Bioresource Technology 101 (2010) 7185–7188

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Bioresource Technology

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Short Communication

Enhanced biodecolorization of azo dyes by anthraquinone-2-sulfonateimmobilized covalently in polyurethane foam

Hong Lu a,b, Jiti Zhou a, Jing Wang a,*, Weilei Si a, Hu Teng a, Guangfei Liu a

a Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), School of Environmental and Biological Science and Technology,Dalian University of Technology, Dalian 116024, Chinab State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, China

a r t i c l e i n f o a b s t r a c t

Article history:Received 1 February 2010Received in revised form 2 April 2010Accepted 7 April 2010Available online 4 May 2010

Keywords:Anthraquinone-2-sulfonatePolyurethane foamAzo dyesImmobilizationBiodecolorization

0960-8524/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.biortech.2010.04.007

* Corresponding author. Tel.: +86 411 84706250; faE-mail address: jwang@dlut.edu.cn (J. Wang).

The effect of anthraquinone-2-sulfonate covalently immobilized in PUF (AQS-PUF) on the decolorizationof azo dyes by Escherichia coli K12 was investigated. The results showed that AQS-PUF mediated biode-colorization rate of azo dye amaranth increased over 5-fold compared with that lacking AQS, and thekinetics of its biodecolorization could be described using Quiroga second order equation. During theabove process, a lot of the cells of E. coli K12 were attached in the walls of AQS-PUF pores. After 10repeated experiments using AQS-PUF, immobilized AQS-mediated biodecolorization efficiency of ama-ranth retained over 98.7% of their original value. Moreover, AQS-PUF could greatly enhance the decolor-ization rates of a broad range of azo dyes. These results indicated that AQS-PUF as a biocarrier exhibitedhigh catalytic activity and good stability for potential applications.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

It has been widely demonstrated that quinone compounds suchas anthraquinone-2-sulfonate (AQS) and anthraquinone-2,6-disulf-onate (AQDS) can function as redox mediators and significantly in-crease the anaerobic biodecolorization rates of xenobioticcompounds azo dyes, especially for highly polar sulfonated or poly-meric azo dyes (Encinas-Yocupicio et al., 2006; Rau et al., 2002;Van der Zee et al., 2001, 2009). But continuous addition of the qui-none compounds will result in an increase of running cost and thesecondary contamination due to continuous discharge of the bio-logically recalcitrant redox mediators. Thus, it is expected that qui-none compounds as redox mediators can be effectivelyimmobilized and used for accelerating the decolorization of azodyes in anaerobic bioreactor.

For this purpose, Van der Zee et al. (2003) studied the role ofactivated carbon, and indicated that activated carbon, which func-tions as a solid redox mediator, could improve the decolorization ofazo dye Reactive Red 2 in upflow anaerobic sludge bed reactors. Inaddition, it was reported (Guo et al., 2007; Su et al., 2009) thatanthraquinone was immobilized in calcium alginate using entrap-ment method. Immobilized anthraquinone could increase 1.5–2-fold on the decolorization rates of azo dyes compared with the con-

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trols lacking anthraquinone. But this method was not suitable forpractical applications owing to weak mechanical strength of cal-cium alginate. Moreover, our previous studies showed that AQDSand AQS were more effective redox mediators than anthraquinoneand activated carbon. Recently, Wang et al. (2009) reported thatAQDS bound into polypyrrole (PPy) on activated carbon felt (ACF)could increase over 3-fold on the biodecolorization efficiencies ofazo dyes than those lacking AQDS. However, it is expensive anddifficult for large scale applications.

With the development of microbial immobilization, polyure-thane foam (PUF) with macropores has attracted much attentiondue to ease of use, good mechanical strength, low toxicity and largesurface area (Duarte et al., 2010; Guo et al., 2010; Zheng et al.,2009). It not only can act as a mobile carrier for active biomass,but also retain the microorganism by incorporating a hybridgrowth system. Hence, PUF was selected as a carrier for quinoneimmobilization. In addition, it was reported that a lot of bacteriawere capable of reducing quinones such as AQS and AQDS underanaerobic conditions, Escherichia coli was one of these bacteriaand ubiquitous in nature (Cervantes et al., 2002; Lovley et al.,2000; Rau et al., 2002). Therefore, E. coli K12 was used as a quinonereducing bacterium.

