Synthesis of reduced graphene Oxide (rgo) for the removal of Tetracycline from aqueous Solutions

Effect of antibiotic residues ... Regulations on antibiotic content in water ... Tetracycline pollution in water ... Technologies of the treatment of antibiotics in water ... Filtration [r]

VIETNAM NATIONAL UNIVERSITY, HANOI

VIETNAM JAPAN UNIVERSITY

HA THI MY TRINH

SYNTHESIS OF REDUCED GRAPHENE OXIDE (rGO) FOR THE REMOVAL OF TETRACYCLINE FROM AQUEOUS

SOLUTIONS

MASTER'S THESIS

VIETNAM NATIONAL UNIVERSITY, HANOI

VIETNAM JAPAN UNIVERSITY

HA THI MY TRINH

SYNTHESIS OF REDUCED GRAPHENE OXIDE (rGO) FOR THE REMOVAL OF TETRACYCLINE FROM AQUEOUS

SOLUTIONS

MAJOR: ENVIRONMENTAL ENGINEERING

CODE: 8520320.01

RESEARCH SUPERVISORS:

Dr TRAN DINH TRINH

Dr NGUYEN THI AN HANG

Hanoi, 2020

i

ACKNOWLEDGEMENTS

This work could not have been completed without the collaboration and help

of many people whom I want to thank

First of all, I would also like to extend my deepest gratitude to Dr Tran Dinh Trinh, who inspired me to develop invaluable insight into a new field to me I’m also deeply indebted to Dr Nguyen Thi An Hang, who provided me with relentless support and constructive advices

Many thanks to Mrs Dao Thi Huong – the laboratory technician – as well as staffs of the Master’s Program in Environmental Engineering for all practical instructions and useful contributions Thanks also go to my classmates and teammates, who enthusiastically supported me during this study

Finally, I cannot begin to express my thanks to my family and friends for their patience and support until I finished this work

I’d like to acknowledge the assistance of the staffs from the Academic, Research and Development Promotion Department of VNU Vietnam Japan University as well as Japan International Cooperation Agency for guiding and supporting me to complete my thesis

