Accepted Manuscript

Title: Synthesized TiO2 /ZSM-5 Composites Used for the

Photocatalytic Degradation of Azo Dye: Intermediates,
Reaction Pathway, Mechanism and Bio-toxicity

Author: Kefu Zhou Xin-Yan Hu Bor-Yann Chen

Chung-Chuan Hsueh Qian Zhang Jiajie Wang Yu-Jung Lin
Chang-Tang Chang

PII: S0169-4332(16)30944-8
DOI: http://dx.doi.org/doi:10.1016/j.apsusc.2016.04.155
Reference: APSUSC 33156

To appear in: APSUSC

Received date: 12-12-2015

Revised date: 20-4-2016
Accepted date: 23-4-2016

Please cite this article as: Kefu Zhou, Xin-Yan Hu, Bor-Yann Chen, Chung-Chuan
Hsueh, Qian Zhang, Jiajie Wang, Yu-Jung Lin, Chang-Tang Chang, Synthesized
TiO2/ZSM-5 Composites Used for the Photocatalytic Degradation of Azo Dye:
Intermediates, Reaction Pathway, Mechanism and Bio-toxicity, Applied Surface
Science http://dx.doi.org/10.1016/j.apsusc.2016.04.155

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Synthesized TiO2/ZSM-5 Composites Used for the Photocatalytic Degradation of

Azo Dye: Intermediates, Reaction Pathway, Mechanism and Bio-toxicity

Kefu Zhoua, Xin-Yan Hua, Bor-Yann Chenb, Chung-chuan Hsuehb*, Qian Zhangc, Jiajie Wanga, Yu-Jung Lina,

Chang-Tang Changd**

a College
of the Environment and Ecology, Xiamen University, Xiamen, China

b Department
of Chemical and Materials Engineering, National I-Lan University, I-Lan, Taiwan

c Department
of Environmental Engineering, National Taiwan University, Taipei, Taiwan

d Department
of Environmental Engineering, National I-Lan University, Ilan, Taiwan
Graphical abstract

Fig. 6 Major photocatalytic pathway of Reactive Black 5, based on the identification of by-products
from GC-MS and IC analysis.
Fig. 7 Time series profiles of cumulative CO2 production during the toxicity tests with azo-dye
Reactive Black 5 samples at different degradation times (regression curves calculated on the basis of
the data obtained between 0 and 44 h).

Highlight

 The major photo-catalytic degradation pathway of azo-dye was elaborated according to the
identification of by-products from GC-MS and IC analysis.

 Comparative assessment on characteristics of abiotic and biotic dye decolorization was

analyzed.

 EDTA (hole scavengers) and t-BuOH (radical scavengers) were used to determine the main
active oxidative species in the system.

 The toxicity effects of degradation intermediates of Reactive Black 5 (RB5) on the cellular
respiratory activity were assessed.
Abstract
In this study, a one-step solid dispersion method was used to synthesize titanium dioxide (TiO2) /
Zeolite Socony Mobil-5 (ZSM-5) composites with substantially reduced time and energy
consumption. A degradation efficiency of more than 95% was achieved within 10 min using 50%
PTZ (synthesized TiO2/ZSM-5 composites with TiO2 contents of 50 wt% loaded on ZSM-5) at pH 7
and 25 °C. The possible degradation pathway of azo-dye Reactive Black 5 (RB5) was investigated
using gas chromatography–mass spectrometry and ion chromatography (IC). The bonds between the
N atoms and naphthalene groups are likely attacked first and cleaved by hydroxyl radicals, ultimately
resulting in the decolorization and mineralization of the azo dye. A comparative assessment of the
characteristics of abiotic and biotic dye decolorization was completed. In addition, the toxicity
effects of the degradation intermediates of azo-dye RB5 on cellular respiratory activity were
analyzed. The bio-toxicity results showed that the decay rate constants of CO2 production from the
azo-dye RB5 samples at different degradation times increased initially and subsequently decreased,
indicating that intermediates of higher toxicity could adhere to the catalyst surface and gradually
destroyed by further photocatalytic oxidation. Additionally, EDTA (hole scavengers) and t-BuOH
(radical scavengers) were used to detect the main active oxidative species in the system. The results
showed that the hydroxyl radicals are the main oxidation species in the photocatalytic process.

