M En

C Miguel

Galvan Arellano

C Francisco Alvarado

Jaime Santoyo Salazar

Dr Maud Barre

Dra Aracelli

Dra Atzin Ferrel

Dr Ashok Adhikari

Dr Onyekachi Michael Nwakanma

Dr Karthik Sekar

Dra Mercyrani Babudhurai

Ayde Fernanda

Rivera Lima

Josué Raúl

Dr Ramírez Paulino

Jaime Vega Pérez

Acknowledgement

I believe I have learned a lot from writing this thesis, searching for the truth of science and life. This thesis writing was the greatest treasure I will cherish in my future academic career and my whole life. I want to take up this opportunity to express my heartfelt recognition to all those who have given their invaluable support and assistance. Throughout my writing of this dissertation, I have received a great deal of backing and support. I want to express my utmost gratitude to my supervisor, Dr. Velumani Subramaniam, for his sincere and selfless support, prompt and helpful advice during my research. He gives me a lifetime unforgettable memory of his benevolence, patience, intelligence, diligence, and erudition. I am very much acknowledged to my co-assessor, Dr. Benoit Schoefs, whose proficiency was valuable for formulating the research questions and methodology. cluster of ZnO nanoparticles formed through the co-precipitation method using zinc acetate as the precursor (c) spindle shaped ZnO nanoparticles were obtained when zinc acetate and sodium hydroxide were used as precursors followed by seven times washing of the precipitate. (dot-dash curve) and 400 µL (double dots-dash curve) of a mother cyanobacterial culture (d) Calibration curves for A750 and ΔA637 measured in the absorbance spectra and the corrected absorbance spectra, respectively. In both cases, the absorbance varies linearly with the culture volume. The insert presents the normalized data showing that the amount of pigment per cell is constant in the range of tested volume (50-400 µL).

List of tables

Introduction to water crisis and nanotechnology

Chapter 1: Introduction to water crisis and nanotechnology

Introduction

Water or availability of potable water is a most discussed topic worldwide, and the management of this water has become a decisive challenge for all the countries across the world due to the reasons that has depicted in Fig 1 .1. Safe water can be defined as any water that does not cause any health risk to humankind after a lifetime consumption [1]. Water requirement for an individual is always a fundamental and human right. But the present plight does not allow this free access to water, as the government has a well-defined protocol to have an equal division of this limited resource among all humankind to ensure equity and well-being. Despite various conflicts regarding water shortage, allotting freshwater supply, preserving the available capital, and improving the ways to develop it more, it remains a question mark in front of man [2].

Water crisis

A global outlook

While developed countries consider the availability of safe drinking water for granted, the other face of the world, i.e., developing, and underdeveloped nations, is struggling to access potable water (Fig 1 .2). As per the World Health Organization (WHO), more than 1.2 billion people live in the water crisis [5]. Due to this reason, more than 5 billion people lost their lives by the intake of polluted or contaminated water and diseases associated with it. The shortage of potable water will not only make humankind and other organisms sick, but it will also lead to various issues. They include an economic crash in the developing countries, thus being a barrier in their growth and creating conflicts between the nations for resource use, diminishing the global security of the states that might collapse [6]. Over the past hundred years, the use of worldwide water has increased by a factor of six. The graph of water intake will continue rising at a rate of 1% per year because of the growing population, economic development, and shifting consumption patterns. The Organization for Economic Co-Operation and Development (OECD) reported that the demand for global water would increase by 55% between 2000 and 2050. This demand is shared between manufacturing (-400%), thermal power generation (+140%) and domestic use (+130%) (OECD,2012) [7]. in the management of the water resource and high risk in the quality of water, i.e., limited availability of clean drinking water. When there is water contamination by pathogens or dangerous pollutants, one significant share of this cause is anthropogenic activities [7]. The reasons behind the increasing taint of fresh and marine water globally include the discharge of organic wastes, pathogens, fertilizer, pesticides, heavy metals, and/or other toxic pollutants. Water pollution due to organic matter is increasing worldwide due to municipal and industrial wastewater discharges into the natural water bodies, increasing agricultural and livestock farming, and high cutback in the river dilution capacity employing reduced runoff and water extractions [9].

In the same way, eutrophication is the aftermath of dispensing human-made nutrientenriched materials from agriculture and industries to water systems. From the records of WHO and UNICEF, pathogen contamination remains the top reason for the low quality of water worldwide. Therefore, pollutants that already prevail and emerging contaminants such as oxadiazon, methiocarb, imidacloprid, etc., remain a significant challenge in developing countries to avoid a serious threat to human health ecosystems [10].

A peep to water availability and quality in Mexico

The United Mexican States extending over 1.964 million km 2 have their 67 % of its territory with arid or semi-arid conditions. The top three environmental issues prevailing in Mexico are air pollution, deforestation, and lack of clean water [11,12].

These facts are dire and an upcoming crisis that directly and indirectly affects millions of Mexicans and their environment (Fig 1 .3). When lack of clean water and the increase of the human population is considered, an immediate urge for safe and clean drinking water for 127 million Mexicans is elevated. Usually, water is obtained from the aquifers of the Basin of Mexico River, which is in the southern part of the Central Volcanic Axis, an upland formation of Late Tertiary origin. The water extracted from these sources is near to three times the average recharge capacity, which leads to a reduction of moisture over time. The water is contaminated with bacteria due to the leaching of wastewater and heavy metals like lead, nickel, mercury, arsenic, chromium, etc. Due to the intake of polluted water, nearly one-fourth of the population is exposed to waterborne diseases. The water that is obtained from the water supplies of Mexico is tinted and odor. For this reason, Mexicans spend 10% of their income to get clean water. The use of clean, polluted, and drinking canned water has become a part of their daily life [13,14].

A peep to water availability and quality in European Union (EU)

France, Sweden, and Germany possess the most considerable number of freshwater resources considering freshwater availability in a country, climate conditions, transboundary water flows, and country size. But the quantity of freshwater divided among each EU member differs from abstraction practices, climate, and sectors (industrial and agricultural). Even though sustainable management of the water in the EU is under control, water scarcity has been faced by specific regions of Europe. This water scarcity is reported for the near future due to less rainfall, population expansion, and industrial and agricultural activities. The water supply is through public or private systems or private drills, but when it comes to the public pool, it relies on the economic status of the country, which is a negligible amount [17].

Current situation

The physical, chemical, and microbiological characteristics of water determine its quality. These properties are either intrinsic to the water or associated with the substances dissolved or dispersed in water. The water which is used for drinking should satisfy various requirements (Table 1.1), which should be decisive and persistent for the customers [18]. The available water treatment methods are collectively termed conventional water treatment techniques. Water is treated from its source through a series of processes to remove suspended particles, followed by disinfection and finally stabilizing the water chemically for future safe use. For the removal of suspended materials, coagulation (coagulating small colloidal particles), flocculation (small particles will form large flocs or aggregates), sedimentation, and sand filtration are the used techniques. Other methods are flotation, micro-filtration, and ultra-filtration. For coagulation and flocculation, chemicals are used to attain this. A specific amount of disinfectant is added to water for a pre-determined period under particular pH and temperature for the disinfection process. Commonly used chemicals for disinfection are chlorine, chlorine gas, ozone, chlorine dioxide, chlorine compounds like calcium hypochlorite (HTH) and, sodium hypochlorite (bleach). Due to the generation of chlorinated byproducts like trihalomethanes (THMs) that can cause adverse health effects, the concentration of this disinfectant is kept at control in drinking water. Ultra-violet (UV)

irradiation is becoming popular due to the formation of no by-products. Like conventional processes, advanced treatment processes for water treatment include reverse osmosis, nanofiltration, ultrafiltration, electrodialysis, activated carbon adsorption, ozonation, and oxidation processes for iron and manganese removal [18].

The significant disadvantages of the current water purification techniques are tabulated in Table 1.2.

Table 1.2: Drawbacks of current water treatment techniques [19].

Process Disadvantages

Chemical precipitation

Usage of chemicals like lime, oxidants, H2S

Coagulation/Flocculation

Utilization of non-reusable chemicals as a coagulant and flocculant.

pH is regulated physiochemically.

The possibility for the management of sludge is low.

Arsenic removal is low.

Flotation

High investment, energy, and maintenance costs.

Chemicals are used.

Selective in the process.

Biological methods

An optimal favorable environment is required.

Management and maintenance of microbes.

Pre-treatment is needed.

The process is slow, and the biodegradability of some pollutants is low.

The generation of biological sludge and management is complex.

Filtration

High cost for the materials and the maintenance of the system.

Depends on the type of materials.

Chemical oxidation

Usage of chemicals.

Production, transport, and management of the oxidants are harrowing.

Activity depends on the type of the oxidant.

Pre-treatment before the process is crucial.

Short life of some of the chemicals.

Some pollutants are resistant to the treatment.

Formation of intermediates.

Salinity matters.

Formation of sludge.

Generation of compounds that are volatile and aromatic amines.

Ion-exchange

Performance is sensitive.

Costly.

Selective in activity.

Incineration

Discharge of pollutants.

Costly.

Difficulty in storage and transportation.

Electrochemistry

The cost of capital and maintenance is high.

Requires chemicals.

Generation of sludge and difficulty for the management of sludge.

Membrane filtration

Costly in the capital, maintenance, and energy.

Slow output.

High specificity.

Management of sludge is a risk.

Evaporation

Costly in every aspect.

Pollution load in the concentrate.

Nanotechnology

Nanotechnology can be defined as the complete package of observing and studying the phenomenon at the nanometer scale and various approaches for manipulating matter on nanometer-scale as they have different properties when they are in larger scale [20]. This is a promising field studied and cited for water treatment. The application of this technology is better due to the structure and large surface area of nanomaterials. When treatment and remediation are considered, long-term water quality, access, and viability of water are promised using nanotechnology.

Sensors that can detect biological and chemical pollutants in water, even at shallow concentrations, are developed with nanotechnology. Nanotechnology is very efficient for monitoring and removing contaminants during water purification with low cost and high specificity [21]. Water treatment can be classified into three different groups (Table 1.

3) [22][23][24][25][26].

Role of nanotechnology in water treatment

When conventional water purification methods display limited efficiency, nanotechnology put forward ingenious remedies to overcome the failures in water purification. The nanomaterials possessing reactivity, tunable pore volume, hydrophilic, hydrophobic, and electrostatic features are considered for better performance in adsorption, catalysis, and optoelectronics. The processes associated to nanotechnology are efficient, modular, and multifunctional. Nanomaterials possess a high specific surface area (SBET), and therefore, a high surface-to-volume ratio, leading to high interaction with the pollutants or pathogens. This nanotechnology is a promising field for the purification of water in an economic way [27,28].

Organic matter as pollutants

With a tremendous increase in the agricultural field and other waste-generating sectors, the contribution to water pollution by the discharge of nutrients, salts, and sediments, livestock became a significant factor for the water pollution by organic matter, pathogens, and other emerging pollutants such as hormones and antibiotics.

Wastes such as animal excreta, animal feed balance, animal-processing industries, and crop residues become the significant sources for organic matter pollution. The presence of organic matter is calculated by the biological oxygen demand (BOD). BOD is defined as the amount of oxygen taken up by microbes to decompose the biodegradable organic matter in the water (Table 1.4) [29]. The rising global population can also contribute to organic pollution [30][31][32][33][34]. Due to the disadvantages mentioned above (see Table 1.2) of the traditional water treatment techniques, advanced oxidation processes (AOPs) are well accepted [35,36] for water purification to make it potable. AOPs are promising due to the generation of reactive oxygen species (ROS) that can react with various pollutants to make the water free from organics, inorganics, and microorganisms [37,38]. AOPs are defined as the oxidation processes that are associated with the generation of hydroxyl radicals (OH . ) and some oxide and superoxide radicals, which aids in water purification, and it was proposed in the 1980s decade for the same. AOPs were later established in their field to oxidative processes using sulfate radicals (SO4 -). ROSmediated disinfection was extended for various types of wastewater treatment due to the formation of strong oxidants, which can cause destruction of organic as well as inorganic pollutants. AO processes include the generation of sufficient quantity of hydroxyl or sulfate radicals to degrade unmanageable organic pollutants, any small amount of organic matter or inorganic contaminants and fasten the wastewater biodegradability helping biological treatment [39]. These types of pollutants can be generally grouped into endocrine disrupting chemicals (EDCs), persistent organic pollutants (POPs), total organic carbon (TOC) and micropollutants [40,41]. Due to the facts mentioned above, during this process there is no conversion of pollutants to another phase (i.e., more hazardous, or toxic) which is the main drawback of traditional methods such as chemical precipitation and adsorption. Another point to be noted is that there is no discharge of any harmful sludge [42,43].

Classification of AOPs

The classified techniques as AOPs (Fig 1 .5) generate ROS that degrades different kinds of pollutants. Sonolysis uses ultrasonic waves having a frequency of 20 kHz to 10 MHz. Due to the ultrasonic, cavitation bubbles are formed and then break down when they reach their resonance size, leading to the liberation of stored energy as explosions degrading the organic molecules in the surroundings. This technique does not use any toxic chemicals as an intermediate and uses only the ultrasound frequency, which is an ecofriendly process [44,45]. Ozonation uses ozone for the oxidation of inorganic and organic pollutants. This technique, widely used in European countries, has a very selective disadvantage due to the electrophilic nature of ozone molecules (ozonation). Ozonation is famous for its activity towards microbes, decolorization, non-protonated amines, micropollutant removal, taste, and odor removal [46]. By absorbing UV light, the killing of microorganisms at the tertiary level of wastewater treatment and destruction of other aquatic organic compound occurs.

Mercury arc lamps transmitting UV light at various intensities are used.

Homogenous and heterogeneous AOPs

AO processes ranges in two categories: (1) homogenous-and (2) heterogeneous-AOPs [48]. The former means that the degradation of compound is performed using the adsorption of UV radiation in the range of 100-400 nm. Whereas heterogeneous AOPs use catalysts for the degradation of compounds on the surface of the catalyst.

In this process, the catalyst generates electron-hole pairs and activates the electrochemical process. The holes and electrons developed through photogeneration can undergo oxidation and reduction processes, respectively. At the presence of an aqueous and aerobic condition, water molecules are adsorbed to the catalyst, which under oxidation generates hydroxyl radicals and superoxide radicals, respectively.

These radicals help in the oxidation of pollutants that have been adsorbed over the catalyst surface.

Photocatalyst as a weapon for water disinfection

Photocatalyst is defined as the catalyst, which is photoactive, photostable, visible or near UV light range, biologically and chemically inert, inexpensive (low cost), and toxic-free. For a semiconductor to be photocatalyst, it should be photochemically active and the redox potential of photogenerated valence band holes must be positive.

The photogenerated conductance band electron must be high enough to generate superoxide radicals. The photocatalysis mechanism (

(a) Graphene oxide (GO)

A two-dimensional honeycomb lattice structure made of carbon material is graphene, with a single layer atomic thickness. Graphene possesses high mechanical properties, excellent electrical properties, high thermodynamic properties, and a large surface area [55]. The existence of strong van der Waals interactions between graphene sheets limits its direct application. Graphene oxide, which is obtained through the chemical oxidation-stripping method, is a prominent derivative of graphene. It has the different oxygen functional groups that cover the basal plane of sp 2 carbon [56]. Due to reactive oxygen functional groups like hydroxyl groups and epoxy groups, GO is used for various applications through chemical functionalization. The oxygen groups make GO hydrophilic [57][58][59]. Lerf and Klinowsky proposed the GO chemical structure model (Fig 1 .7), where it is described as a structure with non-oxidized aromatic areas which are separated by aliphatic 6-membered rings having hydroxyl groups, epoxide groups, and double bonds. These groups lie below and above the basal plane providing the polar nature and hydrophilic nature of GO [56].

