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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 removal
of 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, and
mutagenic 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. Various
mechanisms, 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 aromatic
amines 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 for
their 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. Introduction
Azo 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 Cerniglia
1992). 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

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 anaerobic
conditions, whereas bacterial biodegradation of aromatic amines is an almost exclusively aerobic
process (Fig. 1).

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

treatment
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 aerobic
conditions 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 in
several 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 Azo
dyes. 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 of
reducing equivalents resulting in the formation of aromatic amines. As aromatic amines are

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 from
wastewater 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 microbial
consortia 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 greatly
enhanced 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 anoxic
decolorization 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 two
aromatic 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 been
conducted 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 treatment
used today (Edwards, 2000; Yang et al., 1998). Because aerobic microbes cannot reduce Azo

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 isolated
during 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. This
organism 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). These
organisms, 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) as
the 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 the
component amines (1-amino 2-naphthol) formed during AO7 decolorization. 4aminobenzesulfonate (4-ABS) degradation, however, required the additional presence of an
unidentified strain, SAD4i (Coughlin et al., 2003). Sphingomonas ICX could also decolorize
several 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 source
were 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 these
strains 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 aerobic
conditions, 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.

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)

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 the
reduction of Azo dyes) or non-specialized enzymes (non-specific enzymes that catalyze the
reduction 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 simple
Azo 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 were
isolated 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 from
aerobically 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 indirectly
reduced 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 flavin
reductases 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-generating
system (Semd 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 reductants
like 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 as
sulphide, hydrosulphite and dithionite. Sulphate also results from neutralization of alkaline dye

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 thermodynamic
stability. 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 aromatic
amines, 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 4aminophenols, 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 et
al., 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 of
these 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 of
aromatic 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 large
decreases of toxicity (mostly suppression of bacterial luminescence or inhibition of respiration)

between the effluent of the anaerobic stage and the effluent of the anaerobic stage (ONeill et al.,
2000b; Frijters et al., 2004) or between the effluent of a completely anaerobic reactor and the
effluent of a combined anaerobicaerobic reactor (Gottlieb et al., 2003) provide indirect evidence
for 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 from
the reactive Azo dyes Reactive Black 5 and Reactive Violet 5 were often reported not to be
removed aerobically (Lourenc-o et al., 2000; Lourenc-o et al., 2001; Shaw et al., 2002; Lourenco 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 of
the 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 bacterial
culture (Tan et al., 2000). Also the observation that introduction of biomass from a textile wastetreating 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 the
studies 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 the
color was much more often observed (An et al., 1996; Kuai et al., 1998; ONeill 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 yield
aromatic 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 industrial
applications, 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. The
available literature seems to indicate that anaerobicaerobic 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 dyecontaining wastewaters so that regulatory standards can be met

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