Keywords: Photocatalyst, Degradation pathway, Biotoxicity, Mechanism

1. Introduction

Textile azo-dyes are commonly used in various industries, and azo-dye waste-water has become one
of the main sources of water pollution because of its high chrominance, high salinity, complex
composition and toxic nature [1]. Removing color from waste has more importance than other
colorless organic substances because small amounts of dyes (below 1 mg L-1) are clearly visible and
considerably influence the water environment. However, these effluents are mostly
non-biodegradable and resistant to destruction by physicochemical treatment methods [2]. Azo-dyes
are readily reduced under anaerobic conditions to potentially hazardous aromatic amines [2]. Such
pollutants should be removed from water before they are discharged into the environment.
AOPs (e.g., Fenton [3], H2O2/UV processes [4], and TiO2 mediated photo-catalyst [5]) are based
on the generation of highly reactive species, such as hydroxyl radicals, to oxidize a broad range of
pollutants. Titanium dioxide is known to demonstrate high photocatalytic activity, strong oxidizing
capability, and long-term operational stability [5]. However, the use of TiO2 has disadvantages in
practical applications. For example, TiO2 has a small particle size and is difficult to regenerate [6].
Furthermore, its poor adsorption capacity at a low pollutant concentrations limits its practical
applicability [7]. Therefore, TiO2 supported on a carrier matrix has gained considerable attention,
and various types of loading materials (e.g., zeolite [8], silica [9], activated carbon [10], and glass
fibers [11]) have been prepared. For example, zeolite has been studied as a support material for TiO2
because of its porosity, specific surface area, high thermal stability, excellent adsorption properties
and environmental friendliness [7]. Zeolites offer the promising characteristics of a high surface area,
unique nanoscaled porous structure and ion exchange properties, resulting in highly efficient
photocatalysis [8]. The small ordered mesopore channels of ZSM-5 result in fast intraparticle
molecular transfer, while the large surface area enhanced its light harvesting and adsorption for
reactant molecules [8].
TiO2-mediated photocatalysis has been studied under a broad range of experimental conditions.
However, there has been a lack of information on the identities of major intermediates to consider
their biotoxicity, a very important aspect of these processes, especially in view of their practical
applications. Therefore, our attention has been focused on the reaction pathway, mechanism and
bio-toxicity, based on the identification of the intermediates. This study primarily aimed to obtain
such information for subsequent further optimization.
In this study, for economic feasibility, a one-step method (i.e., solid dispersion method) was used
to synthesize TiO2/ZSM-5 composites. The composites were characterized to demonstrate the
stability and reusability. Furthermore, the findings revealed the effects of the loading content of TiO 2
over ZSM-5, initial pH value, RB5 concentration and environmental temperature on the
photocatalytic performance to obtain the optimal operation conditions. In addition, the possible
degradation pathway of azo-dye RB5, toxicity effects of degradation intermediates, along with the
possible mechanism of the photodegradation reaction were also investigated [12].

2. Materials and methods

2.1. Reagents

Unadulterated ZSM-5 was purchased from American ACROS. The specific surface area (SBET)
and pore size of ZSM-5 were determined using a nitrogen adsorption–desorption analyzer. Titanium
dioxide (P25, 80% anatase and 20% rutile) was purchased from American ACROS. Sodium
hydroxide, hydrochloric acid and ethyl alcohol with a purity of 95% were purchased from Japanese
SHIYAKU. All reagents were analytical reagent grade and used without further purification. All
aqueous solutions were freshly prepared in deionized water.

2.2. Preparation of catalysts

Composites were synthesized using the solid dispersion method, as shown in Fig. S1 [13]. First,
combinations of TiO2 and ZSM-5 of different mass ratios were prepared. This mixture was then
added to 95% ethanol to allow gel formation. Titanium dioxide was completely loaded on the surface
of the zeolite at ambient temperature with 1 h of gentle stirring. Subsequently, the gels were dried at
100 °C for 12 h to remove the solvent. Finally, the catalysts were calcined at 400 °C for 2 h in a
muffle furnace before cooling, grinding and collecting through 200 mesh sieves. For a comparative
study, TiO2 contents of 7, 12.5, 25, and 50 wt% were loaded on ZSM-5 and were defined as 7% PTZ,
12.5% PTZ, 25% PTZ, and 50% PTZ, respectively.

2.3. Characterization techniques

The crystal phases of the synthesized PTZ composites were measured using an X-ray
diffractometer (XRD, Rigaku Ultima IV). The surface morphology of the composites was observed
using a scanning election microscope (SEM, Mac Science, MXP18) and energy dispersive
spectroscopy (EDS). The specific surface areas of the PTZ composites were measured and calculated
by the Brunauer-Emmett-Teller (BET) method from nitrogen adsorption-desorption data with an
automated adsorption apparatus (Micromeritics, ASAP 2020) [14]. The specific chemical bond
vibration of the compounds was examined by Fourier Transform Infrared Spectroscopy
(FT-IR, Bruker, Germany). The particle size analyzer (Zetasizer ver. 6.20, Malvern) was used to
measure the size distribution of nanoparticles. Zeta potential was also measured to analyze the
stability of the compounds. The total organic carbon (TOC) was measured with a Tekmar Dohrmann
carbon analyzer model Phoenix 8000. Gas chromatography–mass spectrometry (GC-MS,
SHIMADZU QP2010 gas chromatography coupled with JEOL JMS-700 mass spectrometry) was
used to detect the photodegradation byproducts. An ion chromatograph (Metrohm, 761 Compact IC)
was used to detect the anions and cations in the final solution.

