Appl Microbiol Biotechnol (2006) 72: 13161321

DOI 10.1007/s00253-006-0418-2

ENVI RON MENTA L BIOTECHNOLO GY

Suizhou Ren . Jun Guo . Guoqu Zeng . Guoping Sun

Decolorization of triphenylmethane, azo, and anthraquinone dyes

by a newly isolated Aeromonas hydrophila strain

Received: 26 October 2005 / Revised: 7 March 2006 / Accepted: 9 March 2006 / Published online: 19 April 2006
# Springer-Verlag 2006

Abstract A broad-spectrum dye-decolorizing bacterium,

strain DN322, was isolated from activated sludge of a
textile printing wastewater treatment plant. The strain was
characterized and identified as a member of Aeromonas
hydrophila based on Gram staining, morphology characters, biochemical tests, and nearly complete sequence
analysis of 16S rRNA gene and the gyrase subunit beta
gene (gyrB). Strain DN322 decolorized a variety of
synthetic dyes, including triphenylmethane, azo, and
anthraquinone dyes. For color removal, the most suitable
pH and temperature were pH 5.010.0 and 2537 C,
respectively. Triphenylmethane dye, e.g., Crystal Violet,
Basic Fuchsin, Brilliant Green, and Malachite Green
(50 mg l1) were decolorized more than 90% within 10 h
under aerobic culture condition and Crystal Violet could be
used as sole carbon source and energy source for cell
growth. The color removal of triphenylmethane dyes was
due to a soluble cytosolic enzyme, and the enzyme was an
NADH/NADPH-dependent oxygenase; For azo and anthraquinone dyes, e.g., Acid Amaranth, Great Red GR,
Reactive Red KE-3B, and Reactive Brilliant Blue K-GR
(50 mg l1) could be decolorized more than 85% within
36 h under anoxic condition. This strain may be useful for
bioremediation applications.

S. Ren
South China Botanical Garden
Chinese Academy of Sciences,
Guangzhou 510650, China
S. Ren . J. Guo . G. Zeng . G. Sun (*)
Guangdong Institute of Microbiology,
Guangzhou 510070, China
e-mail: guopingsun@163.com
Tel.: +86-20-87684471
Fax: +86-20-87684471
S. Ren
China Graduate School
Chinese Academy of Sciences,
Beijing 100039, China

Introduction
Synthetic dyes are extensively used in textile dyeing, paper
printing, color photography, pharmaceutical, food, cosmetic, and other industries (Rafii et al. 1990). Over 10,000
different dyes with an annual production of over 7105
metric tones worldwide are commercially available
(Padamavathy et al. 2003). Two percent of dyes that are
produced are discharged directly in aqueous effluent and
10% are subsequently lost during the textile coloration
process (Pearce et al. 2003). Major classes of synthetic
dyes include azo, triphenylmethane, and anthraquinone
dyes, some of them are known to be very toxic and
mutagenic to living organisms. With the increasing use of a
wide variety of dyes, pollution by dye-wastewater is
becoming increasingly alarming. Color removal, in
particular, has recently become a major scientific interest.
Although several physico-chemical methods have been
used to eliminate the colored effluents in wastewater, these
methods are rather costly and sometimes produce hazardous byproducts, and therefore other alternatives, such as
microbial biodegradation, have attracted interest. Microbial
decolorization and degradation is an environmentally
friendly and cost-competitive alternative to chemical
decomposition processes (Verma and Madamwar 2003).
To develop an efficient dye degradation biotechnology, the
key step is to obtain broad-spectrum and highly efficient
dye-decolorizing bacteria. Although many dye-decolorizing microorganisms have been reported (Banat et al. 1996;
Azmi et al. 1998; Xu et al. 2005), with exception of the
decolorization of dyes by Pseudomonas pseudomallei
13NA and Citrobacter sp. which decolorize both triphenylmethane and azo dyes by a single species of bacterium
(Yatome et al. 1981; AN et al. 2002), there are no reports on
decolorization of triphenylmethane, azo, and anthraquinone dyes by a single strain of bacterium, and no bacterium
that has been reported was able to utilize Crystal Violet as
sole carbon source and energy source for growth up to now.
In the present study, we report the isolation and
characterization of a highly efficient decolorizing bacterium, Aeromonas hydrophila strain DN322, which having

1317

decolorizing ability for triphenylmethane, azo, and anthraquinone dyes.

