TiO2âHorseradish Peroxidase Hybrid Catalyst Based on Hollow

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A TiO2-Horseradish Peroxidase Hybrid Catalyst Based on Hollow Nanofibers for Simultaneous PhotochemicalEnzymatic Degradation of 2,4-Dichlorophenol Xiaoyuan Ji, Zhiguo Su, Mengfang Xu, Guanghui Ma, and Songping Zhang ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b00075 • Publication Date (Web): 14 Jun 2016 Downloaded from http://pubs.acs.org on June 21, 2016

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ACS Sustainable Chemistry & Engineering

A TiO2-Horseradish Peroxidase Hybrid Catalyst Based on Hollow Nanofibers for Simultaneous Photochemical-Enzymatic Degradation of 2,4-Dichlorophenol

Xiaoyuan Ji†, Zhiguo Su†, Mengfang Xu†, Guanghui Ma†, Songping Zhang†,* †

National Key Laboratory of Biochemical Engineering, Institute of Process

Engineering, Chinese Academy of Sciences, Beijing 100190, China

* Corresponding author at: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China. Tel: +86 10 82544958; fax: +86 10 82544958 E-mail: [email protected]

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ABSTRACT Degradation of 2,4-dichlorophenol (2,4-DCP) using TiO2/UV photochemical and horseradish peroxidase (HRP) enzymatic treatments, as well as simultaneous photochemical-enzymatic treatments by combining these two processes were systematically investigated and compared. When free HRP were used in the simultaneous process, a negative synergetic effect was observed due to serious inactivation of the HRP caused by UV irradiation in the presence of TiO2. A hybrid catalyst system was then developed by in situ encapsulating HRP inside nano-chamber of TiO2-doped hollow nanofiber through co-axial electrospinning. Such encapsulation effectively avoided UV-induced deactivation of the enzymes, thus the 2,4-DCP degradation efficiency was improved significantly as compared with the that using HRP or TiO2/UV either separately or simultaneously in free formation. Furthermore, the higher concentration of 2,4-DCP, the more remarkable enhancement was achieved, such that 90% removal ratio was obtained within only 3 h for the degradation of 10 mM 2,4-DCP using the integrated TiO2-HRP hybrid catalysts system. While the removal ratio obtained with dispersed TiO2/UV, TiO2 doped in PU hollow nanofibers, free HRP, combination of dispersed TiO2 and free HRP under UV, as well as the encapsulated HRP, were only 31.37%, 27.98%, 49.71%, 36.53% and 58.32%, respectively. The hybrid catalysts system also showed excellent recycling capability and thermal-stability.

KEYWORDS: 2,4-dichlorophenol, degradation, horseradish peroxidase, TiO2, hollow nanofiber

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INTRODUCTION Chlorophenols is a type of highly toxic and potentially carcinogenic compounds existing in waste waters and non-industrial soils as permanent pollutant to our environment.1-3 Over the past few decades, photochemical method with TiO2 as photocatalysts4-8 and biochemical method with microorganism9-11 or enzymes12-15 remained as the most popular methods for degradation of chlorophenols. Nevertheless, due to some disadvantages, these conventional chlorophenols degrading technologies sometimes do not show efficacious for widely chlorophenols removal.16 For example, the photonic efficiency of the photochemical method will be seriously affected when was applied to treating concentrated chemical industry wastewaters or wastewater with low transparency17-19; while the biological degradation with microorganism method will suffer from strong inhibitory effects from concentrated chlorophenols, also the treatment period is usually rather long20. Different from microorganism biological methods, enzyme strategy is more advantageous. The applying and storing of enzymes are much easier, and the high specificity of enzymes also ensure more selective removal of the hazardous compounds. However, poor stability of enzyme under reaction condition and relatively high price are the biggest obstacle limiting enzyme method.1, 21-23 In order to achieve more efficient degradation of hazardous compounds, a diversity of hybrid strategies by combining photochemical and biochemical processes have been reported

5, 19, 24-25

. Such hybrid strategies were usually performed in

sequential order, with the photochemical process as the initial step to degrade the pollutants hardly biodegradable to more biodegradable molecules, which can then be further degraded in the following biological process.19 However, because of the slow rate of biological degradation process, such sequential process usually take relatively long time, and the cost on both construction of reactor and manpower operation was also increased.5, 19 Although these above mentioned problems was solved by coupling photochemical

and

biological

processes

simultaneously,26

the

photochemical-enzymatic process has been rarely reported since coupling of solution-based enzyme with TiO2/UV will lead to fast inactivation of the enzyme. In 3



