Removal of Dyes From the Effluent

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Removal of Dyes from the Effluent ofTextile and Dyestuff ManufacturingIndustry: A Review of EmergingTechniques With Reference to BiologicalTreatmentHARPREET SINGH RAI a , MANI SHANKAR BHATTACHARYYA b , JAGDEEPSINGH c , T. K. BANSAL d , PURVA VATS e & U. C. BANERJEE fa Department of Chemical Engineering, Thapar Institute ofEngineering and Technology, Patiala, Punjab, Indiab Department of Pharmaceutical Technology, National Institute ofPharmaceutical Education and Research, Sector 67, S.A.S. Nagar,Punjab, Indiac Department of Civil Engineering, National Institute of Technology,Jalandhar, Punjab, Indiad Department of Chemical Engineering, Thapar Institute ofEngineering and Technology, Patiala, Punjab, Indiae Institute of Microbial Technology, Sector 39-A, Chandigarh, Indiaf Department of Pharmaceutical Technology, National Institute ofPharmaceutical Education and Research, Sector 67, S.A.S. Nagar,Punjab, India

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To cite this article: HARPREET SINGH RAI, MANI SHANKAR BHATTACHARYYA, JAGDEEP SINGH, T. K.BANSAL , PURVA VATS & U. C. BANERJEE (2005): Removal of Dyes from the Effluent of Textile andDyestuff Manufacturing Industry: A Review of Emerging Techniques With Reference to BiologicalTreatment, Critical Reviews in Environmental Science and Technology, 35:3, 219-238

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Critical Reviews in Environmental Science and Technology, 35:219–238, 2005Copyright © Taylor & Francis Inc.ISSN: 1064-3389 print / 1547-6537 onlineDOI: 10.1080/10643380590917932

Removal of Dyes from the Effluent of Textileand Dyestuff Manufacturing Industry:

A Review of Emerging Techniques WithReference to Biological Treatment

HARPREET SINGH RAIDepartment of Chemical Engineering, Thapar Institute of Engineering and Technology,

Patiala, Punjab, India

MANI SHANKAR BHATTACHARYYADepartment of Pharmaceutical Technology, National Institute of Pharmaceutical Education

and Research, Sector 67, S.A.S. Nagar, Punjab, India

JAGDEEP SINGHDepartment of Civil Engineering, National Institute of Technology, Jalandhar, Punjab, India

T. K. BANSALDepartment of Chemical Engineering, Thapar Institute of Engineering and Technology,

Patiala, Punjab, India

PURVA VATSInstitute of Microbial Technology, Sector 39-A, Chandigarh, India

U. C. BANERJEEDepartment of Pharmaceutical Technology, National Institute of Pharmaceutical Education

and Research, Sector 67, S.A.S. Nagar, Punjab, India

Biological removal of dyes from effluents of textile and dyestuffmanufacturing industry offers some distinct advantages over thecommonly used chemicals and physicochemical methods. Theseinclude possible mineralization of the dyes to harmless inorganiccompounds like carbon dioxide and water, and formation of alesser quantity of relatively harmless sludge. Removal of dyes fromthese wastewaters has been reviewed with respect to biological decol-orization as well as complete biodegradation of the dye molecules.Emerging techniques with reference to biological treatment of thesewastewaters have been discussed under aerobic, anaerobic, andcombined anaerobic–aerobic systems. Advantages and limitations

Address correspondence to U. C. Banerjee, Department of Pharmaceutical Technology,National Institute of Pharmaceutical Education and Research, Sector 67, SAS Nagar-160 062,Punjab, India. E-mail: [email protected]

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220 H. S. Rai et al.

of different biological methods have been highlighted, and futurestudies to establish these techniques for their applications on indus-trial scale have been suggested.

KEY WORDS: bioaugmentation, biodegradation, decolorization,synthetic effluent, textile and dyestuff effluent, textile dyes, triph-enylmethane and azo dyes

