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Biodegradation of Azo Dyes a Review

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Biodegradation of Azo Dyes a Review

Waleed Mohammed Sheet Alabdraba Mohammed Burhan Ali Albayati

Assistant Prof. Assistant Lecturer

Environmental Engineering Department, College of Engineering, Tikrit University

Abstract

One of the main problems associated with the treatment of textile wastewater is the removalof dyes. Most (60-70%) of the more than 10,000 dyes applied in textile-processing industries are

Azo compounds, i.e. molecules with one or more Azo (N=N) bridges linking substituted

aromatic structures. Discharge of Azo dyes is undesirable, not only for aesthetic reasons, but also

 because many Azo dyes and their breakdown products are toxic toward aquatic life, andmutagenic for humans.  Reductive cleavage of Azo bond, leading to the formation of aromatic

amines, is the initial reaction during the biological metabolism of Azo dyes. Anaerobic/anoxic

Azo dye decolorization by several mixed and pure bacterial cultures have been reported. Under

these conditions, this reaction is nonspecific with respect to organisms as well as dyes. Variousmechanisms, which include direct enzymatic and indirect (mediated) and chemical Azo dye

reduction. Only few aerobic bacterial strains that can utilize Azo dyes as growth substrates have

 been isolated. These organisms generally have a narrow substrate range. Degradation of aromaticamines depends on their chemical structure and the conditions. It is now known that simple

aromatic amines can be mineralized under methanogenic conditions. Sulfonated aromatic

amines, on the other hand, are resistant and require specialized aerobic microbial consortia fortheir mineralization. This review is focused on the biodegradation of Azo dyes and

mineralization of aromatic amines

Key Word: Azo dyes, Biodegradation, Aromatic amines, Anaerobic/aerobic treatment 

1. IntroductionAzo dyes are one of the oldest man-made chemicals and they are still widely used in textile  

 printing and the food industries. The annual production worldwide is approximately 700,000 tons(Zollinger 1987). Of this amount, approximately 10 -15% of the dyes are released into the

environment during manufacturing and usage (Vaidya and Datye 1982). Some dyes and some of

their N-substituted aromatic biotransformation products are toxic and/or carcinogenic and,

therefore, the dyes are considered as important environmental pollutants (Chung and Cerniglia1992). During conventional aerobic wastewater treatment, Azo dyes are not degraded (Pagga and

Brown 1986;  Shaul et al. 1991). However, Azo linkages are easily reduced under anaerobic

conditions (Brown and Laboureur 1983b), with digester sludge (Carliell et al. 1995; Carliell et al.

1994), anaerobic granular sludge (Donlon et al. 1997; RAzo-Flores et al. 1997) or sediments(Weber 1991). The Azo dyes act as electron acceptors for reduced flavin nucleotides and redox

mediators (Gingell and Walker 1971; Keck et al. 1997; Kudlich et al. 1997) enhance reduction.

The reduction of Azo dyes results in the formation of aromatic amines. Most aromatic amines,which accumulate after Azo cleavage, are not mineralized anaerobically (Brown and Hamburger  

1987), with the exception of a few aromatic amines substituted with hydroxyl and carboxyl

groups which were fully degraded under methanogen conditions (RAzo-Flores et al. 1996).However, the aromatic amines are readily degraded aerobically (Brown and Laboureur 1983a;

Konopka 1993; Loidl et al. 1990). Generally, bacterial Azo dye biodegradation proceeds in two

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stages. The first stage involves reductive cleavage of the dyes’ Azo linkages, resulting in the

formation of generally colorless but potentially hazardous aromatic amines. The second stage

involves degradation of the aromatic amines. Azo dye reduction usually requires anaerobicconditions, whereas bacterial biodegradation of aromatic amines is an almost exclusively aerobic

 process (Fig. 1).

