ARTICLE IN PRESS

International Biodeterioration & Biodegradation 59 (2007) 73–84

www.elsevier.com/locate/ibiod

Review

Bacterial decolorization and degradation of azo dyes

Anjali Pandey, Poonam Singh, Leela Iyengar
Department of Chemistry, Biotechnology Laboratory, I.I.T., Kanpur 208016, India
Received 11 May 2006; received in revised form 25 August 2006; accepted 26 August 2006
Available online 27 October 2006

Abstract

Azo compounds constitute the largest and the most diverse group of synthetic dyes and are widely used in a number of industries such
as textile, food, cosmetics and paper printing. They are generally recalcitrant to biodegradation due to their xenobiotic nature. However
microorganisms, being highly versatile, have developed enzyme systems for the decolorization and mineralization of azo dyes under
certain environmental conditions. Several genera of Basidomycetes have been shown to mineralize azo dyes. Reductive cleavage of azo
bond, leading to the formation of aromatic amines, is the initial reaction during the bacterial 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 non-
specific with respect to organisms as well as dyes. Various mechanisms, which include enzymatic as well as low molecular weight redox
mediators, have been proposed for this non-specific reductive cleavage. 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 bacterial decolorization of azo dyes and mineralization of aromatic
amines, as well as the application of these processes for the treatment of azo-dye-containing wastewaters.
r 2006 Elsevier Ltd. All rights reserved.

Keywords: Azo dyes; Decolorization; Biodegradation; Aromatic amines; Anaerobic/aerobic treatment

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
2. Decolorization of azo dyes by bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
2.1. Azo dye decolorization under anaerobic conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
2.2. Azo dye decolorization under anoxic conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
2.3. Azo dye decolorization under aerobic conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
2.4. Mechanism of azo dye reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
2.4.1. Direct enzymatic azo dye reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
2.4.2. Mediated biological azo dye reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
2.4.3. Azo dye decolorization by biogenic inorganic compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
3. Degradation of aromatic amines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
3.1. Degradation of aromatic amines under anaerobic conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
3.2. Aerobic biodegradation of aromatic amines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
3.3. Anaerobic–aerobic biological treatment for azo dye decolorization and degradation . . . . . . . . . . . . . . . . . . . . . . . . . 80
4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

Corresponding author. Tel.: +91 512 2597160; fax: +91 512 2597437.
E-mail address: anjali@iitk.ac.in (A. Pandey).

0964-8305/$ - see front matter r 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ibiod.2006.08.006
 ARTICLE IN PRESS
74 A. Pandey et al. / International Biodeterioration & Biodegradation 59 (2007) 73–84
1. Introduction
Azo dyes, which are aromatic compounds with one or
more –NQN– groups, constitute the largest class of
synthetic dyes used in commercial applications (Zollinger,
1991). In 1994 estimates, the world production of dyes was
around 1 million tons, of which more than 50% were azo
dyes (Ollgaard et al., 1999; Stolz, 2001). These dyes are
widely used in a number of industries, such as textile
dyeing, food, cosmetics, paper printing, with the textile
industry as the largest consumer. All dyes do not bind to
the fabric; depending on the class of the dye, its loss in
wastewaters could vary from 2% for basic dyes to as high
as 50% for reactive dyes, leading to severe contamination
of surface and ground waters in the vicinity of dyeing
industries (Ganesh et al., 1994; O’Neill et al., 1999). Many
dyes are visible in water at concentrations as low as
1 mgl1. Textile processing wastewaters with dye contents
in the range of 10–200 mgl1 are highly colored. Some of
the dyes and their degradation products are carcinogenic in
nature (Levine, 1991). A review of the mutagenicity of
effluents showed that textile and other dye-related indus-
tries produce consistently more potent wastewaters when
compared to other industrial discharges (Houk, 1992).
Recent studies by Rajaguru et al. (2002) and Umbuzeiro
et al. (2005) have shown that azo dyes contribute to
mutagenic activity of ground and surface waters polluted
by textile effluents. Further, their discharge into surface
water leads to aesthetic problems and obstructs light
penetration and oxygen transfer into water bodies, hence
affecting aquatic life. Thus, the removal of color from
textile effluents has been a major concern.