I. INTRODUCTION

Highly colored substances, widely known as colorants, can be used to im-part color to an infinite variety of materials described technically as substrates.Colorants can be subdivided into dyes, which are soluble in the medium inwhich they are applied, and pigments, which are insoluble in the applica-tion medium. Dyes are defined as colored substances that when applied tofibers give them a permanent color that is resistant to action of light, water,and soap. Practically every dyestuff is made from one or more of the com-pounds obtained by the distillation of coal tar. The chief of these are benzene(C6H6), toluene (C6H5CH3), naphthalene (C10H30), anthracene (C14H10), phe-nol (C6H5OH), cresol (C7H7OH), acridine (C13H9N), and quinoline (C9H7N).These compounds are different from the actual dyestuffs, and they must firstbe changed into other compounds called intermediates. These intermediatesare hydrocarbons in which one or more of the hydrogen atoms are replacedby groups such as the nitro group (-NO2), amino group (-NH2), hydroxylgroup (-OH), sulfonic acid group (-OSO3H), and others. Examples of suchcompounds are nitrobenzene (C6H5·NO2), aniline (C6H5·NH2), β-naphthol(C10H7·OH), and β-naphthalenesulfonic acid (C10H7·SO3H). There are twoimportant conditions for a colored compound to act as a dye5,79:

1) Presence of a chromophore: A chromophore is a group responsible forproducing a color because of its capability to absorb light in the nearultraviolet region. Some important chromophores are N O, -NO2, -N N-, -C O, C S, -C N, and (CH-CH)n, and the compounds bearing chro-mophores are known as chromogens.5,79

2) Presence of auxochromes: Dye should get attached to the fiber by meansof stable chemical bonds. These chemical bonds are formed by groupsthat are either acidic or basic in nature. Such groups are known as aux-ochromes, and examples are -OH, -COOH, -SO3H (all acidic), and -NH2,-NHR, -NR2 (all basic). Some dyes require no mordant for fixing them withthe fibers. These dyes are termed direct dyes. The color of direct dyes areduller than those provided by fiber-reactive dyes, and the wash-fastnessis poor—expect anything dyed with them to “bleed” forever. The oneadvantage is that direct dyes may be more lightfast, that is, resistant to

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Removal of Dyes from Textile Manufacturing Effluent 221

fading in the light, than fiber-reactive dyes. On the basis of the chemicalstructure of the chromophore group, dyes are classified as azo dyes, triph-enylmethane dyes, anthroquinone dyes, phthalocyanine dyes, etc.5,79,112

Since 1856, when the first synthetic dye was reported, to this day, theuse of dyes in industries and households has increased remarkably. There aremore than 10,000 dyes available commercially, and more than 7 × 105 tonsof dyestuffs are produced annually.112 The main consumers of dyes are thetextile, tannery, paper and pulp, and electroplating industries. Dyes are alsoused as additives in petroleum products. In addition, a number of dyes anddyestuffs are widely used in the food, pharmaceutical, and cosmetic indus-tries. The huge growth in the textile dyeing and dyestuff manufacturing in-dustries has resulted in an increase in the volume and complexity of thewastewater discharged to the environment. During textile processing, inef-ficiencies in dyeing result in a large amount of dyestuff being directly lostin the wastewater, which ultimately finds way into the environment. It isestimated that 5–10% of the dyes is lost in the effluent during the dyeingprocess,91 while in the case of reactive dyes, as much as 50% of the initialdye load is present in the dye bath effluent.37,82

The release of textile and dye-house effluent may cause abnormal col-oration of surface waters that captures the attention of both the public andthe authorities. Apart from the aesthetic problems, the greatest environmentalconcern with the dyes is their absorption and reflection of sunlight enteringthe water. This interferes with the growth of bacteria and plants, causing adisturbance of the ecology of the receiving water. Thus, the loss of dyes tothe environment has become an environmental hazard. This is because ofthe low biodegradability and the toxic nature of the dyes. In addition, thehuman health impact of dyes has caused concern for a number of years.Some dyes, dye precursors, and their biotransformation products such asaromatic amines have been found to be toxic, mutagenic, and carcinogenicin nature, apart from having the potential of bioaccumulating in the foodchain.6,25,57,61,77 As a result of this, legislation controlling the use of suchsubstances is being developed in various countries.45 As the world has be-come more cautious about the environment and also due to ever-increasingand stringent laws, the textile industry around the world has begun usinginnovative methods for wastewater remediation, more so for the water con-taining the residual color from the dyes that remain almost unaffected by theconventional aerobic treatment systems.71,81 In light of these facts, attentionhas come to the efficient removal of dyes from the environment. However,as compared to the growth of the dye industries and dye products, a littlegrowth has taken place towards their removal. Here we give an overviewof the emerging techniques for the treatment of dye- waste from the dyeindustries and textile industries, giving a special emphasis on its biologicaltreatment procedures.