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one of our previous study, a simultaneous photochemical-enzymatic process by using laccase covalently immobilized to porous glass was developed26. The resistance of laccase against TiO2/UV induced inactivation was improved to certain degree, and a higher 2,4-DCP removal ratio was achieved compared with the process using TiO2/UV and laccase alone. Nevertheless, the preparation of immobilized enzyme was complicated, activity recovery of immobilized enzyme was low, and recovery of the TiO2 nanoparticle catalyst from reaction system remained difficult. Nanofibers also were used to immobilize enzyme to degrade pollutions in water. Horseradish peroxidase (HRP) was successfully immobilized in nanofibers by emulsion electrospinning.27 Benefited from the strong adsorption capacity of nanofibers for chlorophenol, the removal of pentachlorophenol was dramatically enhanced. However, this process was sensitive to pH values, such that a degradation efficiency of 83% was obtained at pH 3, while nearly no adsorption and degradation of pentachlorophenol were observed at pH higher than 4.7 due to deprotonation of the pentachlorophenol.27 Recently, hollow nanofibers-based multienzyme systems have been fabricated successfully in our lab through co-axial electrospinning technology, through which enzymes can be in situ encapsulated inside the nano-chamber of hollow nanofibers.28-31 Compared to the other enzyme immobilization method, encapsulation of enzymes inside hollow nanofibers have been proven to offer several of distinct advantages like high activity recovery, nearly 100% encapsulation efficiency, high stability due to the confinement effect of nano-scaled compartments. Besides these advantages, the problem of rapid UV-induced enzyme inactivation may also be better solved sine the shell of the nanofiber is expected to provide protection to the encapsulated enzymes from direct UV irradiation. And most importantly, by simply dispersing TiO2 nanoparticle into outer phase solution containing polyurethane (PU) forming shell of the hollow nanofibers, the TiO2 nanoparticle will be doped in the shell during co-axial electrospinning, thus a photochemical-enzymatic hybrid catalysts can be fabricated. Furthermore, the nanofibers having formation of woven-membrane enable easy recycling and operation of the hybrid catalysts. Here in the present study, we attempted to fabricate and employ such integrated 4


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TiO2-HRP hybrid catalyst based on hollow nanofibers to degrade 2,4-DCP through simultaneous photochemical-enzymatic processes. The activity and stability of the HRP before and after encapsulation were thoroughly investigated and compared. The results will show that due to significantly enhanced stability of encapsulated HRP against TiO2/UV induced inactivation, the hybrid catalysts could degrade 2,4-DCP more efficiently, especially for concentrated 2,4-DCP. Furthermore, the TiO2-HRP hybrid catalyst system showed excellent reusability and thermal stability.

EXPERIMENTAL SECTION Materials. TiO2 (Degussa P25) was supplied by Degussa (Essen, Germany). Polyurethane A85E (PU) pellet with bulk density about 700 Kg/m3 was supplied by Xiamen Jinyouju Chemical Agent Co. (Xiamen, China). Horseradish peroxidase (HRP, EC 1.11.1.7) were obtained from Beijing Apis Biotechnology Co. (Beijing, China). purchased

2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic from

Sigma.

Glycerol,

acid)

2,4-dichlorophenol

(ABTS)

were

(2,4-DCP),

N,N-dimethylacetylamide (DMAc) and hydrogen peroxide (H2O2) were obtained from Beijing Chemical Reagents Company (Beijing, China). All chemicals mentioned above were analytical grade. Preparation of TiO2-HRP hybrid catalyst based on hollow nanofibers. General procedure for fabrication of photochemical-enzymatic hybrid catalyst: shell-phase solution was formulating through dispersing TiO2 nanoparticles in DMAc to get a concentration of 50 mg/mL. After 3 h ultra-sonication treatment, PU was added and mixed overnight to reach a concentration of 25 wt.%. Core-phase solution was formulated by adding 0.5 mg HRP to 100 µL of 0.1 M pH 7.0 phosphate buffer solution, the solution was then well mixed with 900 µL glycerol. The co-axial electrospinning spinneret and the other electrospinning parameters were as same as one of our previous works.31 Characterization. Scanning electronic microscopy (SEM, JSM-6700F, JEOL, Japan) was used to characterize the surface morphologies and structure of the fibers.31 To characterize the enzymes distribution in hollow chamber, HRP was labeled by 5



