Study on development of visible light active photocatalyst g c3n4 comoo4 for removal of antibiotic and inactivation of antibiotic resistant bacteria

Technologies to remove antibiotics and inactivate antibiotic-resistant bacteria residues in wastewater ...9 2.2.1.. Removal efficiency of synthesized materials with tetracycline antibiot

VIETNAM NATIONAL UNIVERSITY, HANOI

VIETNAM JAPAN UNIVERSITY

NGUYEN THI THU HUONG

VIETNAM NATIONAL UNIVERSITY, HANOI

VIETNAM JAPAN UNIVERSITY

NGUYEN THI THU HUONG

I have read and understood the plagiarism violations I pledge with personal honor that this research result is my own and does not violate the Regulation on prevention of plagiarism in academic and scientific research activities at VNU Vietnam Japan University (Issued together with Decision No 700/QD-ĐHVN dated 30/9/2021 by the Rector of Vietnam Japan University)

Author of thesis

Nguyen Thi Thu Huong

I would also like to extend my sincere gratitude to my co-supervisor – Associate Professor Nguyen Minh Phuong for her precise advice and feedback during my research progress Her insightful suggestions have contributed greatly to this master’s thesis

I would like to convey my special appreciation to Associate Professor Kasuga Ikuro, who also guided me through this research with critical suggestions and provided me with his connections so I could conduct my bacterial study

I must also thank Dr Takemura Taichiro for providing me the chance to carry out molecular biology experiments at NIHE-Nagasaki Friendship Laboratory I must also thank all the staff at NIHE-Nagasaki Friendship Laboratory, especially Duong san for their guidance and useful advice during my internship there

This work is fully supported by the project with the code number of VJU.JICA.21.03, from VNU Vietnam Japan University, under the Research Grant Program of Japan International Cooperation Agency

I would like to acknowledge lecturers at the Master’s Program in Environmental Engineering - VNU Vietnam Japan University for giving constructive criticism to improve the quality of my research

Last but not least, the warmest thanks also go to my classmates, my lab mates, as well

as staff at Vietnam Japan University, with whom I have the pleasure to work while doing the thesis

Sincerely thank

TABLE OF CONTENTS

LIST OF TABLES i

LIST OF FIGURES ii

LIST OF ABBREVIATIONS iii

CHAPTER 1: INTRODUCTION 1

1.1 Research background 1

1.2 Research significance 1

1.3 Research objectives 2

1.4 Thesis structure 3

CHAPTER 2: LITERATURE REVIEW 4

2.1 Issue of antibiotic and antibiotic-resistant bacteria residues in wastewater 4

2.1.1 Issue of antibiotic residues in wastewater 4

2.1.2 Tetracycline antibiotic 5

2.1.3 Issue of antibiotic-resistant bacteria residues in wastewater 6

2.1.4 Escherichia coli (E coli) antibiotic-resistant bacteria 7

2.2 Technologies to remove antibiotics and inactivate antibiotic-resistant bacteria residues in wastewater 9

2.2.1 Physical technologies 9

2.2.2 Biological technologies 10

2.2.3 Chemical technologies 11

2.3 g-C3N4 and CoMoO4 photocatalyst 14

2.3.1 g-C3N4 photocatalyst 14

2.3.2 CoMoO4 photocatalyst 16

2.4 Development of g-C3N4/CoMoO4 heterostructure photocatalyst 17

CHAPTER 3: MATERIALS AND METHODOLOGY 19

3.1 Chemicals and apparatus 19

3.2 Photocatalyst preparation 19

3.2.1 Synthesis of g-C3N4 19

3.2.2 Synthesis of CoMoO4 20

3.2.3 Synthesis of g-C3N4/CoMoO4 20

3.3 Photocatalyst characterization 21

3.3.1 Scanning electron microscopy (SEM) 21

3.3.2 Energy-dispersive X-ray analysis (EDX) 22

3.3.3 Brunauer-Emmett-Teller analysis (BET) 24

3.3.4 X-ray powder diffraction analysis (XRD) 25

3.3.5 Fourier transform infrared spectroscopy (FTIR) 26

3.3.6 UV–vis diffuse reflectance spectroscopy (UV-DRS) 28

3.3.7 Photoluminescence spectroscopy (PL) 29

3.4 Experimental setup 31

3.4.1 Photocatalytic removal of tetracycline antibiotic 31

3.4.2 Photocatalytic inactivation of E coli antibiotic-resistant bacteria 32

3.4.3 Determination of photocatalyst's pH point of zero charge 34

3.4.4 Reactive oxygen species trapping experiments 34

3.5 Statistical analysis 34

CHAPTER 4: RESULTS AND DISCUSSION 36

4.1 Optimization of photocatalyst synthesis conditions 36

4.2 Characterization of synthesized materials 37

4.2.1 Scanning electron microscopy (SEM) 37

4.2.2 Energy-dispersive X-ray analysis (EDX) 38

4.2.3 Brunauer-Emmett-Teller (BET) analysis 39

4.2.4 X-ray powder diffraction analysis (XRD) 40

4.2.5 Fourier transform infrared spectroscopy (FTIR) 41

4.2.6 UV–vis diffuse reflectance spectroscopy (UV-DRS) 42

4.2.7 Photoluminescence spectroscopy (PL) 43

4.3 Removal efficiency of synthesized materials with tetracycline antibiotic 44

4.3.1 Enhancement of tetracycline removal efficiency of g-C3N4/CoMoO4 composite 44 4.3.2 Effect of photocatalyst dosage on tetracycline removal efficiency 45

4.3.3 Effect of pH on tetracycline removal efficiency 46

4.3.4 Effect of initial pollutant concentration on tetracycline removal efficiency 48

4.4 Inactivation efficiency of synthesized materials with E coli bacteria 49

4.5 Proposed photocatalytic mechanism 50

CHAPTER 5: CONCLUSION AND RECOMMENDATIONS 53

5.1 Conclusion 53

5.2 Recommendations 54

REFERENCES 55

LIST OF TABLES

Table 2.1 g-C3N4 and CoMoO4-based heterojunction photocatalyst 18

Table 3.1 Optimization of preparation conditions of g-C3N4/CoMoO4 composite 21

Table 4.1 Surface area and total pore volume of the synthesized materials 39

1 LIST OF FIGURES

Figure 2.1 (a) Structure and (b) speciation diagram of tetracycline antibiotic 6

Figure 2.2 Schematic diagram of the photocatalytic oxidation process of organic pollutants in the aqueous environment 13