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II. BIOLOGICAL TREATMENT

Biological treatment, either aerobic or anaerobic, is generally considered tobe the most effective means of removing the bulk of pollutants from com-plex and high-strength organic wastewater. Also, microorganisms are knownto play a crucial role in the mineralization of biopolymers and xenobioticcompounds.59 Biological removal of the dyes from textile and dyestuff man-ufacturing industry can be broadly classified into three categories: aerobictreatment, anaerobic treatment, and combined anaerobic–aerobic treatment.However, only the biological process of treatment of dye waste and textileeffluent is not sufficient. The process requires the involvement of other phys-ical, chemical, and physicochemical operations. Some of the physical pro-cesses are filtration and separation, dilution, and gamma irradiation. Physicalmethods are mainly used for the primary treatment of the dye wastewater.Physicochemical processes for the dye wastewater treatment include adsorp-tion, flocculation, ion-pair extraction, reverse osmosis, ion exchange, coag-ulation (by alum, ferric chloride, ferrous ammonium sulphate, lime, naturalpolymers like tamarind, seed extract, etc.), and clarification. Chemical treat-ments are oxidation, reduction, and chlorination. Activated carbon, charcoal,anhydrous sodium silicate, minerals, rice husk, tick wood bark, cotton waste,hair coal, fly ash, ground nut shell powder, red soil, bauxite, gypsum, andclays were used for color removal from textile wastewater. Activated carbonis very effective in removing color, but it is capable of treating only smalleffluent volume, operates at slow speed, and has high capital cost.

A. Aerobic Treatment

Most dyes have long been considered nonbiodegradable or nontrans-formable under aerobic conditions.21,71,106,107 In 1986, Pagga and Brown71

performed one of the most comprehensive studies on aerobic degradabilityof dyes. Eighty-seven dyestuffs were tested for their susceptibility to aerobicbiodegradation. The dyestuffs chosen for the study were typical commercialproducts, and bacterial inocula were derived from a conventional (aerobic)effluent treatment plant. The results showed that the dyestuffs were most un-likely to show any significant biodegradation under these conditions. How-ever, efforts to isolate aerobic microorganisms capable of transforming dyesand dye related compounds have continued. Bacteria and fungi are the twomicroorganism groups that have been most widely studied for their abilityto treat dye wastewaters.

1. TREATMENT BY FUNGI

Fungal strains capable of decolorizing azo and triphenylmethane dyeshave been studied in detail.19,73,78,79,94,103 Lignin-degrading white rot fun-gus, Phanerochaete chrysosporium, was investigated extensively during the

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last decade for its capability to degrade many recalcitrant pollutants likechlorophenols, nitrotoluenes, and polycyclic aromatic hydrocarbons.11,58 Fur-thermore, its ability to decolorize a wide range of dyes is now well es-tablished. Phanerochaete chrysosporium was reported to be able to decol-orize three azo dyes, orange II, Congo red, and tropaeolin O, under aerobicconditions.31 Lignin peroxidase, an extracellular enzyme of P. chrysosporium,was found to be responsible for the decolorization process. Later studiesrevealed the potential of P. chrysosporium to decolorize direct dyes, reac-tive dyes, acidic dyes, and disperse dyes.23 Sani and Banerjee79 reportedthe decolorization of acid green 20 at 30◦C by P. chrysosporium in a low-cost medium where the dye was initially converted to an unknown product,showing λmax at 522 nm and then transformed into another colorless com-pound. Ollikka et al.68 further established the potential of this fungus orits enzymes (such as lignin peroxidase) to decolorize various azo, hetero-cyclic, and polymeric dyes. Some other strains of fungi like Kurthia sp., Cy-athus bulleri, Coriolus versicolor, Funalia trogii, Laetiporous sulphureus, andStreptomyces sp.73,79,94,103 have also been reported to decolorize some triph-enylmethane and azo dyes. Phanerochaete chrysosporium, however, clearlyholds an advantage over the other strains because of the nonspecific natureof the enzyme “lignin peroxidase” present in it; because of this, it can de-colorize a diverse array of dyes. Lignin peroxidase has already been shownto be able to catalyze a wide variety of reactions, including benzylic oxida-tion, phenol dimerization, carbon–carbon bond cleavage, hydroxylation, andO-demethylation.71,72,88

However, Wong and Yu98 identified two major problems in the appli-cation of P. chrysosporium to the treatment of real wastewater. First, ligninperoxidase, the key enzyme for dye degradation is released by fungal cellsfollowing a strict secondary metabolism under either carbon or nitrogen lim-itation. This means that the presence of carbon or nitrogen nutrient in theindustrial effluent would prohibit the release of this enzyme by the fungalcells.111 Second, dye degradation by lignin peroxidase consumes consid-erable amounts of hydrogen peroxide and veratryl alcohol as reagents.104

Although veratryl alcohol is a metabolite released by the fungus, a largeamount of hydrogen peroxide, veratryl alcohol, and lignin peroxidase maynot be produced simultaneously in most industrial effluents.

