bioremediation of wastewater containing azo dyes through sequential anaerobic–aerobic bioreactor...

Bioremediation of wastewater containing azo dyesthrough sequential anaerobic–aerobic bioreactorsystem and its biodiversity

Nishant Dafale, Satish Wate, Sudhir Meshram, and Nageswara Rao Neti

Abstract: Wide range of dyes and dyestuffs used in textile manufacturing are xenobiotic compounds and attract stricter tostrict environmental regulations. The ability of microbial consortia to decolorize and metabolize dyes has long beenknown, and the use of bioremediation based technologies for treating textile wastewater has attracted interest. These dyesare decolorized by microbial consortia but technologies for their complete mineralization are still not developed. The mostlogical concept for the removal of azo dyes in biological wastewater treatment systems is based on anaerobic treatment,for the reductive decolorization, in combination with aerobic treatment, for the degradation of the by-products (aromaticamines) generated in the anaerobic bioreactor. Several research and review articles were published on anaerobic decolor-ization; however, research on complete mineralization of dyes through sequential anaerobic–aerobic bioreactors has re-ceived greater attention recently. Bioremediation through sequential anaerobic–aerobic bioreactor system has beenreviewed in this article with critical appraisal using data generated through our experiments. While reviewing this work,we realized the importance of microbial diversity in a treatment unit to better understand the functional status to enhancethe mineralization activity of the bioreactor.

Key words: azo dyes, biodiversity, bioremediation, two-stage, sequential bioreactor.

Resume : Un ensemble de teintures et substances tinctoriales utilisees dans la fabrication des textiles constituent des com-poses xenobiotiques et appellent des reglementations environnementales severes a tres severes. On connaıt depuis long-temps la capacite des populations microbiennes a decolorer et metaboliser les teintures, et l’utilisation de technologiesbasees sur la bioremediation pour traiter les eaux usees des industries textiles souleve beaucoup d’interet. Les populationsmicrobiennes arrivent a decolorer ces teintures, mais les technologies pour les mineraliser completement ne sont pas en-core developpees. Le concept le plus logique pour debarrasser les colorants de type azo des systemes de traitement deseaux usees implique un traitement anaerobie, pour assurer la decoloration reductive, en combinaison avec un traitement ae-robie pour degrader les sous-produits (amines aromatiques) generes pendant la phase anaerobie. Il existe plusieurs articleset revues sur la decoloration anaerobie; cependant, ce n’est que recemment que la recherche sur la mineralisation completedes teintures avec des reacteurs sequentiels anaerobie-aerobie a recu une attention soutenue. Les auteurs passent ici en re-vue la bioremediation en systemes de bioreacteurs anaerobie-aerobie, avec une evaluation critique des donnees genereesdans ces experiences. Tout en realisant cette revue, les auteurs ont constate l’importance de la biodiversite microbiennedans une unite de traitement, pour mieux comprendre le statut fonctionnel permettant d’accelerer l’activite de mineralisa-tion du bioreacteur.

Mots-cles : teintures de type azo, biodiversite, bioremediation, deux etapes, bioreacteur sequentiel.

[Traduit par la Redaction]

Introduction

Bioremediation is a pollution-control technology that usesnatural biological species to catalyze the degradation ortransformation of various toxic chemicals to less harmfulforms. Xenobiotic compounds are not naturally availableand hence the locally occurring microorganisms cannotreadily degrade them. Dyes widely used in textile industry

are considered as xenobiotic. Synthetic dyes are extensivelyused by various industries such as textile (Sokolowska-Gajda et al. 1996), leather tanning (Tunay et al. 1999), paperproduction (Ivanov et al. 1996), food technology (Bhat andMathur 1998), agriculture research (Cook and Linden1997), light harvesting array (Wagner and Lindsey 1996),photo electrochemical cells (Wrobel et al. 2001), hair color-ing (Scarpi et al. 1998), and pharmaceuticals (Zhao et al.2007).

Over one million tonnes of synthetic dyes are producedworldwide every year for dyeing and printing and out ofthis upto 5%–10% are discharged with wastewater (Padma-vathy et al. 2003; Pandey et al. 2007). More than 2000structurally different azo dyes are currently in use (Vijay-kumar et al. 2007). The amount of dye lost depends uponthe type of dye application, varying from 2% loss while us-ing basic dyes to 50% loss in certain reactive sulfonated

Received 24 April 2009. Accepted 4 January 2010. Published onthe NRC Research Press Web site at er.nrc.ca on 18 February2010.

N. Dafale, S. Wate,1 and N.R. Neti. National EnvironmentalEngineering Research Institute, Nehru Marg, Nagpur, India.S. Meshram. Post Graduate Department of Microbiology,R.T.M. University, Nagpur, India.

1Corresponding author (e-mail: [email protected]).

21

Environ. Rev. 18: 21–36 (2010) doi:10.1139/A10-001 Published by NRC Research Press

Env

iron

. Rev

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

YO

RK

UN

IV o

n 06

/10/

14Fo

r pe

rson

al u

se o

nly.

dyes (Pearce et al. 2003). Textile industries produce multi-component wastes (dyes, starches, enzymes, fats, grease,wax, surfactants, resins, and chlorinated organic compoundsetc.) depending upon the wet processes involved (Correia etal. 1994). Textile dyes are of synthetic origin and consist ofcomplex aromatic molecules (Banat et al. 1996). In India,57% consumption is of reactive dyes among all the textiledyestuffs

The Ecological and Toxicological Association of the Dye-stuffs Manufacturing Industry (ETAD) was established tominimize environmental damages, protect users and con-sumers, and to co-operate fully with government and publicconcerns over the toxicological impacts of their products. InIndia, wastewater generated from textile processing is450 000 m3 d–1 and 32 000 m3 d–1 by dyeing facility alone(Census of Textile Power Processing Industry, Ministry ofTextiles, GOI). Even a small amount of dye in water (10–15 mg L–1) is highly visible, affecting the aesthetic merits,water transparency and gas solubility of water bodies (Banatet al. 1996; Bae and Freeman 2007). In particular, the re-lease of colored effluents into the environment is undesir-able, not only due to their color, but also many dyes andtheir breakdown products are carcinogenic or toxic toaquatic life (Vandevivere et al. 1998; Weisburger 2002; Oz-turk and Abdullah 2006).

In addition, as a result of growing environmental aware-ness, upgrading of environmental legislation, and publicpressure, the textile industry in India and elsewhere is seek-ing to develop effective wastewater remediation technolo-gies, especially those which allow color removal that islargely unaffected by conventional treatment systems(O’Neill et al. 2000b). Despite the existence of a variety ofphysicochemical treatment processes, bioremediation of tex-tile effluent is still seen as an attractive solution due to itsreputation as a low-cost, environmental friendly, and pub-licly accepted treatment technology (Banat et al. 1996). Anumber of research papers have been published with focuson decolorization. Decolorization generates many by-prod-ucts, some of which are highly toxic and carcinogenic.Therefore, complete mineralization, which implies decolor-ization and degradation of azo dyes, is desired. Significantresearch on complete degradation of azo dyes over the pastdecade has been published. In this review, we attempted toconsolidate research on complete decolorization and degra-dation of azo dyes using a sequential anaerobic–aerobic sys-tem. This system has been projected to possess ability todegrade the azo dyes completely and can emerge as futuretechnology to solve environmental problems concerning azodyes.

Current technologies for decolorization oftextile wastewater

Physicochemical methods (Fig. 1) such as coagulation andflocculation (Slokar and Le Marechal 1998), adsorption(Choy et al. 1999), ozonation (Xu et al. 1999), photochemi-cals (Yang and Wyatt 1998), membrane filtration (Xu et al.1999) and electrochemical process (Pelegrini et al. 1999)have been used for the treatment of dye containing waste-water (Vandevivere et al. 1998). The main limitations ofthese processes are the cost and they generate large quanti-

ties of sludge leading to secondary pollution (Stolz 2001).The advantages and drawbacks of some nonbiological treat-ment processes as applied in textile wastewater treatment aregiven in Table 1. On the other hand, biological methodssuch as activated sludge processes and anaerobic treatmenthave been applied to control pollution of aquatic environ-ment. Lower cost of treatment and amenability to scale upeasily in field are the merits of biological methods. Some ofthese dye, particularly reactive azo dyes are known to resistbiodegradation. Therefore, color removal from textile efflu-ents through conventional wastewater treatment systems isstill a major environmental problem (Seshadri et al. 1994).Hence, enrichment and augmentation of especially efficientbacterial culture to the biological treatment process appearsto be a techno-economically viable option with respect toconventional technologies.

Effect of various factors on decolorizationDecolorization of azo dyes under anaerobic conditions is

thought to be a relatively simple and nonspecific process.Bacteria such as Pseudomonas and Bacillus have been usedto degrade the azo or reactive dyes from textile industry ef-fluents; this process is referred to as biobleaching (Ashokaet al. 2002).The anaerobic decolorization of azo dyes wasfirst investigated using intestinal anaerobic bacteria (Walkerand Ryan 1971). This was later confirmed by other authors(Stolz 2001) and was shown to be catalyzed by azoreductaseenzymes in the liver as well as anaerobic microorganisms ofthe intestinal tract. Later these compounds were found to be-come readily decolorized with various other anaerobic mi-crobes. Microbial decolorization of dye under anaerobicconditions affected by various components such as:

Dye structureSynthesis of most azo dyes involves diazotization of a pri-

mary aromatic amine, followed by coupling with one ormore nucleophiles (Khalid et al. 2008). Amino- and hy-droxy- groups are commonly used coupling components andhence a large number of structurally different azo dyes existand are used in industry. The azo groups are generally con-nected to benzene and naphthalene rings, but can also be at-tached to aromatic heterocycles or enolizable aliphaticgroups (McCurdy et al. 1991). These side groups are neces-sary for imparting the color to dye, with many differentshades and intensities being possible. A dye molecule con-tains nucleophiles, which are referred to as auxochromes,and aromatic groups called chromophores. Due to their com-plex structures azo dyes are generally recalcitrant to biode-gradation. The influence of dye structure on the colorremoval efficiency of different azo dyes was frequently ob-served under similar conditions (Rajaguru et al. 2000; Isikand Sponza 2004a; Albuquerque et al. 2005).

Biomass concentration and hydraulic retention timeSeveral studies have reported a positive correlation be-

tween the hydraulic retention time of the anaerobic stageand the color removal efficiency (Panswad et al. 2001; Isikand Sponza 2004a; Albuquerque et al. 2005). Lowering thebiomass concentration from 2 to 1.2 g VSS/L and the solidsretention time from 15 to 10 d in a dye-treating sequential

22 Environ. Rev. Vol. 18, 2010

Published by NRC Research Press

Env

iron

. Rev

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

YO

RK

UN

IV o

n 06

/10/

14Fo

r pe

rson

al u

se o

nly.

batch reactor (SBR) resulted in a considerable decrease,from ~90% to 30%, of the color removal efficiency (Lour-enco et al. 2000). Systems with a higher biomass retentioncapacity (upflow anaerobic reactors) are better suited forazo dye decolorization than systems with a lower biomassretention capacity (e.g., SBR). For example, higher ReactiveBlack 5 (RB-5) color removal efficiency was observed in anupflow anaerobic sludge blanket (UASB) reactor in compar-ison to those of the anaerobic phases of SBR systems oper-ated with similar dye concentrations and reaction times(Panswad and Luangdilok 2000; Panswad et al. 2001;Sponza and Isik 2002). We found more than 90% decolor-ization of RB-5 at 1.6 g L–1 MLSS in 24 h using specificenriched bacterial consortia (Mohanty et al. 2006; Dafale etal. 2008a, 2008b).