There are many reports on the use of physicochemical
methods for color removal from dyes containing effluents
(Churchley, 1994; Vandevivere et al., 1998; Swaminathan
et al., 2003; Behnajady et al., 2004; Wang et al., 2004;
Golab et al., 2005; Lopez-Grimau and Gutierrez, 2005).
Extensively used coagulation/ flocculation techniques
produce large amounts of sludge, which requires safe
disposal. Adsorption and, to a certain extent, membrane
filtration techniques lead to secondary waste streams which
need further treatment. These constraints have led to the
consideration of advanced oxidation processes (AOP) and
biological methods as attractive options for the treatment
of dye-containing wastewaters. AOP are defined as those
processes that use strong oxidizing agents (H2O2, Fenton’s
reagent) or heterogenous photocatalysts such as TiO2,
ZnO2, Mn and Fe in the presence or absence of an
irradiation source. These involve mainly the generation of
(OH) radical for the destruction of refractory and
hazardous pollutants (Vandevivere et al., 1998; Alaton
et al., 2002; Al-Kdasi et al., 2004). These methods do not
produce solid waste. However, both AOP and membrane
filtration methods are energy and cost intensive. Biological
methods are generally considered environmentally friendly,
as they can lead to complete mineralization of organic
pollutants at low cost. Azo compounds are xenobiotic in
nature; only one natural azo compound (4–40 dihydroxy
azo benzene) has been reported so far (Gill and Strauch,
1984). Thus they can be expected to be recalcitrant to
biodegradation. It is generally observed that dyes resist
biodegradation in conventional activated sludge treatment
units (Stolz, 2001). It is now known that several micro-
organisms, including fungi, bacteria, yeasts and algae, can
decolorize and even completely mineralize many azo dyes
under certain environmental conditions. Many reviews are
available on the physicochemical and microbiological
methods for decolorization of azo dyes (Banat et al.,
1996; Delee et al., 1998; Vandevivere et al., 1998; O’Neill
et al., 1999; McMullan et al., 2001; Stolz, 2001; Rai et al.,
2005;Van der Zee and Villaverde, 2005). This review
focuses on the pathways and mechanisms by which aerobic
and anaerobic bacteria decolorize azo dyes and degrade
the aromatic amines generated by this reaction. We
also briefly discuss the setting up of potential dual aerobic
and anaerobic processes to remediate azo-dye-containing
wastewaters.
2. Decolorization of azo dyes by bacteria
Reductive cleavage of the –NQN– bond is the initial
step of the bacterial degradation of azo dyes. Decoloriza-
tion of azo dyes occurs under anaerobic (methanogenic),
anoxic and aerobic conditions by different trophic groups
of bacteria. Decolorization of azo dyes under these
different conditions is briefly discussed in subsequent
sections.
2.1. Azo dye decolorization under anaerobic conditions
Methanogenesis from complex organic compounds
requires the coordinated participation of many different
trophic groups of bacteria, including acidogenic, aceto-
genic and methanogenic bacteria (Kasper and Wuhrmann,
1978). Dye decolorization under these conditions requires
an organic carbon/energy source. Simple substrates like
glucose, starch, acetate, ethanol and more complex ones,
such as whey and tapioca, have been used for dye
decolorization under methanogenic conditions (Chinwet-
kitvanich et al., 2000; Willetts et al., 2000; Talarposhti
et al., 2001; Yoo et al., 2001; Isik and Sponza, 2005a; Van
der Zee and Villaverde, 2005).
Extensive studies have been carried out to determine the
role of the diverse groups of bacteria in the decolorization
of azo dyes. Carliell et al. (1996) and Razo-Flores et al.
(1997a) have associated the decolorization with methano-
gens, whereas studies by other investigators showed that
acidogenic as well as methanogenic bacteria contribute to
dye decolorization (Chinwetkitvanich et al., 2000; Bras
et al., 2001). Use of molecular methods to characterize the
microbial populations in anaerobic-baffled reactors treat-
ing industrial dye waste showed that members of the
g-proteobacteria, together with sulfate reducing bacteria
(SRB), were prominent members of mixed bacterial
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A. Pandey et al. / International Biodeterioration & Biodegradation 59 (2007) 73–84
populations. Along with these, a methanogenic population
dominated by Methanosaeta species and Methanomethylo-
vorans hollandica contributed to the treatment of industrial
wastewater (Plumb et al., 2001).
