956 & IWA Publishing 2011 Water Science & Technology 9 63.

5 9 2011

The decolorisation capacity and mechanism of Shewanella

oneidensis MR-1 for Methyl Orange and Acid Yellow 199
under microaerophilic conditions
Y. Y. Yang, L. N. Du, G. Wang, X. M. Jia and Y. H. Zhao

ABSTRACT

Shewanella oneidensis MR-1 was found to reach 99.36% and 78.25% decolorisation for Methyl Y. Y. Yang
G. Wang
Orange and Acid Yellow 199 in solutions, respectively. The suitable pH range for decolorisation of Y. H. Zhao (corresponding author)
College of life science,
Methyl Orange and Acid Yellow 199 by S. oneidensis MR-1 was 4.0–7.0 and 6.0–8.0, respectively, Zhejiang University,

The azo dyes’ removal by S. oneidensis MR-1 was slightly enhanced by addition of Mg2 þ , but Hangzhou 310058,
China
inhibited by Pb2 þ , Cd2 þ , Cu2 þ , Fe3 þ and Fe2 þ . The enzyme activities of NADH-DCIP reductase E-mail: jinxiuchuanyang@gmail.com;
dlg1314@126.com;
and azoreductase were 2.67 and 3.0 times higher, and 1.92 and 2.48 times higher, respectively, in jxfx_0164582@yahoo.com.cn;
jiaxiaoming@21cn.com;
the Methyl Orange treatment and in the Acid Yellow 199 treatment as compared to the control yhzhao58@yahoo.com

treatment. These findings indicated that the azo dyes’ decolorisation by S. oneidensis MR-1 was
via reduction mechanism.
Key words 9 Acid Yellow 199, decolorisation, Methyl Orange, reduction mechanism,
Shewanella oneidensis MR-1

INTRODUCTION

Large amounts of dyes are produced annually and extensively
used in the textile, cosmetic, plastic, food, and pharmaceutical
industries (McMullan et al. 2001; Esther Forgacsa & Gyula
2004). Dyes can be classified into azo, triphenylmethane and
anthraquinone dyes based on the chemical structures, in
which azo dyes are most widely used. Azo dyes are char-
acterised by the presence of one or more azo groups sub-
stituted with aromatic amines. They are produced with stable
molecule structure to be recalcitrant during use. Due to the
mutagenicity of azo dyes, they have been implicated in
pollution and known to be toxic to humans (Banat et al.
1996; Pearce et al. 2003; Olaganathan & Patterson 2009).
Therefore, removal of azo dyes from environment using
different methods has become a focus for environmental
scientists. Physical and chemical methods are not suitable
to treat dye effluent due to their operational costs or second-
ary sludge disposal problems (Kaushik & Malik 2009; Rodri-
guez et al. 2009). Even the new developments in the physical
and chemical approaches, such as electrocoagulation and
electrochemical treatment, have faced some difficulties.
However, bioremediation has become a promising way for
treatment of pollution from various dyes, including azo dyes,
due to its eco-friendly nature and low cost.
Shewanella are metabolically versatile and effective to
degrade recalcitrant organics and reduce heavy metal ions.
Some Shewanella strains were found to be able to decolorise
anthraquinone dye (Xu et al. 2006), azo dye (Xu et al. 2007a;
Khalid et al. 2008; Wu et al. 2009), and triphenylmethane dye
(Chen et al. 2008). S. oneidensis MR-1, which is currently being
used as a model organism, has been extensively studied on its
bioremediation ability of heavy metal ions and humic sub-
stances (Mugerfeld et al. 2009). However, relatively little work
has been done on the dye decolorisation ability of S. oneidensis
MR-1. In this study, the main objective was to investigate the
decolorisation capacity and mechanism of S. oneidensis MR-1
for two azo dyes, Methyl Orange and Acid Yellow 199.