2.4. Photocatalytic measurements

The photocatalytic activities of catalysts were evaluated by measuring the degradation of RB5.
Catalysts (0.2 g L−1) were added to 10 mg L−1 azo-dye RB5 solutions. For controlled experiments, 14
W UV lamps (254 nm, 9.7 W m-2) were used as the light source. After the TiO2/ZSM-5 composite
was added, the solution was magnetically stirred for 30 min in the dark to reach equilibrium prior to
exposure to light. The suspensions were filtered using a centrifugal filter (0.45 μm) to remove the
photo-catalyst particles prior to the measurement. A quantitative analysis of azo-dye RB5 was
performed using a UV– vis spectrophotometer (λmax = 600 nm) [15,16]. All experiments were
conducted in triplicate to confirm data reproducibility.
2.5. GC-MS analysis

All samples were analyzed using GC-MS (SHIMADZU QP2010 gas chromatography coupled
with JEOL JMS-700 mass spectrometry). The fused silica capillary column was J&W Scientific
DB-5 (30m × 0.25 mm ID and 0.25 µm film thickness). The carrier gas was helium introduced at a
rate of 2.11 mL min-1. The injection volume and temperature were 1.0 µL and 150 °C, respectively.
The oven temperature was increased from 60 to 250 °C at a rate of 10 °C min-1 and was maintained
isothermally for 10 min. The temperature and energy of the electron impact ion source were 200 °C
and 70 eV, respectively. A spectral match of at least a 90% with a compound listed in the databank
of the GC-MS spectral library (e.g., NIST 05 and FFNSC 1.2) was required to confirm the structural
identification of a reaction mixture component. Detailed degradation pathways of azo-dye RB5 were
then proposed based on the identity of confirmed components [17].

2.6. Inhibition examination through respirometry

Toxicity effects of azo-dye RB5 samples on the cellular respiratory activity associated with
microbial cells at various degradation times were assessed using an automated Columbus
Micro-Oxymax Respirometer equipped with CO2 sensors. The measurement relied on the circulation
of air through closed-system testing bottles, where the liquid in the bottles remained constant (i.e.,
mobile liquid–flowing gas respirometer) [18]. As the amount of CO2 present in the bottle was nearly
negligible initially, the time course of CO2 production could be attributed to the absence or presence
of the existing toxicant (s) in the cultures. For respirometric experiments, an Escherichia coli DH5α
seed was taken from an isolated colony and precultured in 50 mL of Bacto LB (Luria–Bertani)
culture, Miller (per liter; 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride) for 12 h
(i.e., late exponential growth phase) at 37 °C in a shaking (125 rpm) water bath. Subsequently, 0.5
mL of the precultured broth was inoculated into 50-mL bottles containing fresh 1 × LB broth and 2.5
mL of azo-dye RB5 samples at different degradation times at 30 °C, and these bottles were shaken at
110 rpm for 30 h. The respirometer was calibrated using standard CO2 (g) at 0.5% [19].
3. Results and discussion

3.1. Material characterization

3.1.1. X-ray diffraction analysis
The XRD profiles of ZSM-5, 7% PTZ, 12.5% PTZ, 25% PTZ, 50% PTZ，and commercial TiO2
are shown in Fig. 1(a). The signal peaks at 25.4°, 37.5°, 48.0°, 54.1°, 54.8° and 62.6° indicated the d
101, d 004, d 200, d 106, d 211 and d 204 planes of anatase, respectively. The peak at 27.1° indicated
the d 110 plane of rutile [20]. The characteristic peaks of ZSM-5 were observed at 13.2°, 13.9°, 14.7°,
15.5°, 23.1°, 23.9°, 29.8°, 45.0°, and 45.2°. The structures of ZSM-5 were not altered by TiO2
loading because no significant shift in the ZSM-5 peaks of TiO2/ZSM-5 was observed compared to
pure ZSM-5. As TiO2 loading was increased, the diffraction peaks of TiO2 became wider and sharper.
These findings are in agreement with the results of Wang et al. (2011) [20].
The XRD patterns of the 50%PTZ before and after reaction under the UV light for the degradation
of RB5 are shown in Fig. 1(b). It can be observed that the phase and structure of the 50% PTZ
composite remained unchanged after the reaction, suggesting that the 50% PTZ is stable during
photocatalytic degradation.

3.1.2. N2 adsorption–desorption isotherm analysis

In this study, the SBET and pore sizes of ZSM-5, 7% PTZ, 12.5 % PTZ, 25% PTZ, and 50% PTZ
were calculated from N2 adsorption–desorption isotherms obtained using the
Barrett–Joyner–Halenda method [14]. The SBET and pore size of ZSM-5 were 311 m2 g−1 and 2.40 nm,
respectively (Table 1). In addition, the SBET of the materials decreased from 286 to 205 m2 g−1 and the
pore size increased from 2.55 to 3.46 nm, with an increase in TiO2 loading content from 7 to 50 wt%.
TiO2 particles were loaded on the surface of ZSM-5 rather than within materials. Compared with
pure TiO2, the synthesized TiO2/ZSM-5 composites that had a larger surface area provided more
active sites for the degradation reaction.

