Yongxing Qian,Bin Yang,Zhongjian Li,Lecheng Lei,Xingwang Zhang*

Key Laboratory of Biomass Chemical Engineering of Ministry of Education,Department of Chemical and Biological Engineering,Zhejiang University,Hangzhou 310027,China

Keywords:Redox mediator Biodecolorization Functional bio-carrier

ABSTRACT In order to enhance the biodecolorization rate and avoid the wash-out problems of redox mediators in continuous systems such as a fluidized bed reactor,polyvinyl alcohol(PVA)beads modified with N-containing function groups were investigated and employed as a new sodium anthraquinone-2-sulfonate(AQS)carrier material.Elementary and XPS analyses confirm the existence of AQS on modified PVA bead.The modified PVA beads suit with immobilizing AQS better in adsorption capability and stability.AQS supported on modified PVA beads shows high catalytic activity for biodecolorization of reactive blue 13 in a long process(＞10 runs).

1.Introduction

Effluents from dye manufacturing industries exist in water and sediments for a long time[1].Their disposal into environment is harmful and will cause death of aquatic life[2–4].On account of the low energy cost and environmental compatibility,biodegradation of electronaccepting pollutants,such as dyes,under anaerobic condition has been widely applied for wastewater treatment[5,6].However,its long-time cost and the low efficiency limit its applications[7].

Recently,many redox mediators(RMs)have been investigated for reductive biotransformation of electron-accepting priority pollutants,such as humic substances[8,9]and quinones[10,11].RMs can accelerate the electron transfer between pollutants and microorganisms,enhancing the removal rate of pollutants.Soluble RMs are usually used,but they will be washed-out with the wastewater,so continuous addition of RMs is needed.This will increase running costs.Moreover,the RMs in effluent water will cause secondary contamination[12].To avoid these problems,RMs immobilized on suitable carriers have been investigated.Guo et al.[13]prepared calcium alginate beads immobilized with anthraquinone,where beads were 3.0–4.0 mm in diameter and the loading of anthraquinone was about 0.00047 g.Their results showed about2-fold denitrification rate and good reusability.Cervantes et al.[14]used anion exchange resins(AER)to adsorb RMs,including 1,2-naphthoquinone-4-sulfonate and anthraquinone-2,6-disulfonate,and obtained 8.8-fold decolorization rate for azo dyes compared with the control group.The desorption tests of immobilized quinones on AER showed that the RM detached from AER was negligible at 25°C,but quinone desorption occurred above 25°C.Moreover,the competition of other anions with quinones also limited the utilization of AER.

With the advantage of uniform particle distribution and sludge reduction, fluidized bed reactors are widely used for degradation of pollutants[15,16].However,the decolorization is time consuming and is not effective,so immobilized RMs are needed[17,18].In an anaerobic fluidized bed system,polyvinylalcohol(PVA)beads are suitable carriers for microorganism immobilization due to their superior mechanical strength and chemical stability[19].The density of PVA beads(1.02–1.05 g·ml-1)is nearly the same as that of water,so they are easily fluidized in the reactor.For this reason,PVA beads are suitable as the carrier for RMs.Taking into account its high redox ability and good water solubility,sodium anthraquinone-2-sulfonate(AQS),a typical RM,is employed in this study.

Here AQS is immobilized on modified PVA beads to improve biodecolorization efficiency.The catalytic activity and stability of biodecolorization of reactive blue 13(RB13),a representative of sulfonated reactive azo dyes,are investigated in an anaerobic batch system.

2.Methods

2.1.Chemicals and PVA beads

All chemical reagents used in this study were of analytical grade and were purchased from Hangzhou Huipu Co.Ltd.,China.AQS and PVA beads were obtained from Sinopharm Chemical Reagent Co.,Ltd.(Beijing,China)and Kuraray Co.(Japan),respectively.The PVA beads contain 80%–95%water,are 3 mm in diameter and 4 mg in dried mass,and could endure the highest temperature about 200°C.

2.2.Preparation of modified PVA beads

PVA beads were first dried at 80°C for 8 h as raw material,then 2 g beads were added into mixture solution of 5 ml N-3-(trimethoxysilyl)propyl ethylenediamine(AEAPS,95%purity)and 95 ml toluene.The solution was heated to 110°C under reflux condition for 24 h with argon atmosphere(Ar).The amine-functioned PVA beads were obtained after washing with excessive toluene three times and dried at 80°C for another 8 h.

