decolorization of the textile dyes by newly isolated
DESCRIPTION
decolorizationTRANSCRIPT
Decolorization of the textile dyes by newly isolatedbacterial strains
Kuo-Cheng Chen a,*, Jane-Yii Wu a, Dar-Jen Liou a, Sz-Chwun John Hwang b
a Department of Chemical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan, ROCb Department of Civil Engineering, Chung Hua University, Hsinchu, Taiwan, ROC
Received 7 January 2002; received in revised form 19 September 2002; accepted 24 September 2002
Abstract
Six bacterial strains with the capability of degrading textile dyes were isolated from sludge samples and mud lakes.
Aeromonas hydrophila was selected and identified because it exhibited the greatest color removal from various dyes.
Although A. hydrophila displayed good growth in aerobic or agitation culture (AGI culture), color removal was the best
in anoxic or anaerobic culture (ANA culture). For color removal, the most suitable pH and temperature were pH 5.5�/
10.0 and 20�/35 8C under anoxic culture (ANO culture). More than 90% of RED RBN was reduced in color within 8
days at a dye concentration of 3000 mg l�1. This strain could also decolorize the media containing a mixture of dyes
within 2 days of incubation. Nitrogen sources such as yeast extract or peptone could enhance strongly the
decolorization efficiency. In contrast to a nitrogen source, glucose inhibited decolorization activity because the
consumed glucose was converted to organic acids that might decrease the pH of the culture medium, thus inhibiting the
cell growth and decolorization activity. Decolorization appeared to proceed primarily by biological degradation.
# 2002 Elsevier Science B.V. All rights reserved.
Keywords: Aeromonas hydrophila ; Azo dyes; Anthraquinone dyes; Indigo dyes; Microbial decolorization
1. Introduction
The first synthetic dye, mauevin, was discovered
in 1856. Since then, over 100 000 dyes have been
generated worldwide with an annual production of
over 7�/105 metric tones. Synthetic dyes are
widely used in textile, paper, food, cosmetics and
pharmaceutical industries (Zollinger, 1987; Carliell
et al., 1995). The inefficiency in dyeing processes
has resulted in 10�/15% of unused dyestuff entering
the wastewater directly (Zollinger, 1987; Spadarry
et al., 1994). Color present in dye effluent gives a
straightforward indication of water being polluted,
and discharge of this highly colored effluent can
damage directly the receiving water. Furthermore,
it is difficult to degrade the mixtures of the
wastewater from textile industry by conventional
biological treatment processes, because their ratio
of BOD/COD is less than 0.3 (Chun and Yizhong,
1999). In some cases, traditional biological proce-
dures were combined with physical- or chemical-
* Corresponding author. Tel.: �/886-3-571-6249; fax: �/886-
3-571-3014.
E-mail address: [email protected] (K.-C. Chen).
Journal of Biotechnology 101 (2003) 57�/68
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0168-1656/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved.
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treatment processes to achieve better decoloriza-tion (Vandevivere et al., 1998), but chemical or
physical�/chemical methods are generally costly,
less efficient and of limited applicability, and
produce wastes, which are difficult to dispose of.
As a viable alternative, biological processes have
received increasing interest owing to their cost
effectiveness, ability to produce less sludge, and
environmental benignity (Banat et al., 1996).Therefore, to develop a practical bioprocess for
treating dye-containing wastewater is of great
significance.
The effectiveness of microbial decolorization
depends on the adaptability and the activity of
selected microorganisms. Over the past decades,
many microorganisms are capable of degrading
azo dyes, including bacteria (Zimmerman et al.,1982; Haug et al., 1991; Sani and Banerjee, 1999),
fungi (Gold and Alic, 1993; Swamy and Ramsay,
1999; Balan and Monteiro, 2001; Novotny et al.,
2001), yeast (Martins et al., 1999), actinomycetes
(Zhou and Zimmermann, 1993) and algae (Dilek
et al., 1999). Most azo dyes are reduced anaero-
bically to the corresponding amines with cleavage
of azo bonds by bacterial azoreductase, but theyare difficult to degrade aerobically (Zimmerman et
al., 1982; Banat et al., 1996). Moreover, fungal
ligninolytic enzyme system (lignin peroxidase
(LiP), manganese peroxidase (MnP) and laccase)
might also be involved in the bio-oxidation of dyes
(Gold and Alic, 1993). However, the low pH
requirement (Swamy and Ramsay, 1999) for an
optimum activity of the enzymes and the longhydraulic retention time for complete decoloriza-
tion (Banat et al., 1996; Swamy and Ramsay,
1999) are the disadvantages of using fungi. In
addition, they may inhibit the growth of other
useful microorganisms. Thus, large-scale applica-
tions of fungal decolorization have been limited.