Materials and methods

Dyes and chemicals
Crystal Violet was purchased from Sigma. Other triphenylmethane dyes (Basic Fuchsin, Brilliant Green, and
Malachite Green), and six azo dyes (Acid Amaranth, Great
Red GR, Reactive Red KE-3B, Reactive Brilliant Red M8B, Reactive Black KN-B, and Reactive Brilliant Orange
X-GN), as well as three anthraquinone dyes (Reactive
Brilliant Blue K-GR, Acid Blue 25, and Acid Blue 56)
were of analytical grade.
Isolation and identification of dye decolorizing
microorganism
The dye-decolorizing bacterial strain was isolated from
activated-sludge of a textile-printing wastewater treatment
plant, Guangzhou, China. Screening of microorganisms for
dye decolorization was carried out on agar plates with the
following medium: 200 ml of tap water, 0.2 g of yeast
extract, 0.5 g of (NH4)2SO4, 2.66 g of KH2PO4, 4.32 g of
Na2HPO4, 4.0 g of agar, and 10 mg of dye. After
purification by successive single colony isolation on
LuriaBertani (LB) plate, strain DN322 was identified
from several aspects including morphology characters,
biochemical tests, and sequence analysis of nearly
complete 16S rRNA gene and the gyrase subunit beta
gene (gyrB). Genomic DNA of this strain that served as
template was isolated using a method described previously
(Zhou et al. 1996). For polymerase chain reaction (PCR)
amplification, two universal primer sets, 27f (5-AGA GTT
TGA TCC TGG CTC AG-3) and 1522r (5-AAG GAG
GTG ATC CAG CCG CA-3) (Edwards et al. 1989) for
16S rRNA gene, and primers gyrB 3f (5-TCC GGC GGT
CTG CAC GGC GT-3) and gyrB 14r (5-TTG TCC GGG
TTG TAC TCG TC-3) (Yez et al. 2003) for gyrB gene
were used. The sequences of the amplified 16S rRNA gene
and gyrB gene were determined by TakaRa Biotechnology.
Physiological characterization of strain DN322 was tested
by using the API 20NE System (BioMerieux French)
according to the manufacturers instructions.
Decolorization of dyes by growing cells
Cells were cultured overnight in LB medium at 30 C with
shaking at 150 rpm. When the absorbance at 600 nm
reached 0.6 (about 108 cell ml1), precultured cells were
used to inoculate at 10% (v/v) into 2 ml M9 synthetic

medium (Sambrook et al. 1989) containing 0.1% (w/v)

yeast extracts and each dye at different concentration. For
sole carbon and energy sources experiments, yeast extracts
and glucose were omitted and the precultured cells were
inoculated at 2% (v/v) into 50 ml M9 synthetic medium
containing 0.12 mM Crystal Violet. The cultivation was
carried out on a shaker at 150 rpm at 30 C or maintained at
30 C under microaerophilic condition. Under microaerophilic conditions, oxygen concentrations vary between
0.01 and 0.5 mg l1 without aeration. After incubation at
shaking or microaerophilic condition to an appropriate
sampling time, the cells were separated by centrifugation at
10,000g for 10 min. After being washed twice with
50 mM NaH2PO4 buffer (pH 7.5), and the precipitated cells
were dried at 105 C for 4 h to measure dry weight of the
cells. The supernatants were used as samples for
decolorization assay. Two types of control were used:
uninoculated sterile control and heat-killed cells control.
The former, containing only medium components, indicated the effect of medium components on decolorization,
and the latter showed adsorption to cells. Dye removal by
these controls was supposed to be due to adsorption alone.
Crude extract preparation
Two hundred milliliter precultured cells were centrifuged
and washed as described above, and then suspended in
30 ml of 50 mM NaH2PO4 buffer (pH 7.5). Cell disruption
was carried out in ice bath by sonication (3 s, 40% output,
80) using a sonification unit (SONICS VC-505, America), with monitoring of the cells breaking progress under
the microscope. The cell debris and unbroken cells were
removed by centrifugation for 30 min at 18,000g, and the
remaining cell extracts were stored at 4 C.
Decolorization assay
Decolorization activity was expressed in terms of the
percentage decolorization by the modified method described previously (Yatome et al. 1993). Decolorization of
dyes was determined by monitoring the decrease in
absorbance at the maximum wavelength of each dye. The
maximum wavelength of each dye was shown in Table 1.
An UVVisible scanning spectrophotometer (BECKMAN
DV640) was used for absorbance measurement and
recording of UVVisible absorption spectra. Decolorization activity was calculated as the following, and all assays
were done in triplicate.