doi: 10.2166/wst.2011.275
 957 Y. Y. Yang et al. 9 The decolorisation capacity and mechanism of Shewanella oneidensis MR-1
MATERIALS AND METHODS
Microorganism and the culture medium
S. oneidensis MR-1 (ATCC 700550), which is a facultative
anaerobic bacterium, was granted by Professor Haichun Gao.
It was maintained and grown on a Luria-Bertani medium.
The experimental medium consisted of 2.0 g/L lactate,
1.5 g/L KH2PO4, 1.0 g/L yeast extract, 0.5 g/L NaCl,
0.1 g/L NH4Cl and dyes were added.
Dyes and chemicals
One anthraquinone dye, two triphenylmethane dyes, four azo
dyes and two other dyes were selected for decolorising tests.
Acid Blue 25, Acid Yellow 199 and Acid Red 337 were kindly
provided by Heng Sheng chemicals Ltd, China; the others
were purchased from Sinopharm Chemical Reagent Co., Ltd,
China. All the information of each dye is shown in Table 1.
Decolorisation analysis of each dye by Shewanella
oneidensis MR-1 under different decolorisation
parameter conditions
Stock solutions of dyes were prepared and diluted to the
desired concentration. Strain S. oneidensis MR-1 was first
cultivated under aerobic conditions at 301C overnight and
cell suspension was obtained as reported previously (Chen
et al. 2008). In the decolorisation parameter experiments,
25 mL of dye solution with various medium components
was added into an Erlenmeyer flask (100 mL) and then cell
suspension was transferred to reach an initial cell mass of
0.4–0.5 g/L and was kept in static conditions for 24 h. After
then, samples were collected from the flask, centrifuged and
supernatant was analysed spectrophotometrically for the
residual dye concentration at absorption peak of each dye.
Controls without inoculation were kept under the same
conditions.
The dye decolorisation, an indicator of the dye bioreme-
diation ability of S. oneidensis MR-1, was calculated as
follows:
Ac  Af
Decolorisation percentage ¼  100%
Ac
where Ac is the final absorbance value of aqueous solution of
controls and Af is the final absorbance value of aqueous
solution of samples at fixed time.
Water Science & Technology 9 63.5 9 2011
The decolorisation parameter experiments included the
following treatments:
(1) pH: The pH of the experimental medium for culturing
the strain was adjusted to 3, 4, 5, 6, 7, 8, and 9 with
0.1 mol/L HCl or 0.1 mol/L NaOH.
(2) Carbon sources: 2% (w/v) formate, sucrose, maltose,
lactose, D-fructose, D-mannose, and galactose were
added to a modified experimental medium, which
included 1.5 g/L KH2PO4, 0.5 g/L NaCl, 0.5 g/L beef
extract and dyes.
(3) Nitrogen sources: 0.3% (m/v) NH4Cl, NaNO3, beef
extract, peptone, glycine, glumatic acid, proline were
added to a modified experimental medium, which
included 1.5 g/L KH2PO4, 0.5 g/L NaCl, 0.5 g/L yeast
extract and dyes.
(4) Metal ions: 0.1 mM CuCl2, FeCl3, FeSO4, NiCl3, MgCl2,
PbCl2, MnCl2 were added to the experimental medium
for culturing the strain.
(5) Initial dye concentrations: Dyes of different initial con-
centrations, ranging from 50 to 500 mg/L, were added to
the experimental medium for culturing the strain of
optimum pH.
Each experiment was carried out in triplicate.
Preparation of cell free extract and enzyme assays
The bacterial cells grown in the LB medium at 301C for 24 h
at aerobic conditions were considered as control. The medium
was centrifuged at 10,000 rpm for 10 min and the biomass
was suspended in a potassium phosphate buffer (50 mM, pH
7.4). Then the suspension was sonicated (5S, 100 amplitude,
50 strokes) at 41C. The homogenate was centrifuged
at 7,000 rpm for 20 min and supernatant was used as a source
of crude enzyme. Similar procedure was used to quantify
enzyme activities for the dye decolorisation of Methyl Orange
and Acid Yellow 199 by S. oneidensis MR-1.
Laccase was measured by monitoring the oxidation of
1 mM ABTS in 100 mM sodium acetate buffer (pH 4.5) at
420 nm (Saratale et al. 2009). NADH-DCIP reductase activity
was assayed as Bhosale reported (Bhosale et al. 2006) The
azoreductase activity was determined by monitoring the
decrease in the Methyl Orange concentration at 480 nm in
ð1Þ a reaction mixture of 3 mL containing 100 mM Methyl
Orange, 50 mM sodium phosphate buffer (pH 5.5) and
20 mM NADH (Jadhav et al. 2008). One unit of enzyme
activity was defined as a microgram of Methyl red reduced
min1 mg1 of protein. All enzyme assays were run in
triplicate.
 958 Y. Y. Yang et al. 9 The decolorisation capacity and mechanism of Shewanella oneidensis MR-1 Water Science & Technology 9 63.5 9 2011

