Chemosphere 57 (2004) 293301

www.elsevier.com/locate/chemosphere

Biodegradation mechanisms and kinetics of azo dye 4BS

by a microbial consortium
HeFang
a

, HuWenrong

b,*

, LiYuezhong

College of Physics and Microelectronics, Shandong University, Jinan 250061, People
s Republic of China
b
The Research Centre of Environmental Science & Engineering Technology, Shandong University,
No. 73 Jingshi Road, Jinan, Shandong Province 250061, People
s Republic of China
c
School of Life Science, Shandong University, Jinan 250061, People
s Republic of China
Received 8 October 2003; received in revised form 11 June 2004; accepted 21 June 2004

Abstract
A microbial consortium consisting of a white-rot fungus 8-4* and a Pseudomonas 1-10 was isolated from wastewater
treatment facilities of a local dyeing house by enrichment, using azo dye Direct Fast Scarlet 4BS as the sole source of
carbon and energy, which had a high capacity for rapid decolorization of 4BS. To elucidate the decolorization mechanisms, decolorization of 4BS was compared between individual strains and the microbial consortium under dierent
treatment processes. The microbial consortium showed a signicant improvement on dye decolorization rates under
either static or shaking culture, which might be attributed to the synergetic reaction of single strains. From the curve
of COD values and the UVvisible spectra of 4BS solutions before and after decolorization cultivation with the microbial consortium, it was found that 4BS could be mineralized completely, and the results had been used for presuming
the degrading pathway of 4BS. This study also examined the kinetics of 4BS decolorization by immobilized microbial
consortium. The results demonstrated that the optimal decolorization activity was observed in pH range between four
and 9, temperature range between 20 and 40 C and the maximal specic decolorization rate occurred at 1000 mg l
1 of
4BS. The proliferation and distribution of microbial consortium were also microscopically observed, which further conrmed the decolorization mechanisms of 4BS.
2004 Elsevier Ltd. All rights reserved.
Keywords: Microbial consortium; 4BS; Decolorization mechanisms; Decolorization kinetics

1. Introduction
Azo dyes, the largest chemical class of dyes with the
greatest variety of colors, have been used extensively for
textile, dyeing and paper printing. Approximately 10
15% of the dyes are released into the environment during
manufacturing and usage (Spadary et al., 1994). The

Corresponding author. Tel./fax: +86 531 8392983.

E-mail address: hefanghhd@hotmail.com (F. He).

majority of these dyes is either toxic or mutagenic and

carcinogenic (Nilsson et al., 1993), and poses a potential
health hazard to all forms of life (Sharma and Sobti,
2000). Some azo dyes have been identied as the most
problematic compounds in textile euents as they are
dicult to remove due to their high water solubility
and low exhaustion (Carliell et al., 1994). So purication
of azo dye wastewater has become a matter of great concern, and several advanced treatment methods, such as
color adsorption by activated carbon, had been suggested. Nevertheless it is not widely applied because of

0045-6535/$ - see front matter 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2004.06.036

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F. He et al. / Chemosphere 57 (2004) 293301

the high cost. In the natural environment, azo dye can be

transformed or degraded by a variety of microorganisms, including aerobic and anaerobic bacteria and fungi
(Chung and Stevens, 1993; Banat et al., 1996; Shin and
Kim, 1998; Wong and Yuen, 1998; Swamy and Ramsay,
1999). However, the long growth cycle and moderate
decolorization rate limit the performance of the fungal
system (Banat et al., 1996). In contrast, bacteria could
reduce the color intensity more satisfactorily, but individual bacterial strain cannot degrade azo dyes completely (Haug et al., 1991; Coughlin et al., 1997), and
the intermediate products are carcinogenic aromatic
amines, which need to be further decomposed. Mixed
culture studies may be more appropriate for decolorization of azo dyes. About 80% of color removal in euent
sample containing mixture of azo- and diazo-reactive
dyes was observed by using mixed bacterial culture
(Nigam et al., 1996). An isolated microbial consortium
removed 6784% of color from textile dye euent after
44 h of cultivation (Banat et al., 1997).
The present study used high water soluble Direct
Fast Scarlet 4BS as the model azo dye substrate to isolate strains having comparably high capacity for decolorization of 4BS, and subsequently selected the
optimal microbial consortium by optimizing combination experiments of the isolated individual strains.
Immobilization of microbial cells has received
increasing interest in the eld of wastewater treatment
(Yang et al., 1995; Zhou and Herbert, 1997; Christopher
et al., 2002). Immobilized cells systems have the potential to degrade toxic chemicals faster than conventional
wastewater treatment systems since high densities of specialized microorganisms are used in immobilized cell systems. Among the various cell immobilization methods
that are available, entrapment in polyvinyl alcohol
(PVA) gel beads had been chosen for its ease of use,
low economic cost, low toxicity and high operational
stability to immobilize the microbial consortium.
The objective of this study was to elucidate the decolorization mechanisms of the microbial consortium and
presume the decomposition pathway of 4BS. In addition, the kinetics of 4BS decolorization by immobilized
microbial consortium was also examined. The results
may give us the insight into the synergistic interaction,
dynamics in degradation activity of the microbial community that is usually treated as a black box.