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Fluorescein isothiocyanate isomer (FITC) prior to be encapsulated by co-axial electrospinning. Confocal laser scanning microscopy (CLSM) was applied to characterize the distribution of encapsulated enzymes. The excitation and emission wavelength of FITC were 488 and 545 nm, respectively. Photochemical and enzymatic removal of 2,4-DCP. A certain amount of TiO2 was well dispersed in 0.1 M, pH 8.0 phosphate buffer to get a concentration of 50 mg/mL by sonication for 5 min. 2,4-DCP solution (10 mg/mL) was prepared by adding definite amount of 2,4-DCP into 0.1 M, pH 8.0 phosphate buffer solution, and dissolved for 3 hours. 2,4-DCP degradations were conducted under four different methods described in following: (1) TiO2/UV photochemical method, TiO2 powder or TiO2 doped in hollow nanofibers was dispersed into 2,4-DCP solutions with definite concentration to obtain a final TiO2 concentration of 0.5 mg/mL. The reactant solution was then exposed to UV irradiation; (2) enzymatic method, free or immobilized HRP (final concentration of 0.6 µg/mL) were put into solutions containing 10 mM H2O2 and definite concentrations of 2,4-DCP; (3) simultaneous photochemical-enzymatic method, 2,4-DCP solutions containing 10 mM H2O2 were incubated with both TiO2 and HRP under irradiated by UV; (4) degradation of 2,4-DCP with TiO2-HRP hybrid catalysts: 2,4-DCP solutions containing 10 mM H2O2 were incubated with 4 mg hollow nanofiber-based hybrid catalysts under the irradiation of UV. All the above mentioned reactions were carried out in 0.1 M, pH 8 phosphate buffer at room temperature. A mercury vapor ultraviolet light source consisting six tubular lamps with the maxima radiation at 365 nm was used to supply the UV irradiation. Operational stability and thermal stability of the hybrid catalyst. Operational stability of hollow nanofibers-based hybrid catalysts was tested by measuring changes in the removal ratio of 2,4-DCP after each using cycle. The reactions were run for 4 h and then the nanofiber membrane was retrieved from reaction solution to stop the reaction. Then the nanofibers membrane was washed with buffer solution for several times, and put into the next new reaction solution. The 2,4-DCP removal ratio of the first time was defined as 100%. Thermal stability of the hybrid catalyst was detected by monitoring differences of the removal ratio of 2,4-DCP after the nanofiber 6


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membranes were placed at 60 °C for a certain time. Analytical protocols. Activities of free and immobilized HRP were detected according the methods reported in one of our previous works.28 The concentration of 2,4-DCP was monitored using HPLC system (Agilent 1200, USA) equipped with a Supelcol C-18 RP-column (4.6 mm×250 mm). The mobile phase consisted of methanol, ultra-pure water and acetic acid (60:38:2, v/v/v) was applied at the flow rate of 0.75 mL/min. The UV–vis detector at 280 nm was used to detect the 2,4-DCP.

RESULTS AND DISCUSSION Fabrication

and

characterization

of

hollow

nanofibers-based

photochemical-enzymatic hybrid catalyst. In our previous work, a co-axial electrospinning technology had been developed to fabricate hollow nanofibers successfully.30

Based

on

the

previous

study,

in

order

to

fabricate

a

photochemical-enzymatic hybrid catalyst, TiO2 nanoparticles showing high activity for a various of photochemical reactions32-35, were doped into the shell of PU nanofibers through dispersing them in the shell solution for co-axial electrospinning (Scheme 1).

Scheme 1. Schematic illustration of the co-axial electrospinning device for preparation of TiO2-HRP hybrid catalysts based on hollow nanofibers, and the mechanism for photochemical-enzymatic degradation of 2,4-DCP with such hybrid catalyst. 7