3.1.3. Scanning electron microscopy analysis

The morphology of raw ZSM-5, 7% PTZ, 12.5% PTZ, 25% PTZ, and 50% PTZ were investigated
using SEM, as shown in Fig. 2(a). TiO2 particles were uniformly dispersed and attached to the outer
surface of ZSM-5 without significant aggregation in contrast to the previous study of Wang et al.
(2015) (Fig. 2(a); 50% GTZ (synthesized by sol-gel method and TiO2 content of 50 wt% was loaded
on ZSM-5)) [21]. Thus, the photocatalytic efficiency of the TiO2/ZSM-5 composite materials was
improved. These findings are consistent with those of Ito et al. (2014) [22]. The EDX spectrum in
Fig. 2(b) reveals the presence of Ti, O, and Si, revealing that TiO2 was successfully combined with
ZSM-5.

3.1.4. Fourier transform infrared spectroscopy (FTIR) analysis

Fourier transform infrared spectroscopy of 50%PTZ before and after three runs under the UV light
are shown in Fig. 3(a). The bands at 3421.04 cm-1 and 1636.06 cm-1 are attributed to hydroxyl groups.
The bands at 1069.09 cm-1 and 1216.39 cm-1 are attributed to the stretching vibrations of the Si-O in
ZSM-5, and the strong absorption at 650 cm-1 is attributed to the stretching vibrations of the Ti-O in
anatase TiO2. No peak shift was observed after the reaction, suggesting that the 50% PTZ composites
were stable during the process of photocatalytic degradation. The intensity of the peak at 1089.09
cm-1 decreased, while the peak t 650 cm-1 increased, suggesting that trace amounts of titanium
dioxide were lost during the process of photocatalytic degradation and that the weight content of
ZSM-5 in the PTZ composites increased slightly after reaction.

3.2 Properties of materials

3.2.1. Different TiO2 loadings
The photocatalytic degradation of azo-dye RB5 aqueous solutions with the 7%, 12.5%, 25%, and
50% PTZ composite materials was investigated. The photocatalytic degradation efficiency of various
TiO2 loadings of composite materials was measured under the same conditions. As shown in Fig.
4(a), all of the composite materials showed a similar tendency for azo-dye RB5 degradation without
a time delay. As the TiO2 content increased, the degradation efficiency correspondingly increased.
The highest degradation rate of azo-dye RB5 can be achieved within 15 min using 50% PTZ with a
95% efficiency. Under the same operation conditions, the removal efficiency of azo-dye RB5 was
96.6%, 67.8%, and 47.5% with 25% PTZ, 12.5% PTZ, and 7% PTZ composite materials,
respectively. This phenomenon could be attributed to the increase in active sites with TiO2 loading
[23-26]. However, a similar degradation efficiency was achieved with 25% PTZ and 50% PTZ,
suggesting that the performance plateaued with a further increase in the TiO2 content. This was
observed because light penetration in the reaction system decreased and the amount of activated TiO2
decreased with an increase in TiO2 loading [27]. Furthermore, activated TiO2/ZSM-5 may be
deactivated through collision with ground-state catalysts as follows [28]:

TiO2 * / ZSM - 5  TiO2 / ZSM - 5  TiO2 # / ZSM - 5  TiO2 / ZSM - 5 , (1)

where TiO2*/ZSM-5 and TiO2#/ZSM-5 are activated and deactivated forms, respectively.
With consideration of the economic feasibility of material preparation, the 25% PTZ composite
material was used for practicability.

3.2.2. Effect of pH value

The initial pH value plays a crucial role in the surface charge properties of catalysts, affecting both
the adsorption and degradation of azo-dye RB5. The effect of pH value on the degradation efficiency
was investigated by adjusting the pH values from 3.0 to 11.0 in reactions of 10 mg·L−1 RB5 with the
photocatalyst (0.2 g L−1). The reaction temperature was maintained at 25 °C. These results are
presented in Fig. 4(b). The degradation rate was the highest at pH 7, higher at pH 5 and 9, lower at
pH 11 and lowest at pH 3.
Fig. 3(b) shows the pH-dependent zeta potential of 50% PTZ material with a zero point of
approximately 9.5. Fifty percent PTZ material surfaces were negatively charged when pH > 9.5 and
positively charged when pH < 9.5 [29]. Due to the low pKa value of the sulfonic acid
group, azo-dye RB5 is in the anionic form within the pH range studied [30]. At neutral conditions
(e.g., pH 7), the absorption process was improved through an electrostatic attraction between the
positively charged 50% PTZ material surfaces and negatively charged azo-dye RB5 [30]. Holes or·•
OH radicals were generated on degraded photocatalyst surfaces [31]. When pH < 9.5, transfer of
photogenerated electrons and holes to the dye molecules was a facile process, resulting in a
photocatalytic degradation efficiency of approximately 100% in 15 min at pH 7. With increasing pH,
the negative charges on the 50% PTZ material repel the azo-dye RB5, and a decrease in efficiency of
photodegradation was thus expected [31]. At basic conditions (e.g., pH 11), a low degradation
efficiency was observed because of the dominant repulsion. Under acidic conditions (e.g., pH 3), the
degradation efficiency decreased because the formation rate of the hydroxyl radicals was decreased
[32]. These results were consistent with those of Taicheng et al. (2011) [31] and Yang et al. (2010)
[32].