Thank you all for everything

Ha Noi, August 7th, 2020

Student

Ha Thi My Trinh

ii

Acknowledgements i

List of figures v

List of tables vi

List of abbreviations vii

Introduction 1

Chapter 1 Literature review 4

1.1 Antibiotics pollution 4

1.1.1.Definition, classification and sources of antibiotics 4

Sources of antibiotics 5

Application of antibiotic in human and veterinary dedicines 6

1.1.2 Occurrence of antibiotics in water and environmental effects 8

Occurrence of antibiotics in water 8

Effect of antibiotic residues 8

1.1.3 Regulations on antibiotic content in water 9

1.1.4 Tetracycline pollution in water 10

1.2 Technologies of the treatment of antibiotics in water 11

1.2.1 Filtration and sorption processes 11

1.2.2 Photodegradation and oxidation 12

1.2.3 Biodegradation 12

1.2.4 Other techniques 13

1.2.5 Technologies applied for the treatment of TC in water 14

1.3 Synthesis and application of rGO in antibiotic adsorption 14

1.3.1 Synthesis of rGO 15

Chemical reduction 15

Thermal reduction 15

Solvothermal/hydrothermal reduction 16

Other methods 16

1.3.2 Application of rGO in antibiotic adsorption 17

Chapter 2 Materials and methods 18

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2.1 Materials 18

2.2 Methods 18

2.2.1 Literature review 18

2.2.2 Sample analysis in laboratory 18

Characterization of materials 18

a) Fourier-transform infrared spectroscopy 18

b) Energy-dispersive X-ray spectroscopy 20

c) Scanning Electron Microscopy 21

d) X-ray diffraction 22

e) Surface area and pore volume calculation 23

f) pH point of zero charge 25

Determination of Tetracycline concentration 25

2.2.3 Data calculation 26

a)Removal efficiency 26

b) Adsorption capacity 26

c) Kinetic parameters 27

d) Isotherm parameters 27

e) Thermodynamic study 28

2.2.4 Statistical analysis 28

2.3 Experiment setup 29

2.3.1 Material synthesis 29

2.3.2 Factors influencing the efficiency of TC adsorption 29

a)Contact time 29

b)pH 30

c) Dosage of rGO 30

d) Initial concentration of Tetracycline 30

2.3.3 Isotherm tests 30

2.3.4 Kinetics tests 30

2.3.5 Thermodynamic tests 31

Chapter 3 Results and discussion 32

3.1.Material characterization 32

iv

3.1.1 Fourier-transform infrared spectroscopy 32

3.1.2 Energy-dispersive X-ray spectroscopy 33

3.1.3 X-ray diffraction 34

3.1.4 Surface area and pore volume 35

3.1.5 pH point of zero charge 37

3.2.Adsorption study 38

3.2.1 Factors influencing TC adsorption 38

a) Contact time 38

b) pH 39

c) Dosage 40

d) Initial concentration 41

e) Temperature 42

3.2.2 Adsorption isotherms 43

3.2.3 Adsorption kinetics 45

3.2.4 Adsorption thermodynamics 46

Conclusions and recommendations 48

Conclusions 48

Recommendations 48

References 50

Appendix 58

v

LIST OF FIGURES

Figure 1.1 Pathways of antibiotics into water 6

Figure 1.2 Global antibiotic consumption in livestock 2010 7

Figure 1.3 Legislation on antibiotics as growth promoters 7

Figure 2.1 FT-IR 4600 Jasco 20

Figure 2.2 Principle of EDX measurement 21

Figure 2.3 JSM-IT100/JED-2300 Analysis Station Plus, JEOL 21

Figure 2.4 MiniFlex 600 23

Figure 2.5 TriStar II Plus, Micromeritics 25

Figure 2.6 Calibration curve for Tetracycline 26

Figure 3.1 FT-IR result comparison of GO and rGO 32

Figure 3.2 SEM images of (a) graphite and (b) rGO 33

Figure 3.3 XRD results of GO and rGO 35

Figure 3.4 (a) N2 adsorption and desorption isotherms, (b) Types of physisorption isotherms, and (c) Types of hysteresis loops (IUPAC) 36

Figure 3.5 Pore size distribution of rGO obtained from DFT method 36

Figure 3.6 The plot of ΔpH versus pHi 38

Figure 3.7 Effect of contact time on TC adsorption by rGO 39

Figure 3.8 Effect of pH on TC adsorption by rGO 40

Figure 3.9 Effect of dosage effect on TC adsorption by rGO 41

Figure 3.10 Effect of initial concentration on TC adsorption by rGO 42

Figure 3.11 Effect of temperature on TC adsorption capacity of TC of rGO 43

Figure 3.12 Comparison of experimental data and modeled data on adsorption isotherms 43

Figure 3.13 Plot of ΔG0 against temperature (K) 46

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LIST OF TABLES

Table 1.1 Antibiotics classification according to chemical structure 4

Table 1.2 Chemical properties of tetracycline 10

Table 3.1 IR Spectrum by frequency range 33

Table 3.2 C/O ratio comparison of GO and rGO 34

Table 3.3 Properties of materials used for TC removal in previous studies 37

Table 3.4 Langmuir and Freundlich isotherm parameters on TC adsorption 44

Table 3.5 First- and Second-order kinetic parameters for TC adsorption 45

Table 3.6 Thermodynamic parameters of TC sorption process by rGO 47

Table S1 Antibiotic Resistance Alliance Science-Based PNEC Targets for Risk Assessments 58 Table S2 Removal of tetracycline antibiotics using different treatment processes 65

vii

LIST OF ABBREVIATIONS

EDX Energy-dispersive X-ray spectroscopy

FT-IR Fourier-transform infrared spectroscopy

IUPAC International Union of Pure and Applied Chemistry

1

INTRODUCTION

Since its discovery in 1928, antibiotics have played a very important role in human health protection and livestock industry It is estimated that millions of people have been saved from bacterial diseases (smallpox, cholera, typhoid fever, syphilis, etc.) thanks to antibiotics Antibiotics have revolutionized the treatment of bacterial diseases, which probably increase the average life span of Americans from 47 years

in the early 20th century to 78.8 years (Chain et al 2016) Antibiotics are also widely known as growth stimulant in fisheries, livestock and cultivation

However, besides the great health and economic benefits, antibiotics are also associated with negatively potential risks to humans and ecosystems Antibiotics were considered a persistent or “pseudo-persistent” substances because its speed of entering the environment is faster than that of its decomposition (Gothwal and Shashidhar, 2015), thus causing harms to the ecosystem One of the biggest problems was antibiotic resistance, which means bacteria were resistant to antibiotics that they are sensitive to Antibiotic abuse behavior and antibiotic production activities created

an environment with sub optimal antibiotics (a dose sufficient to kill bacteria) that helped "train" bacteria that develop antibiotic-resistant individuals Many studies warned the increasing occurrence of antibiotics in the environment (soil, sediment, groundwater, surface water, waste water) For instance, in China, the concentration

of quinolones in domestic sludges of wastewater treatment plants (WWTPs) was reported to be up to 29,647μg/kg, and 4,916ng/L in a municipal wastewater reclamation plant in Beijing (Gothwal and Shashidhar, 2015) In many cases, antibiotic levels even exceeded concentrations of industrial wastewaters For example, effluence of a wastewater treatment plant in India was reported to contain ciprofloxacin up to 31 mg/L (Larsson et al 2007) However, there are so far very few regulations for antibiotic concentrations in the water, so that the treatment plants usually do not take this kind of contaminants seriously

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Many studies have been carried out with the purpose of eliminating antibiotics from environments The preferred methods used today are: adsorption; degradation (photo-degradation, catalysis, bio-degradation); and oxidation processes (ozonation,

UV raddiation, ) In particular, adsorption is a commonly applied method thanks to its advantages such as high efficiency and ease of operation Many materials have been used for adsorption process such as activated carbon (Rivera-Utrilla et al., 2013) graphene oxide (Gao et al., 2012), activated sludge (Prado et al 2009) However, studies showed that these materials are time consumed to reach high efficiency Therefore, reducing processing time is one of major approaches to widespread the application of this method