A combination of anaerobic and aerobic conditions is therefore proposed as a feasible

 biological treatment strategy for Azo compounds (Field et al. 1995). Anaerobic and aerobicconditions can be applied in a sequential anaerobic/aerobic bioreactor system but also in a single

reactor in which anaerobic and aerobic micro niches occur side by side. Sequential

anaerobic/aerobic treatment for Azo dyes and textile industry wastewater has been evaluated inseveral studies (An et al. 1996; Brown and Hamburger 1987;  FitzGerald and Bishop 1995; 

Seshadri et al. 1994;  Zaoyan et al. 1992). Also a simultaneous anaerobic/aerobic treatment

methodology for an Azo dye was recently described. (Esenceli and Tiyek 2014).

2. Decolorization of Azo dye by bacteria

Reductive cleavage of the  –  N=N- bond is the initial step of the bacterial degradation of Azodyes. Decolorization of Azo dyes occurs under anaerobic (methanogenic), anoxic and aerobic

condition by different groups of bacteria. Decolourization Azo dyes under these different is

 briefly discussed in subsequent sections.

2.1. Azo dye decolorization under anaerobic conditions

Anaerobic Azo dye reduction is the reductive cleavage of Azo linkages, i.e. the transfer ofreducing equivalents resulting in the formation of aromatic amines. As aromatic amines are

Fig. 1 General overview of the fate of Azo dyes and aromatic amines during anaerobic/aerobic 

treatment

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generally colorless, Azo dye reduction is also referred to as Azo dye decolorization. The first

study on Azo dye reduction was published as early as 1937, when the decolorization of food Azo

dyes by lactic acid bacteria isolated from the human gut was reported (Brohm and Frohwein,1937). Hence, as the formation of toxic aromatic amines in humans is a matter of concern,

research on bacterial Azo dye reduction has traditionally mostly been focused on the activity of

(facultative) anaerobic bacteria from mammalian intestines (Brown, 1981; Chung et al., 1978; ;Chung et al., 1992; Rafii et al.1990; Walker, 1970). Later, when the removal of dyes fromwastewater became a topic, also bacteria from other origins were used to investigate anaerobic

Azo dye reduction, e.g. pure cultures (Wuhrmann et al., 1980), mixed cultures (Haug et al., 

1991), anaerobic sediments (Weber and Wolfe, 1987), digester sludge (Brown and Laboureur, 1983), (Carliell et al., 1994), anaerobic granular sludge (RAzo Flores, 1997) and activated sludge

under anaerobic conditions (Bromly-Challenor, 2000  ; Bell and Buckley, 2003; Albayati, 2010

and Aldoury et al., 2014 ).

2.2 Azo dye decolorization under anoxic conditions

Anoxic decolorization of various Azo dyes by mixed aerobic and facultative microbialconsortia has been reported ( Nigam et al., 1996; Kapdan et al., 2000; Padmavathy et al., 2003;

Khehra et al., 2005; Moosvi et al., 2005). Although many of these cultures were able to grow

aerobically, decolorization was achieved only under anaerobic conditions. Pure bacterial strains,

such as Pseudomonas sp. and Proteus mirabilis, decolorized Azo dyes under anoxic conditions(Chang et al., 2001; Chen et al., 1999; Yu et al., 2001). Azo dye decolorization by mixed, as well

as pure, cultures generally required complex organic sources, such as yeast extract, peptone, or

a combination of complex organic source and carbohydrate(Chen et al., 2003;  Khehra et al., 2005). Glucose is the preferred substrate in anaerobic dye decolorization under methanogenic

conditions, but its suitability for anoxic dye decolorization by facultative anaerobic and

fermenting bacteria seems to vary, depending on the bacterial culture.