Alternate electron acceptors as competitors for azogroup

Reduction of azo dyes is a redox reaction in which theazo group acts as an electron acceptor. The presence of al-ternative electron acceptors may compete with the azo dyefor reducing equivalents. The presence of nitrate, a normalconstituent of textile processing wastewater, was shown toslow down decolorization (Lourenco et al. 2000; Panswadand Luangdilok 2000). These results are in agreement withpreviously published data from batch experiments of azodye decolorization in the presence of nitrate (Carliell et al.1995) and nitrite (Wuhrmann et al. 1980).

In contrast, the presence of sulfate, also a normal constit-uent of textile-processing wastewater, was not found to sig-nificantly affect dye decolorization (Panswad and

Fig. 1. Biological and nonbiological decolorization processes applied to treatment of textile wastewater.

Table 1. Advantages and drawbacks of some nonbiological decolorization processes applied to textile wastewaters.

Physical–chemical methods Method description Advantages DisadvantagesFenton’s reagent Oxidation reaction using mainly

H2O2-Fe(II)Effective decolorization of

both soluble and insolubledyes

Sludge generation. Very expen-sive

Ozonation Oxidation reaction using ozonegas

Application in gaseous state:no alteration of volume

Short half-life (20 min). Veryexpensive

Photochemical Oxidation reaction using mainlyH2O2-UV

No sludge production Formation of by-products

Sodium hypochlorite(NaOCl)

Oxidation reaction using Cl+ toattack the amino group

Initiation and acceleration ofazo-bond cleavage

Release of aromatic amines

Electrochemical destruction Oxidation reaction using electri-city

Breakdown compounds arenon-hazardous

High cost of electricity

Activated carbon Dye removal by adsorption Good removal of a widevariety of dyes

Very expensive, Saturation ofcarbon

Membrane filtration Physical separation Removal of all dye types Concentrated sludge productionIon exchange Ion exchange resin Regeneration: no adsorbent

lossNot effective for all dyes

Electrokinetic coagulation Addition of ferrous sulphate andferric chloride

Economically feasible High sludge production

Dafale et al. 23


Env

iron

. Rev

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

YO

RK

UN

IV o

n 06

/10/

14Fo

r pe

rson

al u

se o

nly.

Luangdilok 2000; Albuquerque et al. 2005). This is in agree-ment with the previously published data from batch experi-ments on azo dye decolorization in the presence of sulfate(Carliell et al. 1998; van der Zee et al. 2003). As sulfatecan be biologically reduced to sulfide, a well-known bulkreductant of azo dyes, it has been suggested that its presencemay rather stimulate than competitively suppress azo dyedecolorization. Albuquerque et al. (2005) supported this andobserved a sharp decline of the anaerobic azo dye decolor-ization efficiency when selectively inhibiting its sulfate-re-ducing activity by the addition of molybdate.

Various decolorization systems

Single microbial speciesMuch of the experimental work involving anaerobic de-

colorization of dyes (predominantly azo dyes) was con-ducted using monocultures. Species of Bacillus andPseudomonas were found to be active in the anaerobic deg-radation of a number of dyes but other microorganisms,such as Aeromonas and purple non-sulphur photosyntheticbacteria, have been found to successfully decolorize a rangeof dyes to some extend at particular conditions.

Wuhrmann et al. (1980) investigated anaerobic reductionof azo dyes in the textile by a strain of Bacillus cereus iso-lated from soil. The biodegradation of azo dyes by Pseudo-monas cepacia 13NA was investigated by Ogawa andYatome (1990) using the dyes, CI Acid Orange 12, CI AcidOrange 20, and CI Acid Red 88. Several reports dealt withdecolorization of dyes using a select isolated bacterial cul-ture. These studies revealed that the isolated cultures areoften specific to the type of dye under consideration butthere is no strain reported so for that has been able to decol-orize a broad range of azo dyes. Therefore, the use of a spe-cific strain or enzymes on reductive decolorization does notmake much sense in treating textile wastewater, which iscomposed of many kinds of dyes. Moreover, pure culturescannot be easily scaled up and maintained in large-scale op-erations typical of actual effluent treatment systems(Coughlin et al. 1997).

Mixed populations of microorganismsActivated sludge is commonly used as an inoculum for ef-

ficient decolorization. These sludges contain a diverse mi-crobial population with significant dominant strains. Khalidet al. (2008) examined 288 strains of azo dye degrading bac-teria community to identify efficient strains for structurallydifferent azo dyes as the sole source of C and N. They ob-served Shewanella putrefeaciens (AS 96) was the most effi-cient strain for complete decolorization of structurallydifferent dye molecules. Kremer (1989) reported that theanaerobic degradation of Acid Red 88 and Acid Orange 7occurred in anaerobic serum bottles inoculated with sludgefrom an anaerobic digester. Ganesh et al. (1994) observedthe degradation of hydrolyzed Reactive Black 5 dye in ananaerobic digester. The use of mixed cultures, such as anae-robic granular sludge, is probably a more logical alternative.Alternatively, it would be interesting to obtain an active andstable microbial consortium through slow adaptation of amicrobial community (e.g., activated sludge from effluenttreatment plant) towards toxic or recalcitrant compounds

making it more efficient to improve the rate of decoloriza-tion process and become suitable for large-scale applica-tions. Indeed, the different microbial consortia present in ananaerobic granular sludge can carry out tasks that no indi-vidual pure culture alone can undertake successfully (Nigamet al. 1996; Pearce et al. 2003; Mohanty et al. 2006; Dafaleet al. 2008b).

Combined anaerobic and aerobic biological systemsAnaerobic decolorization of dyes is inadequate with re-

spect to mineralization of the degradation products. More-over, aerobic decolorization of dyes is practically notfeasible in wastewater treatment plants. Therefore, two-phase reactor systems that incorporate an anaerobic stagefor decolorization and a subsequent aerobic stage for miner-alization of the degradation products have been developed(Stolz 2001; Mohanty et al. 2006; Dafale et al. 2008b).

Haug et al. (1991) achieved mineralization of the sulpho-nated azo dye, Mordant Yellow 3, by 6-aminonaphthalene-2-sulphonate-degrading bacterial association in a two-phaseanaerobic–aerobic system. Zaoyan et al. (1992) reportedthat the anaerobic rotating biological contactor (RBC) wasefficient in removing the color from the effluent and also indegrading some complex organic matter. The aerobic acti-vated sludge unit was responsible for removing the remain-ing organic matter and intermediates from the effluent.Harmer and Bishop (1992) investigated the decolorizationand degradation of an azo dye, Acid Orange 7 (AO 7), usinglaboratory-scale rotating drum biofilm reactors. The biomasswas originally obtained from an activated sludge plant. Theprocess was operated under aerobic bulk-liquid conditions,as it was proposed that the biofilm would provide both anae-robic and aerobic zones that would allow a two-phase min-eralization of AO 7. They also found that both acclimatizedand unacclimatized biomass possessed the ability of decolor-ization.

Combined chemical–biological processesCombinations of chemical pretreatment techniques with

conventional biological treatment systems to optimize colorand total organic carbon (TOC) removal from textile efflu-ents have been used. The chemical pretreatment followedby biological treatment was chosen with the principal aimof removing color from the textile effluent. The reducingagents tested were sodium hydrosulphite, thiourea dioxideand sodium borohydride (McCurdy et al. 1991). Powell etal. (1992) reported on the use of oxidizing agents to pretreattextile effluents containing reactive dyes and the subsequenttreatment of this effluent in a conventional aerobic biologi-cal treatment system.

The use of chemical pretreatment for biological processhas many advantages and limitations. Pretreatment of thewastewater with ozone or Fenton’s reagent did not seem tosignificantly enhance or inhibit subsequent biological degra-dation of the dissolved organic carbon (DOC). Wastewaterpretreated with Fenton’s reagent appeared to show a lowerrate of DOC removal than that pretreated with ozone, how-ever, this was probably due to the fact that Fenton’s reagentremoved a high percentage of non-colored labile organicmatter, resulting in less DOC being readily available to thewastewater microorganisms.



Env

iron

. Rev

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

YO

RK

UN

IV o

n 06

/10/

14Fo

r pe

rson

al u

se o

nly.

Azo dyes removal in sequential anaerobic–aerobic bioreactors

Different microbial consortia present in anaerobic biomasscan carry out tasks that no individual pure cultures under-take successfully (Nigam et al. 1996; Pearce et al. 2003). Ef-ficiency of azo dye reduction, as well as the fate of aromaticcompounds in both anaerobic and aerobic bioreactors hasbeen investigated. van der Zee and Villaverde (2005)studied azo dye reduction mechanisms in batch and continu-ous flow systems. Interestingly, anaerobic systems are foundto decolorize azo dyes to a greater extent (Chen et al. 2003);but the degradation metabolites are reported to be even moretoxic than dye (Stolz 2001). The batch experiments showedthat azo dye MO 1 was 200-fold toxic to the anaerobic mi-croorganisms whereas its expected reduction products (ami-nosalicylic acid and 1,4 phenyledediamine) observed to be2140-fold more toxic. Additionally, low rates of reductionand severe toxicity of the azo dye Reactive Red 2 werefound in a UASB reactor operated at a HRT of 6 h and fedwith a volatile fatty acid (VFA) mixture as an electron do-nor.

A precondition for the reduction of azo dyes is the pres-ence and availability of a co-substrate, because it acts as anelectron donor. Many co-substrates were found to be suit-able as electron donors, viz. glucose (Carliell et al. 1995;Nigam et al. 1996; Dafale et al. 2008a, 2008b), hydrolyzedstarch (Willetts and Ashbolt 2000), tapioca (Chinwetkitva-nich et al. 2000), yeast extract (Nigam et al. 1996), a mix-ture of acetate, butyrate and propionate (Donlon et al. 1997)and the azo dye reduction product 5-ASA as well (Razo-Flores et al. 1997). It was also observed that the extent ofdecolorization of an azo dye like Remazol Black B variesdepending on the co-substrates used, e.g., 82% with glucose,71% for glycerol and lactose, 51% for starch and 39% for adistillery waste (Nigam et al. 1996). This is most likely dueto the growth of microbes in these conditions. Moreover, therate of azo-dye reduction process depends on the type of co-substrate used and (or) on the chemical structure of the azodyes (Van der Zee et al. 2000b). Mordant Orange 1 (MO 1)reduction could be achieved in a UASB reactor fed withglucose or VFA, with efficiencies up to 99% (Razo-Floreset al. 1997). Glucose has a significant impact on the colorremoval rates, probably because of the concentrations of H2(or formate) that are likely to be formed in glucose fermen-tation, a maximum of 4 mol H2 per mol of glucose. Thepresence of an electron donor is a prerequisite for azo dyereduction. In theory, the required amount of electron donat-ing primary substrate is low, 4 reducing equivalent per azolinkage i.e., 32 mg COD per mmol monoazo dye increasethe required amount of primary substrate.

The addition of catalytic concentrations of the redox me-diator enhanced recovery of reactor in terms of both colorremoval and COD removal. Cervantes et al. (2001) showedthat decolorization efficiencies around 90% were achievedby testing the Acid Orange 7 in a UASB reactor operated atan HRT of 2 h, fed with VFA and 3 mmol/L of anthraqui-none-2,6-disulfonate (AQDS). It was observed that the effectof decreasing the HRT was more apparent in the absence ofAQDS. This indicates that AQDS is assisting the transfer of

reducing equivalents to the dye molecules, which apparentlyis the rate-limiting factor.

Biotechnological approaches formanagement of textile mill effluents

The biotechnological approaches to decolorize azo-dyecontaining wastewater are very broad. However, only a fewapplications have been reported so far dealing with the colorand aromatic amines removal. Azo dyes contain mainly ni-tro and sulfonic groups and are quite recalcitrant to aerobicbacterial degradation. This fact is probably related either tothe electron withdrawing nature of the azo bond and theirresistance to attack by oxygenase, or because oxygen is amore effective electron acceptor, therefore having morepreference for reducing equivalents than the azo dye(Knackmuss 1996). The decolorization is observed due toazoreductase enzymes and its efficiency can be increasedby using biotechnological approach viz. biostimulation andbioagumentation of bioreactor. However, in the presence ofspecific oxygen-catalysed enzymes called azoreductase,some aerobic bacteria are capable of reducing azo com-pounds (Stolz 2001).