In the light of these facts, Wong and Yu98 decolorized azo and indigodyes successfully using another fungus, Trametes versicolor. The responsi-ble enzyme for dye decomposition was laccase, an extracellular oxidase,released by the fungus. Compared to P. chrysosporium, T. versicolor offeredsome distinct advantages. First, unlike P. chrysosporium, T. versicolor couldproduce the oxidative enzyme (laccase) even in the presence of nitrogenand carbon nutrients.62 More importantly, laccase is an oxidase, with a re-dox potential of 78 mV, and can catalyze the oxidation of organic pollutantseven in the absence of hydrogen peroxide or other secondary metabolites.87

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Another interesting observation in this study was that while anthraquinonedye was directly oxidized by laccase, the decolorization of (nonsubstrate)azo and indigo dyes required mediation by some synthetic organic com-pounds or anthraquinone dye. This could be of considerable significance incolor removal from the actual industrial effluents, as they are likely to havethe substrate dyes along with the nonsubstrate ones. Laccase needs O2 tocatalyze the oxidation reaction, and various redox mediators have been re-ported to play an important role in the oxidation of phenolic compounds.Campos et al.20 reported that the pure fungal laccase, obtained from a com-mercial source formulation used in the textile industry, when applied withoutany redox mediator did not decolorize Remazol brilliant blue R (RBBR).79,80

But when a low-molecular-weight redox-mediator was added together withthe laccase, decolorization was observed; violuric acid was found to be themost effective mediator, with almost complete decolorization within 20 min.Other mediators like 1-hydroxybenzonitriazole (HOBT) were found to beinefficient in comparision to the violuric acid, and a higher concentrationof HOBT was inhibitory due to inactivation of laccase by the toxic (HOBT)radical. Other mediators like 2-methoxyphenothiaazine, acetosyringone, and4-hydroxybenzenesulfonic acid (PHBS) were also found to be effective forthe decolorization of various classes of dyes by laccase.

2. TREATMENT BY BACTERIA

Similar efforts to identify and isolate aerobic bacteria capable of degradingvarious dyes have been going on since more than two decades ago. Ogawaet al.67 isolated a bacterium (from the sewerage system of a dyestuff factory)that could degrade various dye compounds. However, growth and respira-tion of the aerobic organisms involved were observed to be inhibited by thedyes used in the study. Idaka et al.50 reported the apparent aerobic reductionof simple azo compounds by Aeromonas hydrophilia. Subsequently, Kullaet al.56 described degradative pathways for sulfonated azo dyes by Pseu-domonas strains previously adapted to carboxylated azo dyes. Later, Idakaet al.49 demonstrated the reductive cleavage of azo dyes by Pseudomonascepacia. In 1991, Yatome et al.102 reported the degradation of five triphenyl-methane dyes by growing cells of Bacillus subtilis IFO 13719. A number ofsoil and water samples were screened for their ability to decolorize some ofthe triphenylmethane group of dyes, and one of the strains of Bacillus sp.MTCC B0006 decolorized 400 mg/L each of malachite green, magenta, crystalviolet, brilliant green, ethyl violet, and pararosaniline.7,9 When the toxicityof dyes was assessed, it was found that crystal violet was toxic to all theorganisms tested (E. coli DH5α, S. cerevisiae, and S.pombe); however, thedye decolorized by Bacillus sp. MTCC B0006 was found to be nontoxic.8

The process of decolorization of azo dyes in most studies highlighted thereduction of azo linkage via enzymatic reaction. However, the azo reduc-tases isolated from Pseudomonas strains had a narrow substrate range.55,56,105

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More recently, Hu46 identified the bacterium Pseudomonas luteola, isolatedfrom the sludge of an “activated sludge treatment” system treating dyeingwastewater, which removed the color of four reactive azo dyes: red G, RBP,RP2B, and V2RP. The sludge from activated sludge system was stabilized for6 months prior to the isolation of P. luteola.48 The results showed that afterundergoing shaking incubation for 48 h followed by static incubation foranother 48 h, P . luteola gave color reduction of 37, 93, 93, and 88% for redG, RBP, RP2B, and V2RP, respectively. More recently, some successful studieson the color removal of certain azo dyes in aerobic condition have beenreported.42,47,99,108 All the strains involved in these studies required an addi-tional source of carbon and energy. Since the availability of this additionalsubstrate could have led to the formation of anaerobic microniches, the oc-currence of anaerobic reduction of azo dyes was very much suspected. Also,the possibility of anaerobic reduction of azo linkage could not be excludedeven in the studies involving P. luteola,46 as the static incubation might haveled to the formation of micro anaerobic zones within the aerobic system. Inaddition, a similar role of anaerobic microniches was also suspected in thedegradation of azo dyes in anaerobic biofilm reactors,28,51 especially whenadditional substrate was provided.