Yoo et al. (2001) showed that decolorization of Orange
96 was not significantly affected by 2-bromoethanesulfonic
acid (BES), an inhibitor specific to methanogens. This
suggests that methanogens took no part in the decoloriza-
tion, and it contradicts the findings of many other
investigators. On the other hand, in the presence of acetate
and sulfate, molybdate, an inhibitor specific to SRB,
caused a significant decrease in the decolorization rate
(Yoo et al., 2001).
Reduction under anaerobic conditions appears to be
nonspecific, as most of a varied group of azo compounds
are decolorized, although the rate of decolorization is
dependent on the added organic carbon source, as well as
the dye structure (Bromley-challenor et al., 2000; Stolz,
2001). Furthermore, there is no correlation between
decolorization rate and molecular weight, indicating that
decolorization is not a specific process and cell permeability
is not important for decolorization. Thus, anaerobic azo
dye decolorization is a fortuitous process, where dye might
act as an acceptor of electrons supplied by carriers of the
electron transport chain. Alternatively, decolorization
might be attributed to non-specific extracellular reactions
occurring between reduced compounds generated by the
anaerobic biomass. (Van der Zee et al., 2001a). First-order
kinetics with respect to dye concentration has been
generally reported for the course of dye decolorization,
although zero order was also observed in few cases (Van
der Zee et al., 2001a; Isik and Sponza 2005 b). With a few
specific dyes, such as acid orange 7 (AO7), autocatalysis by
quinone-like compounds, formed during azo dye reduc-
tion, contributes to a significant extent to the overall
reduction process (Van der Zee et al., 2000; Mendez-Paz
et al., 2005).
2.2. Azo dye decolorization under anoxic conditions
Anoxic decolorization of various azo dyes by mixed
aerobic and facultative anaerobic 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 luteola, Aeromonas hydrophila, Bacillus sub-
tilis, Pseudomonas sp. and Proteus mirabilis, decolorized
azo dyes under anoxic conditions (Chang et al., 2001; Chen
et al., 1999, 2003; 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 carbohy-
drate (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
75
decolorization by facultative anaerobes 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,
whereas a significant decrease in azo dye 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
reduced 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
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 –NQN– 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. 4-aminobenzesulfonate
(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
 ARTICLE IN PRESS
76 A. Pandey et al. / International Biodeterioration & Biodegradation 59 (2007) 73–84
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. 1.
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 –NQN– bond. This reduction may involve different
mechanisms, such as enzymes, low molecular weight redox
Fig. 1. Structures of a few azo dyes degraded under aerobic conditions.
mediators, chemical reduction by biogenic reductants like
sulfide, or a combination of these (Fig. 2). Additionally, the
location of the reactions can be either intracellular or
extracellular.
2.4.1. Direct enzymatic azo dye reduction
This mechanism involves enzyme-mediated transfer of
reducing equivalents, generated from the oxidation of the
substrate/coenzyme to azo dyes. These enzymes can be
specific, catalyzing only azo dye reduction, or nonspecific.
The latter enzymes catalyze the reduction of a wide range
of substrates. Due to their nonspecific nature, these
enzymes gratuitously reduce azo dyes.
2.4.1.1. Under anaerobic conditions. The presence of
azoreductases in anaerobic bacteria that decolorized
sulfonated azo dyes during growth on solid or complex
media was first reported by Rafii et al. (1990). These strains
belonged mainly to the genera Clostridium and Eubacter-
ium. Azoreductases from these strains were oxygen–
sensitive and were produced constitutively and released
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A. Pandey et al. / International Biodeterioration & Biodegradation 59 (2007) 73–84
Fig. 2. Schematic representation of different mechanisms of anaerobic azo dye reduction. RM ¼ Redox mediator; ED ¼ electron donor; b ¼ bacteria
(enzymes).
extracellularly. Later investigations made with C. perfrin-
gens showed that azo dye reduction is catalyzed by an
enzyme presumed to be flavin adenine dinucleotide
dehydrogenase, which can also reduce nitro aromatic
compounds (Rafii and Cerniglia, 1995). Immunoelectron
microscope analyses showed that the enzyme was secreted
as it was synthesized (Rafii and Cerniglia, 1995). The gene
for this enzyme from C. perfringens has been cloned and
expressed in Escherichia coli (Rafii and Coleman, 1999).