Figure 2.3 Structure of graphitic carbon nitride 15

Figure 2.4 Crystal structure of cobalt molybdate 16

Figure 3.1 SEM instrument 22

Figure 3.2 EDX instrument 24

Figure 3.3 BET instrument 25

Figure 3.4 XRD instrument 26

Figure 3.5 FTIR instrument 28

Figure 3.6 UV-DRS instrument 29

Figure 3.7 PL instrument 30

Figure 3.8 Standard calibration curve of Tetracycline 32

Figure 4.1 Change in tetracycline removal efficiency of synthesized materials at different preparation conditions 36

Figure 4.2 Effect of pristine mixing ratio on antibiotic removal efficiency 37

Figure 4.3 SEM image of the synthesized a) g-C3N4, b) CoMoO4 and 38

Figure 4.4 EDX spectrum of the synthesized a) g-C3N4, b) CoMoO4 and c) g-C3N4/CoMoO4 composite 39

Figure 4.5 EDX elementary mapping of the synthesized a) g-C3N4, b) CoMoO4 and c) g-C3N4/CoMoO4 composite 39

Figure 4.6 XRD patterns of the synthesized g-C3N4, CoMoO4, and g-C3N4/CoMoO4 composite 40

Figure 4.7 FTIR spectra of the synthesized g-C3N4, CoMoO4, and g-C3N4/CoMoO4 composite 41

Figure 4.8 UV-vis diffuse reflectance absorption spectra of the synthesized g-C3N4, CoMoO4, and g-C3N4/CoMoO4 composite 42

Figure 4.9 Tauc plot of the synthesized g-C3N4, CoMoO4, and g-C3N4/CoMoO4 composite 43

Figure 4.10 PL spectra of the synthesized g-C3N4, CoMoO4, and g-C3N4/CoMoO4 composite 44

Figure 4.11 Tetracycline removal efficiency of the synthesized g-C3N4, CoMoO4, and g-C3N4/CoMoO4 composite 45

Figure 4.12 Effect of photocatalyst dosage on tetracycline removal efficiency 46

Figure 4.13 Effect of pH condition on tetracycline removal efficiency 47

Figure 4.14 pH point of zero charge of the synthesized g-C3N4/CoMoO4 composite 47 Figure 4.15 Effect of tetracycline's initial concentration on the removal efficiency 48

Figure 4.16 E coli inactivation efficiency with different dosages of photocatalyst 49

Figure 4.17 Effect of different scavengers on the photocatalytic efficiency of the synthesized 50

Figure 4.18 The proposed photocatalytic mechanism for degradation of antibiotics and antibiotic-resistant bacteria 51

2 LIST OF ABBREVIATIONS

AMR: Antimicrobial resistance

AOP: Advanced Oxidation Processes

ARB: Antibiotic-resistance bacteria

ARGs: Antibiotic-resistance genes

BET: Brunauer-Emmett-Teller

EDX: Energy dispersive X-ray

E coli: Escherichia coli

FTIR: Fourier transform infrared spectroscopy

HOMO: Highest occupied molecular orbital

LUMO: Lowest occupied molecular orbital

PL: Photoluminescence spectroscopy

SEM: Scanning electron microscopy

UV-DRS: UV–vis diffuse reflectance spectroscopy

WHO: World Health Organization

WWTP: Wastewater treatment plants

XRD: X-ray powder diffraction analysis

1 CHAPTER 1: INTRODUCTION

1.1 Research background

Antibiotics are one of the crucial discoveries of the last century that changed the treatment of a variety of infections in a significant way However, in recent years, the unprecedented issue of antibiotic residues in environmental matrices has been receiving great attention from both academia and the public Several studies reported critical resistance of several kinds of antibiotics in surface and groundwater, sediments, soils, and even foodstuffs (Bombaywala et al., 2021; Daghrir & Drogui, 2013; K Wang et al., 2021) These residues may cause various consequences in the ecological system such as antibiotic-resistant bacteria and human health effects e.g allergy, mutation, and reproductive disorder (Monahan et al., 2021) Most critically, the abuse

of antibiotics has directly resulted in the prevalence of antibiotic-resistant bacteria and antibiotic-resistant genes The rapid growth of antibiotic-resistant bacteria threatens the efficiency of medicines, which have revolutionized medicine and saved millions of lives In 2021, World Health Organization (WHO) listed antimicrobial resistance (AMR) as one of ten global health issues that urgently need collective efforts to tackle (WHO, 2020)

However, reports have shown that both antibiotics and antibiotic-resistant bacteria can not be completely removed by conventional wastewater treatment plants (WWTP), which mostly deploy physical and biological technologies (Baquero et al., 2008; Manoharan et al., 2022; K Wang et al., 2021) Hence, it is vital to develop supplement treatment technologies for the efficient removal of those critical pollutants in wastewater

1.2 Research significance

In recent years, photocatalytic materials have emerged as a highly efficient and economic strategy for both antibiotic treatment and disinfection in the water environment Many photocatalysts such as TiO2, ZnO2, In2O3, and CdSe have been studied for the treatment of organic pollutants in the environment (Manaia et al., 2018; Noor et al., 2021; Philippopoulos & Nikolaki, 2010)