2. Materials and methods

2.1. Azo dye and chemicals
Direct Fast Scarlet 4BS (over 90% purity) was supplied by Printing and Dyeing Co. (Laoling, Shandong,
China). The dye concentration was estimated from the
standard curve of dye concentration versus optical den-

sity at its maximum absorption wavelength (kmax = 500

nm) using a UV-365 scanning spectrophotometer
(UVvisible, Shimadzu, Kyoto, Japan). PVA with a
grade of 99.9% saponication and 2000 degree of polymerization was purchased from Shanghai Petrochemical
Co. (Shanghai, China). All other chemicals were analytical grade.
2.2. Strain isolation and cultivation
The microbial source was obtained from wastewater
treatment facilities of a local dyeing house and was cultivated in enrichment culture media either for bacteria or
for fungi. The previous medium contained (in grams per
liter) peptone 5, yeast extract 2.5, NaCl 5, 4BS 0.05
(pH = 7.0, 2% agar); and the latter contained (in grams
per liter) peptone 5, glucose 5, 4BS 0.05 (pH = 7.0, 1.5%
agar), respectively. 4BS was used as the indicator of
microbial activity. Cultures around which clean zones
expand quickly were further isolated with agar plates
of the same enrichment medium by streak plating. Eight
comparably high eective strains consisting of four bacteria and four fungi were isolated. The single strains
were cultivated in 500 ml asks containing 150 ml
growth medium at 30 C in a rotary shaking bath at
150 rpm for 24 h. Equal volume of a mineral solution
and an enrichment solution was mixed to make the
growth medium. The mineral solution contained (in
grams per liter): Na2HPO4 2, NaH2PO4 1, KNO3 2,
MgSO4 7H2O 0.2, NaCl 5, CaCl2 0.02, 1 ml trace element solution. The trace element solution contained
(in grams per liter): CaCl2 2H2O 2.0, FeSO4 1,
NaWO4 2H2O 0.5, MnSO4 0.5. The pH of the medium
was adjusted to 7.0. The biomass was harvested with
centrifugation at 8000 r min
1 for 10 min and re-suspended in 0.2 mol l
1 phosphorous buer solution with
the same concentration for decolorization experiments.
The optimal microbial consortium was obtained by optimizing combination decolorization experiments of the
above eight comparably high eective single strains.
2.3. Immobilization
Equal quantity of pure white-rot fungus 8-4* and
Pseudomonas 1-10 was washed twice with sterile water
and re-suspended in 50 ml 0.2 mol l
1 phosphorous buffer solution. 10 g Polyvinyl alcohol (PVA) and 1 g sodium alginate were heated to dissolve in 50 ml distilled
water and then cooled down to 3040 C, which was
mixed with microbial cells suspension. The nal mixture
was added drop by drop, through syringe, into saturated
boric acid solution (pH = 6.7, adjusted by Na2CO3 solution) containings 1.0% CaCl2 to yield PVA gel beads
with 23 mm mean diameter. The gel beads were
cross-linked for 24 h at room temperature, then washed