In this study, AQS as a model quinone compound was covalentlyimmobilized in PUF, and the effect of PUF containing AQS (AQS-PUF) on the decolorization of azo dyes by E. coli K12 was investi-gated under anaerobic conditions.

7186 H. Lu et al. / Bioresource Technology 101 (2010) 7185–7188

2. Methods

2.1. Chemicals

Amaranth was a sulfonated azo dye and mainly used in thisstudy. All the azo dyes were purchased from Tianjin TianshunChemical Co., Ltd. Anthraquinone-2-sulfonic sodium salt (AQS)was purchased from Sigma Co., Ltd. All other reagents used wereof analytical grade.

2.2. Strain, media and culture conditions

E. coli K12 used was obtained from Agricultural Culture Collec-tion of China (ACCC) with the accession number 10034. Strain K12was aerobically cultivated at 37 �C with 150 r min�1 in 150 ml Lur-ia–Bertani (LB) medium, which contained: peptone 10 g l�1, yeastextract 5 g l�1, NaCl 10 g l�1 (pH 7.2). For the decolorization ofazo dyes, strain K12 was cultivated at 37 �C in 135 ml anaerobicmedium, which contained: Na2HPO4�12H2O 4.26 g l�1, KH2PO4

2.65 g l�1, NH4Cl 3 g l�1, MgSO4�7H2O 0.2 g l�1, and glucose 2 g l�1

(pH 8.0).

2.3. Preparation of AQS-PUF

Polyurethane foam cubes (0.139 ± 0.005 g/cube) were providedby Dalian-Landa Bio-environment & Tech. Co. Ltd in Dalian City ofChina. Its specific surface area was (1.6–2.2) � 105 m2 m�3. AQS-PUF was obtained by a two-step following procedure: first, PUFcubes reacted with 4.9 g diethylenetriamine in the solution of2 mol L�1 NaOH for 3 h at room temperature, and then PUF cubestaken were washed with distilled water and dried at 100 �C; sec-ond, NH2-PUF cubes reacted with 0.077 g anthraquinone-2-sulfo-nyl chloride (ASC) in the solution of 2 mol L�1 NaOH for 1 h at30 �C, and then NH2-PUF cubes taken were also washed with dis-tilled water and dried at 100 �C for the following decolorizationexperiments. ASC was synthesized using AQS as described by Fenget al. (2002).

2.4. Batch decolorization experiments

Strain K12 was cultivated under aerobic conditions describedabove until they reached the late exponential growth phase. Cellswere harvested by centrifugation at 8000 g for 10 min, washedtwice with physiological saline, and resuspended in anaerobicmedium containing certain concentration of azo dyes and AQS-PUF cubes to an optical density (OD660nm) of about 0.4–0.45. Thenthese cells were transferred into rubber-stopped serum bottles(135 ml) under anaerobic incubator. Finally serum bottles werefilled with the anaerobic medium, and sealed with butyl rubberstoppers, and statically cultured at 37 �C.

The kinetics of AQS-mediated decolorization of azo dyes wasdescribed using the following Quiroga second order equation (Qui-roga et al., 1999), where S is the concentration of azo dye(mmol l�1), m is the decolorization rate of azo dye (mmol l�1 h�1),K2 (mmol�1 l h�1), K1 (h�1) and K0 (mmol l�1 h�1) are kineticconstants.

mð�dS=dtÞ ¼ K2S2 þ K1Sþ K0 ð1Þ

Decolorization efficiency (%) was determined using the previousmethod (Wang et al., 2009). The average specific decolorizationrate of azo dye was determined using the following Eq. (2), whereS0 and St are the concentrations of azo dye (mmol l�1) at time zeroand time t (h), respectively, mt is the cell dry weight (g l�1) at timet. The reaction time t was set as 11 h.

