Design and application of photocatalysts for enhanced ...

Design and application of photocatalysts for enhanced environmental remediation

1

Table of Contents

Chapter 1 General introduction ..................................................................................................... 3

1-1 Background ......................................................................................................................... 3

1-2 Photoelectrochemistry and powdered photocatalyst ........................................................... 6

1-3 Photocatalytic reaction for environmental remediation ...................................................... 9

1-4 Photocatalytic oxidative decomposition of organic compounds ....................................... 11

1-5 Photocatalytic inactivation of pathogens using TiO2 photocatalyst .................................. 13

1-6 Design of photocatalyst showing efficient photocatalytic activity ................................... 15

1-6-1 Efficient charge separation of photocatalyst supported by metal nanoparticles ........ 15

1-6-2 Design of visible-light-driven photocatalyst with narrow band gap .......................... 17

1-7 The correlation between photocatalytic activity and light intensity ................................. 20

1-8 Dissertation outline ........................................................................................................... 22

Chapter 2 Photocatalytic environmental remediation with TiO2 hollow photocatalyst .............. 30

2-1 Different hollow and spherical TiO2 morphologies have distinct activities for the

photocatalytic inactivation of chemical and biological agents ................................................ 30

2-1-1 Introduction ............................................................................................................... 32

2-1-2 Results and discussion ............................................................................................... 34

2-1-3 Conclusions ............................................................................................................... 46

2-1-4 Experimental section ................................................................................................. 47

2-2 Fabrication of efficient visible light responsive TiO2-WO3 hollow particles photocatalyst

by electrospray method ........................................................................................................... 55

2-2-1 Introduction ............................................................................................................... 56


2-2-3 Conclusions ............................................................................................................... 62


2

Chapter 3 Photocatalytic environmental remediation with Rh-doped SrTiO3 under visible light

irradiation .................................................................................................................................... 68

3-1 Photocatalytic degradation of gaseous acetaldehyde over ground Rh-doped SrTiO3 and

Rh-Sb co-doped SrTiO3 under visible light irradiation ........................................................... 68

3-1-1 Introduction ............................................................................................................... 70


3-1-3 Conclusions ............................................................................................................... 91


3-2 Selective inactivation of bacteriophage using visible light-driven Rh-doped SrTiO3

photocatalyst ........................................................................................................................... 98

3-2-1 Introduction ............................................................................................................. 100

3-2-2 Results and discussion ............................................................................................. 102

3-2-3 Conclusions ............................................................................................................. 119

3-2-4 Experimental section ............................................................................................... 120

Chapter 4 Sporicidal performance induced by photocatalytic production of organic peroxide

under visible light irradiation .................................................................................................... 129

4-1 Introduction ..................................................................................................................... 130

4-2 Results and discussion .................................................................................................... 131

4-3 Conclusions ..................................................................................................................... 148

4-4 Experimental section ....................................................................................................... 149

Chapter 5 Summary ................................................................................................................... 155

Publication list ........................................................................................................................... 157

Additional research products ................................................................................................. 158

Acknowledgements ................................................................................................................... 159

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Chapter 1 General introduction 1-1 Background

The pollutant of air and water is the global problem, which is harmful for human

health. The volatile organic compounds in air and the organic compounds in water

should be eliminated. The filter and oxidants are utilized for the removals of organic

compounds. However, they have the problems of difficulty of reuse. In addition, while

the oxidants have been generally used for the sterilization of bacteria and elimination of

organic compounds in the present environmental remediation, many oxidants possibly

produce the carcinogenic chloro-organic compounds because of chlorine-containing

chemicals.1-10

The photocatalyst has gained much attention as the material of environmental

remediation which does not produce the harmful chemical agent and is able to reuse.

Titanium dioxide (TiO2) has been the most used photocatalyst in the world, leading to

sterilization of bacteria and degradation of organic compounds under light

irradiation.11-13 TiO2 greatly received attention as the attractive photocatalyst, owing to

Honda-Fujishima effect reported in 1972.14 They constructed the electrochemical cell in

which TiO2 electrode was connected with a platinum electrode as shown in Figure 1.

When TiO2 is illuminated with solar light, electrons in valence band being composed of

oxygen 2p orbital were excited to conduction band that is composed of Ti 3d orbital.

The excited electrons reduce water to form hydrogen on a Pt electrode (eq. 1), and holes

oxide water to oxygen on the TiO2 electrode (eq. 2) with much less external bias than

that without illumination. Also, high chemical and physical stability is the great

advantage of TiO2. Although ZnO and CdS showing photocatalytic activity had been

studied, photo-corrosion was induced by self-oxidation under light irradiation (eqs. 3,

4).15-16 This technology is much attractive in terms of converting light energy into

chemical energy, but the quantum efficiency is quite low, which is not still at the stage

of practical use.

4

TiO2 electrode : H2O 1/2 O2 + 2H+ + 2e (1)

Pt electrode : H+ + e H2 (2)

Photocorrosion of CdS : CdS + 2h+ Cd2+ + S (3)

Photocorrosion of ZnO : ZnO + 2h+ Zn2+ + 1/2 O2 (4)

Figure 1. Scheme of Honda-Fujishima effect (Electrochemical cell in which the TiO2

and Pt electrode connected through an external circuit)

On the other hand, in 1980s, decomposition of organic compounds (e.g. chlorinated

aromatics,17 chlorinated aliphatic,18 and olefinic compounds,19 nitrogenous

compounds,20 hydrocarbons,21 carboxylic acids,22 alcohols,23 halocarbons,24 and

heteroatom compounds25) using TiO2 photocatalyst has been in parallel studied in the

world because it shows strong oxidative power.16,26 In addition, the destruction of

bacteria and virus was achieved using TiO2 photocatalyst.27-29 The air purification

device and anti-bacterial film based on TiO2 is now in practical use. However, the recent

market of photocatalyst for environmental remediation remains at the same level.

5

Therefore, it is required to develop more efficient photocatalyst in order to spread the

usage of photocatalyst widely.

6

1-2 Photoelectrochemistry and powdered photocatalyst One of the most important factors to improve photocatalytic reaction is the

inhibition of recombination of excited electrons and holes. Since the recombination rate

is fast when the oxidation and reduction sites are in proximity to each other, it is of great

importance to separate their sites. In the case of semiconductor photo-electrode based

on TiO2, TiO2 electrode is connected with metal counter electrode through external

circuit. TiO2 is n-type semiconductor of which Fermi-level exists just under the edge of

conduction band. When TiO2 was contacted with metal or solution, the electrons

transfer until those Fermi-levels become equivalent and the space-charge layer was

formed on the surface of semiconductor as shown in Figure 2, which is called as

Schottoky-barrier.

Figure 2. Space-charge layer of TiO2 semiconductor electrode.

7

The excited electron transfers to Pt counter electrode through the bulk of TiO2,

while the holes move towards the surface of TiO2, that is, the oxidation and reduction

sites successfully separate in photo-electrode reaction. The efficiency of charge

separation improves with increase of external bias, attributed to the promotion of band

bending of space-charge layer. However, such effective charge separation does not

occur in powdered photocatalytic system because it is difficult to apply the external bias.

In particulate system, it is generally known that space-charge layer is difficult to form

on the surface of semiconductor since the depth of the space-charge layer is calculated

to be larger than a few micrometers, which is larger than the particle size.30 The charge

separation of particulate photocatalyst is more difficult to proceed than semiconductor

electrode, but it occurs to some extent. The excited electrons move towards the dark site,

suggesting that the oxidative and reductive reactions occur on the irradiated and dark

site, respectively, as shown in Figure 3.30 In the case of TiO2 supported Pt metal, the

oxidative and reductive reactions occur on the surface of TiO2 and Pt, respectively, and

the effective charge separation was achieved, which was reported by Bard et al.31

Although the charge separation of particulate photocatalyst is less than that of

semiconductor electrode system, it has advantages of much simpler system, less

expensive and higher efficiency of light absorption.

8

Figure 3. Charge separation of particulate TiO2 photocatalyst.

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1-3 Photocatalytic reaction for environmental remediation When the light which exceeds the band gap is irradiated on the surface of TiO2,

electron in valence band consisting of oxygen orbital of 2p is excited to the conduction

band consisting of titanium orbital of 3d (Figure 4).

Figure 4. Scheme of photocatalytic reaction with TiO2 photocatalyst.

Excited electrons are trapped at titanium atoms on the surface of TiO2 or oxygen

molecules absorbed on the surface, and holes are trapped at the oxygen atoms in the

crystalline lattice near the surface of or at hydroxyl groups on the surface.31 In aerobic

condition, various reactive oxygen species (e.g. O2 , OH , HO2 and O ) are

produced by photocatalytic oxidative and reductive reaction as shown in following

reactions (eqs. 5-9).26,31-33 Presence of those reactive oxygen species is confirmed by a

number of researchers using electron spin resonance (ESR) spectroscopy.32,34-36

O2 + e− + O2 (5)

O2 +H+ HO2 (6)

h+ + OH− OH (7)

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h+ + H2O OH + H+ (8)

h+ + O2 2O (9)

Various organic compounds and pathogens can be degraded by those reactive oxygen

species and direct oxidation of generated holes.

In this dissertation, I describe the photocatalytic oxidative reaction of organic

compounds (such as acetaldehyde, 2-propanol and toluene) and pathogens (such as

bacteria and phage).

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1-4 Photocatalytic oxidative decomposition of organic compounds Photocatalytic oxidative decomposition of acetaldehyde and toluene which are

harmful chemical agents are extensively studied by a number of researchers.

Acetaldehyde and toluene are known as representative volatile organic compound,

which are recognized as origin of the sick building syndrome. Firstly, I describe the

photocatalytic degradation of acetaldehyde with TiO2 photocatalyst in this section. The

hydroxyl groups formed on the surface of TiO2 photocatalyst react with acetaldehyde,

forming carbonyl radicals and acetic acid (eqs. 10-12).37,38

CH3CHO + OH∙ → CH3CO∙ + H2O (10)

CH3CO∙ + OH∙ → CH3COOH (11)

CH3COOH + OH∙ → CO2 + H2O∙ + CH3∙ (12)

Furthermore, superoxide radicals generated on the surface of TiO2 also react with

acetaldehyde as follows (eqs. 12-16).38

CH3CHO + O2−∙ → CH3CO∙ + HO2

− (13)

CH3CHO + HO2− → CH3CO− + H2O2 (14)

H2O2 + CH3CO∙ → CH3COOH + OH∙ (15)

CH3CO− + OH∙ → CH3COOH + e− (16)

Acetic acid is produced as main intermediate of photocatalytic oxidative decomposition

of acetaldehyde with TiO2 photocatalyst and it finally decomposes to carbon dioxide in

following reaction (eqs. 17,18).

CH3CHO + H2O + 2h+ → CH3COOH + 2H+ (17)

CH3COOH + 2H2O + 8h+ → 2CO2 + 8H+ (18)

The total reaction is

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CH3CHO + 3H2O +10h+ → 2CO2 + 10H+ (19)

Next, the photocatalytic degradation of toluene was described as follows. The

reaction of degradation of toluene mainly occurs on two pathways as shown in Figure

5.39-41 When the reactive oxygen species react with methyl group of toluene, it is

oxidized to intermediate compounds such as benzyl alcohol, benzoic aldehyde, benzoic

acid and benzene. In the case of direct reaction with aromatic rings, the intermediate

compounds such as cresol, hydroxyl benzoic aldehyde, hydroxyl methyl phenol and

phenol are produced. In addition, the further photocatalytic oxidative decomposition of

intermediates causes aromatic ring opening, producing aliphatic acids and organic acids.

Finally, toluene is oxidized to final product, carbon dioxide, as well as acetaldehyde.

Figure 5. Proposed pathways of photocatalytic oxidative decomposition of toluene.39-41

13

1-5 Photocatalytic inactivation of pathogens using TiO2 photocatalyst Bacteria can be classified Gram-negative and Gram-positive bacteria, which are

distinguished by their ability to hold back the dye-iodine complex.42 Gram-negative

bacteria has the outer membrane consisting of major components lipopolysaccharide

and thin peptidoglycan layer (ca. 2-3 nm), while the cell wall of Gram-positive bacteria

are composed of thick peptidoglycan layer (ca. 30 nm), polysaccharides, teichoic acid

and teichuronic acid in the absence of outer membrane as shown in Figure 6.42,43

Figure 6. The cell structure of Gram-negative and Gram-positive bacteria.43

Virus is a very small infectious agent (20-1000 nm), whose RNA or DNA is

covered with capsid protein. It cannot grow itself without host, while bacteria can

increase the number of cell itself. The virus that infect to bacteria is called

bacteriophage, which is generally used as photocatalytic inactivation of virus, because it

does not infect to humans. In my research, I used bacteriophage Qβ for photocatalytic

inactivation of virus.

Photocatalytic inactivation of various pathogens is extensively studied by many

researchers. One of great advantages of TiO2 photocatalyst is that broad range of

microorganisms are effectively inactivated by photocatalytic oxidation.44,45 Sunada et al.

revealed that inactivation rate of E. coli, Gram negative bacteria, was observed two-step

decay dynamics, suggesting that the oxidation of outer membrane firstly occurred,

following the oxidation of the inner membrane (cytoplasmic membrane).46 However,

such decay dynamics was not observed with virus because of no outer membrane, which

14

was reported by Nakano et al.47 They also demonstrated that the decomposition of viral

capsid protein was observed by SDS-PAGE analysis, finally leading to viral death.

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1-6 Design of photocatalyst showing efficient photocatalytic activity 1-6-1 Efficient charge separation of photocatalyst supported by metal nanoparticles

Co-catalyst is an effective way to enhance photocatalytic activity, attributed to

promote the charge separation. Abe et al. successfully developed tungsten oxide (WO3),

a representative visible-light-driven photocatalyst, supported by Pt nano-particle

showing high photocatalytic activity.48 They supported Pt nano-particle onto the surface

of WO3 photocatalyst by photo-deposition method. Firstly, Pt ion was dispersed in a

solution containing methanol with WO3 photocatalyst. Next, the solution was irradiated

with visible light in order to excite electrons of WO3, which causes the reduction of Pt

ion on the surface of WO3, leading to the deposition of Pt nano-particles. The decrease

of photocatalytic activity of bare WO3 is observed because of reduction itself (W6+ →

W5+). Pt metal plays an important role of inhibiting self-reduction and the increase of

multi-electron reduction of oxygen (O2 + 2H+ + 2e− → H2O2 ).

In addition, Irie et al. suggested that Cu(Ⅱ) grafting also gives an effective

co-catalyst.49 They fabricated TiO2 and WO3 grafted with Cu(Ⅱ) by reflux method. The

photocatalyst was dispersed with the aqueous solution of CuCl2 and heated at 90 °C.

The samples were obtained after calcination at 650 °C. In the case of TiO2, Cu(Ⅱ)

grafting enhances the single-electron reduction of oxygen, due to direct excitation to the

redox potential of Cu2+/Cu+ as shown in Figure 7, which is called interfacial charge

transfer (IFCT). Cu+ reduced by excited electrons from valence band of TiO2 works as

reduction center of oxygen and forms O2−∙. On the other hand, Cu2+ of WO3 grafted

with Cu(Ⅱ) works as multi-electron reduction center of oxygen. Cu2+ is reduced to Cu0,

and forms hydrogen peroxide (O2 + 2H+ + 2e− → H2O2). The difference of reduction of

oxygen between TiO2 and WO3 is attributed to their potential of conduction band. The

potential of conduction band of TiO2 is more negative than Cu2+/Cu+ redox potential

which is negative than single-electron reduction of oxygen, whereas that of WO3 is

more positive than Cu2+/Cu+ but is more negative than Cu2+/Cu0 and multi-electron

reduction of oxygen.

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Furthermore, Arai et al. successfully fabricated WO3 supported Pd metal by simple

grinding method, which is capable of complete decomposition of volatile organic

compounds (VOCs).50 Pd co-catalyst also effectively proceeds the multi-electron

reduction of oxygen (O2 + 2H+ + 2e− → H2O2) as well as WO3 supported Pt.

As mentioned above, co-catalyst plays an important role of efficient photocatalytic

activity.

Figure 7. Energy diagram of TiO2 and WO3 photocatalysts grafted with Cu(Ⅱ) under

visible light irradiation.49,51

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1-6-2 Design of visible-light-driven photocatalyst with narrow band gap

TiO2 photocatalyst responds to UV light, due to its band gap of ca. 3.2 eV. TiO2 is

difficult to show efficient photocatalytic performance under sunlight or room light

because their major light component is visible light that is weak energy. Thus,

developing of visible light responsive photocatalyst is of great importance to improve

photocatalytic activity. It is necessary to make the band gap narrower for design of

visible-light-driven photocatalyst. So far, various visible-light-driven photocatalysts

have been developed by many researchers using the strategies of doping, formation of

new valence band and solid solution as shown in Figure 8.52

Figure 8. Strategies of design of visible-light-driven photocatalyst (band engineering).52

Doping means that the elements of semiconductor are substituted to foreign

elements. When doped elements form the impurity levels in the forbidden band of

semiconductor, the band gap becomes narrower as shown in Figure 8a. The increase of

doping amounts contribute to strong visible light absorption, whereas the photocatalytic

activity gradually decreased, ascribed to increase the recombination centers. Therefore,

doping amounts should be optimized to develop suitable visible-light-driven

photocatalyst. Nitrogen doped TiO2,53,54 sulfur doped TiO2,55 rhodium doped SrTiO3,56

iridium doped SrTiO3,57 cupper doped ZnS58 and so on have ever been developed as

doped photocatalyst that responds to visible light. In addition, recently, it has been

found that co-doping is also a suitable strategy to enhance photocatalytic activity. For

instance, rhodium doped TiO2 (TiO2:Rh) shows no photocatalytic activity of oxygen

evolution from water containing sacrificial agent while rhodium and antimony co-doped

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TiO2 (TiO2:Rh,Sb) is capable of oxygen evolution. The rhodium in TiO2:Rh are doped

as Rh4+ ions, which works as recombination centers. On the other hand, antimony is

doped as Sb5+ ion after calcination in air, which changes Rh valence state from Rh4+ to

Rh3+ because of charge compensation. Therefore, TiO2:Rh,Sb is effective for the

improvement of the photocatalytic activity.

Figure 8b shows the band engineering of the photocatalyst forming new valence

band. The energy levels of orbitals of Pb, Bi, Sn and Ag (Pb6s, Bi6s, Sn5s and

Ag4d)58-61 form above valence band of O2p orbital, which shows visible light

absorption. BiVO4 has gained much attention as efficient visible-light-driven

photocatalyst of oxygen evolution from water containing sacrificed agent (AgNO3

aqueous solution). The great advantage of BiVO4 is not only showing efficient

photocatalytic activity but simple preparation by an aqueous process at room

temperature.62 The absorption edge of BiVO4 is estimated 2.4 eV and band structure of

BiVO4 is revealed by density functional theory (DFT) calculation, indicating the

formation of valence band by Bi6s orbitals.63 Overall water splitting was achieved by

combination of BiVO4 (oxygen evolution photocatalyst) and Rh doped SrTiO3

(hydrogen evolution photocatalyst) under visible light irradiation, which is called

Z-scheme system as shown in Figure 9. Z scheme system consists of an oxygen

evolution photocatalyst, a hydrogen evolution photocatalyst and an electron mediator,

which is quite similar to photosynthesis of green plants.