The GO-based nanocomposites like GO/TiO2 are synthesized through different methods and approaches like electrochemical co-deposition [60], hydrothermal method [61], microwave-assisted method [62], in situ polymerization [63], vacuum impregnation, and sol-gel technique [64]. By adjusting the abundance of functional groups over the GO, the electronic properties and the affinity towards bacteria cells can be tuned [65]. Due to the excellent conductivity of GO, the photo-electrons that were generated from the light irradiated TiO2 can migrate towards GO, thereby enhancing the charge separation, thus leaving behind the photo-holes and leading to Even though photocatalysis was discovered over the 1990s, its applications came into major existence after discovering photocatalytic splitting of water and hydrogen generation on TiO2 photoelectrodes by Fujishima and Honda in 1972 [70]. This opened a separate research field to improve the technique, photocatalysis [71][72][73][74][75][76]. Due to the ability for decomposing organic pollutants, corrosion resistance, non-toxicity, durability, and low cost, TiO2 has been explored in various fields [77,78]. Due to the strong redox power by the photogenerated electrons and holes, TiO2 can inactivate the pathogenic organisms [79].

(c) Bismuth vanadate nanoparticles (BiVO4 nps)

Bismuth-related nanoparticles like BiOCOOH, Bi2WO6, (BiO)2CO3, BiVO4, BiPO4, Bi2O3, and BiOCl are used effectively for various applications due to their high photocatalytic performance, low toxicity, low cost, and high stability. These bismuth-related nanoparticles are good in photocatalysis, anti-flogging, self-cleaning, disinfection, organic pollutant degradation, carbon dioxide reduction, air purification, hydrogen generation, etc [80]. Bismuth vanadate (BiVO4) has three main phases: tetragonal scheelite, monoclinic scheelite, and tetragonal zircon. The monoclinic scheelite phase proved to be active in visible light because of its narrow bandgap, i.e., 2.4 eV, and due to the presence of pair distortion of the Bi 6s orbital. Moreover, the valence band of BiVO4 has an overlap of O 2p and Bi 6s orbitals, thereby enhancing the mobility of photogenerated charge carriers, which improves the photocatalytic ability of this semiconductor [81]. Due to this feature, BiVO4 is widely used in photocatalysis, as it is non-toxic, low cost, environmentally friendly and highly resistant to photo corrosion [82]. The first photocatalytic report on BiVO4 was put forward in 1999 by Kudo et al. The publication rate has increased till date [83]. BiVO4 is an ntype semiconductor displaying high stability, with its flexible optical and electronic properties [84]. Due to the fast recombination of photoinduced charge carriers caused by the narrow bandgap, enormous efforts have been made to improve the separation of photogenerated charge carriers like controlling morphology, semiconductor coupling, doping with metal and non-metal elements, and exposed reactive facets of BiVO4.

In the field of water disinfection, BiVO4 based materials are gaining recognition due to their effectiveness. Using Z-scheme structures, like AgI/BiVO4, which was synthesized through a chemical deposition precipitation route, the semiconductor applied for the inactivation of Escherichia coli, and has inactivated 7 x 10 7 CFU/mL in 50 min under visible light irradiation, with superoxide radical and holes as the ROS responsible for the disinfection [85]. The Z-scheme is inspired from the natural photosynthetic process. In photocatalysis, Z scheme is based on an oxidation semiconductor and a reduction semiconductor. A low valence band position and strong oxidation ability is associated with oxidation semiconductor while a reduction semiconductor possess high conduction band and strong reduction ability. Due to this charge transfer property, a particular photocatalyst with above mentioned characteristics can have various benefits like separation of photogenerated charge carriers, enhanced light harvesting, extended range of photocatalysts, etc [86]. Pseudomonas aeruginosa, E. coli, and Staphylococcus aureus bacterial growth was inhibited 100% within 30 mins of irradiation of the Bi2WO6/BiVO4 heterostructure [87].

Photocatalytic disinfection

Photocatalysis and its applications have gained widespread attention as an alternative for several environmental issues. Among these issues, bacterial disinfection has its priority in the application due to the disadvantages of existing conventional methods (see Table 1.2). To be more specific, chlorination, a well-known technique, can generate by-products like choro-organic compounds, some of them are carcinogenic in nature [88]. At the same time, such as viruses, Legionella pneumophilla (bacteria), and cysts of Cryptosporidium parvum and Giardia lamblia (protozoa) are highly resistant to chlorination. Ozonation and the use of germicidal lamps can cause a reduction in residual effect or can induce the formation of colony mutants [89][90][91]. One of the hot topics under discussion is photocatalytic applications linked to solar photocatalysis for water and wastewater treatment [92][93][START_REF] Goswami | New and emerging developments in solar energy[END_REF][START_REF] Malato | Photocatalytic decontamination and disinfection of water with solar collectors[END_REF][START_REF] Bahnemann | Photocatalytic water treatment: Solar energy applications[END_REF]. Photocatalytic disinfection applies to resistant organisms to traditional methods [START_REF] Otaki | Aqueous microorganisms inactivation by photocatalytic reaction[END_REF][START_REF] Kato | Degradation of norovirus in sewage treatment water by photocatalytic ultraviolet disinfection[END_REF].

ROS generation take place at the semiconductor particle-solution interface. The hydroxyl radical, followed by superoxide radical anion, hydroperoxyl radical, and hydrogen peroxide, are the essential ROS that causes disinfection. Upon forming ROS inside a biological system, various systematic defense mechanisms using an enzymatic pathway takes place to convert the generated ROS to non-toxic substances, as ROS in high concentration is lethal to living cells. Due to the absence of site-specific activity of ROS, the uses of photocatalyst have a significant impact on pathogens for their destruction. The point to be noted is that the concentration of ROS produced by a photocatalyst must be large enough to counteract the defense mechanism for the photocatalytic disinfection [START_REF] Goulhen-Chollet | Monitoring the bactericidal effect of UV-A photocatalysis: A first approach through 1D and 2D protein electrophoresis[END_REF]. Escherichia coli (E. coli) is the most used organism for antibacterial study due to its ease of culture and detection. A typical bacterium possesses a cytoplasmic matrix which is covered by the cell membrane and outer layers. The cell contains genetic material and the biochemical system diseases, and they are excreted out in the feces of mammals [START_REF] Fewtrell | Water Quality: Guidelines, Standards and Health: Assessment of Risk and Risk Management for Water-Related Infectious Disease[END_REF][START_REF] Hunter | Drinking water and infectious disease: Establishing the links[END_REF][START_REF] Paneth | A rivalry of foulness: Official and unofficial investigations of the London cholera epidemic of 1854[END_REF][START_REF] Edberg | Escherichia coli: The best biological drinking water indicator for public health protection[END_REF][START_REF] Enriquez | Direct exposure to animal enteric pathogens[END_REF]. Some pathogens, especially chlorine-resistant parasitic protozoa, such as oocysts of Cryptosporidium parvum and some enteric viruses, are disinfection resistant and environmentally persistent. But the universal indicator for drinking water pollution is considered to be thermo-tolerant fecal E. coli [START_REF] Hambidge | Reviewing efficacy of alternative water treatment techniques[END_REF][START_REF] Li | Mechanisms of inactivation of hepatitis A virus by chlorine[END_REF].

When there is an imbalance in environmental conditions, such as temperature fluctuations and increased nutrient concentrations like nitrogen and phosphorous, an outbreak of algae occurs to form algal bloom or harmful algal bloom (HAB). This HAB in lakes, rivers, and estuaries severely threatens human and aquatic ecosystem health due to toxins (certain species) by these blooms and cause economic damage. HABs are characterized by color change (light to dark green, yellow, red, and brown) and odor and appear in water as scums, paint-like slicks, clotted mats, or foams. Even if there is no discoloration of water, the presence of algal toxins leads to health effects [START_REF] Lopez | Scientific Assessment of Freshwater Harmful Algal Blooms[END_REF].

The most frequently occurring HABs in freshwater bodies is cyanobacteria.

Cyanotoxins cause hepatic, neurologic, respiratory, and dermatologic ill-effects to exposed organisms. The diseases can be acute or chronic and sometimes fatal.

Consumption of drinking water, fish, or shellfish contaminated with these toxins, close contact in the recreational water bodies polluted with toxins, or inhalation of aerosolized toxins can become reasons for the adverse health issues for human [START_REF] Graham | Cyanobacterial Harmful Algal Blooms and U.S. Geological Survey Science Capabilities[END_REF][START_REF] Grattan | Harmful algal blooms and public health[END_REF][START_REF] Chakraborty | Trapping effect analysis of AlGaN/InGaN/GaN Heterostructure by conductance frequency measurement[END_REF] .

Based on the light quality and quantity, cyanobacteria and algae were subjected to a change in their system. To adapt to these conditions, they have developed efficient photosynthetic apparatus to safeguard photosynthetic efficiency and photoprotection.

During reduced light intensity, light capturing, and photosynthetic yield are performed by reorganizing its light-harvesting system. On the other hand, when there is increased light intensity, the excess light energy absorbed is dissipated as heat through nonphotochemical quenching (NPQ) to avoid photodamage or oxidative damage.

Photodamage is more prone to evident in photosystem II (photosynthetic complex) [START_REF] Rochaix | The Dynamics of the Photosynthetic Apparatus in Algae[END_REF].

Objectives, motivation, novelty, and outline of thesis

The primary aim of this research work is to develop graphene oxide, which will be incorporated with TiO2 and BiVO4 nanoparticles to obtain nanocomposite for the applications in the removal of micro-organisms and microalgae from drinking water.

The research study started with the selection of materials i.e., multiwalled carbon nanotube (MWCNTs), zinc oxide (ZnO), titanium dioxide (TiO2) and bismuth vanadate (BiVO4), from which the basement of the work was laid. The short summary of the objectives followed after the fixation of materials is described below: Based on these objectives, the thesis is divided into four chapters with the following brief descriptions (Table 1.5). Graphene oxide (GO) was purchased from Graphenemex, Mexico, and was maintained under appropriate conditions. All the chemical reagents were of analytical grade and used without any purification. The media necessary for the growth of cyanobacteria and microalgae, i.e., BG11 and F2, respectively, was prepared. The deionized (DI) water was produced in a Millipore Milli-Q water purification system.

Table 1.5: Structure of the thesis

Chapter

This chapter consists of the methodology, and protocols followed through the research work and demonstrate the trial-and-error methods to satisfy the objectives of this work.

The work was divided into two stages, i.e., the first stage, where the selection and optimization of the materials and methodology were performed while the second stage consisted of the detailed study and applicative performance of the confirmed materials.

Therefore, the methodology and techniques followed for the above-mentioned stages are discussed in this chapter. The steps that were undertaken during the process of

Cell cultivation

Bacterial culture (Escherichia coli K12)

The bacterial strains of Escherichia coli K12 (E. coli K12) (ATCC ® 10798™) (CBDB-

Fig 2.2: (a) Escherichia coli K12 (E. coli K12) (CBDB-11-1538), (b) E. coli K12 as green metallic sheen in Eosin Methylene Blue agar (EMB agar), (c) E. coli K12 as white translucent colonies in Nutrient agar

Algal culture

The cyanobacterium Anabaena flos-aquae PCC 7120 was cultured in a Blue Green (BG11) medium, whereas the diatom Phaeodactylum tricornutum UTEX 644 (Pt4) [1] were maintained in F2, in a 50 mL flask (Sarstedt). The gas exchange was performed through a membrane in the cap (Fig 2 .3).

Synthesis methods -First stage 2.3.1 Functionalization of MWCNTs and synthesis of MWCNTs/TiO2 composite

MWCNTs underwent a functionalization process to convert them into more active-sited material. Oxidation of MWCNT was carried out by adding mixtures of concentrated nitric/sulphuric acid (HNO3/H2SO4) followed by ultrasonication for 6 h at 60 °C. The functionalization process can be explained in short as follows: the measured quantity of MWCNT was added to a 100 mL mixture of HNO3 (69 %) and H2SO4 (95-98%). The reaction mixture was kept in a reaction flask, coupled to a condenser. The whole mixture was refluxed and sonicated in an ultrasonic water bath at 60°C. After the process, the suspension was diluted using deionized water followed by centrifugation and filtration through a Millipore membrane (0.1 µm). The filtrate was washed using deionized water to attain a neutral pH and finally dried in the oven at 80°C for 6 h. The functionalized MWCNT material was put in contact with a definite amount of refluxed by a definite amount of TiO2 anatase, synthesized through the sol-gel method, and refluxed [2,3]. The synthesized composite was characterized.

Photocatalytic dye degradation study using MWCNT/TiO2 composite

The photocatalytic efficiency was studied by the reaction suspensions containing the MWCNT/TiO2 nanoparticles with the Orange G dye, which is an azo dye that is grouped under the synthetic dyes that have -N=N-groups (azo group) (Fig 2 .4). This dye is particularly used in the textile industry. The composite dosage was fixed at 1 g/L and the suspension of the composite and orange G dye was kept in the dark for 30 mins under stirring to obtain the adsorption-desorption isotherm equilibrium. After the dark incubation, the suspension was exposed to visible light irradiation using a homemade reactor for a period of 2 h. The samples of 1 mL were taken every 10 min and measured using a UV-Vis spectrophotometer to determine the reduction in the concentration of Orange G through the photocatalysis test. The measurements were performed at 476 nm using a calibration curve [4].

Zinc oxide nanoparticle synthesis

Zinc oxide nanoparticles were synthesized through different methodologies adapted from the literature focusing on obtaining spherical nanoparticles. The various methodologies followed are discussed below.

(a) Precipitation method

This zinc oxide synthesis used zinc chloride as the precursor. In this methodology, a solution of zinc chloride was prepared by dissolving zinc chloride in deionized water.

Followed by the preparation of zinc chloride solution, ammonium hydroxide was dropwise added to this suspension using a burette ( ). The supernatant was dried at 100°C for overnight, and then it was calcinated [7]. In this methodology, different parameters were changed as per the need, and they were separated into three batches:

i. 20 g of zinc chloride is dissolved in deionized water at 90°C, 7.27 g of NaOH were dissolved, and 58 ml of sodium hydroxide were used for the reaction to take place, filtered using Whatman filter paper, dried in at 160°C for 7 h, and calcinated at 200°C for 2 h in an oven.

Chapter 2

ii. 5.5 g of zinc chloride were dissolved in deionized water at 90°C, 20 g of NaOH were dissolved in deionized water, and 16 ml solution of sodium hydroxide was used during the process, washing with distilled water 5 times, and dried at 100°C for 30 min.

iii. 5.5 g of zinc chloride were dissolved in deionized water at room temperature, 20 g of NaOH were dissolved, and 16 ml of sodium hydroxide were added. The material was washed with distilled water and methanol 5 times, and dried at 100°C overnight, and then it was calcinated either at 300˚C, 400˚C, or 500˚C. The microwave along with the holder used for the hydrothermal method

Synthesis of graphene oxide/zinc oxide composite

From the zinc oxide nanoparticles, graphene oxide/zinc oxide (GO/ZnO) composite was synthesized by the simple blending process. In 200 ml of deionized water definite amount of graphene oxide was dispersed, and in another 200 mL of deionized water, the synthesized ZnO nanoparticles were dispersed. The dispersion was ultrasonicated and mixed for the better dispersal of the nanoparticles. After 1.5 h of ultrasonication, the dispersion was kept for magnetic stirring overnight and filtered. The filtrate was dried to obtain the GO/ZnO composite for future applications.