�m ¼ ðS0 � StÞ=mt � t ð2Þ

2.5. Analytical methods

The morphologies of bacteria immobilized in PUF cubes werecharacterized by an environmental scanning electron microscope(ESEM Quanta 200 FEG). The infrared spectra of the samples wererecorded by using IR (EQUINOX55, German).The concentrations ofnitrogen and sulfur elements were determined using elementaryanalysis instrument (varioEL III, Italy). Thus, the concentration ofimmobilized AQS (CAQS, mmol l�1) in reaction system could bedetermined using the following Eq. (3), where mAQS-PUF is the massof AQS-PUF cubes (g), CS is the concentration of sulfur element(mmol g�1) in AQS-PUF, V is the volume of the medium (l) in reac-tion system.

CAQS ¼ mAQS-PUFCS=V ð3Þ

Biomass concentration was determined by optical density (OD)at 660 nm, and the relationships between the bacterial cell concen-tration and OD660nm for strain K12 were 1.0 OD = 3.16 g dry cell l�1.The concentrations of azo dyes were detected using the previousmethod (Wang et al., 2009). The assays were performed in tripli-cate and the mean values of the data were presented.

3. Results and discussion

3.1. Characterization of AQS-PUF

Initial PUF contained –OH group at 3280 cm�1. Based on this,PUF was aminated by using diethylenetriamine. It was observedfrom the infrared spectrum of aminated PUF that the characteristicbands at 3208 cm�1 (–NH2) and 1081 cm�1 (C–N) became broaderand stronger compared with those of initial PUF (data not shown).And elementary analysis showed the concentration of nitrogen ele-ment increased 0.228 mmol g�1 PUF. This indicated that –NH2

group had been grafted in PUF. The presence of quinone was welldemonstrated by the characteristic adsorption bands of highly con-jugated C@O groups at 1668 cm�1 (data not shown). Moreover, ele-mentary analysis showed that the concentration of sulfur elementincreased 0.097 mmol g�1 PUF. This means that the concentrationof the immobilized AQS was 0.097 mmol g�1 PUF.

3.2. Performance of AQS-PUF on amaranth biodecolorization

Our study showed that the average specific decolorization rateof amaranth was increased as the number of AQS-PUF cubes in-creased to four. Further increase of the number of AQS-PUF cubescould not obtain higher decolorization rates. Thus, 4 AQS-PUFcubes (0.552 g), which contained about 0.054 mmol AQS, were se-lected for the following experiments.

AQS-PUF mediated biodecolorization process of amaranth(0.4 mM AQS) was studied. Fig. 1 showed that only less than 3%decolorization occurred in E. coli K12-free controls, which indi-cated that the effect of amaranth adsorption to AQS-PUF cubeswas negligible. AQS-mediated decolorization of amaranth followedthe first order reaction with respect to dye concentration(m = 0.704S � 8.5848, R = 0.9928, Supporting information Fig. S1).But the kinetics of AQS-PUF-mediated decolorization of amaranthcould be well described using Quiroga second order equation(Quiroga et al., 1999) (m = �0.0011S2 + 0.7294S � 8.9118,R = 0.992, Supporting information Fig. S2). As shown in Fig. S2,the short lag phase (0.8% decolorization efficiency,K2 = �0.0011 mmol�1 l h�1) appeared during the initial 4 h. It wasassumed that the cells of E. coli K12 need some time to adequatelycontact and adapt AQS immobilized in PUF. After 4 h, amaranthconcentration decreased quickly. It was obvious that AQS-PUF cat-alytic behavior was dominant during the phase. Compared with

0 3 6 9 12 15 180.00

0.02

0.04

0.06

0.10

Con

cent

ratio

n of

am

aran

th (m

mol

l-1)

Time (h)

(a) (b) (c) (d) (e)

0.08

Fig. 1. Anaerobic decolorization of amaranth by E. coli K12 under differentconditions. (a) Cells and 4 AQS-PUF cubes; (b) cells, AQS and 4 PUF cubescoexisting; (c) cells and 4 PUF cubes; (d) cells; (e) 4 AQS-PUF cubes. Reactionconditions: 0.4 mM AQS, 0.16 g l�1 cells, pH 8.0, temperature 37 �C, 150 rpm.