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It has reported that the structure of hollow nanofibers influenced on the activity recovery of enzymes greatly in our previous work.30 Herein, we firstly investigated the effect of core-shell solution flow rate on the structure of TiO2-doped hollow PU, as well as the activity of the encapsulated HRP prepared at different core-shell solution flow rate. By fixing the flow rate of the shell phase solution at 0.5 mL h-1 and varying the core phase solution flow rate range from 0.05 to 0.2 mL h-1, a series of TiO2-doped hollow PU nanofibers encapsulating HRP were fabricated, and the cross-sectional SEM images showing different structures of the hollow nanofibers were provided as Supplementary Information (Figure S1). Nanofibers prepared at a core-shell phase flow rate of 0.07:0.5 mL h-1 exhibited the best defined cylindrical channels with relatively uniform size distribution in the diameter, which was similar to the result from our previous work about hollow nanofiber prepared in absence of TiO231. With ABTS and H2O2 as the substrate, the activity of HRP encapsulated inside hollow nanofiber fabricated at different core-shell phase flow rates was monitored by measuring the changes of ABTS concentration at OD420nm. The HRP encapsulated inside hollow nanofiber with best defined cylindrical channels that was prepared at core-shell phase flow rate of 0.07:0.5 mL h-1 retained the highest enzyme activity recovery about 80% (Figure S1). The SEM images of the both pure PU hollow nanofibers (Figure 1a) and the TiO2-doped PU nanofibers (Figure 1d) fabricated at the core-shell phase flow rate of 0.07 : 0.5 mL h-1 showed that the morphology of the hollow nanofibers were not affected by the doping of TiO2. Both of them exhibited quite uniform size distribution with average inner and outer diameter about 600 nm and 1.2 µm, respectively. According to TEM characterization, plenty of TiO2 nanoparticles are visible on the shell of TiO2-doped PU hollow nanofibers (Figure 1e), indicating a successful doping of TiO2 nanoparticles. The encapsulation of HRP in the chamber of the hollow nanofibers was further confirmed by CLSM observation, strong green fluorescence can be clearly seen indicating uniform dispersion of HRP in the chamber of the hollow nanofibers (Figure 1c and f). 8


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Figure 1. Characterizations of the PU hollow nanofibers with (lower panel) and without (upper panel) doping TiO2 nanoparticles fabricated through co-axial electrospinning technology. (a, d) Cross-sectional SEM images of PU hollow nanofibers (d) with and (a) without TiO2; (b, e) TEM images of PU hollow nanofibers (e) with and (b) without TiO2; (c, f) CLSM images of the FITC-labeled HRP encapsulated in the chamber of PU hollow nanofibers (f) with and (c) without TiO2. All these hollow nanofibers were fabricated at core-shell phase solution flow rates of 0.07:0.5 mL/h.

Effect of H2O2 concentration on the degradation of 2,4-DCP catalyzed with HRP. Because of the wildly availability and high activity, HRP is the most popular enzyme within the peroxidase family for degradation of aromatic substrates, including phenols and aromatic amines from waste waters with H2O2 as oxidant.36-38 The activity of HRP depends on the concentration of H2O2. Here, effect of H2O2 concentrations on the degradation of 2,4-dichlorophenol were investigated by varying its concentration from 0.5 to 15 mM. In all assays, abundant free (1 µg/mL) or immobilized HRP (30 mg nanofiber membrane containing 5.25 µg HRP for 5 mL system) were supplied to reaction solutions, and the 2,4-DCP removal ratio at 4 h was measured. From the results shown in Figure 2, for each of the three tested 2,4-DCP concentration, effect of H2O2 concentration on its degradation efficiency was similar. 9



The 2,4-DCP removal ratio at 4 h increased sharply with increasing of H2O2 concentration until reached an optimum point, indicating that H2O2 was a limiting factor in this range; afterwards, adding excess H2O2 had no significantly effect on the removal efficiency. The higher of the 2,4-DCP concentrations, higher concentration of H2O2 needed to get the highest 2,4-DCP removal efficiency, and the optimum H2O2 concentration happened to be approximately the same as the initial 2,4-DCP concentrations. When free HRP was used, the 2,4-DCP removal ratio decreased at H2O2 concentration of 10 mM. Reasons for this phenomenon might be ascribed to formation of inhibitory intermediate products at high concentration of H2O2, or H2O2 itself acted as inhibitor of HRP activity. For the HRP encapsulated inside hollow nanofiber, however, there was no decrease in 2,4-DCP removal ratio even when the concentration of H2O2 reached to 15 mM, suggesting improved stability than free enzyme at excess of H2O2.

Removal ratio of 2,4-DCP(%)

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90 80 70 60 50 40 30 20 10 0

2

4

6

8

10

12

14

16

Concentration of H2O2 (mM) Figure 2. Effects of H2O2 concentration on 2,4-DCP removal ratio catalyzed by free and immobilized HRP. Concentrations of 2,4-DCP: (■, □) 1mM, (●, ○) 5 mM, and ( ▲ , △ ) 10 mM. The filled and open symbols represent results with the immobilized and free HRP, respectively.

