aerobic fluidized beds system with a Pseudomonas sp. isolate

Bioresource Technology 101 (2010) 34–40

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Biodegradation of Reactive blue 13 in a two-stage anaerobic/aerobic fluidized beds system with a Pseudomonas sp. isolate Jun Lin, Xingwang Zhang, Zhongjian Li, Lecheng Lei * Institute of Industrial Ecology and Environment, Yuquan Campus, Zhejiang University, Hangzhou 310027, China

a r t i c l e

i n f o

Article history: Received 26 March 2009 Received in revised form 5 July 2009 Accepted 16 July 2009 Available online 26 August 2009 Keywords: Fluidized bed Pseudomonas Reactive blue 13

a b s t r a c t Pseudomonas sp. strain L1 capable of degrading the azo textile dye Reactive blue 13, was isolated from activated sludge in a sequencing batch reactor. A continuous two-stage anaerobic/aerobic biological fluidized bed system was used to decolorize and mineralize Reactive blue 13. The key factors affecting decolorization were investigated and the efficiency of degradation was also optimized. An overall color removal of 83.2% and COD removal of 90.7% was achieved at pH 7, a residence time of 70 h and a glucose concentration of 2 g/L, HRT = 70 h and Cglucose = 2000 mg/L. Oxygen was contributing to blocking the azo bond cleavage. Consequently, decolorization occurred in the anaerobic reactor while partial mineralization was achieved in the aerobic reactor. A possible degradation pathway based on the analysis of intermediates and involving azoreduction, desulfonation, deamination and further oxidation reactions is presented. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Colored effluents of the dye-consuming industries are a consequence of dye loss during the textile coloration process. The fixation of dye onto the fiber is mainly dependent upon dyestuff types. Reactive dyes, however, have rather low rates of fixation (60–70%). Therefore, up to 40% of the color is discharged in the effluent, resulting in high color of effluent from reactive dyeing operations (O’Neill et al., 1999). These reactive dyes, no matter what form they appear in (the original or the hydrolyzed form), are difficult to degrade biologically and are potentially toxic to animals and humans. The problem is particularly associated with those reactive azo dyes that are used for dyeing cellulose fibers, since these dyes make up approximately 30% of the total dye market (Pearce et al., 2003). Though many physicochemical techniques have been reported to mineralize reactive azo dyes effectively (Feng et al., 2000; He et al., 2008; Rajkumar et al., 2007), some limitations still exist in practical application, due to the expense and the occurrence of toxic (Kalyani et al., 2008). Biological treatment of dyes, in contrast, is a costeffective alternative to the physicochemical methods. A large number of studies indicate that a combination of an anaerobic with an aerobic system is the most logical strategy for azo dye degradation (Libra et al., 2004; Mohanty et al., 2006; O’Neill et al., 2000). The reductive cleavage of the azo bond under anaerobic conditions is a non-specific and presumably extracellu* Corresponding author. Tel./fax: +86 571 88273090. E-mail address: [email protected] (L. Lei). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.07.037

lar process, resulting in the formation of colorless but toxic aromatic amines (van der Zee and Villaverde, 2005). Biodegradation of aromatic amines, however, is an aerobic process. The feasibility of this strategy was first demonstrated for the sulfonated azo dye, Mordant Yellow 3 (Haug et al., 1991). Since then, different reactor configurations have been used for anaerobic/aerobic systems. These include anaerobic high-rate reactors, such as upflow anaerobic sludge blanket, fixed film, rotating biological contactors and anaerobic baffled reactors for anaerobic processes and activated sludge and rotating biological contactors for aerobic treatment (dos Santos et al., 2007; Kudlich et al., 1996; Tan et al., 1999). In simultaneous treatment systems, decolorization takes place in anaerobic zones, and color removal levels ranging from 70–95% (Pearce et al., 2003). Since mixed culture studies are more comparable to practical situations, degradation of reactive azo dyes is regularly carried out by mixed cultures (Ekici et al., 2001; Mohanty et al., 2006). However, mixed cultures provide an average macroscopic view of the system and the results of experiments with such cultures are difficult to interpret. To determine the mechanisms of biodegradation and to simplify the processes, some studies on the subject of color removals have been carried out using pure culture system (Jadhav et al., 2007; Parshetti et al., 2007). The C.I. Reactive blue 13, a representative of copper-containing reactive azo dyes, was chosen as target in this work. Given the advantage of uniform particle distribution, fluidized bed reactors were employed in the current study. Pseudomonas sp. strain L1, a novel strain with high efficiency in azo dye decolorization, was isolated from a sludge batch reactor and then inoculated into