Disk diffusion assay

One day prior to the test, isolated colonies of E. coli K12 were chosen from the plates to obtain pure line colonies and grow them in 5 mL of Luria Bertani medium overnight to obtain a stationary phase. On the day of assay, the cultures were diluted to obtain a log phase of the bacteria after the standardization of the inoculum to 0.5 by observing the absorbance at 600 nm. At the same time, 100 mg/ml of the GO/ZnO composite dispersion was prepared using deionized water. By using Whatman filter paper, small circles of discs were made to be that can be placed in the agar plates. Carefully spread plating the nutrient agar plates with the optimized bacterial inoculum, load different dosages of the composite dispersion on the disc, and place them over the middle of the nutrient agar plate. The plates were kept under irradiation for 2 h, followed by the overnight incubation at 37°C to observe the inhibition zone [8].

GO/TiO2 nanocomposite synthesis

The synthesis of GO/TiO2 nanocomposites at different loading ratio, i.e., 0.2/0.8, 0.3/0.7, 0.5/0.5, 0.7/0.3, and 0.8/0.2, was done through a one-step process mixing the TiO2 nanoparticles, GO, and deionized water. In 200 ml of deionized water, the desired amount of GO was suspended to obtain an aqueous dispersion. In the same way, TiO2

nanoparticles were also dispersed in deionized water and added to the GO dispersion.

The whole mixture was sonicated for 1.5 h (

Synthesis of TiO2 nanoparticles (optimizations)

Titanium dioxide (TiO2) anatase nanoparticles were synthesized through the sol-gel process because it uses low-cost precursors, and the hydrolysis process can be easily controlled. To 15 ml of ethanol at 60˚C, 1.5 ml of TiCl4 were added dropwise with continuous magnetic stirring. During the process, a large amount of HCl gas was liberated. A sol-gel with a light-yellow tint was formed after a period.

The parameters were fluctuated to reach the optimized conditions to obtain spherical TiO2 anatase nanoparticles.

a. The reaction temperature was room temperature, and calcination temperature was 450°C.

b. The reaction temperature was room temperature, and the calcination temperature was 550°C.

c. The reaction temperature was 50°C, and calcined temperature was 450°C.

d. The reaction temperature was 60°C, and calcined temperature was 450°C.

Synthesis methods

Synthesis of BiVO4 nanoparticles

Bismuth vanadate nanoparticles (BiVO4 nps) synthesis through the sol-gel route involved two solutions prepared from the precursors as starters. Solution A was formed by dissolving (BiNO3)3.H2O in nitric acid, to which citric acid was added.

NH4VO3 was dissolved at 80°C, and citric was added to have Solution B. Thereafter, Meanwhile, pH was adjusted. The whole mixture was then heated at 80°C to form a gel. Once the gel was formed, it was dried at 100°C overnight. The dried sample was calcinated at 500°C for 2 h in an oven [11].

Nanocomposite pellets

For the formation of pellets, 0.975 g of the synthesized powder composite was transferred to a pellet mold of diameter 5 mm and thickness of 3 mm. The mold, along with the weighted composite, was given a uniaxial pressure of 2 Torr. Thus, formed pellets were covered completely with a stretch wrap, followed by the placement of a

Ultrasonication Magnetic stirring

Filtration Drying balloon to cover the pellets to avoid moisture. The isostatic pressure was applied in a holder, where hydrostatic pressure and compressed gas function as a combined action. A pressure of approximately -4500 torr was provided. Once the process was finished, the wrapped pellet was taken out to dry for future applications (Fig 2 .12). The study on the structural and elemental composition of composites was performed using the X-ray diffraction (XRD) technique. From the XRD measurements, the structure, oxidation state, and local symmetry were identified for the detailed study of crystalline structure, which is very important for a photocatalyst. This X-ray technique has an important role in the estimation of the size and distribution of the loading sites, which evaluate the catalytic site dispersion for the catalytic process [12][13][14]. This In most cases, XRD patterns depict low intensity as well as broad diffraction peaks, which may be due to the small size and the amorphous nature of synthesized materials [19].

For the characterization of solid carbon matters, the XRD technique is very common now. The non-destructive XRD method, Debye functions, and pair distribution combine to give novel carbon particles with desired properties. XRD technique is considered as a "global eye" to perform characterization of carbon-related materials to the atomic level [20]. Through XRD, structural strains, detection of impurities, microscopic folding, defects, finding the number of atomic layers, thickness, calculating the interlayer distances, defining the functional groups of fabricated graphene sheets, determining the lattice size and quality, and analyzing graphene-related composites [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34]. For

2.6.2(b) Transmission electron microscope (TEM)

Being one of the most powerful tools in the material science, high energy electron beams can fall through a very thin sample. The interaction between the electrons and atoms due to the electron beam can help in the observation of features like crystal structure, dislocations, and grain boundaries. Similar to SEM, chemical analysis can also be performed. To put into a nutshell, TEM analysis helps in the study of growth of layers, compositions, defects in semiconductors, quality, shape, size of nanoparticles (Fig 2 .19) [49] .

Band gap energy analysis

2.6.3(a) UV-visible spectroscopy

The diffuse reflectance data obtained from the UV-visible spectroscopy was used for the calculation or estimation of the band gap energy using the Kubelka -Munk (K-M) equation, where the outcome is F(R). This theory is based on the light absorption and partial scattering. The theory modelled that the resultant light is emerging from translucent and opaque media, assuming that, there are only two light channels traveling in opposite directions [50]. Considering the solid samples, UV-Vis diffuse reflectance spectroscopy uses the reflectance of the visible radiation spectrum because they cannot penetrate the sample [51]. The optical properties were evaluated using an Agilent-carry-5000 spectrophotometer. For the transmittance and absorbance studies of filters and microalgal concentration, respectively, double beam UV-visible spectrophotometer was used following the Beer-Lambert's law.

Model reaction study

The standard test for the photocatalytic activity of the synthesized TiO2 and 1.5 GOT pellet was studied with the help of methylene blue dye degradation. The conditions, mainly photolysis with and without nitrogen influx, dark condition, photocatalysis with and without nitrogen influx was performed. The experiments were carried out in a 3 mL cuvette with an initial concentration of 20 mg/L along with a single pellet of TiO2 or 1.5 GOT. Prior to the irradiation using an Oriel Xenon arc lamp source, the sample was kept in dark under stirring for 30 mins to obtain the adsorption-desorption equilibrium followed by 2 h of continuous light irradiation. About 100 µL aliquots were withdrawn at every 10 min, which was diluted with 2900 µL of deionized water. The absorbance measurements at regular intervals were performed at 664 nm absorbance using a double beam spectrophotometer (Perkin Elmer Lambda 75). The initial pH was maintained the same throughout the experiment [11,52].

Experimental set up

Experimental set-up for microbial disinfection

The bacterial concentration was determined after the bacterial growth curve study (Fig The changes in the sample induced during photolysis or photocatalysis processes were either followed using a double beam spectrophotometer (Perkin Elmer) after sampling or online through the changes of the chlorophyll fluorescence yields using a Dual PAM-100 (Walz, German) piloted with the software provided by the supplier (https://www.walz.com/products/chl_p700/dual-pam-100/downloads.html). Prior to measurements, the sample, in plain on all four sides cuvette, was fed with sodium bicarbonate (NaHCO3) to avoid the carbon starvation during the experiment [57].

(b1) Sampling for in situ absorbance measurements

The experiments were carried out in 3 mL cuvette with an initial concentration of 400 µL of Anabaena culture (OD750 = 6) and mixed with 2600 µL of BG11 media. The sample was kept in dark under stirring for 30 min to reach the adsorption-desorption equilibrium, followed by 2 h of continuous light irradiation. A 100 µL aliquot of the sample was taken every 10 min. The subsampling was diluted with 2900 µL of BG11 media before recording the in-situ absorbance spectra. To determine the changes in chlorophyll a at any time of the photocatalytic treatment, the difference between the absorbance spectra recorded after t min of illumination and the corresponding spectra recorded during photolysis treatment was calculated.

(b2) Online measurements of chlorophyll fluorescence

The device is equipped by two light emitter-detector heads [58]. Before measurement, the sample was placed in the darkness for 30 min in the presence of low intensity farred light (FR-L, λmax: 720 nm, intensity: 13 µmol photons/m -2 s) to empty the electron transport chain. After switching off FR-L, the sample was irradiated with a low intensity modulated light (FML, λmax: 620 nm, frequency: 800 Hz, intensity: < 15 µmol photons/ m -2 s) for 2 mins. The level of fluorescence recorded was denoted as F0 (Fig. 2.23 (e)).

FR-L was switched on again to favor the PSI activity. Then a saturating white light pulse (L-P, duration: 800 ms, intensity: > 1500 µmol photons/m -2 s) was delivered to the sample. The level of fluorescence recorded was denoted as FM (Fig. 2.23 (e)). The sample was then illuminated by L-ACT for different times, ranging between 0.5 and 4h (Fig. 2.23 (e)).

During the illumination, the fluorescence reached a stationary level denoted as FS (Fig. [58,60] No. tetrasodium salt) dye was used to determine the viability of cells after the photocatalytic and photolytic studies [61]. When biotic or abiotic stress occurred, the response of the cells was presented as membrane damage. The health of a cell is lying on the cell membrane and can be used as a biomarker for the living or dead cells.

Model

Evans's blue is considered an azo dye that can penetrate through the ruptured or destructed cell membrane, and this dye is used as an assay for cell viability. This dye assay principle is that the stressed or destructed cells permeate the dye and, therefore, take up the dye coloration, i.e., a blue protoplasmic stain. In contrast, healthy and live cells have better membrane integrity, blocking the entry of the dye [62]. The counting of algal cells was performed using a Neubauer hematocytometer.

Chapter 3 Characterization of materials and optimization of nanocomposites

Results and Discussion

XRD analysis of MWCNT/TiO2 composite

Following the acid treatment, the purchased MWCNTs were functionalized or oxidized as an initial step for incorporating TiO2 anatase nanoparticles bought from Sigma Aldrich (Refer Chapter 2, section 2. 105) and (211) crystallographic planes of TiO2 anatase phase, respectively. However, with functionalized MWCNTs, the crystalline quality of TiO2 anatase was seemingly decreased, observed from the XRD spectra. The same effect occurred with the TiO2 anatase phase over MWCNTs that reduced the intensity of the characterization peak of MWCNTs, which was attributed to the intense peaks of TiO2 anatase [3][4][5]. When the MWCNT/TiO2 composite was analyzed, the reflection planes of (002) and ( 100), associated to the MWCNTs and (101) related to TiO2 anatase were observed [5,6]. These observations gave a preliminary confirmation on the composite formation and suggested that the anatase phase was conserved during the chemical route of composite synthesis. The crystallite size of the MWCNTs and anatase nanoparticles was calculated using the Scherrer equation (Refer to Chapter 2, section 2.4.1) and was found to be 175.7 nm and 17.03 nm, respectively.

Morphological confirmations

Morphological studies were carried out by SEM analysis to prove the incorporation of TiO2 nanoparticles over the functionalized MWCNTs.

Dye degradation studies

The photocatalytic degradation study of Orange G by the synthesized MWCNT/TiO2 composite was investigated using visible light irradiation (See Chapter 2, section 2.3.2). The degradation percentage was calculated by using the formula:

% = * 1

Justification for the selection of graphene oxide (GO)

An atom-thick sheet of carbon atoms, arranged in the form of a nanoscale chain-link fence, is graphene oxide (GO). GO is claimed to be a fundamental enabler for the development of new generation nanocomposites. When compared with chemical makeup and mechanical properties, GO, and MWCNTs are identical. But, for lending its properties to other materials when they are in a composite form, GO is a better candidate than MWCNTs even though both are strong. To be numbered for the advantages of GO over MWCNTs are [16][17][18][19]:

i. They are rough and possess a wrinkled surface texture due to high-density surface defects caused by thermal exfoliation.

ii. As GO is a planar sheet with a large surface area, the contact area for the materials will be larger than the tube-shaped MWCNTs.

iii. GO geometry is two-dimensional compared to the one-dimensional carbon nanotubes, which helps to maximize the energy that enhances its applications.

The results obtained for the degradation of Orange G dye, as part of a preliminary step for the photocatalytic disinfection study by MWCNT/TiO2 composites, were not satisfied compared with the works of literature. Therefore, GO is considered for proceeding with the disinfection studies. 002), ( 101), ( 110), ( 103), ( 112), (201), and

(202) reflection planes and were in good agreement with that previously reported in literature. All the ZnO nanoparticles formed by various methodologies possessed the wurtzite phase (cubic) [20]. Every sample had its strongest peak for ( 101), (002), and (101) planes, confirming the formation of ZnO nanoparticles, but not the expected hexagonal phase. Fig 3.6 shows the effect of various calcination temperatures after the coprecipitation method, and the XRD spectra revealing the narrowing of the peaks with increased intensity. The presence of additional peaks around 43° attributes towards the unreacted precursors during the synthesis process [21,22]. By the microwave-assisted hydrothermal method, the final amount obtained after every single synthesis was 0.8 g, which was a time-consuming process. The precipitation methods followed for synthesis were of long duration, at the time of sedimentation or washing process. The formation of graphene oxide/zinc oxide (GO/ZnO) composite (Fig 3 .8) is confirmed with the presence of peaks from ZnO, whereas there were no observable peaks of graphene oxide due to the dominant peaks of ZnO [23]. . The nanoparticles showed a large size, which can significantly affect the efficiency of the photocatalytic disinfection process. All these variations in the size and shape of the synthesized ZnO nanoparticles were attributed to the precursors, and the synthesis steps [20,24].

The synthesized GO/ZnO composite was tested for preliminary identification of the efficiency in photocatalytic disinfection. For this, a disk diffusion assay was performed.

After the overnight incubation, the inhibition zone was observed but in nominal diameter (Fig 3 .9). The reduced inhibition zone shows that the ZnO nanoparticles could not perform the photocatalytic disinfection compared with the reported literature, which can be due to the phase formed, as well as to the large particle size blocking the active sites and low absorption of light irradiation.

Optimization of TiO2 nanoparticle synthesis

To obtain nanoparticles of better quality and foreseeing the efficiency in the application, i.e., photocatalytic disinfection, TiO2 nanoparticles were synthesized using titanium chloride as the precursor (see Chapter 2, section 2.6). The conditions were optimized by changing various parameters. The annealing temperature was fixed at 450°C, as the focus of the study is in the anatase phase, and the increase in the calcination temperature can cause the conversion of the anatase phase to the rutile phase. The synthesis temperature during the sol-gel route was varied as room temperature, 50°C, and 60°C, and the XRD spectra of the final powder obtained after these synthesis conditions are depicted in Fig 3 .12. From the results, the spectra at room temperature and 60°C show promising crystalline quality nanoparticles. 60°C

was chosen as the optimized parameter because the synthesis by room temperature was time-consuming. The morphology of the TiO2 nanoparticles obtained after the sol-gel route with synthesis temperature 60°C was found to be spherical, which was confirmed with SEM analysis (Fig 3

Conclusion

To be concluded from this chapter, various synthesis methodologies were adopted to develop highly efficient nanoparticles for photocatalytic disinfection studies. The main objective was to optimize the multiple conditions necessary to improve or enhance the photocatalytic activity. From the literature survey, GO was better in photocatalytic

Results and Discussions

XRD analysis

xGOT and xGOB nanocomposites (0.5, 1.5, and 2.5 wt.%) were synthesized (refer to Chapter 2, section 2.9) through a simple blending process previously reported with minor modifications [14]. From XRD analysis, crystalline properties of graphene oxide could be identified, with an intense peak at 2θ = 26.42°, corresponding to the (002) plane, it was related to the higher crystalline nature of pristine graphite having a dspacing of 3.37 Å. At 2θ = 11.95°, a diffraction peak indicated GO with a d-spacing of 7.4 Å. The presence of oxygen functional groups is the reason behind larger d-spacing of GO [15,16]. TiO2 anatase possesses a tetragonal structure, and the TiO6 octahedron is distorted more. There are reports as the reactivity of anatase crystals is higher for (001) facets when compared to (101) facets [17]. BiVO4 has three crystal structures, namely, tetragonal zircon phase, monoclinic scheelite phase, and tetragonal scheelite phase in which, monoclinic scheelite phase has got major applications [18]. In to the crystallization peak of residual graphite oxide that was not oxidized during the process [19,20]. The interplanar or d-spacing of GO was found to be 0.656 Å for d001 [14].