Decolorization rate % A
B

A  100
A
initial absorbance
B
observed absorbance

1318

Results

Decolorization of triphenylmethane dyes

by strain DN322

Isolation and identification of dye-decolorizing

bacteria
A broad-spectrum dye-degrading bacterial strain, named
strain DN322, was isolated after screening and single
colony purification. The strain is Gram-negative, rodshaped, motile with a single polar flagellum, and facultative anaerobic, oxidase and catalase positive, and Voges
Proskauer positive. The strain produces gas from glucose
and produces H2S from cysteine. Colonies grown on LB
plate are light yellow to pink, smooth on the surface, and
trim on the edge. The API 20NE system identification was
to the species level, and the result of identification was
compared with that of type species A. hydrophila
ATCC7966T. Biochemical and physiological profiles of
strain DN322 is consistent with those of A. hydrophila
ATCC7966T.
To further identify this strain, the nucleotide sequences
of 16S rRNA gene and gyrB gene were analyzed and
compared with those of related strains in GenBank. 16S
rRNA gene and gyrB gene strain DN322 shared 99 and
97% similarity with A. hydrophila ATCC7966T. These
results agree with that of API 20NE system identification,
showing that this new isolate is a member of A. hydrophila.
The sequences of the 16S rRNA gene and the gyrB gene
had been submitted to GenBank under accession number
AY686711 and AY968042, respectively. The strain has
been deposited in China Center for Type Culture Collection
(CCTCC AB205001T) and the National Collection of Type
Cultures (NCTC13358, London).

Table 1 Decolorization (D) of triphenylmethane, azo, and anthraquinone dyes by the growing cells of strain DN322 under
microaerophilic condition
Dye

max Conc. D (%) Time (h)

(mg/l)

Crystal Violet
Crystal Violet
Crystal Violet
Malachite Green
Brilliant Green
Basic Fuchsin
Acid Amaranth
Great Red GR
Reactive Red KE-3B
Reactive Brilliant Red M-8B
Reactive Black KN-B
Reactive Brilliant Orange X-GN
Reactive Brilliant Blue K-GR
Acid Blue 25
Acid Blue 56

590
590
590
617
545
630
477
503
511
540
595
477
603
562
637

Decolorization of various triphenylmethane, azo, and

anthraquinone dyes by the growing cells of strain DN322
were shown in Table 1. The optimum decolorization
activity was observed at pH 5.010.0 and 2537 C in M9
decolorization medium, which was also the optimum
condition for growth of this strain (Figs. 1 and 2).
Compared with azo and anthraquinone dyes, this strain
had a higher decolorization rate for triphenylmethane dyes.
In the case of triphenylmethane dyes tested, strain DN322
showed the highest decolorization rate against Crystal
Violet (Figs. 3 and 4). Crystal Violet was nearly completely
decolorized for initial dye concentration less than 50 mgl1
after 6 h cultivation. With the dye concentration increased,
the decolorization activity was inhibited obviously. Other
triphenylmethane dyes, e.g., Malachite Green, Basic
Fuchsin, and Brilliant Green, were also decolorized
effectively by this strain (Table 1). The four triphenylmethane dyes tested have similar decolorization rate, which
could be due to the similarity in structure of these dyes.
Furthermore, strain DN322 could decolorize four triphenylmethane dyes tested under both shaking and microaerophilic conditions, and the decolorizing speed is a little
faster under shaking condition. However, when the
decolorization mixture was flashed with N2 for 10 min,
no decolorization was observed, indicating that the
decolorization reaction was depended upon the presence
of molecular oxygen. Decolorization of the four triphenylmethane dyes was also observed with cell extracts. When
the cell extracts treated with 1.0 mg proteinase K ml1 for
30 min, more than 85% of decolorization activity was lost.
After heated at 100 C for 10 min, the cell extracts lost all
of its decolorization activity. When 4 l of NADH or
NADPH (25 mM) was added to the cell extracts (50 l)
containing 50 mg l1 dyes, complete decolorization was
observed within 5 min. Compared with the control in
which neither NADH nor NADPH was added, the
decolorization rate was increased over 50 times. When
2