Effect of pH on the degradation of 2,4-DCP catalyzed by HRP and TiO2/UV. Activity of enzymes is usually influenced by the pH values. For the HRP activity 10


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towards degradation of 2,4-DCP, as shown in Figure 3, there was an optimal pH at about 8.0 to obtain the highest 2,4-DCP removal ratio for both free and the immobilized enzymes. While compared with the free HRP, activity of the immobilized one was relatively less affected under acid and alkaline conditions. This stabilization effect might be ascribed to the nano-confinement effect from nano-scale chamber of hollow nanofibers. The effect of pH on phenol removal catalyzed by TiO2 under UV irradiation was also investigated. Figure 3 shows that the 2.4-DCP removal ratio was high under both acidic and alkaline conditions, while lowest degradation efficacy was observed at pH 6.0.

Removal ratio of 2,4-DCP(%)

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80 70 60 50 40 30 20 10 2

3

4

5

6

7

8

9

10

11

pH Figure 3. Effects of pH on the removal of 2,4-DCP with (○) free HRP, (●) the HRP encapsulated inside TiO2-doped hollow nanofibers, and (■) TiO2/UV. The reaction volume was 5 mL and 2,4-DCP concentration was 1 mM. For the enzymatic test, the final concentration of free and immobilized HRP was 0.3 µg/mL, concentration of H2O2 was 10 mM. For the TiO2/UV test, final concentration of TiO2 was 0.5 mg/mL. Removal ratios presented were measured at 3 h for all tests. Effect of UV on the stability of HRP. To obtain high degradation efficiency of 2,4-DCP catalyzed by simultaneous photochemical-biochemical method, the most important issue is how to maintain the activity of HRP under UV irradiation in the presence of TiO2. Immobilizing enzymes to solid carrier or interlinking enzymes to UV-absorbing molecules by multipoint-covalent binding has been reported for 11



improving enzymes resistance to UV induced inactivation.39-40 However, processes for UV-absorber synthesis and the interlinking reaction were complicated, activity recovery of enzymes were very low and the stability enhancement was rather limited. Here in the present work, TiO2-doped hollow nanofibers was fabricated and HRP was

in situ encapsulated inside hollow chamber of the fiber through co-axial electrospinning. It was expected that the shell of hollow nanofiber would protect the enzymes from direct UV irradiation, thus the deactivation can be avoided. Results shown in Figure 4 proved above assumption well. The stability of HRP encapsulated in TiO2-doped hollow nanofibers was significantly improved; more than 90% of the original activity was retained after 6 h UV irradiation in the presence of doped TiO2.

Rusidual activity of HRP (%)

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120 100 80 60 40 20 0 0

2

4

6

8

10

Time (h) Figure 4. The stability of HRP against UV and TiO2. (■) Deactivation of free HRP against TiO2/UV, (●) deactivation of free HRP against UV in the absence of TiO2, ( ▲ ) deactivation of free HRP dissolved glycerol solution against UV, ( ▼ ) deactivation of HRP encapsulated inside TiO2-doped hollow nanofibers against UV.

Photochemical and enzymatic degradation of 2,4-DCP. The efficacy of the photochemical method by UV/TiO2, the enzymatic process with free and immobilized HRP, the simultaneous photochemical-enzymatic process with free HRP and UV/TiO2, as well as the photochemical-enzymatic process with TiO2-HRP hybrid catalysts based on hollow nanofibers were examined by measuring the degradation of 12