On the other hand, very recent reports have demonstrated that some bac-terial strains can mineralize various dyes under aerobic conditions.13,29,30,65,78

A sulfonated azo dye was decolorized and subsequently used as a carbonand energy source by a bacterial strain S5 derived from Hydrogenophagapalleronii S1.13 Also, bacterial strain MI2, isolated from an aerobic biofilmreactor, was reported to mineralize acid orange 7 and acid orange 8.30 Fur-thermore, the ability of Sphingomonas sp. strain 1CX to mineralize acid or-ange 8, acid orange 10, acid red 4, and acid red 88 as sole carbon sourcewas demonstrated.29 Most recently, successful degradation of acid 151 us-ing an aerobic sequenced biofilm reactor was reported.74 The dye served asthe sole carbon source for the microorganisms, and 73% of the carbon wastransformed into carbon dioxide.74 A number of triphenylmethane dyes, suchas magenta, crystal violet, pararosaniline, brilliant green, malachite greenand ethyl violet, were very efficiently decolorized (96–100%) by the cells ofKurthia sp.78 After biotransformation, the extent of COD reduction of thecell-free extract of triphenylmethane dyes was more than 88%, except inethyl violet, where it was approximately 70%.78

B. Anaerobic Treatment

Although some efforts in the recent past to decolorize dyes under aerobicconditions have met with success, the general perception of nonbiodegrad-ability of most azo dyes in conventional aerobic systems still persists.21,71,81

On the other hand, the potential of anaerobic microorganisms to decolorizedyes is well documented and established.17,21,43,65,66,76,93

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The investigations on anaerobic decolorization of azo dyes were startedway back in the early 1970s. Walker and Ryan95reported decolorization of azodyes using intestinal anaerobic bacteria. This potential of intestinal anaerobesto decolorize azo dyes was further established by other researchers.3,18,22

Later studies revealed that these compounds are readily decolorized by vari-ous other anaerobic cultures.17,22,26 However, as the phenomenon of azo dyereduction is not clearly understood yet, the term “azo dye reduction” may in-volve different mechanisms or locations, like enzymatic,43 nonenzymatic,41

mediated,93 intracellular,101 and extracellular.21 It has been established inthese studies that the azo dye, instead of being biodegraded by the microor-ganisms, acts as an oxidizing agent for the reduced flavin nucleotides of themicrobial electron transport chain. Thus, the azo dye is reduced and de-colorized concomitantly with reoxidation of the reduced flavin nucleotides.An important feature of the reductive cleavage of these compounds is thatthe electrons required by the anaerobes for this process are derived fromcosubstrates.41,43,66 It is thought that the presence of cosubstrates enhancesthe reduction of azo compounds by increasing the rate of formation of re-ducing equivalents.41,76 Many different cosubstrates have been found to suitas electron donors, such as glucose,21 hydrolyzed starch,97 tapioca,24 yeastextract,65 acetate, and a mixture of acetate, butyrate, and propionate.76 Alongwith the cosubstrate maintenance of proper environmental conditions interms of pH, temperature, etc. also plays an important role in the decol-orization process. The nature of the cosubstrate has considerable effect onthe extent of decolorization of these dyes.66,76 In addition, the rate of azodye reduction has also been observed to be dependent on the type of co-substrates used, along with the chemical structure of the dye.93 Apart fromelectron donors, the role of “mediators” (compounds that facilitate the trans-port of electrons) to considerably enhance the azo reduction rate has alsobeen observed.54,93 In a study conducted by Frank P. van der Zee et al.92

it was shown that redox-mediating compounds like anthraquinone disul-fonate greatly enhanced both the chemical and biological dye reduction. Itwas also shown that redox-mediating enzyme cofactors released by cell lysiscontribute as a stimulatory effect to the dye reduction.40

