Biodegradation Mechanisms and Kinetics of Azo Dye 4BS by Microbial Consortium

aial

ong

a College of Physics and Microelectronics, Shandong University, Jinan 250061, Peoples Republic of China

manufacturing and usage (Spadary et al., 1994). The and low exhaustion (Carliell et al., 1994). So purication

of azo dye wastewater has become a matter of great con-

cern, and several advanced treatment methods, such as

color adsorption by activated carbon, had been sug-

gested. Nevertheless it is not widely applied because of

0045-6535/$ - see front matter 2004 Elsevier Ltd. All rights reserved.

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

E-mail address: [email protected] (F. He).

Chemosphere 57 (2004)b The Research Centre of Environmental Science & Engineering Technology, Shandong University,

No. 73 Jingshi Road, Jinan, Shandong Province 250061, Peoples Republic of Chinac 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 mech-

anisms, 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 micro-

bial 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 mgl1 of4BS. The proliferation and distribution of microbial consortium were also microscopically observed, which further con-

rmed 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

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 solubilityBiodegradation mechanismsby a microb

HeFang a, HuWenrdoi:10.1016/j.chemosphere.2004.06.036nd kinetics of azo dye 4BSconsortium

b,*, LiYuezhong c

293301

www.elsevier.com/locate/chemosphere

system (Banat et al., 1996). In contrast, bacteria could

reduce the color intensity more satisfactorily, but indi-

for fungi. The previous medium contained (in grams per

fer solution. 10 g Polyvinyl alcohol (PVA) and 1 g so-

294 F. He et al. / Chemosphere 57 (2004) 293301vidual bacterial strain cannot degrade azo dyes com-

pletely (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 decoloriza-

tion 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 iso-

late strains having comparably high capacity for decol-

orization of 4BS, and subsequently selected the

optimal microbial consortium by optimizing combina-

tion 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 poten-

tial to degrade toxic chemicals faster than conventional

wastewater treatment systems since high densities of spe-

cialized microorganisms are used in immobilized cell sys-

tems. 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 decol-

orization mechanisms of the microbial consortium and

presume the decomposition pathway of 4BS. In addi-

tion, 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 com-

munity 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 sup-

plied by Printing and Dyeing Co. (Laoling, Shandong,

China). The dye concentration was estimated from thethe high cost. In the natural environment, azo dye can be

transformed or degraded by a variety of microorgan-

isms, 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 fungalstandard curve of dye concentration versus optical den-dium alginate were heated to dissolve in 50 ml distilled

water and then cooled down to 3040 C, which wasmixed 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 solu-

tion) containings 1.0% CaCl2 to yield PVA gel beads

with 23 mm mean diameter. The gel beads wereliter) 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 bac-

teria 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 at150 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 ele-ment 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 mediumwas adjusted to 7.0. The biomass was harvested with

centrifugation at 8000 rmin1 for 10 min and re-sus-pended in 0.2 mol l1 phosphorous buer solution withthe same concentration for decolorization experiments.

The optimal microbial consortium was obtained by op-

timizing 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 l1 phosphorous buf-sity at its maximum absorption wavelength (kmax = 500nm) using a UV-365 scanning spectrophotometer

(UVvisible, Shimadzu, Kyoto, Japan). PVA with a

grade of 99.9% saponication and 2000 degree of poly-

merization was purchased from Shanghai Petrochemical

Co. (Shanghai, China). All other chemicals were analy-

tical grade.

2.2. Strain isolation and cultivation

The microbial source was obtained from wastewater

treatment facilities of a local dyeing house and was cul-

tivated in enrichment culture media either for bacteria orcross-linked for 24 h at room temperature, then washed

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 Er-

lenmeyer asks with 150 ml mineral solution containing

4BS (nally contained 1.5 g l1 wet biomass, 50 mgl1 of4BS). Cultures were incubating under dierent treat-

ments (see Table 1). All the experiments were operated

in shake asks at an agitation rate of 0 rpm (static incu-

bation, anaerobic condition) and 180 rpm (shaking incu-

bation, aerobic condition) on a rotary shaker (Haer Bin

Dong Lian Dian Zi Device works, Haerbin, China).