19

Figure 9. Z-scheme system consisting of oxygen evolution photocatalyst, hydrogen

evolution photocatalyst and electron mediator.

Solid solution photocatalyst is able to control the energy level of valence band and

conduction band by combination of wide and narrow band gap of semiconductor. Metal

sulfide photocatalysts, such as ZnS and CuS, are used for fabrication of solid solution

photocatalyst. Kudo et al. have successfully developed very efficient solid solution

photocatalyst that responds to visible light.52 In particular, AgInS2-CuInS2-ZnS solid

solution photocatalysts show high photocatalytic performance of hydrogen evolution

from water containing sacrificial agents (S2- and SO32- ions) under visible light

irradiation.64 This photocatalyst has gained much attention because the photocatalytic

activity was higher than that of Pt loaded CdS photocatalyst, which was known as the

visible-light-driven photocatalyst showing high photocatalytic activity for hydrogen

evolution from water containing sacrificial agents (S2- and SO32- ions) under visible light

irradiation.

20

1-7 The correlation between photocatalytic activity and light intensity Ohko et al. reported the correlation between photocatalytic degradation rate of

2-propanol and photon of incident light using TiO2 thin film under very weak UV light

irradiation.65 They described the reason why 2-propanol was selected as a model

reactant in their research as follows. 2-propanol is efficiently decomposed to acetone

that is negligible self-oxidation. In addition, photo-decomposition of 2-propanol to

acetone is involved a single photon in contrast to photo-decomposition of acetaldehyde

that proceeds as a radical-chain reaction, which cannot be accounted for the reaction of

a single photon.

The molecule of 2-propanol reacts with reactive oxygen species that are generated

on the surface of TiO2, producing the molecule of acetone (eqs. 19-22).

CH3CH(OH)CH3 + OH → CH3C (OH)CH3 + H2O (19)

CH3C (OH)CH3 → CH3COCH3 + H+ + e― (20)

CH3C (OH)CH3 + HO2 → CH3COOH(OH)CH3 (21)

CH3COOH(OH)CH3 → CH3COCH3 + H2O2 (22)

Ohko et al. investigated the dependence of the apparent quantum yield for acetone

generation on absorbed photons. As a result, they demonstrated that the apparent

quantum yield was determined by the ratio between the number of adsorbed 2-propanol

molecules on the photocatalyst and the number of absorbed photons of incident light as

shown in Figure10.

21

Figure 10. The correlation between the number of reactant molecules and the number of

photons of incident light.

That is, when the concentration of 2-propanol was low, the number of 2-porpanol

molecules was predominant. Conversely, recombination reaction (eqs. 23,24) proceeded

less efficiently with the decrease of the number of photons, leading to the increase of the

apparent quantum yield for 2-propanol decomposition.

OH + HO2 H2O + O2 (23)

OH + OH H2O2 (24)

Therefore, not only light intensity but the concentration of reactant molecular is of great

importance in photocatalytic reaction.

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1-8 Dissertation outline As mentioned above, many researchers have sought to fabricate efficient

photocatalysts. In Chapter 1, I mentioned about the previous studies on “photocatalysts

supported by metal nanoparticles for improvement of charge separation” and “increase

of efficiency of light absorption by making band gap narrow” as the methods to improve

the photocatalytic activity. In my dissertation, I focused on two topics to improve

photocatatlyic activity, “control of morphology of particle” and “production of

oxidation agent with strong oxidation power”. In particular, I studied “morphology of

particle” and “milling treatment for increasing surface area” in “control of particulate

morphology”. I attempted to improve photocatalytic activity for environmental

remediation using these two concepts.

Chapter 2 is the section focused on “morphology of particle” in control of

morphology of particle. In 2-1 section, I fabricated TiO2 hollow and spherical particles

prepared by combination of electrospray and hydrothermal treatment, developed by my

research group, and investigated the dependence of photocatalytic bactericidal and

anti-phage performance on TiO2 morphologies. In the section 2-2, I prepared

visible-light-driven TiO2-WO3 hollow photocatalyst by combination of electrospray and

hydrothermal treatment and evaluated the photocatalytic oxidative decomposition of

acetaldehyde under visible light irradiation.

Chapter 3 is the section focused on “milling treatment for improvement of surface

area” in control of morphology of particle. In this chapter, I focused on Rh doped

SrTiO3 (STO:Rh) and Rh-Sb co-doped SrTiO3 (STO:Rh,Sb) which have gained much

attention as efficient visible light responsive photocatalysts. I pulverized both

photocatalysts in order to increase the surface area using ball-milling device and

evaluated photocatalytic degradation of acetaldehyde with ground STO:Rh and

STO:Rh,Sb in 3-1 and 3-2 section, respectively. In 3-3 section, anti-pathogenic activity

with both ground photocatalysts was evaluated.

Chapter 4 is the section focused on “production of oxidation agent with strong

oxidation power”. Various bacteria can be sterilized by photocatalytic reaction of TiO2,

23

but it is difficult to inactivate the sporulate bacteria having high resistance. In this

section, I produce organic peroxide, a strong oxidation agent, by photocatalytic reaction,

and expected to inactivate the sporulate bacteria by the chemical agent. Production of

organic peroxide is required to the generation of hydrogen peroxide and organic acid. I

selected WO3 as the visible-light-driven photocatalyst that can possibly produce both

chemical agents, and evaluated the sporicidal performance under visible light

irradiation.

24

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30

Chapter 2 Photocatalytic environmental remediation with TiO2 hollow photocatalyst

2-1 Different hollow and spherical TiO2 morphologies have distinct activities for the photocatalytic inactivation of chemical and biological agents

Abstract The inactivation of Escherichia coli and Qβ phage was examined using their

photocatalytic treatment with TiO2 hollows and spheres that had been prepared by

electrospray, hydrothermal treatment, and calcination. The crystal structures of the

hollows and spheres were assigned to TiO2 anatase, and the surface areas of the hollows

and spheres were determined to be 91 and 79 m2 g−1, respectively. Interestingly, TiO2

spheres exhibited higher anti-pathogen performance than TiO2 hollows. The decrease in

31

anti-pathogen performance treated with hollows were ascribed to the prevention of light

multi-scattering by microorganisms covering the surfaces of the TiO2 hollows. The

photocatalytic decomposition of dimethyl sulfoxide (DMSO) in the presence of TiO2

hollows and spheres was examined in order to study the dependence of photocatalytic

activity on TiO2 morphology for the size scale of the reactants. TiO2 hollows provided

greater photocatalytic decomposition of DMSO than did TiO2 spheres, in contrast to the

pattern seen for pathogen inactivation. Morphology of photocatalyst particles should be

optimized depending on what substance (e.g., organic compound or biological agent) is

being targeted for environmental remediation.

32

2-1-1 Introduction Bacteria and viruses can serve as the causes of food poisoning and infectious

disease; inactivation of these agents is necessary to protect human health. Although

chlorine-containing chemicals are often used as disinfectants, the use of these chemicals

can produce carcinogenic chloro-organic compounds.1–10 Recently, TiO2 photocatalysts,

which are nontoxic, chemically stable, and low cost, have gained much attention as

attractive environmental remediation materials.11–27 Reactive oxygen species (ROS)

such as hydrogen peroxide, superoxide radical, and hydroxyl radicals are generated by

oxidative and reductive reactions of oxygen and water on TiO2 surfaces under UV

irradiation.22,28–32 Bacterial cell membranes and viral capsid proteins are attacked by

ROS, leading to bacterial killing and viral inactivation.33–36 Bacterial killing occurs with

both standard bacteria and drug-resistant microorganisms, making TiO2 photocatalysis

highly appealing. Nakano et al. examined the photocatalytic inactivation, using

TiO2-coated glass, of a number of drug-resistant pathogens, including Gram-positive

bacteria (methicillin-resistant Staphylococcus aureus, vancomycin-resistant

Enterococcus faecalis, and penicillin-resistant Streptococcus pneumoniae),

Gram-negative bacteria (Escherichia coli and multidrug-resistant Pseudomonas

aeruginosa), an enveloped virus (influenza virus), and a nonenveloped virus (feline

calicivirus).37 These researchers demonstrated that TiO2 photocatalysis effectively

inactivates a broad range of microorganisms. TiO2-mediated photocatalysis was

especially effective for the inactivation of Gram-positive bacteria and enveloped virus.

Additional studies have focused on optimizing the photocatalytic performance of TiO2

for applications in environmental remediation. Recent strategies to enhance the

efficiency of TiO2 photocatalysis have included the fabrication of TiO2 in various

morphologies (fibers, spheres, hollows, and so on); the improved photocatalytic

performance of some of these structures is presumed to reflect the freer diffusion of

reactants within the material, resulting in increases in effective surface areas.38–43 Liu et

al. reported that TiO2 hollow materials prepared by electrospray and hydrothermal

treatment showed higher photocatalytic performance in methylene blue degradation

33

than that seen with spheres and fibers, a distinction attributed to multi-scattering inside

the hollow structure and the increased light absorption.42 That report demonstrated that

TiO2 hollows are suitable photocatalysts for decomposition of organic compounds. For

applications in environmental remediation, TiO2 would need to inactivate bacteria and

viruses as well as decompose organic compounds. However, it is not clear whether TiO2

hollows show effective antipathogen performance. To determine whether TiO2 hollows

are a suitable morphology for the inactivation of microorganisms, I investigated the

photocatalytic ina tivation of microorganisms in the presence of TiO2 hollows and

spheres.

34

2-1-2 Results and discussion 2-1-2-1 Preparation of TiO2 photocatalysts

TiO2 hollows and spheres were obtained by electrospray, hydrothermal treatment,

and calcination. In the first step, a PVP–TBT solution was mixed with a small amount

of water to promote TBT hydrolysis, resulting in the formation of a strong TBT 3-D

network. Amorphous TiO2–PVP hollow particles were then obtained due to quick

shrinking of the outer surface compared to the inner surface.42 Hydrothermal treatment

was carried out in order to obtain high crystallinity and a large surface area. I carried out

hydrothermal treatment rather than direct annealing because Liu et al. have previously

shown that directly annealed TiO2 particles exhibited lower photocatalytic performance

than hydrothermally treated samples. Direct annealing resulted in decreased surface area

and pore volume, making it difficult for reactants to infiltrate the TiO2 particles, leading

to an effective decrease in active sites on the surface of these TiO2 particles.42,44 Figure

1 and 2 show field emission scanning electron microscopy and transmission electron

microscopy images of TiO2 hollows and spheres fabricated by hydrothermal treatment

and calcination at 773 K. Imaging confirmed that I had successfully prepared TiO2

hollows and spheres, with a particle size of hollows and spheres of 0.5–2 μm, and

nano-particles (20–80 nm) aggregated together and formed mesoporous structures.

35

Figure 1. Field emission scanning electron microscope (FE-SEM) images of a), c) TiO2

hollows and b), d) TiO2 spheres. c) and d) are provided at increased magnification

compared to a) and b).

36

Figure 2. Transmission electron microscope image of (a and b) TiO2 hollows and (c and

d) TiO2 spheres.

Figure 3 shows pore size distributions of TiO2 hollows and spheres. The pore size

distribution of the TiO2 hollows and spheres ranged from 60 to 90 nm, which does not

indicate the size of hollow. The TiO2 hollows and spheres exhibited surface areas of 91

m2 g−1 and 79 m2 g−1, respectively, demonstrating that these preparations had large

surface areas. Figure 4 shows the diffuse reflectance spectra of TiO2 hollows and

spheres. The two morphologies did not differ in band edges. Figure 5 shows the X-ray

diffraction patterns of TiO2 hollows and spheres after calcination at 773 K. The peaks

observed with both morphologies suggested that in both cases TiO2 assumed an anatase

37

structure.

The crystalline anisotropy seems to be different between TiO2 hollows and spheres

as shown in Figure 2b and 2d. However, the XRD patterns of both TiO2 hollows and

spheres in Figure 5 are quite similar, indicating no difference of crystalline anisotropy of

TiO2 hollows and spheres.

Figure 3. Pore size distributions of TiO2 hollows and spheres (calculated by

Barrett-Joyner-Halenda method).

38

Figure 4. Diffuse reflectance spectra of TiO2 hollows and spheres.

39

Figure 5. X-ray diffraction patterns of a) TiO2 spheres and b) hollows after calcination

at 773 K.

2-1-2-2 TiO2-mediated photocatalytic inactivation of E. coli and Qβ phage

Figure 6 shows the survival rate of E. coli in the presence of TiO2 hollows and

spheres under UV irradiation. In a first control experiment, I confirmed that E. coli was

not inactivated by TiO2 particles under dark conditions, demonstrating that TiO2

particles alone were not bactericidal in the absence of light induction. In a second

control experiment, we investigated the effect of UV irradiation (0.25 mW cm−2) alone,

demonstrating that (in the absence of TiO2) UV light irradiation for up to 120 min was

not bactericidal (bacterial killing of <0.1 log unit). Next, photocatalytic bacterial

inactivation was studied in the presence of TiO2 hollows and spheres under UV light

irradiation for up to 120 minutes. Under these conditions, TiO2 hollows and spheres

showed 1 log reduction and 4 log reduction (respectively) in cell number after 120 min,

40

which indicated that the bactericidal performance of TiO2 spheres was superior to that

of TiO2 hollows. This result is in contrast to that of Liu et al.,42 who reported that

decomposition of methylene blue by TiO2 hollows was superior to that by TiO2 spheres.

In parallel with the evaluation of anti-bacterial activity, I investigated the anti-phage

activity of TiO2 particles in combination with UV illumination. As shown in Figure 7,

illumination with UV (up to 120 min) in the absence of TiO2, or exposure to TiO2

hollows or spheres (without illumination), did not inactivate the Qβ phage. However,

exposure to TiO2 hollows or spheres with illumination yielded an 6 log decrease in

phage density after 120 min. Notably, the rate of phage inactivation with TiO2 spheres

with UV illumination was higher than that with TiO2 hollows, a pattern similar to that

observed for antibacterial performance. It is assumed that the sizes of the targets

(bacteria vs. phage) correlate with the degree of photocatalytic inactivation. In this

context, I note that the length of an E. coli cell is 1–2 μm, a size quite similar to that of

TiO2 particles.

41

Figure 6. Time-dependent photocatalytic inactivation of E. coli in the presence of TiO2

hollows and spheres. The log survival rate of E. coli was defined as log10(100×C/C0). C0,

concentration of E. coli on the control (only UV light irradiation without TiO2) or TiO2

hollows and spheres under UV light irradiation for 0 h; Ct, concentration of E. coli

exposed to UV light irradiation; t, irradiation time. The control sample without TiO2

exposed to 0.25 (×) mW cm-2 UV light irradiation. TiO2 hollows exposed to 0.0 (○) and

0.25 (●) mW cm-2 UV light irradiation. TiO2 spheres exposed to 0.0 (□) and 0.25 (■)

mW cm-2 UV light irradiation. The initial concentration of E. coli was adjusted at

5.0×107 CFU mL-1.

Figure 7. Time-dependent photocatalytic inactivation of Qβ phage in the presence of

TiO2 hollows and spheres. The log survival rate of Qβ phage was defined as

log10(100×C/C0). C0, concentration of Qβ phage on the control (only UV light

irradiation without TiO2) or TiO2 hollows and spheres under UV light irradiation for 0 h;

Ct, concentration of Qβ phage exposed to UV light irradiation; t, irradiation time. The

42

control sample without TiO2 exposed to 0.25 (×) mW cm-2 UV light irradiation. TiO2

hollows exposed to 0.0 (○) and 0.25 (●) mW cm-2 UV light irradiation. TiO2 spheres

exposed to 0.0 (□) and 0.25 (■) mW cm-2 UV light irradiation. The initial concentration

of Qβ phage was adjusted at 5.0×107 PFU mL-1.

As I suggest schematically in Fig. 8, it would be difficult for E. coli to remain in contact

with the surface of a TiO2 hollow because of the presence of a concavity. Furthermore,

since the size of an E. coli cell would be sufficient to cover a concavity, UV light would

be excluded from the inside of the hollow structure. This model would explain why the

degree of bacterial inactivation by TiO2 hollows was much lower than that of TiO2

spheres. In contrast, the Qβ phage is 20–30 nm in size,45 much smaller than E. coli. I

imagine that the phage particles are therefore able to enter the concavity of the hollow

structure. However, the Qβ phage absorbs a broad range of UV light, as shown in Fig. 9,

thereby preventing multi-scattering of UV light inside of the hollows. In addition, Tong

et al. examined the comparison of bactericidal performance (Escherichia coli and

Aeromonas hydrophila) with various TiO2 morphologies (nanotubes, nanorods,

nanosheets, nanospheres and nanopowder: P25).46 As a result, the bacterial inactivation

rates of nanotubes and nanosheets were negligible compared to spherical morphologies.

They proposed that the limited contact area with the bacterial surface led to low

photocatalytic bactericidal performance of nanotubes and nanosheets, which means that

direct contact of TiO2 with bacteria is required for their bacterial inactivation. Gogniat et

al. also reported the importance of the contact area between TiO2 particles and

bacteria.47

In my study, the inactivation of E. coli with TiO2 hollows indicated lower

bactericidal performance than that with spheres. The size of bacteria is larger than that

of TiO2 hollows, which suggested that bacteria could not come into contact with the

concavity of the hollow structure. On the other hand, the size of Qβ phage is much

smaller than E. coli. It is easy to enter the concavity of the hollow structure, in which

Qβ phage can come into contact with TiO2 surfaces. It is therefore that the inactivation

43

rate of the Qβ phage was comparable with hollows and spheres. My results further

suggested the great importance of considering the contact area between TiO2 particles

and reactants in order to effectively inactivate pathogens. This model suggests that TiO2

hollows are not a suitable morphology for the inactivation of microorganisms on this

scale of size.

Figure 8. Schematic diagram of light multi-scattering of TiO2 hollows, adsorption of E.

coli, and Qβ phage on TiO2 hollows, respectively.

Figure 9. UV-Vis absorption spectra of (a) dimethyl sulfoxide (DMSO) and (b) Qβ

phage, plotted along with (c) wavelength distribution of the UV light source.