Decolorization of Mordant Yellow 3 by Sphingomonas xenophaga strain BN6 was greatlyenhanced by glucose, where as a significant decrease in Azo decolorization in its presence was

reported for P leuteola, Aeromonas sp. and few other mixed cultures (Haug et al., 1991; Kapdan 

et al., 2000;  Chang et al., 2001; Chen et al., 2003). The negative effect of glucose on anoxicdecolorization has been attributed either to a decrease in pH due to acid formation, or to

catabolic repression (Chen et al., 2003). HPLC and mass spectrometery data from culture

filtrates after the decolorization of reactive red 22 by P leuteola, showed the presence of twoaromatic amines, as well as a partially redused product (Chang et al., 2001). This is in

accordance with the two-step reduction mechanism of the Azo bond proposed by Gingell and

Walker (1971).

2.3. Azo dye decolorization under aerobic conditions

Conventional activated sludge treatment of wastes is often an effective and highly economic

system for reducing organic pollutants in wastewater. A fair amount of research has beenconducted assessing the viability of using activated sludge to treat textile effluents (Zissi et al., 

1997; Loyd, 1992; Shaul et al., 1991; Pagga and Brown, 1986). However, aerobic treatment of

Azo dye wastes has proven ineffective in most cases, but is often the typical method of treatmentused today (Edwards, 2000; Yang et al., 1998). Because aerobic microbes cannot reduce Azo

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linkages, their ability to destroy dye chromogens is less than anaerobic bacterium. However,

aerobic sludges have been successfully used to stabilize dye metabolites (Brown and Laboureur, 

1983). Several bacterial strains that can aerobically decolorize Azo dyes have been isolatedduring the past few years. Many of these strains require organic carbon sources, as they cannot

utilize dye as the growth substrate (Stolz, 2001). P. aeruginosa decolorized a commercial tannery

and textile dye, Navitan Fast blue S5R, in the presence of glucose under aerobic conditions. Thisorganism was also able to decolorize various other Azo dyes (Nachiyar and Rajkumar, 2003). There are only very few bacteria that are able to grow on Azo compounds as the sole carbon

source. These bacteria cleave  –  N=N –   bonds reductively and utilize amines as the source of

carbon and energy for their growth.Such organisms are specific towards their substrate. Examples of bacterial strains with this

trait are Xenophilus Azovorans KF 46 (previously Pseudomonas sp. KF46) and Pigmentiphaga

kullae K24 (previously Pseudomonas sp. K24), which can grow aerobically on carboxy-orange I

and carboxy-orange II, respectively (Zimmermann et al., 1982; Kulla et al., 1983). Theseorganisms, however, could not grow on structurally analogous sulfonated dyes, acid orange 20

(Orange I) and AO7. Long adaptation of 4-aminobenzenesulfonate (4-ABS) degrading

Hydrogenophaga intermedia strain S1 for growth on 4-carboxy-40-sulfoAzobenzene (CSB) asthe sole organic carbon source led to the isolation of strain S5, which reduced CSB and utilized

the two amine metabolites (Blumel et al., 1998). Coughlin et al. (1999)  have reported the

isolation of a Sphingomonas sp, strain 1CX, an obligate aerobe, which can grow on an Azo dye,

AO7, as sole carbon, energy and nitrogen source. This strain degraded only one of thecomponent amines (1-amino 2-naphthol) formed during AO7 decolorization. 4-

aminobenzesulfonate (4-ABS) degradation, however, required the additional presence of an

unidentified strain, SAD4i (Coughlin et al., 2003). Sphingomonas ICX could also decolorizeseveral Azo dyes consisting of either 1-amino-2-naphthol or 2-amino-1- naphthol coupled via the

Azo bond to a phenyl or naphthyl moiety (Coughlin et al., 1999). Similar Azo dyes, such as AO6

or AO20, which lack these structures, were not decolorized.