consortium

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 dif-

ferent treatment processes were investigated. The results

s and

a100 ml mineral solution

containing 50 mgl1 of 4BS b

ncuba

F. He et al. / Chemosphere 57 (2004) 293301 295The 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

Table 1

Components of decolorization solution for freely suspended cell

Serial number Microorganisms

Group 1 Pseudomonas 1-10



Group 4 White-rot fungus 8-4*



Group 7 White-rot fungus 8-4* + Pseudomonas 1-10



* a: continuous static incubation (30 C); b: continuous shaking iEqual quantity of immobilized beads were added into

300 ml Erlenmeyer asks with 150 ml mineral solution

containing 4BS (concentration of immobilized wet bio-

mass was 1.5 g l1) and shaking on the rotary shakerat 30 C and 180 rpm. All pH measurements were madewith 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 observationDuring incubation, culture uid without inoculation

was used as the control. Cultures were centrifuged at

7000 rpm for 10 min and the supernatants were evalu-

ated via a light absorption method and percentage

reduction rates were calculated.

2.5. Decolorizing cultivation by immobilized microbialchange to shaking incubation (30 C, 180 rpm).c

a

b

c

tion (30 C, 180 rpm); c: static incubation for 18 h (30 C), thenwere 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 ob-

tained 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 ad-

sorbed 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 de-

colorized 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 consor-

tium under dierent treatment processes were also inves-

tigated (see Fig. 1). It could be seen that the microbial

consortium composed of Pseudomonas 1-10 and white-

rot fungus 8-4* had a higher decolorization rate due to

synergistic reaction of each other. After 4 d of static incu-

bation, the surface of Group 7 solution was covered with

dierent treatment processes

Solution of 4BS Treatment processes*

a

b

c

Col

or re

mov

al (%

)

icrob

296 F. He et al. / Chemosphere 57 (2004) 293301an 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 decolor-

ization rate of Group 8 increased evidently and it elimi-

nated 4BS completely within only 30 h. After 18 h of

static culture, the mycelia net of the surface became my-

celial 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 sha-

ken culture gave similar results, but for the individual

strains dierences were seen. The above phenomena

0 20 40 60 80 100 1200

20

40

60

80

100

120

6

5

4

3

2

1C

olor

rem

oval

(%)

Time (h)

Fig. 1. Color removal of 4BS by single strains (left) and the m

corresponding to that in Table 1, at 30 C and pH = 7).might be attributed to the following decolorization mech-

anisms analysis: The azo bonds are reduced and cleaved

by azoreductase under anaerobic conditions to form cor-

responding amines, which is the key step for decoloriza-

tion of azo dyes (Tan et al., 1999; ONeill et al., 2000).Although decolorization can occur under aerobic condi-

tions, it can be promoted remarkably under anaerobic

conditions. The activity of azoreductase from Pseudo-

monas 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 Pseudo-

monas 1-10, but it was not indispensable, which indicated

that Pseudomonas 1-10 was a facultative strain. In addi-

tion, it should be noted that Pseudomonas 1-10 could

not degrade 4BS completely, which indicated that decom-

position of 4BS needs synergistic reaction of versatile en-

zymes coming from dierent individual strains.Group 5 had higher color removal under shaking cul-

ture, which showed that the decolorization ability and

activity of white-rot fungus 8-4* depended on the envi-

ronment 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 col-

or removal of Group 7 was only 5% higher than that in

Group 1, its decolorization rate was 6 h faster than that

0 10 20 30 40 50 60 70 800

20

40

60

80

100

120

8

97

Time (h)

ial consortium (right) under dierent processes (serial numbersof Group 1, which might be attributed to the synergistic

reaction between Pseudomonas 1-10 and white-rot fun-

gus 8-4*. The presence of Pseudomonas 1-10 might stim-

ulate 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 sy-

nergistic eect. The results also indicated that develop-

ment 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 extra-

cellular enzymes of white-rot fungus 8-4*, and this re-

sulted 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.

3.1.4. Presuming the degrading pathway of 4BS in the

microbial consortium

The structure of 4BS is given as following:

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

Fig. 2b. In this gure, the culture had weaker absorb-

ance at 310 nm but stronger absorbance at 250 nm.