44

2-1-2-3 Photocatalytic decomposition of dimethyl sulfoxide (DMSO) upon UV

illumination of TiO2 particles

To provide an example of chemical degradation (rather than biological inactivation)

by TiO2 particles, I investigated the photocatalytic decomposition of DMSO. This

compound was selected in part because DMSO does not absorb in the range of UV light

wavelengths. Additionally, DMSO is of environmental concern, given that this organic

compound is found among pollutants released by industrial facilities and thus is a target

for environmental remediation. DMSO is oxidized to methanesulfinic acid (MSI),

methanesulfonic acid (MSA), and sulfonic acid (SA) by ROS generated on the surface

of TiO2 photocatalysts as follows (eqs. 1-3).48

(CH3)2SO (DMSO) + OH → CH3S(O)OH (MSI)+ CH3 (1)

CH3S(O)OH (MSI) + OH → CH3S(O)(OH)2 + O2 → CH3S(O)2OH (MSA)+ HO2 (2)

CH3S(O)2OH (MSA) + OH → H2SO4 (SA) + CH3 (3)

Figure 10 shows the individual concentrations of MSI, MSA, and SA, as well as the

total production (the sum of the concentrations) of these compounds following exposure

to TiO2 hollows or spheres and UV irradiation. In our experiment, the primary product

was MSA; MSI and SA were detected at lower levels. MSI is the first product of

oxidation of DMSO and readily decomposes to MSA. On the other hand, SA is the final

product of oxidation of DMSO and is generated only with difficulty. Therefore, MSA

was the primary product in our experiment. The production rate of MSA + SA with TiO2

hollows was higher than that with spheres under UV irradiation. The UV-Vis absorption

spectrum of DMSO, as shown in Figure 9, indicated that DMSO does not absorb the

light in the wavelengths of UV; that is, multi-scattering inside TiO2 hollows was not

prevented, consistent with the superior photocatalytic performance of this morphology.

This observation is consistent with previous work indicating that TiO2 hollows exhibit

high photocatalytic performance in chemical decomposition due to multi-scattering

inside the hollow structure.42,43,49,50 In contrast, our research revealed that TiO2 hollows

45

are not suitable for the inactivation of microorganisms. Thus, our work suggested that I

need to design TiO2 photocatalysts with consideration of which reactant is being

targeted for environmental remediation.

Figure 10. Concentration of products (MSA, methanesulfonic acid; SA, sulfonic acid;

Total, total concentration of MSA and SA) by photocatalytic decomposition of dimethyl

sulfoxide (DMSO) in the presence of TiO2 hollows and spheres. Photocatalyst: 5 mg;

light source: 10 W black light (0.85 mW cm-2); volume: 40 mL DMSO aqueous solution

(10 ppm).

46

2-1-3 Conclusions The photocatalytic inactivation rate of E. coli in the presence of TiO2 spheres was

higher than that observed in the presence of TiO2 hollows; I hypothesize that this

difference is due to the large size of E. coli. The size of E. coli is 1–2 μm, a size that is

quite similar to TiO2 particles. Specifically, when E. coli adsorbs to the surface of TiO2

hollows, the cells have difficulty remaining in contact with the TiO2 particles, resulting

in decreased contact efficiency. In the case of inactivation of the Qβ phage, TiO2 spheres

also exhibited superior inactivation of the phage under UV irradiation. I hypothesize

that Qβ phages are small enough to enter the concavities of TiO2 hollows. However,

since the Qβ phage absorbs light in the wavelength of UV, phage particles prevent the

multi-scattering of UV light inside of the hollow structure, resulting in impaired

promotion of photocatalytic activity. On the other hand, UV-illuminated TiO2 hollows

showed a higher rate of DMSO decomposition than did illuminated spheres. I

hypothesize that multi-scattering inside TiO2 hollows could occur because DMSO does

not absorb in the wavelength of the light source. Our findings indicated that the

fabrication of photocatalytic TiO2 materials should take into account the size scale of

the reactants being targeted.

47

2-1-4 Experimental section Preparation of TiO2 hollow particles

0.3 g polyvinylpyrrolidone (PVP) K30 (Wako; Mw: 25000) was dissolved in 2.8

mL ethanol and 3.5 mL acetic acid with magnetic stirring for 10 min. Tetrabutyl titanate

(TBT) monomer and 200 μL ultra-pure water were added to the PVP solution with

stirring for 1 h to obtain PVP–TBT yellow sols. After stirring for 1 h, PVP–TBT sols

were transferred to a syringe and loaded on the syringe pump. The flow rate was set at

1.0 mL h−1, and a metal target was placed on the opposite side of the syringe pump at 15

cm distance. The needle in which the solution flowed was applied at 10 kV to form a

stable Taylor cone. The obtained powder was collected and dried overnight at room

temperature in air. Dried powder (0.2 g), ultra-pure water (20 mL), and ethanol (10 mL)

were combined in an autoclavable vessel and treated hydrothermally at 433 K for 24 h.

After hydrothermal treatment, the treated powder was sintered at 773 K for 2 h in order

to obtain high crystallinity.

Preparation of TiO2 sphere particles

0.3 g polyvinylpyrrolidone (PVP) K30 (Wako; Mw: 25000) was dissolved in 2.8

mL ethanol and 3.5 mL acetic acid with magnetic stirring for 10 min. Tetrabutyl titanate

(TBT) monomer was added to the PVP solution with stirring for 1 h to obtain PVP–TBT

yellow sols. After stirring for 1 h, PVP–TBT sols were transferred to a syringe and

loaded on the syringe pump. The flow rate was set at 1.0 mL h−1, and a metal target was

placed on the opposite side of the syringe pump at 15 cm distance. The needle in which

the solution flowed was applied at 10 kV to form a stable Taylor cone. The obtained

powder was collected and dried overnight at room temperature in air. Dried powder (0.2

g), ultra-pure water (20 mL), and ethanol (10 mL) were combined in an autoclavable

vessel and treated hydrothermally at 433 K for 24 h. After hydrothermal treatment, the

treated powder was sintered at 773 K for 2 h in order to obtain high crystallinity.

Material characterization

48

The morphologies of TiO2 photocatalysts were checked using a field emission

scanning electron microscope (FE-SEM JEOL-7600F). TiO2 photocatalysts were coated

Au layer (10 nm) by sputtering device (SANYU ELECTRON SC-701HMCII) before

observation. The crystal structures of the TiO2 photocatalysts were determined using

powder X-ray diffraction patterns (RIGAKU UltimaIV with Cu Kα radiation). Diffuse

reflectance spectra of TiO2 photocatalysts and ultraviolet-visible absorption spectra

were obtained using a UV-visible spectrophotometer (JASCO V-670). Diffuse

reflectance spectra were converted from reflection to absorbance using the Kubelka–

Munk formula. Nitrogen adsorption and desorption isotherms were recorded at 77 K

using a gas adsorption and desorption analyzer (Quantachrome Autosorb-3B). The

specific surface areas and pore size distributions of the TiO2 photocatalysts were

calculated by the Brunauer–Emmett–Teller (BET) and Barrett–Joyner–Halenda (BJH)

methods, respectively.

Photocatalytic inactivation of E. coli and Qβ phage with TiO2 photocatalysts

E. coli (IAM12119T) was used in our experiment for the evaluation of

photocatalytic bacterial inactivation. Cells of this strain were incubated in nutrient broth

liquid medium at 37 °C for 16 h. The culture was centrifuged at 3500 rpm for 10 min,

and washed with saline solution. After washing, the pellets were resuspended in saline.

The E. coli suspension was adjusted to a density of 5.0 × 107 colony forming units

(CFU) mL−1 with sterilized water. Qβ phage (NBRC20012) was used for the evaluation

of viral inactivation. Qβ phage infectivity was assayed by plaque formation on the E.

coli strain NBRC106373 using the double agar layer method. Qβ phage and E. coli were

mixed with lysogeny broth media (DifcoTM) and agar (Wako Pure Chemicals) by

adjusting the agar concentration of the bottom layer to 3.5% and the top layer to 0.7% at

37 °C. After the plates were incubated at 37 °C overnight, the top agar layer including

the Qβ phage was collected and eluted in 2 mL nutrient broth (Nissui) at 4 °C overnight.

The solution of Qβ phage was centrifuged at 10000g at 24 °C for 20 min. Then, the

supernatant was collected, filtered (0.20 μm) and stocked. Before evaluation, the Qβ

49

phage was diluted with sterilized water to 5 × 107 plaque forming units (PFU) mL−1.

Photocatalytic inactivation was assessed as follows. TiO2 photocatalysts (5 mg) were

suspended in 50 mL of a suspension of E. coli or Qβ phage. Light illumination was

carried out using a UV black light source at 0.25 mW cm−2. The survival rates of E. coli

and Qβ phage were estimated by counting the number of colonies and plaques formed

(respectively).

Photocatalytic decomposition of dimethyl sulfoxide (DMSO) with TiO2 photocatalysts

TiO2 photocatalysts (5 mg) were dispersed with 5 mL ethanol and dried at 373 K

for 1 h to form a coating on the bottom of a glass Petri dish (Φ: 80 mm). The glass Petri

dish was then filled with 40 mL of a 10 ppm DMSO aqueous solution. The

photocatalytic evaluation was conducted under 0.85 mW cm−2 UV irradiation (Toshiba

FL10BLB). The concentrations of methanesulfinic acid (MSI), methanesulfonic acid

(MSA), and sulfonic acid (SA) produced by decomposition of DMSO were measured

using an ion chromatograph equipped with a conductivity detector (Shimadzu

CDD-10A VP) and a column (Shimadzu Shim-pack IC-SA2) at 303 K. The mobile

phase was a mixed solvent of sodium hydrogen carbonate and sodium carbonate

aqueous solution (0.14% NHCO3 and 0.19% Na2CO3) at 1.0 mL min−1.

Measurements of UV-Vis spectra of Qβ phage

A mixed solution of Qβ phage (1 mL) and E. coli strain NBRC106373 (0.1 mL)

was added to a tube with soft LB growth medium (3 mL) and immediately poured onto

LB monolayer in a plate. After the resulting solution of Qβ phage and E. coli was

incubated overnight at 310 K, the top agar layer containing Qβ phage was collected and

eluted with liquid medium (2 mL) at 277 K overnight. The mixture was centrifuged

(10000g, 277 K, 20 min), and the supernatant was collected and filtered using a 0.20

mm filter (Advantec). The absorbance at 250–450 nm of purified Qβ phage (5 × 1010

PFU mL−1) was measured using a UV-Vis spectrometer (Hach DR 5000

spectrophotometer).

50

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55

2-2 Fabrication of efficient visible light responsive TiO2-WO3 hollow particles photocatalyst by electrospray method

Abstract

A visible light responsive TiO2-WO3 hollow particle photocatalyst was

successfully fabricated by an electrospray method, followed by hydrothermal treatment

and calcination at 873 K. The photocatalytic decomposition of acetaldehyde with

pristine TiO2 and TiO2-WO3 hollow particles was evaluated under visible light

irradiation (λ>400 nm). The decomposition of acetaldehyde did not readily proceed with

hollow TiO2 particles under visible light irradiation, whereas the hollow TiO2-WO3

particles showed high photocatalytic activity for the decomposition of acetaldehyde.

56

2-2-1 Introduction Sick building syndrome caused by volatile organic compounds (VOCs) causes

serious health problems. To solve these problems, photocatalyst materials for

environmental purification have been developed by many researchers.1-10 Titanium

dioxide (TiO2) is a representative photocatalyst that exhibits high photocatalytic

performance. Mesoporous TiO2 materials fabricated with large surface areas have been

reported to exhibit high photocatalytic activity.11-14 The preparation of mesoporous

materials primarily involves methods such as direct hydrothermal treatment, sol-gel

reaction and the use of co-polymer templates.11-13,15,16 However, the morphology of the

material is not easily controlled with these methods. Recently, I have fabricated TiO2

photocatalysts with different morphologies, such as spheres, hollow particles and fibers,

by the electrospray method.17-19 In particular, hollow TiO2 particles had a higher activity

for the photocatalytic degradation of methylene blue than the other morphologies, which

is due to multiple scattering of light, resulting in increase of light absorption.18 However,

a TiO2 photocatalyst responds only to UV light, which is limited in practical use inside a

building whose environment is surrounded under visible light originated from light bulb

and fluorescent light etc. Thus, the development of a photocatalyst that responds to

visible light is necessary. TiO2-WO3 composites have been shown to be efficient

visible-light-driven photocatalysts, which is attributed to the high degree of charge

separation.20-23 In this research, visible-light-driven TiO2-WO3 hollow particles were

fabricated by the electrospray method, followed by hydrothermal treatment and

calcination at 873 K. The photocatalytic oxidative decomposition of VOCs under visible

light irradiation (λ>400 nm) was then evaluated over TiO2-WO3 hollow particles and

over pristine TiO2 hollow particle for comparison.

57

2-2-2 Results and discussion A small amount of water was added to the PVP-TTBM solution to obtain hollow

PVP-TiO2 particles by the electrospray process. In this situation, the hydrolysis of

TTBM is promoted by the addition of water and a three dimensional PVP-TTBM

complex is formed.18 When the droplets of PVP-TTBM solution containing ethanol and

acetic acid are formed by the electrospray process, the surface of the droplets are

evaporated and hydrolyzed. The three dimensional PVP-TTBM network has strong

connections, so that the solid surface layer of a droplet is gradually deformed by

shrinking of the inner portion of the droplet during solvent evaporation and TTBM

hydrolysis, which leads to formation of the hollow structure.18

The morphology of the obtained PVP-TiO2 powder was observed using field

emission-scanning electron microscopy (FE-SEM), as shown in Figure 1a. It was

confirmed that the obtained powder particles had hollow structures. TiO2-WO3 hollow

particles were prepared by transferring hollow PVP-TiO2 particles to a Teflon vessel for

hydrothermal treatment with an aqueous solution of ammonium metatungstate hydrate,

followed by calcination at 873 K for 2 h to obtain crystalline structures. After

calcination, the hollow particle structure remained intact, as shown in Figure. 1b. Figure.

1c shows a magnified FE-SEM image of hollow TiO2-WO3 particles, where 20-30 nm

diameter particles are aggregated to form hollow TiO2-WO3 particles.

58

Figure 1. FE-SEM images of (a) TiO2 hollow particles after the electrospray process

and (b) TiO2-WO3 hollow particles after calcination at 873 K. (c) Magnified FE-SEM

image of TiO2-WO3 hollow particles.

59

Diffuse reflectance spectra (DRS) of the pristine TiO2 and TiO2-WO3 hollow

particles are shown in Figure. 2a. The hollow TiO2-WO3 particles have a wider visible

light absorption band than the pristine TiO2 hollow particles, which is attributed to the

narrow bandgap of WO3. Figure. 2b shows X-ray diffraction (XRD) patterns for pristine

TiO2 and TiO2-WO3 hollow particles. The crystal structure of the pristine TiO2 hollow

particles was assigned to the anatase TiO2 phase, and that of the TiO2-WO3 hollow

particles was assigned to both the anatase TiO2 phase and the monoclinic WO3 phase.

Figures. 2c and 2d show a nitrogen adsorption-desorption isotherm and the pore size

distribution for the TiO2-WO3 hollow particles, respectively. The isotherm indicates that

the pores in the TiO2-WO3 hollow particles are cylindrical. Therefore, the

Barrett-Joyner-Halenda (BJH) method was adopted to characterize the pore size

distribution. The results showed that the cylindrical pores mainly had widths of 4-7 nm.

In addition, the surface area of the TiO2-WO3 hollow particles was calculated by the

Brunauer-Emmett-Teller (BET) method to be 54 m2 g−1, which is less than that of 88 m2

g−1 of the pristine TiO2 hollow particles.

60

Figure 2. (a) Diffuse reflectance spectra and (b) XRD patterns for TiO2 and TiO2-WO3

hollow particles, (c) nitrogen adsorption-desorption isotherm and (d) pore size

distribution for TiO2-WO3 hollow particles.

The photocatalytic decomposition of acetaldehyde under visible light irradiation (5

mW cm−2, through a cut-off filter (λ<400 nm)) was investigated over pristine TiO2 and

TiO2-WO3 hollow particles, and the results are shown in Figure 3. The initial

concentration of injected acetaldehyde was 150 ppm. The adsorption of acetaldehyde

under dark conditions was evaluated for 1 h, which indicated that the amount of

acetaldehyde adsorbed on TiO2 hollow particles was higher than that on TiO2-WO3

hollow particles due to the difference in surface area. After visible light irradiation, the

rate for photocatalytic decomposition of acetaldehyde was higher over TiO2-WO3

hollow particles than that of TiO2 hollow particles.

61

Although TiO2 is difficult to decompose acetaldehyde efficiently under visible light due

to its wide band gap, I successfully fabricated visible light responsive heterostructure of

TiO2-WO3. Furthermore, the conduction band level for pristine WO3 is more positive

than that required for multi-electron reduction of oxygen (O2 + 2H+ +2e- = H2O2 (aq),

+0.682 V; O2 + 4H+ + 4e- = 2H2O, +1.23 V), and it is not sufficiently negative for

single-electron reduction of oxygen (O2 + H+ + e- = O2-, -0.56 V; O2 + H+ + e- = HO2,

-0.13 V) to proceed, which leads to the low photocatalytic activity for WO3.24-26

However, heterostructure of TiO2-WO3 contributes high photocatalytic activity due to

charge separation between TiO2 and WO3.21-23. It was clarified that hollow structure of

TiO2 was suitable morphology showing higher photocatalytic performance than various

morphologies such as sphere and fiber, ascribed to the multiple scattering of light.18

Therefore, TiO2-WO3 with hollow morphology should allow superior photocatalytic

activity.

Figure 3. Photocatalytic decomposition of acetaldehyde over TiO2 hollow particles and

TiO2-WO3 hollow particles under visible light irradiation (λ > 400 nm).

62

2-2-3 Conclusions TiO2-WO3 hollow particles were fabricated by an electrospray process, followed by

hydrothermal treatment and calcination. After calcination at 873 K, the hollow

structured particles were not collapsed but remained intact. The hollow particles were

identified as TiO2 anatase and WO3 monoclinic composites by XRD analysis. The

cylindrical pores in TiO2-WO3 have widths of 4-7 nm and a BET surface area of 54 m2

g-1, as measured by nitrogen adsorption-desorption isotherms. Photocatalytic

decomposition of acetaldehyde was evaluated using pristine TiO2 and TiO2-WO3 hollow

particles. Although it was difficult to decompose acetaldehyde to carbon dioxide over

pristine TiO2 hollow particles, the TiO2-WO3 hollow particles exhibited higher

photocatalytic activity for the decomposition of acetaldehyde under visible light (λ>400

nm). This section is expected to contribute to the development of various visible

light-driven hollow particle photocatalysts.