In general, the wastewater from textile industry
contains many various dyes. To gain a widespreadreception, the azo-degrading bacteria should ex-
hibit decolorizing ability for a wide range of dyes.
This study aimed to isolate some bacterial strains,
which possessed the ability to decolorize 24 kinds
of dyes, including azo, anthraquinone, and indigo
dyes. A bacterium displaying the greatest decolor-
izing ability was chosen for further study to
illustrate the factors influencing its efficiency. Inaddition, the major cause of the inhibition of
glucose on the reduction of azo dye was identified.
2. Materials and methods
2.1. Chemicals
Twenty-four dyes were used and chosen from
various types (azo, anthraquinone and indigo) of
important commercial dyes. Azo, anthraquinone
and indigo, containing various substituents such as
nitro and solfonic groups, are the major classes of
dyes with the greatest variety of colors. Acid Blue
74, Acid Orange 7, Acid Red 106, Direct Yellow 4
and Direct Yellow 12 were purchased from theSigma Chemical Company, MO, USA. The other
dyes (Acid Black 172, Acid Blue 264, Acid Yellow
42, Direct Black 22, Direct Orange 39, Direct Red
224, Direct Red 243, Direct Yellow 86, Reactive
Black NR, Reactive Black 5, Reactive Blue 160,
Reactive Blue 171, Reactive Blue 198, Reactive
Blue 222, Reactive Green 19, Reactive Red 120,
Reactive Red 141, Reactive Red 198 and ReactiveYellow 84) were obtained from Everlight Chemical
Industrial Co., Taoyuan, Taiwan. All other che-
micals were reagent grade.
2.2. Screening of decolorizers
Sludge samples were obtained from various
sources including the lake-mud in Tsing Hua
University (Hsinchu, Taiwan) and the sludge ofwastewater treatment plant in Chang Chun Petro-
chemical Co. (Miaoli, Taiwan). In order to obtain
a high-performance bacterial decolorizer, RED
RBN, the most commonly used dye, was first
chosen as the target for screening azo-degrading
bacteria. The mixed bacterial cultures from the
sludge samples were acclimated for 3 months, and
then served as the stock culture. The bacteria-isolating procedures and the test procedures later
used for each dye were carried out in a screening
medium (SM medium). The medium contained the
following components: yeast extract, 10 g; NaCl,
5.0 g in 1 l of distilled water with 0.1 g (except that
described else) of selected dye. Ten milliliter of the
K.-C. Chen et al. / Journal of Biotechnology 101 (2003) 57�/6858
stock culture was added to a 500 ml conical flaskcontaining 100 ml of SM medium and pH was
adjusted to 7.0. The isolates were cultured routi-
nely in an incubator at 30 8C. Next, the broth of
the decolorized flask culture was then transferred
to a fresh SM medium to screen strains that have
color removal ability. The screening procedures in
the liquid culture with SM medium were con-
ducted repeatedly until a decolorizing cultureappeared. An aliquot (0.1�/1 ml samples) of each
supernatant fluid of the isolated cultures was
spread on a SM agar medium, and then incubated
at 30 8C. Colonies surrounded by a decolorized
zone were selected. Isolates were then tested for
their color removal ability in a submerged culture.
Finally, six promising isolates were selected.
2.3. Dye assays and decolorizing cultures
The stock cultures for these isolates were pre-
cultured for 20 h at 30 8C by growing a singlecolony in an anoxic static condition. The same
initial cell concentrations of dye-degrading micro-
organisms were used to decolorize all the dyes.