J. Lin et al. / Bioresource Technology 101 (2010) 34–40

two-stage anaerobic/aerobic fluidized bed reactors. Serial tests were run to assess the pure culture’s decolorization and further degradation capacity in anaerobic and aerobic reactors. Operational parameters of this system were optimized, too. The degradation of azo dye and its metabolites in both anaerobic and aerobic reactors were detected by UV–Vis, HPLC and GC–MS. 2. Methods

35

flasks containing 200 ml of sterilized and colored (200 mg/L Reactive blue 13) medium. The flasks were incubated at 35 °C under static conditions. After 48 h of incubation, the effective bacteria that decolorized the medium were placed on the nutrient agar plates again for further isolation and purification. The ability of decolorization was tested repeatedly. The isolate (L1) with the highest decolorization rate was chosen. An identification of the culture by 16S-rRNA analyses was done by Sangon Biological Engineering Technology & Science Corporation in Shanghai, China.

2.1. Dye and chemicals 2.3. Immobilization of the culture The commercially used textile azo dye C.I. Reactive blue 13 was purchased from the local market and used for the study without any further purification. The medium used in this study was composed of glucose (1000 mg/L), NH4Cl (200 mg/L), KH2PO4 (15 mg/ L), K2HPO4 (10 mg/L), MgSO4 (12.5 mg/L), FeSO4 (12.5 mg/L) and maintained a constant pH of 7 by the addition phosphate buffer. All other chemicals used were of the highest purity available. 2.2. Isolation and screening of dye degrading microorganism The mixed culture was obtained from a sequencing batch reactor (V = 8.5 L) which had been seeded originally with activated sludge from the aerobic part of the SiBao sewage treatment plant in Hangzhou, China. At the acclimatization stage, the culture had been gradually exposed to an increasing concentration of dye for two months. Then, 1.0 ml of a sample from the culture was serially diluted and placed on to nutrient agar containing 300 mg/L dye. Bacterial isolates were picked based on the colony color and their texture and inoculated separately into cotton-plugged 250-ml

Granular activated carbon (GAC) was mixed with the culture in a beaker. The medium supplemented with dye was added. After agitation at 35 °C for 72 h, the surface of the GAC was coated with a thin layer of biofilm which consisted of rod-shaped bacteria. 2.4. Experimental set-up and operation The experimental set-up is depicted in Fig. 1. Two fluidized beds (RA and RO) equipped with three-phase separators were employed as the anaerobic and aerobic reactors, respectively. Both bioreactors, made of Plexiglas, were composed of a main column that was 50 cm in height, 5 cm in diameter. The flow-increasing zone and flow-decreasing zone were separated by a draft-tube, so that the stability of internal circulation was greatly enhanced. In the upper section, a three-phase separator was built to minimize outflow of the immobilized particles. A nozzle was placed on the bottom of RA while an air-distributor was placed in RO. Both RA and RO had a reaction-zone volume of 1500 ml.

Fig. 1. Schematic diagram of the two-stage anaerobic/aerobic fluidized beds system.

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J. Lin et al. / Bioresource Technology 101 (2010) 34–40

GAC coated with Pseudomonas sp. L1 was placed in the bioreactors to reach a solid volume of 5% (v/v). The temperature of the reactors was controlled at 35 °C. To maintain the circulation of particles, the liquid recycle rate was set at 1.7 L/min in RA, while the air input was set at 0.4 L/min in RO. The reactors were connected in series. The system was operated in batch mode for 24 h to activate the immobilized cells before it was subsequently switched to continuous mode. The experiment data were collected after the system had achieved a stable status under the given condition and the adsorption of GAC had already reached balance and other interferences, such as temporary biological inadaptation and variation in biomass caused by sudden operating condition change, were minimized.