A recent report by Razo-Flores et al.,75 however, has gone a step be-yond these studies. In this study, it was shown that it is possible not only todecolorize but also to completely mineralize the azo dye (mordant orange1 MO1) in a lab-scale “upflow anaerobic sludge blanket” (UASB) reactoreven in the absence of a cosubstrate. The azo cleavage product of MO1 wasidentified as 5-azodisalicylate (5-ASA). It was seen that the presence of thecosubstrate was necessary only in the initial phase in order to establish an ac-tive methanogenic consortium during adaptation to the dye. The cosubstratesupplementation was obviated once 5-ASA-degrading bacteria developed inthe consortium. This was because the reduction equivalents required for theazo cleavage were generated from metabolism of 5-ASA. This is contrary

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to the popular perception that the azo cleavage products are recalcitrant toanaerobic degradation.15,16,43

A recent report by Bromley-Challenor et al.14 adds another interestingaspect to the perspective just described. In this study, it was found thatunadapted activated sludge could quite effectively decolorize an azo dyeunder anaerobic conditions in the absence of any external cosubstrate. Fromthe linear correlation between the dye decolorization rate and its concentra-tion, without any external source of carbon and electrons, it was concludedthat the sludge utilized its endogenous energy reserves, and these were suf-ficient for decolorizing even higher concentration (up to 400 mg L−1) ofazo dye. This offers a distinct advantage in cost and flexibility over pro-cesses using defined bacterial cultures or anaerobic digester sludge, whichrequires addition of a carbon source.21,66 Furthermore, the same sludge de-colorized three other structurally dissimilar azo dyes, suggesting that de-colorization was not a dye-specific process. It was concluded that this“gratuitous” anaerobic reduction (process in which microbes obtain no ben-efit from reduction) may be applicable to other xenobiotic compounds aswell. On an industrial scale, an advantage of this normally hidden metabolicpotential of the microflora may be found in cycling of activated sludge orin the use of submerged biological filters between aerobic and anaerobicconditions.14

C. Treatment in Combined Anaerobic–Aerobic System

The general perception that has emerged over the years is that most dyesare generally recalcitrant to aerobic degradation21,71,106 but can be reduc-tively decolorized under anaerobic conditions.17,22,35 The biotransformationproducts generated in this process however, are not further susceptible toanaerobic attack, but are readily biodegraded under aerobic conditions.53,60,84

Hence, anaerobic decolorization followed by aerobic posttreatment is gener-ally recommended for treating colored wastewater from textile and dyestuffmanufacturing industries.16,38 This condition can be implemented either byspatial separation of the anaerobic and aerobic sludge using a sequentialanaerobic–aerobic reactor system110or within one reactor, commonly termedan integrated anaerobic–aerobic reactor system.38

1. SEQUENTIAL ANAEROBIC--AEROBIC TREATMENT

This treatment system involves spatially separated sequential exposure of dyewastewater to anaerobic condition followed by aerobic environment. Numer-ous researchers have studied this system during the last decade.4,39,62,69 Forexample, in the study by An et al.,4 three dye solutions consisting of CI acidyellow 17(AY 17), CI basic blue 3 (BB 3), and CI basic red 2 (BR 2) weretreated in a UASB reactor followed by a semicontinuous activated sludgetank. It was observed that in the anaerobic stage, the dyes such as AY 17,

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BB 3, and BR 2 were decolorized by 20, 72, and 78%, respectively, with nosignificant color removal in the aerobic stage. Later on, an actual effluent froma dye-manufacturing factory was used for the experiment. The effluent hadCOD and color concentration of 1200 mg/L and 500 degrees (dilution factor)respectively. The COD and color removal of 83 and 90%, respectively, wereachieved in the combined anaerobic–aerobic system. It was concluded fromthese studies that the anaerobic stage of the combined system removes bothcolor and COD. In addition, it also improves the biodegradability of dyes forfurther aerobic treatment. Likewise, most of these studies report appreciablecolor and organic matter removal. However, in most cases clear evidence forcomplete biodegradation of dyes is missing. This is primarily because thereis no concrete proof of mineralization of the biotranformed products of thedyes. Only in one case43 is a clear proof of mineralization of an azo dye bybacterial coculture provided.

