Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 138157

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Journal of the Taiwan Institute of Chemical Engineers

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Review

Bacterial decolorization and degradation of azo dyes: A review

R.G. Saratale a,b, G.D. Saratale b,c, J.S. Chang b,d,e,*, S.P. Govindwar a,**
a
Department of Biochemistry, Shivaji University, Kolhapur 416004, M.S., India
b
Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan
c
Department of Biological Environment, College of Agriculture and Life Sciences, Kangwon National University, Chuncheon 200-701, South Korea
d
Research Center for Sustainable Environment, National Cheng Kung University, Tainan 701, Taiwan
e
Center for Biosciences and Biotechnology, National Cheng Kung University, Tainan 701, Taiwan

A R T I C L E I N F O A B S T R A C T

Article history: A variety of synthetic dyestuffs released by the textile industry pose a threat to environmental safety.
Received 21 April 2010 Azo dyes account for the majority of all dyestuffs, produced because they are extensively used in the
Received in revised form 4 June 2010 textile, paper, food, leather, cosmetics and pharmaceutical industries. Existing efuent treatment
Accepted 17 June 2010
procedures are unable to remove recalcitrant azo dyes completely from efuents because of their color
fastness, stability and resistance to degradation. Bacterial decolorization and degradation of azo dyes
Keywords: under certain environmental conditions has gained momentum as a method of treatment, as these are
Azo dyes
inexpensive, eco-friendly and can be applied to wide range of such dyes. This review mainly focuses on
Decolorization
Biodegradation
the different mechanisms of decolorization and discusses the effect of various physicochemical
Azoreductase parameters on the dye removal efciency of different bacteria. The enzymatic mechanisms involved in
Anaerobicaerobic treatment the bacterial degradation of azo dyes, the identication of metabolites by using various analytical
Mineralization techniques, and the nature of their toxicity has been investigated. This review provides an overview of
bacterial decolorization/degradation of azo dyes and emphasizes the application of these processes for
the treatment of azo dye-containing wastewaters.
2010 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
2. Importance of biological treatment relative to physicochemical methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
2.1. Physical and chemical methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
2.2. Biological methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
2.2.1. Decolorization and degradation of azo dyes by fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
2.2.2. Decolorization and degradation of azo dyes by yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
2.2.3. Decolorization and degradation of azo dyes by algae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
2.2.4. Decolorization and degradation of azo dyes by plants (phytoremediation) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
2.2.5. Bacterial decolorization and degradation of azo dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
3. Mechanisms of microbial color removal. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
4. Factors affecting bacterial decolorization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
4.1. Effects of oxygen and agitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
4.2. Effects of carbon and nitrogen sources supplements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
4.3. Effects of temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
4.4. Effects of pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
4.5. Effects of dye concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
4.6. Effects of dye structure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
4.7. Effects of electron donor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
4.8. Effects of redox mediator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150

* Corresponding author at: Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan. Tel.: +886 6 2757575x62651;
fax: +886 6 2357146/2344496.
** Corresponding author. Tel: +91 231 2609152; fax: +91 231 2691533.
E-mail addresses: changjs@mail.ncku.edu.tw (J.S. Chang), spg_biochem@unishivaji.ac.in (S.P. Govindwar).

1876-1070/$ see front matter 2010 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.jtice.2010.06.006
 R.G. Saratale et al. / Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 138157 139

5. Enzymes involved in the bacterial decolorization and degradation of azo dyes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
5.1. Reductive enzymes involved in the bacterial degradation of azo dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
5.2. Oxidative enzymes involved in the bacterial degradation of azo dyes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
6. Analytical methods employed for the decolorization studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
7. Microbial toxicity studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154