Decolorization in an individual dye solution could
be seen visually, and was measured at its max-
imum adsorption wavelength (lmax) on culture
supernatants using a scanning spectrophotometer
(UV/vis, Shimadzu, Kyoto, Japan). To ensure thatthe pH change in dye solution did not influence
decolorization, the visible absorption spectra were
recorded between pH 4.0 and 11.0 and the pH did
not affect spectrum. Biomass concentration was
determined by dry cell weight after 24 h drying at
105 8C. All assays were conducted in triplicates
with uninoculated controls.
2.4. Analysis of color removal in the medium
containing mixture of dyes
All 24 dyes, each at a concentration of 50 mgl�1, were dissolved together in SM medium. The
mixture of dyes did not have a well-defined peak at
the visible absorption spectra. Therefore, the
detection of color level was made using the
American Dye Manufacturers Institute (ADMI)
Tristimulus Filter Method (Eaton et al., 1995).
2.5. Identification of selected azo dye-degrading
bacteria
Bacterial isolates with the greatest decoloriza-
tion abilities were first examined by Gram stain-
ing, and further identifications were performed by
the Culture Collection and Research Center, Food
Industry Research and Development Institute
(Hsinchu, Taiwan).
2.6. Decolorization at different culture conditions
The effects of the various culture conditions
such as agitation, aeration, anoxic state and
anaerobic state on decolorization of RED RBN
were examined owing to their various concentra-
tions of dissolved oxygen (DO). Agitation culture(AGI culture), the only culture at shaking condi-
tion, was operated in a rotary incubation shaker
running at 200 rpm. All the other cultures were
under a static condition with no shaking at all.
Anaerobic culture (ANA culture) was bubbled
with pure nitrogen only at the beginning until the
DO became zero, but anoxic culture (ANO
culture) had never been bubbled at all. Aerobicculture (AER culture) was maintained in a con-
tinuous aeration condition (airflow rate of 3 l
min�1). All the experiments were operated at
30 8C and pH 7.0 under a constant initial dye
concentration (RED RBN) of 50 mg l�1. The
concentration of cells, RED RBN, and DO were
monitored as a function of time.
2.7. Glucose analysis
Reducing sugar was analyzed and determined as
glucose by the DNS (3, 5-dinitrosalicylic acid)
method (Miller, 1959). The color tests were made
with 3 ml aliquots of reagent added to 3 ml
aliquots of sample in tubes. The reagent contained
1% dinitrosalicylic acid, 0.2% phenol, 0.05%
sodium sulfite, and 1% sodium hydroxide. Themixtures were heated for 15 min in a boiling water
bath, and then cooled and adjusted to ambient
temperature under running tap water. The color
intensities were measured in a scanning spectro-
photometer (UV/vis, Shimadzu, Kyoto, Japan) at
575 nm.
K.-C. Chen et al. / Journal of Biotechnology 101 (2003) 57�/68 59
Table 1
Decolorization of textile dyes by various bacteria
Type of dyes (C. I. Number) Chemical
structure of
dye
lmax (nm) Color removal (%) at initial concentration (100 mg l�1)
DEC1 DEC2 DEC3 DEC4 DEC5 DEC6
1 day 7 days 1 day 7 days 1 day 7 days 1 day 7 days 1 day 7 days 1 day 7 days
Azo
Acid Orange 7 (15510) Monoazo 485 799/2 1009/1 479/3 1009/2 389/1 1009/2 759/2 1009/3 449/2 1009/3 279/2 1009/2
Acid Red 106 (18110) Monoazo 533 709/2 1009/1 529/2 1009/1 629/5 1009/2 859/6 1009/1 459/5 1009/2 639/3 1009/3
Direct Orange 39 (40215) Monoazo 414 739/3 1009/1 509/2 1009/1 429/4 1009/1 759/2 1009/2 519/4 1009/5 399/5 1009/6
Reactive Red 198 (unpublished) Monoazo 515 659/1 1009/2 859/2 1009/2 869/3 1009/3 899/3 1009/1 829/3 1009/3 829/5 1009/3
Acid Yellow 42 (22910) Diazo 412 159/1 649/3 69/1 449/3 79/3 459/1 119/2 589/3 39/2 349/1 29/1 69/1