2. INTEGRATED ANAEROBIC--AEROBIC TREATMENT

The basis of these systems lies in the fact that anaerobic and aerobic mi-croorganisms can coexist beneficially in a single biofilm.52,109 Supplying oxy-gen to an oxygen-tolerant anaerobic consortium86 can create an integratedanaerobic–aerobic system. Alternatively, exposing a biofilm to low concen-tration of oxygen along with a cosubstrate28,38 can also create such con-ditions. Study conducted by Tan et al.86 demonstrated that two azo dyes,4-phenylazophenol (4-PAP) and mordant yellow 10 (MY10), were apprecia-bly degraded in an integrated anaerobic–aerobic treatment system. Exposinganaerobic granular sludge to oxygen implemented the integration of anaer-obic and aerobic conditions. The system observed temporary accumulationof aromatic amines, that is, 4-aminophenol (4-AP) and aniline from 4-PAP,and 5-aminosalicylic acid (5-ASA) and sulfanilic acid (SA) from MY10, re-sulting from the reduction of the dye molecules. These compounds weresubsequently mineralized either by facultative aerobic bacteria present in theanaerobic sludge or by addition of inocula from aerobic enrichment cul-ture (developed from aromatic amines) to the anaerobic sludge. The studyconcluded that aerobic enrichment culture developed from aromatic amines,combined with oxygen tolerant anaerobic granular sludge, could potentiallybe used to completely biodegrade azo dyes under integrated anaerobic–aerobic conditions. Anaerobic digestion was enhanced in efficiency by ap-propriate bioaugmentation. This was followed by aerobic posttreatment. Thecosubstrate ethanol was successfully applied as an electron donor for azo dyereduction. It also created anaerobic microniches to facilitate anaerobic azodye reduction in the presence of oxygen.

A recent study by Tan et al.85 aimed at studying the mineralization ofthree azo dyes, mordant orange 1 (MO1), 4-phenylazophenol (4-PAP), andmordant yellow 10 (MY10), under integrated as well as sequential anaerobic–aerobic conditions. The results of this study demonstrated that azo dyes were

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mineralized in both integrated and sequential anaerobic–aerobic conditions.However, the sequential anaerobic–aerobic system was recommended fortwo reasons. Firstly, faster azo dye reduction rates were observed under se-quential anaerobic–aerobic conditions as compared to the integrated ones.This was because of the fact that in the anaerobic stage of the sequentialsystem, the cosubstrate is used only for donating electrons for azo dye re-duction, whereas in the case of an integrated system, part of the cosubstrateis used in creating anaerobic microniches. Second, aerobic degradation ofthe cosubstrates under integrated conditions usually gives rise to deficiencyof either oxygen or cosubstrate, which eventually is unfavorable for decol-orization and mineralization of azo dyes. Where an excess of cosubstratecaused shortage of oxygen, resulting in reduced rate of aerobic degradationof aromatic amines, excess of oxygen resulted in a severe decline in the azodye reduction efficiency in the bioreactor. These negative effects obviouslydo not occur in the sequential anaerobic aerobic system as observed in thisstudy. Thus, a good balance between cosubstrate and oxygen is very muchrequired, which was not easily achieved in the integrated anaerobic–aerobicconditions.

Another study conducted by Van der Zee93 focused on optimizing theanaerobic phase of an integrated anaerobic–aerobic treatment system whenused for azo dye degradation. It states that the system, though effective, issluggish due to slowness of the anaerobic phase. The aim was to speedup the anaerobic phase of the treatment. It was shown that the anaerobicphase can be optimized by using redox mediators, either by continuousdosing of soluble quinones or by incorporation of AC (activated carbon) inthe sludge blanket. Thus, an efficient as well as fast integrated anaerobic–aerobic treatment system can also now be developed.

III. FUTURE WORK

A. Other Important Classes of Dyes

Most of the reports published in literature are either on azo or reactive azodyes. This is because azo dyes account for 50% of all the dyestuffs pro-duced and the azo bond is the most common chromospheres of reactivetextile dye.96 Furthermore, the toxicity, mutagenicity, and carcinogenicity ofthese dyes and their precursors and biotransformation products are widelyreported.25,57,61,77 Reports are also available on other important classes ofdyes like triphenylmethane, anthraquinone, and indigoid, and their degra-dation is generally mediated by the enzyme laccase.1,10,20,27,62,83 Some dyesfrom the triphenylmethane group have also been reported to be toxic, muta-genic, and carcinogenic to biota.12,44 Therefore, more studies are required toevaluate and establish biological methods for degradation and mineralizationof these and other important classes of dyes.