3.2.3. Effects of temperature

The effects of temperature on the performance of catalysts were also assessed with 0.2 g L−1 of
50% PTZ at the initial concentration of 10 ppm RB5 at pH 7. The degradation efficiency of the 50%
PTZ photo catalyst nearly reached completion within 15 min at 25–45 °C (Fig. 4(c)). The
photodegradation efficiency of the 50% PTZ composite material increased with an increase in
temperature from 15 to 25 °C. Low temperature caused aggregation of dye molecules, thereby
affecting the photocatalytic efficiency. Both dye molecules and catalysts possessed higher energy
with increasing temperature, triggering a more intense Brownian motion that increased collision for
dye decolorization. However, no considerable difference in the photodegradation efficiency was
observed at 25, 35, and 45 °C. This insignificant photocatalytic decolorization efficiency at these
temperatures was possibly due to the mass-transfer rate-limited number of active sites on the catalyst
for degradation [33]. Temperature can affect both the adsorption and desorption of dye molecules on
the catalyst [34]. At a high temperature, the adsorbed dye molecules were desorbed to reach different
equilibria of the adsorption-desorption process.

3.2.4. Effect of initial azo-dye RB5 concentrations

At pH 7, the initial concentrations of 5, 10, 20, and 40 mg L−1 with 0.2 g L−1 catalyst loading at 25
°C were investigated. The degradation efficiency decreased with the increasing initial concentration
(Fig. 4(d)). At 5 mg L−1 azo-dye RB5, 10 min was required for a 100% degradation efficiency. The
time for complete degradation increased to 15 and 30 min for 10 and 20 mg L−1 azo-dye RB5,
respectively. The efficiency was only 80% for a 30-min reaction with 40 mg L−1 azo-dye RB5. A
possible reason for this reduction is that a significant amount of UV light was absorbed by dye
molecules rather than the TiO2 particles at a high azo-dye RB5 concentration, reducing the
degradation efficiency [35]. In addition, the degradation efficiency decreased with the light intensity
because of the masking effect of light penetration. The degradation rate decreased at high
concentrations because the number of active sites on TiO2 reduced, limiting azo-dye RB5
degradation. The photocatalytic capability of TiO2 decreased because the generation of OH radicals
on the catalyst surface decreased and the active sites were potentially masked by azo-dye RB5 at a
higher concentration [36]. As shown in Fig. 5, catalyst species most significantly affected the
efficiency of the photodegradation.

3.3 Mineralization

The mineralization of the azo-dye RB5 solution with 50%PTZ was monitored with a TOC
analyzer, and the results are depicted in Fig. 5(a). The results showed that the azo-dye RB5 had been
completely degraded, while only 74% TOC was removed after 20 min of irradiation, indicating that
some intermediate organic species remained in solution and complete mineralization was difficult.
Comparison of the results of different studies on the removal of azo-dye RB5 pollutant were shown
in Table 2.

3.4. Effect of holes and radicals in photocatalytic experiments by scavenger agents (EDTA/t-BuOH)

During photocatalysis, both the photogenerated holes and electrons can generate oxidative
species for degradation of azo-dye RB5. To further understand the mechanism of the photocatalytic
process, hole scavengers and radical scavengers were added (with 10 mM of EDTA or t-BuOH,
respectively) to the reaction to degrade azo-dye RB5 with 50%PTZ nanocomposites [16].
The results of 50% PTZ without of any scavengers and in the presence of t-BuOH and EDTA are
shown in Fig. 5(b). Both EDTA and t-BuOH as hole and radical scavengers decreased the efficiency
of azo-dye RB5 photo-degradation. The poorest performance was observed with addition of t-BuOH,
which could capture reactive species (e.g., hydroxyl (OH) and hydroperoxyl (OOH) radicals) to
inhibit oxidation. Moreover, the poorest azo-dye RB5 degradation efficiency indirectly suggested the
major role of OH for photodegradation. Compared with t-BuOH, photocatalytic degradation in the
presence of EDTA was superior, suggesting that the reactive species, (e.g., superoxide anion (O2−)
radicals) also played vital functions in photocatalysis, although the OH involved in the reaction was
still the major active oxidant for photodegradation.

3.5 Route assessment

 The mechanism of azo-dye RB5 degradation can be described as follows in equations (2)-(6).
Initially, the RB5 molecule is adsorbed onto the surface of 50% PTZ material. The conduction band
electron (e-) and valence band holes (h+) are generated when an aqueous 50% PTZ suspension is
irradiated with UV light. The generation of (e−/h+) pair leads to the formation of reactive oxygen
species. The OH radical and O2− can oxidize RB5 molecules, resulting in the degradation and
mineralization of the organics.