1. Introduction
In 1856 William Henry Perkin accidentally discovered the
worlds rst commercially successful synthetic dye. Such dyes are
dened as colored substances that when applied to bers give
them a permanent color which is able to resist fading upon
exposure to sweat, light, water and many chemicals, including
oxidizing agents and microbial attack (Rai et al., 2005). More than
ten thousand synthetic dyes were developed and used in
manufacturing by the end of the 19th century (Robinson et al.,
2001). Moreover, the growth of the worldwide textile industry in
the years since then had seen a commensurate increase in the use
of such synthetic dyes, and this has been accompanied by a rise in
pollution due to wastewater contaminated with dyestuff (Pandey
et al., 2007). The water consumption and the wastewater
generation from the textile industry (dry processing mill and
woven fabric nishing mills) depend upon the processing
operations employed during the conversion of ber to textile
fabric (Dhanve et al., 2008). The textile industry is one of the
greatest generators of liquid efuent pollutants, due to the high
quantities of water used in the dyeing processes. Moreover, the
processing stages and types of synthetic dyes applied during this
conversion determine the variable wastewater characteristics in
terms of pH, dissolved oxygen, organic and inorganic chemical
content (Banat et al., 1996). It is estimated that 280,000 t of textile
dyes are discharged in such industrial efuents every year
worldwide (Jin et al., 2007). Azo dyes make up approximately
70% of all dyestuffs used worldwide by weight (Zollinger, 1987),
making them the largest group of synthetic colorants and the most
common synthetic dyes released into the environment (Chang
et al., 2001b; Saratale et al., 2009a; Zhao and Hardin, 2007).
Azo dyes absorb light in the visible spectrum due to their
chemical structure, which is characterized by one or more azo
groups (N5 5N) (Chang et al., 2000). The azo group is substituted
with benzene or naphthalene groups, which can contain many
different substituents, such as chloro (Cl), methyl (CH3), nitro
(NO2), amino (NH2), and hydroxyl (OH), carboxyl (COOH),
which give different types of azo dyes (Zollinger, 1991). Azo dyes
accounts for the majority (more than 3000 different varieties) of all
textile dyestuffs produced because of the ease and cost effectiveness
of their synthesis, their stability and the variety of colors available
compared to natural dyes (Chang et al., 2004). They are extensively
used in the textile, paper, food, leather, cosmetics and pharmaceu-
tical industries (Telke et al., 2008). Improper discharge of textile dye
efuent containing azo dyes and their metabolites in aqueous
ecosystems is aesthetically unpleasant and leads to a reduction in
sunlight penetration, which in turn decreases photosynthetic
activity, dissolved oxygen concentration, and water quality, and
had acute toxic effects on aquatic ora and fauna, causing severe
environmental problems worldwide (Vandevivere et al., 1998). In
addition, azo dyes also have an adverse impact in terms of total
organic carbon (TOC), biological oxygen demand (BOD) and
chemical oxygen demand (COD) (Saratale et al., 2009b). Many
synthetic azo dyes and their metabolites are toxic, carcinogenic, and
mutagenic (Myslak and Bolt, 1998). Moreover, numerous reports
indicate that textile dyes and efuents have toxic effects on the
germination rates and biomass of several plant species which have
important ecological functions, such as providing a habitat for
wildlife, protecting soil from erosion and providing the organic
matter that is so signicant to soil fertility (Ghodake et al., 2009a;
Kapustka and Reporter, 1993). Therefore, treatment of industrial
efuents containing azo dyes and their metabolites is necessary
prior to their nal discharge to the environment.
Several physicochemical methods have been used for the
removal of dyes from wastewater efuent. However, implementa-
tion of physical/chemical methods have the inherent drawbacks of
being economically unfeasible (as they require more energy and
chemicals), being unable to completely remove the recalcitrant azo
dyes and/or their organic metabolites, generating a signicant
amount of sludge that may cause secondary pollution problems,
and involving complicated procedures (Forgacs et al., 2004; Zhang
et al., 2004). However, microbial or enzymatic decolorization and
degradation is an eco-friendly cost-competitive alternative to
chemical decomposition process that could help reduce water
consumption compared to physicochemical treatment methods
(Rai et al., 2005; Verma and Madamwar, 2003). In this review we
investigate various methods for the treatment of textile efuent
containing azo dyes, and describe the importance of biological
(mainly bacterial) methods. In addition we also focus on the effect
of physicochemical conditions, the enzymatic mechanisms of
bacterial decolorization, and the toxicity of the degradation
products.
2. Importance of biological treatment relative to
physicochemical methods
Dye-house efuent typically contains only 0.60.8 g L 1 dye,
but the pollution it causes is mainly due to durability of the dyes in
the wastewater (Jadhav et al., 2007). Therefore it is necessary to
search for and develop effective treatments and technologies for
the decolorization of dyes in such efuents. Various physical/
chemical methods, such as adsorption, chemical precipitation,
photolysis, chemical oxidation and reduction, electrochemical
treatment, have been used for the removal of dyes from
wastewater (Fig. 1). Moreover, there are many reports on the
use of physicochemical methods for the color removal from dye-
containing efuents (dos Santos et al., 2007; Vandevivere et al.,
1998; Wang et al., 2009a,b,c).
[()TD$FIG]
Fig. 1. Treatment methods for the removal of dyes from wastewater efuent.
140 R.G. Saratale et al. / Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 138157
2.1. Physical and chemical methods
Physical methods based on coagulationocculation of dyes are
effective for the removal of mainly sulphur and disperse dyes, but
show very low coagulationocculation capacity for acid, direct,
reactive and vat dyes. In addition, the low color removal efciency
and large amount of sludge produced limits the application of
these techniques (Vandevivere et al., 1998). Adsorption methods
have attracted considerable interest due to their higher efciency
for the removal of a wide range of dyes. The selection of an
adsorbent is based on characteristics such as high afnity, capacity
for target compounds and the possibility of adsorbent regeneration
(Subramaniam et al., 2009). Although activated carbon (AC) is a
very effective adsorbent for various types of dyes, it is not often
used due to its high cost (Robinson et al., 2001). To make the
process more economically feasible, some investigators use low-
cost adsorbent materials like peat, bentonite clay, y ash,
polymeric resins, ion exchangers and many biological materials
such as, corn/maize cobs, maize stalks, and wheat straw for the
color removal of dye wastewater (Ramakrishna and Viraraghavan,
1997). However, the practical application of these adsorbents has
been limited by problems associated with their regeneration or
disposal, high sludge production, low effectiveness with regard to
wide range of dyes and high cost (Anjaneyulu et al., 2005; Karcher
et al., 2001). Filtration methods such as ultraltration, nanoltra-
tion and reverse osmosis have been used for water reuse and
chemical recovery. In the textile industry, the use of membranes
provides interesting possibilities for the separation of hydrolysed
dyestuffs and dyeing auxiliaries that simultaneously reduce the
color, BOD and COD of wastewater, and they have also been found
useful for mercerizing and bleaching it. With this approach, the
selection of the type and porosity of the lter depends upon the
chemical composition of the wastewater and the specic
temperature required for the process (dos Santos et al., 2007).
However, membranes have some signicant drawbacks, including
high investment costs, potential membrane fouling and the
production of secondary waste streams which need further
treatment (dos Santos et al., 2007; Robinson et al., 2001).
Chemical oxidation methods enable the destruction or decom-
position of dye molecules, and such approaches use various
oxidizing agents, such as ozone (O3), hydrogen peroxide (H2O2)
and permanganate (MnO4). Modication in the chemical compo-
sition of a compound or a group of compounds takes place in the
presence of these oxidizing agents, and thus the dye molecules
become susceptible to degradation (Metcalf, 2003). Ozonation has
been found to be effective due to its high reactivity with many azo
dyes, the lack of alteration of the reaction volume due to its
gaseous state, and good color removal efciencies (Alaton et al.,
2002). However its short life time, ineffectiveness towards
dispersed dyes and those insoluble in water, low COD removal
capacity, as well as the high cost of ozone, limits the practical
application of this technique (Anjaneyulu et al., 2005). In advanced
oxidation processes (AOP) (photochemical and photocatalytic),
oxidizing agents such as O3 and H2O2 or heterogeneous photo-
catalysts are used with catalysts, such as TiO2, ZnO2, Mn and Fe, in
the presence or absence of an irradiation source which generates
(OH ) radicals for the destruction of hazardous dye pollutants
(Anjaneyulu et al., 2005; Forgacs et al., 2004; Vandevivere et al.,
1998; Wang et al., 2009a,b,c).
The Fenton reaction method is relatively cheap and also has
high COD removal and decolorization efciencies for both soluble
as well as insoluble dyes, but the high sludge generation associated
with this approach also limits its use (Robinson et al., 2001). The
H2O2/UV process is the most effective AOP technology, mainly
because of high color removal (up to 95%), lack of sludge formation
and high COD removal in a short retention time (Alaton et al.,
2002). However it is less effective for highly colored wastewater
and dispersed or vat dyes and it also leads to the formation of
undesirable byproducts, while the inefcient use of UV light
increases the cost of this process (Pearce et al., 2003). Electro-
chemical oxidation is very effective in destroying organic
compounds and producing non-hazardous products, but the high
cost of the electricity required has also limited the use of this
process (Morawski et al., 2000). The majority of color removal
techniques work either by concentrating the color into sludge or by
the complete destruction of the colored molecules. Consequently,
an aerobic granular sludge method for the treatment of color
containing wastewater has been proposed (Adav et al., 2009).
The brief review of the various physical/chemical methods for
dealing with dye wastewater presented above reveals that all of
them have some drawbacks, such as being economically unfeasi-
ble; unable to completely remove the recalcitrant azo dyes and/or
their organic metabolites because of the color fastness, stability
and resistance of azo dyes to degradation (Anjaneyulu et al., 2005);
generating a signicant amount of sludge that may cause
secondary pollution problems; substantially increasing the cost
of these treatment methods; and involving complicated proce-
dures (Eichlerova et al., 2007; Forgacs et al., 2004; Zhang et al.,
2004).
2.2. Biological methods
Bioremediation, or the use of microbial techniques to deal with
pollution, is a key research area in the environmental sciences. In
such approaches microbes acclimatize themselves to the toxic
wastes and new resistant strains develop naturally, which then
transform various toxic chemicals into less harmful forms. The
mechanism behind the biodegradation of recalcitrant compounds
in the microbial system is based on the action of the biotransfor-
mation enzymes (Saratale et al., 2007a). Several reports demon-
strate the degradation of complex organic substances which can be
brought about by enzymatic mechanisms, such as those associated
with laccase (Hatvani and Mecs, 2001), lignin peroxidase (Duran
and Esposito, 2000), NADH-DCIP reductase (Bhosale et al., 2006),
tyrosinase (Zhang and Flurkey, 1997), hexane oxidase (Saratale
et al., 2007b) and aminopyrine N-demethylase (Salokhe and
Govindwar, 2003). A number of biotechnological approaches have
attracted interest with regard to tackling azo dye pollution in an
ecoefcient manner, mainly with the use of bacteria and often in
combination with physicochemical processes. Azo dyes are
xenobiotic in nature and recalcitrant to biodegradation, and the
use of microbial or enzymatic treatment method for the complete
decolorization and degradation of such dyes from textile efuent
has the following advantages: (1) being environmentally-friendly,
(2) being cost-competitive, (3) producing less sludge, (4) yielding
end products that are non-toxic or have complete mineralization;
and (5) requiring less water consumption compared to physico-
chemical methods (Banat et al., 1996; Rai et al., 2005). The
effectiveness of microbial decolorization depends on the adapt-
ability and the activity of the selected microorganisms. Conse-
quently, a large number of species has been tested for the
decolorization and mineralization of various dyes in recent years
(Pandey et al., 2007). The isolation of potent species and thereby
their degradation is one of the interesting biological aspects of
efuent treatment (Chen et al., 2008). A wide variety of
microorganisms are capable of decolorizing of a wide range of
dyes including; bacteria (Dawkar et al., 2008; Jadhav et al., 2007;
Kalyani et al., 2008; Saratale et al., 2009b; Telke et al., 2008), fungi
(Fournier et al., 2004; Humnabadkar et al., 2008; Saratale et al.,
2006), yeasts (Jadhav et al., 2007; Lucas et al., 2006; Saratale et al.,
2009a), actinomycetes (Machado et al., 2006), algae (Acuner and
Dilek, 2004; Daeshwar et al., 2007; Gupta et al., 2006; Parikh and
 R.G. Saratale et al. / Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 138157
Madamwar, 2005; Yan and Pan, 2004) and plants (phytoremedia-
tion) (Aubert and Schwitzguebel, 2004; Ghodake et al., 2009a;
Kagalkar et al., 2009). Moreover, these are even capable of
completely mineralizing many azo dyes under certain environ-
mental conditions.
2.2.1. Decolorization and degradation of azo dyes by fungi
Filamentous fungi are ubiquitous in the environment, inhabit-
ing ecological niches such as soil, living plants and organic waste
material. The ability of fungi to rapidly adapt their metabolism to
varying carbon and nitrogen sources is important for their survival.
This metabolic activity is achieved through the production of a
large set of intra and extracellular enzymes able to degrade various
complex organic pollutants such as polyaromatic hydrocarbons,
organic waste, dye efuents and steroid compounds (Gadd, 2001;
Humnabadkar et al., 2008; McMullan et al., 2001; Saratale et al.,
2007a,b). Fungal systems appear to be most appropriate in the
treatment of colored and metallic efuents (Ezeronye and
Okerentugba, 1999). The ability of such fungi to degrade such a
range of organic compounds results from the relatively non-
specic nature of their ligninolytic enzymes, such as lignin
peroxidase (LiP), manganese peroxidase (MnP) and laccase
(Christian et al., 2005).
Most studies on the azo dye biodegradation have focused on
fungal cultures from white rot fungi which have been used to
develop bioprocesses for the mineralization of azo dyes (Machado
et al., 2006). Phanerochaete chrysosporium is the most widely
studied white-rot fungi, but others have also received considerable
attention, such as Trametes (Coriolus) versicolor, Bjerkandera adusta,
Aspergillus ochraceus, species of Pleurotus, and Phlebia, and a variety
of other isolates (Heining et al., 1998; Humnabadkar et al., 2008;
Pointing and Vrijmoed, 2000; Saratale et al., 2006). However,
application of white-rot fungi for the removal of dyes from textile
wastewater has some inherent drawbacks such as the long growth
cycle and the need for nitrogen limiting conditions. In addition,
white-rot fungi are not naturally found in wastewater, and hence
the enzyme production may be unreliable (Robinson et al., 2001).
Moreover, the long hydraulic retention time required for complete
decolorization also limits the performance of the fungal decolori-
zation system (Banat et al., 1996; Chang et al., 2004), and the
preservation of fungi in bioreactors is also a matter of concern
(Stolz, 2001).
2.2.2. Decolorization and degradation of azo dyes by yeast
Very little work has been done to explore the decolorization
ability of yeast, and it has mainly been studied with regard to
biosorption. Some ascomycetes yeast species, such as Candida
tropicalis, Debaryomyces polymorphus, Candida zeylanoides (Yang
et al., 2003), and Issatchenkia occidentalis (Ramalho et al., 2004),
have been used to carry out putative enzymatic biodegradation
and consequent decolorization of different azo dyes. Recently,
Saccharomyces cerevisiae MTCC-463 was reported to have a role in
the decolorization of Malachite Green and Methyl Red (Jadhav and
Govindwar, 2006; Jadhav et al., 2007). In addition Saccharomyces
cerevisiae cells have also shown bioaccumulation of reactive textile
dye (Remazol Blue, Remazol Black B and Remazol Red RB) during
growth in molasses (Aksu and Donmez, 2003). Some studies also
show that yeast species act as a promising dye adsorbent and are
able to uptake higher dye concentration (Safarkova et al., 2005),
while Galactomyces geotrichum MTCC 1360 can decolorize
triphenylmethane, azo and reactive high exhaust textile dyes
(Jadhav et al., 2008a). More recently, a detailed study was
conducted on the decolorization of Navy Blue HER by using
Trichosporon beigelii NCIM-3326, with the enzymatic mechanism
and toxicity of the degradation products also reported (Saratale
et al., 2009a).
141
2.2.3. Decolorization and degradation of azo dyes by algae
Photosynthetic organisms, such as cyanobacteria or algae, have
ubiquitous distribution and are observed in many habitats around
the world, and are receiving increasing attention in the eld of
wastewater decolorization. A survey of the literature suggests that
algae are capable of degrading azo dyes through an induced form of
an azoreductase (Vijayaraghavan and Yun, 2008). Color removal by
algae is due to three intrinsically different mechanisms of
assimilative utilization of chromophores for the production of
algal biomass, CO2 and H2O transformation of colored molecules to
non-colored ones, and adsorption of chromophores on algal
biomass. Several species of Chlorella and Oscillatoria are capable
of degrading azo dyes to their aromatic amines, and can further
metabolize the aromatic amines to simpler organic compounds or
CO2 (Acuner and Dilek, 2004). Mohan et al. (2002) attributes the
decolorization to biosorption followed by bioconversion and
biocoagulation using algae. It has been reported that more than
30 azo compounds can be biodegraded and decolorized by Chlorella
pyrenoidosa, Chlorella vulgaris and Oscillateria tenuis, with the azo
dyes decomposed into simpler aromatic amines (Yan and Pan,
2004). Thus the foregoing results could mean that algae can play an
important role in the removal of azo dyes and aromatic amines in
stabilization ponds. Moreover, this biosorption process could be
adopted as a cost-effective and efcient approach for the
decolorization of efuents, and it may be a viable alternative to
more costly materials (Banat et al., 1996; Daeshwar et al., 2007).
2.2.4. Decolorization and degradation of azo dyes by plants
(phytoremediation)
Phytoremediation is an emerging technology that promises the
effective and inexpensive approach for the remediation of soils and
groundwater contaminated with heavy metals and organic
pollutants (Patil et al., 2009). The main advantages of phytor-
emediation are that it is an autotrophic system with a large
biomass that requires little nutrient input, is easier to manage, and
is generally approved by the public due to both its aesthetic appeal
and environmental sustainability (Ghodake et al., 2009a; Kagalkar
et al., 2009). More specically, narrow-leaved cattails have been
studied in synthetic reactive dye wastewater treatment under
caustic conditions (Nilratnisakorn et al., 2007). In addition,
Mbuligwe (2005) describes a 7277% reduction in color in
wetlands vegetated with coco yam plants. Moreover, the three
plant species (Brassica juncea, Sorghum vulgare and Phaseolus
mungo) of different agronomic consequence have been evaluated
for the decolorization of the reactive group of azo dyes and textile
efuent. These plants, B. juncea, S. vulgare and P. mungo, achieved
textile efuent decolorization of up to 79, 57 and 53%, respectively
(Ghodake et al., 2009a). Similarly, an herb Blumea malcommi was
found to degrade textile dye Reactive Red 5B (Kagalkar et al., 2009).
Extensive research has been undertaken to develop effective and
efcient phytoremediation techniques. It was reported that hairy
root cultures of Tagetes patula L. (Marigold) are effective in the
decolorization of Reactive Red 198, and the enzyme system in the
plant responsible for this was determined (Patil et al., 2009).
However, large scale application of phytoremediation presently
faces a number of obstacles, including the level of pollutants
tolerated by the plant, the bioavailable fraction of the contami-
nants and evapotranspiration of volatile organic pollutants, as well
as requiring large areas to implant the treatment (Ghodake et al.,
2009b).
2.2.5. Bacterial decolorization and degradation of azo dyes
Generally the decolorization of azo dyes occurs under
conventional anaerobic, facultative anaerobic and aerobic condi-
tions by different groups of bacteria. The mechanism of microbial
degradation of azo dyes involves the reductive cleavage of azo
 142
Table 1
Decolorization of various azo dyes by pure bacterial cultures.