63

2-2-4 Experimental section Preparation of photocatalyst

0.3 g Polyvinylpyrrolidone (PVP, K30, Wako Pure Chemical Industries, Japan) was

dissolved in 2.8 mL of ethanol and 3.5 mL of acetic acid while stirring for 10 min. After

stirring, 4.28 mL of titanium tetrabutoxide monomer (TTBM, Wako Pure Chemical

Industries, Japan) was added to obtain transparent solution of PVP-TTBM. 200 μL of

ultrapure water was added to the PVP-TTBM solution and the resulting solution was

stirred for a further 14 h. The PVP-TTBM solution was then loaded into a syringe

connected with a metal needle and pumped at a flow rate of 1.0 mL h−1. A high voltage

of 15 kV was applied between the needle and a metal target (aluminum) for the

electrospray process. The distance between the needle and target was set at 15 cm. The

electrospray process was performed for several hours, and hollow PVP-TiO2 particles

were collected and dried at room temperature for 24 h. Ammonium metatungstate

hydrate (Stream Chemicals, USA) as a tungsten source was dissolved in 20 mL of

ultrapure water and 10 mL of ethanol while stirring for 10 min, to which 0.6 g of hollow

PVP-TiO2 particles was added. The solution was transferred into a 50 mL Teflon lined

autoclave and heated at 473 K for 24 h. After this hydrothermal treatment, the resultant

powder was washed three times with ethanol and then collected. The obtained powder

was then calcined at 873 K for 2 h to form hollow TiO2-WO3 particles.

Material characterization

The morphologies of photocatalysts were checked using a field emission scanning

electron microscope (FE-SEM JEOL-7600F). TiO2-WO3 and TiO2 photocatalysts were

coated Au layer (10 nm) by sputtering device (SANYU ELECTRON SC-701HMCII)

before observation. The crystal structures of photocatalysts were determined using

powder X-ray diffraction patterns (RIGAKU UltimaIV with Cu Kα radiation). Diffuse

reflectance spectra were obtained using a UV-visible spectrophotometer (JASCO V-670).

Diffuse reflectance spectra were converted from reflection to absorbance using the

Kubelka–Munk formula. Nitrogen adsorption and desorption isotherms were recorded at

64

77 K using a gas adsorption and desorption analyzer (Quantachrome Autosorb-3B). The

specific surface areas and pore size distributions of the TiO2 photocatalysts were

calculated by the Brunauer–Emmett–Teller (BET) and Barrett–Joyner–Halenda (BJH)

methods, respectively.

Evaluation of photocatalytic degradation of acetaldehyde.

A glass petri dish was covered with photocatalyst (0.1 g) in a closed glass reactor

(500 mL). The photocatalyst was irradiated at 5 mW cm−2 light intensity through a

cutoff filter (λ<400 nm) using a 200 W Xe lamp. The initial concentration of

acetaldehyde was adjusted at 150 ppm in a closed glass reactor. The concentration of

acetaldehyde and carbon dioxide were measured using a gas chromatograph (Shimadzu:

GC-2014) with a flame ionization detector (FID) equipped with a methanizer

(Shimadzu; MTN-1). The detail condition of gas chromatograph is shown as follows:

column (4.0 m × 3.0 mm), column temperature (100 oC), mobile phase (nitorgen), flow

rate (30 mL min-1).

65

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P., Enhanced Photocatalytic Performance of Brookite TiO2 Macroporous Particles

Prepared by Spray Drying with Colloidal Templating. Adv. Mater. 2007, 19 (10),

1408-1412.

16. Smarsly, B.; Grosso, D.; Brezesinski, T.; Pinna, N.; Boissière, C.; Antonietti, M.;

Sanchez, C., Highly Crystalline Cubic Mesoporous TiO2 with 10-nm Pore Diameter

Made with a New Block Copolymer Template. Chem. Mater. 2004, 16 (15), 2948-2952.


Fujishima, A., Mesoporous TiO2 Core–Shell Spheres Composed of Nanocrystals with

Exposed High-Energy Facets: Facile Synthesis and Formation Mechanism. Langmuir

2011, 27 (13), 8500-8508.

18. Liu, B.; Zhao, X.; Nakata, K.; Fujishima, A., Construction of hierarchical titanium

dioxide nanomaterials by tuning the structure of polyvinylpyrrolidone-titanium butoxide

complexes from 2- to 3-dimensional. J. Mater. Chem. A 2013, 1 (16), 4993-5000.


Fujishima, A., Hierarchical TiO2 spherical nanostructures with tunable pore size, pore

volume, and specific surface area: facile preparation and high-photocatalytic

performance. Catal. Sci. Technol. 2012, 2 (9), 1933-1939.

20. Liu, K.-I.; Hsueh, Y.-C.; Chen, H.-S.; Perng, T.-P., Mesoporous TiO2/WO3 hollow

67

fibers with interior interconnected nanotubes for photocatalytic application. J. Mater.

Chem. A 2014, 2 (15), 5387-5393.

21. Bai, S.; Liu, H.; Sun, J.; Tian, Y.; Chen, S.; Song, J.; Luo, R.; Li, D.; Chen, A.; Liu,

C.-C., Improvement of TiO2 photocatalytic properties under visible light by WO3/TiO2

and MoO3/TiO2 composites. Appl. Surf. Sci. 2015, 338, 61-68.

22. Pan, J. H.; Lee, W. I., Preparation of Highly Ordered Cubic Mesoporous WO3/TiO2

Films and Their Photocatalytic Properties. Chem. Mater. 2006, 18 (3), 847-853.

23. Miyauchi, M.; Nakajima, A.; Watanabe, T.; Hashimoto, K., Photoinduced

Hydrophilic Conversion of TiO2/WO3 Layered Thin Films. Chem. Mater. 2002, 14 (11),

4714-4720.







26. Nakata, K.; Liu, B.; Goto, Y.; Ochiai, T.; Sakai, M.; Sakai, H.; Murakami, T.; Abe,

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WO3 Nanoparticles. Chem. Lett. 2011, 40 (10), 1161-1162.

68

Chapter 3 Photocatalytic environmental remediation with Rh-doped SrTiO3 under visible light irradiation

3-1 Photocatalytic degradation of gaseous acetaldehyde over ground Rh-doped SrTiO3 and Rh-Sb co-doped SrTiO3 under visible light irradiation

Abstract Rh doped SrTiO3 (STO:Rh), a unique photocatalyst producing hydrogen in

photocatalytic water splitting under visible light irradiation, was examined for the

degradation of acetaldehyde as a volatile organic compounds under visible light

irradiation. Pristine STO:Rh showed slow degradation of acetaldehyde, attributed to low

surface area (2.6 m2 g−1). After ball milling for 60 min, particle size of STO:Rh was

drastically decreased and the surface area increased significantly (46 m2 g−1).

Degradation rate of acetaldehyde over ground STO:Rh showed much higher values than

that over pristine STO:Rh. It is indicated that the surface area of photocatalyst is a quite

important factor for degradation of acetaldehyde. In order to investigate the carbon mass

69

balance of injected acetaldehyde, I evaluated the amounts of formed carbon dioxide and

acetic acid. As a result, the amounts of carbon in formed carbon dioxide and acetic acid

were 0.517 and 3.34 μmol. Therefore, the total amounts of carbon in carbon dioxide and

acetic acid were 3.86 μmol. The amounts of carbon in injected acetaldehyde was 3.88

μmol. It is thus that mass balance of carbon was comparable. It is interestingly shown

that ground STO:Rh can selectively produce acetic acid by photocatalytic

decomposition of acetaldehyde. The predominant production of acetic acid is possibly

due to the stabilization of acetic acid on the basic surface of STO:Rh and/or the

insufficient oxidation potential of holes for complete oxidation of CO2.

In addition, The photocatalytic degradation of gaseous acetaldehyde under visible

light was examined with Rh-Sb-co-doped SrTiO3 (denoted as STO:Rh,Sb) synthesized

by solid state reaction. The obtained photocatalyst was pulverized at 800 rpm by

ball-milling to increase the surface area. Ground STO:Rh,Sb drastically exhibited higher

photocatalytic degradation rate of acetaldehyde, whereas that of pristine STO:Rh,Sb

was negligible. The optimum grinding time for photocatalytic degradation of

acetaldehyde was investigated, suggesting that STO:Rh,Sb grounded for 60 min showed

the highest photocatalytic performance. Although the increase of pulverizing time

contributed to larger surface area, the crystallinity of STO:Rh,Sb was descended,

resulting in fast recombination between photogenerated electrons and holes. In addition,

I revealed that the optimum doping amounts of Rh and Sb was STO:Rh(1%),Sb(1%).

Ground STO:Rh(1%),Sb(1%) for 60 min showed the highest photocatalytic

performance, and it was clarified that the photocatalytic performance was quite stable

for 3 cycles, suggesting that it is reusable. I demonstrated that ground STO:Rh,Sb was a

suitable visible-light-driven photocatalyst for degradation of acetaldehyde.

70

3-1-1 Introduction Photocatalytic reactions under light irradiation have gained much attention not only

for water splitting for hydrogen fuel production, but also for air and water cleaning.1-8

TiO2 is a material that performs photocataltyic reactions under ultraviolet (UV) light

irradiation. However, only 3% of the solar spectrum is composed of UV light. In order

to accelerate photocatalytic reactions, the whole range of solar light including visible

(VIS) should be accessed. Therefore, it is important to develop materials that respond to

VIS light, comprising 42% of sunlight. The band alignment of doping photocatalyst is

an efficient strategy for reducing band gap, showing visible light responsive

photocatalytic performance. Many visible-light-driven photocatalysts have been

developed, such as Bi2WO6, Bi2MoO6, Ag3VO4, and BiVO4, which have demonstrated

oxygen evolution from water.9-14 Also, Numerous metal doping SrTiO3 (Cr-, Ni-, Ir-,

Mn-, Ru-doped SrTiO3)15-20 photocatalysts were developed, which showed

photocatalytic performance under visible light irradiation. In recent years, Kudo et al.

reported a unique p-type photocatalyst, rhodium-doped SrTiO3 (STO:Rh), that was

active for hydrogen evolution from water under VIS light irradiation.18, 21 In addition,

BiVO4/STO:Rh composites and photoelectronchemical cells can accomplish water

splitting without an external bias under VIS light irradiation.22, 23 Thus, STO:Rh is

available for water splitting as a visible-light-driven photocatalyst. In addition, Kudo et

al. developed rhodium-antimony co-doped SrTiO3 (STO:Rh,Sb). STO:Rh,Sb can

produce not only hydrogen but oxygen from water containing a sacrificial reagent24, 25

under VIS light irradiation in contrast to STO:Rh. The Rh valence state in STO:Rh,Sb

can be stabilized as Rh3+ by doping with Sb because antimony can be oxidized to Sb5+,

leading to inhibition of the recombination center of Rh4+.26

As mentioned above, STO:Rh and STO:Rh,Sb photocatalysts are active for

photocatatlyic water splitting under VIS light irradiation. However, there have been no

reports on the capability of STO:Rh for decomposition organic compounds for

environmental purification.

In this work, I evaluate the degradation of acetaldehyde, a volatile organic

71

compounds recognized as an origin of the sick building syndrome, using STO:Rh and

STO:Rh,Sb under VIS irradiation. Furthermore, I examined the effect of grinding those

photocatalysts by ball milling on their activities for acetaldehyde degradation.

72

3-1-2 Results and discussion The samples are denoted STO:Rh(y%) where y% refer to the amount of doped Rh.

Figure 1 shows the X-ray diffraction (XRD) patterns for pristine and ground

STO:Rh(1.0%). The peaks of the pristine STO:Rh(1.0%) were assigned to cubic SrTiO3.

On the other hand, the impurity peaks assigned to SrCO3 were observed, which is

ascribed to the reaction between SrTiO3 and atmospheric CO2.27 After grinding, the

crystal phase of STO:Rh(1.0%) was not changed while the full width at half-maximum

of the peaks was broadened because the crystalline size was decreased by the

ball-milling process.

Figure 1. XRD patterns of (A) pristine and (B) STO:Rh(1.0%) grounded for 60 min.

The samples are denoted STO:Rh(y%),Sb(z%), where y% and z% refer to the

amounts of doped Rh and Sb, respectively. The XRD patterns of pristine and ground

STO:Rh(1.0%),Sb(1.0%) were assigned to cubic STO, as shown in Figure 2A. A peak

73

around 25° and 36.5° that was inconsistent with the peaks of STO appeared as the

milling time was increased. The peak was attributed to anatase TiO2 and SrCO3,

respectively. The crystalline phase transformation of STO to anatase TiO2 and SrCO3 by

mechanical grinding using a ball-milling device has been reported, which may be

attributed to a reaction between the SrTiO3 and atmospheric CO2.27 Additionally, the

STO peaks were broadened by the grinding process, as shown in Figure 2B. The longer

the grinding time was, the higher the value of the full width at half maximum (FWHM)

of the STO was, indicating that crystallinity of STO was descended by pulverization.

74

Figure 2. A: X-ray diffraction patterns of (a) cubic SrTiO3 (JSPDS No. 01-073-0661),

(b) pristine STO:Rh(1.0%),Sb(1.0%), (c) grounded for 30 min, (d) 60 min and (e) 90

75

min.(*: anatase TiO2 and ●: SrCO3), B: The enlarged peaks of STO:Rh(1.0%),Sb(1.0%)

grounded for 30, 60 and 90 min.

The field emission scanning electron microscope (FE-SEM) images for pristine and

ground STO:Rh(1.0%) were shown in Figure 3. I estimated the specific surface area

from nitrogen adsorption at 77 K, using the Brunauer-Emmett-Teller (BET) formula.

The small surface area (2.6 m2 g−1) and the large particle size (3-5 μm) for pristine

STO:Rh(1.0%) were attributed to sintering during high temperature calcination. After

ball milling for 30 and 60 min, the surface areas increased significantly to 24 and 46 m2

g−1, respectively, while the particle size decreased to 100-500 nm.

76

Figure 3. FE-SEM images of (A) pristine, (B) STO:Rh(1.0%) grounded for 30 min, and

(C) STO:Rh(1.0%) grounded for 60 min.

77

Figure 4 shows the FE-SEM images of pristine STO:Rh(1.0%),Sb(1.0%) and

STO:Rh(1.0%),Sb(1.0%) ground for 30, 60, and 90 min at 800 rpm. The pristine

STO:Rh(1.0%),Sb(1.0%) had a large particle size (ca. 1–3 μm) and its surface area was

2.0 m2 g−1, ascribed to its high temperature sintering. Ball-milling successfully reduced

the particle size of STO:Rh(1.0%),Sb(1.0%). The surface areas of

STO:Rh(1.0%),Sb(1.0%) ground for 30, 60, and 90 min drastically increased to 30, 42,

and 47 m2 g−1, respectively.

Figure 4. FE-SEM images and surface areas of a) pristine STO:Rh(1.0%),Sb(1.0%) and

STO:Rh(1.0%),Sb(1.0%) b) ground for 30 min, c) 60 min, and d) 90 min at 800 rpm.

78

Diffuse reflectance spectra of ground STO:Rh(1.0%) are shown in Figure 5. The

absorption at 580 nm was caused by electronic transition from the valence band of STO

to an unoccupied d state of Rh4+, and the absorption at 420 nm was ascribed to

electronic transition from an occupied state of Rh3+ to the conduction band of STO.18, 28

The absorption related to Rh4+ of STO:Rh more strongly appeared than the absorption

related to Rh3+. The absorption around 580 nm decreased after ball milling, possibly due

to the reduction of Rh4+ to Rh3+ induced by oxygen defects during ball milling to

compensate the charge valance.29 It was suggested with X-ray photoelectron spectra

(XPS) as shown in Figure 6.

Figure 5. Diffuse reflectance spectra of (A) pristine, (B) STO:Rh(1.0%) grounded for

60 min and (C) STO:Rh grounded for 30 min.

79

Figure 6. X-ray photoelectron spectra of Rh value of (A) pristine and (B)

STO:Rh(1.0%) grounded for 60 min.

Diffuse reflectance spectra of ground STO:Rh(1.0%),Sb(1.0%) are shown in Figure

7. The spectra confirmed that all of the samples absorbed visible light. The absorption

related to Rh4+ of STO:Rh more strongly appeared than the absorption related to Rh3+,

whereas the absorption related to Rh4+ of STO:Rh,Sb was decreased and the absorption

related to Rh3+ increased. Rh atoms in STO:Rh were doped at Ti4+ sites as Rh4+ to

compensate the charge balance.18, 21 In contrast, Rh4+ in STO:Rh,Sb was reduced to

Rh3+ owing to the presence of Sb that was oxidized to Sb5+ during the calcination in air.

80

Figure 7. Diffuse reflectance spectra of (a) pristine STO:Rh(1.0%), (b) pristine

STO:Rh(1.0%),Sb(1.0%) and that (c) ground for 30 min, (d) 60 min, and (e) 90 min.

I studied the photocatalytic degradation of acetaldehyde by pristine and ground

STO:Rh(1.0%) under visible light irradiation (λ>420 nm), as shown in Figure 8. After

equilibrium in the dark condition for 2 h, the samples were illuminated by visible light

(100 mW cm-2) through an L-42 filter (HOYA; λ<420 nm) from a 200 W Xe lamp. The

initiate concentration of acetaldehyde (85 ppm) slightly decreased on pristine

STO:Rh(1.0%), which indicated low photocatalytic activity. On the other hand, it

drastically decreased on ground STO:Rh(1.0%). STO:Rh(1.0%) ground for 60 min

showed higher photocatalytic activity than STO:Rh(1.0%) ground for 30 minutes. It is

indicated that the difference in the degradation rate of acetaldehyde between pristine

and ground STO:Rh(1.0%) is attributed to the high specific surface area. Furthermore, I

investigated the amounts of formed carbon dioxide and acetic acid using gas

chromatography and HPLC, respectively. As a results, the amounts of formed carbon

81

dioxide and acetic acid were 0.517 and 1.67 μmol, respectively. The amounts of carbon

in acetic acid was 3.34 μmol (1.67 μmol × 2). Thus, the total amounts of carbon in

carbon dioxide and acetic acid were 3.86 μmol. The initial concentration of

acetaldehyde was 85 ppm, that is, the amount of carbon was calculated to be 3.88 μmol,

indicating that the mass balance of carbon was comparable. It is shown that ground

STO:Rh(1.0%) can selectively produce acetic acid by photocatalytic decomposition of

acetaldehyde. Acetic acid was mainly detected rather than carbon dioxide as an

oxidation product of acetaldehyde. The predominant production of acetic acid is

possibly due to the stabilization of acetic acid on the basic surface of STO:Rh(1.0%)30

and/or the insufficient oxidation potential of holes for complete oxidation to carbon

dioxide. Produced acetic acid easily adsorbed on the surface of ground STO:Rh(1.0%),

which is due to the basic surface of STO.

Figure 8. Photocatalytic degradation of acetaldehyde on (A) pristine, (B)

82

STO:Rh(1.0%) grounded for 30 min, and (C) STO:Rh(1.0%) grounded for 60 min under

visible light irradiation (λ > 420 nm). Photocatalyst: 0.5 g, light source: 200 W Xe lamp

with L-42 cutoff filter, gas phase volume: 500 mL.