Table 1 9 Dye removal of each dye (100 mg/L) used in this study by Shewanella oneidensis MR-1 after 24 h

Decolorisation
Chemical structure class Dye Chemical Structure C.I. name lmax (nm) (%)

Anthraquinone Acid blue 25 C.I. 62055 598 49.1578.60

Triphenylmethane Malachite Green C.1.Basic 618 56.32710.42

Green 4

Crystal Violet C.I.Basic 584 58.5176.77

Violet 3

Azo Methyl Orange C.I.13025 470 97.6470.21

Acid Yellow 199 C.I. Acid 455 77.9273.76

yellow 199

Acid Red 337 C.I. 17102 494 78.0270.26

Congo Red C.I. Direct 497 85.8670.32

red 28

Other Methylene Blue C.I. Basic 662 35.1476.97

blue 9

Safranine T C.I. basic 532 55.3679.34

red 2
 959 Y. Y. Yang et al. 9 The decolorisation capacity and mechanism of Shewanella oneidensis MR-1
The decolorisation manner of azo dyes,
decolorisation by Shewanella oneidensis MR-1
Each dye (100 M) was inoculated with S. oneidensis MR-1
for 10 h at optimum pH and temperature. After incubation
for 10 h, samples from the culture were centrifuged at
12,000 rpm for 10 min. Then the supernatant was scanned
from 400 nm to 800 nm using UV-visible spectrophotometer
to detect any transformation of each dye in the system. After
that, the supernatant was used to detect the products of
decolorisation using HPLC analysis. The RP-HPLC column
was C-18 reverse phase column (150.00 mm  4.60 mm) and
at the temperature of 251C. The mobile phase was methanol:
water (90:10, v/v). The flow rate was 0.8 mL/min and 5 mL
was injected in the sampler. The metabolites were detected at
480 nm and 254 nm for Methyl Orange, and 455 nm and
280 nm for Acid Yellow 199, respectively.
Statistical analysis
A SPSS software package was used for Dunnett-t test
and regression analysis in this research. P-value 0.05 was
used to determine the significance between different
treatments.
RESULTS AND DISCUSSION
Decolorisation ability of Shewanella oneidensis MR-1
Ten different dyes belonging in different categories were used
to investigate the decolorisation ability of S. oneidensis MR-1.
The results indicated that S. oneidensis MR-1 could decolor-
ise a wide range of dyes, including acidic and basic dyes,
azo dyes, and triphenylmethane dyes (Table 1). However,
S. oneidensis MR-1 decolorised different dyes to different
extents. Methyl Orange was decolorised faster than the
other tested dyes by S. oneidensis MR-1. Methylene Blue,
which was oxidised from Methyl Blue and more poisonous,
was the least decolorised among the tested dyes. For azo dye
decolorisation, approximately 50% decolorisation was
obtained by S. decolorationis S12 after 10 h under micro-
aerophilic conditions (Xu et al. 2007a). However, 97.3% and
68.2% decolorisation were obtained after 10 h for Methyl
Orange and Acid Yellow 199, respectively, by S. oneidensis
MR-1. Based on the decolorisation results and structure of
dyes, Methyl Orange and Acid Yellow 199 were chosen to
further investigate the similarity and difference of azo dyes,
decolorisation by S. oneidensis MR-1.
Water Science & Technology 9 63.5 9 2011
Effect of initial pH on decolorisation
As an important factor affecting the decolorisation process,
optimum pH value is not only determined by the optimum
growth pH of bacterium, but also by the dye state in the tested
system. In this research, we identified that the suitable pH for
decolorisation of Methyl Orange by S. oneidensis MR-1 was
4.0–7.0, and the optimum pH for Acid Yellow 199 was about
6.0–8.0 (Figure 1(a)). This was a little different from that of
S. decolorationis S12, Shewanella strain J18 143 and
Shewanella sp. NTOU1, for which pH 6.0–8.0 was the
optimum pH value for decolorisation (Pearce et al. 2006;
Xu et al. 2007b; Chen et al. 2008). The situation of S.