Direct Red 224 (unpublished) Diazo 520 59/1 669/3 179/2 499/3 159/4 699/5 129/2 569/2 89/2 579/2 99/1 329/2
Direct Red 243 (29315) Diazo 523 409/2 849/2 229/2 659/2 139/1 689/4 399/1 809/2 159/1 519/2 129/3 669/3
Direct Yellow 4 (24890) Diazo 396 469/2 1009/2 249/2 1009/2 199/2 839/3 609/2 1009/3 229/2 1009/2 149/1 1009/2
Direct Yellow 12 (24895) Diazo 395 719/3 1009/1 519/4 1009/3 409/2 1009/2 779/1 1009/2 629/1 1009/1 399/2 1009/2
Direct Yellow 86 (29325) Diazo 393 519/2 669/3 289/2 669/2 209/1 649/2 429/2 609/4 209/2 569/2 69/1 369/6
Reactive Black 5 (20505) Diazo 597 469/2 959/2 679/3 539/4 629/1 599/5 689/3 859/2 669/3 559/5 639/2 549/5
Reactive Blue 222 (unpublished) Diazo 613 639/2 1009/2 389/2 719/2 439/3 709/4 679/2 1009/1 399/4 649/2 459/5 769/2
Reactive Red 141 (unpublished) Diazo 532 509/2 879/2 179/2 799/3 139/1 829/6 469/2 829/2 109/2 649/6 59/2 399/3
Reactive Red 120 (25810) Diazo 512 419/1 829/2 169/2 669/2 129/2 629/2 409/4 809/5 119/2 599/2 109/1 809/5
Direct Black 22 (35435) Polyazo 482 429/2 699/2 259/2 649/2 199/2 659/3 159/2 459/2 79/2 349/2 59/2 239/3
Acid Black 172 (unpublished) Azo 571 139/1 519/3 89/1 389/4 99/1 409/5 99/1 359/6 29/1 349/2 0 0
Reactive Blue 160 (unpublished) Azo 616 659/1 1009/1 609/2 1009/3 589/1 1009/4 679/2 1009/2 659/2 1009/4 629/2 1009/4
Reactive Blue 171 (unpublished) Azo 608 529/1 809/2 159/2 699/2 139/1 699/2 469/5 729/3 159/2 629/2 219/3 809/5
Reactive Blue 198 (unpublished) Azo 625 99/1 209/2 69/3 209/1 59/1 229/1 109/2 109/1 39/1 119/2 39/2 59/2
Reactive Black NR (unpublished) Azo 598 819/2 1009/2 789/3 1009/2 789/1 1009/2 839/3 1009/2 779/5 1009/3 779/5 1009/2
Reactive Green 19 (unpublished) Azo 630 439/1 839/2 149/2 709/3 109/3 659/2 509/2 779/5 79/2 589/2 209/1 709/3
Reactive Yellow 84 (unpublished) Azo 411 459/2 639/3 199/2 629/2 159/2 609/3 439/4 559/2 149/2 509/5 119/2 449/1
Anthraquinone
Acid Blue 264 (unpublished) Anthraquinone 608 459/2 809/1 279/2 779/3 239/2 689/2 469/3 679/2 259/2 659/2 39/1 199/2
Indigoid
Acid Blue 74 (73015) Indigoid 609 609/2 849/3 509/4 819/3 469/5 859/3 409/1 779/3 309/3 729/5 269/2 709/3
The names of all the dyes above are recognized by the Color Index.
K.-C
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3. Results and discussion
3.1. Isolation and identification
White and pink colonies surrounded by an
almost decolorized zone were isolated and then
tested for color removal capability using sub-
merged cultures. Among these colonies, six of
them with the highest decolorization ability in
SM medium, designated as DEC1-6, were selectedfor a further study.
Decolorization of various dyes by the growing
cells of the six isolates were shown in Table 1.
Among the 24 dyes, Acid Orange 7, Acid Red 106,
Direct Orange 39, Direct Yellow 4, Direct Yellow
12, Reactive Black NR, Reactive Blue 160 and
Reactive Red 198 were reduced completely by all
the strains, DEC1�/6, while Reactive Blue 198 (5�/
22%) and Acid Black 172 (0�/51%) were reduced
only slightly even after 7 days of incubation. The
effectiveness of all the six isolates in decolorizing
these 24 dyes may depend on the structure and
complexity of the dyes, particularly on the nature
and position of substituent in the aromatic rings
and the resulting interactions with the azo bond
(Zimmerman et al., 1982; Sani and Banerjee,1999). However, no clear relationship can be
observed between the position of substituent in
the aromatic rings from published structure of dye
and the decolorization efficiency using dye-degrad-
ing microorganisms in this study, except that most
monoazo dyes tested had color removal higher
than the diazo dyes and anthraquinone dyes tested
under the same initial biomass. The differentefficiency may be due to the number of azo groups.