In order to clarify the stability of ground STO:Rh(1.0%), I carried out the

photocatalytic evaluation for 5 cycles over STO:Rh(1.0%) grounded for 60 min as

shown in Figure 9. It is observed that the decomposition rate of acetaldehyde gradually

decreased, ascribed to be produced acetic acid on the surface of ground STO:Rh(1.0%)

by decomoposition of acetaldehyde. Hence, it is assumed that the photocatalytic

decomposition of acetaldehyde over ground STO:Rh(1.0%) could not be proceeded for

several cycles. Then, I washed the photocatalyst to remove acetic acid adsorbed on the

surface of ground STO:Rh(1.0%) after photocatalytic reaction and I investigated the

photocataytic activity for 5 cycles again. It was clarified that the photocatalytic reaction

proceeded quite stable for 5 cycles as shown in Figure 10, suggesting that ground

STO:Rh(1.0%) is reusable.

83

Figure 9. Photocatalytic degradation of acetaldehyde on ground STO:Rh for 60 min

under visible light irradiation (λ > 420 nm). Photocatalyst: 0.5 g, light source: 200 W Xe

lamp with L-42 cutoff filter, gas phase volume: 500 mL.

0

20

40

60

80

100

0 5 10 15 20

Con

cent

ratio

n of

ace

tald

ehyd

e / p

pm

Irradiation time / hour

84

Figure 10. Photocatalytic degradation of acetaldehyde on ground STO:Rh for 60 min

washed with ultrapure water under visible light irradiation (λ > 420 nm). Photocatalyst:

0.5 g, light source: 200 W Xe lamp with L-42 cutoff filter, gas phase volume: 500 mL.

Moreover, I estimated the turnover number (TON) of reacted acetaldehyde to

doped Rh [eq. 1] .

TON= molar quantities of reacted acetaldehyde

molar quantities of Rh (1)

The TON was 2.2, indicating that the degradation of acetaldehyde proceeded

photocatalytically, accompanied with electronic excitation from Rh3+ of an impurity

level in a band gap to the conduction band of SrTiO3 by visible light illumination. The

conduction band level of STO:Rh(1.0%) is ca. -0.2 V vs. NHE, while the electron donor

level in which holes are generated is around 2.2 V.18 Therefore, oxygen reduction

0

20

40

60

80

100

0 5 10 15 20

Con

cent

ratio

n of

ace

tald

ehyd

e / p

pm

Irradiation time / hour

85

(O2/HO2 = −0.13 V vs. NHE, O2/H2O2 = +0.68 V vs. NHE, O2/H2O = +1.23 V vs.

NHE)31 and acetaldehyde oxidation should simultaneously proceed on the surface of

STO:Rh(1.0%).

Next, I investigated the photocatalytic degradation of acetaldehyde using pristine

and ground STO:Rh(1.0%),Sb(1.0%) under visible light irradiation (λ > 420 nm) as

shown in Figure 11. Before irradiation, the glass reaction vessel was kept in the dark to

allow the acetaldehyde to adsorb onto the surface of the photocatalyst. Adsorption of

acetaldehyde was observed with ground STO:Rh,Sb, while negligible absorption was

observed with pristine STO:Rh,Sb owing to its small surface area. Under visible light

irradiation, the concentration of acetaldehyde drastically decreased in the presence of

ground STO:Rh,Sb, whereas pristine STO:Rh,Sb only slightly decomposed the

acetaldehyde, suggesting that ball milling is effective to enhance the photocatalytic

VOC degradation of STO:Rh,Sb. The optimal grinding time was also examined.

STO:Rh,Sb ground for 60 min showed the highest photocatalytic performance,

exhibiting a reaction rate constant of 0.088 min−1, compared with 0.022 and 0.056 min−1

for STO:Rh,Sb ground for 30 and 90 min, respectively. Despite its largest surface area,

STO:Rh,Sb ground for 90 min exhibited a lower reaction rate constant than that ground

for 60 min. A high surface area is important in improving photocatalytic performance

because the organic compounds must adsorb onto the surface of the photocatalyst.

Meanwhile, crystallinity is also significantly related to photocatalytic performance.

Crystal defects work as recombination centers of excited electrons and holes, resulting

in a decrease in photocatalytic performance. Highly crystalline samples contain few

defects, and therefore show high photocatalytic performance. It is the reason why

STO:Rh,Sb ground for 60 min showed the highest photocatalytic performance.

86

Figure 11. (A) Photocatalytic degradation of acetaldehyde under visible light irradiation

(λ>420 nm) on pristine STO:Rh,Sb and STO:Rh,Sb ground for 30, 60, and 90 min. (B)

Kinetic linear fitting curves. Photocatalyst: 0.3 g, light source: 200-W Xe lamp with

L-42 filter cutoff filter (100 mW cm−2), initial concentration of acetaldehyde: 150 ppm,

gas phase volume: 500 mL.

87

Figure 12 shows the correlation between Rh and Sb doping amounts, and

photocatalytic performance. The reaction rate constant of STO:Rh(1.0%),Sb(1.0%)

ground for 60 min was 0.088 min−1, while those of STO:Rh(0.1%),Sb(0.1%),

STO:Rh(0.5%),Sb(0.5%), STO:Rh(2.0%),Sb(2.0%), and STO:Rh(1.0%) ground for 60

min were 0.020, 0.081, 0.048, and 0.070 min−1, respectively. STO:Rh(1.0%),Sb(1.0%)

ground for 60 min showed the highest photocatalytic performance. The initially injected

acetaldehyde completely decomposed within 150 min of irradiation. Increases in the

amount of doping contribute to visible light absorption but also work as recombination

sites, resulting in low photocatalytic performance. Thus, STO:Rh(1.0%),Sb(1.0%)

showed the highest photocatalytic performance. The photocatalytic performance of

STO:Rh(1.0%),Sb(1.0%) ground for 60 min was higher than that of STO:Rh(1.0%)

ground for 60 min. Time-resolved infrared absorption spectroscopy by Furuhashi et al.

revealed that Rh4+ in STO:Rh works as an efficient recombination center.26 In contrast,

STO:Rh,Sb had a retarded recombination rate, suggesting the limited recombination

ability of Rh3+. Therefore, STO:Rh,Sb showed a higher photocatalytic performance than

STO:Rh. The conduction band level of STO:Rh,Sb is estimated to be ca. −0.2 V vs.

NHE, while the electron donor level in which holes (Rh3+) are generated is ca. +2.1 V vs.

NHE.15 Therefore, oxygen reduction (O2/HO2 = −0.13 V vs. NHE, O2/H2O2 = +0.68 V

vs. NHE, O2/H2O = +1.23 V vs. NHE) and acetaldehyde oxidation should

simultaneously proceed on the surface of STO:Rh,Sb.31, 32

88

Figure 12. (A) Photocatalytic degradation of acetaldehyde under visible light irradiation

(λ > 420 nm) on STO:Rh(1.0%), STO:Rh(0.1%),Sb(0.1%), STO:Rh(0.5%),Sb(0.5%),

STO:Rh(1.0%),Sb(1.0%), and STO:Rh(2.0%),Sb(2.0%) ground for 60 min. (B) Kinetic

linear fitting curves. Photocatalyst: 0.3 g, light source: 200-W Xe lamp with L-42 filter

89

cutoff filter (100 mW cm−2), initial concentration of acetaldehyde: 150 ppm, gas phase

volume: 500 mL.

Finally, to investigated the stability of STO:Rh,Sb, I conducted cycling evaluation

of photocatalytic decomposition of acetaldehyde. I washed the photocatalyst after

photocatalytic reaction with ultrapure water to remove the organic compounds adsorbed

on its surface during cycling evaluation. As a result, the photocatalytic performance of

the sample was found to be quite stable over 3 cycles, indicating that the photocatalyst

is stable and reusable (Figure 13).

Figure 13. Photocatalytic degradation of acetaldehyde on STO:Rh(1.0%),Sb(1.0%)

ground for 60 min under visible light irradiation (λ > 420 nm) for 3 cycles.

Photocatalyst: 0.3 g, light source: 200-W Xe lamp with L-42 filter cutoff filter (100 mW

cm-2), initial concentration of acetaldehyde: 150 ppm, gas phase volume: 500 mL.

90

The amounts of carbon dioxide and acetic acid formed after 4 h photocatalytic

reaction over STO:Rh(1.0%),Sb(1.0%) ground for 60 min after 4 h photocatalytic

reaction were determined using GC and HPLC, respectively. The amount of carbon

dioxide and acetic acid formed were 1.08 μmol and 2.87 μmol, respectively. The

amounts of carbon in the acetic acid was 5.74 μmol (2.87 μmol × 2). Therefore, the total

amount of carbon in the generated carbon dioxide and acetic acid was 6.82 μmol. The

initial concentration of acetaldehyde in the reaction was 150 ppm, corresponding to 6.70

μmol of carbon, suggesting that the mass balance of carbon was satisfactory. These

results also indicated that the predominant product of the photocatalytic oxidative

degradation of acetaldehyde was acetic acid. Because the surface of STO is basic,30 it is

assumed that acetic acid is easily adsorbed and stabilized on the surface of STO.

91

3-1-3 Conclusions Pristine STO:Rh and STO:Rh,Sb that were prepared by a solid-state-rection

exhibited negligible photocatalytic degradation of acetaldehyde, ascribed to small

surface area. To increase the surface area of both photocatalysts, they were pulverized at

800 rpm for 30, 60 and 90 min using ball-milling device. I revealed that the surface area

of ground STO:Rh and STO:Rh,Sb drastically increased and they improved the

photocatalytic performance under visible light irradiation. Ground STO:Rh,Sb showed

higher photocatalytic performance than ground STO:Rh, possibly ascribed to slower

recombination rate of ground STO:Rh,Sb than that of ground STO:Rh. The

photocatalytic performance of ground STO:Rh and STO:Rh,Sb were quite stable for

several cycles, indicating that both photocatalysts are reusable. Our work suggested that

ground STO:Rh and STO:Rh,Sb showed high photocatalytic degradation of

acetaldehyde under visible light irradiation.

92

3-1-4 Experimental section Preparation of photocatalyst

STO:Rh and STO:Rh,Sb were synthesized by a conventional solid-state reaction.34

The starting materials SrCO3 (99.9%, Kanto Chemical Co., Inc.), TiO2 (99.9%,

Soekawa Chemical), Rh2O3 (95%, Wako Pure Chemical), and Sb2O3 (98%, Nacali

Tesque Inc.) were mixed at the required ratios to achieve the composition of

Sr/Ti/Rh/Sb = 1.07:(1.00-2x):x:x in an aluminum crucible with the addition of a small

amount of methanol (99.8%). The powder was firstly calcined in air at 900 °C for 1 h,

and then at 1100 °C for 10 h. The resulting powder (1.0 g) was transferred to a 45-mL

container containing ultrapure water (5.0 mL) and zirconia balls (10.0 g; diameter = 1.0

mm). The powder was milled at 800 rpm for required minutes in a ball-milling device

(Fritsch Pulverisette 7). Finally, the ground particles were collected by filtration, washed

with ultrapure water, and dried at 60 °C for 24 h. In the present report, the samples are

denoted as g-STO:Rh(y%), where y% refers to the Rh doping amount, and

g-STO:Rh(z%),Sb(z%), where z% refers to the Rh and Sb doping amounts.

Characterization of photocatalyst

The morphologies of prepared photocatalysts were observed using filed emission

scanning microscope (FE-SEM; JEOL-7600F) accelerated at 10 kV. The photocatalysts

were coated Au layer (10 nm) by sputtering device (SANYU ELECTRON

SC-701HMCII) before observation. The crystal structures of the prepared photocatalysts

were determined by X-ray diffraction (XRD; RIGAKU Ultima IV) using Cu Kα

radiation. Diffuse reflectance spectra were obtained using a UV-visible spectrometer

(JASCO V-670); the reflection data were converted into absorbance values by the

Kubelka–Munk formula. Nitrogen adsorption and desorption isotherms were measured

at 77 K using a gas adsorption and desorption analyzer (MicrotracBEL BELSORP-max).

The specific surface areas were determined by the Brunauer–Emmett–Teller method.

X-ray photoelectron spectra were measured using an X-ray photoelectron spectrometer

(KRATOS Analytical; AXIS-NOVA).

93

Evaluation of photocatalytic oxidative degradation of acetaldehyde

Photocatalyst was put into a glass petri dish (ɸ: 35 mm) and it was transferred to a

500 mL closed glass reactor. The air containing 50 % humidity was flowed into the

reactor. Before evaluation of photocatalytic performance, the photocatalyst was

illuminated using Xe lamp (100 mW cm−2) without cutoff filter to decompose organic

compounds adsorbed on the surface of photocatalyst. Visible light irradiation was

carried out at 100 mW cm−2 using a 200 W Xe lamp through an L-42 cutoff filter (λ <

420 nm). The concentration of acetaldehyde and carbon dioxide were measured using a

gas chromatograph (Shimadzu; GC2014) with a flame ionization detector (FID)

equipped with a methanizer in order to convert carbon dioxide to methane that is

flammable. To calculate the concentration of formed acetic acid (intermediate of

photocatalytic oxidative degradation of acetaldehyde), the photocatalyst after

photocatalytic reaction were added to water (10 mL) and sonicated for 5 min. After

filtration, the concentration of acetic acid was detected using high-performance liquid

chromatograph (HPLC; Hitachi; Chromaster) with a UV detector (Hitachi; Chromaster

5410) at a wavelength of 210 nm. The HPLC measurement were carried out as

following conditions; column 250 × 4.6 mm (Hitachi; LACHROM 2 C18), column

temperature 25 °C, mobile phase 85% ethanol/15% water (v/v), flow rate 1.0 mL min−1.

94

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and SrTiO3 Photocatalysts Codoped with Antimony and Chromium. J. Phys. Chem. B

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aqueous solutions. Phys. Chem. Chem. Phys. 2005, 7 (10), 2241-2245.



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98

3-2 Selective inactivation of bacteriophage using visible light-driven Rh-doped SrTiO3 photocatalyst

Abstract Bacteriophage (denoted as phage) infection in the food bacterial fermentation

industry is a major problem, leading to the loss of fermented products such as alcohol

and lactic acid. Currently, the prevention of phage infection is limited to biological

approaches, which are difficult to apply in the industry. Herein, I report an alternative

chemical approach using a visible-light-driven Rh-doped SrTiO3 (STO:Rh)

photocatalyst as a material that selectively inactivates phage without affecting bacteria.

The ground powder photocatalyst (g-STO:Rh), which has a large surface area, showed

efficient anti-phage performance without bactericidal activity when irradiated with

visible light (λ > 440 nm). After the photocatalytic reaction, the color of g-STO:Rh

changed from grey to purple, suggesting that the Rh valence state partially changed

from 3+ to 4+, as confirmed by diffuse reflectance spectroscopy. To study the effect of

doped Rh4+ on phage inactivation under visible light irradiation, the survival rate of

phage for g-STO:Rh was compared to ground Rh,Sb-co-doped SrTiO3 where most Rh

ions should be Rh3+ due to charge compensation by Sb5+. Only g-STO:Rh effectively

inactivated phage, which indicated that the Rh4+ ion particularly contributed to phage

inactivation under visible light irradiation. In contrast, neither g-STO:Rh nor

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g-STO:Rh,Sb showed significant bacterial inactivation. These results suggested that

g-STO:Rh has potential as an anti-phage material in food bacterial fermentation.

100

3-2-1 Introduction Probiotic organisms are essential for human life, and are regularly employed in the

food fermentation industry, such as in the production of alcohol and lactic acid.1-12

However, the dairy industry has faced increasing bacteriophage (denoted as phage)

problems, notably phage infection, leading to losses of fermented products.1,2,13-16

Therefore, the development of facile and safe technologies that inactivate phage without

damaging probiotic organisms is essential for the fermentation industry. To date, studies

on addressing phage infection have been limited to employing microbiological

approaches. Specifically, various anti-phage strategies based on phage-sensitive mutants

and the use of genetically modified anti-phage systems have been employed

successfully.2,10,13,14 However, the practical application of such biological approaches is

difficult because of the complex processes involved.

Recently, as an alternative strategy, photocatalysis has gained much attention; this

process uses materials that exhibit reductive and oxidative performance under light

irradiation, and has been proposed for the development of anti-phage materials for facile

and safe application in clinical and industrial settings.17-20 TiO2 is widely used as an

environmental purification material that shows high photocatalytic performance.

However, because of its wide band gap, this compound responds only to UV light. Since

DNA and RNA of phage and bacteria are seriously damaged under UV light irradiation, 21,22 it is difficult to achieve selective inactivation of phage. Therefore, photocatalysis

using materials that respond to UV light is of limited use in the food fermentation

industry.17, 23-25 Herein, I propose a new technology that uses a photocatalyst that

responds to visible light; light of these wavelengths is of lower energy, and hence less

destructive to the probiotic bacteria whose viability is essential to fermentation.

In recent years, doped SrTiO3 has gained attention as a visible-light-driven

photocatalyst. Pristine SrTiO3, which is an n-type semiconductor with a wide band gap

of 3.2 eV, does not exhibit photocatalytic activity (as assessed by water splitting) under

visible light irradiation. In contrast, Rh-doped SrTiO3 (STO:Rh), a p-type

semiconductor, has been employed for hydrogen generation via photocatalytic water

101

splitting to generate hydrogen.26-30 The research has shown that metal doping endows

SrTiO3 with visible-light-responsive behavior; doping permits photocatalysis by

generating impurity levels in the forbidden band of the semiconductor without shifting

the conduction band level.31-35. Substituting a small amount of Rh atoms into the Ti sites

of SrTiO3 stabilizes the valence state of Rh to 4+ upon calcination in air. In the

photocatalytic reaction, the Rh valence state in the STO:Rh photocatalyst reversibly

changes from 4+ (purple) to 3+ (yellow) under visible light irradiation in the presence

of an electron donor such as methanol. Thus, the Rh valence state in STO:Rh is a key to

photocatalysis.26, 28

Another important factor in photocatalysis is a large surface area, since contact

between the target substances and the photocatalyst is required for reaction. I previously

demonstrated that ball-milled (ground) visible-light-driven STO:Rh photocatalyst

(g-STO:Rh) can be employed for environmental remediation, specifically, for the

decomposition of volatile organic compounds (VOCs) upon irradiation with visible

light,36 whereas pristine STO:Rh shows negligible photocatalytic performance owing to

smaller surface area.36 Ball milling thus is a suitable method for the preparation of

materials with enhanced photocatalytic performance.

Herein, I present a chemical approach using g-STO:Rh photocatalyst to selectively

inactivate phage without decreasing bacterial numbers. The anti-phage and bactericidal

activities under visible light irradiation were examined by evaluating the difference in

the inactivation rate of phage and bacteria in the context of the Rh valence state of

g-STO:Rh.