In the protonated process,1 g dried amine-functioned PVA beads were protonated with 100 ml 0.1 mol·L-1H2SO4solution for 24 h,and washed with abundant deionized water until the pH of wash solution was near neutral,then dried for further use.

2.3.Immobilization of AQS on PVA beads

Immobilization of AQS on modified PVA beads was quantified by adsorption isotherms.Various concentrations(100,150,200,250 and 300 mg·L-1)of AQS were used with 0.04 g PVAbeads in 40 mlofdeionized water for the adsorption process ata constant temperature of 33°C with continuous shaking(200 r·min-1).The loading of adsorbed AQS on PVA beads is calculated with

where qeis the loading of AQS on PVA(μmol·g-1),C0is the initial AQS concentration in the solution(μmol·L-1),Ceis the liquid phase concentration of AQS at equilibrium(μmol·L-1),V is the volume of solution(L),and M is the mass of PVA beads(g).

2.4.Desorption of immobilized AQS on modified PVA beads

To evaluate the adsorption of AQS on PVA beads,AQS immobilization beads were exposed to different temperatures and bacteria growth solutions(NH4Cl,KH2PO4and their mixture).0.04 g AQS-saturated PVA beads were put in 50 ml vials containing 40 ml basal medium,which was the same as the inoculum given in the inoculum part.For the effect of growth medium,the basal medium only contained the same concentration of NH4Cl,KH2PO4and their mixture without other nutrients.

Initial and final concentrations of AQS were measured to evaluate AQS immobilization ability.The desorption rate of the AQS–PVA bead is calculated with

where rdesorptionis the AQS desorption rate from PVA beads(%),Cdesorptionis the liquid phase AQS concentration at desorption equilibrium(μmol·L-1),Cinitialis the initial AQS concentration in the solution(μmol·L-1),Vwis the volume of solution(L),and Cadsorptionis the total concentration of AQS on the PVA beads in the solution(μmol·L-1).

2.5.Inoculum

Bacteria(Pseudomonas sp.isolate)used in all batch experiments were cultivated in serumbottles(115 ml)and the medium used for bacterial growth had the following composition[16]:glucose 500 mg·L-1,NH4Cl 191 mg·L-1,KH2PO415 mg·L-1,K2HPO410 mg·L-1,MgSO412.5 mg·L-1,FeSO412.5 mg·L-1,and phosphate buffer with 6.8 g·L-1KH2PO4and 1.135 g·L-1Na OH,with the pH value of solution maintaining at 7.The serum bottles were sealed with a rubber septum by a screw cap and N2was aerated at the beginning to maintain anaerobic condition.Glucose was used as co-substrate(carbon source)and electron donor.1 mol·L-1NaOH and 1 mol·L-1HCl were needed for adjusting pH whenever necessary.All incubations were carried out in a temperature controlled incubator at 33°C and pH 7[16].

2.6.Batch biodecolorization of azo dye RB13

The degradation experiments were performed in serum bottles(115 ml)with a 99 ml inoculum,and 1 ml bacteria culture(original OD600=0.35)was added into the bottles and sealed with a rubber septum by a screw cap.After bacteria grew for 24 h,RB13 was injected into these bottles at an initial concentration of 25 mg·L-1,and different amounts of AQS/PVA beads were added into the bottles whenever necessary.The controls without bacteria or AQS/PVA beads were also used.All the experiments were carried out in triplicate.

2.7.Analytical methods

Samples were periodically collected and centrifuged at 10000 r·min-1for 10 min,and the filtrate was analyzed with a UV–Vis spectrophotometer.The concentrations of RB13 and AQS were characterized at a maximum wavelength λmaxof 570 and 330 nm,respectively.The elementary analysis was performed by Elementar Analysensysteme GmbH(Vario Micro,Germany).The XPS analysis were performed using an X-ray photoelectron spectrometer(Escalab 250Xi,UK),and the morphologies of unmodified and modified PVA beads were examined by using scanning electron microscopy(SU8010,Japan).