oneidensis MR-1 for Methyl Orange may be similar to that
of yeast reported (Ramalho et al. 2004). The protonisation of
N, N-dimethylamino-based dyes at an azo nitrogen occurred
under lower pH, which was easier to degrade (formulas 2 and
3). At pH 7 and afterwards, the decolorisation of Methyl
Orange decreased consistently whereas the decolorisation of
Acid Yellow 199 decreased little. Electron-withdrawing
groups (SO42) of Methyl Orange might lead to a decreased
electron density at the N ¼ N double bond, especially with
para position (Maier et al. 2004), which enhanced the deco-
lorisation rate for Methyl Orange. This indicated that pH 4.0
not only had a positive effect on shifting the following
equilibrium toward decreasing decolorisation of Methyl
Orange (formulas 2 and 3), but also decreased the electron
density at the N ¼ N double bond. Methyl group in the ortho-
position and three benzene rings structure of Acid Yellow
199, which enhanced the electron cloud density at N ¼ N
double bond, attenuated the decolorisation rate(Maier et al.
2004). Thus, pH has little effect on the dye state of Acid
Yellow 199 in the tested system. Extreme acid environment
had a negative effect on dye decolorisation by S. oneidensis
MR-1 due to its being poisonous to cell growth.
Effect of medium components on decolorisation
Effect of nitrogen and carbon sources on decolorisation
Nitrogen and carbon sources were required to promote the
degradation of azo dyes. Peptone was the best nitrogen source
 960 Y. Y. Yang et al. 9 The decolorisation capacity and mechanism of Shewanella oneidensis MR-1
Figure 1 9 Effect of various factors on the dye removal of Methyl Orange and Acid Yellow 199.
while NaNO3 was the worst. Organic nitrogen was more
effective for degradation of azo dyes than inorganic nitrogen.
Glycine was the best amino acid tested as an energy source
for dye removal of Methyl Orange. However, no significant
differences were found among the tested amino acids for the
dye removal of Acid Yellow 199. It was observed that nitro-
gen sources had greater effect on dye removal of Methyl
Orange than that of Acid Yellow 199 (Figure 1(b)). This study
also revealed that yeast extract was the best carbon source for
dye removal, with 99.36% of Methyl Orange and 78.25% of
Acid Yellow to be decolorised, respectively (Figure 1(c)).
Yeast extract and peptone may be the best choice in practice
with regard to costs and dye removal efficiency.
Effect of metal ions on decolorisation
Several metal ions were chosen to study their effects on
decolorisation because many metal ions could affect the
Water Science & Technology 9 63.5 9 2011
activities of enzymes. It was observed that dye removal was
slightly enhanced by addition of Mg2 þ (Figure 1(d)). The dye
removal was 97.21% in the presence of Mg2 þ and 96.82%
without addition of metal ions. The presence of Mn2 þ had a
greater effect on the dye removal of Methyl Orange than that
of Acid Yellow 199 (Figure 1(d)). Heavy metal ions (e.g.
Pb2 þ , Cd2 þ and Cu2 þ ) which influenced the cell growth and
viability had a significant (Po0.05) negative influence on the
dye removal (Figure 1(d)). It was reported that the activity of
azoreductase, which can decompose azo dyes, appears to be
unaffected by Mg2 þ , Mn2 þ , Ca2 þ and Zn2 þ but is almost
completely inhibited by Fe2 þ and moderately inhibited by
Cu2 þ and Hg2 þ (Nachiyar & Rajakumar 2005). Xu reported
that addition of external Fe3 þ enhanced azo reduction by
S. decolorationis S12 under anaerobic conditions, but that
chemically or bacterially produced Fe2 þ did not accelerate
the decolorisation rates (Xu et al. 2007a). However, both