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2,4-DCP. Although the degradation efficacy for high concentration 2,4-DCP was a little higher by increasing the H2O2 concentration, H2O2 is another contaminant. Therefore 10 mM of H2O2 was used for in all of the experiments with HRP. When 4 mg hollow nanofiber membrane-based TiO2-HRP hybrid catalysts was applied in 5 mL reaction solution, the corresponding concentration of HRP and TiO2 was 0.6 µg/mL and 0.5 mg/mL, respectively. Therefore, to make better comparison, dosages of HRP applied in free formation were all adjusted to 0.6 µg/mL, and dosages of TiO2 applied in dispersed formation were all set as 0.5 mg/mL. The pH of the reaction solution for all tests was 8.0. Figure 5 shows the degradation curves of 2,4-DCP with initial concentration of 1 mM, 5 mM and 10 mM, respectively. It can be seen that in all these three tested 2,4-DCP concentrations, the TiO2/UV process with dispersed TiO2 and that doped in shell of hollow nanofibers exhibited the lowest efficacy in 2,4-DCP removal, and photocatalytic activity of TiO2 after immobilization was affected only very slightly. A more efficient degradation of 2,4-DCP was obtained in the enzymatic process with free HRP, and that with the encapsulated HRP was even faster, which could be ascribed to the enhanced stability of the HRP against 10 mM H2O2 as discussed before. The simultaneous photochemical-enzymatic process with dispersed TiO2 and free HRP under UV irradiation, however, was less efficient than that with enzyme only. Such phenomenon was similar to what was observed in our previous work with laccase and TiO2/UV for simultaneous photochemical-enzymatic degradation of 2,4-DCP.26 As shown in Figure 4, the irradiation UV in the presence of TiO2 would lead rapid deactivation of free HRP; therefore, the degradation of 2,4-DCP under this condition was less efficient than that of the enzymatic process. When HRP was encapsulated inside the nano-chamber of hollow nanofibers, photochemical-enzymatic process using the TiO2-HRP hybrid catalyst was remarkably faster than that using TiO2/UV and HRP alone, or the simultaneous photochemical-enzymatic process with free HRP and UV/TiO2. The same trends were seen for all these tested three different 2,4-DCP concentrations. And what was more important, enhancement in the removal efficacy of high concentration 2,4-DCP (10 13



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mM) was more significant than that of 1 mM and 5 mM. By using the hollow nanofibers-based TiO2-HRP hybrid catalyst, about 90% of 10 mM 2,4-DCP was degraded within 3 h; while the removal ratio obtained with dispersed TiO2/UV, TiO2 doped in PU hollow nanofibers, free HRP, combination of dispersed TiO2 and free HRP under UV, as well as the encapsulated HRP, were only 31.37%, 27.98%, 49.71%, 36.53% and 58.32%, respectively. The efficiency of TiO2-HRP hybrid catalyst for 2,4-DCP degradation was also much higher than some other reported studies. Xu et al.41 crosslinked HRP to PAN-based beads modified with ethanediamine and chitosan. It took at least 10 hs to obtain 90% removal ratio of 3 mM 2,4-DCP with 3 mM H2O2 using this immobilized HRP at high dosage of 0.2 mg/mL. A composite of graphene oxide and nano-Fe3O4 (GO/Fe3O4) and HRP was used as binary catalyst for simultaneous degradation of 2,4-DCP, a significant synergistic effect between GO/Fe3O4 and HRP was observed, such that a removal as high as 93% was obtained in their simultaneous use as compared with 2,4-DCP removal of 35% and 9% by single use of HRP and GO/Fe3O4, respectively.42 However, the preparation of the GO/Fe3O4 catalyst was rather complicated and the GO/Fe3O4-HRP binary catalyst was not integrated. Another simultaneous photocatalytic-enzymatic process was using TiO2/UV and laccase.26 Immobilizing of laccase covalently to controlled porous glass enhanced the stability of laccase against TiO2/UV induced inactivation, therefore 90% removal percentage for the degradation of 5 mM 2,4-DCP was achieved within 2 hs by coupling the laccase with the TiO2/UV. However, the TiO2 nanoparticles was used in dispersed formation, which leads to difficulties in recycling and may raise safety concerns. In contrast to these two binary catalysts system, our TiO2-HRP hybrid catalyst was integrated in hollow nanofiber membrane, which will greatly facilitate its operation and recycling, and concerns on potential environmental hazardous effects from nanomaterials could also be avoided.

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Figure 5. Degradation of different concentration of 2,4-DCP with (□) dispersed TiO2/UV, (■) TiO2 doped PU hollow nanofibers with UV, (△) dispersed TiO2/UV and free HRP, (○) free HRP, (●) HRP encapsulated inside PU hollow nanofibers, and (▲) hollow nanofibers based TiO2-HRP hybrid catalysts under UV. In all cases, concentrations of TiO2 and HRP were adjusted to the same level, at 0. 5 mg/mL and 0.6 µg/ mL, respectively.