Graphene is a single layer of carbon with thickness as a carbon molecule, dense with carbon molecules containing sp2 in honeycomb lattice (Fitzer, et al 1995, Choi et al., 2010; Ray, 2015) Graphene Oxide (GO) and Reduced Graphene Oxide (rGO) is oxidized graphene and has the presence of epoxy and hydroxyl functional groups; There were differences in functional groups or C:O ratio (Haubner et al., 2010) Although the presence of oxygen-containing groups made GO able to be hydrophilic which is suitable for water treatment, these functional groups weakened the π-electron activity linked to high fraction of sp3 C atoms which is important interaction for adsorption process (Ai et al., 2019) On the other hand, rGO with the large specific surface area (Nidheesh, 2017), significantly fewer functional groups than GO, and its powder form made it more efficient and economic

The main objectives of this research are to:

1) Develope rGO as adsorbent for the removal of TC from aqueous solutions 2) Investigate the effect of pH, adsorbent dose, initial TC concentration, contact time, and temperature on the adsorption of TC by rGO in the batch experiments 3) Estimate the TC adsorption capacity and removal efficiency of the as-synthesized rGO and compare to other adsorbents

4) Elucidate the TC adsorption mechanisms by rGO

The thesis consists of 4 main chapters as follows:

a) Chapter 1: Literature review: Overviews on antibiotics in the aspects of classification, current applications in human and veterinary, pollution situation,

c) Chapter 3: Results and discussion: Refers the main research results, including characterization of rGO, factors influencing the adsorption of TC, adsorption isotherms, and adsorption kinetics

d) Chapter 4: Conclusions and recommendations: Summarizes the main findings of the present study and suggests future perspectives

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CHAPTER 1 LITERATURE REVIEW

1.1 Antibiotics pollution

1.1.1 Definition, classification and sources of antibiotics

According to IUPAC, antibiotic is a substance produced by, and obtained from, certain living cells (especially bacteria, yeasts and moulds), or an equivalent synthetic substance, which is biostatic or biocidal at low concentrations to some other form of life, especially pathogenic or noxious organisms (Duffus, 2007)

Antibiotics can be classified based on their chemical structure, action mechanism, action spectrum, and the route of administration (Gothwal and Shashidhar, 2015) For further study of kinetics and mechanism, classification according to chemical structure is preferred in Table 1.1

Table 1.1 Antibiotics classification according to chemical structure (Kebede et al.,

3 Macrolactam antibiotics

Erythromycin Amphotericin

Oxygen-containing

Heterocyclic

antibiotics

1.Non-condensed(single) heterocycles 2.Condensed (fused) heterocycles

5

Sources of antibiotics

Antibiotics mostly demonstrated degradation performance <30% over 7 days, except of tetracycline is <44% over 7 days (Wilkinson et al 2019) Tetracycline is relatively inert within human body (Hirsch et al., 1999) Then, remaining antibiotic

in environment would become long-term contaminants and accumulate in biomass Antibiotic residues enter the environment primarily through human and animal waste and from manufacturing (Gelband and Miller-petrie, 2016) Antibiotics used in human and veterinary care are excreted which sometimes are used as agricultural fertilizer, most of times are discharged to environment Up to 90% of antibiotic dose can be excreted in animal urine and up to 75% in their feces Combined with expired industrial drugs, all of un-used antibiotic go into WWTPs and landfill Several studies reported that conventional treatment techniques cannot remove these persistent compounds completely, therefore significant amounts enter the aqueous environment, and end up to be influent of water treatment plants with inefficient treatment techniques

1.Furan derivatives 2.Pyran derivatives

Alicyclic

antibiotics

1.Cycloalkane derivatives 2.Small terpenes 3.Oligoterpene antibiotics

Streptovitacins

Aromatic

antibiotics

1.Benzene compounds 2.Condensedaromatic comp

3.Non-benzene aromatic

comp

Chloramphenicol Grisefulvin Novobiocin

Aliphatic

antibiotics

1.Alkane derivatives 2.Aliphatic carbocyclic acid

derivatives

Varitin

But over-use of these chemical compounds for both human and veteran care were attending concern due to its consequence Antibiotic use in humans was increasing worldwide for first-line and some last-resort antibiotics Only about 20% was used in hospitals, and non-prescription use of antibiotics is approximated 90% outside USA and Europe High-income countries tend to had higher in consumption

in per capita, but low and middle income countries had the greater increase in antibiotic use (Gelband and Miller-petrie, 2016)

Although antimicrobial consumption for human care was general decreased in 2018-2019 in EU (European Centre for Disease Prevention and Control, 2019), expanding use antimicrobials in livestock, one of consequences of increased meat demanding, is global trend With estimated 80 percent of all antibiotics consumed in the United States are used in food animals (Gelband and Miller-petrie, 2016) As Van Boeckel et al estimated global consumption of antimicrobials in food animal production was 63,151 (±1,560) tons in 2010, doubled in 2013 (~131.109 tons) and projected to reach 200,235 tons by 2030 (Van Boeckel et al., 2017, 2015)

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Figure 1.1 Global antibiotic consumption in livestock 2010 (Van Boeckel et al.,

2015)

Figure 1.3 Legislation on antibiotics as growth promoters illustrated according to

(Organization for Economic Co-operation and Development, 2015)