Recently, graphitic carbon nitride (g-C3N4) has drawn great attention from academia as

a facile, metal-free, non-toxic, and photochemically stable semiconductor, which can drive photo-oxidation reactions even under visible light However, the photocatalytic efficiency of g-C3N4 is limited by its high rate of recombination between photo-induced electrons and holes, low light absorption efficiency, and low specific area (Huang et al., 2014; Ismael, 2020; Kong et al., 2019) Hence, it is critical and essential

to study the synthesis of g-C3N4-based photocatalyst to tackle its current drawbacks and deploy its high potential in organic pollutant removal in the aqueous environment CoMoO4 - a transition metal molybdate with narrow band gap energy (2.1 – 2.8 eV), great redox activity, and strong catalytic electrochemical characteristics - is also considered a promising visible-light-driven photocatalyst (Umapathy & Neeraja, 2016; Veerasubramani et al., 2014; Zagorac et al., 2017) The CoMoO4 photocatalyst has been successfully applied for organic pollutant removal as well as bacterial inactivation (Adabavazeh et al., 2021; Feizpoor et al., 2019; Gao et al., 2019; Umapathy & Neeraja, 2016) However, the application of CoMoO4 for organic compound photodegradation is limited because of the low potential energy of the conduction band and fast recombination of photo-induced electrons and holes

With their potential yet drawbacks to act as an individual photocatalyst, together with the compatibility in their bandgap energy, g-C3N4 and CoMoO4 could be coupled to promisingly form a heterojunction displaying enhanced photocatalytic activity

To date, there has been no research into the development of this target composite for

the treatment of tetracycline antibiotics and E coli antibiotic-resistant bacteria Hence,

this study is conducted to fill the research gap in the development of the high potential photocatalyst material to address the urgent issues of antibiotic and antibiotic-resistant bacteria residues in wastewater

1.3 Research objectives

This research is implemented to achieve the following objectives:

(1) Synthesize g-C3N4/CoMoO4 heterojunction photocatalyst exhibiting high photocatalytic activity under visible light

(2) Characterize the synthesized g-C3N4/CoMoO4 heterojunction photocatalyst (3) Investigate the synthesized material’s photocatalytic removal efficiency with the Tetracycline antibiotic

(4) Investigate the synthesized material’s photocatalytic inactivation efficiency

with E coli antibiotic-resistant bacteria

1.4 Thesis structure

This thesis is structured into 5 chapters The main contents of each chapter are presented below:

Chapter 1 introduces the research context and describes the significance of the topic,

as well as points out the research objectives

Chapter 2 highlights the issues of the antibiotic and antibiotic-resistant bacteria

residue in wastewater, current technologies to address these issues, and introduces the photocatalytic oxidation process utilizing photocatalyst as an emerging strategy

Chapter 3 describes the materials and methods to prepare and characterize the target

photocatalysts Experimental designs to investigate the photocatalytic activity of the synthesized photocatalyst in removing antibiotic and antibiotic-resistant bacteria are also elaborated

Chapter 4 presents and discusses the results obtained from all the characterization

analyses and experiments conducted

Chapter 5 concludes the key results of this research and recommendations for further

research to be conducted

2 CHAPTER 2: LITERATURE REVIEW

2.1 Issue of antibiotic and antibiotic-resistant bacteria residues in wastewater

2.1.1 Issue of antibiotic residues in wastewater

An antibiotic is a type of antimicrobial substance active against bacteria It is the most important type of antibacterial agent for fighting bacterial infections through mechanisms that either kill or inhibit the growth of bacteria (Waksman, 1947)

In the decades after the discovery of antibiotics, it has been recognized that the use of antibiotics in human health, veterinary medicine, and agriculture is associated to the contamination of several environmental compartments Depending on the class, 40–90% of the prescribed antibiotic dosage is excreted in the feces and urine as the parent chemical - in the active form, ultimately damaging soils, waterways, plants, etc (Baquero et al., 2008)

When existing in the environment, antibiotic residues can have negative effects on biota at different levels and cause various human health effects e.g allergy, mutation, and reproductive disorder through the consumption of contaminated food and water (M Wang et al., 2017)

In addition, the misuse of antibiotics boosts bacteria or genes that are resistant to antibiotics and may be passed from the environment to people It is hypothesized that greater antibiotic usage may fail the treatment in human medicine, lengthening sickness duration, morbidity, and death (World Health Organization, 2018)

A significant concern arises from the improper disposal of unused medicinal products

by discharging them into the sewage systems WWTPs can treat antibiotic-tainted wastewaters, but traditional treatment plants are incapable of removing antibiotics entirely The antibiotic classes most often found in WWTPs include macrolides, sulfonamides, trimethoprim, quinolones, and tetracyclines (J Wang et al., 2020) The WWTP's sludge and ultimate effluent may include antibiotics In addition, the sludge might be utilized as a manure fertilizer, and the effluent is released into surface water (Baquero et al., 2008)

The situation is also critical in Vietnam by the careless use of antibiotics in several facets of human health care and agriculture Additionally, the absence of effective wastewater treatment systems makes it possible for antibiotic residues in the aquatic environment to spread widely Other activities also emit a significant amount of antibiotics into the water environment, in addition to the common source of antibiotics from aquaculture (Binh et al., 2018)

2.1.2 Tetracycline antibiotic

Tetracycline is a frequently used antibiotic due to its broad-spectrum activity against both Gram-positive and negative bacteria, fungus, rickettsia, and parasites Tetracycline is the second most manufactured and ingested antibiotic in the world due

to its cheap cost, low toxicity, broad-spectrum action, and ability to be administered orally It is the most common antibiotic used in human medicine, veterinary medicine, and as a feed ingredient in agriculture (Daghrir & Drogui, 2013)

Based on the preparation techniques, tetracycline is classified into three categories based on its manufacturing methods: natural, semi-synthetic, and synthetic tetracycline Natural tetracyclines like tetracycline, oxytetracycline, and chlortetracycline are

obtained through the fermentation of Streptomyces sp bacteria, while

semi-synthetically produced tetracyclines and artificially prepared tetracycline like are obtained through the chemical transformations of the precursor compounds (Borghi & Palma, 2014)

Tetracycline has three pKa values i.e the negative base -10 logarithm of the acid dissociation constant (Ka) of a solution At pH 3.3, 7.7, and 9.7, respectively, these pKa values comprise protonation of oxygen bound in C3 site, protonation of dimethyl functional group in C4 site, and protonation of oxygen bound in C10 and C12 sites Hence, tetracycline exits in three forms depending on the pH of the solution: cationic

at pH 3.3, zwitterionic at pH range of 3.3 – 7.7, and anionic at pH > 7.7 (see Figure 2.1) As a result, the increased pH will amplify tetracycline's negative charge; when

pH exceeds 7.0, 25% of tetracycline residues in the anionic form tetracycline is soluble in alcohols but not in organic solvents (Po-Hsiang et al., 2009) The chemical structure and speciation diagram of tetracycline is given in Figure 2.1:

Figure 2.1 (a) Structure and (b) speciation diagram of tetracycline antibiotic

(Gopal et al., 2020) The high hydrophilic and low volatile nature of tetracycline allows it to persist in the environment for longer periods which can facilitate gene mutation or gene lateral transfer in aquatic microbes, resulting in "antibiotic resistance genes" (ARGs), which harm is produced by ARGs is larger than the chronic toxicity of anthelmintics, and which have unfavorable characteristics such as persistence and ease of movement, translation, and dissemination between bacteria (Kümmerer, 2009)

2.1.3 Issue of antibiotic-resistant bacteria residues in wastewater

Antibiotic resistance is defined as bacteria that are not inhibited by the usually achievable systemic concentration of an agent with a normal dosage schedule and/or fall in the minimum inhibitory concentration ranges Hence, the bacteria survive and continue to multiply causing more harm Widespread use of antibiotics promotes the spread of antibiotic resistance Bacterial susceptibility to antibacterial agents is achieved by determining the minimum inhibitory concentration that inhibits the growth of bacteria (Guilfoile & Alcamo, 2007)

Likewise, multiple drug resistance is defined as the resistance to two or more drugs or drug classes Cross-resistance is the acquisition of resistance to one antibiotic that confers resistance to a different antibiotic that the organism has not been exposed to Today, the majority of antibiotics used to treat hospital infections are no longer effective against at least 70% of the germs that cause them (Wang et al., 2020) Some organisms are resistant to all approved antibiotics and can only be treated with experimental and potentially toxic drugs (Bisht et al., 2009)

ARBs and ARGs can currently be discovered in a wide range of habitats, such as wastewater treatment systems, hospital effluents, and pharmaceutical effluents These locations are unusual for having high densities of bacteria and low levels of antibiotics, which encourages the release of ARB and ARGs into the environment (Levy & Marshall, 2004) Multiple factors contribute to the spread of ARB and ARGs, including the selection pressure that antibiotics exert even at very low antibiotic doses and the acquisition of resistance by horizontal gene transfer from other bacteria The global emergence and spread of antibiotic-resistant bacteria drive humans to face the lack of available effective treatment for the infection caused by antibiotic-resistant bacteria This issue is so serious that it is predicted to bring around 10 million deaths

in 2050 (O’Neill, 2014)

Wastewater treatment plants receive sewage from various sources, including hospitals and households, representing important sources of antibiotics as well (Nagulapally et al., 2009; Zhang et al., 2009)

The WWTP's current technologies hardly ever effectively reduce or eradicate all bacteria Instead, the biological wastewater treatment process provides perfect temperature, favorable conditions for the multiplication of bacteria and antibiotic-resistant genes, and increased occurrence of horizontal gene transfer Antibiotics are continuously in contact with susceptible bacteria at low, sub-inhibitory doses, which may put antibiotic-resistant bacteria under selective pressure As a result, the main pathway for the spread of antibiotic resistance in the aquatic environment is through WWTP effluents

The predominant bacterial species that are found in WWTPs include E coli, Coliforms, Enterococci, Enterobacteria, Pseudomonads, and Acinetobacter, vancomycin-resistant Enterococcus spp, and methicillin-resistant Staphylococcus aureus (Noor et al., 2021)

2.1.4 Escherichia coli (E coli) antibiotic-resistant bacteria

E coli is a bacterium with a special position in the microbiological world due to its

potential to cause serious infections in both humans and animals as well as its important function in the innate microbiota of the various hosts The possible

transmission of virulent and resistant E coli between animals and humans through

various pathways, including direct contact, contact with animal excretions, or via the food chain is causing critical concern in both academia, government, and the public (Blount, 2015)

E coli strains can be generally classified into 3 groups: (i) harmless commensal strains

that are a part of the normal microbiota of the gastrointestinal tract, strains that cause diarrheal 12 intestinal diseases, and (iii) strains that cause extraintestinal infections

(Chang et al.) E coli are the main agent causing diarrheal diseases, which accounts

for around 9% of children's death worldwide (Poolman, 2016)

Additionally, E coli is a significant source of resistance genes that may be the cause of

medical treatment failures in both humans and animals Over the past few decades, a

rising number of resistance genes have been found in E coli isolates, and many of these resistance genes were acquired through horizontal gene transfer (Laurent et al., 2018) E coli participates in the enterobacterial gene pool as both a donor and a

recipient of resistance genes, allowing it to both take-up and provide other bacteria resistance genes (Blount, 2015)

Antimicrobial resistance in E coli is generally regarded as one of the biggest problems

in both people and animals on a global scale and should be taken seriously as a public

health issue (Reinthaler et al., 2003) Multidrug resistance in E coli can cause

difficult-to-treat infections in animals, but it also serves as a substantial and common reservoir of resistance determinants for most antimicrobial families in a wide range of animal species, including humans

Even though the various routes by which resistant E coli isolates are transmitted from

animals to humans have not yet been elucidated and their relative significance quantified, some data suggests that the food chain may be involved because these bacteria are frequently found colonizing foods sold at retail in numerous nations and continents (Laurent et al., 2018)

2.2 Technologies to remove antibiotics and inactivate antibiotic-resistant bacteria residues in wastewater

2.2.1 Physical technologies

This approach includes the collection and removal of organic pollutants from wastewater, utilizing absorbent materials to evacuate hazardous impurities, thus purifying and disinfecting the effluent in the process Numerous studies have been conducted on the use of various adsorbents, including activated carbon, metal, and silica gel, to remove antibiotics and ARB from wastewater (Lu et al., 2020)