ZSM - 5 / TiO2  RB5  ZSM - 5 / TiO2  - RB5 ,

Adsorption
(2)

 ZSM - 5 / TiO2 e / h  ,

ZSM - 5 / TiO2  hv  (3)

e   O2 
 O2 , (4)

h   H 2O 
OH  H  , (5)

RB5  OH O2 h   intermedia te products  CO2   H 2O , (6)

The intermediate compounds of the photocatalytic degradation of azo dye Reactive Black 5 were
analyzed using GC-MS and IC, as shown in Table 3. Figure 6 shows the major photocatalytic
pathway of azo dye Reactive Black 5 at pH 7 based on the identification of by-products from
GC-MS and IC analyses. Sites with a high electron density are more available for attack by hydroxyl
radicals [45]. Therefore, sites 4, 9, 15, and 21 of RB5 were easily attacked by OH radicals, as shown
in Fig. 6. The electron density of the naphthalene ring is higher than that of the benzene ring. The
bonds C(8) -N(9) and C(15) -N(19) were likely cleaved first, and the intermediates of naphthalene
were formed after rearrangement [46]. The azo bond was not destroyed, and 4-diazobenzenesulfonic
acid was formed at the beginning of the photocatalytic degradation process, demonstrated by the
hydrazine and diazene intermediates identified by GC-MS, as shown in Table 3. Naphthalene was
easily attacked by OH to form 1,2-naphthalenediol and 1,2-Naphthoquinone, primarily through the
substitution of hydroxyl groups [47]. These intermediates were then oxidized to phenylacetaldehyde
and oxalic acid [48], which were detected by GC-MS and IC, respectively. Subsequently,
phenylacetaldehyde was oxidized to carboxylic acids. The electron withdrawing sulphonate group
inhibits reactivity towards OH of the ring to which it is attached, and thus, the ring with the amino
group is the first target for the hydroxy radicals [49]. The nitrate anion, nitrite anion or ammonium
cation was not detected using IC, indicating that N-containing species remain adsorbed on the
photocatalyst surface or, most probably, that significant quantities of N2 have been produced and
transferred to the gas-phase [50]. In the azo bonds, each nitrogen atom is in its +1 oxidation state,
which favors the evolution of gaseous dinitrogen by the following equations (7,8):

R  N  N  R  H   R  N  N   RH , (7)

R  N  N  R  N  N , (8)

Therefore, 4-diazobenzenesulfonic acid was oxidized to nitrogen and benzene sulfonic acid [2].
Benzene sulfonic acids were then oxidized to benzoquinone, which was subsequently oxidized to
carboxylic acid. Photodegradation can prevent the accumulation of intermediates. Finally, the
organic acids present were further mineralized.
A comparative assessment of the characteristics of abiotic and biotic dye decolorization was
performed. Photocatalytic degradation of azo dyes was carried out through oxidation; however,
biodegradation of azo dyes occurred by reductive decolorization [49]. Both the rate of
photodegradation and biodegradation were significantly affected by the steric hindrance and electron
density near the azo bond. Photodegradation proceeds at higher rates than biodegradation. For
photodegradation, azo dyes containing electron-releasing groups (e.g., amino group) around an azo
bond are more easily degraded [49].

3.6 Biological respiration inhibition test

In this study, the saturated cumulative amount of CO2 (denoted as mCO2) and the decay rate
constant for CO2 production (α) were considered to be toxicity indicators for the toxicant(s) present
in the environment. Rankings of mCO2|∞ and α could be directly used for comparison of the toxicity
potency for different conditions [19]. According to the data of respirometric CO 2 production by
E.coli DH5α (Fig. 7), the toxicity rankings of RB5 at various levels of degradation intermediates for
the saturated cumulative CO2 production (mCO2|∞) and the decay rate constant of CO2 production (α)
can be shown as follows (notes in Table 4). As shown in the time courses of the CO2 respirometric
data (Fig. 7), the rankings of the biotoxicity potency of azo-dye RB5 samples at different degradation
times (unit: mg L−1) were Blank (LB) < No.5 (C/C0 = 0.1) < No.4 (C/C0 = 0.25) < No.1 (C/C0 = 1.0)
< No.2 (C/C0 = 0.75) < No.3 (C/C0 = 0.5). The results showed that the decay rate constants of CO2
production from the azo-dye RB5 samples at different degradation times increased at first and then
decreased, indicating that the higher toxic intermediates that were generated could be firmly adhered
to the surface of catalysts and gradually destroyed by further photocatalytic oxidation. Addition of
azo-dye RB5 samples at different degradation times did not significantly inhibit the respiratory
activity required for the growth of E. coli DH5α cells. In fact, the decay rate constant of CO2
production for azo-dye RB5 samples at different degradation times did not greatly deviate from that
for the blank sample. Therefore, azo-dye RB5 exhibited low toxicity and the toxicity of intermediates
played the most crucial role. However, the concentration of degradation intermediates of azo-dye
RB5 seemed to be too low to cause obvious toxic effects. To determine the changes in the biological
toxicity of wastewater in the process of photocatalytic degradation, additional biological respiration
inhibition tests must be conducted with pure degradation intermediates.