Fe3 þ and Fe2 þ inhibited the dye removal by S. oneidensis
 961 Y. Y. Yang et al. 9 The decolorisation capacity and mechanism of Shewanella oneidensis MR-1
MR-1 in this report. Further study was needed to understand
the mechanisms associated with the effects of the metal ions
on the decolorisation of the azo dyes by S. oneidensis MR-1 in
this study.
Effect of initial dye concentration on decolorisation
The dye removal by S. oneidensis MR-1 generally decreased
with increasing initial concentrations of Methyl Orange and
Acid Yellow 199. This could be due to a combination of
factors including the toxicity of the dyes to cell growth at
higher concentrations, and the ability of the enzyme to
recognise the substrate efficiently at very low concentrations
(Pearce et al. 2003). Dye removal decreased gradually
with increase in the initial dye concentrations after 24 h.
It indicated that the decolorisation of Methyl Orange and
Acid Yellow 199 were still kept at a high level at moderate
concentration (200 mg/L) (data not shown). However,
high concentration was more recalcitrant and difficult to
decolorise. The negative relationship between the dye
removal and initial dye concentration could be well described
with a linear model for Methyl Orange (y ¼ 106.510.14x,
R2 ¼ 0.98) and Acid Yellow 199 (y ¼ 80.340.09x, R2 ¼ 0.83).
From the equation, we may conclude that Acid Yellow 199
could not be decolorised completely (ymax ¼ 80.34). Acid
Yellow 199 was more recalcitrant than Methyl Orange.
Analysis of enzymes related to the dye decolorisation
and degradation
Azoreductase, which catalyses reductive cleavage of azo
bonds (-N ¼ N-), has been extensively investigated in the
biodegradation of azo dyes (Banat et al. 1996; Stolz 2001).
Recently, oxidative enzymes and NADH-DCIP reductase
were found to be responsible for the dye decolorisation in
bacteria (Wu et al. 2009). Laccase, NADH-DCIP reductase
and azoreductase were detected to investigate whether
these enzymes were responsible for the decolorisation of
Methyl Orange and Acid Yellow 199 by S. oneidensis MR-1.
In the present study, significant increase (P o0.05) in the
enzyme activity of NADH-DCIP reductase and azoreduc-
tase was observed over period of azo dyes, decolorisation
(Table 2), while laccase activity decreased significantly after
decolorisation. Reductive cleavage of azo bonds (-N ¼ N-)
with the help of azoreductase was the initial and critical
step for the azo dyes, biodegradation (Hrmova et al. 1984).
The reductase activities of NADH-DCIP reductase and
azoreductase increased significantly for Methyl Orange
(267% and 300%) than that of Acid Yellow 199 (192%
Water Science & Technology 9 63.5 9 2011
Table 2 9 Intracellular enzyme activities of Shewanella oneidensis MR-1 cells after
decolorisation compared to cells in control
Enzyme assay Control Methyl Orange Acid Yellow 199
Laccase| 0.03770.003 0.010**70.001 0.007a**70.0009
NADH-DCIP|| 17.7271.15 47.41**71.07 34.12b**70.49
Azoreductase||| 0.7670.07 2.28**70.10 1.89c**70.06
Values are mean of three experiments 7SEM. significantly different from the control cells
at *Po0.01, **Po0.001 by Dunnett-t test. Significantly different from the decolorisation
of two dyes at
a
P40.05, bPo0.01, cPo0.001 by one way ANOVA with Tukey-Kramer multiple
comparisions test.
|
Enzyme activity, units ml1 min1.
||
g DCIP reduced, mg1 min1.
|||
g Methyl red reduced, mg1 min1.
and 248%) compared to control, which indicated that
Methyl Orange was more available and easy to decolorise
by S. oneidensis MR-1. This was in accord with the deco-
lorisation percentage in the experiments. From these
results, we proposed that the strain S. oneidensis MR-1