F. He et al. / Chemosphere 57 (2004) 293301

twice with sterile water and stored in refrigerator for

subsequent use.
2.4. Decolorizing cultivation by freely suspended cells
Fresh cells solutions were introduced into 300 ml Erlenmeyer asks with 150 ml mineral solution containing
4BS (nally contained 1.5 g l
1 wet biomass, 50 mg l
1 of
4BS). Cultures were incubating under dierent treatments (see Table 1). All the experiments were operated
in shake asks at an agitation rate of 0 rpm (static incubation, anaerobic condition) and 180 rpm (shaking incubation, aerobic condition) on a rotary shaker (Haer Bin
Dong Lian Dian Zi Device works, Haerbin, China).
During incubation, culture uid without inoculation
was used as the control. Cultures were centrifuged at
7000 rpm for 10 min and the supernatants were evaluated via a light absorption method and percentage
reduction rates were calculated.
2.5. Decolorizing cultivation by immobilized microbial
consortium
Equal quantity of immobilized beads were added into
300 ml Erlenmeyer asks with 150 ml mineral solution
containing 4BS (concentration of immobilized wet biomass was 1.5 g l
1) and shaking on the rotary shaker
at 30 C and 180 rpm. All pH measurements were made
with a pHS-3 digital pH-meter (Shang Hai Lei Ci Device
works, Shanghai, China) with a combined glass-calomel
electrode. The other conditions were the same as the
above for free cells.
2.6. Morphological observation
The gel beads were rinsed with distilled water and
xed with 4.0% (v/v) glutaraldehyde solution overnight
to allow for complete penetration into the gel. The xed
gel beads were then dehydrated by sequential immersion
in increasing concentration of ethanol to removal the

295

nal traces of water. The dehydrated beads were then

dried in a CO2 atmosphere under critical conditions.
The subsequent samples were cut in halves with a sterile
scalpel, coated with gold, and examined using a scanning
electron microscope (Jeol JSM T-300, Hitachi, Japan).

3. Results and discussion

3.1. Biodegradation of 4BS by free cells
3.1.1. Decolorization by single strains
The decolorization of 4BS by single strains under different treatment processes were investigated. The results
were shown in Fig. 1.
It could be seen that Group 1 exhibited high activity
and could get 90% color removal within 36 h. However,
the decolorization rate of Group 2 was very slow and it
only got 65% color removal after 5 d. Group 3 nally obtained about 84% color removal, 6% lower than that in
Group 1. The cells of Pseudomonas 1-10 in all of the
above groups remained white, indicating no 4BS was adsorbed into the cell surface. It seemed that Group 4
showed no biodegradation occurrence, and the cells of
white-rot fungus 8-4* became red in color, indicating
the adsorption of 4BS into the cell surface. Group 5 decolorized 4BS almost completely, but its decolorization
rate was also slow and reached good color removal after
23 d of incubation. Group 6 caused 89% color removal,
about 10% lower than that of Group 5, due to the absence
of oxygen for 18 h. The color of cells of white-rot fungus
8-4* in Group 5 and 6 maintained its original white.
3.1.2. Decolorization in the microbial consortium
The decolorization of 4BS in the microbial consortium under dierent treatment processes were also investigated (see Fig. 1). It could be seen that the microbial
consortium composed of Pseudomonas 1-10 and whiterot fungus 8-4* had a higher decolorization rate due to
synergistic reaction of each other. After 4 d of static incubation, the surface of Group 7 solution was covered with

Table 1
Components of decolorization solution for freely suspended cells and dierent treatment processes
Serial number

Microorganisms

Group
Group
Group
Group
Group
Group
Group
Group
Group

Pseudomonas 1-10
Pseudomonas 1-10
Pseudomonas 1-10
White-rot fungus 8-4*
White-rot fungus 8-4*
White-rot fungus 8-4*
White-rot fungus 8-4* + Pseudomonas 1-10
White-rot fungus 8-4* + Pseudomonas 1-10
White-rot fungus 8-4* + Pseudomonas 1-10

1
2
3
4
5
6
7
8
9

Solution of 4BS

Treatment processes*

100 ml mineral solution

containing 50 mg l
1 of 4BS

a
b
c
a
b
c
a
b
c

a: continuous static incubation (30 C); b: continuous shaking incubation (30 C, 180 rpm); c: static incubation for 18 h (30 C), then
change to shaking incubation (30 C, 180 rpm).