Pure TiO2 prepared through TiCl4 hydrolysis showed an XRD diffraction pattern of anatase phase (ICDD:03-065-5714) with the tetragonal structure without other TiO2 polymorphs [21][22][23][24]. Pure BiVO4 (ICDD:01-075-1866) synthesized by sol-gel route displayed monoclinic scheelite phase [25,26] and the iconic twin peaks of BiVO4 [27].

The GO peaks were not observed in the XRD pattern of nanocomposites ( and (b)) due to the overlapping intense peaks of anatase/monoclinic scheelite phase or the low detection limit of XRD technique or maybe by the reduction of GO to reduced GO [22,28]. Still, the formed MOx nanocomposites were highly crystalline with no phase transformation even after the physical synthesis route. In both nanocomposites, the intensity of the (101) plane of GOT and the (103) plane of GOB has significantly reduced with increasing GO content, resulting in the crystallinity enhancement as mentioned in Table 1. Conversely, for 2.5 GOT and GOB, the crystallinity has been increased, which can be assumed due to the increment in the GO content [29].

The crystallinity obtained for 1.5 GOT and GOB (Table 4.1) can result in boosted photocatalytic disinfection application. This is due to the increment of the surface area, light absorption, surface energy, and lattice distortion of the formed nanocomposites.

Moreover, the energy of photo-generated radicals is increased with the decrement of the crystallite size, in association to the quantum confinement effect, thereby enhancing the photocatalytic disinfection process [30]. show the enlarged view of the [START_REF] Wu | Visible-light-induced photocatalytic inactivation of bacteria by composite photocatalysts of palladium oxide and nitrogen-doped titanium oxide[END_REF] anatase peak in GOT and (103) monoclinic phase of GOB nanocomposites. In GOT nanocomposite, there was a shift towards a higher 2θ angle, while a shift to lower 2θ angle in 0.5 GOB, and 1.5 GOB nanocomposites; for 2.5 GOB (the shift occurred toward higher angles). These results can be attributed to the decrement of the lattice parameters by increasing the GO content. The crystallite size was obtained using the Scherrer equation [31].

= Eq: (4.1)
Where, K -Scherrer constant (0.94 for spherical particles), λ -Wavelength of Cu -Kα1 (approximately 1.5405 Å), β -Full width at half maximum (FWHM) value, θ -Bragg's angle (in radians).

Raman studies

Raman spectroscopy is one of the powerful techniques to analyze the crystal structure of carbon-based materials [32]. In oxide nanoparticles, several factors like lattice dynamics modifications are detailed by the transitional invariance, which is broken up due to the extension in the crystal structure (1-100 nm range), along with the explanation of atomic vibrations in a domain of that kind in terms of phonons is not possible with a well-defined wave vector and this forms the basis for the phonon confinement model (PCM). PCM was developed for the description of the intensity in first-order Raman scattering of semiconductor nanocrystals [33,34].

Raman spectra are based on vibrational modes, and therefore, graphene possesses six normal modes and two atoms per unit cell at Brillouin zone center (Г), where (Г) = A2u + B2g + E1u + E2g, in which E2g phonons are more active. The three prominent peaks in the Raman spectra of graphene are D peak at 1320-1350 cm -1 , G peak at 1580-1605 cm -1 , and 2D or G' peak at 2640-2680 cm -1 , sometimes at 1602-1625 cm -1 .

Defect and disorder level within the graphite layers is associated with the D mode, and the two-dimensional hexagonal lattice is related to G and 2D bands [35]. G band can be attributed to the first order scattering of the E2g phonon of sp 2 carbon atoms. D band is the result of distension in the hexagonal symmetry of graphitic lattice due to the edge defects, internal structural defects, and dangling bonds, whereas the G band is attributed to the in-plane stretching motion of symmetric motion or the symmetric sp 2

C-C bonds. The narrowing of the G band is because there are fewer functional groups in the atomic lattice [36,37]. These peaks form the characteristic feature for the graphene-based materials, but slight variation can be observed for the parameters like linewidths, intensities, and peak position for the graphene-based material because of graphene (support) interaction, heteroatom doping, strain, number of graphene layers, etc.

During the oxidation of graphite to form graphite oxide, the G band may get broadened, and the D band is narrowed, or the intensity is increased, which are attributed to the considerable decrease in the size of in-plane sp 2 domains due to the introduction of oxygen-containing groups. Also, the shift in the G band is ascribed to the presence of isolated double bonds that prevails on the carbon network of graphite oxide [38]. The intensity ratio of D and G peaks, i.e., ID/IG is calculated for the comparison of structural defects. Raman spectroscopy is used for the study of the level of alteration on the GO surface. As per the group theory, TiO2 anatase possesses six Raman active modes, namely, Eg (144 cm -1 ), Eg (197 cm -1 ), B1g (399 cm -1 ), B1g (513 cm -1 ), A1g (519 cm -1 ), and Eg (638.2 cm -1 ) [39][40][41]. When there is a shift and broadening of the Raman peak (144 cm -1 ), it can be due to the surface pressure or confinement effects. The highintensity peak at 144 cm -1 can be the result of the long-range order of the anatase.

Broadening of the peak indicates the contribution of phonons away from the Brillouin zone center [42].

TiO2 anatase nanoparticles are well characterized and studied by Raman scattering spectroscopy. The size effects are interpreted by phonon confinement or nonstoichiometry [40,[43][44][45]. BiVO4 nanoparticles are excited by a red laser with a wavelength of 648 nm, and the typical Raman vibrational bands are 210 cm -1 , 324 cm - 1 , 366 cm -1 , 640 cm -1 , 710 cm -1 , and 826 cm -1 . Raman band at 210 cm -1 can provide the structural information of BiVO4, while bands centered at 324 cm -1 and 366 cm -1 are the asymmetric and symmetric VO4 tetrahedron formations, respectively. The asymmetric stretching vibration of the shorter V-O bond is assigned at 640 cm -1 . Bands centered at 710 cm -1, and 826 cm -1 are the stretching modes of two vibrational modes of V-O bonds. The information regarding the variations in the structure is given by the two bands, 710 and 826 cm -1 [46][47][48].

The D (disordered or sp 3 defect) peak in the GO spectrum around ∼1350 cm -1 is due to the occurrence of -OH and epoxy groups, while G (graphitic or sp 2 defect) peak ∼of nanoparticles, where the stretching and bending vibrations, modes result from the VO4 3-tetrahedron. In the spectra, the intense peak at 780.7 cm -1 was dominated by the symmetric V-O stretching mode (vs), and a weak shoulder at 661.1 cm -1 was associated to the antisymmetric V-O stretching mode (vas). 314.99 cm -1 was associated to the symmetric bending mode (δs (VO4) 3-), and the peak at 280.11 cm -1 , was related to the antisymmetric bending mode (δas (VO4) 3-). The external rotational mode was observed at 235 cm -1 , which belongs to the vanadate ion. The twin band, δs (VO4) 3-and δas (VO4) 3-was merged, showing a single peak as the GO content is increased due to the phase transition from monoclinic to tetragonal. The decrease in the intense peak vs. was attributed to the increase in the loading of GO [52].

All these modes were typical for pure monoclinic scheelite phase of BiVO4

nanoparticles [53]. was increased [54][55][56]. This was clear evidence on the nanocomposite formation in both cases without any crystalline phase changes.

UV-Vis DRS analysis

Due to the presence of π-π* sp 2 clustered C=C chromophore units, a predominant absorption maximum was observed at approximately 230 nm. At about 300 nm, the presence of a shoulder was shown due to its n-π* plasmon peak of autochrome, which has got carboxyl, carbonyl, and hydroxyl functional groups. Absorption at approximately 212 nm is caused by the π-π* aromatic transition of the C-C bond, and this defines the sp 3 feature of GO. There can be a change in this behavior if there is a reduction in the oxygen groups of graphene oxide [57,58]. TiO2 was found to be a large bandgap semiconductor, with a bandgap value of 3.2 eV. The valence band of TiO2 consists of 2p orbitals where oxygen is hybridized with 3d orbitals of titanium, and the conduction band is composed of 3d orbitals of titanium

Morphological analysis

(a) SEM and elemental mapping

The GO showed the wrinkle-curtain-like structure, where the folding can be due to the stacking (Fig 4 .5). In MOx nanoparticles, the particle aggregation and bulkiness of the material were seen but with the uniform distribution of the corresponding elements (carbon-C, oxygen-O, titanium-Ti, bismuth-Bi, vanadium-V). The elemental analysis confirmed the GO/MOx nanocomposite formation, depending on the wt.% ratios with no other impurities. These results were consistent with the optical and structural characterizations discussed in the above sections. BiVO4 and C of GO contribute to the construction of GOB nanocomposite [78]. This confirmed the successful formation of nanocomposites and their stability. The copper-Cu was also detected in both the spectra, due to the Cu grid used for the sample analysis. nanoparticles, around 20% disinfection was seen in 30 min. Interestingly, in the presence of nanocomposites, disinfection activity was enhanced by more than 60%, which was influenced by the loading of GO and TiO2. Among the three GOT nanocomposite ratios, 1.5 GOT showed better bactericidal activity towards E. coli K12, resulting in more than 99% disinfection in 30 min, which was decreased when GO content was increased in the nanocomposite (Fig 4 .11 (a)). In logarithmic terms, 3-log reduction of E. coli K12 was observed using 1.5 GOT. After 120 min of light exposure, no significant disinfection activity was found for any of the composites. It is possible that, the availability of active sites might have reduced after a certain period. The decrement in the photocatalytic activity can be ascribed to excess GO, which can increase the opacity and light scattering of the nanocomposites, impeding the MOx nanoparticles from light absorption. A decrease in the activity can also be because of the short half-life period of radicals generated, usually in the order of microseconds or ROS formed in a low concentration. As a result, bacteria can have a defense mechanism to protect themselves, thereby causing bacterial recovery and re-growth [80][81][82][83]. pairs, which is contributed by GO as it is both an electron acceptor and transporter [76]. Apart from the other facts, high crystallinity enhances photocatalytic efficiency, confirmed by the XRD (Fig 4 .2). Therefore, in conclusion, 1.5 GOT with a dosage of 1.05 g/L, and 1.5 GOB with a dosage of 0.1 g/L were chosen for further studies.

(b) TEM micrographs

Applications of nanocomposites (a) Photocatalytic disinfection experiments

(b) Bacterial morphology studies

The damage of bacterial cells concerning the various ROS species upon contact with 1.5 GOT and GOB was investigated by TEM. Electron micrographs show a dramatic change between control bacteria and nano catalyst-treated ones. For instance, the decomposition of the cell membrane leads to the leakage of internal cell components resulting in the cell becoming white or translucent. The two-way synergistic mechanism of nanocomposites causes this. The MOx nanoparticles produce ROS, while GO can act as nano-knives that penetrate into the bacterial cells, resulting in the membrane disruption [84,85]. Thus, the TEM investigation proved the impact of GO/MOx composite altering cell membrane permeability and intracellular leakage of components, killing bacteria cells.

(c) Scavenger studies

From the ROS scavenger study (see Chapter 2, section 2.5.2) of 1.5 GOT nanocomposite with the addition of chromium (VI), the photocatalytic disinfection was not affected. This proves that the role of e -for disinfection is low. Due to the addition of C6H4O2 for the removal of O2•⁻ radicals, the disinfection was inhibited more when compared to chromium (VI) scavenger, which shows that O2•⁻ radicals have a crucial The results also pointed out the importance of low dosage as there was a decrease in the reaction rate constant when the dosage was increased. This is due to two main reasons: The mechanism of photocatalytic disinfection of a GO/MOx nano catalyst can be explained in brief as follows. When 1.5 GOT/GOB and E. coli K12 are in contact, the catalysts got adhered over the bacterial cells. Then, the catalyst gains energy greater than its bandgap to generate ROS, which causes seizures on the microbial cell membrane and thereby allows the entry of particles that possibly reacts with the major cellular components. Once the cellular integrity and the nucleic acid or protein synthesis system are damaged, the cells are lysed, leaving behind the cellular debris [92,93].

Conclusions

GO/MOx nano catalysts were synthesized by adopting a green, facile, eco-friendly, and cost-effective method (one-step blending). Several characterization techniques were performed to demonstrate the formation of the nanocomposites. The morphological analysis (TEM and EDAX) revealed the homogeneous distribution of TiO2 and BiVO4 nanoparticles decorated over the GO sheets, having a curtain-like structure with wrinkles. According to Raman results, the corresponding D and G bands were identified at around 1323 and 1600 cm -1 , respectively, indicating the crystalline quality of the synthesized nanocomposites and strong evidence of composite formation. The band gap values were decreased with increasing the GO content as verified from the DRS analysis, suitable for the visible-light-driven photocatalysts, which is one of the foremost objectives of the incorporation of GO with TiO2 or BiVO4 nanoparticles. In addition to the formation of GOT and GOB nanocomposites, the enhancement in catalytic efficiency under illumination was reflected by the photocatalytic inactivation test. The disinfection was achieved to its maximum by 1.5

GOT at an optimum dosage of 1.05 g/L, providing 99.9% inactivation of E. coli K12 (10 7 CFU/mL) in 30 min under visible-light irradiation, while for 1.5 GOB 89% of disinfection with a very low dosage of 0.1 g/L, for a bacterial concentration of 10 7 CFU/mL, in 60 min. The kinetics of photocatalytic inactivation fitted well with the Weibull deactivation function and followed pseudo-first-order kinetics. The antibacterial reaction using photocatalysts is confirmed with the interaction of ROS by scavenger study.

Moreover, it was found that •OH radicals are responsible for the disinfection, which is followed by O2•⁻ and e -, while for GOB nanocomposites, it was found to be driven majorly by superoxide (O2•⁻) radicals. TEM image revealed the bacterial inactivation validation caused by 1.5 GOT and 1.5 GOB. There was a 3-log reduction and 1-log reduction of E. coli K12 for 1.5 GOT and 1.5 GOB nanocomposites, respectively. This result demonstrates the synergistic behavior of GO and nanoparticles that could enhance the adherence towards the bacteria for boosted disinfection. The in-depth study delivered a new insight and provided great potentiality in various biomedical applications.

Chapter 5

Study on micro algal photocatalytic destruction Chapter 5 : Study on micro algal photocatalytic destruction

Introduction

Strictly, aquatic oxygenic photosynthetic microorganisms divide in two groups, denoted as cyanobacteria and microalgae. The former groups prokaryotic organisms whereas the latter groups eukaryotic organisms. Both groups have colonized every type of ecological niches [1], including water reservoirs [2]. Both groups use nitrogen, phosphorus, sunlight, carbon dioxide, and water to generate biomass according to abiotic constraints. Under certain circumstances, cyanobacteria and microalgae can divide out of control, forming blooms [3][4][5]. Despite recent attempts to valorize the biomass produced during the blooms [6], cyanobacteria and microalgal blooms have generally a negative impact on economic activities, directly, for example by killing fishes [7] or indirectly, by rendering the water unfit for consumption. The increase in bloom frequency and the change in their geographic distribution makes bloom a very worrying events [8], especially regarding the phenomenon occurring in potable water reservoirs because water constitutes an increasing crucial resource [9]. In this frame, it is crucial to find adequate ways to efficiently treat the water contaminated by cyanobacteria and/or microalgae. To reach this goal, several possibilities of physical treatments such as plasma treatments have been tested. Recently, the availability of nanomaterials opened new avenues for water decontamination as illustrated in chapter 4.