Name of strain Name of dye and Condition (pH, temp. Time (h) Decolorization (%) Type of enzymes References
concentration (8C), agitation) involved

Pseudomonas sp. Reactive Blue 13; (200 mg L 1) 7.0, 35, static 70 83.2 NA Lin et al. (2010)
Micrococcus glutamicus Reactive Green 19 A; (50 mg L 1) 6.8, 37, static 42 100 Oxidative and reductive Saratale et al. (2009c)
NCIM 2168
Enterobacter EC3 Reactive Black 5; (1 g L 1) 7.0, 37, anaerobic 36 92.56 NA Wang et al. (2009a,b,c)
Mutant Bacillus sp. ACT2 Congo Red; (3 g L 1) 7.0, 37, static 3748 1230 Reductive Gopinath et al. (2009)
Lactobacillus acidophilus and Water and oil soluble azo NA, 37, anaerobic 36 86100 NA Chen et al. (2009a)
Lactobacillus fermentum dyes; (6 mg L 1)
Geobacillus stearothermophilus Orange II; (0.050 mM) 56, 50, aeration 24 9698 NA Evangelista-Barreto
(UCP 986) (150 rpm) et al. (2009)

R.G. Saratale et al. / Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 138157
Aeromonas hydrophila Reactive Red 198, Reactive Black 5, 30, agitation 60.2; 80.9; 66.5; Reductive Hsueh et al. (2009)
Reactive Red 141, Reactive Blue 171, (125 rpm), 7.5 36.0; 33.7a
Reactive Yellow 84 (300 mg L 1)
Aeromonas hydrophila Reactive Red 141; (3.8 g L 1) 7.0, 30, 200 rpm, 48 100 NA Chen et al. (2009b)
Chemostat pulse
technique
1
Escherichia coli JM109 Direct Blue 71; (150 mg L ) 9.0, 30, anaerobic 12 100 Reductive Jin et al. (2009)
(pGEX-AZR)
Bacillus sp. VUS Navy Blue 2GL; (50 mg L 1) 7.0, 40, static 18 94 Oxidative and reductive Dawkar et al. (2009)
Citrobacter sp. CK3 Reactive Red 180; (200 mg L 1) 7.0, 32, anaerobic 36 96 NA Wang et al. (2009a,b,c)
Acinetobacter calcoaceticus Direct Brown MR; (50 mg L 1) 7.0, 30, static 48 91.3 Oxidative and reductive Ghodake et al. (2009a)
NCIM-2890
Bacillus genus C.I. Reactive Orange 16; 78, 30, static 24 88 Reductive Telke et al. (2009a)
(100 mg L 1)
Pseudomonas sp. SUK1 Reactive Red 2; (5 g L 1) 6.27.5, 30, static 6 96 Oxidative and reductive Kalyani et al. (2008)
Pseudomonas aeruginosa Remazol Orange; (200 mg L 1) 7.0, 30, static 24 94 Reductive Sarayu and Sandhya (2010)
Enterococcus gallinarum Direct Black 38; (100 mg L 1) NA, NA, static 20 days 100 Reductive Bafana et al. (2009)
Pseudomonas sp. SU-EBT Congo red; (1 g L 1) 8.0, 40, static 12 97 Oxidative Telke et al. (2009a)
Brevibacillus laterosporus Golden Yellow HER; (50 mg L 1) 7.0, 30, static 48 87 Oxidative and reductive Gomare et al. (2009)
MTCC 2298
1
Rhizobium radiobacter Reactive Red 141; (50 mg L ) 7.0, 30, static 48 90 Oxidative and reductive Telke et al. (2008)
MTCC 8161
Comamonas sp. UVS Direct Red 5B; (1.1 g L 1) 6.5, 40, static 13 100 Oxidative Jadhav et al. (2008)
1
Exiguobacterium sp. RD3 Navy Blue HE2R; (50 mg L ) 7.0, 30, static 48 91 Oxidative and reductive Dhanve et al. (2008)
Proteus mirabilis RED RBN; (1 g L 1) 6.57.5, 3035, static 20 95 Reductive followed by Chen et al. (1999)
biosorption
Aeromonas hydrophila Red RBN; (3000 mg L 1) 5.510.0, 2035, NA 8 90 NA Chen et al. (2003)
Pseudomonas aeruginosa NBAR12 Reactive Blue 172; (500 mg L 1) 7.08.0, 40, static 42 83 Oxidative and reductive Bhatt et al. (2005)
Bacillus sp. Congo Red; (100300 mg L 1) 7.0, 37, NA 2427b 12c 100b 100c Effect of sonication Kannappan et al. (2009)
K1ebsi1e11a pneumoniae R5-13 Methy1 Red; (100 mg L 1) 6.08.0, 30, 200 rpm 168 100 Reductive Wong and Yuen (1996)
Rhodopseudomonas Reactive Brilliant Red; X-3B; 8, 3035, anaerobic 24 90 Reductive Liu et al. (2006)
palustris AS1.2352 (50 mg L 1)
Citrobacter sp. Azo and Triphenylmethane 79, 3540, static 1 100 Adsorption An et al. (2002)
dyes; (5 mM)
Shewanella decolorationis S12 Acid Red GR; (150 mM) NA, 30,d ande 68d 100d Reductive Xu et al. (2007)
10e 100e
Paenibacillus azoreducens sp. nov. Remazol Black B; (0.1 g L 1) NA, 37, static 24 98 NA Meehan et al. (2001)
Bacteroides fragilis Amaranth, Orange II and 8, 35, static NA 95 NA Bragger et al. (1997)
Tartrazine; (0.1 mM)
Desulfovibrio desulfuricans Reactive Orange 96 and NA, 28, Anaerobic 2 95 Reductive Yoo et al. (2000)
Reactive Red 120
Bacillus fusiformis KMK5 Disperse Blue 79 and Acid 9, 37, anoxic 48 100 Reductive Kolekar et al. (2008)
Orange 10; (1.5 g L 1 each)
1
Aeromonas hydrophila var 24 B Various azo dyes; (10100 mg L ) NA 24 5090 Reductive Idaka and Ogawa (1978)
Sphingomonas sp. BN6 Acid azo dyes, Direct azo dyes NA NA Flavin Reductase Russ et al. (2000)
and Amaranth; (0.1 mmol)
 R.G. Saratale et al. / Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 138157 143

bonds (N5 5N) with the help of azoreductase enzymes under

anaerobic conditions that resulted in the formation of colorless
Chang and Lin (2000)

solutions containing potentially hazardous-aromatic amines

Chen et al. (2009a,b)

Ogawa et al. (1986)

Kulla et al. (1983)

(Chang and Kuo, 2000; Van der Zee and Villaverde, 2005). The
Yoo et al. (2000)
resulting intermediate metabolites (e.g., aromatic amines) are
further degraded aerobically or anaerobically (Joshi et al., 2008).
Many recent studies focus on the utilization of microbial
biocatalysts to remove dye from the efuent (Chang et al., 2004;
Hu, 2001). Extensive studies have been carried out to determine
the role of the diverse groups of bacteria in the decolorization of
azo dyes (Pandey et al., 2007). The bacterial decolorization and
degradation of these dyes has been of considerable interest since it
can achieve a higher degree of biodegradation and mineralization,
is applicable to a wide variety of azo dyes, is inexpensive and
environmentally-friendly, and produces less sludge (Khehra et al.,
Reductive

Reductive
Reductive

2006; Rai et al., 2005; Saratale et al., 2009c; Verma and Madamwar,
2003).
NA

NA

2.2.5.1. Using pure bacterial culture. The efuents from the textile
industry are complex, containing a wide variety of synthetic dyes
and other products, such as dispersants, acids, bases, salts,
1

detergents, humectants, oxidants, and so on. Discharge of these

h
1

colored efuents into rivers and lakes results in a reduced

113.7 mg
dye g cell

7080

dissolved oxygen concentration, thus creating anoxic conditions

that are lethal to resident organisms. Biological processes provide
95
90

90

an alternative to existing technologies because they are more cost-

effective, environmentally-friendly, and do not produce large
Within 2 h from

quantities of sludge. Moreover, bacterial decolorization is normally

360 to 362 h

faster compared to fungal systems with regard to the decoloriza-

tion and mineralization of azo dyes. It has been observed that
mixed cultures are particularly useful in this area, as some
NA

68

35

24

microbial consortia can collectively carry out biodegradation tasks

that no individual pure strain can undertake successfully (Nigam
et al., 1996). However, mixed cultures only provide an average
a constant dye loading

macroscopic view of what is happening in the system, and the

28, 100 rpm without

rate of 200 mg h 1

results are not easily reproduced, making thorough, effective

aeration and with

7.0, 30, 125 rpm

7.0, 30, 110 rpm

interpretation of the results quite difcult. Efforts to isolate pure

11.3, 37, NA

bacterial cultures capable of degrading azo dyes started in the

1970s with reports of a Bacillus subtilis, Aeromonas hydrophila and
in a Bacillus cereus (Wuhrmann et al., 1980). Recently a substantial
NA

amount of research on the subject of color removal has been

carried out using single bacterial cultures like Proteus mirabilis,
Pseudomonas luteola, and Pseudomonas sp., and isolated Pseudomo-
nas sp. SUK1 has shown very promising results for the azo dye
degradation under anoxic conditions (Chang et al., 2001b; Chen
Reactive Orange 96 Reactive

Reactive Red 141; (3.8 g L 1)

Acid Orange 12 Acid Orange

Orange I, Orange II; (1 g L 1)

et al., 1999; Kalyani et al., 2008; Yu et al., 2001). In addition, there

are also several studies describing the decolorization of reactive
Red 120; (0.3 mmol L 1)

azo dyes mediated by pure bacterial culture and the results are
summarized in Table 1. Pseudomonas aeruginosa decolorized a
(200600 mg L 1)
Reactive Red 22;

20 Acid Red 88;

Decolorization n mg L 1 h 1 ODU 1 NA: not available.

commercial tannery and textile dye, Navitan Fast Blue S5R, in the
Microaerophilic, DO range 0.20.5 mg L 1, 150 rpm.

presence of glucose under aerobic conditions. This organism was

also able to decolorize various other azo dyes (Nachiyar and
Rajkumar, 2005). The use of a pure culture system ensures
reproducible data, and thus interpretation of experimental
observations becomes easier. It also becoming easier to determine
Without sonication pretreatment.

the detailed mechanisms of biodegradation using the tools of

With sonication pretreatment.

biochemistry and molecular biology, and this information could be

useful to regulate the enzyme system in order to produce modied
Pseudomonas cepacia 13NA

Desulfovibrio desulfuricans

strains with enhanced enzyme activities. The quantitative analysis

Anaerobic condition.
Aeromonas hydrophila

of the kinetics of azo dye decolorization by a particular bacterial

Pseudomonas luteola

culture has been studied in detail (Chang and Kuo, 2000; Telke
Pseudomonas sp.

et al., 2008). In our laboratory, the metabolic pathway of particular

dyes using pure culture was determined using oxidoreductive
enzymatic studies and using various analytical techniques such as
UVvis spectroscopy, fourier transform infrared spectroscopy
d
b
a

e
c

(FTIR), gas chromatography mass spectrometry (GC/MS), high

 144
Table 2
Decolorization of various azo dyes by co-cultures of pure bacterial strains.

Name of strain Name of dye and Condition (pH, temp. Time (h) Decolorization Type of enzymes References
concentration (8C), agitation) (%) involved

Bacterial consortium-GR (Proteus Green HE4BD, mixture of 6 8.0, 37, static 24 100 Oxidative and Saratale et al. (2010a)
vulgaris and Miccrococcus reactive dyes; (50 mg L 1 each) reductive
glutamicus)
Penicillium sp. QQ and bacterial Reactive Brilliant red X-3B and 3.0, 30, static 72 87.8 NA Gou et al. (2009)
Sphingomonas xenophaga QYY Acid Red B; (50 mg L 1)
Consortium SKB-II (Bacillus vallismortis Congo Red, Brodeaux, Ranocid 7.0, 37, agitation 120 96, 89.6, NA Tony et al. (2009a,b)
1
and B. Megaterium) fast Blue and Blue BCC; (10 mg L ) (130 rpm) 81, 82.7
Consortium TJ-1 (Aeromonas caviae, Acid Orange 7 and many other 7.0, 37, 16 90 NA Joshi et al. (2008)