In spite of such extensive studies on oxygen-sensitive azo
reductases from anaerobic bacteria by Rafii and cow-
orkers, the source of NADH necessary for the extracellular
enzyme activity is still not clear.
Another mechanism of dye decolorization could involve
cytosolic flavin-dependent reductases, which transfer elec-
trons via soluble flavins to azo dyes. However, recent
studies of Russ et al. (2000) with a recombinant Sphingo-
monas strain-BN6 have shown that the reduction of
sulfonated azo dyes by cytosolic flavin-dependent azo
reductases is mainly observed in vitro and is of little
importance in vivo. A further indication that flavin-
dependent azoreductases are almost completely laboratory
artifacts was shown by the addition of flavins to resting
cells of strain BN6, which resulted in no significant increase
in azo dye reduction. These findings led Russ et al. (2000)
to suggest that, in living cells with intact cell membranes,
other enzyme systems and/or other redox mediators are
responsible for the reduction of azo dyes. In bacteria that
possess electron transport systems in their membranes, as
in the case of aerobic or facultatively anaerobic bacteria,
such as Sphingomonas strain BN6, the transfer of electrons
from the respiratory chain to appropriate redox mediators
could take place directly. If intracellular reductases are
involved in the process, it may be assumed that mediators
different from flavin cofactors, with a higher ability to pass
through the membranes, must be involved.
77
2.4.1.2. Under aerobic conditions. The existence of azo
reductases in obligate aerobic bacteria was first proven
when two azo reductases were isolated and characterized
from carboxy-orange-degrading strains Pseudomonas K22
(reclassified as Pigmentiphaga kullae K24) and Pseudomonas
KF46 (reclassified as Xenophilus azovorans KF46F) (Zim-
mermann et al., 1982, 1984). These intracellular azoreduc-
tases showed high specificity to dye structures and
reductively cleaved their carboxylated, as well as their
sulfonated, structural analogs. Purified azo reductases from
these two organisms were distinctly different, although both
were small polypeptides without any bound flavin and
required NADPH for activity. A survey of various orange
dyes as substrates for carboxy orange II azo reductase
showed that a hydroxy group in the ortho position to the
azo bond was required. In contrast, the enzyme from
P. kullae KF24 required a hydroxy group at the para
position to the azo group. Although AO7 and AO20 could
be decolorized by the respective azoreductases and even
serve as inducers, the organisms could not utilize sulfonated
dyes as the carbon source. The azoreductase from Bacillus
sp. strain OYl-2 decolorized AR88, AO7 and a series of
proprietary reactive dyes (Suzuki et al., 2001). This enzyme
was also found to be a small monomeric protein with a
molecular mass of approximately 20 KDa. Sequences of
genes encoding these three specific azoreductases are now
known (Suzuki et al., 2001; Blumel et al., 2002; Blumel
and Stolz, 2003). Amino acid sequence alignments did not
show any noticeable homology between this azoreductase
and two other well-characterized azoreductases from
X. azovorans KF 46 and P. kullae (Blumel and Stolz, 2003).
Nonspecific enzymes catalyzing azo bond reduction have
been isolated from aerobically grown cultures of Shigella
dysenteriae (Ghosh et al., 1992), E. coli (Nakanishi et al.,
2001), Bacillus sp. (Maier et al., 2004), Staphylococcus
aureus (Chen et al., 2005) and P. aeruginosa (Nachiyar and
 ARTICLE IN PRESS
78 A. Pandey et al. / International Biodeterioration & Biodegradation 59 (2007) 73–84
Rajkumar, 2005). Where characterized, these enzymes have
been shown to be flavoproteins.
It has been reported that intracellular sulfonated azo dye
reduction requires not only the presence of azo reductases
but also a specific transport system (s), which allows the
uptake of the dye into the cells (Russ et al., 2000).