efficiency was obtained at pH 6. Negative effects were observed at pH 5 and pH 9. At pH 5, the decolorization process was inhibited and it took 120 h for the decolorization rate to reach above 80%. At pH 9, the biomass decreased greatly. The decolorization rate varied with pH from 80.8% to 94%. Fig. 2b displays the removal of Reactive blue 13 at different initial dye concentrations. The time of decolorization did not increase when the concentration increased from 50 to 400 mg/L. The color was reduced rapidly in the first few hours and then the reduction slowed. This phenomenon is possibly due to the toxicity of the metabolites that were formed during dye reduction, as previously observed by Wuhrmann et al. (1980). Higher dye concentrations (above 500 mg/L) showed a toxic effect since decolorization was decreased.

2.5. Analytical methods 3.2. Effect of oxygen 2.5.1. Dye concentration 5 ml sample was centrifuged at 8873g for 10 min and the absorbance of the supernatant was determined by UV–Vis spectrophotometer (UV-1200, Shimadzu). The concentration of Reactive blue 13 was derived from a standard curve, based on observations of absorbance obtained at kmax (596 nm). 2.5.2. HPLC analysis HPLC analysis was carried out on an Agilent 1200 chromatograph, which was equipped with a UV detector and an ODS-C18 reversed phase column. To determine the dye fragments produced upon decolorization, the treated samples were centrifuged (8873g for 10 min), clarified by 0.22 lm filters and then used directly for HPLC analysis. A mobile phase composed of 30% acetonitrile, 0.1% H3PO4, and 69.9% water was used at a flow rate of 1 ml/ min. The UV detector wavelength was set at 250 nm. 2.5.3. GC–MS analysis Spectra of intermediate compounds were obtained with GC–MS (Agilant 6890N/5975B), equipped with a HP-5 capillary column (30 m  0.25 mm  0.25 lm film thickness). 200-mL samples were centrifuge at 8873 g and the supernatant was filtered through a 0.22 lm membrane filter. The filtrate was extracted three times with methylene dichloride and evaporated to 1 ml in a rotary vacuum evaporator with 45 °C water bath. The column oven was initially set at 70 °C for 5 min, and then programmed to reach 150 °C at a rate of 5 °C min1. Both injector and detector were operating at 260 °C. The compounds were identified on the basis of mass spectra and retention time using the NIST library.

A comparative experiment between anaerobic and aerobic fluidized bed seeded with Pseudomonas sp. L1 was done. Color removal was barely observable in aerobic reactor. The similar phenomenon was observed in the two-stage fluidized beds system. According to the data in Table 1, the decolorization capacity of Pseudomonas sp. L1 in RA changed with varying HRT, pH and feed concentration. The color removal efficiency fluctuated between 51.98% and 86.88%. In contrast, the decolorization in RO was inefficient (less than 5%) under each operating condition tested. The inhibition caused by oxygen was also proved by comparison of the decolorization effect under static and shaking conditions (Kalme et al., 2007; Kalyani et al., 2008; Saratale et al., 2009). Inefficiency of decolorization was found at shaking condition which allowed for better oxygenation. Furthermore, in many reports on aerobic degradation, the bacterial strains were incubated without shaking in the presence of azo dyes (Chang and Lin, 2000; Chen et al., 1999). These resting cell cultures presumably become rapidly oxygen depleted, and the reactions observed should therefore be viewed as an anaerobic incubation (Stolz, 2001). As a high-redox-potential electron acceptor, oxygen inhibits the dye reduction in aerobic environment. This is because the sequence of electron-accepting processes is differentiated according to the free energy gained in the respective catabolism (Yoo et al., 2001). As shown in the equations below, the redox potential level of oxygen reduction is much higher than that of azo bond reduction. Thus, the electrons liberated from the oxidation of electron donors by the cells are preferentially used to reduce oxygen rather than the azo dye (Pearce et al., 2003).