However, natural or modified adsorbents have disadvantages in terms of economic feasibility, applicability, removal efficiency, and regeneration Among them, due to its great adsorption capability, activated carbon has been extensively utilized to remove antibiotics and ARB by physical adsorption Activated carbon powder is an amorphous carbon material with a large surface area and a high porosity Zhang et al (2016) assessed the effectiveness of powdered activated carbon in eliminating six typical groups of 28 antibiotics from water, namely tetracyclines, macrolides, chloramphenicols, penicillins, sulfamides, and quinolones The results reveal that powdered activated carbon displayed greater antibiotic adsorption capability The greatest recorded removal effectiveness was 91.9% in deionized water under optimal circumstances with a dose of 20 mg/L of powdered activated carbon and a contact duration of 120 minutes The absorption efficiency of antibiotics may be impacted by a number of factors, including the type of activated carbon, the initial concentration of target compounds, pH, temperature, and adsorbent concentration, even though the use

of adsorption filtration by adsorbents like activated carbon is suitable for eliminating high doses of many antibiotic compounds (Le-Minh et al., 2010)

Hiller et al (2019) also studied on the removal of related ARGs from digested swine wastewater The system can remove 95% of antibiotics and ARGs from swine effluent Although adsorption is important in wastewater treatment because it results in the fast mass transfer of pollutants from one medium to another, it does not lead to the total elimination or biodegradation of contaminants In addition, the disposal of contaminated adsorbents is one of the most severe issues that restrict the application of

this approach to efficiently remove antibiotics and ARB in wastewater (Sharma et al., 2016)

Prior research has revealed that biological treatment units might be hotspots for ARGs propagation due to the presence of abundant nutrients and dense bacteria in activated sludge, which may facilitate horizontal gene transfer across various bacterial species (Guo et al., 2017)

2.2.3 Chemical technologies

Chemicals are used during wastewater treatment in an array of processes to expedite disinfection These chemical processes, which induce chemical reactions, are called chemical unit processes, and are used alongside biological and physical cleaning processes to achieve various water standards Current chemical technologies to remove antibiotics and ARB from wastewater include chlorination, ozonation, and photocatalysis

Chlorination is the process of adding chlorine to drinking water to kill parasites, bacteria, and viruses Chlorine is typically utilized in disinfection units of water and wastewater refineries due to its inexpensive cost as an oxidant Antibiotics may oxidize during chlorination and transform into inactive small molecules The studied antibiotics are reported to be removed at a rate of 50 - 90% by chlorination technology (Acero et al., 2010) For bacteria in general, a log-3 inactivation of several commonly found strains in wastewater was obtained with contact time ranging from 0.032 to 42.9

mg min/L (Helbling & VanBriesen, 2007) However, this method holds several limitations such as the need for a high concentration of free chlorine, the need to adjust the pH, and the possibility of creating the byproducts, even more, harmful than the initial compounds (Acero et al., 2010)

Ozonation is a chemical water treatment technique based on the infusion of ozone into water Ozone is a gas composed of three oxygen atoms (O3), which is one of the most powerful oxidants Ozone is a highly reactive oxidant that demonstrates selectivity for moieties commonly found in antibiotic molecules The oxidation of compounds is a process that occurs thanks to the emission of ozone that spontaneously transforms into oxygen; the process consists of joining three oxygen molecules to form ozone, but the third oxygen molecule is very unstable, and it binds with the polluting particles in water until they are eliminated (Carbajo et al., 2015) Complete treatment of antibiotics and ARB obtained by the ozonation process has been reported by some studies (Cuerda-Correa et al., 2019; Iakovides et al., 2019) The results of other research, however, showed that although this method's degrading efficiency is great, the mineralization and purification of wastewater appear to be low In fact, this method

is not recognized as suitable for the treatment of environments contaminated by

pharmaceuticals because it depends on pH fluctuations and requires expensive equipment and high energy (Esplugas et al., 2007; Lüddeke et al., 2015)

Photocatalysis is also one of the advanced oxidation processes (AOP) deploying photocatalyst materials that generate strong oxidation compounds as being stimulated With its desirable properties such as being recyclables and having low toxication risk, photocatalysts have been intensely studied for the application of treating not antibiotics and ARBs in wastewater (Baaloudj et al., 2021; Chen et al., 2022; Hiller et al., 2019; Zhu et al., 2020)

The background theory of the mechanism behind the photocatalytic oxidation process has been thoroughly studied In photocatalyst, the chemical reaction usually happens due to the transfer of electrons from the valance band (VB) to the conduction band (CB) As the number of orbitals (N) in the VB, or the highest occupied molecular orbital (HOMO) and the CB, or also the lowest unoccupied molecular orbital (LUMO) increases, the energy needed to transfer the electrons from the VB to the CB will decrease (J Zhang et al., 2018)

In this process, upon irradiation of the light with the energy equal to or more than the bandgap of the semiconductor photocatalyst, the electrons at the surface of the photocatalysts are excited and jump from its VB to the CB, resulting in the formation

of the holes These photo-generated electrons and holes, in the aqueous environment, catalyze the formation of reactive oxygen species, commonly superoxide anion (.O2-) and hydroxyl radical (.OH-), which drive the degradation of organic pollutants into carbon dioxide, water, and less harmful products

According to Hofmann et al., these photo-generated electrons and holes can recombine

by releasing heat for 10 to 100 nanoseconds The photocatalyst's low quantum efficiency is due to this recombination of electrons and holes If these charge-carrier species are separated by the inclusion of suitable scavengers or the incorporation of part of the trap sites on the surfaces as a result of creating flaws, surface adsorbents, or other sites, this process can be greatly sped up (Wenderich & Mul, 2016)

If sufficient time can be provided for the holes and electrons before their recombination, these photo-generated charge carriers transfer to the surface of the

photocatalyst and can undergo the charge transfer to initiate the redox reactions with the pollutants adsorbed on its surface A VB hole, denoted as h+, possesses a strong oxidation power by having a redox potential ranging from +1.0 to +3.5 V (measure vs normal hydrogen electrode (NHE) at room temperature), depending on the photocatalyst and the conditions such as pH Therefore, the existence of holes is crucial for the photocatalytic destruction of organic molecules on the catalyst surface The oxidation can occur directly through h+ connected to surfaces before it is trapped within the particle or at its surface, or indirectly through interaction with the surface-bound hydroxyl radical The process can be illustrated in Figure 2.2:

Figure 2.2 Schematic diagram of the photocatalytic oxidation process of organic

pollutants in the aqueous environment

(Zhang et al., 2018) The generated surface hydroxyl radicals have the potential to totally mineralize the contaminants by oxidizing them To prevent the buildup of extra charges within the catalytic particles, the photoexcited electrons must also respond Therefore, effective electron removal can improve the photocatalytic oxidation of contaminants

2.3 g-C 3 N 4 and CoMoO 4 photocatalyst

2.3.1 g-C 3 N 4 photocatalyst

Graphitic carbon nitride or g-C3N4 is a non-metallic semiconductor material, the polymer form of carbon nitride has a layered structure like graphite This material has the advantage of having small bandgap energy, about 2.7 eV, which can work under

sunlight, can synthesize large quantities, and is stable g-C3N4 is becoming increasingly important due to the theoretical predictions of their unusual properties and promising applications ranging from photocatalysts, and heterogeneous catalysts, to substrates

Recently, a variety of nanostructures and nanocapillary g-C3N4 materials have been developed for a variety of new applications With many attractive properties such as chemical and thermal stability, low density, non-corrosion, and impermeable to water, g-C3N4 is gradually becoming one of the most promising materials The chemical stability of g-C3N4 was discovered by Gillan, and research has shown that g-C3N4 is almost insoluble in water, ethanol, toluene, diethyl ether, and Tetrahydrofuran This may be due to Van der Waals's forces between the overlapping layers

Graphitic carbon nitride is known to have a graphite-like layered structure (see Figure 2.3) In the same monolayer of g-C3N4, both triazine and the original tri-s-triazine or s-heptazine are considered structural units due to their high stability Under normal conditions, g-C3N4 has the most stable allotrope, g-C3N4 consists of layers stacked along the axis forming graphite faces These graphite faces are composed of hexagonal rings of triazine structural units (C3N3) In this structure, the bonds between the rings are fastened by the nitrogen atom With that said, g-C3N4 is known to have a layered structure like graphite and is also proven by X-ray powder diffraction(XRD) results in studies In the same monolayer of g-C3N4, both the triazine and the original tri-s- triazine or s-heptazine are thought to be the structural units Later, however, only tri-s-triazine was considered as the structural unit due to its high stability

Figure 2.3 Structure of graphitic carbon nitride

(Cao & Yu, 2014) With its low bandgap (~2.7 eV), g-C3N4 can drive photo-oxidation reactions even under visible light (Mamba & Mishra, 2016; C Zhang et al., 2019; Zhao et al., 2014) However, the pure g-C3N4 has some drawbacks such as its low redox potential and high rate of recombination between photo-induced electrons and holes, which dramatically limits its photocatalytic efficiency Several strategies have been investigated, including modification of the material’s size and structure (Darkwah &

Ao, 2018; Xu et al., 2018), nonmetal and metal doping (Dai et al., 2020; Oh et al., 2017; Z.-T Wang et al., 2019), and coupling with other photocatalysts (Nithya & Ayyappan, 2020; Ren et al., 2019; Tian et al., 2015; Tian et al., 2020; Ye et al., 2013) For example, Liu et al (Liu et al., 2017) improved bulk g-C3N4‘sperformance in terms

of Rhodamine B degradation from 30% to 100% by synthesizing mesoporous g-C3N4

nanorods through the nano-confined thermal condensation method Dai et al (Dai et al., 2020) doped g-C3N4 with Cuthrough a thermal polymerization route and acquired

a degradation rate of 90.5% with norfloxacin antibiotic Nithya and Ayyappan (Nithya

& Ayyappan, 2020) synthesized hybridized g-C3N4/ZnBi2O4 for reduction of nitrophenol and reached an optimal removal efficiency of 79.0%

4-Among all, the construction of heterostructure photocatalysts by coupling g-C3N4 with other semiconductors seems to be an effective strategy to prevent electron and hole recombination, hence improving photocatalytic efficiency for contaminant treatment

2.3.2 CoMoO 4 photocatalyst

CoMoO4 is also considered a promising visible-light-driven photocatalyst with a narrow band gap energy (2.1 – 2.8 eV), great redox activity, and strong catalytic electrochemical characteristics (Umapathy & Neeraja, 2016; Veerasubramani et al., 2014; Zagorac et al., 2017) To date, several searches have studied the structure of cobalt molybdate Cobalt molybdate (CoMoO4) exists in three polymorphic modifications The α-CoMoO4 modification crystallizes in the monoclinic space

group C2/m, No.12, as well as the β-CoMoO4 phase but, is clearly distinguished from the α-phase by tetrahedral coordination of Mo6+ atoms (Smith, 1962)

In previous studies, CoMoO4 has been synthesized by various methods such as precipitation, sol-gel, solid-state reaction, hydrothermal, and complete evaporation (Adabavazeh et al., 2021; Gao et al., 2019; Rosić et al., 2018; Umapathy & Neeraja, 2016)

Figure 2.4 Crystal structure of cobalt molybdate

(Rosić et al., 2018) The CoMoO4 photocatalyst has been successfully applied for organic pollutant removal as well as bacterial inactivation (Adabavazeh et al., 2021; Feizpoor et al., 2019; Gao et al., 2019; Umapathy & Neeraja, 2016) However, the application of CoMoO4 for organic compound photodegradation is limited because of the low potential energy of the conduction band and fast recombination of photo-induced electrons and holes Some studies have been conducted to address this issue, for

instance, Umpathy and Neeraja (Umapathy & Neeraja, 2016) enhanced the photocatalytic activity of CoMoO4 with 4-chlorophenol degradation by preparing CoMoO4/TiO2 nanocomposites, which showed higher efficiency (97.5%) as compared

to that of pure CoMoO4 (88.0%)