Three bacterial strains that could utilize Azo dye (AO 7 or acid red 88) as sole carbon sourcewere isolated from soil and sewage samples and were identified as Bacillus sp. OY1-2,

Xanthomonas sp. NR25-2 and Pseudomonas sp. PR41-1. (Sugiura et al., 1999). Recently, four

 bacterial species have been isolated using methyl red as the sole carbon source. Two of thesestrains have been identified as Vibrio logei and P. nitroreducens. Amine products were not

detected in the culture medium, indicating their degradation (Adedayo et al., 2004). The

structures of few Azo dyes that are mineralized under aerobic conditions are presented in Fig. 2.

2.4. Mechanism of Azo dye reduction

The first step in the bacterial degradation of Azo dyes, in either anaerobic or aerobicconditions, is the reduction of the  –  N=N –   bond. This reduction may involve different

mechanisms, such as enzymes, low molecular weight redox mediators, chemical reduction by

 biogenic reductants like sulfide, or a combination of these (Fig. 3). Additionally, the location of

the reactions can be either intracellular or extracellular.

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Fig. 2 Structures of a few Azo dyes degraded under aerobic conditions.

Fig. 3 Schematic representation of the different mechanisms of anaerobic Azo dye reduction

RM = redox mediator; ED = electron donor; b = bacteria (enzymes)

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2.4.1. Direct enzymatic Azo dye reduction

According to the first mechanism of biological Azo dye reduction, enzymes transfer the

reducing equivalents originating from the oxidation of organic substrates to the Azo dyes.

Enzymes that catalyze Azo dye reduction may either be specialized enzymes (catalysing only thereduction of Azo dyes) or non-specialized enzymes (non-specific enzymes that catalyze thereduction of a wide range of compounds, including Azo dyes). Evidence for the existence of

specialized Azo dye reducing enzymes, so-called .Azoreductases., has so far only been found in

studies with some aerobic and facultative aerobic bacteria that could grow with mostly simpleAzo compounds as sole source of carbon and energy. These strains grew under strict aerobic

conditions by using a metabolism that started with reductive cleavage of the Azo linkage (Ince 

and Gonenc, 1997). The existence of enzymes catalysing Azo dye reduction in aerobic bacteria

was for the first time proven when two Azoreductases from obligate aerobic bacteria wereisolated and characterised (Zimmermann et al., 1984: Zimmermann et al., 1982). These

intracellular Azoreductases showed high specificity to dye structures. Aside from these specific

Azoreductases, also non-specific enzymes catalysing Azo dye reduction have been isolated fromaerobically grown cultures of Shigella dysenteriae (Ghosh et al., 1992) Escherichia coli (Ghosh 

et al., 1993) and Bacillus sp. (Suzuki et al., 2001) . Where characterized enzymes were found to

 be flavoproteins (Ghosh et al., 1993; Ghosh et al., 1992;  Rafii and Cerniglia, 1995) 

2.4.2. Indirect (mediated) biological Azo dye reduction.

According to the second mechanism of biological Azo dye reduction, Azo dyes are indirectlyreduced by enzymatically reduced electron carriers. Early research has hypothesised that reduced

flavins (FADH2, FMNH2, riboflavin) generated by flavindependent reductases can reduce Azo

dyes in a non-specific chemical reaction (Gingell and Walker, 1971; Roxon, 1967 ). Flavins were

indeed often found to stimulate Azo dye reduction and recent research has revealed that flavinreductases are indeed .anaerobic Azoreductases (Russ et al., 2000). Also other reduced enzyme

cofactors capable of direct Azo dye reduction have been reported, e.g. NADH ( Nam and 

Renganathan, 2000), NADH and NADPH (Hernandez et al., 1967) and an NADPH-generatingsystem (Semdé et al., 1998). Aside from enzyme cofactors, also various artificial redox

mediating compounds are important stimulants of biological Azo dye reduction (methyl

viologen, benzyl viologen, Riboflavin, FAD, FMN, Phenosaphranin, Menadione, Neutral Red,2-hydroxy-1,4-naphthoquinone) (Chung et al., 1978).

