dyes as sole carbon source. Microbios, 84, 171-185. Nigam, P., Singh, D. & Marchant, R. (1995b). An investigation of the biodegradation of textile dyes by aerobic and anaerobic microorganisms. In Environmental Biotechnology: Principles and Applications, ed. M. Moo-Young. Kluwer Academic, The Netherlands, pp. 278-292. Nigam, P. & Marchant, R. (1995). Selection of a substratum for composing biofilm system of a textile-effluent decolorizing bacteria. Biotechnol. Lett., 17, 993-996. Ogawa, T., Fuji, I., Kawal, K., Yatome, C. & Idaka, E. (1989). Growth inhibition of Bacillus subtilis upon interaction between basic dyes and DNA. Bull. Environ. Contain. Toxicol., 42, 402-408. Ogawa, T., Shibata, M., Yatome, C. & Odala, E. (1988). Growth inhibition of Bacillus subtilis by basic dyes. Bull. Environ. Contain. Toxicol., 40, 545-552. Ogawa, T. & Yatome, C. (1990). Biodegradation of azo dyes in multistage rotating biological contactor immobilized by assimilating bacteria. Bull. Environ. Contam. Toxicol., 44, 561-566. Ogawa, T., Yatome, C., Idaka, E. & Kamiya, H. (1986). Biodegradation of azo acid dyes by continuous cultivation of Pseudomonas cepacia 13 NA. J. Soc. Dyers and .. Colourists, .2., 12-14. Og/itveren, U. B. & Koparal, S. (1994). Color removal from textile effluents by electrochemical destruction. J. Environ. Sci. Health,/t29, 1-16. Ohmomo, S., Itoh, N., Watanabe, Y., Kaneko, Y., Tozawa, Y. & Ueda, K. (1985). Continuous decolorization of molasses wastewater with mycelia of Coriolus versicolor Ps4a. Agric. BioL Chem., 49, 2551-2555. Ollikka, P., Alhonmaki, K. Leppanen, V-M, Glumoff, T., Raijola, T. & Souminen, I. (1993). Decolorization of azo, triphenyl methane, heterocyclic and polymeric dyes by lignin peroxidase isoenzymes from Phanerochaete chrysosporium. Appl. Environ. Microbiol., 59, 4010-4016. Oxspring, D. A., McMullan, G., Smyth, W. F. & Marchant, R. (1996). Decolorization and metabolism of the reactive textile dye, Remazol-Black-B, by an immobilized microbial consortium. Biotechnol. Lett., 18, 527-530. Pasti-Grigsby, M. B., Paszczynski, A., Goszczynski, S., Crawford, D. L. & Crawford, R. L. (1992). Influence of aromatic substitution patterns on azo dye degradability by Streptomyces spp. and Phanerochaete chrysosporium. Appl. Environ. Microbiol., 58, 3605-3613. Paszczynski, A. & Crawford, R. L. (1991). Degradation of azo compounds by ligninase from Phanerochaete chrysosporium: involvement of veratryl alcohol. Biochem. Biophys. Res. Comm., 178, 1056-1063. Paszczynski, A., Pasti-Grigsby, M. B., Goszczyanski, S., Crawford, R. L. & Crawford, D. L. (1992). Mineralization of sulfonated azo dyes and sulfanilic acid by Phanerochaete chrysosporium and Streptomyces chromofuscus. Appl. Environ. Microbiol., 58, 3598-3604. Perie, F. H. & Gold, M. H. (1991). Manganese regulation of manganese peroxidase expression and lignin degrada- Microbial decolorization of textile dyes: a review tion by the white-rot fungus Dichom#us squalens. Appl. Environ. Microbiol., 57, 2240-2245. Poots, V. J., McKay, G. & Heakt, J. J. (1976). The removal of acid dye from effluent using natural absorbents--I1. Water Res., 10, 1067-1070. Prasad, D. Y. & Joyce, T. K (1991). Color removal from kraft bleach-plant effluents by Trichoderma sp. Tappi J. (January), 165-169. Raghukumar, C., Chandramohan, D., Michel, F. C. & Reddy, C. A. (1996). Degradation of lignin and decolorization of paper-mill bleach plant effluent (Bpe) by marine fungi. Biotechnol. Lett., 18, 105-106. Richardson, M. L. (1983). Dyes--the aquatic environment and the mess made by metabolises. J. Soc. Dyers and Colorists, 99, 198-200. Ryu, B. H. & Weon, Y. D. (1992). Decolorization of azo dyes by Aspergillus sojae B-10. J. Microbiol. Biotechnol., 2, 215-219. Sayadi, S. & Ellouz, R. (1995). Roles of lignin peroxidase and manganese peroxidase from