Similar observation was obtained on the investiga-
tion of the degradability in different structures of
azo dyes by Phanerochaete chrysosporium (Pod-
gornik et al., 1999).
On the other hand, Table 1 also shows that the
decolorization rate of the six isolates were
DEC1�/DEC4�/DEC2X/DEC3�/DEC5�/
DEC6 after 1 day of incubation under the same
initial cell concentrations. However, if we want to
consider an isolate favorable for development of a
practical bioprocess for decolorization, the deco-
lorization rate is very important. Therefore, sev-
eral biochemical and physiological investigations
were conducted to identify the best strain, DEC1.
The strain was identified as Aeromonas hydrophila
according to the GN microplate (Biolog, CA,
USA), API 20E (BioMerieux SA, Marcy l’etoile,
France), API 50 CHE (BioMerieux) and partial
sequencing of 16S rRNA gene. The characteriza-
tion of strain DEC1 was summarized in Table 2.
From phylogenetic analysis based on 16S rRNA
sequence, strain DEC1 was also identified as a
strain that is most related to A. hydrophila .
Table 2
Biochemical and physiological profiles of strain DEC1
Characteristics A. hydrophila (DEC1)
Morphology Rod
Motile �/
Gram staining �/
Aerobic growth �/
Anaerobic growth �/
Nitrate reduction �/
Catalase �/
Gas from glucose �/
H2S from cysteine �/
Acetoin from glucose �/
Indole production �/
Acid from glucose �/
Arginine dihydrolase �/
b-galactosidase �/
Cytochrome oxidase �/
Hydrolysis of
Esculin �/
Gelation �/
Assimilation of
Adipate �/
Arabinose �/
Citrate �/
Gluconate �/
Glucose �/
Malate �/
Maltose �/
Mannitol �/
Mannose �/
N -acetyl-glucosamine �/
Phenyl-acetate �/
Acid from
Arabinose �/
Maltose �/
Mannitol �/
Xylose �/
K.-C. Chen et al. / Journal of Biotechnology 101 (2003) 57�/68 61
In addition, large quantities of RED RBN and
Remazol Black B are now used in textile and
dyestuff industries in Taiwan. RED RBN and
Remazol Black B are denoted as Reactive Red 198
and Reactive Black 5 in Color Index, respectively.
Thus, RED RBN and Remazol Black B were
chosen as the target dyes for further study on
microbial characteristics and the causes of degra-
dation by the representative strain DEC1, A .
hydrophila .
3.2. Characteristics of microbial decolorization
Bacterial degradation of azo dyes is often an
enzymatic reaction linked to anaerobiosis, because
it is inhibited by oxygen that is in competition with
the azo group as the electron receptor in the
oxidation of the reduced electron carrier, i.e.
NADH (Wuhrmann et al., 1980; Zimmerman et
al., 1982; Banat et al., 1996). Seldom are bacteriaable to decolorize azo compounds in the presence
of oxygen (Wuhrmann et al., 1980). Although the
strong oxygen effect on bacterial decolorization
has been proved definitely, the quantitative corre-
lation between DO and color removal has seldom
been reported. A. hydrophila was propagated in
Fig. 1. Effect of various culture conditions on decolorization
by A. hydrophila at 30 8C in SM medium containing 50 mg l�1
RED RBN. (j) AER culture (air flow rate of 3 l min 1�1); (m)
AGI culture (rotary agitation at 200 rpm); (') ANO culture
(no aeration, no agitation); (^) ANA culture (gassing the flasks
with pure nitrogen before static culture).
Fig. 2. Effect of pH on color removal of RED RBN by A.
hydrophila in SM medium at 30 8C under ANO culture. Initial
dye concentration: 100 mg l�1. (j) 1 day of cultivation; (m) 2
days of cultivation; (') 3 days of cultivation.
