www.elsevier.com/locate/chemosphere
, HuWenrong
b,*
, LiYuezhong
College of Physics and Microelectronics, Shandong University, Jinan 250061, Peoples Republic of China
b
The Research Centre of Environmental Science & Engineering Technology, Shandong University,
No. 73 Jingshi Road, Jinan, Shandong Province 250061, Peoples Republic of China
c
School of Life Science, Shandong University, Jinan 250061, Peoples 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 l1 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
0045-6535/$ - see front matter 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2004.06.036
294
295
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*
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).
296
120
80
6
60
100
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).
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.
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
70
60
50
40
30
20
10
0
10
15
20
25
30
Time (h)
298
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.
50
40
30
20
10
0
0
100
95
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
-1 -1
Specific decolorization rate (mg l h )
100
rdye,max
80
60
40
20
Km
0
0
500
1000
1500
2000
2500
3000
299
rdye;max 4BS
K m 4BS
The maximum specic decolorization rate (rdye,max) estimated from the experiment data was 81.2 mg l1 h1 and
the value of apparent Michaelis constant (Km) was 337.2
mg l1. 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 l1. However, azo dye concentration at 300 mg l1 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
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 l1
090%
15 d
Pasti-Grigsby et al.
(1992)
Amaranth
0.5 mM
3 lM min1 g
of protein1
80%
4d
Textile dyes
6784%
44 h
4BS
50 mg l1
99.1%
24 h
This work
4BS
50 mg l1
99.6%
6h
This work
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 l1 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
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Microbial decolorization of textile-dye-containing euent:
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301