2.4 Development of g-C 3 N 4 /CoMoO 4 heterostructure photocatalyst

When using g-C3N4 as a hybridization agent with CoMoO4 material, the photocatalytic activity of the material can be improved by forming a conjugated photocatalyst system The conduction band of g-C3N4 is more negative than that of CoMoO4 (-1.24 eV and 0.67 eV respectively), while CoMoO4 possesses a relatively positive valance band (2.63 eV) compared to the conduction band of g-C3N4, would theoretically facilitate the electron transition within the coupled photocatalyst to prolong the electron-hole separation When the conjugated hybrid system between g-C3N4 and CoMoO4 is formed, the electrons in the conduction band of the CoMoO4 catalyst can move easily

to the valence band of the g-C3N4 photocatalyst, thereby limiting the inhibits the recombination of electrons and holes in both catalysts In the conventional catalytic system of g-C3N4 and CoMoO4, electrons will move from the conduction band to the valence band Therefore, the recombination of photogenerated electrons and holes occurs rapidly leading to a sharp decrease in conversion efficiency

In detail, under the illumination of sunlight, both CoMoO4 and g-C3N4 can be excited

to generate pairs of electrons and holes The photoinduced electrons in the conduction band of CoMoO4 tend to transfer and recombine with the holes in the valence band of g-C3N4 In this way, the larger number of photogenerated electrons accumulated in the conduction band of g-C3N4 can reduce the adsorbed O2 to form more •O2- Meanwhile, the photo-generated holes left behind in the valence band of CoMoO4 can oxidize the adsorbed H2O to give •OH Therefore, the photocatalytic activity of the g-

C3N4/CoMoO4 system would be significantly increased, leading to the decomposition

of organic compounds by •O2- and •OH reactive species

Till now, some previous studies have been conducted on the synthesis of g-C3N4 and CoMoO4-based heterojunction photocatalyst, with the target pollutant and performance summarized in Table 2.1 Notably, Habibi-Yangjeh et al reported synthesizing visible-light-driven g-C3N4/Fe3O4/CoMoO4 photocatalysts with removal

efficiency over rhodamine B, methylene blue, and fuchsine of 62.9%, 72.8%, and 71.5% respectively Zhang et al further studied the photocatalytic ability of composite including g-C3N4 and a variation of CoMoO4 ratio for methylene blue treatment, where the optimal composite showed a 94.0% degradation rate However, in these previous works, influence factors on the synthesis of g-C3N4/CoMoO4 haven’t been systematically studied

To date, there has been no research into the development of this target composite for

the treatment of tetracycline antibiotic and E coli antibiotic-resistant bacteria

Table 2.1 g-C3N4 and CoMoO4-based heterojunction photocatalyst

g-C3N4/Fe3O4/CoMoO4

Rhodamine B (RhB)

Methylene blue (MB)

RbB: 72%, 60

(2020) MB: 84%, 60 min

TiO2/g-C3N4

Ciprofloxacin antibiotic 93.4%, 60 min

Hu et al (2020)

TiO2/CoMoO4/PANI Rhodamine B 99%, 90 min Feizpoor et

al (2019)

CoMoO4-Fe-g-C3N4 Methylene blue 99.5%, 90 min

Yangjeh et al (2018)

Habibi-g-C3N4-ZnBi2O4

4-Nitrophenol 4-Nitrophenol Nithya and

Ayyappan (2020)

3 3 CHAPTER 3: MATERIALS AND METHODOLOGY

3.1 Chemicals and apparatus

In this research, melamine (Tianjin Damao Chemical Reagent Factory, China, 99.5%) was used as the precursor for g-C3N4 synthesis To prepare CoMoO4, cobalt nitrate (Guangdong Guanghua Sci-Tech Co., Ltd, China, 98.5%), and sodium molybdate (Dezhou Jinmao Chemical Co., Ltd, China, 99.0%) were used as precursors Ethanol absolute (Duc Giang Chemicals Group JSC Co., Vietnam, 99.7%) was used to wash the synthesized materials

Tetracycline powder (AK Scientific Inc., United States, 95.3%) was used to prepare

solutions, and p-benzoquinone (Xiya Reagent Co., Ltd, China, 99.0%) together with

isopropyl alcohol (Xilong Scientific Co., Ltd, China, 99.7%) was used as scavengers

in the reactive oxygen species trapping experiments Sodium hydroxide (Xilong Scientific Co., Ltd, China, 96.0%) and hydrochloric acid (Xilong Scientific Co., Ltd, China, 38.0%) were deployed to adjust the pH in the reactor

In microbiological experiments, Trypto-Soy Broth (Eiken Chemical Co., Ltd, Japan)

was used as the cultivation medium of E Coli, and Tryptone Bile X-glucuronide

(TBX) agar (Merck Millipore Corp., Germany) supplemented with Tetracycline powder (AK Scientific Inc., United States, 95.3%) was used to prepare agar for enumeration of bacteria colonies Phosphate Buffer Saline (PBS) powder (Wako Pure Chemical Industries, Ltd., Japan) was used to prepare buffer solution in the

photocatalytic reactor with the presence of E coli bacteria, isolated from To Lich river,

3.2.2 Synthesis of CoMoO 4

For the preparation of pristine CoMoO4, cobalt nitrate and sodium molybdate precursors were firstly dissolved in distilled water under the magnetic stirring condition for 30 minutes to obtain 1 M solutions of each chemical

After that, the prepared cobalt nitrate solution was slowly added to sodium molybdate solution under a constant stirring condition for 1 hour

Then, the obtained mixture was transferred into a stainless-steel autoclave for the hydrothermal process at 180 oC for 6 hours The product was then centrifuged to remove the supernatant and dried at 60 oC for 24 hours to get the purple CoMoO4

powder

3.2.3 Synthesis of g-C 3 N 4 /CoMoO 4

The g-C3N4/CoMoO4 composite was synthesized by the hydrothermal - calcination method Firstly, 1 g of the prepared g-C3N4 was added into distilled water and magnetically stirred for 30 minutes The mixtures of cobalt nitrate and sodium molybdate were obtained by stirring certain amounts of these precursors in distilled water for 30 minutes