R.G. Saratale et al. / Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 138157
Protues mirabilis and Rhodococcus azo dyes; (200 mg L 1) microaerophilic
globerulus)
Consortium-GR (P. vulgaris and Scarlet R and mixture of 8 dyes; 7.0, 37, static 3 100 Reductive Saratale et al. (2009b)
M. glutamicus) (50 mg L 1 each)
Bacterial consortium DMC Direct Black 22; (100 mg L 1) 7.0, 45, static 12 >91 NA Mohana et al. (2008)
Consortium of E.coli DH5a and Reactive Red 22; (1 g L 1) 7.0, 28, 200 rpm 25 100 NA Chen and Chang (2007)
Pseudomonas luteola
1
Four bacterial strains consortium Acid Red 88; (100 mg L ) 7.0, using UFCR 3 months 98 Reductive Khehra et al. (2006)
Bacillus cereus, Pseudomonas anoxicaerobic
putida, Pseudomonas uorescence sequential bioreactor
and Stenotrophomonas acidaminiphila
1
Consortium of Enterobacter sp., Reactive Red 195; (30 mg L ) 7.0, 37, 150 rpm 48 90 NA Jirasripongpun
Serratia sp., Yersinia sp., Erwinia sp et al. (2007)
Consortium of two isolated strains Direct Yellow 86, Basic Orange 2, 910.5, 28, 120 rpm 168 80 NA Resmi et al. (2004)
(BF1, BF2) and a strain of Reactive Green 19, Direct Blue 54,
Pseudomonas putida (MTCC-1194) Reactive Blue 171, Reactive Red 141,
Acid Red 260 (50 mg L 1 each)
Microbial consortium (white-rot Direct Fast Scarlet 4BS; (50 mg L 1) 49, 2040, NA 24 99.1 NA Fang et al. (2004)
fungus 84 and Pseudomonas 110)
Four bacterial isolates consortium Acid Red 88 7.0, 35, 100 rpm 24 78 Reductive Khehra et al. (2005)
Bacillus cereus (BN-7), Acid Red 119, 24 99
Pseudomonas putida (BN-4), Acid Red 97, 24 94
Pseudomonas uorescence Acid Blue 113, 24 99
1
(BN-5) and Stenotrophomonas Reactive Red 120; (60 mg L each) 24 82
acidaminiphila (BN-3)
Mixed bacterial consortium JW-2 Reactive Violet 5R; (100 mg L 1) 6.58.5, 2537, static 36 100 NA Moosvi et al. (2007)
Bacterial consortium SV5 Ranocid Fast Blue; (100 mg L 1) 7.0, 37, static 24 100 NA Mathew and
Madamwar (2004)
Bacterial consortium Direct Red 81; (200 mg L 1) 7.0, 37, NA 35 90 NA Junnarkar et al. (2006)
Cyanobacterial cultures isolated Acid Red 97, FF Sky Blue, Amido NA, 27, static 26 days 7890 Oxidative Parikh and
(Gloeocapsa pleurocapsoides Black 10B; (100 mg L 1) Madamwar (2005)
and Chroococcus minutus)
1
Acclimatized Microbial consortium Reactive Black 5; (100 mg L ) NA, 37, static, 48 7090 Enzyme secretion Dafale et al. (2008)
Pseudomonas aeruginosa and anaerobic batch
Bacillus circulans, and
(NAD1 & NAD6) isolates
1
Five-member bacterial consortium; Direct Blue-15; (50 mg L ) NA, 37, static 24 92.14 NA Kumar et al. (2007)
Alcaligenes faecalis, Sphingomonas
sp. EBD, Bacillus subtilis, Bacillus
thuringiensis and Enterobacter
cancerogenus
1
Bacterial consortium RVM11.1 Reactive Violet 5; (200 mg L ) 6.58.5, 2540, static 37 94 NA Moosvi et al. (2005)
 R.G. Saratale et al. / Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 138157 145

performance liquid chromatography (HPLC) nuclear magnetic

Oxspring et al., 1996

resonance spectroscopy (NMR), and so on. The related studies of

Tony et al. (2009a,b)

the effects of various physicochemical parameters and enzymatic
mechanisms using pure bacterial culture on the decolorization of
azo dyes are summarized in Table 1.

2.2.5.2. Using co-culture and mixed bacterial cultures. Bacterial

decolorization is efcient and fast, but individual bacterial strains
usually cannot degrade azo dyes completely, and the intermediate
products are often carcinogenic aromatic amines, which need to be
further decomposed (Joshi et al., 2008). Thus, treatment systems
composed of mixed microbial populations achieve a higher degree
of biodegradation and mineralization due to the synergistic
metabolic activities of the microbial community, and have
considerable advantages over the use of pure cultures in the
NA

NA

degradation of synthetic azo dyes (Chen and Chang, 2007; Khehra

et al., 2005; Saratale et al., 2010b). In a microbial consortium, the
individual strains may attack the dye molecule at different
positions or may utilize metabolites produced by the co-existing
strains for further decomposition (Chang et al., 2004; Forgacs et al.,
2004; Jadhav et al., 2008b; Saratale et al., 2009b). In general, during
azo dye degradation initial cleavage of the azo bonds takes place,
95

93

which results into the production of aromatic amines which are

toxic in nature, however in the presence of microbial consortium
these aromatic amines get degraded by complementary organisms,
making the process more effective and efcient (Moosvi et al.,
10 days

2005, 2007; Tony et al., 2009a,b). In addition, it is time consuming

and difcult to isolate a single bacterial strain from dye-containing
48

wastewater samples, long-term adaptation procedures are then

required for effective decolorization and degradation of azo dyes.
Several studies concerning the biodegradation of colored waste-
anaerobic Filter (UAF)

rate of 60 ml/h (PBR)

7.0, 37, aeration rate

water using co-cultures and mixed bacterial cultures are given in

at batch condition

along with a ow

Tables 2 and 3, respectively.

of 0.6 mmol/min
between 12 and
7.0, room temp

20, Upow

3. Mechanisms of microbial color removal

Azo compounds are susceptible to biological degradation under

both aerobic and anaerobic conditions (Khehra et al., 2005). In
general microbial degradation of azo dyes involves the reductive
cleavage of azo bonds (N5 5N) with the help of an azoreductase
enzyme under anaerobic conditions, and this involves a transfer of
four-electrons (reducing equivalents). This then proceeds through
two stages at the azo linkage, and in each stage two electrons are
)
1

transferred to the azo dye, which acts as a nal electron acceptor,

)
1
Remazol Black B; (0.5 g L

resulting in dye decolorization and the formation of colorless

Direct Red 28 (10 mg L

solutions (Chang et al., 2000). The resulting intermediate

metabolites (e.g., aromatic amines) are then further degraded
aerobically or anaerobically (Chang et al., 2004; Pandey et al.,
2007). Decolorization of azo dyes under anaerobic conditions is
thought to be a relatively simple and non-specic process. The
efcacy of various anaerobic treatment applications for the
degradation of a wide variety of synthetic dyes has been
demonstrated many times (Wang et al., 2009a,b,c). It has been
reported that under anaerobic conditions a low redox potential
(50 mV) forms which causes the effective decolorization of the
azo dyes (Bromley-Challenor et al., 2004). Some investigators
Consortium of Alcaligenes faecalis,

Five isolates (Bacillus vallismortis,

B. pumilus, B. cereus, B. subtilis

suggest that the presence of oxygen usually inhibits the azo bond
Commomonas acidovorans

reduction activity, since aerobic respiration may dominate

utilization of NADH, thus impeding the electron transfer from
and B. megaterium)

NADH to azo bonds (Chang et al., 2001b). It was also reported that
anaerobic azo dye decolorization is a fortuitous process, where azo
NA: not available.

dye might act as an electron acceptor supplied by the carriers of the

electron transport chain (Bromley-Challenor et al., 2004; Stolz,
2001). Alternatively, decolorization might be attributed to non-
specic extracellular reactions occurring between reduced com-
pounds generated by the anaerobic biomass. (Van der Zee et al.,
 146
Table 3
Decolorization of various azo dyes by mixed bacterial cultures.

Name of strain Name of dye and Condition (pH, temp. Time (h) Decolorization Type of enzymes References
concentration (8C), agitation) (%) involved
1
Mixed culture Reactive Black 5; (1003000 mg L ) 6.07.0, RT, NA 48 90 Two stage Mohanty et al. (2006)
anaerobicaerobic
treatment
1
Mixed, mesophilic methanogenic Reactive Red 2; (502000 mg L ) 7.0, 35, static 250 days 9287 NA Beydilli and
culture (mg L 1 day 1
) Pavlostathis (2005)
Granulated anaerobic mixed Reactive Black 5 and Direct Anaerobic process, 35, 300 90 Reductive Isik and Sponza (2004)
culture Brown 2; (0.2 and 3.2 g L 1) agitation
Mixed culture of bacteria Remazol Brilliant Orange 3R, 30, 200 rpm, combined 24 78.9 NA Supaka (2004)
Remazol Black B and Remazol anaerobicaerobic system

R.G. Saratale et al. / Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 138157
Brilliant Violet 5R; (100 mg L 1)
Mixed anaerobic bacterial Acid Orange 7, and Direct Red NA, 35, 100 rpm 50 days 88 Reductive Bras et al. (2001)
consortia 254; (60 and 300 mg L 1)
Dried activated sludge Reactive Black 5; (116 mg L 1) 2.0, 20, NA 30 min 50 Adsorption Gulnaz et al. (2006)
Mixed bacterial cultures Mixture of azo-and NA 96 80 NA Nigam et al. (1996)
diazo-reactive dyes;
Mixed bacterial cultures Diazo-linked chromophore; (NA) 7.0, use of triple mix gas 48 85 Flavin coenzymes Knapp and Newby (1995)
H210%, CO210% N280%
at 30 8C strictly anaerobic
conditions
Mixed cultures Reactive and disperse textile 6, 37, 120 rpm 120240 100 NA Asgher et al. (2007)
dyes; (0.5 g L 1)
1
Methanogenic and Mixed bacteria Acid Orange 7; (60300 mg L ) NA, 37, NA 140 96 NA Bras et al. (2001)
cultures
Isolated halophilic and Remazol Black B, Maxilon Blue, 511, 2540, static 96 100 Reductive Asad et al. (2007)
halotolerant bacteria Sulphonyl Scarlet BNLE,
Sulphonyl Blue TLE, Sulphonyl
Green BLE, Remazol Black N,
and Entrazol Blue; IBC; (5 g L 1)
Mixed culture of bacteria Remazol Brilliant Orange 3R, NA, 30, 200 rpm, sequential 24 78.9 NA Supaka (2004)
Remazol Black B and Remazol anaerobicaerobic treatment
Brilliant Violet 5R; (0.51.0 g L 1) process
24 90 NA
Mixed culture Methyl Red; (2030 mg L 1) 6, 30, 180 rpm 16 82 Reductive Adedayo et al. (2004)
Mixed microbial culture Direct Black-38; (100 mg L 1) NA, 37, static 240 100 NA Kumar et al. (2007)
1
Activated sludge obtained from Reactive Red 3.1; (2030 mg L ) Anaerobic packed bed reactor 51 9093 NA Bromley-Challenor
domestic and industrial followed by an aerobic et al. (2004)
efuent treatment plants stirred tank reactor
Original seed sludge collected from Acid Red 42, Acid Red 73, NA 80 90 NA Goncalves et al. (2001)
a municipal wastewater Direct Red 80, Disperse
treatment plant Blue 56; (200 mg L 1)
Mixed bacterial population, with Wastewater containing An anaerobic bafed reactor NA NA NA Plumb et al. (2001)
sulphate-reducing bacteria, and up to 15 different
a methanogenic population sulfonated azo dyes
Sulphate-reducing bacteria, methane Hydrolysed Reactive NA 40 95 NA Yoo et al. (2000)
producing bacteria and fermentative Orange 96
bacteria in an anaerobic mixed culture 30 90
An uncharacterized aerobic biolm. Acid Orange 7 A rotating drum bioreactor 1 90 NA Coughlin et al. (2002)
Strain 1CX (Sphinogomonas sp.) and containing the biolm
Strain SAD4I (Gram-negative bacterium)
Bacillus cereus, Sphaerotilus natans, Azo dyes NA, NA, Anoxic conditions NA NA Reductive Wuhrmann et al. (1980)
Arthrobacter sp., activated sludge
Anaerobic digester sludge and aeration tank Acid Orange 10, Acid 2-stage anaerobic-aerobic NA 6590 NA Fitzgerald and
mixed liquor Red 14 Acid Red 18 (xed lm uidised Bishop (1995)
bed/suspended growth
activated sludge) reactor
 R.G. Saratale et al. / Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 138157 147

2001). Anaerobic decolorization of various azo dyes achieved with

mixed aerobic and facultative anaerobic microbial consortia has
been reported (Moosvi et al., 2005; Nigam et al., 1996), and
Nigam et al. (1996)

ONeil et al. (1999)