K.-C. Chen et al. / Journal of Biotechnology 101 (2003) 57�/6862
SM medium using AER, ANO, ANA, or AGIculture to observe cell growth, DO concentration
and decolorization (Fig. 1). In both AGI and AER
cultures, the presence of oxygen would normally
inhibit the activity of decolorization, resulting in a
low efficiency of color removal with A. hydrophila .
As a matter of fact, AGI or AER culture was only
run for 1.5 days and then switched to anoxic static
condition. Color disappeared due to the DOconcentration dropping to almost zero. The above
results suggest that decolorization of azo dye
would not take place at a DO concentration higher
than 0.45 mg l�1 and a slight increase in cell mass
at the initial stage would enhance the efficiency of
color removal (Fig. 1). Therefore, the results
indicated that the decolorization by A. hydrophila
was very sensitive to DO level. To achieve aneffective color removal, agitation and aeration
should be avoided.
Fig. 2 shows that the suitable pH for decolor-
ization of RED RBN ranged from 5.5 to 10.0 with
a sharp change toward both ends of the pH range.
At the two extreme pH values (i.e. pH 4.5 and
11.0), a strong negative effect occurred signifi-
cantly on the growth of bacteria and the stabiliza-tion of pH. These results show that decolorization
of various types of dyes with A. hydrophila
occurred over an extensive range of pH. In other
words, they are favorable for developing a prac-
tical bioprocess for a dye-containing wastewater.
Additionally, when the initial pH of the culture
was at 4.5, the cell mats were deeply colored by
adsorbed dyes only. The adsorption of dye on thecell surface may be related to the mechanism of
charge neutralization. Normally, the dyes tested
are negatively charged. In contrast, the cells in
solution tend to possess relatively positive charges
at lower pH. Thus, the cells may have relatively
higher affinity for the dyes.
Whether RED RBN was used as a substrate for
A. hydrophila , a proper color removal, specificdecolorization rate and cell growth under ANO
culture was observed in the range of 20�/35 8C(data not shown). The low color removal at a
temperature beyond 35 8C may be attributed to
the thermal deactivation of the decolorization
enzymes and the low biomass. According to the
above results, the following decolorization experi-
ments using A. hydrophila were performed at
30 8C and pH 7.0 under ANO culture.
To determine the maximum RED RBN con-
centration tolerated by A. hydrophila , experiments
with different initial dye concentrations (1000�/
8000 mg l�1) were performed. The decolorization
efficiency was above 90% for initial dye concen-
tration less than 3000 mg l�1 after 8 days
cultivation, but it decreased with further increase
in dye concentration. When the dye concentration
was as high as 8000 mg l�1, almost 60% of the dye
was removed after 10 days of cultivation (data not
shown). This means that an acceptable high color
removal can be achieved by the A. hydrophila
strain in an extensive range of azo dye concentra-
tions. In addition, a substrate inhibition effect was
observed at dye concentrations higher than 3000
mg l�1. Reduction in color removal and cell
growth may result from the toxicity of dyes to
bacteria through the inhibition of metabolic activ-
ities. Azo dyes generally contain one or more
sulphonic-acid groups on aromatic rings, which
might act as detergents to inhibit the growth of
microorganisms (Wuhrmann et al., 1980). On the
other hand, it was also reported that dyes were the
Fig. 3. Effect of various nitrogen sources on decolorization of
RED RBN by A. hydrophila at 30 8C under ANO culture.
Initial nitrogen sources concentration: 10 g l�1. (I) blank
(without any nitrogen); (j) peptone; (m) tryptone; (')
monosodium glutamate; (%) meat extract; (") beef extract;
(k) urea; (^) yeast extract.
K.-C. Chen et al. / Journal of Biotechnology 101 (2003) 57�/68 63
inhibitors for nucleic acid synthesis (Ogawa et al.,
1988) or for cell growth (Ogawa et al., 1989).
Furthermore, the color removal exhibited a
growth-associated pattern (data not shown). Themaximum cell growth yield was about 1.2�/1.6 and
0.7�/1.0 g l�1 for dye concentrations between 1000
and 3000 and 4000�/8000 mg l�1, respectively. Our
works on the association of growth (kinetic para-
meters) and decolorization by A. hydrophila are
now in progress. Similar results were obtained
using Remazol Black B instead of RED RBN.