Various biotechnological approaches are used to over-come difficulties in conventional treatment plants, and en-hance the efficiency of azo dye decolorization. Firstly,different reactor configurations like the widely used UASBsystem and expanded granular sludge bed (EGSB) systemare used to immobilize high concentration of biomass (Let-tinga 1995; van Lier et al. 2001). Secondly, the redox medi-ators are used as enhancers for decolorization as theyaccelerate the electron transfer from primary electron donorto terminal electron acceptor. However, co-substrates areused as electron donor for azo dye reduction and creation ofanaerobic condition for dye reduction in the presence of freeavailable oxygen. It also provides energy for multiplicationof bacterial cultures.

The usual low efficiency of both color and chemical oxy-gen demand (COD) removal of conventional techniqueshave been overcome by the development of sequential anae-robic–aerobic system. Prerequisite for the mineralization ofmany azo dyes is a combination of reductive and oxidativesteps (Tan et al. 1999). A way to stimulate the biodegrada-tion of dye and aromatic amines is to bioaugment the anae-robic and aerobic reactors with strains or enriched culturescapable of decolorizing and degrading desired compounds.Mixed microbial consortia offers considerable advantagesover the use of pure cultures in the degradation of syntheticdyes. The individual strains may attack the dye molecule atdifferent positions or may use decomposition products pro-duced by another strain for further decomposition (Forgacset al. 2004). Hence, it would be interesting and beneficial toobtain an active microbial culture towards toxic or recalci-trant compounds. Thurnheer et al. (1988) used a chemostatwith a co-culture of five different bacteria to degrade sevensulfonated aromatic compounds. The bacterial strains usedwere Comamonas testosteroni T-2, C. testosteroni PSB-4,Alcaligenes sp. O-1, strain M-1, and strain S-1. In anotherreport, Kolbener et al. (1994) investigated the degradationof 3-nitrobenzenesulfonic acid (3-NBS) and 3-ABS tricklingfilter using six activated sludge samples of different origin,

Dafale et al. 25


Env

iron

. Rev

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

YO

RK

UN

IV o

n 06

/10/

14Fo

r pe

rson

al u

se o

nly.

four sludges originated from domestic wastewater and twofrom an industrial wastewater treatment plants. Interestingly,only the last two sludges were able to degrade both the com-pounds.

However, knowledge of biological degradation is still in-adequate, especially with reference to reactive dyes. Reac-tive Black 5 dye is widely used in the textile dyeingindustry and is reported to be toxic (Reutergadh and Lang-phasuk 1997). Aromatic amines, an anaerobic reductionproduct of dye decolorization are generally not degraded(Field et al. 1995); with the exception of a few aromaticamines; characterized by the presence of hydroxyl and (or)carboxyl groups (Razo-Flores et al. 1997; Kalyuzhnyi et al.2000). Mineralization of the aromatic amines by aerobicbacteria is more common and therefore aerobic conditionsare preferable to degrade the accumulated aromatic aminesto CO2, H2O, and NH3 (Isik and Sponza 2003). The aerobicstage of combined anaerobic–aerobic treatment of dye wastealso eliminated the COD attributed to removal of aromaticamines, which are anaerobicaly recalcitrant (Isik and Sponza2004b).

Acclimatization and enrichment of bacterial communityAdaptation of microbial community to degrade a previ-

ously recalcitrant compound, through prior exposure of themicroorganisms to desired compound, is known as enrich-ment or acclimation. Anaerobic decolorization of textiledyes can occur with unacclimated microorganisms too,although prior exposure of microorganism to the particularazo dyes has been shown to play a major role in enhancingdegradation. These adapted microorganisms may also haveincreased tolerance to previously inhibitory concentrationsof dyes. It is possible that exposure of biomass to dyes mayeven facilitate the development of microorganisms that areable to degrade dye metabolites. Kremer (1989) includedspecific enrichment programmes to improve the dye degra-dation ability of anaerobic microorganisms with the aim of:

(1) improving the rate of decolorization(2) improving degradation and possibly mineralizing the de-

gradation products resulting from the decolorization ofdye.

(3) improving microbial tolerance to inhibitory concentra-tions of dye and degradation products.

Reductive decolorization in the presence of redoxmediators

Redox mediators are compounds that accelerate the elec-tron transfer from a primary electron donor to a terminalelectron acceptor, which may increase the reaction rates byone to several orders of magnitude (Cervantes et al. 2002).The enzymes biologically reduce the mediator, as the directelectron acceptor of the primary electron donor. Secondly,the electrons are chemically transferred to the azo dye actingas terminal electron acceptor with consequent mediator re-generation. Redox mediators have shown to be effective notonly for reductive decolorization, but also for the reductivetransformation of iron (Lovley et al. 1998), nitroaromatics(Dunnivant et al. 1992), polyhalogenated compounds(O’Loughlin et al. 1999) and radionuclides (Fredrickson etal. 2000). These redox mediators are very effective for azo

dye reduction, and very likely due to the nature of the azochromophore –N=N–, which is electrochemically unstableand has the capacity to receive electrons from the reducedform of the mediator. The transfer of reducing equivalentsfrom a primary electron donor (co-substrate) to a terminalelectron acceptor generally acts as the rate limiting step inanaerobic azo dye reduction (van der Zee et al. 2003). Fla-vin based compounds like FAD, FMN and riboflavin, aswell as quinone based compounds like AQS, AQDS andlawsone, have been extensively reported as redox mediatorsduring azo dye reduction (Field and Brady 2003). Reducedflavins can act as an electron shuttle from nicotinamide ad-enine dinucleotide phosphate (NADPH)-dependent flavopro-teins to dye molecules as electron acceptor (Dos Santos etal. 2003).

The enhancing decolorization through cloning andexpression of gene encoding the azoreductase activity

Azoreductase activity has been identified in several bacte-ria, viz. Xenophilus azovorans KF46F (Blumel et al. 2002),Pseudomonas luteola (Hu 2001), Rhodococcus (Chang andLin 2001), Shigella dysenteriae Type I (Ghosh et al. 1992),Klebsiella pnumoniae RS-13 (Wong and Yuen 1996), Clos-tridium perfringens (Rafii et al. 1997). The genes encodingazoreductase from some bacteria viz. Geobacillus stearo-thermophilus OY-2 (Suzuki et al. 2001), Xenophilus azovor-ans KF46F (Blumel et al. 2002), Escherichia coli(Nakanishi et al. 2001), B. anthracis Ames (Read et al.2003) and Bacillus cereus ATCC10987 (Rasko et al. 2004)have been identified. The expression of recombinant azore-ductase and characterization of the gene encoding azoreduc-tase from Rhodobacter sphaeroides AS1.1737 and B.latrosporus RRK1 were studied for decolorization by severalauthors (Bin et al. 2004; Sandhya et al. 2008). Rafii and Co-leman (1999) compared the azoreductase gene of C. perfrin-gens with other azoreductases, the amplified fragmentcontaining the azoreductase gene was hybridized with thechromosomal DNA from other azoreductase-producing Clos-tridium species, a Eubacterium sp., Enterobacter cloacae,Citrobacter amalonaticus, E. coli and the recombinantphage. The hybridization indicated sequence homology be-tween the azoreductase gene of C. perfringens and the azor-eductases from these strict and facultatively anaerobicbacteria. There was low, barely detectable hybridizationwith DNA from Peptostreptococcus anaerobius, Peptostrep-tococcus productus, Bifidobacter infantis, Bacteroides the-taiotaomicron and unidentified strain 3 (another anaerobicGram-positive rod) having azoreductase activity (Rafii et al.1990). This indicates genetic differences in the types ofazoreductases produced in these bacteria.

Degradation of aromatic aminesSeveral bioreactor studies have demonstrated partial or

complete removal of many aromatic amines in the aerobicstage. Some evidences were based on quantitative detectionof individual compounds using sophisticated analytic techni-que like LC-MS or HPLC, if standards of the expectedamines are available. Most of the evidence was based onquantitative detection of total aromatic amines as a parame-ter using a diazotization-based method (Rajaguru et al.2000; Isik and Sponza 2003; Isik and Sponza 2004b; Sponza



Env

iron

. Rev

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

YO

RK

UN

IV o

n 06

/10/

14Fo

r pe

rson

al u

se o

nly.

and Isik 2005), based on the appearance of non-identifiedpeaks in HPLC chromatograms (Lourenco et al. 2000; Lour-enco et al. 2003; Albuquerque et al. 2005) or based on eval-uation of UV-vis spectra (Isik and Sponza 2004b; Cabral etal. 2005). Moreover, an increase in toxicity after anaerobictreatment of azo dyes (Libra et al. 2004; Isik and Sponza2004a) that is often observed can be interpreted as indirectevidence for the formation of aromatic amines. Complete re-moval of the expected amines from hydrolyzed ReactiveBlack 5 reduction in the expected stoichiometric ratio of2 mol p-aminobenzene-2-hydroxylethylsulfonic acid and1 mol 1,2,7-triamino-8-hydroxynaphthalene-3-,6-sulfonicacid per mol hydrolyzed Reactive Black-5 could be demon-strated by LC-MS analysis (Libra et al. 2004).

The aerobic biodegradation of aromatic aminesComplete mineralization of dye molecules is possible if

the anaerobic azo-reduction is followed by aerobic oxida-tions of the amines formed in the previous steps (Mou-taouakkil et al. 2004; Nachiyar and Rajakumar 2004). Theconversion of these compounds generally requires enrich-ment of specialized aerobic bacterial population. Many ofthe aromatic compounds were found to be degraded underaerobic conditions, e.g., compounds like aniline, carboxy-lated aromatic amines, chlorinated aromatic amines, andsubstituted benzidines (Pinheiro et al. 2004). Thurnheer etal. (1988) operated a chemostat at high HRT inoculatedwith five isolated cultures, could degrade seven benzenesul-fonic acids, including three ABS isomers. The aerobic bio-degradation of many aromatic amines have been extensivelystudied, however, a group of aromatic amines that remaindifficult to degrade are the sulfonated aromatic amines,which are released back into the environment (Tan and Field2000).

The autoxidation of aromatic aminesSome aromatic amines like phenylenediamines, amino-

phenols, aminonaphthol and o-aminohydroxynaphthalenesul-fonic acid tend to autoxidize under aerobic conditions(Kudlich et al. 1999). Autoxidation implies a process inwhich oxygen reacts with the aromatic products via free rad-ical reactions. This process results in the formation of col-ored oligomers and polymers, which is obviouslyundesirable. Moreover, the initially formed oligomers mayhave toxic and mutagenic effects (Field et al. 1995). Conse-quently, the autoxidation process eliminates the aromaticamines and the compounds produced are more recalcitrantto biological degradation (Van der Zee et al. 2000a).

Mechanisms for the reduction of azo dyesThe reductive decolorization of azo dyes under anaerobic

conditions is a combination of both biological and chemicalmechanisms. The biological contribution can be divided intospecialized enzymes called azoreductase, which are presentin bacteria that are able to grow in the presence of dye mol-ecules. However, to date there is no clear evidence of anae-robic azoreductase or nonspecific enzymes that catalyze thereduction of a wide range of electron-withdrawing contami-nants, including azo dyes (Stolz 2001). Thus a co-metabolicreaction is probably the main mechanism of dye reduction in

which the reducing equivalents or reduced cofactors viz.,NADH, NAD(P)H, FMNH2 and FADH2 acting as secondaryelectron donor, channel electrons to cleave the azo bond(Dos Santos et al. 2003).