Banat et al. (1996)

although many of these cultures were able to grow aerobically,

decolorization was achieved only under anaerobic conditions.
Much of the experimental work involving the anaerobic decolori-
zation of dyes (predominantly azo dyes) was conducted using
mono cultures. Species of Bacillus, Pseudomonas, Aeromonas,
Proteus, Micrococcus and purple non-sulphur photosynthetic
bacteria were found to be effective in the anaerobic degradation
of a number of azo dyes (Table 1). It was also reported that in
anaerobic conditions the permeation of the azo dyes through
biological membrane into the microbial cells acts as the principal
rate-limiting factor for the decolorization (Kodam et al., 2005).
Several bacterial strains that can aerobically decolorize azo dyes
NA

NA

NA

have been isolated during the past few years. Under aerobic
conditions mono- and di-oxygenase enzymes catalyze the incorpo-
ration of oxygen from O2 into the aromatic ring of organic
compounds prior to ring ssion (Sarayu and Sandhya, 2010). Some
aerobic bacteria are able to reduce azo compounds with the help of
6884

oxygen catalysed azoreductases and produce aromatic amines (Lin

100

78

et al., 2010). It was also reported that the aerobic azo reductases were
able to use both NAD(P)H and NADH as cofactors and reductively
cleaved not only the carboxylated growth substrates of the bacteria,
but also the sulfonated structural analogues (Nachiyar and
Rajkumar, 2005). This type of azoreductase activity was found in
NA
48

48

Pseudomonas species strains K22 and KF46, and after purication

and characterization it was observed that this enzyme system was
avin-free (Zimmermann et al., 1982, 1984). These bacteria cannot
utilize azo dye as the growth substrate, and require additional
Upow Anaerobic Sludge

organic carbon sources (Stolz, 2001). Moreover, there are few

Anaerobic conditions

bacteria that are able to grow on azo compounds as the sole carbon
source. These bacteria cleave N5 5N bonds reductively and utilize
Blanket (UASB)

amines as the source of carbon and energy for their growth. Such
organisms are specic towards their substrate. Examples of bacterial
strains with this trait are Xenophilus azovorans KF 46 (previously
Pseudomonas sp. KF46) and Pigmentiphaga kullae K24 (previously
NA

Pseudomonas sp. K24), which can grow aerobically on carboxy

orange I and carboxy orange II, respectively (McMullan et al., 2001).
Only few bacteria with specialized azo dye reducing enzymes have
been found to degrade azo dyes under fully aerobic conditions
(Nachiyar and Rajkumar, 2003; Zimmerman et al., 1984). Finally,
Blumel and Stolz (2003) cloned and characterized the genetic code of
Reactive dyes, diazo dyes,

and phthalocyanine dyes

azo dyes, disperse dyes

the aerobic azo reductase from Pagmentiphaga kullae K24.

wastewater containing
diazo-reactive dyes

Procion Red HE7B

Simulated textile
Various azo and

4. Factors affecting bacterial decolorization

In the textile industry different structures of synthetic dyes are

often used during ber processing, and therefore the efuents
produced are markedly variable in chemical composition, includ-
ing organics, nutrients, sulphur compounds, salts and different
toxic substances (Chen et al., 2003; Ghodake et al., 2009b; Pearce
Alcaligenes faecalis, Commomonas acidovorans

et al., 2003). In biological treatment processes, various physico-

plant (Inclined Tubular Digester) granules
Sludge from textile wastewater treatment

chemical operational parameters, such as the level of agitation,

Thermophilic anaerobic bacterial culture

oxygen, temperature, pH, dye structure, dye concentration,

(Upow Anaerobic Sludge Blanket)
from paper pulp processing plant

supplementation of different carbon and nitrogen sources, electron

donor and redox mediator, directly inuence the bacterial
decolorization performance of azo dyes. Thus, to make the process
more efcient, faster and practically applicable, prior determina-
tion of the effect of each factor on the bacterial decolorization of
azo dyes is essential.
NA: not available.