However, the use of nanomaterials should be taken with caution because when dispersed in water, they can enter any type of organisms, and possibly be toxic to nontarget organisms. In all cases, internalized nanomaterials risk being returned to the environment upon the death of the organisms that absorbed them, which could constitute a source of pollution [10,11]. To avoid these possibilities, compressed nanomaterials, i.e., pellets (see chapter 2 for preparation) have been used in this chapter because pellets can resemble the films in terms of activity, films being the most common application for nanoparticles generating ROS. In addition, the use of dispersed nanoparticles, may result in ROS generation both internally and externally because nanomaterials have been internalized. The latter possibility vanishes using pellets, offering the possibility to study the effects of externally generated ROS alone on microalgae and determined the potential of externally generated ROS for water decontamination.

The photosynthetic process can be seen as the combination of reactions relying on different principles. In the biochemical phase responsible for carbon fixation, relies on biochemistry and is characterized by slow constant rates (in the range of s to min). In contrast, photo biochemical phase responsible for light absorption and, electron generation and transport, relies on physics and chemistry with fast constant rates (slowest in the ms range). From these statements arises the view that both types of reactions will be saturated very differently. According to the chemistry and physics rules, the oxidative risk, i.e., the generation of ROS in a photosynthetic cell, is significantly elevated because this process manipulates light energy, electrons, and oxygen, the latter being produced through the photooxidation of water. Living for several billion years in a cellular environment with a high oxidative risk, photosynthetic organisms have developed inducible mechanisms aiming at limiting the extent of the risk. Those mechanisms are powerful. Besides the classical enzymatic tools such as superoxide dismutase, catalases, and peroxidases, the photosynthetic cells can eliminate the excess of light energy absorbed as heat using several nonphotochemical quenching mechanisms. They play crucial roles in decreasing the internal ROS generation [12,13].

The photosynthetic apparatus contained two different types of pigment-protein complexes, namely photosystem I and photosystem II. Each photosystem is composed of a light-harvesting complex, meant to harvesting photons and transferring the associated energy to a reaction centre. The light-harvesting complex contains several pigments, including chlorophylls and carotenoids. For this work, only PSII is considered, because the chlorophyll fluorescence is mostly arising from this complex (see however [14]). Two extreme models have been created to explain how the photosystems are cooperating in the thylakoid membrane. At one extreme, the puddle model proposes that each photosynthetic unit is independent from the neighbouring photosynthetic units. At the other extreme, one finds the lake model according to which the photosynthetic units are cooperating to generate oxygen. Actually, many intermediate models have been proposed (reviewed in [15]). The degree of connexion between the photosynthetic units is given by the connectivity factor [16].

The performance of the photosynthetic apparatus has been studied by different means, including the variations of the chlorophyll fluorescence yield. Since the pioneering publication of Kautsky et al,1960 [17], the analysis of the modifications of the chlorophyll fluorescence yield has been developed until it becomes a major and popular method of the study of the photosynthetic activity [18]. Several protocols are available, including the saturation pulse method (for reviews, see [18][19][20]). The saturation pulse method can provide information during steady-state illumination, e.g., under field conditions and applied to every type of photosynthetic organisms, including lichen and phytoplankton. The method presents several other advantages, such as its sensitivity, easiness to manage, great flexibility and a rather simple mathematical formalism. It is based on the recording of the variation of two chlorophyll fluorescence yields of dark-adapted samples and in the light. It is out of the scope of this contribution to review the experimental procedures leading to the measurement of the different chlorophyll fluorescence yields. The interested readers are referred to excellent recent reviews on the topic [17][18][19][20]. Briefly, the time course of chlorophyll fluorescence yields is recorded in dark-adapted samples and in the light. These chlorophyll fluorescence yields are F0 and FM and, Ft and FM', respectively (for nomenclature see [13]). Based on these basic yields, a myriad of fluorescence parameters has been defined (see Chapter 2, Table 2.2). They are used to characterize the photochemical and nonphotochemical mechanisms occurring in the photosynthetic apparatus during an illumination. They are only described shortly here -for a review see [18,19,21]. The definitions of the parameters presented in Table 2.2 does not need to know the level of connectivity between photosynthetic units [21]. Most of the studies on this topic have been dedicated to the photosynthetic apparatus of land plants and it has been concluded that the lake model generally prevails [21], even though sometimes the connectivity was not observed during the first hours of bean leaf greening [22]. Even though several studies claimed that the model is also valid in algae, the type of organization of the photosynthetic apparatus in phytoplankton, including diatoms, is far to be investigated thoroughly. Nevertheless, a formal puddle model seems unrealistic because it is established that PSII reaction centres form functional dimers in the diatom thylakoids [23]. Several elegant measurements have shown that the photosynthetic units are well approximated by the lake model, but are most accurately described by intermediate models [15]. However, studying the marine diatom


Thalassiosira pseudonana [24], Xu et al found rather low value for the PSII connectivity [25]. Interestingly, the values were increasing with the light intensity (30 versus 350 µmol/m -2 s), suggesting that an increase of proportion of the photosynthetic units in the configuration of the lake model would occur at high light intensities. Unfortunately, very high light intensities such as those used in this study were not tested and therefore the limit of this increase cannot be evaluated. Wu et al also working with T.

pseudonana reported an opposite variation i.e. a decrease of the connectivity under an increase of blue light irradiation (30 versus 450 µmol/m -2 s) [26]. Lavaud et al reported that the connectivity might greatly differ between diatoms [27]. They reported that the connectivity was almost zero in Phaeodactylum tricornutum (0.02) and much higher (0.16) in Skeletonema costatum. Being unable to conclude about the type of model prevailing in P. tricornutum, both puddle and lake models were considered. It is important to note that the mathematical derivation have demonstrated that the parameter YII has the same equations in both models [28].

In aquatic environments, nanoparticles can aggregate, dissolve or precipitate depending on their properties and their liquid medium, like size, coating material, shape, pH, ionic strength, and dissolved organic matter. Nanoparticles can interact with the algae surrounding them, similar to that occurring in bacteria [29][30][31][32][33][34][35][36]. The toxicity of nanoparticles or algal killing by the photocatalytic activity is by the physical restraints and oxidative stress [37] (Fig 2 .2). When there is a large aggregation of nanoparticles over and around the microalgae, the availability of light for photosynthesis is reduced and blocks the nutrient uptake [38,39]. By the exposure of algae with the nanoparticles, oxidative stress occurs due to the over-accumulation of the ROS generated [40][41][42][43][44]. The exposure to nanoparticles results in ROS production, thus causing lipid peroxidation of the membrane and activating antioxidative enzymes, The image shows the mechanism in which the nanoparticles cause the detrimental effect for the microalgae [37] This chapter discusses the effect of nanoparticles on cyanobacteria and microalgae in various conditions as a preliminary step towards microalgal disinfection.

Results and Discussion

Shaping the light beam to allow photosynthesis and photocatalytic ROS generation

The two objectives of this work were to synthesize innovative photocatalyst generating ROS under visible light irradiation (see Chapter 2) and using the generated ROS for decontaminating water from bacteria, cyanobacteria, and microalgae. From the above sections, a description of the characteristics of the innovative photocatalysts in terms of bandgap, corresponding at an absorbance at 405 nm, was reported (refer to Chapter 4, section 4.2.3). On the other hand, cyanobacteria and microalgae are photosynthetic organisms, i.e., they convert the energy associated with photons into biochemical energy [45,46] using a range of pigments, i.e., molecules able to absorb light [47]. For instance, the pigment set of the diatom P. tricornutum absorbs light in the visible range of electromagnetic spectrum, including the blue region. Therefore, the light beam used for ROS generation can also be used for photosynthesis. The

Photocatalytic ROS generation of pellets

To demonstrate the capacity of pellets to generate ROS, methyl blue degradation was followed by recording the variation of the absorbance at 665 nm (ΔA665 = A665t=0 -A665t=t) in different conditions (Fig. 5.3 (a)). Methylene blue is considered as a standard dye for the photocatalytic experiments. The absorbance spectra were corrected as explained in chapter 2, section 2.8.3 (a). Regardless the presence of nanomaterials, no significant variation of the absorbance was observed in the absence of light (yellow squares and black diamonds in Fig. 5.3 (a). In the presence of light and nanomaterials (1.5 GOT), the absorbance at 665 nm decreased linearly before to saturate after 80 min of illumination (black squares in Fig. 5.3 (a)). To demonstrate that the decrease in the absorbance at 665 nm is due to photocatalysis, the experiment was repeated with nitrogen bubbling inside the cuvette, limiting ROS formation. In this condition no significant variation of ΔA665 was recorded (Fig 5 .3 (b)). The mechanism involved is as follows [48]:

/ + ℎ → / ( + ℎ ) ℎ + MB → ( ) + ℎ → . + . + → +

Photocatalytic disinfection of Anabaena

Anabaena is a filamentous cyanobacteria (Fig 5 .4). Some taxa are famous for forming blooms and toxins [49]. Traditionally, the evaluation of disinfection requires enumeration of the living organisms as explained in the previous chapter. Unfortunately, even if they are bacteria, cyanobacteria, especially filamentous cyanobacteria, grow much slower than non-photosynthetic bacteria. In addition, the enumeration of filamentous cyanobacteria requires a specific chamber allowing the sample to settle. Once sedimented, the individuals are counted by direct counts under the microscope. This method is called the Utermöhl method [50]. Because this method is very timeconsuming, it was decided to follow the variations of an internal marker of the cyanobacteria, such as phycocyanin or chlorophyll a. To reach this goal, in situ absorbance spectroscopy was chosen because it is fast and relatively sensitive.

Measuring absorbance spectra in situ is always tricky because of the featureless nonselective light scattering of the sample [51] This scattering depends on cell size and shape and results from changes in the refractive index at the interphase boundaries e.g., between medium, cell, cytosol, and intracellular structures [52]. In addition, a selective scattering with prominent spectral features related to sharp changes in the refractive index in the region of light absorption by aggregated pigments embedded in thylakoid membranes is observed [53][54][55][56]. Scattering is also wavelength dependent, and proportional to the inverse of the fourth power of the wavelength. To summarize, the absorbance spectrum of a scattering sample presents two typical characteristics: and phycobilisomes (637 nm) (Fig. 5.5). Similar spectra were obtained by Mezlyak et al [51]. The correction was not perfect because the corrected spectrum presented a weak distortion zone around 550 nm and was not valid for the blue region (Fig. 5.5 (b)). Because the blue region was not used in this work, the small distortion around 550 nm was considered as negligible. The absorbance was measured far from this distortion zone, i.e., between 600 and 700 nm.

From the above discussion on the damping effect of scattering on the amplitude of the absorbance bands, it was first determined how many algae are needed to record a signal in the range of 600 to 700 nm. For such a purpose, absorbance spectra of increasing amount of cyanobacteria were recorded by diluting increasing volume of a mother culture (OD750 = 6) in the recording cuvette (3 mL) (Fig 5 .5 (c)) and corrected according to the procedure explained above. The absorbance at 750 nm in noncorrected absorbance spectra (A750) and the absorbance at 637 nm in corrected absorbance spectra (ΔA637) were taken as proxies for the number of cyanobacteria and the phycocyanin amount, respectively. the correction procedure was adequate and (2) the ratio between the proxies reflected the cellular quota in phycocyanin. Among the tested dilutions, the highest absorbance variations were obtained with 400 µL of mother culture, this volume was used for further investigations. Higher volumes were not tested because the values of ΔA637 started to saturate (data not shown). Surprisingly, the kinetic presented a positive trend suggesting that during the treatment chlorophyll a was produced. GOT composites. This can be assumed due to the following reasons [57,58]. This alga possesses a one-dimensional multicellular chain structure, as shown in Fig 5 .4, with a diameter between 3 and 5 µm. Therefore, i. Anabaena cells are much larger when compared to Escherichia coli.

ii. The quanta of generated reactive oxygen species (ROS) by the interaction of 1.5 GOT pellet photocatalyst, and the light irradiation was not enough for the disinfection process.

iii. The age of the culture is an important parameter as the resistivity of the cells increases with time due to the protective coating which is formed outside the cell.

iv.

The method used to follow the disinfection may be not adequate. Actually, irradiation using UVA, UVB or a combination of both during 24 h only slightly decreased the cellular quota in chlorophyll a [59]. For this reason, another method was used to follow the disinfection of media containing diatoms.

Photocatalytic disinfection of Phaeodactylum tricornutum

Phaeodactylum tricornutum is a marine unicellular diatom famous for its morphological modifications depending on the culture conditions [60,61]. In most conditions, it occurs under pennate morphotype as displayed in Fig 5 .7. P. tricornutum was chosen for this work because under axenic conditions in artificial seawater e.g., f2 medium, cells divide approximately once a day. In addition, its genome has been sequenced [61] and the literature about its physiology, biochemistry, stress response and biotechnological applications is abundant [62][63][64]. Using this diatom will extend the data about the potential of using the nanoparticles prepared in this study for decontamination of marine organisms, that also form blooms [3].

(a) Effect of light intensity on the diatom inactivation by photocatalysis

When working with microalgae and a photocatalytic material excited in the blue region, light play a dual role: it is used for generating ROS and to drive photosynthesis. If the former activity is positively impacted by light, the second one is not always because excess of photons and may increase the internal oxidative risk. To find a compromise between these two opposite actions of light, the effect of two light intensities on the efficiency of photocatalytic algal removal in freshwater media at 22 °C was studied in the absence (photolysis) and in the presence of the photocatalytic material.

(c) Effect of time on the destruction of algal cells

The data described in the previous sections indicated that increasing the light intensity to boost externally ROS production might have a counter-productive effect on microalgal cell destruction because it would enhance the intensity of the defence mechanisms. If we take into account that (1) these defence mechanisms are costly from the cell energy and NAD(P)H/NAD(P) + consumption point of views [67], (2) under prolonged light irradiation, photosynthetic activity is saturated and even reduced, and -Dead: In addition to scarcity, those diatoms that were observed no longer viable (Fig 5.13 (d)). This conclusion is because cell viability is lying on plasmalemma integrity. Therefore, cell staining with a dye that is not penetrating naturally in cell unless plasmalemma has been disrupted can be used as a proxy of cell viability. Evans's blue, an azo dye exhibits this property [66], was used for such a purpose. The samples were observed after photocatalysis for maximum uptake of blue coloration, pointing out to the lost membrane integrity of the algal cells.

These results heavily contrast with those obtained without the photocatalytic treatment, with diatoms viable from the physiological point of view ( formation of the nanocomposite. The antibacterial ability of all the synthesized five different composites was tested using a visible-light-driven homemade photoreactor.

The results revealed the efficiency of the 1.5 GOB nanocomposite at 0.1 g/L, giving 89% disinfection in 60 mins under visible light irradiation for an initial bacterial loading of 10 7 CFU/mL.

Finally, the optimized nanocomposite used for the bacterial disinfection was extended to apply the microalgal destruction by the photocatalysis process. The preliminary studies on photocatalytic disinfection using microalgae were performed against Anabaena sp. and Phaeodactylum tricornutum. For this study, we have used the nanocomposite in pellet form when compared to the bacterial disinfection. A brief description of the parameters responsible for the photocatalytic disinfection mechanism was also discussed.