China, USA, India and Brazil were estimated to be top 4 countries in term of the most antibiotics consumption in livestock in 2010 and 2030, as well as largest increasing in antibiotic consumption Interestingly, comparing between Figure 1.2 antibiotic consumption and Figure 1.3 legislation on antibiotics as growth promoters, regions with highest antibiotic use in animal husbandry include all four difference legislation areas: ban, partial ban, voluntary withdrawal, and no ban This raises the question of effectiveness of the current solutions on policy of limiting the use of antibiotics

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1.1.2 Occurrence of antibiotics in water and environmental effects

Occurrence of antibiotics in water

Aus der Beek et al (2016) collected data of more than 1000 publications about pharmaceutical concentrations and found out that pharmaceuticals in the environment

is truly topic of global concern when it detected in 71 countries all around the world According to Bu et al (2013), sulfonamides, fluoroquinolones, macrolides, tetracyclines and other antibiotics were detected in surface water, rivers, sea waters and sediments with concentrations mostly at the level of µg/L up to dozens of µL They implied that the concentration of these contaminants is related with high population density cities Some extremely high concentrations were found in certain areas indicating local point sources or inefficiency of WWTPs to remove these contaminants in sewage (Bu et al., 2013) (David, 2017) reported presence of 18 pharmaceuticals in surface waters in lower Great Lakes with highest concentrations were noted at 0.79µg/L (ibuprofen), 0.55µg/L (naproxen), and 0.65µg/L (carbamazepine) This review also said that in Grand River watershed, southern Ontario, 14 in 28 surveyed pharmaceuticals were detected with highest concentrations belonged to monensin and sufamethazine which are used for livestock

In Tinkers Creek, 12 antibiotics were detected at 18 upstream and downstream from WWTP discharges to the mainstream In summarizing, pharmaceutical compounds are most detected near the discharge of WWTPs or agricultural production, in less diluted water bodies Wilkinson et al (2019)were detected 31 active pharmaceutical ingredients including antibiotics in tap, surface, wastewater treatment plant (WWTP) influent and WWTP effluent water collected globally

The statistics show that the more regions surveyed, such as Europe, USA, China, the more antibiotic contaminations are detected, also meaning that the less surveyed areas, like low- and middle-income countries are less contamination, but it

is data limitations

Effect of antibiotic residues

Previous studies revealed that some pharmaceutical compounds have concentrations in the range of chronic and acute toxicity in ecosystem

5-in a male fish fem5-inization experiment 5-in Canada (Kidd et al., 2007)

The widespread of antimicrobials use has created selection pressure, promote the process of formation and spread of antimicrobial resistant pathogens worldwide Resistant microbes and resistance genes can come forth and back between human, animals, food, water and the environment antibiotic resistance genes formed in animals due to growth stimulation can be transferred directly to humans via food route or indirectly through the environment It is estimated that about 33,000 death each year from drug-resistant bacteria in the EU-EEA (European Centre for Disease Prevention and Control, 2019) Resistant strain, H58, originated in Asia and Africa was increased from 7% to 97% prevalence rate in 5 years The resistance rates and trends are becoming global concerning 77% of E faecium healthcare-associated infections in the United States were resistant to vancomycin (Lahsoune et al., 2007) Among of high-income countries, United States was reported having higher rates of resistance to many Gram- positive bacteria, while resistance rates of Gram-negative bacteria were high in Southern and Eastern Europe In Asia, median resistance of K pneumoniae to ampicillin was 94%, and to cephalosporins, 84%, these contaminants

in Africa was 100 and 50%, respectively Multi-resistance appeared in 30% of strains

in Asia and 75% of strains in Africa (Gelband and Miller-petrie, 2016)

Antibiotic-resistant infections also contribute to the financial burden on healthcare systems Europe cost an estimated €1.5 billion annually, including healthcare expenditures and productivity losses (America and America, 2009); as United States is as much as $20 billion, and productivity losses total another $35 billion (Lahsoune et al., 2007)

1.1.3 Regulations on antibiotic content in water

At the present, EU and South Korea are only regions have completely banned the use of antibiotics as growth promoters, but EU is still one of the regions using

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most antibiotics for animal husbandry The reason is the large part of antibiotics are overlapping between use of disease prevention and growth promoters Due to limited quantitative data, toxicity assessments for antibiotics cannot lead to a standard of legal concentration in the environment, just Predicted No Effect Environmental Concentration (PNEC) (Table S1) values have been suggested as maximum levels in

an environmental matrix

1.1.4 Tetracycline pollution in water

Tetracycline antibiotics are one of the primarily antibiotics groups used for veterinary purposes, for human therapy and in agriculture sector as feed additive (Daghrir and Drogui, 2013) According to (Xie et al 2010; Cheng et al 2005) show that tetracycline antibiotics are ranked second in the production and usage of antibiotics worldwide and are ranked first in China Tetracycline, one of three most commonly used in the tetracyclines, showed that they are high water solubility (0.041mg/L) and low volatility (low log KOW) Physic-chemical properties of tetracycline are listed in Table 1.2 Therefore, these antibiotics likely stay persistence

in the aquatic environment Removal efficiency of conventional wastewater treatment plant was range from 12% to 80% (Daghrir and Drogui, 2013)

Table 1.2 Chemical properties of tetracycline (Rivera-Utrilla et al., 2013)