Currently there appears to be no available information
about the transport systems for these dyes, although there
are few reports on systems which are involved in the
transport into bacterial cells of other sulfonated substrates,
such as p-toluene sulfonate, taurine and alkane sulfonates
(Locher et al., 1993; Eichhorn et al., 2000). The ability of
X. azovorans KF46F and Sphingomonas sp. strain ICX to
take up AO7 and reduce the dye in vivo shows the presence
of transport, as well as azoreductase enzymes, in these
organisms. Construction of recombinant organisms with
sulfonated dye decolorizing ability, therefore, may require
the transfer of the aerobic azoreductase gene into bacterial
strains that are able to grow on sulfonated aromatics.
These organisms generally exhibit narrow substrate speci-
ficity. Studies on 4-ABS degrading strains have also shown
that they are highly specific, as they can utilize only 4-ABS
and not other benzenesulfonates. (Feigel and Knackmuss,
1993; Singh et al., 2004). The 2-ABS degrading Alcaligenes
sp. strain O-1 can utilize two other aromatic sulfonates,
benzene and toluene sulfonate, for growth. However, cell
extracts of this strain can desulfonate at least six substrates
(Thurnheer et al., 1986). This suggests the presence of
highly specific transport systems for the uptake of aromatic
sulfonates in these cultures. Thus the derived recombinants
may still have restricted substrate specificity.
2.4.2. Mediated biological azo dye reduction
As highly polar sulfonated, as well as high molecular
weight, polymeric azo dyes are unlikely to pass through the
Fig. 3. Proposed mechanism for the redox mediator (RM)—dependent reduction of azo dyes by strain BN6 (adapted from Keck et al., 1997).
cell membrane (Levine, 1991), it was suggested that
reduction of these dyes could also occur through mechan-
isms that are not dependent on their transport into the cell.
There are now many reports on the role of redox mediators
in azo bond reduction by bacteria under anaerobic
conditions (Keck et al., 1997; Van der Zee et al., 2001b;
Dos Santos et al., 2003). Riboflavin in catalytic amounts
significantly enhanced the reduction of mordant yellow
10 by anaerobic granular sludge (Field and Brady, 2003).
1-amino 2-napthol, one of the constituent amines of the
azo dye, AO7, increased its decolorization rate, possibly by
mediating the transfer of reducing equivalents (Mendez-
Paz et al., 2005). The addition of synthetic electron carriers
such as anthraquinone-2,6-disulphonate could also greatly
enhance the decolorization of many azo dyes (Van der Zee
et al., 2001b). Keck et al. (1997) reported the first example
of the anaerobic cleavage of azo dyes by redox mediators
formed during the aerobic degradation of a xenobiotic
compound. Cell suspensions of Sphingomonas sp. strain
BN6 grown aerobically in the presence of 2-naphthyl
sulfonate (NS), exhibited a 10–20 fold increase in
decolorization rate of an azo dye, amaranth, under
anaerobic conditions, over those grown in its absence.
Even the addition of culture filtrates from these cells could
enhance anaerobic decolorization by cell suspensions
grown in the absence of NS. Based on these observations,
a mechanism was proposed for the mediated reduction of
azo dyes by S. xenophaga (Fig. 3). Other bacterial cultures
generating redox intermediates during the aerobic degra-
dation of aromatic compounds can also lead to the
enhancement of dye decolorization in anaerobic conditions
(Keck et al., 1997). Recently, Chang et al. (2004) also
showed that the addition of culture supernatants contain-
ing metabolites of a dye-decolorizing strain, E. coli strain
NO3, enhanced azo dye decolorization rates.
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A. Pandey et al. / International Biodeterioration & Biodegradation 59 (2007) 73–84
Van der Zee et al. (2003) have reported that activated
carbon, which is known to have quinone groups at its
surface, enhanced dye decolorization. This is probably one
of the first examples of biocatalysis mediated by activated
carbon.
2.4.3. Azo dye decolorization by biogenic inorganic
compounds
Azo dye decolorization can occur from purely chemical
reactions with inorganic compounds such as sulfide and
ferrous ion that are formed as end products of metabolic
reactions under anaerobic conditions. It has been shown
that H2S generation by SRB resulted in the extracellular
decolorization of azo dyes (Yoo et al., 2000; Diniz et al.,
2002). Sulfate-influenced dye reduction correlated with
biogenic sulfide formation under methanogenic conditions.