Future prospects

With the promising outcomes obtained from the above investigations, future work is aimed to assess these nanoparticle systems in actual samples, and to scrutinize the effects and applications in the field would be interesting. To achieve this, it would be ideal using the synthesized nanocomposites to produce a film or pellet to gain a better microbial disinfection activity considering future applications. By keeping in mind, the application on a large-scale wastewater treatment process and avoiding the further contamination of drinking water by the powdered nanocomposite or avoiding the negative interaction with the waste elements in polluted water, better understanding of the waste nanocomposite is essential. So, prospects of microbial disinfection activity aim to reach an efficient model for the application in the sewage water treatment process and largescale drinking water treatment systems.

The preliminary results obtained from the investigation of microalgal disinfection activity are optimistic for the fight against harmful algal blooms and their toxins. As mentioned before in the microbial disinfection, more optimizations in dosage and a detailed study in the mechanism and the interaction of these nanocomposites with the salinity are essential for the active exploration of the material activity, paving the way in the destruction of microalgal cells. The effect of the ROS activity due to the nanoparticles has to be discovered for various types of microalgae, as it has got complex structure compared to the bacteria. We attained disinfection of E. coli K12 having a concentration of 10 7 CFU/mL with a small quantity of 1.5

GOT (1.05 g/L), obtaining 99.9 % of inactivity in 30 min.

Conversely, for 1.5 GOB (0.

1 Fig 1 . 1 Fig 1 . 2 Fig 1 . 3 Fig 1 . 4 Fig 1 . 5 Fig 1 . 6 2 Fig 2 . 1

 1111213141516221 Fig 1.1 The causes of water pollution in the world Fig 1.2 An index for level of water crisis from significantly affected to less stressed countries Fig 1.3 Water stress index of various parts in Mexico Fig 1.4 Reasons behind the cause of diseases and increase in death rate of Mexico Fig 1.5 The general classification of advanced oxidation processes Fig 1.6 The general mechanism of photocatalysis by a semiconductor Fig 1.7 Lerf-Klinowsky model of GO showing the epoxy and hydroxyl bonds Fig 1.8 The mechanism of the photocatalytic disinfection by GO/TiO2 composite Fig 1.9 Mechanism of photocatalytic inactivation of microbes by a semiconductor through the generation of reactive oxygen species Chapter 2

Fig 2. 2 Fig 2 . 3

 223 Fig 2.2 (a) Escherichia coli K12 (E. coli K12) (CBDB-11-1538), (b) E. coli K12 as green metallic sheen in Eosin Methylene Blue agar (EMB agar), (c) E. coli K12 as white translucent colonies in Nutrient agar. Fig 2.3 Culture of microalgae (a) Phaeodactylum tricornutum b) Anabaena flos aquae with air circulation through the cap.

Fig

  Fig 2.4 Orange G dye molecular structure Fig 2.5 (a) Dropwise addition of ammonium hydroxide to zinc chloride solution to obtain the precipitate (b) the image displays the occurance of precipitation after the reaction of ammonium hydroxide and zinc chloride Fig 2.6 (a) To the zinc chloride solution, controlled addition of ammonium hydroxide was performed through a burette to mark the formation of preciptate (b) milky white or cloudy precipitate by the reaction of zinc chloride and ammonium hydroxide Fig 2.7 (a) The microwave sample holder, where zinc acetate, methanol, ethylene glycol, and sodium hydroxide mixture were added for the hydrothermal

Fig 2. 9 Fig 2 . 12

 9212 Fig 2.9 (a) During the addidtion of TiCl4 to ethanol, large amount of HCl gas was generated (b) a light yellow tinted gel was formed through the sol-gel route Fig 2.10 Dropwise addition of solution A ((BiNO3)3.H2O in nitric acid to which citric acid was added) to solution B (ammonium vanadate and citric acid solution) Fig 2.11 The four steps involved for nanocomposite synthesis, i.e., ultrasonication, magnetic stirring, filtration, and drying, are depicted for GO/TiO2 composite synthesis. Fig 2.12 The step-by-step procedures that were carried out for the formation of pellets where successive techniques, i.e., uniaxial pressure and isostatic pressure was used.

Fig 2 .Fig 2 .Fig 2 . 16 Fig 2 . 18 Fig 2 . 19 Fig 2 .

 222162182192 Fig 2.13 (a) Pellets of the thickness of ≈ 3mm and diameter of 5 mm(b) pellets dissolved in 15 min (formed by uniaxial pressure) (c) pellets are stable in PBS or deionized water for three days (formed by successive uniaxial and isostatic pressure) Fig 2.14 The XRD representation and the basic principle for the working during the analysis of samples Fig 2.15 Bruker D2 phaser diffractogram (MREB, CINVESTAV) Fig 2.16 Graphene crystal structure (a) Zigzag, and (b) Armchair Fig 2.17 Raman spectroscopy analysis (SEES, CINVESTAV) Fig 2.18 Scanning electron microscope (SEM) (MREB, CINVESTAV) Fig 2.19 Transmission electron microscope (TEM) (Physics department, CINVESTAV) Fig 2.20 (a) Graph plotting the triplicate bacterial growth curve, (b) photographs after the nutrient agar plates was spread plated after the growth curve study.

Fig 2 . 21 3 Fig 3 . 1 Fig 3 . 4 Fig 3 . 6

 2213313436 Fig 2.21 The sequential protocol followed for the microbial photocatalytic disinfection study using E. coli K12 as the model organism Fig 2.22 Procedure used for correcting the in vivo absorbance spectra Fig 2.23 Experimental set up used for measuring the effect of photolysis and photocatalysis.

Fig 3. 8 Fig 3 . 9

 839 Fig 3.8 XRD spectra of GO/ZnO composite synthesized through simple blending process, depicting defined peaks of ZnO nanoparticles. Fig 3.9 Photographs depicting the disk diffusion assay performed in nutrient agar plates spreaded with E.coli K12 suspension. It was identified that the zone of inhibition was formed by the effect of GO/ZnO composite after visible-light irradiation followed by overnight incubation.

Fig 3 .Fig 3 .Fig 3 . 4 Fig 4 . 1

 333441 Fig 3.10 The XRD diffractometer of five different ratios namely, 0.2/0.8, 0.3/0.7, 0.5/0.5, 0.7/0.3, and 0.8/0.2 GO/TiO2 compoite showing the intense peaks of TiO2 anatase phase. Fig 3.11 SEM analysis of GO with wrinkled structure and five different synthesized GO/TiO2 composite (0.2/0.8, 0.3/0.7, 0.5/0.5, 0.7/0.3, and 0.8/0.2)

Fig 4. 3

 3 Fig 4.3 Raman spectra of pristine GO, TiO2 and BiVO4 nanostructures and (a) 0.5, 1.5, 2.5 GOT nanocomposites, (b) 0.5, 1.5, 2.5 GOB nanocomposites, D and G band of GO region-of-interest from 1100-1700 cm -1 (a*) GOT and (b*) GOB nanocomposites.

Fig 4. 4

 4 Fig 4.4 UV-Vis-DRS spectra of pristine and nanocomposites (a) TiO2, 0.5, 1.5, 2.5GOT, (b) BiVO4, 0.5, 1.5, 2.5GOB.

Fig 4. 5

 5 Fig 4.5 SEM images and the corresponding elemental mapping of pristine nanostructures, such as GO, TiO2, BiVO4 Fig 4.6 SEM images and the corresponding elemental mapping of 0.5, 1.5 and 2.5 wt.% of GOT nanocomposites Fig 4.7 SEM images and the corresponding elemental mapping of 0.5, 1.5 and 2.5 wt.% of GOB nanocomposites.

Fig 4. 8

 8 Fig 4.8 (a), (b) Low-resolution, (c) high-resolution TEM image, (d) and (e) corresponding FFT pattern of 1.5 GOT, respectively

Fig 4. 9

 9 Fig 4.9 (a), (b) Low-resolution, (c) high-resolution TEM image, (d) and (e) corresponding FFT pattern of 1.5 GOB, respectively Fig 4.10 Energy dispersive analysis of (a) 1.5 GOT (b) 1.5 GOB. Fig 4.11 E. coli K12 survival after photocatalytic disinfection is depicted as the ratio of viable to total cells (Nt/N0). (a) GOT nanocomposites (b) GOB nanocomposites.

Fig 4 . 12 Fig 4 . 14 Fig 4 . 16 5 Fig 5 . 1 Fig 5 . 4 FilamentousFig 5 . 5

 4124144165515455 Fig 4.12 Photographs of photocatalytic disinfectant activity of pristine nanoparticle and the corresponding E. coli K12 colonies on EMB agar plate Fig 4.13 Images of E. coli K12 colonies on EMB agar medium after irradiation with GO/MOx nanocomposites respectively.

Fig 5. 6 Fig 5 . 7 Fig 5 . 8 Fig 5 . 9 Fig 5 . 10

 6575859510 Fig 5.6 Kinetic of the absorbance at 665 nm changes specific to the photocatalytic treatment Fig 5.7 Microscopic pictures of the marine diatom Phaeodactylum tricornutum. Fig 5.8 Effects of light intensity of the continuous illumination on the chlorophyll fluorescence recorded in the absence (green) or in the presence (orange) of photocatalytic material

Fig 1 . 1 :

 11 Fig 1.1:The causes of water pollution in the world[3,4]

Fig 1 . 2 :

 12 Fig 1.2: An index for level of water crisis from significantly affected to less stressed countries (The image is modified information from reference [8])

Fig 1 . 3 :

 13 Fig 1.3: Water stress index of various parts in Mexico (reproduced from [15])

Fig 1 . 4 :

 14 Fig 1.4: Reasons behind the cause of diseases and increase in the death rate of Mexico [16].

Fig 1 . 5 :

 15 Fig 1.5: The general classification of advanced oxidation processes [47].

Fig 1 . 6 )

 16 Fig 1.6: the general mechanism of photocatalysis by a semiconductor[54].

Fig 1 . 7 :

 17 Fig 1.7: Lerf-Klinowsky model of GO showing the epoxy and hydroxyl bonds[56]

  an improved photocatalytic activity of TiO2. Charge separation occurs due to the formation of superoxide radicals by the electrons in the GO sheets by the reaction of dissolved oxygen. The extraction and transfer of the electrons from TiO2 towards GO lead to efficient charge carrier separation, thus enhancing the photocatalytic disinfection [66-68] (Fig 1.8).

Fig 1 . 8 :

 18 Fig 1.8: The mechanism of the photocatalytic disinfection by GO/TiO2 composite[69]

  photocatalytic disinfection is still unknown. Various kinds of literature reports disintegration of peptidoglycan, formation of pores in the cell wall and cell membrane, lipid peroxidation, DNA damage, etc., as the outcome of photocatalytic inactivation [100-105]. Fig 1.9 shows a proposed mechanism of photocatalytic disinfection

Fig 1 . 9 : 1 . 7

 1917 Fig 1.9: Mechanism of photocatalytic inactivation of microbes by a semiconductor through the generation of reactive oxygen species (Modified from [106])

( i )

 i photosynthetic bacteria E. coli, the cyanobacteria Anabaena, sp., and the microalga Phaeodactylum tricornutum. The obtained results are confirmed with the advanced

Brief description Chapter 2

 2 photocatalytic disinfection study and the various stages of optimizations considering the application.Chapter 4The fourth chapter discusses the characterization and applicationlevel study of photocatalytic disinfection of microbes using GO/TiO2 and GO/BiVO4 nanocomposite. The results confirmed with advanced characterizations are also discussed here.Chapter 5 This chapter reports the preliminary studies on the photosynthetic microorganism's disinfection study by the nanocomposite. It goes through various optimizations, and stages followed to accomplish the destruction of cyanobacteria and microalgae.

Fig 2 . 1 :

 21 Fig 2.1: Flowchart showing the selection of functional materials. As a preliminary step, the materials were examined for their efficiency in the disinfection research. The optimized materials and conditions proceeded for the photocatalytic disinfection study as the second stage.

11 -

 11 1538) were obtained from the Colección Nacional de Cepas Microbianas Celulares (CDBB) of CINVESTAV, Mexico (Fig 2.2 (a)). E. coli K12 was grown at 37°C overnight in LB broth under aerobic conditions with constant shaking. For the preparation of the bacterial stock suspension, 3 µl of E. coli K12 was added into 3 mL of Luria Bertani (LB) medium and incubated for 16 h at 37 ˚C. The initial concentration of the bacterial suspension was around 10 9 CFU/mL. Then, bacterial cells were collected by centrifugation at 4000 rpm (RCF = 1792) for 20 min. The pellets were resuspended and washed with sterile phosphate buffer saline (PBS) solution three times. The bacterial stock suspension was enumerated by surface plating in triplicate samples on nutrient agar after serial dilution in PBS. The plates were incubated for 24 h at 37°C (Fig 2.2 (b) and (c)).

Fig 2 . 3 :

 23 Fig 2.3: Culture of microalgae (a) Phaeodactylum tricornutum b) Anabaena flos aquae with air circulation through the cap.

Fig 2 . 4 :

 24 Fig 2.4: Orange G dye molecular structure[5]

Fig 2 . 5

 25 (a)). The reaction mixture was continuously stirred in a magnetic stirrer at room temperature till the complete precipitation occurred (Fig 2.5 (b)). The precipitate was washed with deionized water and then dried in a hot air oven at 100° C for complete drying. The dried precipitate was crushed, milled, and then sieved to get uniform-sized particles. After sieving, the precipitate was calcined in a muffle furnace at 450° C for 2 h by the heating rate at 5°C[6].

Fig 2 . 5 :

 25 Fig 2.5: (a) Dropwise addition of ammonium hydroxide to zinc chloride solution to obtain the precipitate (b) the image displays the occurrence of precipitation after the reaction of ammonium hydroxide and zinc chloride

Fig 2 . 6 :

 26 Fig 2.6: (a) To the zinc chloride solution, controlled addition of ammonium hydroxide was performed through a burette to mark the formation of precipitate (b) milky white or cloudy precipitate formation by the reaction of zinc chloride and ammonium hydroxide

Fig 2 .

 2 Fig 2.7: (a) The microwave sample holder, where zinc acetate, methanol, ethylene glycol, and sodium hydroxide mixture were added for the hydrothermal treatment (b)

  Fig 2.8 (a)) and then magnetically stirred at room temperature for 12 h (Fig 2.8 (b)), so that a homogeneous suspension was obtained. The solid obtained after filtration (Fig 3.8 (c)) was further dried (Fig 2.8 (d))in an oven at 50 °C for 4 h[9].

Fig 2 . 8 :

 28 Fig 2.8: (a) The desired or weighed amount of GO and metal oxide was mixed in deionized water for ultrasonication (b) the ultrasonicated suspension was magnetically stirred for 12 h in room temperature (c) Overnight sample was filtered using a nylon membrane (d) the filtrate was dried to obtain the powder.

-Second stage 2 . 4 . 1

 241 Synthesis of TiO2 nanoparticlesTitanium dioxide (TiO2) anatase nanoparticles were synthesized through TiCl4 hydrolysis in ethanol because it uses low-cost precursors, and the hydrolysis process can be easily controlled. Briefly, to 15 mL of ethanol at 60˚C, 1.5 mL of TiCl4 were added dropwise with continuous magnetic stirring. During the process, a large amount of HCl gas was released due to the hydrolysis of TiCl4 with water (Fig 2.9 (a)). A gel with a light-yellow tint was formed after a period (Fig 2.9 (b)). The obtained gel was dried overnight at 100˚C and calcined at 450˚C [10].

  Solution A was added dropwise to Solution B under continuous stirring (Fig 2.10).

Fig 2 .