4.1. Effects of oxygen and agitation

Decolorization of azo dyes occurs under strictly anaerobic

(methanogenic), facultative anaerobic and aerobic conditions by
148 R.G. Saratale et al. / Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 138157
different trophic groups of bacteria. It has been reported that under
anaerobic conditions the feeding of a carbon source, such as simple
substrates to a bacterial culture like glucose, starch, acetate, and
ethanol, and more complex ones, such as whey and tapioca, could
affect the decolorization process and that most of the reduction of
azo dyes to amines occurs during active bacterial growth (Banat
et al., 1996; Van der Zee and Villaverde, 2005; Yoo et al., 2000). It
was also observed that the decolorization of azo dyes was far
superior under strictly anaerobic conditions, although it also
occurred under semi-anaerobic ones (Knapp and Newby, 1995).
Similar results were observed in studies on pure bacterial strains
such as Pseudomonas luteola, Proteus mirabilis, Pseudomonas sp.
SUK1, Micrococcus glutamicus NCIM-2168 (Chang et al., 2001b;
Chen et al., 1999; Kalyani et al., 2008; Saratale et al., 2009c). It is
assumed that under anaerobic conditions reductive enzyme
activities are higher, however a small amount of oxygen is also
required for the oxidative enzymes which are involved in the
degradation of azo dyes. Some studies have reported that during
bacterial degradation of azo dyes both oxidative and reductive
enzymes play a role (Tables 1 and 2). These earlier results suggest
that for efcient color removal aeration and agitation, which
increases the concentration of oxygen in the solution, should be
avoided (Chang and Lin, 2001). It was also observed that the effect
of oxygen on azoreduction is irreversible (Bragger et al., 1997;
Pearce et al., 2003). Quantitative analysis of the correlation
between DO and the decolorization rate in the decolorization of
C.I. Reactive Red 22 by Escherichia coli NO3 has been studied in
detail (Chang and Kuo, 2000). In this study under static conditions
(no agitation) the DO level in the culture immediately dropped to
nearly zero, and thus decolorization occurred, whereas under
agitation at a rate of 200 rpm the DO level only decreased to
0.5 mg L 1, and no signicant color removal was observed. Similar
results were observed in the decolorization of azo dyes by pure
culture, and the results are summarized in Table 1. The
intermediates formed during azo dye reduction reaction, like
the simple aromatic compounds, are degraded via hydroxylation
and ring-opening in the presence of oxygen (Chang et al., 2001b;
Pandey et al., 2007). The aerobic condition is required for the
complete mineralization of the azo dye molecules. Thus, for the
most effective efuent treatment an anaerobic process with
subsequent aerobic treatment can be used to decolorize waste-
waters containing dyes and improve their biodegradability
(Khehra et al., 2006; You and Teng, 2009).
4.2. Effects of carbon and nitrogen sources supplements
Azo dyes are decient in carbon sources, and the biodegrada-
tion of dyes without any supplement of carbon or nitrogen sources
is very difcult (Sani and Banerjee, 1999). Azo dye decolorization
by mixed as well as pure cultures generally requires complex
organic sources, such as yeast extract, peptone, or a combination of
complex organic sources and carbohydrates (Chen et al., 2003;
Khehra et al., 2005). During decolorization of azo dyes via reduction
of azo bonds, it was reported that reducing equivalents from
various carbon sources are transferred to the dye. It was also
observed that in anaerobic consortia, acidogenic bacteria convert
the soluble substrates, such as carbohydrates, to volatile organic
acids or alcohols, such as acetic acid and methanol, which act as
competitive substrates for methanogenic, sulfate reducing, and
acetogenic bacteria (Georgiou et al., 2004; Yoo et al., 2000). Some
studies performed the azo dye decolorization in the presence of
additional carbon and nitrogen sources. In these, the addition of
carbon sources seemed to be less effective in promoting
decolorization, probably due to the preference of the cells in
assimilating the extra carbon sources over using the dye
compound as the carbon source (Saratale et al., 2009b). In contrast,
addition of the organic nitrogen sources, such as peptone, beef
extract, urea, yeast extract and so on, can regenerate NADH, which
acts as an electron donor for the reduction of azo dyes by
microorganisms, and thus effective decolorization was observed
(Chang et al., 2000). To make the process economically feasible and
practically applicable, some investigators have used lignocellulosic
agricultural waste as an additional supplement for effective
decolorization. Lignocelluloses constitute a major portion of
agricultural and forest wastes, and industrial efuents from the
global pulp/paper, food and other industries produce up to
0.85  1011 t these substances per annum (Saratale et al., 2008).
It was observed that the presence of lignocellulosic substrates
enhances decolorization by effective production of lignolytic
enzymes. Similar results were also reported for Comamonas sp.
UVS, which shows effective decolorization of Direct Blue GLL along
with the production of lignolytic enzymes (Jadhav et al., 2008c).
With regard to the decolorization of Scarlet R by bacterial
consortium GR and Reactive Green 19A by Micrococcus glutamicus
NCIM-2168, we have studied various agricultural wastes, such as
extract of sugarcane baggase powder, wood straw, rice husk and
rice straw, to enhance the decolorization performance. In these
studies supplementation of rice husk and rice straw extract acts as
a nitrogen source which enhances the decolorization performance
in synthetic media (Saratale et al., 2009b,c).
4.3. Effects of temperature
In microorganisms the environmental temperature directly
establishes organismal temperature, as the microbial cell responds
to temperature changes by adaptation via biochemical or enzymatic
mechanisms. Consequently, temperature is a factor of paramount
importance for all processes associated with microbial vitality,
including the remediation of water and soil. Some studies dealing
with the activation energy of microbial decolorization of azo dyes
has been undertaken (Chang and Kuo, 2000; dos Santos et al., 2007),
in which narrow temperature ranges were determined as being
necessary for the decolorization of azo dyes by extremely complex
consortia of microorganisms inhabiting active sludge. In addition it
has also been reported that in microbial physiology temperature
changes lead to a sudden alteration of the activation energy (Yu et al.,
2001). Moreover, the effects of temperature on the growth rate,
biomass yield and reaction mechanism have also been reported
(Blaga et al., 2008). It was observed that the decolorization rate of azo
dyes increases up to the optimal temperature, and afterwards there
is a marginal reduction in the decolorization activity. This decline at
higher temperatures can be attributed to the loss of cell viability or
the denaturation of an azo reductase enzyme (Chang et al., 2001a;
Saratale et al., 2009c). However, it has been shown that with certain
whole bacterial cell preparations the azoreductase enzyme is
relatively thermostable and can remain active up to temperatures
of 60 8C over short periods of time (Pearce et al., 2003).
4.4. Effects of pH
The medium pH is also an important factor with regard to
decolorization. The pH has a major effect on the efciency of dye
decolorization, and the optimal pH for color removal is often
between 6.0 and 10.0 (Chen et al., 2003; Guo et al., 2007; Kilic et al.,
2007). The rate of color removal is higher at the optimum pH, and
tends to decrease rapidly at strongly acid or strongly alkaline pH. It is
thought that the effects of pH may be related to the transport of dye
molecules across the cell membrane, which is considered as the rate-
limiting step for the decolorization (Chang et al., 2001b; Kodam et al.,
2005). In our laboratories similar results were observed with many
microbial strains (Table 1). Biological reduction of the azo bond can
result in an increase in the pH due to the formation of aromatic
 R.G. Saratale et al. / Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 138157
amine metabolites, which are more basic than the original azo
compound (Willmott, 1997). Generally altering the pH within a
range of 7.09.5 has very little effect on the dye reduction process.
However Chang et al. (2001b) found that the dye reduction rate
increased nearly 2.5-fold as the pH was raised from 5.0 to 7.0, while
the rate became insensitive to pH in the range of 7.09.5. Jadhav et al.
(2008a) showed that with the decolorization of Brilliant Blue G by
consortium-GB (combination of Galactomyces geotrichum and
Bacillus sp.) the decolorization was not pH dependent, and complete
decolorization was observed in the pH range from 5 to 9. The
effective decolorization of Reactive Red 190 by isolated Citrobacter
sp. CK3 in strongly acidic (at pH 4) and strongly alkaline (at pH 12)
conditions has also been reported (Wang et al., 2009b). This pH
tolerance of decolorizing bacteria is quite important, as it makes
them suitable for practical bio-treatment of dyeing mill efuents
(Aksu and Donmez, 2003; Wang et al., 2009a).
4.5. Effects of dye concentration
A survey of the literature suggests that increasing the dye
concentration gradually decreases the decolorization rate, proba-
bly due to the toxic effect of dyes with regard to the individual
bacteria and/or inadequate biomass concentration (or improper
cell to dye ratio), as well as blockage of active sites of azoreductase
by dye molecules with different structures (Jadhav et al., 2008a;
Sani and Banerjee, 1999; Saratale et al., 2009c; Tony et al., 2009a,b).
Similar results were observed in the bacterial decolorization of
various reactive azo dyes (Dhanve et al., 2008; Kalyani et al., 2008;
Saratale et al., 2009b). It was also observed that reactive group azo
dyes with sulfonic acid (SO3H) groups on their aromatic rings
greatly inhibited the growth of microorganisms at higher dye
concentrations (Chen et al., 2003; Kalyani et al., 2008). However,
Saratale et al. (2009b) found that the increasing concentration
effect was reduced when bacterial co-culture was used instead of
pure culture, and this might be due to the synergistic effect of both
microorganisms. Moreover, Dubin and Wright (1975) reported the
absence of any effect of dye concentration on the reduction rate.
This observation is compatible with a non-enzymatic reduction
mechanism that is controlled by processes that are independent of
the dye concentration. A detailed kinetic study and the relation
between substrate dependent specic decolorization rate and
equilibrium conversion have been reported by a number of studies
(Chang et al., 2000; Pearce et al., 2003; Telke et al., 2008).
4.6. Effects of dye structure
There are diverse structures present in the synthetic azo dyes,
and changes in the chemical structures, i.e. isomers or the presence
of different functional groups, signicantly affects the decolorization
capability in the form of biodegradability and reduction. It has been
reported that dyes with simple structures and low molecular
weights exhibit higher rates of color removal, whereas the removal
rate is lower in the case of dyes with substitution of electron
withdrawing groups such as SO3H, SO2NH2 in the para position of
the phenyl ring, relative to the azo bond and high molecular weight
dyes (Hsueh et al., 2009; Pearce et al., 2003; Sani and Banerjee, 1999).
Similarly, it has also been reported that the color removal rate is
faster in the case of monoazo dyes compared to diazo and triazo ones
(Hu, 2001). It was also observed that the enzyme (azoreductase)
production was related to particular dye structures (Kulla et al.,
1983). Specically, azo compounds with hydroxyl or amino groups
are more likely to be degraded than those with methyl, methoxy,
sulpho or nitro groups. With regard to the reduction of azo dyes,
reduction to the anion radical occurs by a fast one-electron transfer
reaction, followed by a second, slower electron transfer event to
produce the stable dianion (Rau et al., 2002; Zimmermann et al.,
149
1982). Thus the functional group of azo dyes with a higher electronic
density might be not favorable to this second electron transfer
forming the dianion, leading to low or no capability for decoloriza-
tion (Pearce et al., 2003). Due to this, the sulfonated reactive group of
azo dyes are normally considered to be more recalcitrant than
carboxylated azo dyes, while the rate-limiting step during bacterial
decolorization of sulfonated azo dyes is the permeation of dyes
through the bacterial cell membrane (Kodam et al., 2005; Lorenco
et al., 2000). In addition, the electron density hydrogen bonding in
the region of the azo bond also has a signicant effect on the
reduction rate (Beydilli et al., 2000). This is likely due to the
contribution of hydrogen bonding directly inuencing the azo-
hydrazone tautomerism of hydroxyazo compounds (Hsueh et al.,
2009). Puried Orange II azoreductase from a Pseudomonas strain
KF46 has been studied for its decolorization efciency with regard to
various orange dyes, in which the specicity of azoreductase was
found to be strongly dependent upon the electron-withdrawing
ability of functional groups in the proximity of the azo linkage by
which the dye is susceptible to biodecolorization (Zimmermann
et al., 1982). It was also noticed that if the substituent is in the para
position the azo linkage becomes more susceptible for decoloriza-
tion compared to the ortho and meta positions of the phenyl ring
(Hsueh et al., 2009). Zimmermann et al. (1982) reported that the
electron-withdrawing groups present on the phenyl ring accelerate
this color removal. The aerobic biodegradability of 25 sulfonated azo
dyes with their chemical structures was reported by Suzuki et al.
(2001), who found that the steric effect of chemical structure
strongly affected the color removal efciency. Moreover, in the case
of the terminal non-enzymatic reduction mechanism, reduction
rates are inuenced by changes in an electron density in the region of
the azo group, causing an increase in the reduction rate (Rau et al.,
2002; Walker and Ryan, 1971).
4.7. Effects of electron donor
The azo dyes and the other organic content of textile
wastewater are too low to act as a sufcient substrate for the
growth of anaerobic bacteria and thus for complete decolorization
to take place. Therefore it is necessary to have an external substrate
(electron-donor) supply to enhance the anaerobic decolorization
performance (Telke et al., 2009a). Various electron donors and
electron acceptors are always present in the environment, but it is
important to study the effects of different donors and acceptors on
bacterial azoreduction (He and Sanford, 2003). The electron donors
sodium acetate, sodium formate, sodium succinate, sodium citrate
and sodium pyruvate have been shown to enhance the azo dye
decolorization of C.I. Reactive Orange 16 with isolated Bacillus sp.
ADR (Telke et al., 2009a) and various azo dyes with isolated
Shewanella decolorationis S12 (Hong et al., 2007). It has been
suggested that the bacterial anaerobic azoreduction is a biochemi-
cal process that oxidizes the electron donors and transfers the
electrons to the acceptors through a multicomponent system
related to the electron transport chain. In addition dehydro-
genases, cytochromes, and menaquinones act as essential electron
transport components for the azoreduction mechanism. It has
been observed that in the presence of some electron donors the
electron transport process is inhibited, and this might be due to the
competition for electrons from the donors (Georgiou et al., 2004). A
thermodynamic study found that the various electron-donating
half-reactions are different due to the diverse reaction rates, which
are likely to be inuenced by the type of electron donor (Pearce
et al., 2003). It has also been observed that the addition of electron
donors, such as glucose or acetate ions, apparently induces the
reductive cleavage of azo bonds (Bras et al., 2001). Moreover,
formate acts as a most effective electron donor for the anaerobi-
cally induced electron transfer pathway to the dye molecules, and
150 R.G. Saratale et al. / Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 138157
this may be because the pathway involves the formate dehydro-
genase enzyme (Lloyd et al., 1997). Amongst these substrates
methanol deserves particular attention, since it is widely used as a
cost-effective electron donor for the biological treatment of
wastewater. It has been noted that the products of cell lysate
residue can function as electron donors for an anaerobic azo dye
reduction, with the active cells metabolizing the lysis products
(Yoo et al., 2000). These ndings aid better understanding of the
mechanism of bacterial anaerobic azoreduction, and have implica-
tions for improving treatment methods for wastewater contami-
nated with azo dyes. It is also important to determine the
physiological electron donors for each biological color removal
process, because the donors not only induce the reduction
mechanism, but also stimulate the enzymatic system responsible
for the reduction process (Pearce et al., 2003; Van der Zee et al.,
2001). Similarly, during decolorization of C.I. Reactive Orange 16
by isolated Bacillus sp., ADR requires NADH as an electron donor for
NADH-DCIP reductase, and in the presence of articial electron
donors, such as sodium acetate, sodium formate, sodium citrate,
and sodium pyruvate, induction in the reduction activity was
observed (Telke et al., 2009a). In contrast, certain chemicals, such
as thiomersal and p-chloromercuibenzoate, inhibit the alcohol
dehydrogenase of the NADH-generating systems required to
produce reducing equivalents for dye reduction (Georgiou et al.,
2004). In addition, coenzyme-reducing equivalents that are
involved in normal electron transport by the oxidation of organic
substances may act as the electron donors for azo dye reduction
(Plumb et al., 2001).
4.8. Effects of redox mediator
The transfer of reducing equivalents from a primary electron
donor (co-substrate) to a terminal electron acceptor (azo dye)
generally acts as the rate-limiting step in the anaerobic azo dye
reduction process (Van der Zee et al., 2001). It has been observed
that the azo dye decolorization activity is also related to the
electron density in the region of the azo bond, and that lowering
the electron density in the azo link could enhance the color
removal rate (Walker and Ryan, 1971). The supplementation of
redox mediators accelerates the transfer of reducing equivalents to
the terminal electron acceptor (i.e. azo dye), and also minimizes the
steric hindrance of the dye molecule (Bragger et al., 1997; Moir
et al., 2001; Rau and Stolz, 2003). Flavin-based compounds, such as
avin adenide dinucleotide (FAD) and avin adenide mononucle-
otide (FMN), and quinone-based compounds such as anthraqui-
none-2,6-disulfonate (AQDS), anthraquinone-2-sulfonate (AQS),
riboavin (vitamin B2), cyanocobalamin (vitamin B12) and
lawsone (2-hydroxy-1,4-naphthoquinone), have been extensively
reported as redox mediators (dos Santos et al., 2004). A very small
concentration of the redox mediator is sufcient for this type of
electron transfer. Redox mediators are characterized by a redox
potential ranging from 200 to 350 mV (Semde et al., 1998). The
stimulating performance of redox mediators is dependent on the
redox potential, which measures the ease with which a molecule
will accept electrons and can be reduced. It has been reported that
the color removal rate is highest when the redox potential of the
system is at its most negative, and the rate falls as the redox
potential of the system rises (Liu et al., 2009; Rau et al., 2002). It was
also reported that quinoide redox mediators, such as lawsone, AQS
and AQDS, utilize cytoplasmic or membrane-bound quinone
reductase by which they can shuttle reducing equivalents from
bacteria to azo dyes (Rau and Stolz, 2003; Rau et al., 2002).
Enhancement in the anaerobic reduction of azo dyes by Escherichia
coli was observed in the presence of a redox mediator, and this is