Walker and Ryan (1971) postulated that decolorizationrates are related to the electron density in the azo bond re-gion. They suggested that color removal rates would in-crease by lowering the electron density in the azo linkage.Therefore use of redox mediators would not only tend to ac-celerate the transfer of reducing equivalents to the terminalelectron acceptor, i.e., azo dye, but also to minimize thesteric hindrance of the dye molecule (Moir et al. 2001).Thus, the reducing equivalents are formed during the con-version of the primary electron donor, i.e., the organic mat-ter, during the different steps of carbon flow underanaerobic conditions.

The reduction of dye molecules involves the cleavage andtransfer of four-electrons, which proceeds through twostages (step 1 & 2) at the azo linkage, i.e., hydrazo inter-mediate and aromatic amine formation. The fates of azodyes in the reactions are presented in two steps. Step 1 isthe anaerobic reductive cleavage of the azo bond and subse-quent production of the aromatic amines, which are anae-robically recalcitrant. Thus, azo dyes cannot be completelybiodegraded but only reduced in step 1. Step 2 is the re-quired post-treatment in which the reduced products are ulti-mately degraded.

½1� R1�N¼N�R2 þ 2e� þ 2Hþ

! R1�NH�NH�R2 ðstep 1Þ

½2� R1�NH�NH�R2 þ 2e� þ 2Hþ

! R1�NH2 þ R2�NH2 ðstep 2Þ

½3� ðR1�N¼N�R2Þ þ 4e� þ 4Hþ

! R1�NH2 þ R2�NH2 ðoverallÞ

The role of bacterial enzyme in azo dye reductionThe exact mechanism of the anaerobic azo dye reduction

is not clearly understood yet. Therefore, the term azo dyereduction may involve different mechanisms or locationslike enzymatic, nonenzymatic, mediated, intracellular, ex-tracellular and various combinations of these mechanismsand locations (Haug et al. 1991; Carliell et al. 1995; Kudlichet al. 1997; Van der Zee et al. 2000b). The process respon-sible for biological decolorization of azo dyes under anaero-bic conditions is subject to debate. Theories proposed for themechanism of azo reduction can be broadly categorized intotwo groups:

(1) intracellular azo reduction(2) extracellular azo reduction

Both intracellular and extracellular decolorization relieson the production of enzymatically-generated reduced flavinnucleotides. In both cases the mechanism proposed for azoreduction is similar, in that reduction of the azo bond is con-current with re-oxidation of enzymatically-generated re-duced flavin nucleotides. The dye acting as an oxidizingagent for the reduced flavin nucleotide of the electron trans-port chain is reduced and consequently, decolorized. These

Dafale et al. 27


Env

iron

. Rev

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

YO

RK

UN

IV o

n 06

/10/

14Fo

r pe

rson

al u

se o

nly.

reduced flavins are formed in catabolic process and there-fore the availability of respiration substrate should also beconsidered as a rate-controlling step in azo reduction (Hauget al. 1991). Intracellular azo dye reduction cannot be re-sponsible for the conversion of all types of azo dyes, espe-cially for sulfonated azo dyes, which have limitedmembrane permeability (Stolz 2001). No correlation be-tween molecular size and rates of decolorization was found,which indicates that dye penetration into the cell wall is notplaying a major role in the reductive decolorization, andtherefore the latter is likely to occur outside the cell or ismembrane-bound. The current hypothesis is that azo dye re-duction mostly occurs by the involvement of extracellular ormembrane-bound enzymes (Stolz 2001).

Anaerobic azo dye reduction can be construed to involveeither a direct enzymatic reaction or indirect reaction withreduced enzyme cofactors. According to the direct enzy-matic azo dye reduction, enzymes transfer the reducingequivalents originating from the oxidation of organic sub-strate (glucose) to the azo dyes. In the indirect mechanism,the azo dyes are reduced by enzymatically reduced electroncarriers (Fig. 2). The role of enzymes in microbial azo dyereduction is uncertain. Kremer (1989) has stated that azo re-duction is enzymatically catalysed by azoreductase. It is alsonot clear whether this azoreductase enzyme directly cataly-ses the final transfer of electrons to the target compound orwhere, as proposed by Wuhrmann et al. (1980) and Haug etal. (1991).

The role of soluble flavins in bacterial azo reductionThe anaerobic microorganisms not only generate the elec-

trons to cleave the azo bond, but also maintain low redoxpotential (< –50 mV), which is required for the transfer ofreducing equivalent to the dye molecules (Bromley-Challe-nor et al. 2000). Although a complete mineralization cannotgenerally be reached anaerobicaly, the reductive transforma-tion increases the susceptibility of the aromatic molecules tooxygenases (Field et al. 1995). The effectiveness of redoxmediators in enhancing the decolorization of textile waste-water is still unclear due to wide range of redox potentialsamong azo dyes (–180 to –430 mV) (Rau and stolz 2003).

Azo reduction occurs via enzymatically-generated reducedflavins, but that the final step in the transfer of electrons oc-curs non-enzymatically. The latter is generally referred to asnon-enzymatic azo reduction and is mediated by a flavopro-tein in the microbial electron transfer chain. This flavopro-tein catalyses the generation of reduced flavins[flavinmononucleotide (FMN) or flavinadeninedinucleotide(FAD)] by the re-oxidation of reduced nicotinamide adeninedinucleotide (NADH) or nicotinamide adenine dinucleotidephosphate (NADPH). These reduced flavins transfer elec-trons to dye molecules (terminal electron acceptor), therebyreducing the azo bonds and concurrently re-oxidized. A pro-posed mechanism for catalysis by azo reductases andNADPH is shown in Fig. 3 based on the studies by Kremer1989.

These azo reducing flavo proteins have been found to dif-fer according to the class of microorganism in which theyare observed. For example, Streptococcus faecalis and Pro-teus vulgaris have a FMN prosthetic group, the enzyme cat-alyzing azo reduction in S. faecalis is activated to a similar

extent by riboflavin, FMN or FAD. As dye reduction is pro-posed to occur through reduction of the dye as a terminalelectron acceptor in the microbial electron transport chain,the presence of competitive electron accepting agents in thesystem would be expected to affect the rate of azo reduc-tion. The sensitivity of the dye reduction to the presence ofoxygen can then be explained as a competition of the oxi-dants (azo dye and oxygen) for the reduced electron carriersin the respiration chain, with respiration of oxygen being thefavored reaction.

Configuration of anaerobic–aerobic systemfor biodegradation of azo dyes

The anaerobic and aerobic reactor was successfully bio-augmented with a dye decolorizing anaerobicaly enrichedbacterial culture and aromatic amines degrading aerobic en-riched bacterial cultures. The use of mixed cultures, such asanaerobic granular sludge, which is composed of stable mi-crobial pellet with a high activity, is probably a better op-tion. Two configurations that combine anaerobic andaerobic are available for complete mineralization of dyewastewater. Tan (2001) investigated the use of integratedand sequential anaerobic–aerobic systems for the biodegra-dation of azo dyes.

(1) the sequential anaerobic–aerobic rector system(2) the integrated anaerobic–aerobic reactor system

In a sequential anaerobic–aerobic reactor system the re-quired condition can be implemented by spatial separationof the two sludges (Stolz 2001). However, these conditionscan also be created in one reactor integrating anaerobic–aerobic reactor system (Field et al. 1995). These two config-urations are the needed technology for the future in the tex-tile industry.

The sequential or integrated anaerobic–aerobicbioreactor system

The integrated anaerobic–aerobic conditions can be cre-ated by using an anaerobic granular sludge with a high tol-erance for oxygen as a carrier material for aerobic biofilm(Zitomer 1998). The integrated condition in practice is pos-sible by using a support material. The anaerobic bacteriawill develop inside the support material and aerobic bacteriaon the outside which in a way forms a protective barrier forthe anaerobic bacteria (Kudlich et al. 1996). Calcium algi-nate beads were used as a support material to create the in-tegrated anaerobic–aerobic conditions. The same co-culturethat has degraded a dye molecule under the sequential anae-robic–aerobic batch conditions was used for degradation ofazo dye under integrated anaerobic–aerobic conditions.

The sequential anaerobic and aerobic degradation has beenstudied for the conversion of azo dyes by numerous research-ers (Lourenco et al. 2000; O’Neill et al. 2000a; O’Neill et al.2000b; Rajaguru et al. 2000). Sequential anaerobic–aerobicconditions can readily be imposed to wastewater by first ex-posing it to anaerobic conditions followed by aerobic condi-tions by using an anaerobic reactor followed by an aerobicreactor. In one of the reactor studies it was shown that thecolor removal by a two-stage anaerobic–aerobic treatmentprocess was 70% higher than that of a one-stage aerobic treat-ment process (Ekici et al. 2001; van der Zee and Villaverde



Env

iron

. Rev

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

YO

RK

UN

IV o

n 06

/10/

14Fo

r pe

rson

al u

se o

nly.

2005). This is due to most aromatic amines, which accumu-late after azo cleavage, are not mineralized anaerobicallywhereas only few aromatic amines substituted with hydroxyland carboxyl groups were fully degraded under methano-genic conditions (Razo-Flores et al. 1997). There is evidencethat linear alkylbenzenesulfonates and other sulfonatedaromatics are utilized and degraded as a sulfur source undersulfur limiting anaerobic conditions (Jensen 1999). Minerali-zation of the aromatic amines by aerobic bacteria and aerobicsludge in treatment plants is more common. The aromaticamines serve as main substrate for the organisms in the aero-bic bioreactor (Fig. 4). However, it should be noted that somearomatic amines are readily autoxidized in the presence ofoxygen to humic like oligomeric and polymeric structures.Figure 5 represents the overview of steps involved inbiodegradation of azo dyes in a sequential anaerobic–aerobicbioreactor system and its biodiversity analyses.

Toxicity assessment

Basically, cytotoxicity of typical dye molecules may berelatively low, but the toxicity of related aromatic amine is

very significant due to their carcinogenicity or mutagenicity(Libra et al. 2004). As aromatic amines are difficult to re-move via traditional wastewater treatment and inevitablytend to persist, the toxicity evaluation upon these amineswill be apparently crucial to operation success in dye decol-orization and degradation. This study is important to investi-gate toxic impacts of aromatic amine for risk assessment inoperation. Several bacterial assays are available for deter-mining the toxicity of environmental samples. Some conven-ient and rapidly determined selected bacterial assays aredisplayed in Table 2. Aromatic amines increase cytotoxicityin anaerobic treatment was eventually showed using respira-tion-inhibition tests (O’Neill et al. 2000b). In addition,Daphnia magna (Moran et al. 2008) was used to determinethe toxicity of dye molecules in aerobic treatment of textilewastewater. Wang et al. (2003) also performed biolumines-cence of Lumistox bacteria (Microtox1) assays to revealthe toxicity of Remazol Black 5 in a baffled reactor. Toxic-ity assessment of by-product to Pseudomonas luteola wasalso performed using plate-count method (Chen 2006). Gen-otoxicity testing based on the effect of DNA-damagingagents on a dark mutant of Photobacterium leiognathi and

Fig. 2. The direct and indirect enzymatic anaerobic reduction of azo dyes.

Fig. 3. Mechanism of catalysis by azo reductase and NADPH.

Dafale et al. 29


Env

iron

. Rev

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

YO

RK

UN

IV o

n 06

/10/

14Fo

r pe

rson

al u

se o

nly.

Fig. 4. Schematic representation of decolorization and mineralization of azo dyes in anaerobic and aerobic bioreactor.

Fig. 5. Synoptical representation of steps involved in biodegradation of azo dyes and biodiversity of sequential bioreactor system.

Table 2. Some short-term bacterial toxicity assays.