 2 Fig 2.9: (a) During the addition of TiCl4 to ethanol, a large amount of HCl gas was generated (b) a light-yellow tinted gel was formed through the sol-gel route

Fig 2 . 12 :

 212 Fig 2.12: The step-by-step procedures that were carried out for the formation of pellets where successive techniques, i.e., uniaxial pressure and isostatic pressure was used.

Fig 2 .

 2 Fig 2.13: (a) Pellets of the thickness of ≈ 3mm and diameter of 5 mm (b) pellets dissolved in 15 min (formed by uniaxial pressure) (c) pellets are stable in PBS or deionized water for three days (formed by successive uniaxial and isostatic pressure)

  technique analyzes molecular and crystalline structures, quantitative resolution of chemical species, qualitative identification of various compounds, isomorphous substitutions, measuring the degree of crystallinity, polymorphisms, stacking faults, particle sizes, and phase transitions[15][16][17][18]. In XRD, X-ray light falls on the crystal leading to the formation of diffraction patterns. These patterns show the physicochemical features of the crystal (Fig 2.14).

Fig 2 .

 2 Fig 2.14: The XRD representation and the basic principle for the working during the analysis of samples[18]

  this study, Bruker D2 Phaser X-ray Diffractometer (XRD) with Cu Kα radiation (λ= 0.15406 Ǻ) operating at 30 kV and 10 mA was used (Fig 2.15).

Fig 2 .

 2 Fig 2.15: Bruker D2 phaser diffractogram (MREB, CINVESTAV)

Fig 2 . 16 :

 216 Fig 2.16: Graphene crystal structure (a) Zigzag, and (b) Armchair

Fig 2 . 17 :

 217 Fig 2.17: Raman spectroscopy analysis (SEES, CINVESTAV)

Fig 2 . 19 :

 219 Fig 2.19: Transmission electron microscope (TEM) (Physics department, CINVESTAV)

2. 20 (

 20 a) and (b)). All experiments were performed in 50 mL batch reactors in triplicates under simulated solar radiation. The suspension was magnetically stirred at 400 rpm and exposed to light for 2 h. The halogen lamp was focused at 15 cm above the reactor and used a solar simulator light source at a light intensity of 500 W/m 2 . The xGO/MOx nanocomposites were sonicated in water for 30 min to ensure stable dispersion prior to each experiment. Subsequently, the bacterial stock solution was diluted in the reactor to the desired initial concentration (10 7 CFU/mL). Before irradiation, the mixture was kept in the dark, stirred for 30 min for reaching adsorption-desorption equilibrium. The bacterial viability was performed by the standard plate count method.Samples were drawn at different intervals during the experiment, after appropriate dilution, 100 μL of the sample were taken and put into Eosin-Methylene blue (EMB) agar plate and incubated overnight at 37°C (Fig 2.21). After optimizing the parameters, three control experiments were conducted under the same conditions. The control experiments were performed in the absence of photocatalyst. The disinfection percentage was determined using the equation (2.2) where, N (control) and N (time) was the number of viable cells counted from control and aliquots obtained at different time intervals, t, respectively.

Fig 2 . 2 . 8 . 2

 2282 Fig 2.20: (a) Graph plotting the triplicate bacterial growth curve, (b) photographs after the nutrient agar plates was spread plated after the growth curve study.

Fig 2 . 21 : 2 . 8 . 3

 221283 Fig 2.21: The sequential protocol followed for the microbial photocatalytic disinfection study using E. coli K12 as the model organism.

Fig 2 . 22 .

 222 Fig 2.22. Procedure used for correcting the in vivo absorbance spectra. (a) Mathematical fit (dashed curve) of the featureless absorbance spectrum recorded in situ (full line) (b) Corrected absorbance spectrum (dotted curve) as the result of the operation 'original spectrum-minus-fitted baseline'

2. 23 (

 23 e)). During this period, the sample was regularly irradiated by saturating light pulses (L-P), eventually triggering a transient increase of the fluorescence level, denoted as (Fig. 2.23 (b)). At the end of the L-ACT period, L-ACT was switched off but FR, FML and L-P during 20 min. The latter illumination eventually triggered a transient increase of the fluorescence level, denoted as " (Fig. 2.23 (e)). At the end of this 20 min period, the sample stayed on the bench overnight under the ambient light. The F0, FM and " levels were then recorded (Fig. 2.23 (e)). From the measurements of the basic chlorophyll fluorescence yields, parameters allowing the characterization of the photosynthetic activity as well as the management of the absorbed light energy were calculated using the equation indicated in Table2.2[59].

Fig 2 . 23 :

 223 Fig 2.23: Experimental set up used for measuring the effect of photolysis and photocatalysis.

  Maximum quantum yield of photosystem II (PSII) photochemistry FV/FM =(FM -F0)/FM Puddle, Lake YII Effective quantum yield of photochemical energy conversion in PSII YII = (FM' -Ft)/FM' = 1 -YNPQ -NPQ+1+qL(FM/Ft-1)) Lake YNO Quantum yield of nonregulated energy dissipation YNO = 1/(NPQ+1+qL(FM /F0-1)) Puddle qN Coefficient of nonphotochemical quenching qN = (FM -FM')/( FM-F0') Puddle qP Coefficient of photochemical quenching qP = (FM -F)/( FM '-F0') Lake qL Coefficient of photochemical quenching qL = qP * F0'/Ft After a period of darkness of 15 min in the presence of low-intensity FR-L, FR-L was switched off, and the sample was irradiated with FML for 2 min, allowing the measurement of the F0 level. The sample was irradiated with a single L-P, and the level FM is recorded. FR-L is switched on together with L-ACT for 0.5 to 4h. The chlorophyll fluorescence progressively decreased until reaching the FS level. During this period, the sample was regularly irradiated by L-P, allowing the measurement of level. At the end of the L-ACT period, all the light sources were switched off but FR, FML, and L-P, allowing the measurement of the levels and having the same meaning. The sample stayed on the bench overnight under the ambient light. The F0, FM, and levels were then recorded. The test using Evans blue (6,6'-[(3,3'-dimethyl[1,1'biphenyl]-4,4'-diyl)bis(azo)]bis[4-amino-5-hydroxy-1,3-naphthalenedisulfonic acid]

Chapter 3 :of materials and optimization of nanocomposites 3 . 1 IntroductionFig 3 . 1 :

 33131 photocatalytic disinfection under visible light irradiation [1,2]. This chapter discusses the scientific background and justification for the challenges faced during the selection of the materials, the preliminary characterization results, and finalization of the dopant nanoparticles and nanocomposites for the photocatalytic antibacterial study. The flow chart shows the steps undertaken during the process of selection (Fig 3.1).

  3.1). The oxidized MWCNTs, TiO2 anatase, and MWCNT/TiO2 composite were characterized by XRD analysis to identify the crystalline quality and purity of the materials. XRD characterization is a better tool for measuring graphitization extent and the degree of the nanotube alignment in MWCNTs. Fig 3.2 shows the XRD spectra of the composite and the individual materials. The intense peak at 2θ = 24° corresponds to the (002) reflection plane, whereas the lower intensity peak at 2θ = 43.6° is typical to the (100) plane. These planes point out to the functionalized MWCNTs. From the literature, peaks at 2θ = 25.2° and 2θ = 44° corresponds to crude MWCNTs, and peaks show a downward shift due to an increase in the sp 2 C=C layer spacing. This literature provides evidence for keeping the crystallinity of MWCNTs after the oxidation or functionalization of MWCNTs by the acid treatment. The characteristic peaks of synthesized TiO2 anatase defined in the XRD spectra, i.e., diffraction peaks at 2θ = 25.28°, 37.8°, 48.05°, 53.89°, and 55.06° corresponded to the (101), (004), (200), (

Fig 3 . 2 :

 32 Fig 3.2: XRD analysis of MWCNTs, TiO2 anatase, and MWCNT/TiO2 heterostructure Graph represents the formation of MWCNT/TiO2 composite through acid treatment.

Fig 3 . 3

 33 shows the SEM images of the TiO2 anatase nanoparticles (Fig 3.3 (a)) and the oxidized MWCNTs (Fig 3.3 (b)).From the SEM image of TiO2 nanoparticles, large pieces or chunks of particles were observed with agglomeration. The SEM images indicated the distribution of TiO2 nanoparticles on the nanotubes without any apparent agglomerations (Fig 3.3 (c)). TiO2 nanoparticles were enclosed within the three-dimensional matrix by the presence of dispersed active sites of MWCNTs. Seen from the image, the accumulation or decoration of TiO2 nanoparticles over the MWCNTs directs the supporting role played by MWCNTs acting as a core for the deposition and growth of TiO2 nanoparticles throughout the nanotubes. This image also indicates the close contact that arises between MWCNTs and TiO2 anatase nanoparticles[7][8][9]. This SEM image pointed to the confirmation for forming MWCNT/TiO2 composite. The average crystallite size agreed with the XRD analysis[10][11][12].

Fig 3 . 3 :

 33 Fig 3.3: SEM images of the (a) agglomerated TiO2 nanoparticles (b) oxidized or functionalized MWCNTs (c) TiO2/MWCNTs composite confirming the incorporation of TiO2 nanoparticles over the carbon nanotubes.

Eq: 3 . 1

 31 photocatalytic activity was attributed to the adsorption properties of TiO2 anatase nanoparticles decorated over the MWCNTs, thereby increasing the surface area. After 30 min of light irradiation, one-fourth of the dye was degraded (Fig 3.4)[14,15]. The low photocatalytic performance of the MWCNTs/TiO2 composite can be due to large quantities of MWCNTs, scattering the incident light, and thus decreasing the photocatalytic process.

Fig 3 . 4 : 3 . 2 . 5

 34325 Fig 3.4: Photocatalytic degradation of orange G dye using MWCNTs/TiO2 composite as a preliminary step for the selection of material for the photocatalytic disinfection study

  The XRD spectra of different ZnO nanoparticles during different synthesis routes are depicted in Fig 3.5. To verify the formation of spherical ZnO nanoparticles, morphological analysis was performed through SEM. Unfortunately, all the formed nanoparticles were not in the expected shapes but obtained rod-shaped (Fig 3.7 (a)), cluster (Fig 3.7 (b)) and spindle-shaped (Fig 3.7 (c))

Fig 3 . 5 :Fig 3 . 6 :

 3536 Fig 3.5: XRD spectra of ZnO nanoparticles obtained through different synthesis methods, i.e., precipitation method and coprecipitation method (with varying parameters)

Fig 3 . 8 :

 38 Fig 3.8: XRD spectra of GO/ZnO composite synthesized through the simple blending process, depicting defined peaks of ZnO nanoparticles.

Fig 3 . 9 :

 39 Fig 3.9: Photographs depicting the disk diffusion assay performed in nutrient agar plates spread with E. coli K12 suspension. It was identified that the zone of inhibition was formed by the effect of GO/ZnO composite after visible-light irradiation followed by overnight incubation.

3. 2 . 6

 26 GO/TiO2 composite optimization XRD pattern of the GO/TiO2 composite is depicted in Fig 3.10. From the graphs, it was evident that the intensity of characteristic peak at the (101) reflection plane was diminishing as the concentration of GO content increased. This increase can be due to the sheet-like structure of GO covering the TiO2 nanoparticles, thereby showing a low signal. But as per the literature, GO content must be restricted towards percentage amount to increase the efficiency of the composite and extract the versatile properties of GO. Therefore, these ratios were eliminated from the present study and were focusing on the low loadings of GO. The SEM analysis of the obtained composites is shown in Fig 3.11. It shows the decoration of TiO2 nanoparticles over the GO sheets but has got large chunks or agglomerations.

Fig 3 . 10 :

 310 Fig 3.10: The XRD diffractometer of five different ratios, namely, 0.2/0.8, 0.3/0.7, 0.5/0.5, 0.7/0.3, and 0.8/0.2 GO/TiO2 composite showing the intense peaks of the TiO2 anatase phase.

13 )Fig 3 . 12 :

 13312 Fig 3.12: XRD spectra of TiO2 nanoparticles synthesized through sol-gel method focused on the synthesis temperature, namely, room temperature, 50°C and 60°C

  photocatalyst that generates ROS during exposure to light. The most preferred photocatalyst is TiO2 due to its low cost, non-toxicity, chemical stability, and activity near-ultraviolet region[10][11][12]. Microorganisms are composed of water (70 -90 %) and cellular components like lipids, polysaccharides, lipids, proteins, and nucleic acids, prone to ROS attack, causing cell death. Therefore, photocatalysis has a significant efficiency in biocidal properties (Fig 4.1).

Fig 4 . 1 :

 41 Fig 4.1: The illustration representing the typical microbial disinfection by a semiconductor photocatalyst (reproduced from [13])

  Fig 4.2 (a), the X-ray diffraction (XRD) patterns of GO, TiO2, and xGOT nanocomposites are shown, while Fig 4.2 (b) shows the patterns corresponding to the GO, BiVO4, and the xGOB nanocomposites. No impurity/secondary peaks and phase transitions were observed, which confirms the purity of the as-synthesized products as well as crystalline phases. The characteristic peak of GO (International Centre for Diffraction Data-ICDD:00-065-1528) around a 2θ value of 10.8° was observed, which corresponds to the (001) plane. Besides, the sharp peak at 2θ = 26.14° that corresponds to interlayer distance (002) between sp2 carbon layers, linked

  Fig 4.2 (a)

Fig 4 . 2

 42 (a*) and Fig 4.2 (b*)

Fig 4 . 2 :

 42 Fig 4.2: XRD patterns of (a) TiO2 and its various nanocomposites, (a*) scanned (101) plane of GOT nanocomposites, (b) BiVO4 and its different wt.% nanocomposites, (b*) scanned (103) plane of GOB nanocomposites.

  Fig 4.3 (a)) [50][51]. Moreover, all the spectra representing the D and G characteristic bands of GO, respectively, with negligible band shift were observed (Fig 4.3 (a*))[36]. Raman spectra modes (Fig 4.3 (b)) revealed the monoclinic phase of the BiVO4

  The presence of the D and G band of GO in the Raman spectra of nanocomposites confirmed the presence of GO which was not observed in the XRD pattern proving the sensitive analysis by Raman spectroscopy. A shift is kept at the positions of D and G bands in the GOB composites (Fig 4.3 (b*)) when GO content

Fig 4 . 3 :

 43 Fig 4.3: Raman spectra of pristine GO, TiO2 and BiVO4 nanostructures and (a) 0.5, 1.5, 2.5 GOT nanocomposites, (b) 0.5, 1.5, 2.5 GOB nanocomposites, D and G band of GO region-of-interest from 1100-1700 cm -1 (a*) GOT and (b*) GOB nanocomposites.

Figs 4 .

 4 Figs 4.6 and 4.7 represent the GOT and GOB nanocomposites with different loading ratios, showing the decoration of MOx nanoparticles onto the GO sheets evenly distributed. When the MOx loading were increased from 0.5 to 2.5, the detection or exposure of C element signal of the GO decreases, proving the MOx nanoparticles homogeneous interaction. In both the composites, particularly 2.5 GOT/B, the distribution of nanoparticles almost covered the GO sheets, thereby giving enhanced signals for the nanoparticles [67-70].

Fig 4 . 5 :

 45 Fig 4.5: SEM images and the corresponding elemental mapping of pristine nanostructures, such as GO, TiO2, BiVO4

Fig 4 . 6 :

 46 Fig 4.6: SEM images and the corresponding elemental mapping of 0.5, 1.5, and 2.5 wt.% of GOT nanocomposites.

Fig 4 . 7 :

 47 Fig 4.7: SEM images and the corresponding elemental mapping of 0.5, 1.5, and 2.5 wt.% of GOB nanocomposites.

Fig 4 . 8 :

 48 Fig 4.8: (a), (b) Low-resolution, (c) high-resolution TEM image, (d) and (e) corresponding FFT pattern of 1.5 GOT, respectively.