because of the induction in the lawsone-dependent azoreductases
activity reported earlier (Rau et al., 2002). Similarly, enhancement
in the decolorization performance of azo dyes in the presence of
lawsone at a concentration of 0.2 mM by Escherichia coli JM109 was
also observed. However, with further increases in the lawsone
concentration over 0.2 mM the stimulating effect on decoloriza-
tion decreased, and it is suggested that this might be due to the
toxicity of a high concentration of lawsone or the lack of sufcient
electron sources (Liu et al., 2009; Sauriasari et al., 2007). Lorenco
et al. (2001) observed some color removal in the presence of
autoclaved cells, suggesting the existence of an active reducing
factor that is capable of dye reduction in the absence of microbial
activity. The effect of redox mediators on the decolorization rates
has generally been investigated with azo model compounds as well
as in the decolorization of textile wastewaters. However, the effect
of redox mediators in enhancing the decolorization of textile
wastewater is still unclear due to the wide range of redox
potentials among azo dyes ( 180 to 430 mV) present in
wastewater, high dye COD concentration (about 1.4 g L 1) and
the different properties of the dyes (dos Santos et al., 2004).
5. Enzymes involved in the bacterial decolorization and
degradation of azo dyes
The enzymatic approach has attracted much interest with
regard to decolorization/degradation of azo dyes from wastewater
as an alternative strategy to conventional physicochemical
treatments. The oxidoreductive enzymes are responsible for
generating highly reactive free radicals that undergo a complex
series of spontaneous cleavage reactions. It has been observed that,
due to the susceptibility of enzymes to inactivation in the presence
of the other chemicals. It is likely that enzymatic treatment will be
the most effective in those streams that have the highest
concentrations of target contaminants and the lowest concentra-
tion of other contaminants that may tend to interfere with the
treatment. The oxidoreductive enzymes, such as lignin peroxidase,
laccases, tyrosinase, azoreductase, riboavin reductase, NADH-
DCIP reductase and aminopyrine N-demethylase, have been
mainly utilized in the bacterial decolorization and degradation
of azo dyes, and these are described in this section (Campos et al.,
2001; Heining et al., 1998; Maier et al., 2004; Ryan et al., 2003;
Suzuki et al., 2001).
5.1. Reductive enzymes involved in the bacterial degradation of azo
dyes
Azoreductases are the enzymes which catalyze the reductive
cleavage of azo bonds (N5 5N) to produce colorless aromatic
amine products (Chang et al., 2001b). Azoreductases are observed
in many organisms, including the rat liver enzyme, rabbit liver
aldehyde oxidase and intestinal microbiota (Chen et al., 2005a,b).
Several studies have been investigated bacterial cytoplasmic
azoreductases, and suggested that they can be applied for the
purpose of environmental biotechnology (Maier et al., 2004;
Ramalho et al., 2004; Zimmermann et al., 1982). On the basis of
their functions, azoreductases are categorized as avin-dependant
azoreductases (Chen et al., 2005a,b) and avin-independent
azoreductases (Blumel and Stolz, 2003). The avin-dependent
azoreductases are further organized on the basis of their cofactor,
NADH only, NADPH only (Chen et al., 2005a,b) or both (Wang et al.,
2007), as these coenzymes serve as electron donors. Based on the
primary amino acid level the classication of azoreductases is
found to be difcult, hence recently a classication based on the
secondary and tertiary amino acid analyses has been developed
(Abraham, 2007). Azoreductases from bacteria represent novel
families of enzymes with little similarity to other reductases. For
example the expression of Bacillus subtilis azoreductases AzoR1
and AzoR2, which have around 30% amino acid sequence identities
 R.G. Saratale et al. / Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 138157
with E. coli AzoR, can both be induced by electrophiles, such as 2-
methylhydroquinone (2-MHQ), catechol and diamide (Leelak-
riangsak et al., 2008). The purication and characterization of
azoreductase enzymes from many bacteria, such as Xenophilus
azovorans KF46F, Pigmentiphaga kullae K24, Enterococcus faecalis,
Staphylococcus aureus, Escherichia coli, Bacillus sp. OY1-2, and
Rhodobacter sphaeroides have been extensively studied (Blumel
and Stolz, 2003; Chen et al., 2005a,b; Suzuki et al., 2001). Substrate
specicity of the azoreductase has been studied by using synthetic
model substrates based on disodium-(R)-benzyl-azo-2,7-dihy-
droxy-3,6-disulfonyl-naphthalene (Maier et al., 2004). The in-
volvement of intracellular azoreductase in bacterial decolorization
has been doubted in recent years. Due to their high polarities and
complex structures, many azo dyes are difcult to diffuse through
cell membranes, although intracellular azoreductase enzyme
activity has been observed in many bacteria (Saratale, 2009; Telke
et al., 2008). It has also been reported that two typical avin-
dependent azoreductases, AZR of Rhodobacter sphaeroides and
AzoR of E. coli, have quinone reductase activities (Liu et al., 2009).
Chen et al. (2005a,b) reported an aerobic avin mononucleotide
(FMN)-dependent azoreductase from Enterococcus faecalis, and
showed its capability for degrading a wide range of azo dyes, while
they also found that a human intestinal bacterium, Pseudomonas
aeruginosa, shows oxygen-insensitive azoreductase activity to-
wards several azo dyes. The decolorization of various azo dyes by
using pure bacterial cultures or co-cultures and the involvement of
reductive enzymes are summarized in Tables 1 and 2.
In addition, some repots have found that NADH-DCIP reductases
are marker enzymes of bacterial and fungal mixed function oxidase
systems, and take part in the detoxication of xenobiotic
compounds (Bhosale et al., 2006; Saratale et al., 2007a). Several
studies in our laboratory reported this as a marker enzyme for the
reduction of azo bonds, because in the presence of this enzyme the
2,6-dichlorophenolindophenol (DCIP) accepts an electron from
NADH to form its leuco variety. Originally DCIP in its oxidized form
is blue in color, and becomes colorless when it is reduced.
Involvement of NADH-DCIP reductase in the degradation of Reactive
Orange 16 by Bacillus sp. ADR, Methy Red by Brevibacillus
laterospores, Red BL1 by isolated Pseudomonas sp. SUK1 and Direct
Brown MR by Acinetobacter calcoaceticus have all been reported
(Ghodake et al., 2009a; Gomare and Govindwar, 2009; Kalyani et al.,
2008; Telke et al., 2009a). In addition, involvement of riboavin
reductase in the degradation of azo dyes has been found (Russ et al.,
2000). In this process, the non-enzymatic reduction of free avins by
NADPH and NADH is rather slow, and requires the organism to
possess a system to catalyze the reaction. As a result, the enzyme
NAD(P)H:avin oxidoreductase or avin reductase are evolved. The
enzyme avin reductase catalyzes the reduction of various avins,
that is riboavin, avin mononucleotide (FMN) and avin adenine
dinucleotide (FAD), at the expense of reduced pyridine nucleotides
(Ingelman et al., 1999). The role of riboavin reductase was
determined in the degradation of Mordant Yellow 10 by using
anaerobic granular sludge and Brilliant Blue G by consortium-GB
(consisting of Galactomyces geotrichum MTCC 1360 and Bacillus sp.
VUS) (Field and Brady, 2003; Jadhav et al., 2008a). Moreover, the
involvement of both these enzymes in the degradation of Scarlet R by
Consortium-GR (consisting of P. vulgaris and M. glutamicus), Reactive
Green 19A by Micrococcus glutamicus NCIM 2168 and Navy Blue GL
by isolated Bacillus sp. VUS has also been reported (Dawkar et al.,
2009; Saratale et al., 2009b,c).
5.2. Oxidative enzymes involved in the bacterial degradation of azo
dyes
Along with the reductive enzymes, some investigators have
demonstrated the involvement of some oxidative enzymes, such as
151
lignin peroxidase, laccase, and tyrosinase, in the bacterial decolori-
zation and degradation of azo dyes. Lignin peroxidase (EC 1.11.1.14;
LiP) was rst discovered based on the H2O2-dependent CaCb
cleavage of lignin model compounds, and subsequently shown to
catalyze the depolymerization of methylated lignin in vitro (Saratale
et al., 2010b). LiP are glycoproteins with molecular weights
estimated at 3846 kDa. LiP has been used to mineralize a variety
of recalcitrant aromatic compounds, such as three and four-ring
polyaromatic hydrocarbons (PAHs), polychlorinated biphenyls and
synthetic dyes (Dawkar et al., 2009; Duran and Esposito, 2000). The
mechanism of azo dye oxidation by peroxidases such as LiP probably
involves the oxidation of the phenolic group to produce a radical at
the carbon bearing the azo linkages. The water attacks this phenolic
carbon to cleave the molecule producing phenyldiazene, and the
phenyldiazene can then be oxidized by a one-electron reaction
generating N2 (Chivukula and Renganathan, 1995). The ligninolytic
enzyme system is widely studied in the white-rot fungi Phaner-
ochaete chrysosporium, which is able to decolorize several recalci-
trant dyes (Verma and Madamwar, 2003). To date, very few studies
have reported LiP activity in the bacteria for the decolorization of azo
dyes, although some have found that it is involved in the asymmetric
cleavage of azo dyes (Dhanve et al., 2008; Saratale et al., 2009c). The
role of oxidative enzymes in the decolorization of sulfonated
reactive group of azo dyes has been extensively studied (Table 1).
Moreover, it has been observed that the presence of lignocellulosic
substrates enhances decolorization by effective production of the
lignolytic enzymes responsible for decolorization (Jadhav et al.,
2008c). Recently, purication and characterization of the lignin
peroxidase activity from the Bacillus sp. strain VUS and from
Acinetobacter calcoaceticus NCIM 2890 have been studied, and it was
observed that puried enzymes have a greater ability to degrade
various azo dyes (Dawkar et al., 2009; Ghodake et al., 2009a).
Laccases (E.C.1.10.3.2), also known as phenol oxidase, are
copper containing enzymes that catalyze the oxidation of several
aromatic and inorganic substances (particularly phenols) with the
concomitant reduction of oxygen to water (Majcherczyk et al.,
1998). The molecular structure of laccases exhibits four neighbor-
ing copper atoms, which are distributed among different binding-
sites, and they are classied into three types: Copper I, II and III,
which are differentiated by specic characteristic properties that
allow them to play an important role in the catalytic mechanism of
the enzyme. The molecular weight of laccase was found to be in the
range of 60390 kDa (Kalme et al., 2009). Laccases and immobi-
lized laccases have been intensively studied with regard to the
degradation of various recalcitrant compounds, such as chlor-
ophenols, PAHs, lignin-related structures, organophosphorous
compounds, phenols and azo dyes (Abadulla et al., 2000; Chivukula
and Renganathan, 1995; Duran et al., 2002; Saratale et al., 2009a).
This enzyme decolorizes some azo dyes without direct cleavage of
the azo bonds through a highly non-specic free radical mecha-
nism, thereby avoiding the formation of toxic aromatic amines
(Kalme et al., 2009). Laccase activity for the decolorization of
various synthetic dyes has mainly been studied in lignolytic fungi
(Abadulla et al., 2000). Although the biochemical characterization
of laccase from bacterial resources has also been studied
(Diamantidis et al., 2000; McMahon et al., 2007), little information
is available related to substrate specicities for dye decolorization.
In addition, very few bacteria (Micrococcus glutamicus NCIM 2168,
Pseudomonas desmolyticum NCIM 2112) express the phenol
oxidase activity that catalyzes the oxidation of azo dyes (Kalme
et al., 2007; Saratale et al., 2009c). Recently Telke et al. (2009a)
reported a novel enzyme, the laccase-like Bacillus sp. ADR phenol
oxidase, which is involved in the degradation of C.I. Reactive
Orange 16. Surprisingly, this phenol oxidase can react with
nonphenolic substrates, which explains the existence of bacterial
phenolic and nonphenolic oxidation systems that exist without
152 R.G. Saratale et al. / Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 138157
lignin peroxidase. Kalme et al. (2009) puried and characterized
the laccase enzyme from Pseudomonas desmolyticum NCIM 2112,
and tested it against various azo dyes. In addition, purication and
characterization of an extracellular laccase from a Pseudomonas sp.
LBC1 and its application for the removal of bisphenol A has also
been reported (Telke et al., 2009b).
Tyrosinase (EC1.14.18.1), also known as monophenol mono-
oxygenase, catalyzes the oxidation of phenol. This enzyme system
has been used for phenol removal, in which molecular oxygen
rather than hydrogen peroxide is the oxidant, theoretically
reducing the potential cost of applying the technology (Wu
et al., 2001). The enzymatic reaction is carried out in two phases;
in the rst phase hydroxylation of monophenols leading to
o-diphenols takes place, with the resulting product often known
as monophenolase (Duran and Esposito, 2000), while in the second
phase oxidation of o-diphenols to o-quinones, which is often
referred to as o-diphenolase, occurs. In these reactions oxygen is
used as an oxidant and o-quinone as a product which can inactivate
the tyrosinase activity. Therefore, phenoloxidases of the tyrosinase
type have both cresolase and catecholase activity (Kalme et al.,
2009). Use of the tyrosinase enzyme from Bacillus thuringiensis for
the decontamination of water polluted with phenols has also been
reported (El-Shora and Metwally, 2008). This enzyme acts as a
marker of the oxidative enzymes involved in the degradation of azo
dyes. Moreover, the involvement of tyrosinase in the degradation
of Direct Blue-6 by Pseudomonas desmolyticum NCIM 2112 (Kalme
et al., 2007) and disperse dye brown 3REL by a microbial
consortium consisting of Galactomyces geotrichum MTCC 1360
and Bacillus sp. VUS (Jadhav et al., 2008b) has been demonstrated.
In addition, purication of the veratryl alcohol oxidase enzyme
from Comamonas sp. UVS during decolorization of Red HE7B and
Direct Blue GL has been reported (Jadhav et al., 2009a,b).
Some investigators checked the marker enzyme of the mixed
function oxidase system aminopyrine-N demethylase activity
during the decolorization of azo dyes. This enzyme has been
widely investigated in toxicological studies (Govindwar et al.,
1983), and requires NADH or NADPH as cofactors in which
aminopyrine is converted into formaldehyde, as detected by Nash
reagent (Bhosale et al., 2006). Involvement of this enzyme in the
degradation of kerosene by Aspergillus ochraceus NCIM-1146
(Saratale et al., 2007a) as well as in the degradation of azo dyes
by Pseudomonas sp. SUK1, Pseudomonas desmolyticum NCIM 2112,
and isolated bacterial consortium PMB11 has been reported
(Kalme et al., 2007; Kalyani et al., 2008; Patil et al., 2008). The
results of these earlier studies suggest reductive enzymes are
initially required for the degradation of azo dyes, and that some
oxidative enzymes are needed if complete mineralization is to take
place.
6. Analytical methods employed for the decolorization studies
In order to uncover the possible mechanism of dye decoloriza-
tion, various analytical techniques were used to identify the
metabolites generated from azo dyes after bacterial treatment.
UVvis spectroscopy (UVvis) is the primary technique to
determine dye decolorization (Knapp and Newby, 1995). The
disappearance of the sharp peak (at lmax) is observed after
decolorization of the azo dye, and further evidence of the removal
of the dye can be observed with an increase in absorbance towards
the UV region (Saratale et al., 2009a). For the mixture of dyes and
industrial efuents, the true color level independent of hue was
measured using the American Dye Manufacturers Institute
tristimulus lter method (ADMI 3WL) and ADMI 31WL using 31
different wavelengths (Chen et al., 2003), thereby opening the way
to a more accurate denition of water and wastewater color (dos
Santos et al., 2007). The thin layer chromatography (TLC) technique
has proved to be an ideal solution with the separation power and
spot/zone capacity necessary to resolve closely related dyes and
their intermediates, and this is made possible by choosing different
stationary phases from the almost unlimited variations in mobile-
phase mixtures. Some previous investigators (Bhatt et al., 2005;
Mohana et al., 2008) used high performance thin layer chroma-
tography (HPTLC) for the study of dye degradation. This technique
also follows the same principles as TLC, and has some advantages
such as automatic application devices, smaller plates and better
sensitivity. Current liquid chromatography generally utilizes very
small packing particles and a relatively high pressure, and this is
referred to as high performance liquid chromatography (HPLC). In
dye degradation studies (Chang et al., 2001b; Lopez-Grimau and
Gutierrez, 2006; Saratale et al., 2010b), the degradation is
conrmed by the occurrence of new HPLC peaks with different
retention times compared to the original dye, and these also
indicate the formation of new structure analogues. The retention
time (Rt) can be dened as the time required for a band to travel
the length of column, and it is generally given in seconds or
minutes. Moreover, Fourier transform infrared spectroscopy (FTIR)
is widely used in infrared spectroscopy. In dye degradation studies
FTIR spectrum enables the determination of both the type and
strength of interactions that occur within azo dyes containing
different functional groups after biodegradation by bacteria, and
thus it acts as a valuable analytical tool. In mass spectrometry, the
mass spectra of the mixture components are acquired, providing a
very powerful qualitative analysis tools. When the mobile phase is
a liquid or gas, the technique is called liquid chromatography
mass spectrometry (LCMS) or gas chromatographymass spec-
trometry (GCMS). Both theses techniques are useful for the
determination of secondary metabolites of azo dyes formed after
bacterial treatment. This technique is also useful for the
determination of molecular weights, structural information of
dye metabolites and can help propose the microbial metabolic
pathways of azo dyes (Saratale et al., 2009c; Telke et al., 2009a). In
addition, nuclear magnetic resonance (NMR) is a powerful and
theoretically complex analytical tool that allows the quantitative
study of compounds in either solution or in the solid state, and is
very efcient in gathering structural information concerning
molecular compounds. The analytical tools used for monitoring
azo dye biodegradation are listed in Table 4. In addition, some
investigators (Saratale et al., 2009b,c; Telke et al., 2008) have found
that the levels of decolorization and biodegradation can be
determined by measuring the percentage of mineralization by
total organic carbon (TOC), chemical oxygen demand (COD) and
biochemical oxygen demand (BOD) removal ratio by measuring
the initial and nal content.
7. Microbial toxicity studies
A variety of synthetic dyestuffs released by the textile industry
pose a threat to environmental safety. It is estimated that 1015%
of such substances are lost in the efuent during the dyeing process
(Pearce et al., 2003; Robinson et al., 2001), and in the case of the
reactive group of azo dyes up to 5075% is lost (Patil et al., 2008).
The release of textile and dye-house efuent may cause abnormal
coloration of the surface water, and this creates the greatest
immediate environmental concern with regard to water quality,
and directly affects the aquatic ora and fauna. In addition, the
human health impact of azo dyes has caused concern for number of
years, and the occupational exposure of workers in dye
manufacturing and dye utilizing industries has received consider-
able attention. It has been found that puried forms of many azo
dyes are directly mutagenic and carcinogenic (Chen, 2002). The
bacterial degradation of azo dyes is carried out with three
mechanisms: (i) azo dyes that are toxic only after reduction and
 R.G. Saratale et al. / Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 138157 153