3.Results and Discussion

3.1.Characterization of AQS/PVA beads

The composition ofmodified PVA and AQS/PVAsamples obtained by an elementary analysis instrument is summarized in Table 1.The PVA beads are mainly composed of elements C and O,consistent with the chemical structure of PVA(Fig.1).In the AEAPS modified PVA sample,elements N and Si are present.Since only–NH2groups contain element Nin AEAPS,the presence ofNconfirms that–NH2groups are introduced on the surface of PVA beads.Since AQS is soluble in water as anion,more–NH2groups lead to better AQS adsorption.With the protonated process for increasing adsorption capacity of AQS,due to the utilization of H2SO4,element S also appears in protonated amine-functioned PVA beads.Comparing with the initial protonated amine-functioned PVA beads,the percentage of S increases when AQS adsorbs on these beads.The elementary analysis identifies that AQS is immobilized on the modified PVA beads.

Fig.2 depicts the XPS analysis of elements O and C in PVA beads.For element O,C–O is the main function group in PVA beads and aminefunctioned PVA beads,and the S–O function group appears in AQS/PVA beads as expected.The same as element O,there is little difference in element C between PVA beads and amine-functioned PVA beads,and AQS/PVA beads have the C–S function group.Elements N,Si and S are also analyzed by XPS,and the results agree with the elementary analysis.

The surface morphologies of PVA beads did not change much in the preparation of AQS/PVA beads[Fig.3(b)and(c)].However,when PVA beads were dried at 80°C,their microstructures were destroyed[Fig.3(a)and(b)].In a preliminary test,the PVA beads could not adsorb AQS regardless of whether they were dried or not,so their modification is necessary.In Fig.3(d),bacteria are clearly found on the AQS/PVA beads,attaching in the decolorization process.

Table 1 The elementary analysis of beads before and after modification and AQS adsorption

Fig.1.Chemical structure of PVA.

3.2.Adsorption of AQS on modified beads

Fig.2.XPS analysis of unmodified and modified PVA beads.a–c:element O;d–f:element C.

Fig.3.SEM micrographs of unmodified and modified PVAbeads.(a)PVAbeads(notdried);(b)PVAbeads(dried);(c)amine-functioned PVAbeads;(d)AQS-amine-functioned PVAbeads after biodecolorization.

Fig.4 shows the adsorption of AQS on modified PVAbeads.Protonated PVA beads present higher adsorption capacity for AQS compared with unprotonated ones[Fig.4(a)].The Langmuir and Freundlich equations are applied to describe AQS adsorption data[20].The results indicate that the Langmuir equation provides much better fitting according to correlation coefficients R2,implying a monolayer of AQS.The maximum biosorption capacity of AQS on protonated and unprotonated beads is 155 and 90.9 μmol·g-1,respectively.Therefore,the following experiments were performed using protonated PVA beads.

The adsorption capacity on AQS–PVA in this study has little improvement compared with previous results.Yuan et al.immobilized AQS on ceramsites and obtained immobilized AQS of 2.3 μmol·g-1[21].They also immobilized AQS on polyurethane foam cubes and the immobilized AQS increased to 97 μmol·g-1[22].There may be two reasons for this difference.Firstly,hydroxyl(–OH)groups are important for the amination process.Without or with little–OH groups in their supports[21,22],the amination process is poor,while abundant–OH groups on PVA beads result in an easy amination process[21].Secondly,in the immobilized method[21,22],chemical adsorption is the main mechanism,while in our study physical adsorption is the main mechanism.A desorption test is needed to evaluate the stability of immobilized AQS.

3.3.Desorption tests for immobilized AQS on modified PVA beads

Fig.4.Adsorption of AQS on modified beads(a)and isotherms fitting with Langmuir(b)and Freundlich(c)models.

Fig.5.Effect of temperature(a)and bacterium growth medium(b)on AQS desorption.

Fig.5 describes the effect of temperature and anions on AQS detachment from PVA beads.AQS desorption rate has little changes at temperatures below 35°C,while it increases when the temperature is above 40 °C,achieving 23.6%at50 °C.The biodecolorization is commonly in 30–35 °C,so the AQS is stable on the modified PVA beads.Fig.5(b)shows that at 33°C,the effect of medium anion is not distinct and the AQS desorption rate with coexisting anions is below 1.3%.Competition may exist for anion-exchange sites on the materials.Cervantes et al.[14]found that HCO3-and Cl-ions did not vary in desorption tests while PO43-and SO42-changed a lot,and pointed out that PO43-was mainly responsible for quinone detachment.In our bacterium growth medium,the main anion was H2PO4-(50 mmol·L-1)and ithad little effect on the detachment of AQS.Thus anion competition did not occur in this desorption process.These results indicate that AQS can be firmly immobilized on the modified PVA beads in the biodecolorization.