No Assay Basis for the test1 Microtox Inhibition of bioluminescence of Vibrio fischerii2 Spirillum volutants Toxicants cause loss of coordination of rotating fascicles of flagella with con-

comitant loss of motility3 Growth inhibition Measure growth inhibition of pure or mixed cultures via absorbance determi-

nation of suspension or zone of inhibition4 Viability assays Viability of bacterial cultures on agar plate (CFU/mL)5 ATP assay Inhibitory effect of toxicant on ATP level in microorganism6 Respirometry Measures microbial respiration in environmental samples7 Toxi-Chromotest Based on inhibition of biosynthesis of b-galactosidase in E. coli



Env

iron

. Rev

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

YO

RK

UN

IV o

n 06

/10/

14Fo

r pe

rson

al u

se o

nly.

genetically engineered Pseudomonas containing the lux op-eron that codes for bioluminescence was used for toxicityassay of wastewater (Wiles et al. 2003). Moreover, ATA-anaerobic toxicity, respiration inhibition and Daphniamagna tests were used to assess the toxicity of Direct Black38 in an anaerobic–aerobic sequential reactor system (Isikand Sponza 2004b; Sponza and Isik 2005).

Biodiversity of bioreactor using culture-based and culture-independent tool

This study attempts to bring out the importance of knowl-edge of microbial diversity in dye decolorization operation.Results obtained may help in identification of more dye de-colorizing species, leading to better understanding of micro-biology of dye decolorization and subsequent efficientprocess optimization. Dye decolorizing bacteria have tradi-tionally been detected by plating on dye decolorizing agarand looking for zones of decolorization (Abd El-Rahim etal. 2003; Mohanty et al. 2006). However, it ignores the syn-ergistic decolorization activity that a bacterial consortiummight exert in the effluent treatment plant (Khehra et al.2005). Whereas, the unculturable bacteria may play a vitalrole in the decolorization process. Hence, culture-basedmethods cannot be directly used to analyze microbial diver-sity, because it recognize only a small proportion of totalmicrobiota in the environmental samples (Kampfer et al.1996). Unexplored microbial diversity represents a vast un-tapped source of novel and unique enzymatic activities andmetabolic pathways (Robertson et al. 2004; Tringe et al.2005; Ferrer et al. 2005). Modern genomic techniques ena-ble the direct and comprehensive extraction of microbialDNA from microbial consortia and the generation of envi-ronmental genomic DNA (cDNA) libraries has solved thisproblem to a great extent (Eschenhagen et al. 2003 Gray etal. 2003; Robertson et al. 2004). It provides a means tomore accurately describe microbial populations and gives aclearer overall picture of microbial diversity.

Activated sludge reactors contain highly dynamic micro-bial communities, which obscure the understanding of theinherent microbial population. The stability of bacterialcommunities in activated sludge systems depends not onlyon operational parameters like HRT, food to microorganismratio (F/M) but also on the type of compounds. A combina-tion of polymerase chain reaction (PCR), hybridization and16S rDNA and physiological analysis has been emphasizedfor detection of the degradation capacity of different sludgeto demonstrate the variation in bacterial population over aperiod of time in response to change in conditions of the bi-oreactor. Rapid shift in the species composition of the bacte-rial population was observed in an anaerobic digesterrunning under constant environmental conditions over a pe-riod of 2 years (Zumstein et al. 2000).

Organisms with the desired decolorization and degrada-tion capacity may or may not be the predominant species ina bioreactor and identifying such a potential in a communityof microorganism in situ is difficult. Hence, special attentionis paid to how it is becoming possible to relate the composi-tion of the community of microbes present in activatedsludge and the in situ function of individual populationsthere and to how such information might be used to manage

and control these systems better (Chua et al. 2003). Se-quencing of 16S rDNA has become a generally acceptedtool in the study of phylogenetic relationships between mi-croorganisms and in species identification. Microbial com-munity characterization was also performed through RAPD,AFLP and RFPL analysis, to determine the nature and quan-tity of microbial diversity constitute the bioreactor. Thesetechniques find their greatest application in detecting poly-morphisms in closely related organisms (low divergence)such as those that compose species complexes, differentpopulations of a single species (Kapley et al. 2007a, 2007b;Purohit et al. 2003). Knowledge of the microbial communitynaturally present can influence the design and treatment ca-pacity of the widely used land based systems.

It has been estimated that only 10%–15% of prokaryotesin the activated sludge can be cultured in the laboratory(Handelsman 2004; Tringe et al. 2005), a phenomenon thatlimits our understanding of microbial physiology, genetics,and community ecology of reactor system. The microbialcommunity in ETPs is responsible for the degradation ofmany organic pollutants. In diversity analysis, different pa-rameters need to be included to minimize the loss in diver-sity and increase the possibility of acquiring novel strainsexisting within the community. There are various reports de-scribing the diversity of bacterial community in ETP, CETPand various bioreactors using the metagenomic and genomictools (Purohit et al. 2003; Kapley et al. 2007a, 2007b; Raniet al. 2008). Recent advances in the field of metagenomicsto provide glimpses into the life of uncultured microorgan-isms, may allow application of modern genomic techniquesto achieve a better understanding of the microbial flora inthe dye degrading bioreactor without the need for isolationor laboratory cultivation of individual species.

ConclusionsThe majority of the textile industries have been following

the physicochemical routes of wastewater treatment withmajor emphasis on water and chemicals recovery. Advancedmembrane technologies applied frequently these days in tex-tile industry are usually uneconomical. The ever increasingstringency of regulatory bodies and greater public awarenesstend to drive industries towards adaptation of low cost inte-grated biotechnological approaches. A switch over to se-quential anaerobic–aerobic treatment processes therefore isimperative. As revealed in this survey, there is increasingemphasis on sequential anaerobic–aerobic bioreactors forachieving complete degradation of dyes in textile effluents.It also appears pertinent that monitoring bacterial diversityin a bioreactor using culturable and culture independent ge-nomic tools can provide better understanding of the func-tional status of wastewater treatment and providefundamental information for the design and operation of bi-oreactors. At present, very few studies complementing dyedecolorization with biodiversity of the decolorizing popula-tion have been performed (Plumb et al. 2001). Thus, an in-tegrated biotechnological approach for completemineralization of dye containing wastewater using sequen-tial anaerobic–aerobic system based on enriched mixed bac-terial consortium appears to hold great technologicalpromise.

Dafale et al. 31


Env

iron

. Rev

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

YO

RK

UN

IV o

n 06

/10/

14Fo

r pe

rson

al u

se o

nly.

AcknowledgmentNishant Dafale gratefully acknowledges the Council of

Scientific and Industrial Research (CSIR), India for theaward of a Senior Research Fellowship (SRF) to carry outthe work. Authors are also thankful to Director, Dr. TapanChakrabarti, NEERI, Nagpur, for providing the facility andconstant support during this study.

ReferencesAbd El-Rahim, W.M., Moawad, H., and Khalafallah, M. 2003. Mi-

croflora involved in textile dye waste removal. J. Basic Micro-biol. 43(3): 167–174. doi:10.1002/jobm.200390019. PMID:12761767.

Albuquerque, E., Lopes, T., Serralheiro, L., Novais, M., and Pin-heiro, M. 2005. Biological sulphate reduction and redox media-tor effects on azo dye decolorization in anaerobic-aerobicsequencing batch reactors. Enzyme Microb. Technol. 36(5–6):790–799. doi:10.1016/j.enzmictec.2005.01.005.

Ashoka, C., Geetha, S., and Sullia, B. 2002. Bioleaching of compo-site textile dye effluent using bacterial consortia. Asian J. Micro-biol. Biotechnol. 4: 65–68.

Bae, J.S., and Freeman, H.S. 2007. Aquatic toxicity evaluation onnew direct dyes to the Daphnia magna. Dyes Pigments, 73(1):81–85. doi:10.1016/j.dyepig.2005.10.015.

Banat, I.M., Nigam, P., Singh, D., and Marchant, R. 1996. Micro-bial decolorization of textile-dye containing effluents- A Re-view. Bioresour. Technol. 58(3): 217–227. doi:10.1016/S0960-8524(96)00113-7.

Bhat, R.V., and Mathur, P. 1998. Changing scenario of food colorsin India. Curr. Sci. 74: 198–202.

Bin, Y., Jiti, Z., Jing, W., Cuihong, D., Hongman, H., Zhiyong, S.,and Yongming, B. 2004. Expression and characteristics of thegene encoding azoreductase from Rhodobacter sphaeroidesAS1.1737. FEMS Microbiol. Lett. 236(1): 129–136. doi:10.1111/j.1574-6968.2004.tb09638.x. PMID:15212802.

Blumel, S., Knackmuss, H.J., and Stolz, A. 2002. Molecular clon-ing and characterization of the gene coding for the aerobic azor-eductase from Xenophilus azovorans KF46F. Appl. Environ.Microbiol. 68(8): 3948–3955. doi:10.1128/AEM.68.8.3948-3955.2002. PMID:12147495.

Bromley-Challenor, C.A., Knapp, J.S., Zhang, Z., Gray, N.C.,Hetheridge, M.J., and Evans, M.R. 2000. Decolorization of anazo dye by unacclimated activated sludge under anaerobic con-ditions. Water Res. 34(18): 4410–4418. doi:10.1016/S0043-1354(00)00212-8.

Cabral, G.I., Penha, S., Matos, M., Santos, A.R., Franco, F., andPinheiro, H.M. 2005. Evaluation of an integrated anaerobic-aerobic SBR system for the treatment of wool dyeing effluents.Biodegradation, 16(1): 81–89.

Carliell, C.M., Barclay, S.J., Naidoo, N., Buckley, C.A., Mulhol-land, D.A., and Senior, E. 1995. Microbial decolorization of areactive azo dye under anaerobic conditions. Water S.A. 21(1):61–69.

Carliell, M., Barclay, J., Shaw, C., Wheatley, D., and Buckley, A.1998. The effect of salts used in textile dyeing on microbial de-colorization of a reactive azo dye. Environ. Technol. 19(11):1133–1137. doi:10.1080/09593331908616772.

Cervantes, F.J., van der Zee, F.P., Lettinga, G., and Field, J.A.2001. Enhanced decolourisation of acid orange 7 in a continuousUASB reactor with quinones as redox mediators. Water Sci.Technol. 44(4): 123–128. PMID:11575075.

Cervantes, F.J., de Bok, F.A.M., Duong-Dac, T., Stams, A.J.M.,Lettinga, G., and Field, J.A. 2002. Reduction of humic sub-

stances by halorespiring, sulphate-reducing and methanogenicmicroorganisms. Environ. Microbiol. 4(1): 51–57. doi:10.1046/j.1462-2920.2002.00258.x. PMID:11966825.

Chang, J.S., and Lin, C.Y. 2001. Decolorization of an azo dye withrecombinant Escherichia coli strain harboring azo-dye-decolor-izing determinants from Rhodococcus sp. Biotechnol. Lett.23(8): 631–636. doi:10.1023/A:1010306114286.

Chen, B. 2006. Toxicity assessment of aromatic amines to Pseudo-monas luteola: Chemostat pulse technique and dose-responseanalysis. Process Biochem. 41(7): 1529–1538. doi:10.1016/j.procbio.2006.02.014.

Chen, K.C., Wu, J.Y., Liou, D.J., and Hwang, S.C. 2003. Decolor-ization of the textile dyes by newly isolated bacterial strains. J.Biotechnol. 101(1): 57–68. doi:10.1016/S0168-1656(02)00303-6.PMID:12523970.

Chinwetkitvanich, S., Tuntoolvest, M., and Panswad, T. 2000.Anaerobic decolorization of reactive dyebath effluents by atwo-stage UASB system with tapioca as a co-substrate. WaterRes. 34(8): 2223–2232. doi:10.1016/S0043-1354(99)00403-0.

Choy, K.K.H., McKay, G., and Porter, J.F. 1999. Sorption of aciddyes from effluents using activated carbon. Resour. Conserv.Recycling, 27(1-2): 57–71. doi:10.1016/S0921-3449(98)00085-8.