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3-2-2 Results and discussion The inactivation rates of phage (bacteriophage Qβ) in the presence of pristine and

ground SrTi0.99Rh0.01O3 (denoted as pristine STO:Rh(1%) and g-STO:Rh(1%),

respectively) in aqueous suspension under dark conditions and visible light irradiation

are shown in Figure 1. Negligible inactivation rates were obtained without photocatalyst

under visible light irradiation and with photocatalyst under dark conditions, indicating

that neither light illumination nor photocatalyst particles alone are sufficient to

inactivate phage. In addition, pristine STO:Rh(1%) showed negligible inactivation rates

under visible light irradiation, suggesting that pristine STO:Rh(1%) photocatalyst is

ineffective for inactivation of phage under visible light illumination. Conversely, the

considerable inactivation of phage was observed in the presence of g-STO:Rh(1%) after

4 h of irradiation. This improved performance was attributed to the larger surface area

of g-STO:Rh(1%) when compared with that of pristine STO:Rh(1%). These results were

consistent with those discussed in the introduction section regarding the superior

photocatalytic performance of g-STO:Rh(1%) to pristine STO:Rh(1%) for the

decomposition of VOCs under visible light irradiation.36 Large contact area between the

target substance and the photocatalyst surface is important for facilitating photocatalytic

activity.

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Figure 1. Survival rate of bacteriophage Qβ in the presence of STO:Rh(1%) and

g-STO:Rh(1%) photocatalysts in aqueous suspension under dark conditions and visible

light irradiation (110 mW cm−2, λ > 440 nm). Photocatalyst: 0.15 g, light source: 200 W

xenon lamp with a Y-44 cutoff filter, volume: 50 mL, initial density of phage: 5.0 × 107

PFU mL−1.

The optimal Rh doping of g-STO:Rh for inactivation of phage was evaluated, as

shown in Figure 2. Negligible phage inactivation was observed without g-STO:Rh

under visible light irradiation and with g-STO:Rh under dark conditions. In contrast, the

survival of phage decreased slightly in the presence of g-STO:Rh(0.5%) and

g-STO:Rh(2%) after 4 h of irradiation. A ~5-log reduction in phage number was

observed with g-STO:Rh(1%) under similar conditions. In the photocatalytic H2

evolution, the best performance was also obtained with STO:Rh(1%).26 Thus, the

optimal Rh doping amounts for inactivation of phage showed a trend similar to that for

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Figure 2. Survival rate of bacteriophage Qβ in the presence of g-STO:Rh(0.5, 1, or 2%)

photocatalysts with various Rh doping amounts in aqueous suspension under dark

conditions and visible light irradiation (110 mW cm−2, λ > 440 nm). Photocatalyst: 0.15

g, light source: 200 W xenon lamp with a Y-44 cutoff filter, volume: 50 mL, initial

density of phage: 5.0 × 107 PFU mL−1.

the sacrificed H2 evolution. Increasing the doping amount enhanced visible light

absorption (Figure 3), while concurrently providing more recombination centers

between the excited electron and holes, thereby leading to reduced photocatalytic

activities. Thus, the optimal Rh doping amount in g-STO:Rh for application to

inactivation of phage was determined to be 1%.

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Figure 3. Diffuse reflectance spectra of g-STO:Rh (0.5, 1, or 2%) photocatalysts.

Next, the bacterial inactivation rates of Escherichia coli (denoted as bacteria) in the

presence of pristine and ground pristine STO:Rh(1%) and g-STO:Rh(1%) in aqueous

suspension under dark conditions and visible light irradiation were investigated, as

shown in Figure 4. As with phage, negligible bacterial inactivation was observed

without photocatalyst under visible light irradiation and with photocatalyst under dark

conditions, indicating neither light illumination nor the photocatalyst particles alone

were sufficient to cause bacterial killing. Additionally, pristine STO:Rh(1%) showed

negligible inactivation of bacteria under visible light irradiation, suggesting that pristine

STO:Rh(1%) photocatalyst is ineffective for bacterial sterilization under visible light

illumination. Conversely, bacterial inactivation was almost negligible in the presence of

g-STO:Rh(1%) after 4 h of irradiation, which is in contrast to inactivation of phage.

Obvious bacterial killing required more than 12 h of irradiation, which suggested that

g-STO:Rh shows superior anti-phage activity to bactericidal activity.

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Figure 4. Survival rate of Escherichia coli in the presence of STO:Rh(1%) and

g-STO:Rh(1%) photocatalysts in aqueous suspension under dark conditions and visible

light irradiation (110 mW cm−2, λ > 440 nm). Photocatalyst: 0.15 g, light source: 200 W

xenon lamp with a Y-44 cutoff filter, volume: 50 mL, initial density of bacteria: 5.0 ×

107 CFU mL−1.

The inactivation of bacteria in the presence of g-STO:Rh with various Rh doping

amounts in aqueous suspension under dark conditions and visible light irradiation was

evaluated to establish the optimal Rh doping amount, as shown in Figure 5.

g-STO:Rh(1%) showed the highest antibacterial activity, yielding a ~4.5-log reduction

in cell density after 24 h of irradiation, whereas g-STO:Rh(0.5%) and g-STO:Rh(2%)

exhibited ~2-log and ~3-log

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Figure 5. Survival rate of Escherichia coli in the presence of g-STO:Rh photocatalysts

with various Rh doping amounts in aqueous suspension under dark conditions and

visible light irradiation (110 mW cm−2, λ > 440 nm). Photocatalyst: 0.15 g, light source:

200 W xenon lamp with a Y-44 cutoff filter, volume: 50 mL, initial density of bacteria:

5.0 × 107 CFU mL−1.

reductions, respectively, following irradiation for the same interval. Interestingly, the

bactericidal activity of a representative photocatalyst, TiO2 (P25, Evonik), which is

employed worldwide as an environmental remediation material, was higher than the

anti-phage activity shown in Figure 6. This result is consistent with that of Cho et al.17

Thus, g-STO:Rh is a unique photocatalyst showing effective anti-phage activity.

108

Figure 6. Survival rate of bacteriophage Qβ and Escherichia coli in the presence of TiO2

photocatalyst under UV light irradiation (0.12 mW cm−2). Photocatalyst: 5 mg, light

source: black light, volume of aqueous solution: 50 mL, initial densities of phage and

bacteria: 5.0 × 107 PFU mL−1 and 5.0 × 107 CFU mL−1, respectively.

After photocatalytic inactivation of phage and bacteria with g-STO:Rh(1%) under

visible light irradiation, the color of the photocatalyst changed from grey to purple,

presumably due to changes in the Rh valence state. Previously, I reported that the Rh

valence state of g-STO:Rh(1%) after ball-milling changes from the higher oxidation

number, 4+, to the lower oxidation number, 3+, because of oxygen defects generated

and charge valence balance.36 Before ball-milling, g-STO:Rh(1%) is purple and

includes a high amount of Rh4+.26 Thus, the color change observed in g-STO:Rh(1%)

upon visible light irradiation may be attributable to changes in the Rh valence states

from 3+ to 4+. To elucidate the change in the Rh valence state of g-STO:Rh(1%) before

and after 4 h photocatalytic inactivation of phage, the associated diffuse reflectance

109

spectra of the photocatalyst were recorded (Figure 7). The absorption features at 580

and 420 nm are ascribed to Rh4+ and Rh3+, respectively.37 The absorption at 580 nm is

caused by electron transition from the valence band of SrTiO3 to the unoccupied d state

of Rh4+, and the absorption at 420 nm is due to electron transition from the occupied

state of Rh3+ to the conduction band of SrTiO3. Following 4-h of irradiation with visible

light, the Rh4+ absorption of g-STO:Rh(1%) increased, whereas the Rh3+ absorption

decreased, indicating that the Rh valence state of g-STO:Rh(1%) changed from 3+ to 4+

Figure 7. Diffuse reflectance spectra of g-STO:Rh(1%) photocatalyst before and after

4-h photocatalytic inactivation of bacteriophage Qβ.

, which is the opposite result in comparison with the report of Konta et al. on which they

described that the Rh valence state of pristine STO:Rh changed from Rh4+ to Rh3+ in the

photocatalytic water splitting. It is assumed that change of the Rh valence state from

Rh4+ to Rh3+ is attributed to the presence of hole scavenge (Figure 6a), ascribed that the

trivalency is the most stable oxidation number among Rh ions in oxides.26 In contrast,

110

change of Rh4+ ion was not observed in the absence of hole scavenger (Figure 6b).26

Therefore, I speculated that change of the Rh valence state of g-STO:Rh from Rh3+ to

Rh4+ should not be observed in the presence of hole scavenge. In order to investigate

change of Rh valence state of g-STO:Rh, I added methanol to the aqueous solution

containing g-STO:Rh powder and irradiated with visible light. As a result, change of the

Rh valence state was not observed in the presence of methanol under visible light

irradiation (Figure 6c), indicating that change of the Rh valence state of g-STO:Rh from

Rh3+ to Rh4+ (Figure 6d) was due to the absence of hole scavenge in photocatalytic

anti-phage evaluation. I inferred that when g-STO:Rh photocatalyst is irradiated with

visible light, the electron of Rh3+ is excited to the conduction band consisting of Ti 3d

orbitals.

Figure 8. Change of the Rh valence state of pristine and ground STO:Rh(1%) in the

presence and absence of hole scavenge.

After excitation of Rh electron, the valence state of Rh changes from 3+ to 4+. It has

been reported that various reactive oxygen species are generated in reductive and

oxidative reactions as follows.38-41

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Reductive reactions

O2 + e− O2−

O2− + H+ ▪HO2

▪HO2 + e− + H+ H2O2

Oxidative reactions

OH− + h+ ▪OH

▪OH + ▪OH H2O2

I confirmed that H2 production was not observed in bacterial suspension after 24 h

under visible light irradiation (λ < 440 nm). Therefore, I inferred that the excited

electrons of g-STO:Rh reduce oxygen molecules, which produces various reactive

oxygen species. In the oxidative reaction, OH− adsorbed on a photocatalyst is oxidized

by holes, resulting in formation of ▪OH. To confirm the presence of ▪OH, I examined

the photocatalytic decomposition of dimethyl sulfoxide (DMSO). DMSO is known to

be oxidized to methanesulfinic acid (MSI) and methanesulfonic acid (MSA) by ▪OH

generated on the photocatalyst.42-44 After visible light irradiation (λ < 440 nm) of

g-STO:Rh, both MSI and MSA products were detected, indicating the production of

▪OH.

To investigate the correlation between the Rh4+ ion and inactivation of phage and

bacteria, the survival rates in the presence of g-STO:Rh and g-STO:Rh,Sb were

compared. Previous reports have suggested that the Rh valence state can be stabilized as

Rh3+ by doping with Sb; because antimony can be oxidized to Sb5+ and the SrTiO3 host

contains equivalent amounts of Rh3+ and Sb5+, Rh4+ formation is largely suppressed in

Sb-doped material.45,46. Thus, the Rh valence states of STO:Rh,Sb would not be

interchangeable under visible light irradiation.45,47. Therefore, the pathogen inactivation

dependence on Rh4+ ion was evaluated by comparing the effects of STO:Rh and

STO:Rh,Sb. X-ray diffraction measurement revealed that both g-STO:Rh and

g-STO:Rh,Sb featured cubic SrTiO3 structures (Figure 9), although small peaks of TiO2

112

anatase (ca. 25º) were observed. I assumed that TiO2 anatase does not affect

photocatalytic performance in our study because anatase TiO2 responds to UV light.

Additionally, the surface areas of g-STO:Rh and g-STO:Rh,Sb were comparable (~41

m2 g-1) (Table 2), suggesting that the photocatalytic activities of the two substances

should not differ due to this parameter. Distinct anti-phage activities were observed with

Figure 9. X-ray diffraction patterns of (a) SrTiO3 (PDF 01-073-0661), (b)

g-STO:Rh(0.5%), (c) g-STO:Rh(1%), (d) g-STO:Rh(2%), (e)

g-STO:Rh(0.5%),Sb(0.5%), and (f) g-STO:Rh(1%),Sb(1%) photocatalysts.

Table 2. Surface areas of g-STO:Rh and g-STO:Rh,Sb photocatalysts

Dopants Surface area / m2 g-1

Rh(0.5%) 42

Rh(1%) 41

113

Rh(2%) 42

Rh(0.5%),Sb(0.5%) 42

Rh(1%),Sb(1%) 40

g-STO:Rh, as shown in Figure 10. Interestingly, both g-STO:Rh(0.5%),Sb(0.5%) and

g-STO:Rh(1%),Sb(1%) exhibited negligible phage inactivation, whereas

g-STO:Rh(1%) provided a 5-log reduction after 2 h of irradiation. These results showed

Figure 10. Survival rate of bacteriophage Qβ in the presence of g-STO:Rh(1%) and

g-STO:Rh(0.5%),Sb(0.5%) or g-STO:Rh(1%),Sb(1%) photocatalysts in aqueous

suspension under dark conditions and visible light irradiation (110 mW cm−2, λ > 440

nm). Photocatalyst: 0.15 g, light source: 200 W xenon lamp with a Y-44 cutoff filter,

volume of aqueous solution: 50 mL, initial density of phage: 5.0 × 107 PFU mL−1.

that the Rh4+ ion under visible light irradiation contributes to anti-phage activity. Figure

11 shows the survival rates of bacteria in the presence of g-STO:Rh and g-STO:Rh,Sb

114

in aqueous suspension under dark conditions and visible light irradiation. As seen with

g-STO:Rh, bacterial inactivation was negligible with g-STO:Rh,Sb under dark

conditions, indicating that the g-STO:Rh,Sb photocatalyst particles does not cause

bacterial killing. In contrast, under visible light irradiation, no obvious differences in

Figure 11. Survival rate of Escherichia coli in the presence of g-STO:Rh(0.5, 1, or 2%)

and g-STO:Rh(0.5%),Sb(0.5%) or g-STO:Rh(1%),Sb(1%) photocatalysts in aqueous

suspension under dark conditions and visible light irradiation (110 mW cm−2, λ > 440

nm). Photocatalyst: 0.15 g, light source: 200 W xenon lamp with a Y-44 cutoff filter,

volume: 50 mL, initial density of bacteria: 5.0 × 107 CFU mL−1.

bacterial killing were observed between g-STO:Rh and g-STO:Rh,Sb. Figure 12 shows

the diffuse reflectance spectra of g-STO:Rh(1%),Sb(1%) before and after 4-h

photocatalytic inactivation of phage. Although most rhodium ions exist as Rh3+ in

prepared g-STO:Rh,Sb, a small amount of Rh4+ ion exists. However, Rh4+ and Rh3+

115

absorption features changed minimally after irradiation, presumably due to the presence

of the antimony dopant. It is therefore suggested that the Rh4+ ion in g-STO:Rh,Sb

under visible light irradiation did not directly contribute to bactericidal performance.

Rh4+ ion in g-STO:Rh under visible light irradiation is shortage of an electron. It

may suggest that the surface of g-STO:Rh positively charges, attracting the substances

whose surface negatively charges. On the other hand, the charges of both bacteria and

phage are negative,48,49 indicating that it is difficult to discuss the differences of charges

between bacteria and phage. Thus, I focused on the surficial compositions of bacteria

and phage. The surface of bacteria mainly contains the lipopolysaccharide (LPS)

consisting of lipid and polysaccharide while that of phage consists of protein. It is

known that the denaturation of protein easily occurs by some ions in solution. Therefore,

it may be assumed that Rh4+ ion in g-STO:Rh under visible light irradiation works as

oxidation or denaturation site of protein, leading to the inactivation of phage.

TiO2 photocatalysts incorporating Cu(I) nano-clusters display both anti-phage and

anti-bacterial activity under visible light irradiation,24,50,51 suggesting that the Cu

valence state plays a role in the anti-pathogen performance of that material. Therefore,

our results suggested that g-STO:Rh(1%), which preferentially exhibits anti-phage

activity over bacterial activity, has promise as an effective visible-light-driven

photocatalyst in addressing phage infection in the food fermentation industry.

116

Figure 12. Diffuse reflectance spectra of g-STO:Rh(1%),Sb(1%) photocatalyst before

and after 4-h photocatalytic inactivation of bacteriophage Qβ. Diffuse reflectance

spectra were converted from reflection to absorbance by the Kubelka-Munk method.

Moreover, I irradiated with visible light to g-STO:Rh(1%) and

g-STO:Rh(1%),Sb(1%) for 4 hours, and then evaluated the photocatalytic anti-phage

performance with g-STO:Rh(1%) and g-STO:Rh(1%),Sb(1%) under dark condition.

Interestingly, phage inactivation of g-STO:Rh(1%) was observed superior to that of

g-STO:Rh(1%),Sb(1%) even under dark condition as shown in Figure 13, suggesting

that the effect of anti-phage performance of Rh4+ ion remains under dark condition.

117

Figure 13. Survival rate of bacteriophage Qβ in the presence of g-STO:Rh(1%) and

g-STO:Rh(1%),Sb(1%) photocatalysts irradiated with visible light (110 mW cm−2, λ >

440 nm) for 4 h in aqueous suspension under dark conditions. Photocatalyst: 0.15 g,

light source: 200 W xenon lamp with a Y-44 cutoff filter, volume of aqueous solution:

50 mL, initial density of phage: 5.0 × 107 PFU mL−1.

In the above studies, the sterilization of phage and bacteria were examined

separately. To investigate the anti-phage performance of g-STO:Rh(1%) in the presence

of bacteria, the inactivation rate of pathogens in a suspension containing both phage and

bacteria in the presence of g-STO:Rh(1%) was evaluated with visible light irradiation.

Distinct phage inactivation was still observed in the presence of bacteria, as shown in

Figure 14; a 3-log reduction was noted after 2 h of irradiation, an interval at which

negligible bacterial killing was observed.

118

Figure 14. Survival rate of pathogens in a mixed suspension of bacteriophage Qβ and

Escherichia coli in the presence of g-STO:Rh(1%) photocatalyst in aqueous suspension

under visible light irradiation (110 mW cm−2, λ > 440 nm). Photocatalyst: 0.15 g, light

source: 200 W xenon lamp with a Y-44 cutoff filter, volume of aqueous solution: 50 mL,

initial densities of phage and bacteria: 5.0 × 107 PFU mL−1 and 5.0 × 107 CFU mL−1,

respectively.

119

3-2-3 Conclusions Visible-light-driven g-SrTiO3:Rh(1%) photocatalyst showed efficient anti-phage

performance at irradiation intervals that did not cause bactericidal activity. The Rh

valence state of g-SrTiO3:Rh(1%) changed from 3+ to 4+, accompanied by a color

change from grey to purple, following visible light irradiation. The large surface area of

the photocatalyst and Rh4+ ion under visible light irradiation contributed to the high

anti-phage performance. Moreover, anti-phage activity was observed even in the

presence of bacteria. I demonstrated that the Rh4+ ion in g-STO:Rh under visible light

irradiation contributed to effective and selective inactivation of phage. The precise

inactivation mechanisms, possibly via attachment inhibition and/or nucleic acid

denaturation, have not yet been determined, in part because of the complexity of such

processes. However, research toward elucidating the mechanism, which is under way, is

expected to be important for the designing and fabricating of efficient anti-phage

materials. Specifically, it will be critical to determine the relationship between

anti-phage activity and the function of Rh4+ ions in g-STO:Rh under visible light

irradiation. The current findings demonstrate the potential of a visible-light-driven

STO:Rh photocatalyst for deactivating phage (a bacterial predator) in the food bacterial

fermentation industry.