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F. He et al. / Chemosphere 57 (2004) 293301

120

120

80

6
60

100

Color removal (%)

Color removal (%)

100

40

80

8
60
40
20

20

4
0

0
0

20

40

60

80

100

120

Time (h)

10

20

30

40

50

60

70

80

Time (h)

Fig. 1. Color removal of 4BS by single strains (left) and the microbial consortium (right) under dierent processes (serial numbers
corresponding to that in Table 1, at 30 C and pH = 7).

an evidently mycelia net (slight red) made up of white-rot

fungus 8-4*. The color removal of Group 7 was only 5%
higher but 6 h faster than that in Group 1. The decolorization rate of Group 8 increased evidently and it eliminated 4BS completely within only 30 h. After 18 h of
static culture, the mycelia net of the surface became mycelial pellets lled with the whole ask of Group 9 due to
changing to shaking culture, which immediately caused
almost 100% color removal. The color of all cells in
Group 8 and 9 remained its original color.
3.1.3. Analysis of decolorization mechanisms
It could be seen interestingly from the decolorization
results that for the microbial consortium, static and shaken culture gave similar results, but for the individual
strains dierences were seen. The above phenomena
might be attributed to the following decolorization mechanisms analysis: The azo bonds are reduced and cleaved
by azoreductase under anaerobic conditions to form corresponding amines, which is the key step for decolorization of azo dyes (Tan et al., 1999; O
Neill et al., 2000).
Although decolorization can occur under aerobic conditions, it can be promoted remarkably under anaerobic
conditions. The activity of azoreductase from Pseudomonas 1-10 was very high under static condition, which
removed 90% of 4BS within 36 h. But the activity was
suppressed partly in the presence of oxygen under shaking
culture. Consequently, its decolorization rate became
slow (see Group 2 and 3). The results also demonstrated
that anaerobic conditions favored the growth of Pseudomonas 1-10, but it was not indispensable, which indicated
that Pseudomonas 1-10 was a facultative strain. In addition, it should be noted that Pseudomonas 1-10 could
not degrade 4BS completely, which indicated that decomposition of 4BS needs synergistic reaction of versatile enzymes coming from dierent individual strains.

Group 5 had higher color removal under shaking culture, which showed that the decolorization ability and
activity of white-rot fungus 8-4* depended on the environment of rich oxygen (Moreira et al., 1998; Zhang
et al., 1999). Decolorization ability of white-rot fungus
8-4* attributed to its extracellular enzymes-ligninolytic
peroxidases. These enzymes were typically produced
during secondary metabolism of the stationary phase
(Bumpus et al., 1985; Eaton, 1985). So its decolorization
rate was slow and it also needs stimulation by other
strains or enzymes.
Due to the synergistic interaction of individual
strains in the microbial consortium, the decolorization
rate of 4BS was increased remarkably. Although the color removal of Group 7 was only 5% higher than that in
Group 1, its decolorization rate was 6 h faster than that
of Group 1, which might be attributed to the synergistic
reaction between Pseudomonas 1-10 and white-rot fungus 8-4*. The presence of Pseudomonas 1-10 might stimulate the production of extracellular enzymes from
white-rot fungus 8-4*, leading to the decolorization rate
of Group 8 increase obviously relative to Group 5. In
Group 9 it might be attributed that the azoreductase
of Pseudomonas 1-10 had high activity under anaerobic
condition and cleaved the azo bonds. Keeping the
cultures under shaking condition subsequently caused
remarkable increase of decolorization rate due to the synergistic eect. The results also indicated that development of mycelia to mycelial pellets led to increased
diusion rate between oxygen, 4BS and cells. The more
the diusion of oxygen, the higher the activity of extracellular enzymes of white-rot fungus 8-4*, and this resulted in considerable increase of decolorization rate.
The color of all cells remained their original color after
cultivation, indicating the color removal was actually
proceeded primarily by biological degradation.