3. Results and discussion CRB13 (mg/L)

The isolated bacterium is Gram-negative, rod-shaped with polar flagella and a facultative anaerobe. Its colonies are dull yellow, circular and slightly convex. An identification of culture based on biochemical tests and 16S-rRNA analysis was done by Sangon Biological Engineering Technology & Science Corporation in Shanghai, China. The sequence of a 1402-bp region of the 16S-rRNA gene of the isolate was determined. The nucleotide alignment showed the highest phylogenetic similarity to the genus Pseudomonas. The similarity scores ranged from 93.5% (Pseudomonas sp. MFY72) to 98.8% (Pseudomonas sp. AHL 2 and 7.5). To evaluate the adaptability of the isolated bacteria, the effect of pH and initial dye concentration on decolorization was tested under static condition (35 °C and Cglucose = 2000 mg/L). Fig. 2a shows the decolorization of Reactive blue 13 by Pseudomonas sp. L1 with the pH varying from 5–9. The results indicated that the optimum

pH=5 pH=6 pH=7 pH=8 pH=9

a

200 150 100 50 0 600

CRB13 (mg/L)

3.1. Identification and evaluation of dye decolorization bacteria

250

500

50 mg/L 100 mg/L 200 mg/L 300 mg/L 500 mg/L

b

400 300 200 100 0 0

20

40

60

80

100

120

Time (h) Fig. 2. The effect of (a) pH and (b) initial dye concentration on decolorization.

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J. Lin et al. / Bioresource Technology 101 (2010) 34–40 Table 1 Effect of different operating conditions on color and COD removal efficiency. Experimental parameter

Concentration of dye (mg/L)

HRT (h)

Concentration of glucose (mg/L)

pH

20 30 40 50 60 70 500 1000 2000 3000 4000 5000 5 6 7 8 9

Influent

RA effluent

RO effluent

194.1335 198.2818 206.1414 203.9321 206.5502 204.6509 198.5758 200.8618 203.9321 205.8881 201.7032 210.9175 199.7326 229.1879 203.9321 216.4965 199.5638

65.8404 53.1078 46.9380 41.4512 37.1708 34.8787 95.3507 62.4009 41.4512 40.5242 43.6568 42.7781 26.2037 47.9919 41.4512 52.8707 30.7868

63.1283 51.1012 47.2579 41.5510 37.3716 34.4039 90.6120 60.8068 41.5510 40.4170 43.5284 42.2109 26.6219 46.4160 41.5510 51.6575 30.0695

O þ 4Hþ þ 4e ! 2H2 O ðE00 ¼ 810 mvÞ 0

þ



R—N@N—R þ 4H þ 4e ! R—NH2 þ R

ð1Þ 0

—NH2 ðE00

< 200 mvÞ

ð2Þ

The inhibitory effect of oxygen on bacterial azo reduction is temporary, since the culture regained its decolorization capacity after transfer to the anaerobic reactor. It can be concluded that for the purpose of color removal, aeration and agitation, which increases the concentration of oxygen in solution, should be avoided (Chang and Lin, 2000). However, mineralization of glucose and dye will not occur in the absence of oxygen.

Overall removal efficiency

COD (mg/L) Influent

RA effluent

RO effluent

67.48 74.23 77.08 79.63 81.91 83.19 54.37 69.73 79.63 80.37 78.42 79.99 86.67 79.75 79.63 76.14 84.93

1960 1850 1870 2020 1920 1930 480 920 2020 2750 3880 4730 1840 1880 2020 1860 1920

1360 1210 1300 1230 1280 1210 210 530 1230 1700 2520 3150 1420 1280 1230 1420 1560

650 420 280 230 200 180 100 110 230 300 400 820 430 180 230 290 620

Overall removal efficiency

66.84 77.30 85.03 88.61 89.58 90.67 79.17 88.04 88.61 89.09 89.69 82.66 76.63 90.43 88.61 84.41 67.71

glucose was enhanced as HRT increased, but the enhancement became lower immediately after the HRT reached 50 h. Regardless of the increased HRT, the constant high remaining COD concentration in the effluent of RA showed that even glucose could not be mineralized completely under anaerobic conditions. In contrast, most of soluble COD was removed in RO. As the HRT went up to 70 h, both the total color and COD removal efficiency increased to 83.2% and 90.7%, respectively.