120

3-2-4 Experimental section Preparation of photocatalysts

Rh-doped SrTiO3 (STO:Rh), and SrTiO3 co-doped with Rh and Sb (STO:Rh,Sb)

were synthesized by a solid-state reaction. The starting materials SrCO3 (99.9%, Kanto

Chemical Co., Inc.), TiO2 (99.9%, Soekawa Chemical), Rh2O3 (95%, Wako Pure

Chemical), and Sb2O3 (98%, Nacali Tesque Inc.) were mixed at the required ratios to

achieve the composition of Sr/Ti/Rh = 1.07:(1.00-x):x. The mixture then was ground in

an aluminum crucible with the addition of a small amount of methanol (99.8%). The

powder was calcined in air at 900 °C for 1 h, and then at 1100 °C for 10 h. Aliquots (1.0

g) of the resulting powder were transferred to 45-mL containers containing ultrapure

water (5.0 mL) and zirconia balls (10.0 g; diameter = 1.0 mm). The powder was milled

at 800 rpm for required minutes in a ball-milling device (Fritsch Pulverisette 7). Finally,

the ground particles were collected by filtration, washed with ultrapure water, and dried

at 60 °C for 24 h. In the present report, the samples are denoted as g-STO:Rh(y%),

where y% refers to the Rh doping amount, and as g-STO:Rh(z%),Sb(z%), where z%

refers to the Rh and Sb doping amounts.

Characterization

The crystal structures of the prepared photocatalysts were determined by X-ray

diffraction (RIGAKU Ultima IV) using Cu Kα radiation. Diffuse reflectance spectra

were obtained using a UV-visible spectrometer (JASCO V-670); the reflection data were

converted into absorbance values by the Kubelka–Munk formula. Nitrogen adsorption

and desorption isotherms were measured at 77 K using a gas adsorption and desorption

analyzer (MicrotracBEL BELSORP-max). The specific surface areas were determined

by the Brunauer–Emmett–Teller method.

Photocatalytic anti-bacteria and anti-phage performance

Bacteriophage Qβ (NBRC20012) and Escherichia coli (IAM12119T) were used in

our experiment as phage and bacteria, respectively. These reagents were incubated in

121

Nutrient Broth liquid medium at 37 °C for 16 h, respectively. The culture was

centrifuged at 1100 g for 10 min, followed by washing with sterile physiological saline

solution (pH 7). After washing, the resulting pellet was resuspended in saline. This

bacterial suspension was adjusted to a concentration of 5.0 × 107 colony-forming units

(CFU) mL−1 with sterilized water. For phage work, the density was determined by

testing infection of Escherichia coli (NBRC106373) using the double-agar-layer plaque

assay. The density of the phage suspension then was adjusted to 5.0 × 107

plaque-forming units (PFU) mL−1 with sterilized water.

The photocatalytic inactivations of phage and bacteria were tested as follows. The

photocatalyst (150 mg) was added to the suspension of phage or bacteria in sterilized

water (50 mL) and sonicated for 5 min. The photocatalyst-bacteria or

photocatalyst-phage suspension then was irradiated at 110 mW cm−2 light intensity

using a xenon lamp equipped with a Y-44 cutoff filter (λ < 440 nm). After irradiation,

serial dilutions of the bacterial suspension were plated to Nutrient Agar, while serial

dilutions of the phage were mixed with Escherichia coli (NBRC106373) and plated

using the double-agar-layer plaque assay as above. The resulting plates were cultivated

at 37 °C for 1 day. The survival rate of phage and bacteria was determined by counting

the colonies and plaques formed, respectively. An aliquot (5 mg) of a TiO2 photocatalyst

(Aeroxide® P-25, Evonik) was used for testing bactericidal activity under UV light

irradiation (0.12 mW cm−2) when added to a suspension (50 mL) of phage or bacteria.

All of the inactivation experiments were repeated for three times. Statistical analysis

(error bar, standard deviation, averaged values and log scale expression) was carried out

in our study by using the Microsoft Excel Program.

Detection of ▪OH produced by photocatalyst

The photocatalyst (25 mg) was dispersed in 10 ppm DMSO aqueous solution (30

mL) and sonicated for 5 min. The suspension was irradiated at 100 mW cm−2 using a Xe

lamp equipped with a Y-44 cutoff filter (λ < 440 nm) for 24 h. MSI and MSA produced

by photocatalytic oxidative decomposition of DMSO were detected using an ion

122

chromatograph equipped with a conductivity detector (SHIMADZU CDD-10A VP) and

a column (SHIMADZU Shim-pack IC-SA2) at 303 K. The mobile phase was a mixed

solvent of 0.14% sodium hydrogen carbonate, 0.19% sodium carbonate aqueous

solution at 10 mL min-1.

Detection of H2 with photocatalyst in bacterial suspension under visible light

irradiation

The photocatalyst (60 mg) was dispersed in a bacterial suspension (30 mL) adjusted

to 5.0 × 107 CFU mL-1 and sonicated for 5 min. The suspension was irradiated for 24 h

at 100 mW cm-2 using a xenon lamp equipped with a Y-44 cutoff filter (λ < 440 nm).

The gas chromatograph equipped with a thermal conductivity detector and a column

(SHIMADU Molecular Sieve 13X) at 313 K was used. Argon was used as a carrier gas

at 1.0 mL min−1.

123

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129

Chapter 4 Sporicidal performance induced by photocatalytic production of organic peroxide under visible light irradiation

Abstract Some bacteria are known to sporulate under conditions of nutrient and water shortage.

The resulting spores have much greater resistance to common sterilization methods,

such as heating at 100°C and exposure to various chemical agents. Because such

bacteria cannot be inactivated with typical alcohol disinfectants, peroxyacetic acid

(PAA) often is used, but PAA is a harmful agent that can seriously damage human

health. Furthermore, concentrated hydrogen peroxide, which is also dangerous, must be

used to prepare PAA. Thus, the development of a facile and safe sporicidal disinfectant

is strongly required. In this study, I have developed an innovative sporicidal disinfection

method that employs the combination of an aqueous ethanol solution, visible light

irradiation, and a photocatalyst. I successfully produced a sporicidal disinfectant one

hundred times as effective as commercially available PAA, while also resolving the

hazards and odor problems associated with PAA. The method presented here can

potentially be used as a replacement for the general disinfectants.

130

4-1 Introduction Under adverse conditions (low nutrient or limiting water), some bacteria enter into

a developmental pathway called sporulation. The resulting spores are a dormant cell

type that possesses very high resistance to common sterilization methods, including

heating at 100 °C and treatment with chemical agents. These resistance properties are

attributed to the impermeability of a thick proteinaceous outer spore coat along with the

stabilization of the cell contents within a spore core, which packages the DNA while

containing high levels of pyridine-2,6-dicarboxylic acid (dipicolinic acid, DPA), and

excluding water.1–5 The spores cannot be inactivated with general alcohol disinfectants,

necessitating the use of harsher agents such as hypochlorite disinfectants. However,

hypochlorite is corrosive to metal and clothing owing to its strong basicity. Recently,

peroxyacetic acid (PAA) has been adopted, but concentrated PAA is a harmful agent that

can seriously damage human skin and respiratory systems, and this agent has a very

strong odor.5,6 Furthermore, concentrated hydrogen peroxide, which must be used to

prepare the disinfectant, is itself dangerous. Thus, the development of a facile and safe

sporicidal disinfectant is strongly required for applications in the food sector, the health

industry, and the home. To resolve the problems described above, I developed a

sporicidal disinfectant that uses a combination of an aqueous ethanol solution

(commonly used as a disinfectant and known to be safe to human health), visible light

irradiation, and a photocatalyst. Photocatalysis is a facile oxidative and reductive

method whereby the photocatalyst compounds are activated only upon irradiation.8–16 I

expected that photocatalytic oxidative decomposition of ethanol would produce organic

acids such as acetic acid and formic acid (known food components that are generally

regarded as safe) along with hydrogen peroxide from the multi-electron reduction of

atmospheric oxygen, such that the organic peroxide would endow the mixture with

sporicidal activity.

131

4-2 Results and discussion To investigate the sporicidal performance of the method proposed in this study, I

used spores of Bacillus subtilis, a model non-pathogenic spore former that is commonly

used to evaluate techniques for the inactivation of bacterial spores. WO3 was used as a

visible-light-driven photocatalyst. The band edge of the WO3 used in this work was

estimated to be ca. 460 nm, as shown in Figure 1, indicating a band gap of ca. 2.7 eV.

The crystal structure of the WO3 was found to be monoclinic phase using X-ray

diffraction (Figure 2A). The particle size of the WO3 was estimated to be ca. 100nm

based on field emission scanning electron microscope (FE-SEM) observations (Figure

3A). The surface area of the WO3 was calculated to be 7.6 m2 g−1 based on gas

adsorption-desorption measurements.

Figure 1. Diffuse reflectance spectra of TiO2 and WO3.

132

Figure 2. X-ray diffraction patterns of (A) WO3, (a) WO3 sample, (b) monoclinic WO3 (JSPDS No. 01-083-0950), (B) TiO2 (P-25), (a) TiO2 sample, (b) rutile TiO2 (JSPDS No. 00-021-1276), (c) anatase TiO2 (JSPDS No. 01-084-1285).

133

Figure 3. Field emission scanning electron microscope images of (A) WO3 and (B)

TiO2.

I examined the survival rate of B. subtilis spores in the presence of WO3 suspended

in aqueous ethanol disinfectant and irradiated with visible light. The dependence of the

obtained sporicidal performance on the ethanol:water ratio was studied as shown in

Figure 4. A decrease in the survival rate of B. subtilis spores was fist observed for the

6:4 (v/v) ethanol/water solution, with a ca. 2.5-log reduction observed after 24 h of

134

irradiation. At ethanol:water ratios of 7:3, 8:2, and 9:1 (v/v), the spores were completely

inactivated after 24, 9, and 12 h visible light irradiation, respectively. Furthermore, I

investigated the sporicidal performance of ethanol:water solutionat the indicated ratios

(3:7, 6:4, 8:2, 9:1, v/v) without WO3. The reduction of survival rate of spore in aqueous

ethanol solution was not observed as shown in Figure 5.

Figure 4. Survival rate of B. subtilis spores in the presence of WO3 suspended in

ethanol:water solution at the indicated ratios ((a) 0:10; (b) 1:9; (c) 3:7; (d) 6:4; (e) 7:3;

(f) 8:2; (g) 9:1, v/v) and illuminated with visible light for the indicated time.

Photocatalyst: 25 mg, light source: Xe lamp (vis) with L-42 filter ( λ> 420 nm),

liquid-phase volume: 50 mL, density of B. subtilis spores: 2.0 × 106 CFU mL−1.

135

Figure 5. Survival rate of B. subtilis spores in ethanol:water solution at the indicated

ratios (3:7, 6:4, 8:2, 9:1, v/v) without WO3.

These results indicated that the optimum sporicidal performance occurred at an

ethanol/water ratio of 8:2 (v/v), and demonstrated that the inactivation of bacterial

spores could be achieved by a facile and safe photocatalytic method. To confirm the

presence of organic peroxide, HPLC measurements were examined according to the

method of Pinkernell et al.17,18 PAA oxidizes methyl p-tolyl sulfide (MTS) to methyl

p-tolyl sulfoxide (MTSO) [eq. 1].

……(1)

136

Therefore, the level of PAA can be quantified measuring the amount of MTSO

generated. Thus method has the great advantage because the detection of PAA is not

inhibited with the coexistence of hydrogen peroxide which typically oxidizes organic

chemicals. A portion of the WO3 suspension after photocatalytic reaction was added to

an MTS solution and the presence of MTSO was detected as shown in Figure 6.

137

Figure 6. HPLC chromatograms of MTSO and MTS. (a) WO3 in ethanol:water

solution (8:2, v/v) and illuminated with visible light for 12 h, TiO2 in ethanol:water

solution (8:2, v/v) and illuminated with UV light for 24 h, and 120 ppm hydrogen

138

peroxide. (b) Expanded view of the time interval from 3.2 to 3.8 min in panel

The amount of organic peroxide produced gradually increased during the

photocatalytic reaction, as shown in Figure 7. The amount of organic peroxide produced

aftr 12 h of visible light irradiation was estimated to be ca. 8.84 μmol (ca. 13.5 ppm),

confiming the successful production of organic peroxide by photocatalytic oxidation of

aqueous ethanol solution by WO3 illuminated with visible light.

Figure 7. Time-dependence of amount of organic peroxide, hydrogen peroxide, acetic

acid, and formic acid produced by WO3 suspended in ethanol:water solution (8:2, v/v)

illuminated with visible light (λ > 420 nm) for the indicated time.

The organic peroxide was generated by an equilibrium reaction between hydrogen

peroxide and organic acids [eq. 2].6,19,20 Firstly, reaction between organic acid and

proton occurs, which generates carbocation [eq. 3].21 Subsequently, the carbocation of

139

organic acid combines with molecule of hydrogen peroxide, leading to formation of

organic peroxide [eq. 4].21 Therefore, the generation of organic peroxide is required the

presence of organic acid and hydrogen peroxide.

……(2)

……(3)

……(4)

I quantified the amounts of generated hydrogen peroxide, acetic acid, and formic acid

using HPLC measurements. As can be seen in Figure 7, the amounts of producs

increased with time of irradiation. After 12 h of irradiation, the amount of hydrogen

140

peroxide, acetic acid, and formic acid generated in the reaction system approached 73.3

μmol (49.8 ppm), 107 μmol (129 ppm), and 64.5 μmol (59.4 ppm), respectively. The

generation of hydrogen peroxide is known to proceed based on the multi-electron

reduction of oxygen (O2 + 2H+ + 2e− = H2O2, +0.68 V). The conduction band level of

WO3 is negative enough (+0.5 V vs. NHE) for this multi-electron reduction to occur21–23

as shown in Figure 8.

Figure 8. Relationship between the potential of oxygen reduction and the band

structures of TiO2 and WO3.

141

The photocatalytic oxidative decomposition of ethanol by photocatalysis proceeds with

the formation of acetic acid and formic acid as intermediate products.24,25 To promote

the multi-electron reduction of oxygen, the presence of protons (H+) is of great

significance. The equilibrium reaction between carboxylic acid and water proceeds as

shown in eq. 5, producing carboxylic species and H+.

……(5)

Thus, this equation indicates that the presence of water molecules is of great importance

to the production of organic peroxide, and suggests that an optimum ethanol:water ratio

exists. The method of Pinkernell et al. was used to estimate the amount of organic

peroxide produced in the system. The cross reactivity between MTS and hydrogen

peroxide also was investigated, in this case using a concentration of hydrogen peroxide

(120 ppm) that was more than twice that produced by WO3 after 12 h of irradiation. As

can be seen in Figure 6, the hydrogen peroxide did not react with the MTS, indicating

that the detected MTSO was derived from the presence of organic peroxide only.

Next, the sporicidal performance of a suspension of WO3 in 8:2 (v/v) ethanol:water

after 12 h of visible light irradiation was compared with that of the commonly used,

commercially available PAA disinfectant. Because the amount of generated organic

peroxide was estimated (see above) to be ca. 13.5 ppm, 15 ppm PAA was used in the

comparison. Figure 9 shows that the bacterial spores were completely inactivated

(~6-log decrease in survival) in the solution treated with WO3 after 4 h of illumination.

In contrast, 15 ppm commercial PAA disinfectant solution did not provide appreciable

killing (<0.5-log decrease) in the same interval. Testing at higher PAA concentrations

revealed that the WO3 system exhibited a sporicidal performance equivalent to 1500

ppm PAA, indicating that our method has a sporicidal effect 100 times that of PAA.

Additionally, I note that the suspension treated with WO3 contained not only acetic acid

142

but also formic acid. I therefore presume that peroxyformic acid, which is a stronger

oxidant than PAA, was produced in the presence of WO3. The generation of

peroxyformic acid may account for the remarkable sporicidal performance of the

suspension treated with WO3 compared to that observed in the suspension treated with

the commercial PAA disinfectant.

Figure 9. Survival rate of B. subtilis spores for the indicated time under dark conditions

aftr the treatment, either of commercially available peracetic acid (PAA) solution at

various concentrations (15, 150, 500, 1000, or 1500 ppm) or of WO3 suspended in

ethanol:water solution (8:2, v/v) after 12 h of visible light irradiation. Photocatalyst: 25

mg, light source: Xe lamp (vis) with L-42 filter (λ> 420 nm), liquid-phase volume: 50

mL, density of B. subtilis spores: 2.0 × 106 CFU mL−1.

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In addition, I evaluated the survival rate of B. subtilis spores in a system containing

TiO2, which is (globally) the most widely used and applied photocatalyst. It is well

known that although TiO2 exhibits high photocatalytic activity, its conduction band level

is much more negative than the potential of the multi-electron reduction of oxygen. As a

result, the single-electron reduction of oxygen (O2 + e− = O2−, −0.284 V; O2 + H+ + e− =

HO2, −0.046 V) preferentially proceeds over TiO2 instead28–30 as shown in Figure 8.

Also, it was reported that O2− and HO2 can transfer to H2O2 through some subsequent

reactions.30–32 However, TiO2 should rapidly consume hydrogen peroxide producing

during irradiation as shown in Figure 10A, which suppressed increasing the

concentration of hydrogen peroxide. Hence, it seems that organic peroxide cannot be

generated via TiO2. The band edge of the TiO2 used in this work was observed at ca.

400 nm, as shown in Figure 1, indicating an estimated band gap of 3.1 eV. The crystal

structure of the TiO2 was a mixture of anatase and rutile phases (Figure 2B), and the

particle size was estimated to be ca. 20 nm (Figure 3B). The surface area of the TiO2

was found to be 54 m2 g−1, larger than that of the WO3.

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Figure 10. (A) Photocatalytic decomposition of hydrogen peroxide (100 ppm) and

formic acid (130 ppm) with TiO2 under UV light irradiation. Photocatalyst: 15 mg, light

source: black light (UV; 1 mW cm−2), liquid-phase volume: 30 mL. (B) Photocatalytic

decomposition of hydrogen peroxide (100 ppm) and formic acid (130 ppm) with WO3

under visible light irradiation. Photocatalyst: 15 mg, light source: Xe lamp (VIS; 110

mW cm−2), liquid-phase volume: 30 mL.

Figure 11 shows that the B. subtilis spores were not inactivated in aqueous ethanol

solution upon illumination with either UV or visible (λ> 420 nm) light in the presence

of TiO2, indicating that TiO2 is not suitable for the photocatalytic inactivation of

bacterial spores.