Volume

nm3

Cross area

nm2

Chemical Structure

Solubility g/L

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tracked in municipal waste water treatment plants effluence samples, in the concentration range of 280 to 540 ng/l in Tehran, Iran (Javid et al., 2016)

Ecological risk and potential toxic effects of tetracycline antibiotics residues

in the environment also paid attention As many antibiotics, their residues in environment promoted the formation and the development of antibiotic resistant microorganisms These antimicrobial agents may disturb the microflora of the human intestinal and increase the risk of certain infections (Heuer et al., 2009)

1.2 Technologies of the treatment of antibiotics in water

Techniques that based on physical reactions (sedimentation, suspension, adsorption/desorption, and gas transfer), biological transformations (biodegradation and co-metabolism), and chemical reactions (hydrolysis, oxidation, photo-degradation) play a important role in water treatment plants But as Gothwal and Shashidhar (2015) reviewed, removal efficiencies of conventional sewage treatment are found to vary substantially due to not designed to deal with new pollutants like antibiotics

scour/re-1.2.1 Filtration and sorption processes

Several studies reported that membrane processes like a reverse ultrafiltration system can reach ≈ 87.5 % efficiency in remove oxytetracycline from pharmaceutical wastewater (Li et al 2004) and 50~80% tetracyclines in synthesis water by nanofiltration (Koyuncu et al., 2008) But these studies also warned that high concentrations of organic substances present in the environment would hinder treatment performance

osmosis-In sorption process, the contaminant is transferred from liquid phase to solid phase A number of materials were applied to remove antibiotics, like activated carbon, clays, carbon nanotubes, ion exchange, sewage sludge, and waste oil sludge-derived adsorbents (Daghrir and Drogui, 2013; Gothwal and Shashidhar, 2015) Accordingly, the sorption of antibiotics onto soils reached equilibrium in few hours and was significantly dependent on various factors (pH, organic matter, and mineral content in soil) and behavior of antibiotic (molecular structure, functional groups)

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Sorption processes had high removal rate in synthesis water (up to 94%) while hardly exceeded 67% in real river water (Choi et al., 2008) Hence, this process was also interfered by organic matter in the same way as that in the filtration process, which has been aforementioned

1.2.2 Photodegradation and oxidation

Photolysis processes using light are simple, clean and less expensive Some antibiotics, which are sensitive with UV irradiations like tetracyclines, can be degraded at the rate of up to 90% Although some studies indicated the presence of intermediate compounds which can be more toxic than original one (Daghrir and Drogui, 2013) In the photodegradation process, parameters such as light source, pH, temperature, time, type of matrix, and the type/amount of impurities in the matrix (salts, organic compounds, soils, etc.) are important In addition, maintenance and electrical cost are usually considered as limiting factors

The process of oxidation involves the use of strong oxidizing agent such as hydroxyl radicals, ozone, potassium permanganate, and chlorine Advanced oxidation processes which enhance the formation of strong oxidant like free radicals are attracting attention from many projects Treatment efficiencies often reach 90%

in an extremely short time with a dosage material in mg/L Some wise used oxidation techniques is ozonation, chlorination, Fenton system Most of the studies are lack of information on by-products which formed during process Belongs on the minority, Li et al (2008) indicated that the first by-product of oxytetracycline was more toxic than the parent compound after ozonation It is possible by-products can be ecotoxic, and operation and optimization of oxidation process should be monitor through toxicity tests and determining transformation products

1.2.3 Biodegradation

The biodegradation of antibiotics by bacteria were rarely reported Alexy et al (2004) have been assessed of degradation of 18 antibiotics in the Closed Bottle Test, result is only a few were slightly degraded in 28 days, with the highest rate at 27% is

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Benzylpenicillin Those antibiotic also was only compound can be biodegradation up

to 87% of ThCO2 degradation in combined test (Gartiser et al., 2007) Many antimicrobials reported that can not be biodegradable or negligible biodegradable by bacteria On the other hand, degradation of antibiotics by fungus show a more delight result Studying in Trametes versicolor, Rodríguez-Rodríguez et al (2012) reported that Sulfapyridine was completely degraded, and sulfathiazole reach 88% after 96h incubation 72h hydraulic retention time successfully eliminated sulfapyridine, sulfamethazine and sulfathiazole in a mixture and their metabolites

1.2.4 Other techniques

Some common water treatment processes were evaluated for antibiotics removal by Adams et al (2002), included metal salt coagulation, excess lime/soda ash softening, powdered activated carbon sorption, ultraviolet photolysis, ion exchange, and reverse osmosis The results demonstrated that metal salt coagulation, excess lime/soda ash softening, ultraviolet photolysis, and ion exchange were not effective for antibiotic removal; powdered activated carbon sorption and reverse osmosis could be used to these compounds, but it only available at selected water treatment plants Conventional biological wastewater treatment processes were reported that it was effective for the removal of some antibiotics, but 10-

1000 ng/L concentrations in secondary treated effluents were occurred Application

of advanced treatment after conventional biological improved the removal of antibiotics, but operation and maintenance cost goes up (Le-Minh et al., 2010)