UVvis: ultravioletvis spectroscopy; TLC: thin layer chromatography; HPLC: high performance liquid chromatography; HPTLC: high performance thin layer chromatography; FTIR: Fourier transform infrared spectroscopy; LCMS:
cleavage of the azo bond linkage to give aromatic amines, mostly

Ghodake et al. (2009a)

Saratale et al. (2009b)

Saratale et al. (2009c)

Adedayo et al. (2004)

Mohana et al. (2008)
Dawkar et al. (2009)

Chang et al. (2001b)

Kalyani et al. (2008)

Moosvi et al. (2005)

Khehra et al. (2006)

via intestinal anaerobic bacteria. The aromatic amines are
Telke et al. (2009a)

Lopez-Grimau and
Bhatt et al. (2005)

liquid chromatography mass spectrometry; GCMS: gas chromatography mass spectrometry; NMR: nuclear magnetic resonancespectroscopy; ESI-MS: electrospray ionization mass spectrometry; ND: not determined.
Gutierrez (2006)
metabolically oxidized to reactive electrophilic species that

Xu et al. (2007)
covalently bind DNA; (ii) metabolic oxidization without azo
References

Hu (2001)
reduction for azo dyes with structures containing free aromatic
amine groups; and (iii) direct oxidation of the azo bond linkage to
highly reactive electrophilic diazonium salts azo dyes. Each
mechanism may be compound specic, and thus azo toxicity is
probably caused by more than one mechanism. It has been
observed that the transformed intermediates of azo dyes are highly

Aniline, 2,4-dimethylaniline, o-toluidine, and 4-nitroaniline

toxic and mutagenic in nature (An et al., 2007; Brown and DeVito,
1993; Pandey et al., 2007). Obviously, lack of adequate toxicity

4 hydroxy benzenesulphonate and 1 diazo 2-naphthol

3-Amino-4-methoxyphenyl-sulfone sulfonic acid ester
Biphenyl amine, 3-amino 6-hydroxybenzoic acid and
4-Amino-3-(2-bromo-4,6-dinitro-phenylazo)-phenol
and acetic acid 2-(-acetoxy-ethylamino)-ethyl ester

gures for contaminants with regard to cell populations makes

6-Nitroso naphthol and dihydroperoxy benzene

bioremediation (e.g., decolorization or degradation) unpredictable

and unreliable for on-site operation. The toxicity of azo dyes and
their metabolic intermediates has been investigated by many
2 amino benzoic acid and N-N, dimethyl

researchers (Kalme et al., 2007; Kalyani et al., 2008; Saratale et al.,

6 amino naphthalene sulfonic acid

2009b; Tan et al., 2005; Telke et al., 2008), and this can be studied
by assessing the microbial toxicity of the sample of before and after
bacterial treatment.
Azo dye degradation by bacterial systems requires relatively
1-4-phenylenediamine
Identied metaboltes

longer residence times, which depends upon many factors, such as

1,4-Benzenediamine

transportation through the cell and, the tendency of the dye to be

Aromatic amines
Naphthanlene

held for a period of time in cells for either degradation or storage

naphthanlene

Sulfonic acid
2-Naphthol

1-Naphthol

(Chen, 2002). Thus dye-decolorization response in a relatively

short time prevents the chronic toxicity and bioaccumulation of
dye, which is a necessary condition for cell survival. In addition, it
ND

has been observed that at high concentrations of azo dyes enzyme

activity is inhibited and cell death occurs. It has been reported that
investigating the toxicity of dyes towards known cultures is a more
UVvis, TLC, HPLC, FTIR and GCMS

sensitive approach that produces results that can be reproduced

and compared. Moreover if the test organism is the biodecolorizer
UVvis, HPLC, FTIR and GCMS
UVvis, TLC, FTIR and GCMS

itself, then the results will also indicate performance of the

Analytical techniques used

UV, HPLC, FTIR and GCMS

UVvis TLC, HPLC, GCMS

UV, TLC, FTIR and GCMS

degradation operation and suitability of the biodecolorizer for this

UV, NMR, ESI-MS, HPLC
UV, TLC and 1H NMR

operation (Chen, 2006). Similarly some investigators have studied

the toxic effect of azo dyes and their metabolites after bacterial
UVvis, HPTLC
UVvis, HPTLC
UVvis, HPTLC

treatment on plate assay. In this they measured the zone of

HPLCLCMS

inhibition in the presence of a control azo dye and their

HPLC, MS
H NMR

metabolites in the same concentration, and also used nitrogen

FTIR

and phosphate solubilizing bacteria because of their importance in

1

agriculture (Gomare and Govindwar, 2009; Kalme et al., 2007;

Examples of different analytical tools used for monitoring of azo dye biodegradation.

Saratale et al., 2009c, 2010b). The results suggest that after

bacterial treatment the extract of degraded metabolites was found
Vibrio logei and Pesudomonas nitroreducens

to be less toxic than the parent compound, which indicates

detoxication of the azo dyes.
Micrococcus glutamicus NCIM 2168

Pseudomonas aeruginosa NBAR12

Pseudomonas sp., and Bacillus sp.

8. Conclusions
Bacterial consortium RVM 11.1

Human Intestinal Microora

Bacterial consortium DMC
Acinetobacter calcoaceticus

Accumulation of dyestuff and dye wastewater creates not only

Pseudomonas sp. SUK1
Name of dye degrader

Stenotrophomonas sp.,

environmental pollution, but also medical and aesthetic problems.

Bjerkandra sp. BOS55
Pseudomonas luteola

Pseudomonas luteola

As regulations are becoming even more stringent, there is an

Bacillus sp. ADR

Bacillus sp. VUS

Consortium-GR

urgent need for technically feasible and cost-effective treatment

methods. Microbial and enzymatic decolorization and degradation
of azo dyes have signicant potential to address this problem due
to their environmentally-friendly, inexpensive nature, and also
because they do not produce large quantities of sludge. In addition,
bacteria have many other advantages such as a fast growth rate
and high hydraulic retention time, and thus they could be efcient
in treating high-strength organic wastewaters. The literature
(RP2B, V2RP, Red 22)

reviewed in this paper indicates that a large number of lab-scale

Reactive Green 19A
Reactive Orange 16

Reactive Blue 172

Direct Brown MR

studies have been conducted on decolorization of azo dye solutions

Reactive Violet 5
Name of azodye

Reactive Red 22

Sudan azo dyes

Direct Black 22
Reactive Red 2

Navy Blue 2GL

using pure and mixed bacterial culture, however there is still a

Reactive dyes

Acid Red 88
Methyl Red

need to generate relative performance data on industrial efuents.

Orange II
Scarlet R

Under anaerobic conditions the reductive cleavage of azo bonds

Table 4

leads to the formation of toxic aromatic amines, so it is still

necessary to assess the extent of mineralization of these by
154 R.G. Saratale et al. / Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 138157
developing efcient aromatic amine bacterial degrader. Anaero-
bicaerobic biological methods may be appropriate for the
treatment of azo dye-containing wastewaters, and since various
physicochemical parameters inuence the decolorization per-
formance, optimization of these is essential. To disclose the
possible mechanisms of dye decolorization, increased understand-
ing of the biochemical basis (enzymatic study) and more detailed
characterization of the intermediates and metabolites produced
during biodegradation using various analytical techniques are
summarized in this review. Further, to ensure the safety of the
decolorized wastewater, studies should be conducted on the
toxicity of the treated efuent/dye solution. Based on the
successful laboratory results, efforts should then be made to
scale-up and apply bacterial decolorization techniques in real
industrial efuents. Moreover, the techniques of molecular biology
and biochemistry coupled with the latest advances in genomics
and proteomics revolutionizing various aspects of fundamental
biological sciences offer a wide range of possibilities for enhancing
the performance of bacterial treatments of azo dye-containing
wastewater. Moreover, as the knowledge base and the funding in
this area of research increases, it is hoped that bacterial treatments
will become the predominant solution to the problem of colored
wastewater in the textile coloration industry.
Acknowledgements
This review is dedicated to the memory of Advocate Mahesh D.
Saratale who passed away on November 3, 2009.
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