Chua, A.S., Takabatake, H., Satoh, H., and Mino, T. 2003. Produc-tion of polyhydroxyalkanoates (PHA) by activated sludge treat-ing municipal wastewater: effect of pH, sludge retention time(SRT), and acetate concentration in influent. Water Res. 37(15):3602–3611. doi:10.1016/S0043-1354(03)00252-5. PMID:12867326.

Cook, S.F., and Linden, D.R. 1997. Use of rhodamine WT to facil-itate dilution and analysis of atrazine samples in short-termtransport studies. J. Environ. Qual. 26: 1438–1441.

Correia, V.M., Stephenson, T., and Judd, S.J. 1994. Characteriza-tion of textile wastewater–a review. Environ. Technol. 15(10):917–929. doi:10.1080/09593339409385500.

Coughlin, M.F., Kinkle, B.K., Tepper, A., and Bishop, P.L. 1997.Characterization of aerobic azo dye-degrading bacteria and theiractivity in biofilms. Water Sci. Technol. 36(1): 215–220. doi:10.1016/S0273-1223(97)00327-2.

Dafale, N., Rao, N.N., Meshram, S.U., and Wate, S.R. 2008a. De-colorization of azo dyes and simulated dye bath wastewaterusing acclimatized microbial consortium – biostimulation andhalo tolerance. Bioresour. Technol. 99(7): 2552–2558. doi:10.1016/j.biortech.2007.04.044. PMID:17566726.

Dafale, N., Wate, S., Meshram, S., and Nandy, T. 2008b. Kineticstudy approach of remazol black-B use for the development oftwo-stage anoxic-oxic reactor for decolorization/biodegradationof azo dyes by activated bacterial consortium. J. Hazard. Mater.159(2-3): 319–328. doi:10.1016/j.jhazmat.2008.02.058. PMID:18394798.

Donlon, B.A., Razo-Flores, E., Luijten, M., Swarts, H., Lettinga,G., and Field, J.A. 1997. Detoxification and partial mineraliza-tion of the azo dye mordant orange 1 in a continuous upflowanaerobic sludge-blanket reactor. Appl. Microbiol. Biotechnol.47(1): 83–90. doi:10.1007/s002530050893.

Dos Santos, A.B., Cervantes, F.J., Yaya-Beas, R.E., and Van Lier,J.B. 2003. Effect of redox mediator, AQDS, on the decolourisa-tion of a reactive azo dye containing triazine group in a thermo-philic anaerobic EGSB reactor. Enzyme Microb. Technol. 33(7):942–951. doi:10.1016/j.enzmictec.2003.07.007.

Dunnivant, F.M., Schwarzenbach, R.P., and Macalady, D.L. 1992.Reduction of substituted nitrobenzenes in aqueous solutions con-taining natural organic matter. Environ. Sci. Technol. 26(11):2133–2141. doi:10.1021/es00035a010.

Ekici, P., Leupold, G., and Parlar, H. 2001. Degradability of se-



Env

iron

. Rev

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

YO

RK

UN

IV o

n 06

/10/

14Fo

r pe

rson

al u

se o

nly.

lected azo dye metabolites in activated sludge systems. Chemo-sphere, 44(4): 721–728. doi:10.1016/S0045-6535(00)00345-3.PMID:11482661.

Eschenhagen, M., Schuppler, M., and Roske, I. 2003. Molecularcharacterization of the microbial community structure in two ac-tivated sludge systems for the advanced treatment of domesticeffluents. Water Res. 37(13): 3224–3232. doi:10.1016/S0043-1354(03)00136-2. PMID:14509710.

Ferrer, M., Martınez-Abarca, F., and Golyshin, P.N. 2005. Mininggenomes and ‘metagenomes’ for novel catalysts. Curr. Opin.Biotechnol. 16(6): 588–593. doi:10.1016/j.copbio.2005.09.001.PMID:16171989.

Field, J.A., and Brady, J. 2003. Riboflavin as a redox mediator ac-celerating the reduction of the azo dye mordant yellow 10 byanaerobic granular sludge. Water Sci. Technol. 48(6): 187–193.PMID:14640217.

Field, J.A., Stams, A.J.M., Kato, M., and Schraa, G. 1995. En-hanced biodegradation of aromatic pollutants in cocultures ofanaerobic and aerobic bacterial consortia. Antonie Leeuwen-hoek, 67(1): 47–77. doi:10.1007/BF00872195. PMID:7741529.

Forgacs, E., Cserhati, T., and Oros, G. 2004. Removal of syntheticdyes from wastewaters: a review. Environ. Int. 30(7): 953–971.doi:10.1016/j.envint.2004.02.001.

Fredrickson, J.K., Kostandarithes, H.M., Li, S.W., Plymale, A.E.,and Daly, M.J. 2000. Reduction of Fe(III), Cr(VI), U(VI), andTc(VII) by Deinococcus radiodurans R1. Appl. Environ. Micro-biol. 66(5): 2006–2011. doi:10.1128/AEM.66.5.2006-2011.2000.PMID:10788374.

Ganesh, R., Boardman, G.D., and Michelsen, D.L. 1994. Fate ofazo dyes in sludges. Water Res. 28(6): 1367–1376. doi:10.1016/0043-1354(94)90303-4.

Ghosh, K., Mandal, A., and Chaudhuri, J. 1992. Purification andpartial characterization of two azoreductases from Shigella dys-enteriae Type I. FEMS Microbiol. Lett. 98(1-3): 229–234.doi:10.1111/j.1574-6968.1992.tb05519.x.

Gray, K.A., Richardson, T.H., Robertson, D.E., Swanson, P.E., andSubramanian, M.V. 2003. Soil-based gene discovery: a newtechnology to accelerate and broaden biocatalytic applications.Adv. Appl. Microbiol. 52: 1–27. doi:10.1016/S0065-2164(03)01001-3. PMID:12964238.

Handelsman, J. 2004. Metagenomics: application of genomics touncultured microorganisms. Microbiol. Mol. Biol. Rev. 68(4):669–685. doi:10.1128/MMBR.68.4.669-685.2004. PMID:15590779.

Harmer, P., and Bishop, E. 1992. Transformation of azo dye AO-7by wastewater biofilms. Water Sci. Technol. 26(3–4): 627–636.

Haug, W., Schmidt, A., Nortemann, B., Hempel, D.C., Stolz, A.,and Knackmuss, H.J. 1991. Mineralisation of the sulphonatedazo dye mordant yellow 3 by a 6-aminonaphthalene-2-sulpho-nate degrading bacterial consortium. Appl. Environ. Microbiol.57(11): 3144–3149. PMID:1781678.

Hu, T.L. 2001. Kinetics of azoreductase and assessment of toxicityof metabolic products from azo dyes by Pseudomonas luteola.Water Sci. Technol. 43(2): 261–269. PMID:11380189.

Isik, N., and Sponza, D.T. 2003. Aromatic amine degradation in aUASB/CSTR sequential system treating Congo Red dye. J. En-viron. Sci. Health, Part A–Toxic/Hazard. Subst. Environ. Eng.38(10): 2301–2315.

Isik, M., and Sponza, D.T. 2004a. Monitoring of toxicity and inter-mediates of C.I. Direct Black 38 azo dye through decolorizationin an anaerobic/aerobic sequential reactor system. J. Hazard.Mater. 114(1-3): 29–39. doi:10.1016/j.jhazmat.2004.06.011.PMID:15511571.

Isik, M., and Sponza, D.T. 2004b. Decolorization of azo dyes under

batch anaerobic and sequential anaerobic/aerobic condition. J.Environ. Sci. Health Part A Tox. Hazard. Subst. Environ. Eng.39(4): 1107–1127.

Ivanov, K., Gruber, E., Schempp, W., and Kirov, D. 1996. Possibi-lities of using zeolite as filler and carrier for dyestuffs in paper.Das Papier. 50: 456–460.

Jensen, J. 1999. Fate and effects of linear alkylbenzene sulphonates(LAS) in the terrestrial environment. Sci. Total Environ. 226(2-3): 93–111. doi:10.1016/S0048-9697(98)00395-7. PMID:10085562.

Kalyuzhnyi, S., Sklyar, V., Mosoloava, T., Kucherenko, I., Russ-kova, J.A., and Degtyaryova, N. 2000. Methanogenic biodegra-dation of aromatic amines. Water Sci. Technol. 42(5–6): 363–370.

Kampfer, P., Erhart, R., Beimfohr, C., Bohringer, J., Wagner, M.,and Amann, R. 1996. Characterization of bacterial communitiesfrom activated sludge: culture-dependent numerical identifica-tion versus in situ identification using group- and genus-specificrRNA-targeted oligonucleotide probes. Microb. Ecol. 32(2):101–121. doi:10.1007/BF00185883. PMID:8688004.

Kapley, A., De Baere, T., and Purohit, H.J. 2007a. Eubacterial di-versity of activated biomass from a common effluent treatmentplant. Res. Microbiol. 158(6): 494–500. doi:10.1016/j.resmic.2007.04.004.

Kapley, A., Prasad, S., and Purohit, H.J. 2007b. Changes in micro-bial diversity in fed-batch reactor operation with wastewatercontaining nitroaromatic residues. Bioresour. Technol. 98(13):2479–2484. doi:10.1016/j.biortech.2006.09.012. PMID:17070036.

Khalid, A., Arshad, M., and Crowley, D.E. 2008. Accelerated deco-lorization of structurally different azo dyes by newly isolatedbacterial strains. Appl. Microbiol. Biotechnol. 78(2): 361–369.doi:10.1007/s00253-007-1302-4. PMID:18084755.

Khehra, M.S., Saini, H.S., Sharma, D.K., Chadha, B.S., andChimni, S.S. 2005. Comparative studies on potential of consor-tium and constituent pure bacterial isolates to decolorize azodyes. Water Res. 39(20): 5135–5141. doi:10.1016/j.watres.2005.09.033. PMID:16289280.

Knackmuss, H.J. 1996. Basic knowledge and perspectives of bioe-limination of xenobiotic compounds. J. Biotechnol. 51(3): 287–295. doi:10.1016/S0168-1656(96)01608-2.

Kolbener, P., Baumann, U., Cook, A.M., and Leisinger, T. 1994. 3-Nitrobenzenesulfonic acid and 3-aminobenzesulfonic acid in alaboratory trickling filter: Biodegradability with different acti-vated sludges. Water Res. 28(9): 1855–1860. doi:10.1016/0043-1354(94)90160-0.

Kremer, F.V. 1989. Anaerobic degradation of monoazo dyes. Ph.D.Thesis, Department of Civil and Environmental Engineering,University of Cincinnati.

Kudlich, M., Bishop, P.L., Knackmuss, H.J., and Stolz, A. 1996.Simultaneous anaerobic and aerobic degradation of the sulfo-nated azo dye Mordant Yellow 3 by immobilized cells from anaphthalenesulfonate-degrading mixed culture. Appl. Microbiol.Biotechnol. 46(5–6): 597–603. doi:10.1007/s002530050867.

Kudlich, M., Keck, A., Klein, J., and Stolz, A. 1997. Localizationof the enzyme system involved in anaerobic reduction of azodyes by Sphingomonas sp. Strain BN6 and effect of artificial re-dox mediators on the rate of azo dye reduction. Appl. Environ.Microbiol. 63(9): 3691–3694. PMID:16535698.

Kudlich, M., Hetheridge, M.J., Knackmuss, H.J., and Stolz, A.1999. Autoxidation reactions of different aromatic o-aminohy-droxynaphthalenes that are formed during the anaerobic reduc-tion of sulfonated azo dyes. Environ. Sci. Technol. 33(6): 896–901. doi:10.1021/es9808346.

Dafale et al. 33


Env

iron

. Rev

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

YO

RK

UN

IV o

n 06

/10/

14Fo

r pe

rson

al u

se o

nly.