F. He et al. / Chemosphere 57 (2004) 293301

3.1.4. Presuming the degrading pathway of 4BS in the

microbial consortium
The structure of 4BS is given as following:

297

Fig. 2b. In this gure, the culture had weaker absorbance at 310 nm but stronger absorbance at 250 nm.
After 30 h for decolorizing cultivation, the culture had

OH
N

OH
NHCONH

SO3Na

NHCOCH3

SO3Na

There are two phenyl and naphthyl rings in the structure

of 4BS.
Fig. 2 displayed a typical example of the changes of
UVvisible spectra of 4BS, using the supernatant of
the culture, before and after decolorization cultivation
with the microbial consortium.
From Fig. 2a, 4BS has the maximum absorbance
wavelength (kmax) at 500 nm and the absorbance at
250 and 310 nm conrm the phenyl and naphthyl
rings-possessing 4BS structure (Ke and Dong, 1998).
After 20 h of incubation, the dye structure changed
markedly and the absorbance at 500 nm disappeared
completely (Fig. 2b). This indicated the cleavage of
azo bonds to form corresponding intermediates with
phenyl and naphthyl rings as the parent matrix. Then
the naphthyl rings were cleaved partly to form phenyl
rings or nally were cleaved completely and aliphatic
hydrocarbon intermediates were formed. So the intermediates with phenyl ring were the major components in
the culture, and its content might be more than that
in the original solution, which could be seen from

no maximum absorbance in the range of 200700 nm

(Fig. 2c). This suggested the opening of all aromatic nuclei. These nal products, without any conjugated bonds
or
, were simple aliphatic hydrocarbons,
amines, alcohols and so on, or were even mineralized
completely to CO2 and H2O. During the decolorization
process, N2 and NH3 might also be formed.
On the other hand, 4BS added to the original dye
solution at time zero was equivalent to a COD value
of 37 mg l
1, which was raised to 70 mg l
1 after 10 h
of cultivation and then reduced to the background level
within 30 h (see Fig. 3).
The changes of COD value could be attributed that
4BS was recalcitrant to oxidation degradation. With
the cleavage of azo bonds and the naphthyl and phenyl
rings by the microbial consortium, however, 4BS was
biodegraded to small organics that were easily degraded
via oxidation, leading to the great increase of COD
value. The microorganisms continued to consume the
obtained small organics until near-complete removal of
COD value of culture. All the above results were
compelling evidences that 4BS was mineralized completely. In contrast, after treating with common activated sludge, which is the existing wastewater
treatment (where we obtained the microbial source),

70

COD (mg l -1)

60
50
40
30
20
10
0

Fig. 2. UVvisible spectra of culture containing 4BS before and

after degradation by the microbial consortium, at 30 C and
pH = 7 under shaking culture. (a) Original dye solution; (b)
incubation for 20 h; (c) incubation for 30 h; (d) spectrum for
control culture.

10

15

20

25

30

Time (h)

Fig. 3. The curve of COD value of culture containing 4BS

before and after decolorization cultivation in the microbial
consortium, at 30 C and pH = 7 under shaking culture.

298

F. He et al. / Chemosphere 57 (2004) 293301

activity under normal and realistic operational temperatures, indicating that the immobilized microorganisms
could acclimatize themselves to a broad range of pH
and temperature of practical dyeing wastewater.

only 6070% of COD and 5060% of color were removed prior to any enrichment procedure. The results
demonstrated that the ordinary activated sludge treatment had relatively low eciency. The removal ability
had increased remarkably after enrichment procedure,
which suggested that the usage of this isolated microbial
consortium is of high values in practical wastewater
process of colored euent.

3.2.2. Performance of the immobilized microbial

consortium
Fig. 5 showed 4BS biodegradation by the immobilized microbial consortium as measured by reduction
in dye concentration versus time of incubation.
It could be seen that, due to the provision of necessary protection from recalcitrant organics 4BS that were
toxic to free cells and manipulation of growth rate of
microorganisms independent of washout along with
the proceeding of operational time, the activity of microorganisms inside the immobilized beads can be increased
remarkably. After shaking incubation, 99% of color removal could be accomplished after as short as 6 h;
whereas for free cells, only 90% of color removal was
achieved and it took 24 h for this to carried out. This
again suggested that the immobilized microbial consortium had the ability to treat practical printing and dyeing wastewater with big uctuation.