Phanerochaete chryso~porium in the decolorization of olive mill wastewaters. Appl. Environ. Mierobiol., 61, 1098-1103. Sayadi, S., Zorgani, F. & Ellouz, R. (1996). Decolorization of olive mill wastewaters by free and immobilized Phanerochaete chrysosporium cultures--effect of the high-molecular-weight polyphenols. Appl. Biochem. Biotechnol., 56, 265-276. Sayadi, S., Zorgani, F., Labat, W Jaoua, M., Aifa, S., Gargouri, A., Zekri, S. & Ellouz, R. (1995). Lignin peroxidase is the key enzyme in the decolorization of olive mill wastewaters by Phanerochaete chrysosporium. J. Cellular Biochem., S21A SIA, 53. Schliephake, K., Lonergan, G. T., Jones, C. L. & Mainwaring, D. E. (1993). Decolorization of a pigment plant effluent by Pycnoporus cinnabarinus in a packed-bed bioreactor. Biotechnol. Lett., 15, 1185-1188. Seshadri, S., Bishop, P. L. & Agha, A. M. (1994). Anaerobic/aerobic treatment of selected azo dyes in wastewater. Waste Mgmnt, 15, 127-137. Shaul, G. M., Holdsworth, T. J., Dempsey, C. R. & Dostall, K. A. (1991). Fate of water soluble azo dyes in the activated sludge process. Chemosphere, 22, 107-119. Shu, H. Y., Huang, C. R. & Chang, M. C. (1994). Decolorization of mono-azo dyes in wastewater by advanced oxidation process: a case study of acid red 1 and acid yellow 23. Chemosphere, 29, 2597-2607. Society of Dyers and Colourists (1976). Color Index, 3rd edn. Society of Dyers and Colourists, Yorkshire, UK. Spadaro, J. T. & Tenganathan, V. (1994). Peroxidasecatalysed oxidation of azo dyes: mechanism of disperse 227 Yellow 3 degradation. Arch. Biochem. Biophys., 312, 301-307. Spadaro, J. T., Gold, M. H. & Renganathan, V. (1992). Degradation of azo dyes by the lignin-degrading fungus Phanerochaete chrysosporium. Appl. Environ. Microbiol., 58, 2397-2401. Srivastava, P. N. & Prakash, A. (1991). Bio-accumulation of heavy metals by algae and wheat plants fed by textile effluents. J. Ind. Poll. Control Fed., 7, 25-30. Ulker, B. & Savas, K. (1994). Color removal from textile effluents by electrochemical destruction. J. Environ. Sci. Health, A29, 1-16. Watanabe, Y., Sugi, R., Tanaka, Y. & Hayashida, Y. (1982). Enzymatic decolorization of melanoidin by Coriolus sp. No. 20. Agric. Biol. Chem., 46, 1623-1630. Weber, E. J. & Wolfe, N. L. (1987). Kinetics studies of reduction of aromatic azo compounds in anaerobic sediment/water systems. Environ. Toxicol. Chem., 6. 911-920. Wilking, A. & Frahne, D. (1993). Textile effluent-treatment methods of the 90s. Melliand English, 74, E325-E328. Wong, P. K. & Yuen, P. Y. (1996). Decolorization and biodegradation of methyl red by Klebsiella pneurnoniae RS- 13. Water Res., 30, 1736-1744. Wuhrmann, K., Mechsner, K. I. & Kappeler, Th. (1980). Investigation on rate determining factors in the microbial reduction of azo dyes. Eur..I. Appl. Mict~biol., 9, 325-338. Yatome, C., Ogawa, T., Hayashi, H. & Ogawa, T. (1991). Microbial reduction of azo dyes by several strains. J. Environ. Sci. Health, A26, 471-485. Yatome, C., Ogawa, T., Hishida, H. & Taguchi, T. (1990). Degradation of azo dyes by cell-free extracts from Pseudomonas stutzeri. J. Soc. Dyers and Colourists, 106, 280-282. Yatome, C., Ogawa, T., Itoh, K., Sugiyama, A. & Idaka, E. (1987). Degradation of azo dyes by cell-free extracts from Aeromonas hydrophila var. 2413. J. Soc. Dyers' and Colourists, 3, 389-395. Zhou, W. & Zimmermann, W. (1993). Decolorization of industrial effluents containing reactive dyes by actinomycetes. FEMS Microbiol. Lett., 107, 157-162. Zimmermann, T., Kulla, H. G. & Leisinger, T. (1982). Properties of purified orange 1I azoreductase, the enzyme initiating azo dye degradation by Pseudomonas KF46. Eur. J. Biochem., 129, 197-203. Zollinger, H. (1987). Colour Chemistry--Synthesis, Properties and Applications of Organic Dyes and Pigments. VCH Publishers, New York, pp. 92-100.