3.4.Effect of AQS/PVA beads on biodecolorization of RB 13

Fig.6(a)shows the effect of AQS concentration on the decolorization of RB13.More AQS in the solution leads to higher RB13 biocolorization rate.In fact,3 μmol AQS–PVA beads(with 3 μmol of total concentration of AQS on PVA beads)are sufficient for the decolorization of RB13(＞96%)in 12 h,compared with 42.1%in an AQS absent system.Fig.6(b)compares the biodecolorization with protonated amine-functioned PVA beads and bacteria(AQS free),AQS-modified beads(bacteria free),coexistence of AQS-modified beads(AQS-Im)and bacteria,and AQS solution(AQS-So)and bacteria.The PVA beads without AQS did not show significant decolorization of RB13(＜3%),indicating that the physical adsorption process is not responsible for the RB13 removal.The homogeneous AQS can significantly enhance the biodecolorization of RB13 due to the improved electron transfer rate[21,23],so AQS-So is better than AQS-Im,and the biodecolorization time is only 4 h with AQS-So.

In this paper,the first-order equation is used as follows[24]:

where Atand A0are the absorbance at time t(h)and initial time t=0,respectively,and k is the first-order rate constant(h-1).

The first-order rate constant k for AQS-Im,AQS-So,and AQS-free is 0.071 h-1,0.93 h-1,and 0.038 h-1,respectively.The detachment of AQS was also determined in the biodecolorization process.The same as in the AQS absent system,AQS could not be detected in the solution of the AQS-Im system,indicating that the enhancing decolorization rate could be attributed to AQS immobilized on PVA beads.

The possible mechanism of the difference between the AQS-So and AQS-Im biodecolorization systems is described in Fig.7.The electron is produced from glucose by bacteria B,and the oxidation AQS will change to reduction AQS,while the reduction AQS will lose the electron to electron-withdrawing compound RB13,then back to oxidation AQS.It will improve the dye decolorization rate and the right black arrow line is the transfer path of electron[8,12].In the AQS-Im biodecolorization process,although the degradation mechanism is the same,the mass transfer is limited.The reaction may occur on different parts on the bead surface and the contact of dye and AQS is limited by biomass,so its activity is slightly lower than that of AQS-So.

Different materials for immobilizing RMs were reported,such as metal-oxide nanoparticles[20],anion exchange resins[14],polyurethane foam cubes[22],and ceramsites[21].Comparing these supports,the AQS/PVA beads in this study present better adsorption stability than anion exchange resins,with the detachment temperature increasing to 35°C.The adsorption capacity of AQS on AQS/PVA beads is above 100 μmol·g-1,a little higher than that on polyurethane foam cubes and ceramsites.Furthermore,the PVA beads as immobilizing material could hardly be disrupted and washed-out from the fluidized bed reactors in our long-running test.

Fig.6.Effect of immobilized AQS loading on RB13 decolorization(a)and comparison of biodecolorization with various systems(b)(C0RB13=25 mg·L-1).

Fig.7.Possible mechanism for AQS-So and AQS-Im biodecolorization systems.

3.5.The stability of AQS on PVA for biodecolorization

It is important to evaluate the stability of catalytic activity in AQS-modified beads for practical applications.To make the immobilized bacteria inactive,the beads after the biodecolorization of azo dyes were autoclaved and washed with distilled water before another process.The next biodecolorization was performed using these beads under the same conditions.The results show that the AQS-modified beads can retain the catalytic activity for more than 10 runs(Fig.8).Even with 10 runs,the decolorization rate was above 95%and the concentration of AQS in the outletsolution was nearly not detected.This indicates thatthe immobilized AQS on modified beads can maintain high catalytic activity and good stability.Guo et al.[23]immobilized anthraquinone on calcium alginate(CA)and found that after 4 cycles,the CA immobilization beads could retain over90%.The AQS/PVA beads we obtained are a little betterthan the calciumalginate concerning the stability ofcatalytic activity.

Fig.8.Continual decolorization of RB13 by immobilized AQS.

4.Conclusions

The AQS was effectively immobilized on modified PVA beads.The results indicate that it can efficiently enhance the biodecolorization rate of azo dye RB13 while maintaining stable activity in the continuous process.Compared with other materials,the modified beads have better AQS immobilized capacity and better stability either in desorption of AQS or catalytic activity in RB13 biodecolorization.