Figure 11. Survival rate of B. subtilis spores in the presence either of TiO2 suspended in

ethanol:water solution at indicated ratio (0:10, 1:9, 4:6, 7:3, 8:2; v/v) and illuminated

146

with UV light for the indicated time, or of TiO2 suspended in ethanol:water solution (8:2,

v/v) and illuminated with visible light for the indicated time. Photocatalyst: 25 mg, light

source: black light (UV), Xe lamp (VIS) with L-42 filter (λ > 420 nm), liquid-phase

volume: 50 mL, density of B. subtilis spores: 2.0 × 106 CFU mL−1.

Next, the quantitative amount of organic peroxide, hydrogen peroxide, acetic acid,

and formic acid produced in the presence of TiO2 in 8:2 (v/v) ethanol/water under UV

light irradiation was investigated. As shown in Figure 6, no MTSO peak detected,

indicating that the TiO2 did not produce organic peroxide. As shown in Figure 12, the

formation of acetic acid and formic acid was confirmed, indicating that the

photocatalytic oxidative reaction proceeded. In contrast, the production of hydrogen

peroxide was not observed over TiO2. These results revealed that WO3 is a more

suitable visible-light-driven photocatalyst for the inactivation of bacterial spores than

TiO2, and suggests that the presence of hydrogen peroxide is key to the production of

organic peroxide. In addition, Figure 12 shows that TiO2 only produced a small amount

of formic acid in comparison with WO3. The photocatalytic decompositions of formic

acid with TiO2 under UV light irradiation and with WO3 under visible light irradiation

were examined as shown in Figure 10. It was suggested that TiO2 rapidly decompose

formic acid rather than WO3, leading to the production of a small amount of formic acid

with TiO2. The efficient sporicidal performance obtained with a low concentration of

organic peroxide in this study resolves the odor problem and danger encountered with

traditional sporicidal disinfectants. Hence, the present method is superior not only for

the food and health industries, but also for use by the general public at home. This result

is of great significance in terms of the production of an efficient sporicidal disinfectant

from a safe chemical agent, aqueous ethanol, using not UV but visible light illumination.

One of the next goals of this sporicidal sterilization method is more rapid inactivation of

bacterial spores. If it could be achieved, this novel method is expected to become

prevalent owing to the possibility that the inactivation of bacterial spores may be

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realized simply by squirting an aqueous ethanol solution onto substrates coated with

photocatalyst under visible light.

Figure 12. Time-dependence of amount of organic peroxide, hydrogen peroxide, acetic

acid, and formic acid produced by TiO2 suspended in ethanol:water solution (8:2, v/v)

and illuminated with UV light for the indicated time.

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4-3 Conclusions The sterilization of bacterial spores were achieved by combination of

visible-light-driven WO3 photocatalyst, ethanol aqueous solution, and visible light

irradiation while TiO2 photocatalyst that is most used and applied photocatalyst could

not inactivate spores in ethanol aqueous solution even under UV light irradiation. It was

clarified that the sterilization of spores was caused by production of organic peroxide

that is strong oxidant. The presences of organic acid and hydrogen peroxide were

strongly required to produce the organic peroxide. Producing organic acid and hydrogen

peroxide were ascribed to the photocatalytic oxidative decomposition of ethanol and

reduction of oxygen, respectively. Although WO3 could produce organic peroxide, TiO2

did not produce organic peroxide because of no production of hydrogen peroxide, due to

rapid consumption of hydrogen peroxide with TiO2 during light irradiation. This

research is of great significance in terms of the production of an efficient sporicidal

disinfectant from a safe chemical agent, ethanol aqueous solution, using not UV but

visible light irradiation.

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4-4 Experimental section Materials

TiO2 and WO3 were purchased from Evonik and Sigma-Aldrich, respectively, and

used as received without any purification.

Characterization

Diffuse reflectance spectra were obtained using a UV-VIS spectrometer (JASCO

V-670) and were converted from reflection to absorbance using the Kubelka-Munk

formula. Crystal structures were determined using X-ray diffraction (XRD; RIGAKU

Ultima IV) with CuKα radiation. Particle morphologies were observed using a field

emission scanning electron microscope (FE-SEM; JEOL-7600F) accelerated at 15 kV.

Samples for FE-SEM were sputtered with 10 nm Au metal by sputtering device

(SANYU ELECTRON SC-701HMCII) before observation. Surface areas were

determined by the Brunauer-Emmett-Teller (BET) method using a gas adsorption and

desorption analyzer (MicrotracBEL; BELSORP-max) with nitrogen gas at 77 K.

Preparation of spores

Bacillus subtilis spores were used for evaluation of sporicidal activity. Vegetative

cells of B. subtilis (IAM12118T) were grown and sporulated by incubation on nutrient

agar (NA) medium for 10 days at 37 °C. The spores were collected with 10 mL

ultra-pure water. Lysozyme (100 mg) was added to the suspension to remove remaining

vegetative bacterial cell wall. The suspension then was incubated for 30 min at 37 °C

and washed three times with 10 mL ultra-pure water with harvesting by centrifugation at

3500 rpm. The formation of B. subtilis spores was confirmed by the Wirtz method using

malachite green and safranin stains. Additionally, no decrease in the survival rate of B.

subtilis spores was observed in ethanol/water (70:30, v/v), suggesting the formation of

spores (i.e., ethanol-resistant cells).

Evaluation of photocatalytic inactivation of spores

150

The density of spores was adjusted to 2.0 × 106 CFU mL−1. Photocatalyst (25 mg)

was added to glass containers containing 50 mL of spores suspended in the

ethanol/water solution. Photo-irradiation was carried out using a black light (2.0 mW

cm−2) for UV irradiation and a Xe lamp (110 mW cm−2) with L-42 cut-off filter (HOYA,

λ < 420 nm) under magnetic stirring for visible irradiation. After illumination for the

indicated time interval, dilutions of the suspension were plated to NA, and the plates

were incubated for 48 h at 37 °C. The spore survival rate was determined using the

colony counting method.

Comparison of sporicidal performance of commercially available PAA (15, 150, 500,

1000, and 1500 ppm) and suspension treated with WO3 in ethanol:water (8:2, v/v)

after 12 h visible light irradiation

The concentration of bacterial spores was adjusted to 2.0 × 106 CFU mL−1. Each

concentration of PAA was obtained by diluting commercially available 6% PAA

(Sigma-Aldrich) with ultra-pure water. Spore suspension was added to the solution of

PAA or the suspension of WO3 in ethanol:water (8:2, v/v) after 12 h of visible light

irradiation. After the irradiation, the WO3-treated suspension was centrifuged at 10000

rpm for 10 min to remove the WO3 powder. The sporicidal performance of the

treatments was evaluated as above.

Determination of organic acid, hydrogen peroxide, and organic peroxide levels

Levels of formic acid and acetic acid were quantified using a HPLC (Shimadzu;

LC-20AD) with a UV detector (Shimadzu; SPD-20A) at a wavelength of 210 nm. The

HPLC conditions were as follows: column 300 × 7.8 mm (Phenomenex; Rezex

ROA-Organic Acid), column temperature 60 °C, mobile phase 0.005 N sulfuric acid,

flow rate 0.5 mL min−1. The photocatalytically treated samples were diluted 10-fold

with ultra-pure water before loading to prevent deterioration of the column.

Hydrogen peroxide was detected using a HPLC (Hitachi; Chromaster) with a UV

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detector (Hitachi; Chromaster 5410) at a wavelength of 210 nm. The HPLC conditions

were as follows: column 250 × 4.6 mm (Hitachi; LACHROM 2 C18), column

temperature 25 °C, mobile phase 0.1% phosphoric acid, flow rate 1.0 mL/min.

Organic peroxide was detected using a HPLC (Hitachi; Chromaster) with a UV

detector (Hitachi; Chromaster 5410) at a wavelength of 254 nm. The HPLC conditions

were as follows: column LACHROM 2 C18, column temperature 40 °C, mobile phase

75% ethanol/25% water (v/v), flow rate 1.0 mL/min. The photocatalytically treated

samples (0.5 mL) were mixed with MTS ethanol solution (50 ppm, 0.5 mL) at 25 °C for

2 min. The concentration of organic peroxide was quantified using the area of the

MTSO peak.

152

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photocatalytic water treatment technology: A review. Water Res. 2010, 44 (10), 2997-3027. 15. Kudo, A.; Miseki, Y., Heterogeneous photocatalyst materials for water splitting. Chem. Soc. Rev. 2009, 38 (1), 253-278. 16. Yamaguchi, Y.; Terashima, C.; Sakai, H.; Fujishima, A.; Kudo, A.; Nakata, K., Photocatalytic Degradation of Gaseous Acetaldehyde over Rh-doped SrTiO3 under Visible Light Irradiation. Chem. Lett. 2016, 45 (1), 42-44. 17. Pinkernell, U.; Karst, U.; Cammann, K., Determination of Peroxyacetic Acid Using High-Performance Liquid Chromatography with External Calibration. Anal. Chem. 1994, 66 (15), 2599-2602. 18. Pinkernell, U.; Effkemann, S.; Karst, U., Simultaneous HPLC Determination of Peroxyacetic Acid and Hydrogen Peroxide. Anal. Chem. 1997, 69 (17), 3623-3627. 19. Alasri, A.; Roques, C.; Michel, G.; Cabassud, C.; Aptel, P., Bactericidal properties of peracetic acid and hydrogen peroxide, alone and in combination, and chlorine and formaldehyde against bacterial water strains. Can. J. Microbiol. 1992, 38 (7), 635-642. 20. Gehr, R.; Cochrane, D., PERACETIC ACID (PAA) AS A DISINFECTANT FOR MUNICIPAL WASTEWATERS: ENCOURAGING PERFORMANCE RESULTS FROM PHYSICOCHEMICAL AS WELL AS BIOLOGICAL EFFLUENTS. Proceedings of the Water Environment Federation 2002, 2002 (1), 182-198. 21. Sawaki, Y.; Ogata, Y., The Kinetics of the Acid-Catalyzed Formation of Peracetic Acid from Acetic Acid and Hydrogen Peroxide. Bull. Chem. Soc. Jpn. 1965, 38 (12), 2103-2106 21. Nosaka, Y.; Takahashi, S.; Sakamoto, H.; Nosaka, A. Y., Reaction Mechanism of Cu(II)-Grafted Visible-Light Responsive TiO2 and WO3 Photocatalysts Studied by Means of ESR Spectroscopy and Chemiluminescence Photometry. J. Phys. Chem. C 2011, 115 (43), 21283-21290. 22. Irie, H.; Miura, S.; Kamiya, K.; Hashimoto, K., Efficient visible light-sensitive photocatalysts: Grafting Cu(II) ions onto TiO2 and WO3 photocatalysts. Chem. Phys. Lett. 2008, 457 (1–3), 202-205. 23. Abe, R.; Takami, H.; Murakami, N.; Ohtani, B., Pristine Simple Oxides as Visible Light Driven Photocatalysts: Highly Efficient Decomposition of Organic Compounds over Platinum-Loaded Tungsten Oxide. J. Am. Chem. Soc. 2008, 130 (25), 7780-7781. 24. Nimlos, M. R.; Wolfrum, E. J.; Brewer, M. L.; Fennell, J. A.; Bintner, G., Gas-Phase Heterogeneous Photocatalytic Oxidation of Ethanol: Pathways and Kinetic Modeling. Environ. Sci. Technol. 1996, 30 (10), 3102-3110.

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•- and OH• Formed in TiO2 Aqueous Suspensions by Photocatalytic Reaction and the Influence of H2O2 and Some Ions. Langmuir 2002, 18 (8), 3247-3254. 29. Bielski, B. H. J.; Cabelli, D. E.; Arudi, R. L.; Ross, A. B., Reactivity of HO2/O-

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Chapter 5 Summary In my dissertation, I focused on “control of morphology of particle” and

“production of oxidation agent with strong oxidation power” and carried out the studies

on the effect of these factors on photocatalytic activity of environmental remediation.

In Chapter 2 focusing on “morphology of particle” in “control of morphology of

particle”, the photocatalyst was fabricated by combination of electrospray and

hydrothermal treatment developed by our research group. In 2-1 section, it was clarified

that TiO2 hollow particles were not effective morphology for bactericidal and

anti-phage performance, whereas it effectively decomposed organic compounds

(dimethyl sulfoxide) because of multi-scattering inside particles. In the bacterial case,

the size is quite similar to TiO2 hollow particles, resulting in the decreased contact

efficiency, and phage absorbs light in the wavelength of UV, preventing

multi-scattering inside of the hollow structure. This research revealed that the size scale

of the reactants should be considered in the fabrication of TiO2 photocatalyst targeted

for application of photocatalytic environmental remediation.

In 2-2 section, visible-light-driven TiO2-WO3 hollow photocatalyst was fabricated

by electrospray, hydrothermal treatment and calcination, which was developed by our

research group. It showed higher photocatalytic oxidative decomposition of

acetaldehyde under visible light irradiation than pure TiO2 hollow photocatalyst. I

successfully fabricated the visible-light responsive photocatalyst using the present

simple method.

In Chapter 3 focusing on “milling treatment for improvement of surface area” in

“control of morphology of particle”, photocatalytic performance for environmental

remediation was evaluated with ground STO:Rh and STO:Rh,Sb photocatalysts. As a

result, the photocatalytic activity of decomposition of organic compounds and

anti-pathogenic performance of ground STO:Rh and STO:Rh photocatalysts improved

under visible light irradiation, indicating that the milling process is a suitable method

for the improvement of photocatalytic environmental remediation. Interestingly, the

156

photocatalytic anti-phage performance of ground STO:Rh photocatalyst drastically

improved in comparison with ground STO:Rh,Sb photocatalyst under visible light

irradiation. Diffuse reflectance spectra revealed that the valence state of Rh atom in

ground STO:Rh photocatalyst changed from Rh3+ to Rh4+ after visible light irradiation,

while that of ground STO:Rh,Sb photocatalyst did not change. The presence of Rh4+ of

ground STO:Rh photocatalyst under visible light irradiation possibly contributed to the

specific anti-phage performance.

In Chapter 4 focusing on “production of oxidation agent whose oxidation power is

strong”, the inactivation of bacterial spores was successfully achieved with WO3

photocatalyst under visible light irradiation, whereas TiO2 did not exhibit the sporicidal

performance under visible light and UV light irradiation. The products of organic

peroxide, hydrogen peroxide and organic acid were observed in the solution of WO3

photocatalyst after photocatalytic reaction using HPLC measurement. On the other hand,

the presences of organic peroxide and hydrogen peroxide were not confirmed with TiO2

photocatalyst. Since the conduction band level of TiO2 was more negative than that of

WO3, the single-electron reduction of oxygen preferentially proceeded rather than

multi-electron reduction of oxygen, resulting in less production of hydrogen peroxide.

In this dissertation, I have found not only the novel phenomenon but also

application of photocatalytic environmental remediation by strategies of “control of

morphology of particle” and “production of oxidation agent with strong oxidation

power”. I strongly believe that the findings of my research contribute to develop the

application of photocatalytic environmental remediation.

157

Publication list Main papers



Visible Light Irradiation.Chem. Lett. 2016, 45 (1), 42-44.

2. Yamaguchi, Y.; Shimodo, T.; Usuki, S.; Torigoe, K.; Terashima, C.; Katsumata, K.-i.;

Ikekita, M.; Fujishima, A.; Sakai, H.; Nakata, K., Different hollow and spherical TiO2

morphologies have distinct activities for the photocatalytic inactivation of chemical and

biological agents. Photochem. Photobiol. Sci. 2016, 15 (8), 988-994.

3. Yamaguchi, Y.; Shimodo, T.; Chikamori, N.; Usuki, S.; Kanai, Y.; Endo, T.;

Katsumata, K.-i.; Terashima, C.; Ikekita, M.; Fujishima, A.; Suzuki, T.; Sakai, H.;

Nakata, K., Sporicidal performance induced by photocatalytic production of organic

peroxide under visible light irradiation. Sci. Rep. 2016, 6, 33715.

4. Yamaguchi, Y.; Liu, B.; Terashima, C.; Katsumata, K.; Suzuki, N.; Fujishima, A.;

Sakai, H.; Nakata, K, Fabrication of Efficient Visible Light Responsive TiO2-WO3

Hollow Particle Photocatalyst by Electrospray Method. Chem. Lett. 2016, 46 (1),

122-124.

158

Additional research products Patent

Award

1. Dissertation award in master grade, Department of Pure and Applied Chemistry,

Faculty of Science and Technology, Tokyo University of Science, March 2014.

2. Presentation award, The 96th Chemical Society of Japan Annual Meeting, Japan,

Kyoto, March, 2016.

3. Poster award, The 13th Research Progress Meeting of Specific Research Grant in

Tokyo University of Science, Japan, Tokyo, June, 2016.

159

Acknowledgements

At first, I would like to owe my deepest gratitude to Professor Hideki Sakai for

educating me how to live as a researcher and giving me a wonderful opportunity to

study this significant PhD research. His professional guidance and evocative

suggestions greatly contributed to the success of my project in Tokyo University of

Science.

I am deeply grateful to Dr. Akira Fujishima, administrator of Tokyo University of

Science, and Professor Masahiko Ikekita for giving me a precious opportunity to study

my research in Photocatalysis International Research Center (PIRC).

I am greatly indebted to Professor Kazuya Nakata, Professor Baoshun Liu,

Professor Chiaki Terashima, Professor Ken-ichi Katsumata, Professor Tomonori Suzuki

and Dr. Norihiro Suzuki, Photocatalysis International Research Center (PIRC) in Tokyo

University of Science, for giving me various significant advices, discussions and

guidances in my research. Nothing could be achieved in my research without their

superb supports.

I owe a very important debt to Professor Masahiko Abe, Professor Kenichi Sakai,

Dr. Takeshi Endo and Dr. Kanjiro Torigoe, Tokyo University of Science, for giving me a

many meaningful advices and supports in my research.

I would like to offer my special thanks to Professor Akihiko Kudo, Tokyo

University of Sicence, giving me a technical supports fabricating the various

photocatalyts and a lot of advices in my research projects.

160

I appreciate the significant technical supports of Dr. Yoshihiro Kanai in my

research. I learned from him how to live as a researcher. I could not overcome my

doctoral life without his heartwarming advises.

I am really grateful to Ms. Yuko Shibayama, Ms. Yuko Yoshihara and Dr.

Rizwangul Ibrahim, PIRC, supporting my projects. I could not concentrate on my

research without their supports.

In addition, I would like to thank all past and present members of Sakai-Sakai

group, Abe-Sakai group, Ikekita-Nakata group, and PIRC in Tokyo University of

Science.

Finally, I really wish to express the sincerest gratitude to my relatives, my sister,

my parents and grandparents throughout my life. If they had not support me, I could not

have accomplish my research.

I am going to live the rest of my life, not forgetting that I am living owing to

various precious supports from many people.

Yuichi Yamaguchi

March 2017

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