Lettinga, G. 1995. Anaerobic digestion and wastewater treatmentsystems. Antonie Leeuwenhoek, 67(1): 3–28. doi:10.1007/BF00872193. PMID:7741528.

Libra, J.A., Borchert, M., Vigelahn, L., and Storm, T. 2004. Twostage biological treatment of a diazo reactive textile dye and thefate of the dye metabolites. Chemosphere, 56(2): 167–180.doi:10.1016/j.chemosphere.2004.02.012. PMID:15120563.

Lourenco, N.D., Novais, J.M., and Pinheiro, H.M. 2000. Reactivetextile dye colour removal in a sequencing batch reactor. WaterSci. Technol. 42(5–6): 321–328.

Lourenco, N.D., Novais, J.M., and Pinheiro, H.M. 2003. Analysisof secondary metabolite fate during anaerobic-aerobic azo dyebiodegradation in a sequential batch reactor. Environ. Technol.24(6): 679–686. doi:10.1080/09593330309385603. PMID:12868522.

Lovley, D.R., Fraga, J.L., BluntHarris, E.L., Hayes, L.A., Phillips,P., and Coates, J.D. 1998. Humic substances as a mediator formicrobially catalyzed metal reduction. Acta Hydrochim. Hydro-biol. 26(3): 152–157. doi:10.1002/(SICI)1521-401X(199805)26:3<152::AID-AHEH152>3.0.CO;2-D.

McCurdy, M.W., Boardman, G.D., Michelsen, D.L., and Woodby,R.M. 1991. Chemical reduction and oxidation combined withbiodegradation for the treatment of a textile dye wastewater.46th Purdue Industrial Waste Conference, May 1991.

Mohanty, S., Dafale, N., and Rao, N.N. 2006. Microbial decolori-zation of reactive black-5 in a two-stage anaerobic-aerobic reac-tor using acclimatized activated textile sludge. Biodegradation,17(5): 403–413. doi:10.1007/s10532-005-9011-0. PMID:16477361.

Moir, D., Masson, S., and Chu, I. 2001. Structure-activating rela-tionship study on the Clostridium paraputrifucum. Environ. Tox-icol. Chem. 20: 479–484. doi:10.1897/1551-5028(2001)020<0479:SARSOT>2.0.CO;2. PMID:11349846.

Moran, C., Hall, M.E., and Howell, R. 2008. Effect of sewagetreatment on textile effluent. J. Soc. Dyes Col. 113: 272–274.

Moutaouakkil, A., Zeroual, Y., Dzayri, F.Z., Talbi, M., Lee, K.,and Blaghen, M. 2004. Decolorization of azo dyes with Entero-bacter agglomerans immobilized in different supports by usingfluidized bed bioreactor. Curr. Microbiol. 48(2): 124–129.doi:10.1007/s00284-003-4143-0. PMID:15057480.

Nachiyar, C.V., and Rajakumar, G.S. 2004. Mechanism of Navitanfast blue S5R degradation by Pseudomonas aeruginosa. Chemo-sphere, 57(3): 165–169. doi:10.1016/j.chemosphere.2004.05.030.PMID:15312732.

Nakanishi, M., Yatome, C., Ishida, N., and Kitade, Y. 2001. Puta-tive ACP phosphodiesterase gene (acpD) encodes an azoreduc-tase. J. Biol. Chem. 276(49): 46394–46399. doi:10.1074/jbc.M104483200. PMID:11583992.

Nigam, P., Mc Mullan, G., Banat, I.M., and Marchant, R. 1996.Decolorization of effluent from the textile industry by a micro-bial consortium. Biotechnol. Lett. 18(1): 117–120. doi:10.1007/BF00137823.

O’Loughlin, E.J., Burris, D.R., and Delcomyn, C.A. 1999. Reduc-tive dechlorination of trichloroethene mediated by humic-metalcomplexes. Environ. Sci. Technol. 33(7): 1145–1147. doi:10.1021/es9810033.

O’Neill, C., Hawkes, F.R., Hawkes, D.W., Esteves, S., and Wilcox,S.J. 2000a. Anaerobic-aerobic biotreatment of simulated textileeffluent containing varied ratios of starch and azo dye. WaterRes. 34(8): 2355–2361. doi:10.1016/S0043-1354(99)00395-4.

O’Neill, C., Lopez, A., Esteves, S., Hawkes, F.R., Hawkes, D.L.,and Wilcox, S. 2000b. Azo-dye degradation in an anaerobic-aerobic treatment system operating on simulated textile effluent.

Appl. Microbiol. Biotechnol. 53(2): 249–254. doi:10.1007/s002530050016. PMID:10709990.

Ogawa, T., and Yatome, C. 1990. Biodegradation of azo dyes inmultistage rotating biological contactor immobilized by assimi-lating bacteria. Bull. Environ. Contam. Toxicol. 44(4): 561–566.doi:10.1007/BF01700876. PMID:2322681.

Ozturk, A., and Abdullah, M.I. 2006. Toxicological effect of indoleand its azo dye derivatives on some microorganisms under aero-bic conditions. Sci. Total Environ. 358(1-3): 137–142. doi:10.1016/j.scitotenv.2005.08.004. PMID:16165191.

Padmavathy, S., Sandhya, S., Swaminathan, K., Subrahmanyam,Y.V., Chakrabarti, T., and Kaul, S.N. 2003. Aerobic decoloriza-tion of reactive azo dyes in presence of various cosubstrates.Chem. Biochem. Eng. Quarterly. 17: 147–151.

Pandey, A., Singh, P., and Iyengar, L. 2007. Bacterial decoloriza-tion and degradation of azo dyes. Int. J. Biodeterioration Biode-gradation. 59(2): 73–84. doi:10.1016/j.ibiod.2006.08.006.

Panswad, T., and Luangdilok, W. 2000. Decolorization of reactivedyes with different molecular structures under different environ-mental conditions. Water Res. 34(17): 4177–4184. doi:10.1016/S0043-1354(00)00200-1.

Panswad, T., Iamsamer, K., and Anotai, J. 2001. Decolorization ofazo-reactive dye by polyphosphate- and glycogen-accumulatingorganisms in an anaerobic-aerobic sequencing batch reactor.Bioresour. Technol. 76(2): 151–159. doi:10.1016/S0960-8524(00)00073-0. PMID:11131799.

Pearce, C.I., Lloyd, J.R., and Guthrie, J.T. 2003. The removal ofcolor from textile wastewater using whole bacterial cells: a re-view. Dyes Pigments, 58(3): 179–196. doi:10.1016/S0143-7208(03)00064-0.

Pelegrini, P., Peralto-Zamora, A.R., De Andrade, J., and Reyers, N.1999. Electrochemically assisted photocatalytic degradation ofreactive dyes. Appl. Catal B- Environ, 22(2): 83–90. doi:10.1016/S0926-3373(99)00037-5.

Pinheiro, H.M., Touraud, E., and Thomas, O. 2004. Aromaticamines from azo dye reduction: status review with emphasis ondirect UV spectrophotometric detection in textile industry waste-waters. Dyes Pigments, 61(2): 121–139. doi:10.1016/j.dyepig.2003.10.009.

Plumb, J.J., Bell, J., and Stuckey, D.C. 2001. Microbial populationsassociated with treatment of an industrial dye effluent in ananaerobic baffled reactor. Appl. Environ. Microbiol. 67(7):3226–3235. doi:10.1128/AEM.67.7.3226-3235.2001. PMID:11425746.

Powell, W.W., Michelsen, D.L., Boardman, G.D., Dietrich, A.M.,and Woodby, R.M. 1992. Removal of colour and TOC fromsegregated dye discharges using ozone and fentons reagent. Che-mical Oxidation forthe Nineties, 2nd Int. Symposium, February1992.

Purohit, H., Kapley, A., Moharikar, A., and Narde, G. 2003. Ex-traction of activated biological sludge for PCR compatible DNAfrom effluent treatment systems. J. Microbiol. Methods, 52:315–323. doi:10.1016/S0167-7012(02)00185-9. PMID:12531500.

Rafii, F., and Coleman, T. 1999. Cloning and expression in Escher-ichia coli of an azoreductase gene from Clostridium perfringensand comparison with azoreductase genes from other bacteria. J.Basic Microbiol. 39(1): 29–35. doi:10.1002/(SICI)1521-4028(199903)39:1<29::AID-JOBM29>3.0.CO;2-W. PMID:10071864.

Rafii, F., Franklin, W., and Cerniglia, C.E. 1990. Azoreductase ac-tivity of anaerobic bacteria isolated from human intestinal mi-croflora. Appl. Environ. Microbiol. 56(7): 2146–2151. PMID:2202258.



Env

iron

. Rev

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

YO

RK

UN

IV o

n 06

/10/

14Fo

r pe

rson

al u

se o

nly.

Rafii, F., Hall, J.D., and Cerniglia, C.E. 1997. Mutagenicity of azodyes used in foods, drugs and cosmetics before and after reduc-tion by Clostridium species from the human intestinal tract.Food Chem. Toxicol. 35(9): 897–901. doi:10.1016/S0278-6915(97)00060-4. PMID:9409630.

Rajaguru, P., Kalaiselvi, K., Palanivel, M., and Subburam, V. 2000.Biodegradation of azo dyes in a sequential anaerobic-aerobicsystem. Appl. Microbiol. Biotechnol. 54(2): 268–273. doi:10.1007/s002530000322. PMID:10968644.

Rani, A., Porwal, S., Sharma, R., Kapley, A., Purohit, H.J., and Ka-lia, V.C. 2008. Assessment of microbial diversity in effluenttreatment plants by culture dependent and culture independentapproaches. Bioresour. Technol. 99(15): 7098–7107. doi:10.1016/j.biortech.2008.01.003. PMID:18280146.

Rasko, D.A., Ravel, J., Økstad, O.A., Helgason, E., Cer, R.Z.,Jiang, L., Shores, K.A., Fouts, D.E., Tourasse, N.J., Angiuoli,S.V., Kolonay, J., Nelson, W.C., Kolstø, A.B., Fraser, C.M.,and Read, T.D. 2004. The genome sequence of Bacillus cereusATCC 10987 reveals metabolic adaptations and a large plasmidrelated to Bacillus anthracis pXO1. Nucleic Acids Res. 32(3):977–988. doi:10.1093/nar/gkh258. PMID:14960714.

Rau, J., and Stolz, A. 2003. Oxygen-insensitive nitroreductasesNfsA and NfsB of Escherichia coli function under anaerobicconditions as lawsone-dependent Azo reductases. Appl. Environ.Microbiol. 69(6): 3448–3455. doi:10.1128/AEM.69.6.3448-3455.2003. PMID:12788749.

Razo-Flores, E., Donlon, B.A., Lettinga, G., and Field, J.A. 1997.Biotransformation and biodegradation of N-substituted aromaticsin methanogenic granular sludge. FEMS Microbiol. Rev. 20(3-4): 525–538. doi:10.1111/j.1574-6976.1997.tb00335.x. PMID:9340000.

Read, T.D., Peterson, S.N., Tourasse, N., Baillie, L.W., Paulsen,I.T., Nelson, K.E., Tettelin, H., Fouts, D.E., Eisen, J.A., Gill,S.R., Holtzapple, E.K., Okstad, O.A., Helgason, E., Rilstone, J.,Wu, M., Kolonay, J.F., Beanan, M.J., Dodson, R.J., Brinkac,L.M., Gwinn, M., DeBoy, R.T., Madpu, R., Daugherty, S.C.,Durkin, A.S., Haft, D.H., Nelson, W.C., Peterson, J.D., Pop, M.,Khouri, H.M., Radune, D., Benton, J.L., Mahamoud, Y., Jiang,L., Hance, I.R., Weidman, J.F., Berry, K.J., Plaut, R.D., Wolf,A.M., Watkins, K.L., Nierman, W.C., Hazen, A., Cline, R., Red-mond, C., Thwaite, J.E., White, O., Salzberg, S.L., Thomason,B., Friedlander, A.M., Koehler, T.M., Hanna, P.C., Kolstø,A.B., and Fraser, C.M. 2003. The genome sequence of Bacillusanthracis Ames and comparison to closely related bacteria. Nat-ure, 423(6935): 81–86. doi:10.1038/nature01586. PMID:12721629.