In the absence of sulfur compounds, dye decolorization
readily occurred in the presence of granular sludge,
demonstrating the importance of enzymatic mechanisms.
An analysis of decolorization kinetics in batch reactor and
in laboratory scale anaerobic sludge bed reactors indicated
that the relative importance of chemical dye reduction
mechanisms in high rate anaerobic bioreactors is small,
due to the high biomass in the reactors (Van der Zee
et al., 2003).
3. Degradation of aromatic amines
Aromatic compounds possess a large negative resonance
energy, resulting in thermodynamic stability. Microorgan-
isms, 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 carbox-
ylation, 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
79
methanogenic conditions. They include the three isomers of
aminobenzoate, 2- and 4-aminophenols, 2, 4-dihydroxyani-
line and 5-aminosalicylic acid (5-ASA). (Connor and
Young, 1993; Razo-Flores et al., 1997b; Kalyuzhnyl
et al., 2000; Yemashova et al., 2004). Complete degrada-
tion of azo disalicylate and partial mineralization of the
azodyes, Mordent Orange 1, AO6, AO7 and AO52, under
methanogenic conditions, has been reported (Donlon et al.,
1997; Razo-Flores et al., 1997b; Savelieva et al., 2004;
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 biodegradation of aromatic amines
Aromatic amines formed from azo dye reduction, have
been reported to be more easily degraded under aerobic
conditions (Brown and Laboureur, 1983; Haug et al., 1991;
Ekici et al., 2001). The aerobic biodegradation of aniline
and p- amino benzoate appears to be ubiquitous (Gheewala
and Annachhatre, 1997; Blumel et al.,1998). Bacteria
capable of biodegrading 5-ASA are less numerous (Stolz
et al., 1992). Tan et al. (1999) have reported that
4-aminophenol (4-AP) and 5-ASA tend to autoxidise in
the presence of oxygen. However, the autoxidation rate for
4-AP was orders of magnitude greater than that for 5-ASA.
Hence biodegradation of 5-ASA was possible, whereas
4-AP removal was mainly due to autoxidation under
aerobic conditions (Tan et al., 1999).
A group of aromatic amines difficult to degrade even
under aerobic conditions are represented by aryl sulfo-
nates, aminobenzene (ABS) and aminonaphthyl sulfonates
(ANS), which are the constituents of many azo dyes.
Among the three isomers of ABS, 4-ABS appears to be
more susceptible to biodegradation than 2- and 3-ABS.
A co-culture consisting of H. palleroni (Strain S1) and
Agrobacterium radiobacter (Strain S2), as well as a few
pure bacterial strains, degrade 4-ABS (Feigel and Knack-
muss, 1993; Perei et al., 2000; Singh et al., 2006). The
uniqueness of these strains is that they can degrade only
4-ABS and not other aromatic sulfonates. 2-ABS-degrad-
ing cultures appear to be rare (Thurnheer et al., 1986; Tan
et al., 2005).There is only one report of the isolation of a
2-ABS-degrading pure culture, which was characterized to
be of the genus Alcaligenes (strain O-1). This strain can
utilize two other sulfonated aromatics, benzene and toluene
sulfonates, as growth substrates. (Thurnheer et al., 1990).
2-ABS degradation is a plasmid-associated trait in this
organism (Jahnke et al., 1990). Not many reports are
available on 3-ABS degradation (Kolbener et al., 1994;
Nachiyar and Rajkumar, 2003). Recent studies by
Nachiyar and Rajkumar (2004) seem to suggest aniline as
an intermediate during the aerobic degradation of 3-ABS
in P. aeruginosa. This appears to be an unusual desulfona-
tion reaction, as aerobic desulfonation reactions generally
involve hydroxylation catalysed by either mono or
dioxygenases and catechol sulfonates have been identified
 ARTICLE IN PRESS
80 A. Pandey et al. / International Biodeterioration & Biodegradation 59 (2007) 73–84
as intermediates during 2- as well as 4-ABS (Feigel and
Knackmuss, 1993; Junker et al. 1994).
Few bacterial cultures utilizing naphthyl amines as
the sole organic carbon source have been reported.