In conclusion, filtration and sorption provided high efficiency and economy in antibiotic removal, but do not destroy the pollutants but transfer it to another phase Photodegradation and oxidation recently proposed as alternative methods for elimination of persistent compounds due to highly degradation rate in shorter time while use very dosage of materials But it raised a caution about by-products which have limited information Biodegradation is favorable in term of economy and environmentally, but rarely applied for antibiotic treatment

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1.2.5 Technologies applied for the treatment of TC in water

Many treatments of tetracycline antibiotics in waters were suggested, included photocatalysis, oxidation, Fenton, adsorption, filtration, gamma radiation (table S2) Adsorption, most wised used in conventional wastewater treatment plants, was reported in previous studies that it was not so effective for TC removal Choi et al (2008) studied about adsorption using granular activated carbon in combination with coagulation process for TC antibiotics removal The results showed that removal rate

of tetracyclines could be up to 94 % from synthetic water, whereas did not exceed

67 % from river water Rivera-Utrilla et al (2013) reported about TC removal by activated carbons, which reached 375.4 mg/g adsorption capacity calculating by Langmuir model, whereas it took about 200h to achieve equilibrium Graphene oxide was reported that the maximum adsorption capacity calculated by Langmuir model could be reach 313 mg/g (Gao et al., 2012), but the materials could not be recoverable Therefore, the material that improved in adsorption capacity while reducing contact time and being able to recovery was necessary to widely apply adsorption technique

in large-scale for TC removal

1.3 Synthesis and application of rGO in antibiotic adsorption

Carbon-based materials always preferred in various fields due to their easy accessibility, hardness, and its ability to exist in various dimensions (Nidheesh, 2017) Graphene, nano-carbon material, is an promising material with many outstanding properties Graphene oxide (GO), a particular branch of graphene material, was recognized by properties which go along with the presence of several functional groups like epoxy, hydroxyl, carboxyl in the surface Obtained from either bottom-

up or top-down approaches, GO consisted exfoliated multi graphene sheets, then possess extremely high theory surface area which is an advantage for sorption technique Several polar functional groups in surface help GO very hydrophilic, but causing it is difficult for revoking after using In addition, electrically insulating character also occurred in result of these groups Therefore, reduced of graphene oxide (rGO) which has relatively surface area with less surface groups could be more favorable due to recoverability

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1.3.1 Synthesis of rGO

GO reduction methods included chemical reduction, thermal reduction, solvothermal/hydrothermal reduction, microwave and photo reduction, and photo catalyst reduction (Nidheesh, 2017) Confirmation of effective reduction process are characterized by Scanning electron microscopy (SEM), Energy-dispersive X-ray spectroscopy (EDX), Fourier-transform infrared spectroscopy (FT-IR), and X-ray diffraction (XRD) technique

Chemical reduction

Chemical reduction is the most popular method to synthesize rGO GO dispersion was reduced with reduction agents, oxygen functional groups are eliminated and the π-electron conjugation within the aromatic ring structure was partially restored Investigated reduction agents were L-ascorbic acid, D-glucose, tea polyphenol (Xu et al., 2015), NaBH4, Hydrazine hydrate (Alibeyli et al 2017), borohydrides (Chua and Pumera, 2013), oxalic acid (Song et al 2012), sodium hydrosulfite (Zhou et al., 2011) All of these studies reported that rGO have been synthesized with appropriate characteristics such as C/O ratio, oxygen functional groups, diffraction peaks Zhou et al (2011) studied about synthesis of PVA/graphite oxide (GO) nanocomposites films by using sodium hydrosulfite as reduction agent They included that a 40% increase in tensile strength and 70% improvement in elongation at break have been obtained with only the addition of 0.7 wt.% of reduced graphite oxide The highest conductivity achieved is 8.9× 10-3 S/m for the composites containing 3 wt.% rGO, and the conductivity achieved is comparable to those achieved with hydrazine Chemical reductions usually propose high reducing efficiency in a short time

Thermal reduction

The GO reduced by heating is known as thermal reduction by annealing This process is carried out in vacuum, or inert, or reducing atmosphere The rapid heating covers the oxygen-containing functional groups on surface to decompose into gases, thus creates pressure between layers The heating also increased the volume of gases stacked between graphene sheets, which lead to exfoliation phenomenon (Singh et al

16

2016) Li et al (2009) developed N-doped-rGO sheets through thermal annealing of

GO in ammonia They concluded that the high temperature is needed to achieve the better reduction of GO Schniepp et al (2006) also reported the similar result, which the C/O ratio would increase (from <7 to >13) when heating temperature increasing (<5000C to 7500C) Therefore, this method exhibited the high reduction performance but it also consumed a lot of energy for heating

Solvothermal/hydrothermal reduction

Solvothermal or hydrothermal reduction occurs at low temperature and high pressure inside the sealed container, basically reach supercritical condition of the solvent which turn into reduction agent Some solvents were studied is water-NH4OH (Johra and Jung, 2015), N,N-dimethylformamide (Wang et al., 2009), N-methyl-2-pyrrolidinone (Dubin et al 2014) rGO, which reduced by solvothermal treatment (1800C at 12h) N,N-dimethylformamide as solvent, was reported that had higher C/O ratio than chemical reduction by hydrazine reduction at normal pressure (Wang et al., 2009) On the other hand, reducing GO in N-methyl-2-pyrrolidinone results confirmed the formation of single sheets of the solvothermal rGO platelet The obtained C/O ratio was low, 5.15 at 2000C, and demands 10000C to achieve 6.36 (Dubin et al., 2014) Similar with thermal method, for water treatment, this method may costly due to required heating in long time