3.2. Kinetics of 4BS decolorization by immobilized

microbial consortium
From Section 3.1.3, it could be seen that the presence
of oxygen can improve remarkably the color removal of
the microbial consortium. In addition, cells entrapment
inside polymeric material might provide a comparable
low-oxygen microenvironment due to diusion resistance,
which was benecial to the stimulation of Pseudomonas 110
s activity. Based on the above consideration, the immobilized microbial consortium was chosen to be cultivated
under continuous shaking incubation (180 rpm).

Concentration of 4BS (mg l-1 )

3.2.1. Environmental factors on decolorization of 4BS

Shake culture experiments were conducted at dierent initial pH values between 3 and 10, and the temperature was controlled at 25 C (room temperature). The
optimal pH for decolorization ranged from 4 to 9 (Fig.
4). More than 90% color removal was still obtained
when solution pH was deviated to basic value. And this
indicated that the immobilized microbial consortium
could treat practical basic dyeing wastewater at normal
operational temperature and largely decreases the economic cost for acidication.
Under optimum neutral pH condition and over a
range of 2040 C, the immobilized cells showed high
activity of decolorization (Fig. 4). At 35 C the immobilized beads became soft slowly with bulgy volume and
consequently shorten their life, so the optimal operational temperature was 30 C. The results showed essentially no thermal deactivation of the decolorization

50
40
30
20
10
0
0

Fig. 5. 4BS biodegradation by the immobilized microbial

consortium as measured by reduction in dye concentration.

100
95

Color removal (%)

Color removal (%)

95
90
85
80
75

90
85
80
75
70
65

70
2

Solution pH

10

11

Time (h)

15

20

25

30
35
Temperature (C)

40

45

Fig. 4. Eect of pH (left) at 25 C and temperature (right) at pH = 7 on the decolorization of 4BS.

-1 -1
Specific decolorization rate (mg l h )

F. He et al. / Chemosphere 57 (2004) 293301

to be toxic to the white-rot fungus Phanerochaete

chrysosporium (Pasti-Grigsby et al., 1992). As a conclusion, immobilized microbial consortium has the ability
to degrade high concentration of 4BS.

100

rdye,max
80

60

3.3. Morphological observation

40

20

Km
0
0

500

1000

1500

2000

2500

3000

initial dye concentration (mg l-1 )

Fig. 6. Dependence of specic decolorization rate to the

concentration of 4BS using cell beads in shaking culture. Bars
indicate standard deviation (n = 3).

3.2.3. Decolorization kinetic model

In order to determine the maximum decolorization
rate and the maximum concentration tolerance of the
immobilized microbial consortium to 4BS in a shaking
culture, experiments with dierent initial 4BS concentration, ranging from 30 to 3000 mg l
1, were performed.
Fig. 6 showed the dependence of specic decolorization
rate to the concentration of 4BS.
It could be seen that the correlation between specic
decolorization rate (rdye) and initial concentration of
4BS ([4BS]) was thus interpreted by MichaelisMenten
model as following:
rdye

299

rdye;max  4BS
K m 4BS

The maximum specic decolorization rate (rdye,max) estimated from the experiment data was 81.2 mg l
1 h
1 and
the value of apparent Michaelis constant (Km) was 337.2
mg l
1. Fig. 6 also indicated that the toxic tolerance of
dye for the immobilized microbial consortium was excellent, that a substrate inhibition eect might occur only
at dye concentration higher than 1000 mg l
1. However, azo dye concentration at 300 mg l
1 was found