962
645
306
945
933
956
963
916
938
875
825
754
857
605
216

6
24
72
10
6
8
6
24
24
24
24
36
36
52
52

1.8
1.6

Absorbance (600nm)

50
200
500
50
50
50
50
50
50
50
50
50
50
50
50

1.4
1.2
1
0.8
0.6
0.4
0.2
0
3

10

11

12

pH

Fig. 1 Effect of initial pH on the growth of strain DN322. The

strain was cultured in M9 synthetic medium containing 0.1% (w/v)
yeast extracts at various pHs at 30 C

1319
1.4

Absorbance (600nm)

1.2
1
0.8
0.6
0.4
0.2
0
4

10

20

25

30

35

40

45

50

Temperature ( C)

Fig. 2 Effect of temperature on the growth of strain DN322. The

strain was cultured in M9 synthetic medium containing 0.1% (w/v)
yeast at various temperatures at pH 7.5

the cell extracts were flashed with N2 for 10 min, no

decolorization was observed in this phenomenon similar to
that of intact cells. All these results indicated that the color
removal of triphenylmethane dyes was due to a soluble
cytosolic enzyme, and the enzyme was an NADH/
NADPH-dependent oxygenase.
Strain DN322 was capable of utilizing Crystal Violet as
sole carbon and energy sources for the cell growth, as
depicted in Fig. 5. During DN322 growth on 0.12 mM
Crystal Violet as a sole source of carbon and energy, cell
mass increased approximately five times after 72 h cultivation. No significant growth occurred in cultures lacking
Crystal Violet, and controls lacking cells did not show any
decolorization of Crystal Violet.

Fig. 4 Time course of the decolorization of Crystal Violet by strain

DN322. Strain was cultured in M9 synthetic medium containing
0.1% (w/v) yeast and 0.12 mM dye at pH 7.5 at 30 C under aerobic
condition

of 50 mg l1 (Fig. 6), whereas it would take 24 h for the

Great Red GR and Reactive Red KE-3B (diazo group) at
the same concentration. Other azo dyes, e.g., Reactive
Brilliant Red M-8B, Reactive Black KN-B, and Reactive
Brilliant Orange X-GN could be decolorized over 70%
within 36 h at the same concentration. Unlike triphenylmethane dyes, all these azo dyes were decolorized more
rapidly under microaerophilic condition than that under
aerobic condition. Shaking culture suppressed the decolorization of azo dyes, indicating that the decolorization
mechanism of azo dyes by strain DN322 is due to azo

Decolorization of azo and anthraquinone dyes

by strain DN322
For azo dyes, strain DN322 decolorized 95% of Acid
Amaranth (mono azo group) in 6 h at the dye concentration

Fig. 5 Decolorization of Crystal Violet by strain DN322 with

companying data for cell growth in M9 medium. The absorbance
(abs) of Crystal violet at the maximum wavelength (590 nm) is
shown for tubes containing a cell inoculum ( ) and control tubes

Fig. 3 UVVisible absorbance spectra of decolorization for Crystal

Violet by strain DN322. Strain was cultured in M9 synthetic
medium containing 0.1% (w/v) yeast and 0.12 mM dye at pH 7.5 at
30 C under aerobic condition

lacking cells (). Cell growth is illustrated for tubes containing

Crystal violet () and for controls () containing a cell inoculum
but lacking Crystal violet. All data represent the mean of three
triplicate tubes, and error bars represent standard deviation.
Lack of error bars for analytical data indicates that the error bars
were smaller than the symbol

1320

Fig. 6 UVVisible absorbance spectra of decolorization for Acid

Amaranth by strain DN322. Strain was cultured in M9 synthetic
medium containing 0.1% (w/v) yeast and 0.12 mM dye at pH 7.5 at
30 C under microaerophilic condition

bound reduction under anaerobic condition, which is

different from that of triphenylmethane dyes.
In the decolorization of anthraquinone dyes Reactive
Brilliant Blue K-GR, a different phenomenon of decolorization was observed. Firstly, a blue flocculate was formed
and precipitated, and the cells of strain DN322 were stained
blue, indicating that Reactive Brilliant Blue K-GR was
adsorbed on the cell surface. Then the dye was decolorized
as the precipitated flocculate turned to white and more than
80% of color was removed in 36 h at 50 mg l1 (Fig. 7).
Other anthraquinone dye, e.g., Acid Blue 25 and Acid Blue
56 could also be decolorized as Reactive Brilliant Blue KGR, but need a little longer time.