2.4.3 Chemical Azo dye reduction

Textile manufacturers are aware that addition of reducing agents to dye containing effluents

leads to considerable decolorization (Glover, 1993). Hence, Azo dye can be reduced by chemical

reductants like dithionite (Davis et al., 1993;  Zbaida and Levine, 1992) and zerovalent iron

( Nam and Renganathan, 2000). Moreover, chemical Azo dye reduction by biogenic reductantslike sulphide, is possible as well (Yoo et al., 2000). Dye-containing wastewaters usually contain

moderate to high sulphate concentrations. Sulphate is often an additive of dye baths or it is

formed by the oxidation of more reduced sulphur species used in dyeing processes, such assulphide, hydrosulphite and dithionite. Sulphate also results from neutralization of alkaline dye

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effluents with sulphuric acid. Sulphide is therefore a relevant compound, as it will be generated

 by sulphate reducing bacteria during treatment of these wastewaters in anaerobic bioreactors.

3. Degradation of aromatic amines

Aromatic compounds possess a large negative resonance energy, resulting in thermodynamicstability. Microorganisms, particularly bacteria, have evolved enzyme systems that degrade the benzene structure under aerobic and anoxic conditions (Gibson and Subramanian, 1984; Schink  

et al., 2000). Common to both oxygen-dependent and anoxic metabolism of aromatic compounds

is a separation into peripheral and central pathways (Heider and Fuchs, 1997). Peripheral pathways convert the large variety of compounds into a few central intermediates. In aerobic

metabolism, the initial reactions involve the replacement of other functional groups of the

aromatic ring with hydroxyl groups, followed by cleavage by incorporating two oxygen atoms.

These reactions are catalysed by hydroxylases and oxygenases. Under anoxic conditions,dearomatization is achieved by ring reduction and also includes other unique reactions such as

carboxylation, reductive dehydroxylation and addition reactions, which are absent in the aerobic

metabolism (Heider and Fuchs, 1997).

3.1. Degradation of aromatic amines under anaerobic conditions

Decolorization of Azo dyes in anaerobic environments leads to the formation of aromaticamines, many of which were assumed to resist further degradation under these conditions (Stolz,

2001). Nevertheless, mineralization of few simple aromatic amines has been reported under

methanogenic conditions. They include the three isomers of aminobenzoate, 2- and 4-aminophenols, 2, 4 dihydroxyaniline and 5-aminosalicylic acid (5-ASA). (Connor and Young, 

1993;  RAzo-Flores et al., 1997b; Yemashova et al., 2004). Complete degradation of Azo

disalicylate and partial mineralization of the Azo dyes, Mordent Orange 1, AO6, AO7 and

AO52, under methanogenic conditions, has been reported (Donlon et al., 1997; RAzo-Flores etal., 1997b; Yemashova et al., 2004). Many reports have shown that sulfonated aromatic amines

(SAA) are nonbiodegradable under methanogenic conditions (Tan et al., 2005).

3.2. Aerobic fate of aromatic amines

The aerobic biodegradation of many aromatic amines has been extensively studied. Many ofthese compounds were found to be degraded under aerobic conditions (Baird et al. 1977; Brown

and Laboureur 1983a, Albayati, 2010 and Aldoury et al., 2014,), e.g. compounds like aniline

(Anson and MacKinnon 1984;  Konopka 1993), carboxylated aromatic amines ( Stolz et al. 

1992), chlorinated aromatic amines (Hwang et al. 1987; Loidl et al. 1990), and (substituted) benzidines (Baird et al. 1977). Several of the studies reveal evidence for partial or complete

removal of many aromatic amines in the aerobic stage. The decrease or disappearance of the,

sometimes unidentified, peaks in HPLC chromatograms (Harmer and Bishop, 1992; FitzGerald 

and Bishop, 1995; Tan et al., 2000; Isik and Sponza, 2004a), the decrease or disappearance ofaromatic amines as detected with a diAzotization-based method ( Isik and Sponza, 2004c; Isik  

and Sponza, 2004a; Sponza and Isik, 2005), as well as the decrease of UV absorbance (Cruz and