3.4. Effect of feed concentration 3.3. Effect of hydraulic retention time (HRT) The effect of HRT on dye decolorization was evaluated when the reactors were operated at a constant pH of 7 and influent concentration of 200 mg/L Reactive blue 13. The COD loading was maintained at 2000 mg/L by adding 1000 mg/L glucose into the medium. Here, we used overall length of time that water samples remained in the two reactors as the HRT. Ratios of HRT in each reactor could be calculated according to the volume of two reactors (7:5). Table 1 depicts the steady state results obtained at a gradually rising HRT from 20 to 70 h. The overall degradation of dye and

B

2.5

Influent RA effluent RO effluent

Absorbance

2.0

Absorbance (mAU)

3.0

The decolorization rate measured under different glucose concentrations at a 50-h HRT is listed in Table 1. In the absence of glucose, decolorization decreased sharply, since Reactive blue 13 cannot be utilized directly as carbon source by Pseudomonas sp. L1. The decolorization performance of biological systems was enhanced by adding glucose. Similar observations were made for different dyes in the past (Kapdan et al., 2000; Yang et al., 2009). The color removal efficiency increased to 80.3% when the feed concentration reached 3000 mg/L; however, further increases in feed concentration resulted in increased residual COD, even in the RO reactor.

1.5

C

A

1.0 0.5 0.0 300

400

500

600

700

Wavelength (nm) Fig. 3. UV–Vis spectra of Reactive blue 13 treated in RA and RO.

800

40 30 20 10 0

Influent

A E

40 30 20 10 0

F

RA effluent D

40 30 20 10 0

C

B

RO effluent D

0

C

5

10

15

20

25

Retention Time (min) Fig. 4. HPLC analysis of Reactive blue 13 and its degradation products.

30

38

J. Lin et al. / Bioresource Technology 101 (2010) 34–40

In the color removal process, glucose not only served as a source of carbon and energy, but also as an electron donor. Although a perfect substrate for cells in reactor studies, glucose employed in the practical wastewater treatment would be costly. However,

the electron-donating primary substrates used in many studies varied from simple substrates like ethanol and glucose to more complex ones, including relevant constituents of textile-processing wastewaters like starch and carboxymethylcellulose (CMC)

Cl Cu

N O

NaSO 3 O

N

HN N

N

N

NH O3SNa

Anaerobic

NaSO 3

NaSO 3

Azoreduction OH

NaSO3 OH

OH

NH2

NH2 H2N

N

O3SNa

NH2 N

N

NaSO 3

[a]

NaSO3

OH

NH2

NH2 H2N

OH

[c]

Deamination OH

[b]

Ring-cleavage

ve n ati latio d i y Ox box r a c de

Aerobic

Deamination

Desulfonation OH

[d]

COOH

COOH CH3

94.1

91.1

9000

9000

8000

8000

7000

7000

[a]

6000

5000

5000 4000

4000

66.1

3000 2000

55.0

1000 0

20

30

40

50

60

2000 75.1 83.8 70

80

103.0 90

117.1

133.1

100 110 120 130 140

65.1

1000 0

93.0

281.0

118.0 40

60

80 100 120 140 160 180 200 220 240 260 280 60.0

9000

8000

8000

7000

7000

[c]

6000

[d]

6000

5000

73.0

5000

4000

4000

66.0

3000 2000

0

136.1

3000

9000

1000

[b]

6000

28.0 18.0

41.1

3000 28.1

2000 39.1

46.6 54.0

77.0

10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100

1000 0

87.0

18.1 10

20

51.0 30

40

50

98.9 60

70

80

90

100

110

120

Fig. 5. Proposed pathway for the degradation of Reactive blue 13 and identification of metabolites by GC–MS: (a) aniline, (b) benzeneacetic acid, (c) phenol and (d) hexanoic acid.