4.Conclusion
A TiO2/zeolite composite was successfully prepared using the solid dispersion method. The
characterization results indicated that composite materials were uniformly dispersed and showed
excellent adsorption properties resulting in the improvement of photodegradation performance. A
degradation efficiency of more than 95% was achieved within 10 minutes with 50% PTZ at pH 7 and
25 °C. The major photocatalytic pathway of azo-dye Reactive Black 5 was proposed, based on the
identification of by-products from GC-MS and IC analyses. Sites with a high electron density are
more easily attacked by hydroxyl radicals. The bonds between nitrogen atoms and naphthalene
groups are likely cleaved first by hydroxyl radicals, ultimately resulting in the decolorization and
mineralization of the azo dye. Toxicity effects of degradation intermediates of azo-dye Reactive
Black 5 on the cellular respiratory activity increased at first and then decreased, indicating that the
high toxicity intermediates generated could be firmly locked on the catalyst surface and gradually
destroyed by further photocatalytic oxidation. Furthermore, EDTA (hole scavengers) and t-BuOH
(radical scavengers) were used to detect the main active oxidative species in the system. The results
showed that the hydroxyl radicals are the main oxidation species in the photocatalytic process.

Acknowledgments

The authors sincerely appreciate the financial support for this research (MOST
103-2622-E-197-009-CC3) provided by the Taiwan Ministry of Science and Technology. This study
was completed as part of cooperative achievements for Academic Exchange Program between
Xiamen University (China) and National I-Lan University (Taiwan) in 2014–2015. The authors
gratefully acknowledge Professor Bor-Yann Chen and Professor Chung-Chuan Hsueh for kindly
suggesting valuable comments and analyzing experimental data.
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Figure captions

Fig. 1 (a) XRD patterns of raw ZSM-5, 7% PTZ, 12.5% PTZ, 25% PTZ, 50% PTZ, and TiO2. (b)
XRD patterns of 50% PTZ before and after three runs.

Fig. 2 (a) Scanning electron microscope and energy-dispersive X-ray spectroscopy images of raw
ZSM-5, 7% PTZ, 12.5% PTZ, 25% PTZ, 50% PTZ, and 50% STZ. (b) The element distribution of
50% PTZ with EDS.

Fig. 3 (a) The Fourier transform infrared spectroscopy of 50% PTZ before and after three runs. (b)
Zeta potential of 50% PTZ composite catalyst.

Fig. 4 Effects of different operation parameters on the degradation efficiency of RB5. (a: effect of
different TiO2 loadings with PTZ material; b: effect of pH value; c: effect of temperature; d: effect of
the initial RB5 concentration. (Basic condition: pH = 7, dosage of catalyst = 0.2 g L-1, temperature =
25 ℃, light strength = 9.7 W m-2)

Fig. 5(a) Comparison of RB5 mineralization and degradation by 50% PTZ. (b) Effect of EDTA and
t-BuOH supplementation on the photocatalytic degradation of RB5.

Fig. 6 Major photocatalytic pathway of Reactive Black 5, based on the identification of by-products
from GC-MS and IC analysis.

Fig. 7 Time series profiles of cumulative CO2 production during the toxicity tests with RB5 samples
at different degradation times (regression curves calculated on the basis of the data obtained between
0 and 44 h).
Table 1 Brunauer–Emmett–Teller characterization and energy band gap value of various materials.
Table 2 Comparison of the results of different studies on the removal of RB5 dye pollutant.

Table 3 Retention time, compound name and molecular structure of intermediate products analyzed
by gas chromatography–mass spectrometry and ion chromatography.

Table 4 Kinetic parameters determined by extrapolating cumulative CO2 production curves.

 a b

Fig. 1 (a) XRD patterns of raw ZSM-5, 7% PTZ, 12.5% PTZ, 25% PTZ, 50% PTZ, and TiO2. (b)
XRD patterns of 50% PTZ before and after three runs.
 a
a

b b C

Ti
Si

Fig. 2 (a) Scanning electron microscope and energy-dispersive X-ray spectroscopy images of raw
ZSM-5, 7% PTZ, 12.5% PTZ, 25% PTZ, 50% PTZ, and 50% STZ. (b) The element distribution of
50% PTZ with EDS.
 0.30 40

1089.09
0.25 50% PTZ
650 30
before reaction
after three runs
0.20
Transmittance (%)

Zeta potential (mV)

20

0.15
10
0.10

0
0.05 1216.39

1635.05 3421.04
-10
0.00
a a b b
-0.05
1000 2000 3000 4000
-20
2 4 b 6 8 10 12
-1
wavenumber (cm ) pH

Fig. 3 (a) Fourier transform infrared spectroscopy of 50% PTZ before and after three runs. (b) Zeta
potential of 50% PTZ composite.
 A B
1.0