Other methods

Microwave reduction is wised used as rapid way to product rGO Using microwave irradiation to rise instantaneous internal temperature is very effective to shorten the reaction time and improves efficiency, although it may consume a massive amount of energy On the other hand, photo reduction using sunlight, ultraviolet light, and Krypton fluoride laser provided a additive free, simple, clean, and flexible approach to reduce GO But similar with microwave reduction, this method may costly due to high-energy radiation (Nidheesh, 2017) An alternative method is microbial reduction, which was report by (Chen et al., 2017) by using Azotobacter chroococcum, considering an eco-friendly method due to avoid the use

of chemical agents and agglomeration of rGO

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1.3.2 Application of rGO in antibiotic adsorption

Thanks to the outstanding properties like large surface area of 2D graphene sheets, porous structure, stability, conductivity, and flexibility, rGO and their modified material can be used as adsorbent (Ali et al 2019; Gao et al 2012; Huízar-Félix et al 2019; Maliyekkal et al 2013; Song et al 2016; Sun et al 2013); and photocatalytic/photochemical oxidation (Moussa et al., 2016; Sun et al., 2014)

Applications of rGO and their deviations in environment for now focus on hazardous pollutants such as heavy metals (Ali et al., 2019) rGO exhibits chlorpyrifos uptake capacity as ~1200mg/g, 10–20% higher than that of GO, as well

as able to regeneration and reuse (Maliyekkal et al., 2013) (Huízar-Félix et al., 2019) reported about removal of TC using magnetic rGO material, which was expected to increase electrostatic interaction between rGO with TC and recoverability However,

it was found that hybrid material exhibited a lower removal efficiency than precursor Keshvardoostchokami et al (2019) concluded that Ag+-reduced rGO could remove 79.17 mg/g sulfamethoxazole in batch system rGO also showed a rather high adsorption capacity for TC/sulfamethazine mixture (277.76 mg/g) than each substance (219.10mg/g for TC and 174.42mg/g for sulfamethazine) (Song et al 2016) And several studies value recoverability and reusability of rGO compared to its precursor GO

Graphite fine powder extra pure were obtained for Merck (Germany)

The other chemicals used in the study included: concentrated sulfuric acid (H2SO4), sodium nitrate (NaNO3), potassium permanganate (KMnO4), hydrogen peroxide (H2O2), hydrochloric acid (HCl), L-ascorbic acid (C6H8O6), nitric acid (HNO3), sodium hydroxide (NaOH).The entire reagents used were of A R grade 2.2 Methods

2.2.1 Literature review

The references that used in this study were domestic and international journals, published studies Website of large organizations (IUPAC, AMR industry alliance, Sigma-aldrich) also reviewed

Information, methods, and explanations were cited and adopted in order to give an overview of the objectives And the data and mechanisms were compared and explained with obtained data

2.2.2 Sample analysis in laboratory

Characterization of materials

a) Fourier-transform infrared spectroscopy

Fourier-transform infrared spectroscopy (FT-IR) based on the interaction between analyte and beam of multi-wavelength within infrared region (400-4000 cm-

1) The result of that would lead to the analyte absorbing part of the energy and reduces the intensity of the incident ray At this time, the molecule will make

IR spectra usually is performed by transmittance against wavelength:

Infrared spectra curve represents the dependence of the intensity of the infrared absorption of the analyte on wavenumbers or wavelengths On the infrared spectrum, the horizontal axis represents the wavelength (μm) or the number of wavenumbers (cm-1), the vertical axis represents the absorption intensity (abs) or transmittance (T%) Each maximum in the IR spectrum featured the presence of a functional group or oscillation of a bond Therefore, it can be based on these characteristic frequencies to determine the presence of links or functional groups in the target analyte Conventionally the IR region is subdivided into three regions, near

IR, mid IR and far IR Most of the IR used originates from the mid IR region

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Figure 2.2 Principle of EDX measurement (Reinoud Lavrijsen, 2010)

EDX is measured by JSM-IT100/JED-2300 Analysis station, JEOL, at nano technology laboratory, VJU (Figure 2.2)

Figure 2.3 JSM-IT100/JED-2300 Analysis Station Plus, JEOL (source:

www.jeol.co.jp) c) Scanning Electron Microscopy

Scanning electron microscopy (SEM) is a method that uses high-energy electron beams to investigate very small objects The results obtained through these surveys reflect the morphology, appearance and crystallography of the target object

d) X-ray diffraction

The principle of the X-ray diffraction (XRD) technique is based on the diffraction phenomenon of X-rays on the crystal lattice When X-ray radiation interacts with some matter, elastic scattering effect would appear with electrons of atoms in crystal structure material, which will lead to X-ray diffraction phenomenon Theoretically, lattices are made up of atoms or ions that are uniformly distributed in space in a certain order When the X-ray beam reaches the crystal surface and goes deep inside the crystal lattice, the lattice acts as a special diffraction grating Atoms

or ions excited by the X-ray beam and form centers that emit reflected rays

Based on the position and intensity of the diffraction peaks on the recorded diagram, crystal lattice parameters and the distance between reflective surfaces in the crystal were determined