Reutergadh, L.B. and Langphasuk, M. 1997. Photocatalytic deco-lourisation of reactive azo dye: A comparison between TiO2

and us photocatalysis. Chemosphere, 35(3): 585–596. doi:10.1016/S0045-6535(97)00122-7.

Robertson, D.E., Chaplin, J.A., DeSantis, G., Podar, M., Madden,M., Chi, E., Richardson, T., Milan, A., Miller, M., Weiner,D.P., Wong, K., McQuaid, J., Farwell, B., Preston, L.A., Tan,X., Snead, M.A., Keller, M., Mathur, E., Kretz, P.L., Burk,M.J., and Short, J.M. 2004. Exploring nitrilase sequence spacefor enantioselective catalysis. Appl. Environ. Microbiol. 70(4):2429–2436. doi:10.1128/AEM.70.4.2429-2436.2004. PMID:15066841.

Sandhya, S., Sarayu, K., Uma, B., and Swaminathan, K. 2008. De-colorizing kinetics of a recombinant Escherichia coli SS125strain harboring azoreductase gene from Bacillus latrosporusRRK1. Bioresour. Technol. 99(7): 2187–2191. doi:10.1016/j.biortech.2007.05.027. PMID:17601730.

Scarpi, C., Ninci, F., Centini, M., and Anselmi, C. 1998. High-per-

formance liquid chromatography determination of direct andtemporary dyes in natural hair colourings. J. Chromatogr. A,796(2): 319–325. doi:10.1016/S0021-9673(97)01015-7. PMID:9540212.

Seshadri, S., Bishop, P.L., and Agha, A.M. 1994. Anaerobic/aero-bic treatment of selected azo dyes in wastewater. Waste Manag.14(2): 127–137. doi:10.1016/0956-053X(94)90005-1.

Slokar, Y.M., and Le Marechal, A.M. 1998. Methods of decolora-tion of textile wastewaters. Dyes Pigments, 37(4): 335–356.doi:10.1016/S0143-7208(97)00075-2.

Sokolowska-Gajda, J., Freeman, H.S., and Reife, A. 1996. Syn-thetic dyes based on environmental considerations. Part 2: Ironcomplexes formazan dyes. Dyes Pigments, 30(1): 1–20. doi:10.1016/0143-7208(95)00048-8.

Sponza, D.T., and Isik, M. 2002. Decolorization and azo dye degra-dation by anaerobic/aerobic sequential processes. Enzyme Mi-crob. Technol. 31(1–2): 102–110. doi:10.1016/S0141-0229(02)00081-9.

Sponza, D.T., and Isik, M. 2005. Reactor performances and fate ofaromatic amines through decolorization of Direct Black 38 dyeunder anaerobic/aerobic sequential. Process Biochem. 40(1):35–44. doi:10.1016/j.procbio.2003.11.030.

Stolz, A. 2001. Basic and applied aspects in the microbial degrada-tion of azo dyes. Appl. Microbiol. Biotechnol. 56(1-2): 69–80.doi:10.1007/s002530100686. PMID:11499949.

Suzuki, Y., Yoda, T., Ruhul, A., and Sugiura, W. 2001. Molecularcloning and characterization of the gene coding for azoreductasefrom Bacillus sp. OY1-2 isolated from soil. J. Biol. Chem.276(12): 9059–9065. doi:10.1074/jbc.M008083200. PMID:11134015.

Tan, G. 2001. Integrated and sequential anaerobic-aerobic biode-gradation of azo dyes. Ph. D. Thesis, Agrotechnology and FoodSciences, Sub-department of Environmental Technology, Wa-geningen University, Wageningen, The Netherlands.

Tan, N.C.G., and Field, J.A. 2000. Biodegradation of sulfonatedaromatic compounds. In Lens, P., Hulshoff-Pol, L. W. (Editors),environmental technologies to treat sulfur pollution. Principlesand Engineering. IWA Publishing, London, 377–392.

Tan, N.C.G., Lettinga, G., and Field, J.A. 1999. Reduction of theazo dye Mordant Orange 1 by methanogenic granular sludge ex-posed to oxygen. Bioresour. Technol. 67(1): 35–42. doi:10.1016/S0960-8524(99)00098-X.

Thurnheer, T., Cook, A.M., and Leisinger, T. 1988. Co-culture ofdefined bacteria to degrade seven sulfonated aromatic com-pounds: efficiency, rates and phenotypic variations. Appl. Mi-crobiol. Biotechnol. 29(6): 605–609. doi:10.1007/BF00260992.

Tringe, S.G., von Mering, C., Kobayashi, A., Salamov, A.A., Chen,K., Chang, H.W., Podar, M., Short, J.M., Mathur, E.J., Detter,J.C., Bork, P., Hugenholtz, P., and Rubin, E.M. 2005. Compara-tive metagenomics of microbial communities. Science,308(5721): 554–557. doi:10.1126/science.1107851. PMID:15845853.

Tunay, O., Kabdasli, I., Ohron, D., and Cansever, G. 1999. Use andminimalization of water in leather tanning processes. Water Sci.Technol. 40(1): 237–244. doi:10.1016/S0273-1223(99)00390-X.

van der Zee, F.P., and Villaverde, S. 2005. Combined anaerobic-aerobic treatment of azo dyes—a short review of bioreactor stu-dies. Water Res. 39(8): 1425–1440. doi:10.1016/j.watres.2005.03.007. PMID:15878014.

Van der Zee, F.P., Lettinga, G., and Field, J.A. 2000a. The role of(auto) catalysis in the mechanism of anaerobic azo reduction.Water Sci. Technol. 42(5–6): 301–308.

Van der Zee, F.P., Lettinga, G., and Field, J.A. 2000b. Azo dye de-

Dafale et al. 35


Env

iron

. Rev

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

YO

RK

UN

IV o

n 06

/10/

14Fo

r pe

rson

al u

se o

nly.

colourisation by anaerobic granular sludge. Chemosphere, 44(5):1169–1176. doi:10.1016/S0045-6535(00)00270-8.

van der Zee, F.P., Bisschops, I.A., Lettinga, G., and Field, J.A.2003. Activated carbon as an electron acceptor and redox med-iator during the anaerobic biotransformation of azo dyes. En-viron. Sci. Technol. 37(2): 402–408. doi:10.1021/es025885o.PMID:12564915.

van Lier, J.B., van der Zee, F.P., Tan, N.C., Rebac, S., and Kleere-bezem, R. 2001. Advances in high-rate anaerobic treatment: sta-ging of reactor systems. Water Sci. Technol. 44(8): 15–25.PMID:11730131.

Vandevivere, P.C., Bianchi, R., and Verstraete, W. 1998. Treatmentand reuse of wastewater from the textile wet-processing indus-try: Review of emerging technologies. J. Chem. Technol. Bio-technol. 72(4): 289–302. doi:10.1002/(SICI)1097-4660(199808)72:4<289::AID-JCTB905>3.0.CO;2-#.

Vijaykumar, M.H., Vaishampayan, A., Shouche, S., and Karegou-dar, B. 2007. Decolorization of naphthalene-containing sulfo-nated azo dyes by Kerstesia sp. Strain VKY1. Enzyme Microb.Technol. 40(2): 204–211. doi:10.1016/j.enzmictec.2006.04.001.

Wagner, R.W., and Lindsey, J.S. 1996. Boron-dipyrromethane dyesfor incorporation in synthetic multi-pigment light-harvesting ar-rays. Pure Appl. Chem. 68(7): 1373–1380. doi:10.1351/pac199668071373.

Walker, R., and Ryan, A.J. 1971. Some molecular parameters influ-encing rate of reduction of azo compounds by intestinal micro-flora. Xenobiotica, 1(4-5): 483–486. doi:10.3109/00498257109041513. PMID:5006111.

Wang, C., Yediler, A., Lienert, D., Wang, Z., and Kettrup, A.2003. Ozonation of an azo dye C.I. Remazol Black 5 and toxi-cological assessment of its oxidation products. Chemosphere,52(7): 1225–1232. doi:10.1016/S0045-6535(03)00331-X.

Weisburger, J.H. 2002. Comments on the history and importance ofaromatic and heterocyclic amines in public health. Mutat. Res.506–507: 9–20.

Wiles, S., Whiteley, A.S., Philp, J.C., and Bailey, M.J. 2003. De-velopment of bespoke bioluminescent reporters with the poten-tial for in situ deployment within a phenolic-remediatingwastewater treatment system. J. Microbiol. Methods, 55(3):

667–677. doi:10.1016/S0167-7012(03)00203-3. PMID:14607409.

Willetts, M., and Ashbolt, J. 2000. Understanding anaerobic deco-lorization of textile dyes wastewater: mechanism and kinetics.Water Sci. Technol. 42: 409–416.

Wong, P.K., and Yuen, P.Y. 1996. Decolorization and biodegrada-tion of methyl red by Klebsiella pneumoniae RS13. Water Res.30(7): 1736–1744. doi:10.1016/0043-1354(96)00067-X.

Wrobel, D., Boguta, A., and Ion, R.M. 2001. Mixtures of syntheticorganic dyes in a photoelectrochemical cell. J. Photochem.Photobiol. Chem. 138(1): 7–22. doi:10.1016/S1010-6030(00)00377-4.

Wuhrmann, K., Mechnsner, K.I., and Kappeler, Th. 1980. Investi-gation on rate determining factors in the microbial reduction ofazo dyes. Appl. Microbiol. Biotechnol. 9(4): 325–338. doi:10.1007/BF00508109.

Xu, Y., Lebrun, R.E., Gallo, P.-J., and Blond, P. 1999. Treatmentof textile dye plant effluent by nanofiltration membrane. Separ.Sci. Technol. 34(13): 2501–2519. doi:10.1081/SS-100100787.

Yang, Y., and Wyatt, D.T. 1998. Decolorization of textile dyestuffsusing UV/H2O2 photochemical oxidation technology. TextileChem. Color. 30(4): 27–35.

Zaoyan, Y., Ke, S., Guangliang, S., Fan, Y., Jinshan, D., and Hua-nian, M. 1992. Anaerobic-aerobic treatment of a dye wastewaterby combination of RBC with activated sludge. Water Sci. Tech-nol. 9: 2093–2096.

Zhao, D., Zhu, C., Sun, S., Yu, H., Zhang, L., Pan, W., Zhang, X.,Yu, H., Gu, J., and Cheng, S. 2007. Toxicity of pharmaceuticalwastewater on male reproductive system of mus musculus. Tox-icol. Ind. Health, 23(1): 47–54. doi:10.1177/0748233707077446.

Zitomer, D.H. 1998. Stoichiometry of combined aerobic andmethanogenic COD transformation. Water Res. 32(3): 669–676.doi:10.1016/S0043-1354(97)00258-3.

Zumstein, E., Moletta, R., and Godon, J.-J. 2000. Examination oftwo years of community dynamics in an anaerobic bioreactorusing fluorescence polymerase chain reaction (PCR) single-strand conformation polymorphism analysis. Environ. Microbiol.2(1): 69–78. doi:10.1046/j.1462-2920.2000.00072.x. PMID:11243264.



Env

iron

. Rev

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

YO

RK

UN

IV o

n 06

/10/

14Fo

r pe

rson

al u

se o

nly.

bioremediation of wastewater containing azo dyes through sequential anaerobic–aerobic bioreactor...

Documents