50% PTZ pH 3
0.8 25% PTZ pH 5
12.5% PTZ pH 7
7% PTZ pH 9
pH 11
0.6

0.4

0.2

0.0
D
C/CO

1.0
C

15 oC 5 mg L-1
0.8 25 oC 10 mg L-1
35 oC 20 mg L-1
45 oC 40 mg L-1
0.6

0.4

0.2

0.0
30 35 40 45 50 55 6030 35 40 45 50 55 60

Time (min)

Fig. 4 Effects of different operation parameters on the degradation efficiency of RB5. (a: effect of
different TiO2 loadings with PTZ material; b: effect of pH value; c: effect of temperature; d: effect of
the initial RB5 concentration. (Basic condition: pH = 7, dosage of catalyst = 0.2 g L-1, temperature =
25 ℃, light strength = 9.7 W m-2)
 100 1.0

Degradation t-BuOH
TOC removal EDTA
80 0.8 only photocatalyst
Relative concentration C/C0 (%)

60 0.6

C/C0
40 0.4

20 0.2

a b
0 0.0
0 5 10 15 20 25 30 0 5 10 15 20 25 30

Radiation time (min) Time (min)

Fig. 5(a) Comparison of RB5 mineralization and degradation by 50%PTZ.

(b) Effect of EDTA and t-BuOH supplementation on the photocatalytic degradation of azo-dye RB5.
Fig. 6 Major photocatalytic pathway of Reactive Black 5, based on the identification of
by-products from GC-MS and IC analysis.

1
Fig. 7 Time series profiles of cumulative CO2 production during the toxicity tests with azo-dye
Reactive Black 5 samples at different degradation times (regression curves calculated on the
basis of the data obtained between 0 and 44 h).

2
Table 1
Brunnauer–Emmett–Teller characterization and energy band gap value of of various materials.

Catalysts Surface area Pore size Pore volume Band gap

(m2 g-1) (nm) (cm3 g-1) (eV)
ZSM-5 311 2.40 0.194 1.24
7% PTZ 286 2.55 0.183 2.99
12.5% PTZ 261 2.69 0.162 3.02
25% PTZ 209 2.79 0.146 3.06
50% PTZ 205 3.46 0.177 3.10

TiO2 42 16.3 0.170 3.20

3
Table 2, Comparison of the results of different studies on the removal of RB5 dye pollutant.

Catalyst k (min-1) Method Reference

UV/GR/TNT 0.025 photodegradation [16]

UV/TiO2/Feo 1.9 Photodegradation [37]

SEG/ZnO 0.02 Photodegradation [38]

SiO2/Fe3O4/O3/H2O2 1.2 Photodegradation [39]

UV/TiO2/Biofilm 0.035 Photodegradation [40]

UV/H2O2 0.21 Photodegradation [41]

N/TiO2 0.02 Photodegradation [42]

BFO 0.94 Photodegradation [43]

UV/MNP/TiO2 0.047 photodegradation [44]

UV/TiO2/Zeolite 0.57 Photodegradation This study

4
Table 3, Retention time, compound name and molecular structure of intermediate products
analyzed by gas chromatography–mass spectrometry and ion chromatography.

Methods
Residence
CAS# Compounds Formula
Time(min) GC-MS (m/z of main
IC
peak in MS)

4.857a 75-07-0 Acetaldehyde √ 44*, 43 ―

5.517a 57-14-7 1,1-dimethylhydrazine √ 60* ―

6.887a 60-34-4 Methylhydrazine √ 45*, 46 ―

7.918a 503-28-6 Dimethyldiazene √ 43*, 58 ―

25.691a 122-78-1 Phenylacetaldehyde √ 91*, 92 ―

26.321a 625-74-1 2-methyl-1-nitropropane √ 41*, 56, 57 ―

9.597b ― Oxalate ions (COO)2 2− ― √

15.475b ― Sulfate ion S04 2- ― √

4.377b ― Sodium ions Na+ ― √

NDb ― Nitrate ions NO3 - ― ×

NDb ― Nitrite ion NO2 - ― ×

NDb ― Ammonium ion NH3 + ― ×

a
: gas chromatography-mass spectrometry (MS) analysis method.

b
: ion chromatography analysis method.

5
Table 4, Kinetic parameters determined by extrapolating cumulative CO2 production curves.

Culture condition m CO2|∞(mg) β (mg) α(×10-2 h-1) r2

Blank (LB) 210 192.1 1.46 0.97

No.1 (C/C0=1.0) 184 156.6 0.96 0.97

No.2 (C/C0=0.75) 151 140.7 1.54 0.97

No.3 (C/C0=0.5) 140 134.1 1.88 0.98

No.4 (C/C0=0.25) 192 167.8 1.04 0.97

No.5 (C/C0=0.1) 200 180.6 1.24 0.96

a Based on equation: mCO2  mCO2   exp( t )

t 

b Based on “asymptotic condition” of equation:

mCO2 t  mCO2    exp( t );   mCO2 