J. Lin et al. / Bioresource Technology 101 (2010) 34–40

(Jadhav et al., 2007; O’Neill et al., 2000). In all cases, azo dye decolorization occurred, which suggests that the process is relatively non-specific with respect to its electron donor (van der Zee and Villaverde, 2005). Thus, one could speculate that decolorization may probably occur with many substrates other than glucose, constituents of wastewater, for example. 3.5. Effect of pH Table 1 shows that the decolorization of Reactive blue 13 with Pseudomonas sp. L1 occurred between pH 5 and 9. Compared to the static condition, the decolorization in fluidized bed was less affected by varying pH, especially at pH 5 or 9. Since the bacteria on GAC were a lot more adapted and tolerant to the unfavourable environment. However, the adverse effect still existed at pH 5 and 9, as the COD removal was decreased, implying that the metabolism of bacteria was inhibited. At a pH between 5 and 9, the color removal efficiency fluctuated from 75.6% to 86.9%, while the total COD removal efficiency varied between 67.7% and 90.4%. Taken all factors into consideration, the optimum pH for degradation is between pH 6 and 7. 3.6. Analysis of decolorized products The UV–Vis spectra (Fig. 3) of the decolorized samples show the disappearance of the peaks (A, B and C) in both visible and UV region. The possibility of decolorization by adsorption only was ruled out, since the UV–Vis absorption peaks (A, B and C) did not decrease in proportion to one another. Unlike peak A, the decrease of peak B and C in UV region was observed in both RA and RO effluent. Given that those metabolites have no absorption in the visible region but do in the UV range (aromatic amines, for example), HPLC was used for further analysis of the biodegradation process. The HPLC chromatograms of decolorized media were obtained from samples derived from steady running system under the constant condition (HRT = 50 h, pH 7, Cglucose = 2000 mg/L). Since some intermediates appeared to be air sensitive, samples were directly used for HPLC analysis after centrifugation at 8873 g and filtration through a 0.22 lm membrane filter. Fig. 4 shows the HPLC chromatograms of the influent and effluents from both reactors. UV-absorbing substances eluting at between 0 and 30 min were examined. A strong absorbance peak (peak A) was observed at a retention time of 25 min, which represents Reactive blue 13. As the decolorization proceeded in RA, the intensity of peak A dramatically decreased and could hardly be seen and other peaks (peak E and F) were also removed. Meanwhile, the appearance of some new peaks (peaks B, C and D) indicates that by-products were formed during the anaerobic treatment. Furthermore, the fact that new peaks shifted towards lower retention times suggests that the by-products of anaerobic treatment were smaller and more polar than their parent compounds (Elizalde-González et al., 2009). Comparison of spectra of Reactive blue 13 from RA and RO shows that further degradation occurred during the aerobic treatment, as the new peak (peak B) found in the effluent of RA was not found in the effluent of RO and the peak C was reduced. Meanwhile, no new peaks were observed and the intensity of peak D increased. Therefore, peak D probably represented one of the products and the rest (small molecular organic acids) could not be detected in the UV region. The GC–MS analysis revealed the probable metabolites produced during biodegradation. Based on the results and relative references (Kagalkar et al., 2009; Kalyani et al., 2009; Valli Nachiyar and Suseela Rajakumar, 2004), a probable degradation pathway for Reactive blue 13 is introduced (Fig. 5). Decolorization in the anaerobic reactor was mainly caused by the cleavage of the azo

39

bond. The degradation was also confirmed by the existence of aniline (Fig. 5a) in the RA effluent. According to our proposal, naphthalene-like compounds were primary degradation intermediates produced by the cleavage of the azo bond. Those intermediates were further oxidized to aromatic intermediates such as benzeneacetic acid (Fig. 5b). The evidence for the formation of phenol (Fig. 5c) indicates that aniline underwent oxidative deamination involving hydroxylation reaction. Ring-cleavage compounds and fragments obtained from digestion of glucose, like hexanoic acid (Fig. 5d), butanoic acid and pentanoic acid were identified as the final products.

4. Conclusion A continuous two-stage anaerobic/aerobic biological fluidized beds system, seeded with Pseudomonas sp. L1 attached to granular activated carbon, was employed to decolorize and degrade the textile dye Reactive blue 13. Reduction in COD of 90.7% and color removal of 83.2% was achieved when the continuous system operated in optimized conditions (HRT = 70 h, pH 7, Cglucose = 2000 mg/L). The potential for industrial application of the isolated bacterium was demonstrated by the observation that decolorization occurred over a range of pHs and dye concentrations. The reductive cleavage of azo-bond chromophore occurred mainly under anaerobic conditions, leading to the production of naphthalene-like compounds and aromatic intermediates. The resulting fragments were subsequently oxidized to small molecular organic acids in the aerobic reactor.

Acknowledgements The authors would like to acknowledge financial support for this work provided by MOST project of China (No. 2007AA06Z339; No. 2008BAC32B06).

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