Sphingomonas sp. strain ICX decolorized AO7 and
degraded 1-amino-2-naphthol (Coughlin et al., 1999)
P. aeruginosa degraded 1,4-diaminonaphthalene (Nachiyar
and Rajkumar, 2004). Sulfonated naphthylamines are
among the most common products of bacterial decoloriza-
tion of azo dyes. Pure cultures have been isolated belonging
to the genera Pseudomonas, Moraxella and Arthrobacter,
which can either degrade 2-aminonaphthyl sulfonate
(2ANS) or utilize it as sulfur source (Nortemann et al.,
1986; Wittch et al., 1988; Ohe et al., 1990; Rozgaj and
Glancer, 1992).
Extensive studies have been carried on the degradation
of 6-aminonaphthyl-2-sulfonate (6A2NS). A co-culture
consisting of 11 bacterial strains was able to utilize
6AN2S. None of the individual strains could degrade
6AN2S, indicating that apparently each strain participates
in the degradation process (Rozgaj and Glancer, 1992).
A mixed bacterial culture utilized 6A2NS as the sole source
of carbon, energy, nitrogen and sulfur. From this culture,
strain BN6 was isolated. This strain could convert 6A2NS
to pyruvate and 5-ASA. 5-ASA was excreted in almost
stiochiometric amounts and was utilized by other members
of the culture (Nortemann et al., 1986). After some years of
cultivation of S. xenophaga strain BN6 and some
accompanying bacteria, strain BN12, which could com-
pletely degrade 6A2NS, was isolated. This strain was
identified as Pseudoaminobacter salicylatoxidans (Kampfer
et al., 1999). To date, this is the only organism that is
known to completely degrade 6A2NS. Based on their
studies with strain BN6, Nortemann et al. (1994) suggested
the following requirements for the productive breakdown
of substituted naphthalene sulfonates (i) an uptake
system and naphthyl sulfonate dioxygenase with low
substrate specificity (ii) counteraction of rapid autoxida-
tion of dihydroxy naphthalenes and other metabolites.
Studies on the degradation of naphthalenesulfonates by
S. xenophaga are reviewed by Stolz (1999).
Available literature on degradation of sulfonated aro-
matic amines (SAA) show that cultures generally have a
narrow substrate range. Mixtures of aromatic sulfonates
can only be degraded by mixed bacterial consortia, which
harbor diverse activities for congeneric substrates and
metabolites (Hopper, 1991).
Few aromatic amines, such as 5-ASA, phenylenedia-
mines, aminophenols and aminonaphthols, tend to auto-
xidize in the presence of oxygen. (Jensen et al., 1993;
Kudlich et al., 1999). Oxygen reacts with the aromatic
products, via free radical reactions, resulting in the
formation of undesirable colored oligomers and polymers
that may be toxic and mutagenic (Field et al., 1995).
Though the autoxidation process eliminates the aromatic
amines, the products formed are more recalcitrant to
biological degradation. Thus, for biological degradation of
these compounds, the rate of degradation must be higher
than the rate of autoxidation.
3.3. Anaerobic–aerobic biological treatment for azo dye
decolorization and degradation
A wide range of structurally diverse dyes is used in the
textile industry in the same unit. The emerging effluent thus
contains many of these dyes. Treatment of such an effluent
requires the application of non-specific processes (Correia
et al., 1994). Owing to this constraint, treatment of textile
and other dye-containing wastewaters is commonly carried
out using chemical coagulation/flocculation, followed by
activated sludge (Pophali et al., 2003; Georgiou et al.,
2005).
Bacterial decolorization of azo dyes under methanogenic
conditions is non-specific. Although azo dye decolorization
under anoxic conditions is also non-specific, limitations of
this method seem to be the requirement for yeast extract
or peptone, thus making the process economically inviable
for industrial-scale application unless alternate cheaper
sources are identified (Nigam et al., 1996; Chen et al., 2003;
Moosvi et al., 2005). Except for a few, the aromatic amines
formed from decolorization of azo dyes are recalcitrant to
biodegradation under anaerobic conditions. These anaero-
bically decolorized effluents can still be hazardous, as many
aromatic amines are toxic. Thus their removal, which
requires aerobic conditions, is essential.