After 8 d decolorizing cultivation, the microbial population development and distribution in the gel beads
were microscopically observed. The results were shown
in Fig. 7.
Fig. 7a showed that most of white-rot fungus 8-4*
was growing in the peripheral surface of the inner layer
of the immobilized beads and developed into long
threadlike hypha in the pores of the beads. This area
was rich in oxygen comparing to other parts of the beads
due to diusion resistance, which conrmed that the
good growth of white-rot fungus 8-4* depended on the
microenvironment of rich oxygen. Most of circle-shaped
Pseudomonas 1-10 was found in the interior part of the
gel matrix (Fig. 7b) and this was an anaerobic zone,
which proved further that anaerobic conditions favored
the growth of Pseudomonas 1-10. The results demonstrated that cells entrapment inside polymeric material
might provide a comparably lower-oxygen microenvironment in the interior part and higher-oxygen microenvironment in the peripheral surface of the inner layer of
the gel beads due to diusion resistance which simultaneously favored the stimulation of activities of Pseudomonas 1-10 and white-rot fungus 8-4*, and contributed
to the synergistic eect in the microbial consortium.
The proliferation and development of microbial consortium inside the beads also further conrmed the decolorization mechanisms by the microbial consortium: azo
bonds were rstly cleaved by the azoreductase of Pseudomonas 1-10 under relatively anaerobic condition; due
to the synergistic eects of the microbial consortium under suitable microenvironment condition, the decolorization rate of 4BS was improved remarkably and
nally mineralization was completed.

Fig. 7. Microbial population development and distribution of the immobilized beads during continuous batch operation.
(a) Peripheral surface of inner layer of beads; (b) interior part of beads.

300

F. He et al. / Chemosphere 57 (2004) 293301

Table 2
Biodegradation characteristics of azo dye
Strain

Dye

Concentration

1

Color removal

Cultivation time

Reference

2799%

15 d

Pasti-Grigsby et al.
(1992)
Hu (1994)

Phanerochaete chrysosporium

Azo dye

150 mg l

Pseudomonas luteola

Red G
RBB
RP2B
V2RP

37.4%
93.2%
92.4%
88%

4
4
4
4

Streptomyces spp.

Azo dye

50 mg l
1

090%

15 d

Pasti-Grigsby et al.
(1992)

Sphingomonas sp. BN6

Amaranth

0.5 mM

3 lM min
1 g
of protein
1

Kudlich et al. (1997)

A mixed bacterial cultures

Mixture of azo- and

diazo-reactive dyes

80%

4d

Nigam et al. (1996)

An isolated bacterial cultures

Textile dyes

6784%

44 h

Banat et al. (1997)

The microbial consortium

4BS

50 mg l
1

99.1%

24 h

This work

The immobilized microbial

consortium

4BS

50 mg l
1

99.6%

6h

This work

3.4. Biodegradation characteristics

Under the experiment conditions, the free microbial
consortium demonstrated markedly higher activity and
got almost 100% color removal within 24 h (50 mg l
1
of 4BS, Group 9). On the other hand, the immobilized
microbial consortium could remove 99% of color after
6 h of shaking incubation (50 mg l
1 of 4BS). In Table
2, the color removals of this paper were compared with
that of previous experiments shown by others.
From Table 2, it could be seen that the isolated
microbial consortium of this paper had higher activity
and better color removal of azo dye than the other
experiments.

4. Conclusions
The study performed decolorization experiments of
individual strains and the microbial consortium under
static and shaking culture. The results showed that the
decolorization mechanisms and degrading pathway as
following: azo bonds were rstly cleaved by the azoreductase of Pseudomonas 1-10 and the rate of producing
extracellular enzymes of white-rot fungus 8-4* were stimulated, and consequently increased due to the synergistic
reaction with Pseudomonas 1-10. The activity of the
extracellular enzymes was also high in the environment

d
d
d
d

of rich oxygen. So the decolorization rate of 4BS was improved remarkably due to all the above synergistic effects, leading to the complete mineralization of 4BS.
The optimal decolorization activity was observed in
pH range between 4 and 9, temperature range between
20 and 40 C. The maximal specic decolorization rate
occurred at 1000 mg l
1 of 4BS. Hence, the immobilized
microbial consortium is able to decolorize high concentration of azo dye eectively. In addition, microscopic
observation revealed that the decolorizing microbial
consortium developed habitat segregation from the
peripheral surface into the interior part of the gel beads,
which can be used to further conrm the decolorization
mechanisms of 4BS.

Acknowledgments
The authors are grateful to the nancial support provided by the Sino-Japan Cooperative program (No.
003250103) and Bonus Fund for Excellent Young Scientists of Shandong Province (No. 9934).

References
Banat, I.M., Nigam, P., Singh, D., Marchant, R., 1996.
Microbial decolorization of textile-dye-containing euent:
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