decolorised azo dyes via enzymatic reduction mechanism.
Wu et al. (2009) reported that laccase was responsible for
the decolorisation of Reactive Black 5 by S. oneidensis WL-
7, indicating the azo dyes, decolorisation by S. oneidensis
WL-7 was via oxidation mechanism. This was different
from what we found in S. oneidensis MR-1. The enzyme
activities were significantly different for Methyl Orange and
Acid Yellow 199 decolorisation due to their different che-
mical structure, indicating substitution was accounting for
the degradability of different dyes and interaction between
dyes and enzyme system.
The decolorisation manner of azo dyes,
decolorisation by Shewanella oneidensis MR-1
Microbial decolorisation is mainly attributed to biosorption
and biodegradation (Knapp & Newby 1995). In the case of
adsorption, dyes are only adsorbed onto the surface of cells
and the cells become deeply coloured, whereas cells were still
kept colourless after degradation. To discover the possible
dye removal mechanism by S. oneidensis MR-1, we analysed
the UV-vis spectral changes and HPLC before and after
microbial decolorisation. Firstly, UV-vis scan (300–800 nm)
of culture supernatants withdrawn at different time intervals
of Methyl Orange and Acid Yellow 199 are shown in Figure
2. For Methyl Orange, peak observed at 470 nm (0 h) was
decreased without any shift in lmax up to nearly complete
 962 Y. Y. Yang et al. 9 The decolorisation capacity and mechanism of Shewanella oneidensis MR-1
Figure 2 9 Variation in the UV-vis spectra of Methyl Orange (a) and Acid Yellow 199 (b) before and after decolorisation by Shewanella oneidensis MR-1.
decolorisation of dye (10 h), while, peak observed at 455 nm
(0 h) was decreased slowly without any shift in lmax for
Acid Yellow 199. The great changes in UV and visible
spectra indicated that dyes in medium disappeared and new
products could come into being. The colour of cells for the
two dyes after 10 h was colourless. Secondly, HPLC was
carried out to detect the biotransformation of the azo dyes by
S. oneidensis MR-1. The peaks for Methyl Orange and Acid
Yellow 199 detected at 480 nm and 455 nm were not found
after decolorisation, indicating the two azo dyes decomposed
to small molecules. The metabolites of Methyl Orange
and Acid Yellow 199 were detected at 254 nm and 280 nm
at retention time 3.014 min and 2.567 min, respectively (data
were not shown). It indicated that the new products
came into being. Hence, the decolorisation of Methyl Orange
and Acid Yellow 199 of S. oneidensis MR-1 was mainly due
to the microbial degradation, rather than inactive surface
adsorption.
CONCLUSIONS
The model microorganism S. oneidensis MR-1 was found to
be able to decolorise efficiently the two azo dyes under the
experimental conditions. Yeast extract and peptone could be
the suitable carbon and N source for culturing the strain in
decolorising the dyes. Most of the heavy metal ions appeared
to have negative effects on the dye removal, although addition
of Mg2 þ could slightly enhance the dye removal. Significant
increase in the enzyme acticities of NADH-DCIP reductase
and azoreductase were observed after the decolorisation,
which implied that the two enzymes were involved in the
decolorisation process.
Water Science & Technology 9 63.5 9 2011
ACKNOWLEDGEMENTS
This study was supported by the National Hi-Tech
Research and Development Program (863) of China (No.
2007AA06Z329), the Science and Technology Project of
Zhejiang Province (No. 2007C23036; 2008C13014-3), the
International Cooperation Project in Science and Technology
of Zhejiang Province (No. 2008C14038).
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