3.3. Effects of nitrogen sources on decolorization
Fig. 3 shows the influence of various organic
nitrogen sources on the efficiency of decolorization
of RED RBN by A. hydrophila . Decolorizationwith peptone or yeast extract was very effective, so
the dye concentration decreased quickly, resulting
in 90% color removal within 2 days of cultivation.
In addition to the organic nitrogen sources, the
inorganic nitrogen sources such as KNO3,
NaNO3, NaNO2, NH4Cl, (NH4)2SO4 were also
selected for decolorization. Similar performances
were observed with control flasks (without any
nitrogen source) but resulting in around 10�/15%
color removal after 6 days cultivation (data not
shown). The results clearly indicate that decolor-
ization of RED RBN by A. hydrophila was greatly
affected by the addition of various nitrogen
sources. The metabolism of yeast extract is con-
sidered essential to the regeneration of NADH
that acts as the electron donor for the reduction of
azo bonds (Carliell et al., 1995). Between these two
nitrogen sources, yeast extract was finally chosen
as a part of culture medium for further experi-
ments because yeast extract is cheaper than
peptone. Similar results were obtained using
Remazol Black B instead of RED RBN.
It had also been found that increasing yeast
extract concentrations (from 0 to 10 g l�1) resulted
in higher decolorization rates, and the decoloriza-
tion rates reached a plateau as yeast extract was
higher than 8 g l�1. However, the color removal
(�/90%) was not enhanced significantly by the
increase in yeast extract from 8 to 10 g l�1 after 1
Fig. 4. Time courses of growth and decolorization of mixture dyes by A. hydrophila at 30 8C under static culture (initial pH 7.0). (m),
Dye concentration; (k), biomass.
K.-C. Chen et al. / Journal of Biotechnology 101 (2003) 57�/6864
day of incubation (data not shown). Therefore,
yeast extract at a concentration of 8 g l�1 as
nitrogen source for decolorization of A. hydrophila
was added in further experiments.
3.4. Decolorization of mixed dyes
Dyes of different structures are often used in the
textile processing industry, and, therefore, the
effluents from the industry are markedly variablein composition. A nonspecific biological process
may be vital for treatment of the textile effluents
containing a mixture of dyes. The rate of decolor-
ization was very fast and the color removal was
almost 90% within 2 days of cultivation, followed
by an insignificant change in decolorization for the
next 10 days (Fig. 4). Fig. 4 also displayed a
growth-associated pattern on color removal.According to the reports (Knapp and Newby,
1995; Sani and Banerjee, 1999) decolorization of
dyes by bacteria can be due to adsorption to
microbial cells or to biodegradation. In adsorp-
tion, examination of the absorption spectrum will
reveal that all peaks decrease approximately in
proportion to each other. If the dye removal is
attributed to biodegradation, either the major
visible light absorbance peak will completely
disappear or a new peak will appear. Dye adsorp-
tion can also be judged clearly by inspecting the
cell mats. Cell mats become deeply colored be-
cause of adsorbing dyes, whereas those retaining
their original colors are accompanied by the
occurrence of biodegradation. The absorbance
peak at 515 nm (A point) disappeared completely
after 7 days cultivation (Fig. 5a). As seen in Fig.
5b, there was a significant decrease in color
intensity or in the peak absorbance at 306, 370
and 597 nm (Points B, C and D, respectively).
Moreover, as the RED RBN and Remazol Black
B were removed, the A. hydrophila strain remained
colorless. A similar result was also observed in a
Fig. 5. Variation in UV�/visible spectra of various dye solu-
tions after decolorizing cultivation with A. hydrophila . (*/)
Original dye solution; (� � �) decolorized dye solution.
Fig. 6. Effect of glucose concentration on decolorization of
RED RBN by A. hydrophila in SM medium at 30 8C under
ANO culture ( initial pH 7.0). Initial glucose concentration, (I)
0 g l�1; (j) 0.15 g l�1; (m) 1.25 g l�1; (") 10.0 g l�1.
K.-C. Chen et al. / Journal of Biotechnology 101 (2003) 57�/68 65
mixture of dyes. Consequently, according to theabove results, the color removal by A. hydrophila
strain might be largely attributed to biodegrada-
tion, and the biosorption onto the bacterial
surfaces was not significant.