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B. Anaerobic High-Rate Reactors

In a study conducted by Donlon et al.,33 degradation of nitrophenols (con-taminants originating from manufacturing of dyes) was investigated in a UASBreactor, which was continuously fed with a volatile fatty acid (VFA) mixture asa primary substrate. The results showed dramatic detoxification of the nitro-phenols in the reactor where the concentration of nitrophenol was 25 timeshigher than the normal values. The overall results indicated that a UASB reac-tor can be used to rapidly detoxify and under certain conditions completelydegrade nitroaromatic compounds. The successful operation of continuousanaerobic reactors based on reductive detoxification of other similar organiccompounds has also been cited in the literature.64,65,100

The main cause of effective treatment of these xenobiotics under anaero-bic conditions in high rate reactors is the rapid facile reduction of these com-pounds to products of lower toxicity.34,83 Furthermore, the immobilization ofanaerobic bacteria and maintenance of a high concentration of biomass inthe high rate reactors are factors that improve the tolerance of the anaerobicsystem to toxic substances.36,70 Also, the use of VFAs, glucose, ethanol, etc.as primary substrates not only builds up a high concentration of biomass inthe reactor, but also increases the number of reducing equivalents (electrondonors), which leads to rapid reduction of these compounds to lesser toxicmetabolites.76,89,90 In the light of this, anaerobic high-rate reactors shouldbe given special attention for future studies on biological treatment of dyewastewaters.

C. Bioaugmentation

Bioaugmentation is the process of obtaining maximum biodegradability bythe addition of nonindigenous microbial cultures to have optimum bacterialbiomass. This offers considerable advantage in dealing with the problems ofbacterial acclimatization, toxicity of compounds, and restart of the system.It was successfully employed in the anaerobic phase of anaerobic–aerobicintegrated treatment of dyes.86 Also, for example, in the study conductedby Razo-flores et al.76 the introduction of 5-ASA-degrading bacteria at thevery beginning would have obviated the need for the cosubstrate in theearly stages of the experiment. In addition, the time lost in developingthe 5-ASA-degrading bacteria in the reactor would have been avoided.

D. Mineralization of Dyes

Decolorization of the dyes often results from the initial degradation of the dyemolecules to some intermediate organic compounds. These compounds con-tribute to biochemical oxygen demand (BOD) and chemical oxygen demand

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(COD) of the wastewater, This means that dye wastewater even if effectivelydecolorized, may more often fall short of the required consent standard for itsdisposal into the receiving bodies. It may be necessary to reveal the biologi-cal mechanisms responsible for the decolorization and degradation of dyes.Besides, the biotranformation products of the dyes may some times be evenmore toxic than the dyes themselves. Therefore, mineralization of these com-pounds should be an integral part of the studies on biological decolorizationof dye wastewater.72 Enrichment studies to isolate organisms responsible forutilizing various dyes as the sole source of carbon and energy would go along way in effective decolorization and mineralization of these pollutants.

IV. CONCLUSION

Accumulation of dyestuff and dye wastewater creates not only environmen-tal pollution but also medical and aesthetic problems associated with hu-man health and society. Though biological means of dye degradation andremoval from the effluents of dye and textile industries hold promises formeaningful addressing the problem, the method relies on success of find-ing out a suitable organism and designing of condition for the process. Atthe same time, biological means of dye degradation alone cannot tackle theproblem successfully. Therefore, along with biological processes, other pre-and posttreatment methods are required. Pretreatment of dyeing effluent byadvanced oxidation processes (AOP) catalyzed by a source of ultraviolet(UV) light and a powerful oxidant is a promising alternative for the effectiveremoval of color and refractory organics from the effluent.2 Metal oxides,mainly TiO2, in combination with a strongly photoreactivating agent like UVrays are used for this process. Zinc oxide appears to be a suitable alternativeto TiO2 for water treatment.32 A crucial feature in designing such system isthe optimization of operating conditions (such as UV and oxidant dosages),to yield maximum removal at acceptable costs. On the other hand, to date,dye degradation and removal of dye waste from the environment are some-what at an immature stage from the microbiological point of view, and abetter understanding of the processes is required to apply a suitable combi-nation of aerobic, anaerobic, pure culture, and mixed culture methodologiesfor effective treatment of dye waste. Processes of mineralization of dye wastealso have to be integrated with the biodegradation so that the products origi-nate from the biodegradation processes do not contaminate the environmentfurther more. An understanding of the enzymology of dye degradation willfurther enhance the efficiencies of these processes towards its successful ap-plication. Application of modern molecular biology techniques for cloningand overexpression is expected to enter the field of dye degradation bybiological means, with significant impact on it.

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Removal of Dyes From the Effluent

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