Operational and thermal stability of the TiO2-HRP hybrid catalyst. Operational stability of TiO2-HRP hybrid catalyst is a significant characteristic for its potential industrial applications. To demonstrate the recycling capability of the hybrid catalyst for photochemical-enzymatic degradation of 2,4-DCP, the reaction was allowed for 4 h before the nanofiber membrane was retrieved from reaction solution with forceps to stop the reaction. The membrane was then applied for next reaction cycles. Results in Figure 6(a) indicated that the TiO2-HRP hybrid catalyst based on hollow nanofiber membrane retained about 90% activity in the tenth cycles compared to the values in the first cycle. Our previous work has demonstrated that the encapsulation of enzymes inside PU hollow nanofibers could significantly improve the long term stability of enzymes, such that the encapsulated glucose oxidase and horseradish peroxidase could retain almost 100% of its original during four months storage 4 oC, and even at 25 oC, above 50% of activity was retained after storage for four months.28 Therefore, here only the thermal-stability of the integrated photochemical-enzymatic system at 60oC was studied. As illustrated in Figure 6(b), the hybrid catalysts not only realized recycling and reusing of the whole system, the encapsulated HRP also exhibited significantly 15



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enhanced thermal-stability. The half-life of the hollow nanofiber encapsulated HRP at 60oC was estimated to be 8 h, which was corresponding to about 16-fold enhancement as compared with that of the free enzyme. Because HRP was confined inside the nano-chamber of the hollow fibers, it was considered that the spatial confining effect provided a distinctive stabilizing mechanism for enzymes. Such excellent stability makes the integrated TiO2-HRP hybrid catalysts attractive for potential applications in industry.

Figure 6. Performance of photochemical-enzymatic hybrid catalyst based on hollow nanofiber for removal 2,4-DCP. (a) Reusability of the system, and (b) thermal stability of the free and hollow nanofibers encapsulated HRP at 60oC.

CONCLUSION In this work, a simultaneous photochemical-enzymatic 2,4-DCP degradation process with TiO2 and HRP as photo- and bio-catalysts was investigated. The resistance of HRP against the TiO2/UV induced deactivation was a determining factor for the effectiveness of such simultaneous process. Encapsulation of HRP inside hollow chamber of the nanofiber was found to protect the HRP from deactivation effectively, made it feasible to combine the photo-oxidation and enzymatic process for degradation of 2,4-DCP. The TiO2-HRP hybrid catalysts based on hollow nanofibers exhibited high efficiency for the degradation 2,4-DCP, especially at high concentration. About 90% of 10 mM 2,4-DCP was degraded within 3 h by using the TiO2-HRP hybrid catalyst based on hollow nanofibers. While the removal ratio 16


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obtained with obtained with dispersed TiO2/UV, TiO2 doped in PU hollow nanofibers, free HRP, combination of dispersed TiO2 and free HRP under UV, as well as the encapsulated HRP, were only 31.37%, 27.98%, 49.71%, 36.53% and 58.32%, respectively. The high efficiency of the integrated photochemical-enzymatic catalysts therefore provide a prospective strategy for removal of high concentration of 2,4-DCP. Besides the high activity, such highly integrated system also demonstrated excellent recycling capability and stability. Moreover, with TiO2 nanoparticles being integrated, concerns on potential environmental hazardous effects from this nanomaterials could be avoided. We expect that the integrated photochemical-enzymatic process will offer a useful platform for pollutant degradation.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: XXX. Cross-sectional SEM images, size distribution and activity recovery of HRP encapsulated inside of TiO2-doped hollow nanofibers produced at various core-shell phase solution flow rates (PDF). AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Fax/Phone: +86 10 82544958. Notes The authors declare no competing ﬁnancial interest. ACKNOWLEDGEMENTS

This work was supported by the National Natural Science Foundation of China (Grant Nos. 21376249, 21336010, 91534126), 973 Program (2013CB733604), and

National

Major

Scientific

Equipment

(2013YQ14040508). 17


Development

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A TiO2-Horseradish Peroxidase Hybrid Catalyst Based on Hollow Nanofibers for Simultaneous Photochemical-Enzymatic Degradation of 2,4-Dichlorophenol

Xiaoyuan Ji, Zhiguo Su, Mengfang Xu, Guanghui Ma, Songping Zhang*

Novel TiO2-HRP hybrid catalyst based on hollow nanofiber for simultaneous photochemical-enzymatic degradation of 2,4-DCP was fabricated by facile co-axial electrospinning.

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TiO2âHorseradish Peroxidase Hybrid Catalyst Based on Hollow

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