3.5. Effect of glucose on the degradation of the
mixed dyes
Glucose has been added to enhance the decolor-ization performance of biological systems in some
studies (Haug et al., 1991; Carliell et al., 1995;
Kapdan et al., 2000). However, others reported
that glucose inhibited the decolorizing activity
(Chung et al., 1978; Knapp and Newby, 1995).
The variability may be due to the different
microbial characteristics. In this study, various
concentrations of glucose (0�/10 g l�1) were firstevaluated for decolorization of RED RBN by A.
hydrophila under ANO culture (Fig. 6). Fig. 6
clearly indicates that glucose concentration of
higher than 0.15 g l�1 inhibited appreciably the
azo reduction of azo dye by A. hydrophila . In
addition, the color of the cell surface became red,
and the color removal, decolorization rate and
biomass decreased significantly with increasingglucose concentration.
Fig. 6 depicts that after 1 day cultivation, only
1.0�/1.9 g l�1 of glucose was consumed in the
medium supplemented with the glucose concentra-
tion at a range of 1.25�/10 g l�1, and the pH of the
media dropped from 7.0 to 4.7�/5.0, followed by a
relatively stable pH value for the next 2 days.
Obviously, this low pH range (4.7�/5.0) had asignificantly negative effect on the growth of
bacteria, so the decolorization of azo groups was
inhibited. These results are also in good agreement
with those found at lower pH as aforementioned
(Fig. 2). Moreover, while the pH of the medium
was adjusted to 7.0 by adding aseptic NaOH after
3 days cultivation, it is worthy of note that color
removal of RED RBN in this culture was in-creased from 25 to 90% within 2 days (data not
shown), and the color of cell surface changed
visually from red to white (original color of the
cell). According to above results, it is inferred that
as consumption of glucose concentration in-
creased, the rate of accumulation of organic acids
in the medium was also increased. The growth and
decolorization of A. hydrophila were inhibited at
lower pH in the medium.
To confirm that glucose inhibited the decolor-
ization activity and the cell growth was due mainly
to the lower pH that was, in part, caused by the
consumed glucose or converted organic acids,
phosphate buffer was added into the medium to
provide pH control during growth and dye deco-
lorization (Fig. 7). The pH of the culture supple-
mented with phosphate buffer dropped much less
than that without buffer because the phosphate
buffer was proved to provide a good pH control as
well as high decolorization activity and cell growth
of A. hydrophila . The results clearly show that the
inhibition of cell growth and bacterial decoloriza-
tion of azo dye by glucose was attributed to the
reduced pH in the surrounding medium through a
Fig. 7. Effect of glucose concentration on decolorization of
RED RBN by A. hydrophila in SM supplemented with or
without buffer at 30 8C under ANO culture after 2 days
cultivation (initial pH 7.0). (j) Supplemented with phosphate
buffer; (I) without buffer.
K.-C. Chen et al. / Journal of Biotechnology 101 (2003) 57�/6866
biological conversion to organic acids. This de-monstrates the importance of pH control to
decolorization if some very biodegradable carbon
sources were present in dye wastewater. Many
aspects of the mechanisms involved in the inhibi-
tion of decolorization by glucose are still scarcely
known.
4. Conclusions
The results indicate that utilization of A. hydro-
phila was suitable for the decolorization of dyes
(RED RBN and Remazol Black B) in the presence
of a nitrogen source such as yeast extract. Cer-
tainly, the use of yeast extract as a nitrogen source
for cell growth would be of low economic effi-ciency in the application of industrial treatment
plant. In order to enhance process efficiency, the
search for cheaper supplementary nitrogen sources
would be essential in future works. In contrast to
nitrogen sources, glucose showed inhibitory effects
on the cell growth and the decolorization activity.
Additionally, to ensure an effective azo dye
decolorization with A. hydrophila required arigorous control of the DO concentration (B/
0.45 mg l�1) in the biological process. High dye
concentrations (�/3000 mg l�1) might have a
toxic effect on the isolate. This strain could also
decolorize synthetic effluent containing a mixture
of different dyes. That is applicable to a wide
variety of individual dyes and mixture of dyes.
Acknowledgements
The authors acknowledge the financial support
of National Science Council of Republic of China
under Grant No. NSC-89-2211-E-007-005.
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