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AQS (K1 = 0.704 h�1), AQS-PUF-mediated biodecolorization rate ofamaranth (K1 = 0.7294 h�1) was slightly higher. Moreover, in thewhole time scale, the average specific decolorization rate of ama-ranth in the presence of 4 AQS-PUF cubes increased over 5-foldcompared with that lacking AQS. These results showed that AQS-immobilizing method was effective and immobilized AQS wasthe active component in PUF.

After amaranth decolorization (Fig. S3), AQS-PUF was analyzedby using SEM. It was observed that a lot of the cells of E. coli K12were attached in the walls of PUF pores (data not shown). More-over, the pores of PUF were much larger than the size (diameter)of the cells of strain K12. This indicated immobilized cells couldmore effectively reduce AQS immobilized in PUF, and reducedAQS could also effectively transfer electron to amaranth due to en-ough large pores in PUF.

3.3. Effect of AQS-PUF on the decolorization of different azo dyes

AQS-PUF-mediated biodecolorization of five acid dyes, tworeactive dyes and one direct dye within 11 h was investigated. Itwas obvious that the average specific biodecolorization rates ofall tested dyes were over 2.6–5-fold higher in the presence ofimmobilized AQS than those lacking AQS. In particular, for multi-sulfonic azo dyes with high molecular weight, AQS-PUF-mediateddecolorization rates of Reactive Red 141 (12.4 mmol g�1 h�1), AcidRed 73 (32.4 mmol g�1 h�1) and Direct Black 22(11.0 mmol g�1 h�1) significantly increased over 2.5-fold comparedwith those lacking AQS (3.7 mmol g�1 h�1, 6.5 mmol g�1 h�1,3.0 mmol g�1 h�1). Thus, immobilized AQS as a solid redox media-tor could greatly enhance biodecolorization rates of a broad rangeof azo dyes.

3.4. The stability of catalytic activity

The reusability of AQS-PUF cubes for anaerobic biodecoloriza-tion of azo dye amaranth was investigated. To avoid the effect ofthe cells immobilized in AQS-PUF on the biodecolorization, 4AQS-PUF cubes were autoclaved and then washed with distilledwater. During next decolorization experiment, the above AQS-PUF cubes were reused and the same amount of the cells of

E. coli was inoculated again into the reaction system. The resultsshowed that the decolorization efficiencies of amaranth could re-tain over 98.7% of their original value during 10 runs (Supportinginformation Fig. S4). And elementary analysis showed that the con-centration of sulfur element did not reduce. This indicated that theimmobilized AQS could keep high catalytic activity and goodstability.

4. Conclusions

To our knowledge, AQS functioning as an effective redox medi-ator was firstly covalently immobilized in PUF. Our results showedthat AQS-PUF could greatly enhance the biodecolorization rates ofazo dyes used in this study. Immobilized AQS exhibited good cata-lytic activity and stability. Thus, AQS-PUF has potential applica-tions in accelerating the anaerobic biodecolorization of azo dyes.Further study about the application of AQS-PUF in bioreactor willbe done.

Acknowledgements

This subject was supported by National Key Scientific and Tech-nology Project for Water Pollution Treatment of China(2008ZX07208-004-2), National Natural Science Foundation ofChina (No. 50978040), Open Project of State Key Laboratory of FineChemicals (KF0818), the 46th Postdoctoral Funds of China andPCSIRT (IRT0813).

Appendix A. Supplementary data

Detailed information includes the amaranth decolorizationkinetics obtained by replotting Fig. 4a and b (Figs. S2 and S1),UV–Vis spectra during the biodecolorization of amaranth (Fig. S3)and AQS-PUF reusage experiments (Fig. S4).

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.biortech.2010.04.007.

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