Buitro´ n, 2001; Shaw et al., 2002) all indicate removal of aromatic amines. Moreover, the largedecreases of toxicity (mostly suppression of bacterial luminescence or inhibition of respiration)

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 between the effluent of the anaerobic stage and the effluent of the anaerobic stage (O’Neill et al.,

2000b; Frijters et al., 2004) or between the effluent of a completely anaerobic reactor and the

effluent of a combined anaerobic – aerobic reactor (Gottlieb et al., 2003) provide indirect evidencefor the removal of aromatic amines. The results taken as a whole suggest that many of the

aromatic amines from anaerobic cleavage of Azo dyes were removed in the consequent aerobic

stage. However, some aromatic amines may not be removed. Especially cleavage products fromthe reactive Azo dyes Reactive Black 5 and Reactive Violet 5 were often reported not to beremoved aerobically (Lourenc-o et al., 2000; Lourenc-o et al., 2001; Shaw et al., 2002; Lourenc-

o et al., 2003). Also a relatively large fraction (50%) of the aromatic amines from the benzidine

 based dye Direct Black 38 resisted removal in the aerobic stage (Sponza and Isik, 2005). Most ofthe studies reporting aromatic amine removal do not reveal the underlying mechanism.

As aerobic biodegradation of aromatic amines requires specific microorganisms, the type of

 biomass may play a role. At least in one laboratory reactor study, the degradation of an aromatic

amine, sulfanilic acid, could only be achieved after bioaugmentation with a proper bacterialculture (Tan et al., 2000). Also the observation that introduction of biomass from a textile waste-

treating water works decreased the toxicity of the effluent from an Azo dye treating baffled

reactor (Gottlieb et al., 2003) suggests the involvement of specific bacteria. In many of thestudies reporting aromatic amine removal, it is not clear whether the removal is due to

 biodegradation, adsorption or chemical reactions. Remarkably, although autoxidation of aromatic

amines during aerobic treatment, as suggested by an increase of color, has been observed in

some studies (Kalyuzhnyi and Sklyar, 2000; Cruz and Buitro´ n, 2001), a slight decrease of thecolor was much more often observed (An et al., 1996 ; Kuai et al., 1998; O’Neill et al., 2000b; 

Shaw et al., 2002; Kapdan et al., 2003; Isik and Sponza, 2004c; Sponza and Isik, 2004; Kapdan 

and Alparslan, 2005; Ong et al., 2005). Since many of the Azo dyes treated in these studies yieldaromatic amines that are expected to autoxidize, the latter observation suggests, in several cases,

removal of these compounds or their autoxidation products from the water phase.

4. Conclusion

Among the synthetic dyes, which are widely used for textile dyeing and other industrialapplications, those containing an Azo chromosphere constitute the largest class. Since dyes are

design to be resistant to microbial and physico-chemical attack, conventional processes of

wastewater treatment, including biological treatment, do not easily destroy most of them. Theavailable literature seems to indicate that anaerobic – aerobic biological methods may be

appropriate for the treatment of dye-containing wastewaters. However, there is a still a need to

assess the extent of mineralization of aromatic amines, as many amines can undergo

autoxidation, leading to the formation of soluble recalcitrant polymers, which may be toxic.Degradation of many amines, including SAA, requires the presence of specialized cultures. SAA

degraders have a very narrow substrate range. Hence, there is a requirement for developing

microbial consortia that harbor genes for the rapid degradation of mixtures of aromatic amines.

Such cultures may have to be used for the bioaugmentation of aerobic treatment units. Molecular biology techniques may also be used to improve the strains so that rapid mineralization of

aromatic amines can be achieved. Their use, however, requires caution. It may also be necessary

to combine AOP with biological processes to achieve the required degree of treatment of dye-containing wastewaters so that regulatory standards can be met

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