After 30 h for decolorizing cultivation, the culture had

no maximum absorbance in the range of 200700 nm

(Fig. 2c). This suggested the opening of all aromatic nu-

clei. These nal products, without any conjugated bonds

N NNHCONH

OH

SO3Na

SO3Na

OH

N N NHCOCH3

Fig. 2. UVvisible spectra of culture containing 4BS before and0 5 10 15 20 25 30

0

10

20

30

40

50

60

CO

D (m

g l-1

)

F. He et al. / Chemosphere 57 (2004) 293301 297after degradation by the microbial consortium, at 30 C andpH = 7 under shaking culture. (a) Original dye solution; (b)

incubation for 20 h; (c) incubation for 30 h; (d) spectrum for250 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 interme-

diates 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 fromcontrol culture.Time (h)

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

before and after decolorization cultivation in the microbialor , 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 mgl1, which was raised to 70 mgl1 after 10 hof 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 com-

pletely. In contrast, after treating with common acti-

vated sludge, which is the existing wastewater

treatment (where we obtained the microbial source),

70consortium, at 30 C and pH = 7 under shaking culture.

only 6070% of COD and 5060% of color were re-

moved prior to any enrichment procedure. The results

demonstrated that the ordinary activated sludge treat-

ment 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. 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

activity under normal and realistic operational tempera-

tures, indicating that the immobilized microorganisms

could acclimatize themselves to a broad range of pH

and temperature of practical dyeing wastewater.

3.2.2. Performance of the immobilized microbial

consortium

Fig. 5 showed 4BS biodegradation by the immobi-

lized microbial consortium as measured by reduction

in dye concentration versus time of incubation.

It could be seen that, due to the provision of neces-

sary 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 micro-

organisms inside the immobilized beads can be increased

remarkably. After shaking incubation, 99% of color re-

moval 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 consor-

tium had the ability to treat practical printing and dye-

ing wastewater with big uctuation.

30

40

50

atio

n of

4BS

(mg

l-1)

298 F. He et al. / Chemosphere 57 (2004) 293301inside polymeric material might provide a comparable

low-oxygenmicroenvironment due to diusion resistance,

which was benecial to the stimulation of Pseudomonas 1-

10s activity. Based on the above consideration, the immo-bilized microbial consortium was chosen to be cultivated

under continuous shaking incubation (180 rpm).

3.2.1. Environmental factors on decolorization of 4BS

Shake culture experiments were conducted at dier-

ent initial pH values between 3 and 10, and the temper-

ature was controlled at 25 C (room temperature). Theoptimal 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 eco-

nomic cost for acidication.

Under optimum neutral pH condition and over a

range of 2040 C, the immobilized cells showed highactivity of decolorization (Fig. 4). At 35 C the immobi-lized beads became soft slowly with bulgy volume and

consequently shorten their life, so the optimal opera-

tional temperature was 30 C. The results showed essen-tially no thermal deactivation of the decolorization

2 3 4 5 6 7 8 9 10 1170

75

80

85

90

95

Col

or re

mov

al (%

)

Solution pHFig. 4. Eect of pH (left) at 25 C and temperature15 20 25 30 35 40 45

65

70

75

80

85

90

95

100

Col

or re

mov

al (%

)

Temperature (C)

0 2 4 6 80

10

20

Conc

entr

Time (h)

Fig. 5. 4BS biodegradation by the immobilized microbial

consortium as measured by reduction in dye concentration.(right) at pH = 7 on the decolorization of 4BS.

to be toxic to the white-rot fungus Phanerochaete

chrysosporium (Pasti-Grigsby et al., 1992). As a conclu-

sion, immobilized microbial consortium has the ability

to degrade high concentration of 4BS.

3.3. Morphological observation

After 8 d decolorizing cultivation, the microbial pop-

ulation 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

0 500 1000 1500 2000 2500 30000

20

40

60

80

100

rdye,max

K m

Spec

ific

deco

loriz

atio

n ra

te (m

g l-1

h-1)

initial dye concentration (mg l-1)

F. He et al. / Chemosphere 57 (2004) 293301 2993.2.3. 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 concentra-

tion, ranging from 30 to 3000 mgl1, 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 rdye;max 4BSKm 4BSThe maximum specic decolorization rate (rdye,max) esti-

mated from the experiment data was 81.2 mgl1h1 andthe value of apparent Michaelis constant (Km) was 337.2

mgl1. Fig. 6 also indicated that the toxic tolerance ofdye for the immobilized microbial consortium was excel-