In this study, XRD measurement was carried out by MiniFlex600, Rigaku, at VNU Key Laboratory of Advanced Materials for Green Growth (KLAMAG) (Figure 2.4)

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Figure 2.4 MiniFlex 600 (source: www.rigaku.com) e) Surface area and pore volume calculation

Brunauer–Emmett–Teller (BET) method

Brunauer–Emmett–Teller (BET) method still is a common method to evaluating the surface area of porous and finely-divided materials Surface area of material is determined by physisorption of a gas on its surface An adsorption isotherm of amount of adsorbed gas (typically N2) against a range of increasing pressures at a constant temperature (77K for N2 liquid) The amount of adsorbed gas are recommended to be in cm3/g units Conversely, desorption isotherm is achieved

by removed gas as pressure is reduced The bet equation is defined as follows (Naderi, 2015):

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The BET specific surface area (ssa) can be calculated by known the average area of value am (molecular cross‐sectional area, equals 0.162 nm2 for an absorbed nitrogen molecule) and implementing the following equation (Naderi, 2015):

This equation is the basis for calculating pore-size distributions, assuming cylindrical pores, from adsorption isotherms When taking into account multi-layer adsorption, rk meaning the radius of the condensation occurring, which is the function

of the pore radius and the statistical thickness of the adsorption film

Density Functional Theory (DFT)

This method calculated the density distribution of adsorbent liquid in pore spaces at a given temperature and equilibrium pressure can be given for pore structures and molecular interactions In contrast to BJH method that only reliable with mesoporous material, the DFT technique yields pore-size distributions of micro-

to meso-porous objects

Surface area and pore volume are measured by TriStar II Plus, Micromeritics Instrument Corporation, America (Figure 2.5)

12 hours Solutions are filtered to re-measure the pH value of the solution (pHf) The difference between the initial pH (pHi) and the equilibrium pH (pHf) is pHf – pHi = ΔpH The pH value where the curve of ΔpH crosses pHi axis is pHzpc

Determination of Tetracycline concentration

Preparing calibration curve for determining Tetracycline concentrations in aqueous solutions: Preparing series of Tetracycline solutions with concentrations range from 1 and 10 mg/L Optical density of TC solutions measured with 1 cm thick cuvette on UV-Vis UNICO S2150UV at 357nm at Environmental engineering laboratory, VJU.

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Figure 2.6 Calibration curve for TetracyclineThe results show that the dependence of Tetracycline concentration on the absorbance in the solution follows the linear formula: y = 0.029x + 0.001, with regression coefficient R2 = 0.9995

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35

Concentration (mg/L)Calibration curve

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where: qe is the adsorption capacity (mg/g); Co is the initial TC concentration

in solution (mg/L); Ce is equilibrium TC concentration in solution (mg/L); V is volume of solution (L); and m is amount of rGO (g)

which qe is equilibrium adsorption capacity (mg/g); qt is adsorption capacity

at time t (mg/g); k1 is the rate constant for pseudo-first-order (min-1); k2 is the rate constant for pseudo-second-order (g·mg-1·min-1)

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where C0 is the initial concentration (mg/l) and b is the Langmuir constant (L/mg) For favorable adsorption process the value of RL should be in the range between zero and one The values of RL were determined at T= (298, 308, and 318)

K (Anirudhan and Radhakrishnan, 2008)

Freundlich isotherm is given by:

e) Thermodynamic study

The experiment was carried out at different temperature conditions and parameters that assumed constant included enthalpy (ΔH), entropy (ΔS), and Gibbs free energy (ΔG) are calculated by following equation:

Where R is the natural gas constant; T is temperature (K); KC is the constant

at equilibrium KC would be obtained from constants of isotherm equations (Húmpola

et al., 2013)

2.2.4 Statistical analysis

Statistical analysis was applied descriptive statistics method through determining mean, variance, standard deviation Numerical data that are normally distributed are analyzed with parametric tests

∼500C Then add 10 ml of 30% hydrogen peroxide solution (H2O2) and continue stirring until the solution changes from brown to light yellow

The mixture is washed with 5% HCl, then the mixture is washed with distilled water until pH~6 The final mixture was ultrasonicated within 3 hours and then dried

at 500C overnight The ultrasonication separate the GO layers being filled with acid molecules before, in addition to removing unwanted mechanical deposits from graphite

Reduced GO is synthesis by mild reduction method, using L-ascorbic acid 1 gram of L-ascorbic acid is dissolved in 100 ml of distilled water After adding 0.1gram dried GO, mixture is ultrasonicated within 45 minutes The mixture is then heated at 90~950C for 1 hour The black precipitate is filtered by vacuum pump and further washed by 1M HCl and distilled water to neutral pH Finally, filter and dry in

a vacuum oven at 500C for 4 hours to obtain rGO

2.3.2 Factors influencing the efficiency of TC adsorption

a) Contact time

To survey effect of contact time, 1 flask which contained 500 ml of tetracycline solution 5mg/L and 50mg rGO were shaking at 120prm in under-exposed