Then, the portions of prepared g-C3N4 were added to the mixtures to obtain the weight ratios of g-C3N4/CoMoO4 of 6:4 and kept being stirred for another 1 hour The final mixtures were transferred into a 100 ml autoclave and reacted at 180℃ for different hydrothermal times of 3 hours and 6 hours The final samples were centrifuged and washed with distilled water and ethanol 2 times

Finally, the samples were dried, and finally, the dried products were heated in a Muffle furnace at different calcination temperatures of 300°C, 400°C, and 500 °C for 4 hours

to get the target composites

To investigate the effect of preparation conditions, including the hydrothermal time, calcination temperature, and mixing ratio of the two components, on the final material’s photocatalytic activities, these conditions were varied in each sample as shown in Table 3.1:

Table 3.1 Optimization of preparation conditions of g-C3N4/CoMoO4 composite

Sample name g-C 3 N 4 :CoMoO 4

mass ratio

Hydrothermal time

Calcination temperature

3.3.1 Scanning electron microscopy (SEM)

Scanning Electron Microscopy (SEM) is a technique that typically produces dimensional images of a sample by scanning it with a high-energy beam of electrons in

two-a rtwo-aster sctwo-an ptwo-attern SEM ctwo-an provide informtwo-ation on the microstructure of cotwo-ated surface, distribution of photocatalyst on the substrate surface, homogeneity, and morphology of particles in the coating In the SEM system, firstly, the primary electron beam is produced under a high vacuum and is scanned across the surface of a specimen When the electron interacts with the different atoms at the sample surface, a variety of signals produce information regarding the surface or elemental composition

of the sample (Pinto et al., 2018; Perret et al., 2005)

In operation, the sample is put on a stub and sputter-coated with gold before SEM investigation Sputter coating is required to acquire good results by providing the

samples under high voltage of SEM a protective layer A computer attached to the instrument manages and controls it By establishing a vacuum, the sample that is fastened to an exchanging rod and electron column is connected to the SEM After inserting the stub into the sample holder inside the electron column, the sample position must be kept straight, and the electron column must be closed After using the controller to set the necessary working distance and magnification, the SEM photos are taken for further analysis (Perret et al., 2005)

In this research, the surface morphology of the as-synthesized materials is observed using an SEM TM 4000 Plus (Hitachi High-Technologies Corp., Japan) (Figure 3.1), conducted at VNU Key Laboratory of Advanced Materials for Green Growth, University of Science, Vietnam

Figure 3.1 SEM instrument

3.3.2 Energy-dispersive X-ray analysis (EDX)

The elemental composition of the material is characterized by energy-dispersive X-ray (EDX) analysis, using a MisF+ instrument (Oxford Instruments plc., UK) coupled

with an SEM analyzer, located at VNU Key Laboratory of Advanced Materials for Green Growth, University of Science, Vietnam

When in operation, the specimen is bombarded with a high-energy electron beam during the analysis procedure The bombardment of electrons causes the atoms on the specimen surface to release X-rays Bremsstrahlung X-rays, also known as continuous X-rays, and characteristic X-rays are the two types of X-rays that come from the interaction

Bremsstrahlung X-rays are produced by the interaction of an incident electron with the specimen's atomic nuclei This takes the form of a background spectrum over which the particular X-ray spectra are overlaid Characteristic X-rays, on the other hand, are produced when high-energy electrons hit lower-energy state vacancies, resulting in the transition of higher-energy electrons to lower-energy state vacancies The energy difference between the higher and lower energy states correlates to the energy of emitted X-rays and is affected by the specimen's properties The X-ray lines in the X-ray spectrum represent the distinctive X-rays The shell holding the inner void is indicated by capital Roman letters such as K, L, or M, whilst the Greek letters and numbers indicate the group to which the line belongs in decreasing order from to and the line's intensity in decreasing order from 1 to 2, respectively

An X-ray detector captures both Bremsstrahlung and typical X-ray emissions, which are shown as a spectrum of X-ray energy versus intensity The energy of the distinctive X-rays allows qualitative analysis to show the components that contribute

to the specimen, whilst the intensity of the corresponding X-rays allows quantitative analysis to reveal the concentration of the elements present (Bell & Garratt-Reed, 2003)

Figure 3.2 EDX instrument

3.3.3 Brunauer-Emmett-Teller analysis (BET)

To study the material’s pore structure, BET analysis was conducted with a BET Nova Touch LX4 (Quantachrome Corp., USA) (Figure 3.3) located at VNU Key Laboratory

of Advanced Materials for Green Growth, University of Science, Vietnam

The physical adsorption of gas molecules on a solid surface is served as the basis for BET analysis for the measurement of the specific surface area of materials Adsorption

is defined as the adhesion of atoms or molecules of gas to a surface The quantity of gas adsorbed is determined by the exposed surface, as well as temperature, gas pressure, and the intensity of the gas-solid interaction

Because of its excellent purity and strong interaction with most materials, nitrogen is often utilized in BET surface area analysis Due to the weak contact between gaseous and solid phases, the surface is chilled with liquid N2 to get measurable levels of adsorption The sample compartment is then gradually filled with known quantities of nitrogen gas Partially vacuum circumstances are used to produce relative pressures lower than atmospheric pressure There is no additional adsorption when the saturation pressure is reached, regardless of how high the pressure is raised Pressure transducers with high precision and accuracy detect pressure changes caused by the adsorption

process After the adsorption layers have developed, the sample is taken out of the nitrogen environment and heated, causing the adsorbed nitrogen to be released and quantified The information gathered is represented by a BET isotherm, which displays the quantity of gas adsorbed as a function of relative pressure

Figure 3.3 BET instrument

3.3.4 X-ray powder diffraction analysis (XRD)

An X-ray diffractometer is used to perform structural characterization on a wide range

of specimens from powder to thin films When using XRD, the constructive interference of monochromatic X-rays and a crystalline sample allows for the analytical investigation of the crystal structure (Bunaciu et al., 2015) In fact, the detailed mechanism of an XRD follows the Bragg equation, which is shown in Equation 1:

In the equation, n is an integer, λ is the characteristic wavelength of the X-ray source,

d is the interplanar spacing between rows of atoms in the specimen, and θ is the angle