Decolorization and degradation of azo dyes in biological
processes based on bacterial activities thus requires
anaerobic–aerobic conditions. These treatments can be
carried out either sequentially or simultaneously. For the
sequential treatment, anaerobic and aerobic environments
can be provided either in a single reactor for different
periods or in two separate reactors. The feasibility of this
strategy was first demonstrated for the sulfonated azo dye,
Mordant Yellow 3 (Haug et al., 1991). In a simultaneous
treatment system, decolorization takes place in anaerobic
zones of the biofilm or in immobilized microbes entrapped
in a gel matrix (Field et al., 1995; Kudlich et al., 1996).
Different reactor configurations used for anaerobic/
aerobic steps, and their efficiencies, have been excellently
reviewed recently by Van der Zee and Villaverde (2005).
These include anaerobic high-rate reactors, such as upflow
anaerobic sludge blanket, fixed film, rotating biological
contactors and anaerobic baffled reactors for anaerobic
processes and activated sludge and rotating biological
contactors for aerobic treatment (Isik and Sponza, 2004;
Ong et al., 2005; Van der Zee and Villaverde, 2005). An
auxiliary substrate is generally needed for the decoloriza-
tion. Theoretically, the required amount of electron-
donating substrate is low, 32 mg COD per mmol monoazo
dye. However, the actual requirement is much higher due
to competition from other reactions for reducing equiva-
lents. In most of the studies, primary substrate COD was
usually in great excess. The major part of the substrate may
be consumed in the anaerobic step. A few amines may also
 ARTICLE IN PRESS
A. Pandey et al. / International Biodeterioration & Biodegradation 59 (2007) 73–84
be mineralized. As a result, a small fraction of the auxiliary
substrate or its metabolites and undegraded aromatic
amines serve as the carbon source for the organisms in
the aerobic reactor. Color removal levels ranging from
70–95% have been reported in anaerobic/aerobic reactors
(Van der Zee and Villaverde, 2005). However, the fate of
the aromatic amines has been specifically addressed by few
investigators. Some of these studies show partial or
complete removal of many aromatic amines in the aerobic
stage. Furthermore, decrease in toxicity (shown by
suppression of bacterial luminescence) between the efflu-
ents of the anaerobic stage and anaerobic–aerobic stage
seems to provide indirect evidence for the removal of
aromatic amines. As reviewed by Pinheiro et al. (2004),
various substituted aminobenzene and aminonaphthalene
and aminobenzidine compounds have been found to be
aerobically biodegradable. However, they generally require
the enrichment of specialized cultures. Biodegradability of
SAA has been demonstrated only for a few simple ABS
and ANS compounds.
Most of these studies are still at the laboratory scale and
with synthetic wastewaters. Reports on pilot-scale and full-
scale implementation of anaerobic–aerobic biological
treatment are still scarce. Carliell et al. (1996) investigated
laboratory and full-scale trials on the treatment of
exhausted reactive dye bath effluent and reported that co-
treatment of the effluent in an existing sewage sludge
digester exhibited promising results. Delee et al. (1998)
have reviewed the reports on full-scale and pilot-scale
plants and some of their limitations. A two-stage fixed bed
pilot plant for on-site anaerobic decolorization of textile
wastewater was reported by Georgiou et al. (2005).
Anaerobic (with facultative anaerobic bacterial culture)–
aerobic sequential system was used for color and COD
removal from real textile wastewater at pilot scale (Kapdan
and Alparslan, 2005). These recent studies with real
wastewaters have shown that the addition of an anaerobic
unit prior to activated sludge significantly improves the
effluent quality with respect to color. There appears to be
only one report on the successful full-scale implementation
of anaerobic technology in combination with aerobic
membrane technology for the treatment of more than
1000 L3 wastewaters per day from textile processing
industry. (Stolz, 2001, VA Tech WABAG, 2003).
4. Conclusion
The presence of dyes imparts an intense color to
effluents, which leads to environmental as well as aesthetic
problems. The treatment of azo-dye-containing waste-
waters still presents a technical challenge. As regulations
are becoming even more stringent, there is urgent need for
technically feasible and cost-effective methods. The avail-
able 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,
81
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 degra-
ders 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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