Fig. 6. Dependence of specic decolorization rate to the

concentration of 4BS using cell beads in shaking culture. Bars

indicate standard deviation (n = 3).lent, that a substrate inhibition eect might occur only

at dye concentration higher than 1000 mgl1. How-ever, azo dye concentration at 300 mgl1 was found

Fig. 7. Microbial population development and distribution of t

(a) Peripheral surface of inner layer of beads; (b) interior part of beahe immobilized beads during continuous batch operation.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 demon-

strated that cells entrapment inside polymeric material

might provide a comparably lower-oxygen microenvi-

ronment in the interior part and higher-oxygen microen-

vironment in the peripheral surface of the inner layer of

the gel beads due to diusion resistance which simulta-

neously favored the stimulation of activities of Pseudo-

monas 1-10 and white-rot fungus 8-4*, and contributed

to the synergistic eect in the microbial consortium.

The proliferation and development of microbial consor-

tium inside the beads also further conrmed the decolor-

ization mechanisms by the microbial consortium: azo

bonds were rstly cleaved by the azoreductase of Pseu-

domonas 1-10 under relatively anaerobic condition; due

to the synergistic eects of the microbial consortium un-

der suitable microenvironment condition, the decolori-

zation rate of 4BS was improved remarkably and

nally mineralization was completed.ds.

ration

1

(1992)

1

(1992)

1

1

300 F. He et al. / Chemosphere 57 (2004) 2933013.4. Biodegradation characteristics

Sphingomonas sp. BN6 Amaranth 0.5 mM

A mixed bacterial cultures Mixture of azo- and

diazo-reactive dyes

An isolated bacterial cultures Textile dyes

The microbial consortium 4BS 50 mgl

The immobilized microbial

consortium

4BS 50 mglTable 2

Biodegradation characteristics of azo dye

Strain Dye Concent

Phanerochaete chrysosporium Azo dye 150 mgl

Pseudomonas luteola Red G

RBB

RP2B

V2RP

Streptomyces spp. Azo dye 50 mglUnder the experiment conditions, the free microbial

consortium demonstrated markedly higher activity and

got almost 100% color removal within 24 h (50 mgl1

of 4BS, Group 9). On the other hand, the immobilized

microbial consortium could remove 99% of color after

6 h of shaking incubation (50 mgl1 of 4BS). In Table2, 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 azore-

ductase of Pseudomonas 1-10 and the rate of producing

extracellular enzymes of white-rot fungus 8-4* were stim-

ulated, and consequently increased due to the synergistic

reaction with Pseudomonas 1-10. The activity of the

extracellular enzymes was also high in the environmentof rich oxygen. So the decolorization rate of 4BS was im-

3 lMmin1gof protein1

Kudlich et al. (1997)

80% 4 d Nigam et al. (1996)

6784% 44 h Banat et al. (1997)

99.1% 24 h This work

99.6% 6 h This work37.4% 4 d Hu (1994)

93.2% 4 d

92.4% 4 d

88% 4 d

090% 15 d Pasti-Grigsby et al.Color removal Cultivation time Reference

2799% 15 d Pasti-Grigsby et al.proved remarkably due to all the above synergistic ef-

fects, 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 rateoccurred at 1000 mgl1 of 4BS. Hence, the immobilizedmicrobial consortium is able to decolorize high concen-

tration 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 pro-

vided by the Sino-Japan Cooperative program (No.

003250103) and Bonus Fund for Excellent Young Scien-

tists of Shandong Province (No. 9934).

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Biodegradation mechanisms and kinetics of azo dye 4BS by a microbial consortiumIntroductionMaterials and methodsAzo dye and chemicalsStrain isolation and cultivationImmobilizationDecolorizing cultivation by freely suspended cellsDecolorizing cultivation by immobilized microbial consortiumMorphological observation

Results and discussionBiodegradation of 4BS by free cellsDecolorization by single strainsDecolorization in the microbial consortiumAnalysis of decolorization mechanismsPresuming the degrading pathway of 4BS in the microbial consortium

Kinetics of 4BS decolorization by immobilized microbial consortiumEnvironmental factors on decolorization of 4BSPerformance of the immobilized microbial consortiumDecolorization kinetic model

Morphological observationBiodegradation characteristics

AcknowledgmentsReferences

Documents

Biodegradation Mechanisms and Kinetics of Azo Dye 4BS by Microbial Consortium