Discussion
Strain DN322 was identified as a member of A. hydrophila
by biochemical analysis and nearly complete sequence
analysis of 16S rRNA gene and gyrB gene. Strain DN322
shared 99% similarity with A. hydrophila subsp. hydro-

Fig. 7 UVVisible absorbance spectra of decolorization for

Reactive Brilliant Blue K-GR by strain DN322. Strain was cultured
in M9 synthetic medium containing 0.1% (w/v) yeast and 0.12 mM
dye at pH 7.5 at 30 C under microaerophilic condition

phila type strain ATCC7966T in the 16S rRNA gene

sequence. The gyrB gene, a gene that encodes the subunit of DNA gyrase, was proven to be an excellent
molecular chronometer for phylogenetic studies of the
genus Aeromonas (Yez et al. 2003). In our study, the
gyrB gene sequence similarity between strain DN322 and
type strain ATCC7966T was 97%. The gyrB gene result
agreed with 16S rRNA gene analysis, and the former
showed a higher capacity to differentiate between species.
Rajesh and Uttam 1999 also found that decolorization
activity of Crystal Violet by Kurthia sp. was inhibited when
the dye concentration was increased to 20.4 mg l1, and
Malachite Green was more easy decolorized than Crystal
Violet. In our study, the decolorization activity of A.
hydrophila DN322 was inhibited only when the dyes
concentration reached 50 mg l1, opposite to Kurthia sp.,
Crystal Violet was easier to be decolorized than Malachite
Green by strain DN322. Yatome et al. 1993 reported that
the growth of Nocardia corallina cells was completely
inhibited at concentration 2.9 mg l1 of Crystal Violet.
However, strain DN322 was capable of utilizing 50 mg l1
Crystal Violet as sole carbon and energy sources for the cell
growth. These results indicate that there are different
mechanisms of decolorization of triphenylmethane dyes in
different bacteria.
Decolorization of dyes by bacteria could be due to
adsorption to microbial cells or to biodegradation (Knapp
and Newby 1995; Sani and Banerjee 1999). In adsorption,
cell mats become deeply colored because of adsorbing
dyes, whereas those retaining their original colors are
accompanied by the occurrence of biodegradation. In our
research, the cells of A. hydrophila strain DN322 remained
colorless during the process of decolorization. Similar
results were also observed in other triphenylmethane and
azo dyes. Consequently, according to the above results, the
color removal by strain DN322 should be largely attributed
to biodegradation. However, in the decolorization of
Crystal Violet by Aeromonas sp. B5, the cells were stained
violet, indicating that Crystal Violet was adsorbed on the
cell surface, and the decolorization of Crystal Violet was
due to the sorption of dye on the cells (Nobuki et al. 2000).
Although a lot of triphenylmethane dye-decolorizing
microorganisms have been reported, the enzyme which is
responsible for the decolorization and degradation of these
triphenylmethane dyes has never been purified from
bacteria, and its characterization has never been described
(Azmi et al. 1998). Therefore, it would be a meaningful and
helpful work to purify the decolorization enzyme and
elucidate the decolorization mechanism of the triphenylmethane dyes. Strain DN322 is the first Aeromonas sp. that
is capable of decolorizing several triphenylmethane dyes
and utilizing Crystal Violet as sole carbon source for
growth. The color removal of triphenylmethane dyes in
strain DN322 was due to a soluble cytosolic enzyme.
Recently, the decolorization enzyme was purified to
homogeneity and identified as an NADH/NADPH-dependent, heme-containing oxygenase with a molecular of
87 kDa in our lab. The decolorization enzyme would be a
good material for further research of the enzymological

1321

mechanism of triphenylmethane dyes decolorization by

bacteria.
Based on these results, A. hydrophila strain DN322
exhibited to be a highly promising microorganism both for
the application in the treatment of dyeing and printing
industrial wastewater and bioremediation to remove
recalcitrant triphenylmethane, azo, and anthraquinone dye
pollutants from contaminated environment.
Acknowledgements This work was supported by the fund of Chinese
National Programs for High Technology Research and Development
(No. 2003AA214040), Guangdong Provincial Natural Science Fund
(No.2004A30404002), Guangdong Provincial Natural Science
Fund (No. 200115017) and Guangdong Provincial Natural Science
Fund (No. 032319).

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