Fig 4 . 9 :

 49 Fig 4.9: (a), (b) Low-resolution, (c) high-resolution TEM image, (d) and (e) corresponding FFT pattern of 1.5 GOB, respectively

Fig 4 .

 4 Fig 4.11 (a) and (b) depict the visible-light-induced photocatalytic disinfection of water using the nanocomposites after 120 min (see Chapter 2, section 2.5.1). During photolysis, nearly 15% of inactivation was observed in 60 min, whereas with TiO2

Fig 4 .

 4 Fig 4.11: E. coli K12 survival after photocatalytic disinfection is depicted as the ratio of viable to total cells (Nt/N0). (a) GOT nanocomposites (b) GOB nanocomposites.

Fig 4 . 12 :

 412 Fig 4.12: Photographs of photocatalytic disinfectant activity of pristine nanoparticle and the corresponding E. coli K12 colonies on EMB agar plate

Fig 4 . 14 :

 414 Fig 4.14: Effect of dosage on photocatalytic inactivation of E.coli K12 by (a) 1.5 GOT (b) 1.5 GOB

Fig 4 .

 4 15 (a) displays an intact and well-defined morphology with a characteristic bacterial shape. The electron-dense region corresponds to the cellular components, including nucleic acids and proteins, whereas the electron-lucent refers to the cell wall or lipopolysaccharide outer membrane.

Fig 4 . 15 :

 415 Fig 4.15: TEM micrographs of (a) E. coli K12 control, bacteria treated with (b) 1.5 GOT (c) 1.5 GOB nanocomposite after 60 min

  photocatalytic disinfection was inhibited completely, showing the importance of •OH radical for the disinfection (Fig 4.16 (a)) [86]. Considering the 1.5 GOB nanocomposite, the photocatalytic inactivation was repressed by the addition of C6H4O2 and CH3CH2OH, proving that the O2•⁻ and •OH radicals have a major role in the disinfection

Fig 4 . 16 :

 416 Fig 4.16: Effect of scavenger study on photocatalytic inactivation of E. coli K12 by (a) 1.5 GOT, (b) 1.5 GOB nanocomposites

Fig 4 . 17 :

 417 Fig 4.17: Mechanism of photocatalytic inactivation of E. coli K12 by 1.5 GO/MOx nanocomposites



  Maximum quantum yield of PS II photochemistry: it is usually called the FV/FM ratio) and quantifies the maximum photochemical efficiency (capacity) of open RCs II. In this work, ΦPo is equivalent to YII in the dark. Photochemical quenching of variable Chl fluorescence: These parameters qP and qL indicate the actual photochemical capacity of PSII during a continuous illumination. It is related to the photochemical energy conversion by charge separation in the reaction center of photosystem II. It quantifies the actual fraction of RCs II being open, i.e., with oxidised QA. Nonphotochemical quenching of variable Chl fluorescence: The coefficients qN, BPQ, YNO and YNPQ reflect the activation of several processes of nonphotochemical nature during the light period and mostly leading to nonradiative deactivation of the excitation energy as heat (thermal dissipation). They include the pH-gradient build up, state-transitions, ATP-synthesis regulation, inactivation of photosystem II, conformational changes within thylakoid membranes, activation of the xanthophyll cycle, etc. The lake model differentiates the regulated mechanisms (YNPQ) from the nonregulated ones (YNO).

  i.e., SOD and POD. The ROS can cause the impairment of photosynthetic activity and deterioration of mitochondrial membrane and DNA. At a certain point, the ROS generation can cause suppression of genes encoding for the reaction center protein of PSII, light-harvesting proteins of photosystem, electron transport chain, and RuBisCO associated with carbon fixation. NADPH synthesis is lowered, inhibiting the CO2 assimilation, and decreasing the production of sugars in the Calvin cycle[37].

Fig 5 . 1 :

 51 Fig 5.1: The image shows the mechanism in which the nanoparticles cause the

chapter 2 ,Fig 5 . 2 .Fig 5 . 3

 25253 photocatalytic materials requires the comparison of experiments performed in the absence (photolysis) and in the presence (photocatalysis) of the photocatalytic material

Fig 5 . 4 .

 54 Fig 5.4. Filamentous Anabaena sp. observed in phase contrast light microscope ©Prof. B. Schoefs (Le Mans University, France)



  a baseline with a strong drift  shrunken absorbance peaks, especially in the blue region. These characteristics were observed in the recorded absorbance spectra of the cyanobacteria Anabaena flos-aquae PCC 7120 (Fig. 2.22). The original and corrected spectra are again presented in Fig 5.5. The baseline-corrected absorbance spectrum presents maxima at 438, 637, and 673 nm, typical of chlorophyll a (673 and 438 nm)

Fig 5 . 5

 55 Fig 5.5(d) displays the calibration curves obtained with both wavelengths. The fitting with linear regression was good in both cases ( = 0.99 and = 0.96). When normalized, both curves superimposed almost perfectly (inset Fig 5.5 (d)), demonstrating that the correct procedure provided coherent results indicating that (1)

Fig 5 . 5 :

 55 Fig 5.5: Calibration curve of Anabaena concentration (a) Mathematical fit (dashed curve) of the featureless absorbance spectrum recorded in situ (full line) (b) Corrected absorbance spectrum (dotted curve) as the result of the operation 'original spectrumminus-fitted baseline' (c) In situ absorbance spectra recorded with 50 (full curve), 100 (dotted curve), 200 (dashed curve), 300(dot-dash curve) and 400 µL (double dots-dash curve) of a mother cyanobacterial culture (d) Calibration curves for A750 and ΔA637 measured in the absorbance spectra and the corrected absorbance spectra, respectively. In both cases, the absorbance varies linearly with the culture volume. The insert presents the normalized data showing that the amount of pigment per cell is constant in the range of tested volume(50-400 µL).

Fig 5 . 6 :

 56 Fig 5.6: Kinetic of the absorbance at 665 nm changes specific to the photocatalytic treatment

Fig 5 . 7 .

 57 Fig 5.7. Microscopic pictures of the marine diatom Phaeodactylum tricornutum.

  2). Consequently, the addition of either filter to the experimental setup modified the light intensity but not the emission spectrum: it was around 1700 µmol photons/m -2 s with the former and around 2500 µmol photons/m -2 s with the latter. Qualitatively, the kinetics of fluorescence are very similar. In each condition, a large chlorophyll fluorescence quenching developed rapidly after the onset of the continuous illumination. Interestingly, a transient variation was observed after the onset of the continuous illumination (open arrow in Fig 5.8). It is generally admitted that this slow and transient change reflects adjustments between the increasing capacity of the Calvin cycle to use the products of the light phase (ATP and NADPH). However, the interpretation might be more complex as shown with the green microalgaHaematococcus pluvialis[65]. Despite its interest, this aspect will not be discussed here. During the period of darkness, the quenching mechanisms relaxed[66], as demonstrated by the decrease of the qN and YNPQ and the increase of qP (Fig 5.8 (c)-(d)). Interestingly, the cells recovered faster after the photocatalytic treatment than after the photolysis one.

Fig 5 . 8 .

 58 Fig 5.8. Effects of light intensity of the continuous illumination on the chlorophyll fluorescence recorded in the absence (green) or in the presence (orange) of photocatalytic material. (a) light intensity of 1700 µmol photons/m -2 s (b) light intensity of 2500 µmol photons/m -2 s (c-d) variations of quenching parameters during a period of darkness (20 min) following a continuous illumination of 1h 40 min in 1700 µmol photons/m -2 s (c) in the absence or (d) the presence of photocatalytic material The open arrow points on the transient change in the chlorophyll fluorescence yield.

Fig 5 . 9 .

 59 Fig 5.9. Impact of the cell density on the variations of the chlorophyll fluorescence levels in the absence and in the presence of the photocatalytic material.(a-b) Typical chlorophyll fluorescence curves recorded with (a) a low and (b) a high cell density in the absence (green) or in the presence (orange) of the photocatalytic material. (c-d) Variations of the chlorophyll fluorescence yield and parameters of (c) the puddle and (d) lake models describing the response of the microalgae to the environmental conditions.

  externally produced antioxidative enzymes have a short half-life time (human-SOD: 7-20 h[70]) and therefore have to be regularly renewed. The following hypothesis was formulated according to which increasing the duration of the continuous light treatment over 1.5 h may have a positive effect on the disinfection process. Consequently, the effects of the duration of the continuous illumination on the destruction of P. tricornutum was studied. Four different timings were selected: 0.5, 1, 2, and 4 h. Slow kinetics of chlorophyll fluorescence were recorded (Fig.5.10) and analysed(Fig 5.11) as explained earlier (see chapter 2, section 2.8.3 (b2)).

Fig 5 . 10 .

 510 Fig 5.10. Effects of the duration of the continuous illumination on the chlorophyll fluorescence induction curves recorded in the absence (green) or on the presence (yellow) of photocatalytic material. (a) 30 min of illumination (b) 1h of illumination (c) 2h of illumination (d) 4h of illumination

Fig 5 . 11 .

 511 Fig 5.11. Variations of the chlorophyll fluorescence during the increasing continuous illumination and after one night of recovery (a): 0.5 h of continuous illumination (b) 1h of continuous illumination (c) 2 h of continuous illumination (d) 4 h of continuous illumination The period of illumination and of recovery are indicated by yellow and black arrows, respectively. The dashed arrows indicate the period of test of the quality of recovery.

Fig 5 . 12 .

 512 Fig 5.12. Variations of the parameters YNO during continuous illumination of diatoms in the absence (YNO-L) or in the presence (YNO-C) of photocatalytic material (a) Variations before (red symbols), during illumination (yellow), after a dark period of 20 min following the illumination (green) and after one night of darkness (black) (b) Variations during illumination (yellow) and after a dark period of 20 min following the illumination (green). The dashed line figures out proportional variations of YNO-L and YNO-C. The dotted and dot dashed lines represent the slopes of the two phases of the variations of YNO-C versus YNO-C.

Fig 5 .

 5 photocatalysis and photolysis was carried out to have a strong confirmation of the destruction of the algal cells. The dark incubation was performed by continuous stirring and with the addition of sodium bicarbonate as the carbon source to avoid the starving of the cells during incubation. After the overnight incubation, the chlorophyll fluorescence measurement was performed to observe absorption peaks representing the presence of chlorophyll pigment. Surprisingly, the dark incubation test shown the destruction or removal of chlorophyll pigment. In contrast, the photolytic samples showed strong peaks of chlorophyll content, indicating the liveness of the diatom cells(Fig 5.14). This also proves that there was no chance of recovery in the photocatalytic sample.

Fig 5 . 13 3 Conclusion

 5133 Fig 5.13 Characterization of the cells illuminated during 4 h in the absence or the presence of photocatalytic materials (a,b) room temperature absorbance spectra in the absence (a) or (b) the presence of photocatalytic material. (c,d) bright field observations of the diatoms stained with Evans blue, a marker of cell viability (e) displays a visual view of the culture after the treatments.Pigment discoloration after photocatalysis (marked C) while no change in the sample after photolysis (marked L) (f) Graph depicting the relative number of alive cells after photolysis and photocatalysis where 376 x 10 4 cells/mL (triplicates) of alive cells was observed after counting photolysis sample which was nill in photocatalytic sample

Titre:

  photocatalytique des microalgues à savoir, Anabaena sp. et Phaeodactylum tricornutum. Les résultats étaient prometteurs et à notre connaissance, il s'agit de la première étude utilisant le nanocomposite GOT pour la destruction des algues susmentionnées. Une brève enquête sur les paramètres responsables de la désinfection photocatalytique aux microalgues a été réalisée

  photocatalytic disinfection of microalgae, namely, Anabaena sp. and Phaeodactylum tricornutum. The results were promising, and to the best of our knowledge, this is the first study using the GOT nanocomposite to destroy these algal cells. A brief investigation on the parameters responsible for the photocatalytic microalgal disinfection was performed.

Chapter 1 Table 1

 11

	.1	Requirements for drinking water
	Table 1.2	Drawbacks of current water treatment techniques
	Table 1.3	Three different groups of water treatment via nanotechnology
	Table 1.4	BOD of various wastes
	Table 1.5	Structure of thesis
		Chapter 2

Table 2

 2

	.1	Summary of the optical properties of the filters used for shaping the
		light beam
	Table 2.2	Description of the parameters used to characterize the
		photosynthetic activity and the equations used for calculation
		Chapter 4

Table 4

 4

	.1	Structural and optical characterization of xGOT and xGOB
		nanocomposites and their components

Table 1 .

 1

		1: Requirements for drinking water [18]
		Property	Condition
			Harmful microorganisms or other toxic
			substances should be removed from water
	1	Safe to drink
			by a definite treatment process before used
			for drinking
			Drinking water must have clean and
	2	Aesthetically pleasing
			clear appearance, taste, and odor.
			Potable water should not act as a corrosion
	3	Chemically stable	element or cause any deposits in pipes or
			fixtures during supply.

Table 1 .

 1

		3: Three different groups of water treatment via nanotechnology
	No	Group	Features
			Organic or inorganic materials are used to
			synthesize these nanoparticles to have high affinity
			to any pollutants. These nanoparticles can remove
			various types of contaminants and have the
	1	Nano-adsorbents	properties like catalytic potential, high reactivity,
			small size, and considerable surface energy. This
			group includes metallic nanoparticles, magnetic
			nanoparticles, nanostructured mixed oxides, and
			metallic oxide nanoparticles.
			These materials, i.e., metallic nanoparticles, can
			interact with light energy-giving photocatalytic
	2	Nano-catalysts	activities that generate radicals to destruct
			pollutants. Nano-catalysis uses semiconductors and
			metal oxides.
			These materials separate particles from wastewater
			and efficiently remove dyes, heavy metals, and other
	3	Nano-membranes	contaminants from water. Nanotubes, nanoribbons,
			and graphene-based membranes, and nanofibers
			are examples of nano-membranes.

Table 1 .

 1

	4: BOD of various wastes [9].
	Source	BOD (mg O2/liter)
	Milk	140000
	Silage effluents	30000-80000
	Pig slurry	20000-30000
	Cattle slurry	10000-20000
	Liquid effluent draining from slurry stores	1000-12000
	Untreated domestic sewage	200-500
	Treated domestic sewage	20-60
	Clean river water

.5 Advanced Oxidation Processes (AOPs)

Table 2 . 1 :

 21 Summary of the optical properties of the filters used for shaping the light beam.

			Transmission at
	Type of filter	λmax (nm)		Supplier
			λmax (%)
	High-pass
	Infra-red filter	-	Circa 75%	Unknown
	filter
	Long-pass	559, cut-off:		Edmund
	Visible filter		58, at 405 nm: 35%
	filter	400		Optics
	Daylight blue
	Band pass	425	68	Lee filters
	filter

Table 2 . 2 .

 22 Description of the parameters used to characterized the photosynthetic activity and the equations used for calculation

Table 4 .

 4

	1. Structural and optical characterization of xGOT and xGOB
	nanocomposites and their components
		Crystallite size	Band gap
	Samples
		(nm)	(eV)
	GO	7.7	-
	TiO2	14.8	3.15
	BiVO4	11.0	2.43
		GOT
	0.5	15.2	3.04
	1.5	11.9	3.01
	2.5	16.1	2.99
		GOB
	0.5	10.4	2.41
	1.5	9.0	2.39
	2.5	12.3	2.38

  To modulate the intensity of the continuous light, an additional band pass filters, was added to the filter set defined above (see chapter 2, section 2.8.3 (b)). Both filters are characterized by a similar maximum transmission (Table 2.1) and differed by their respective transmission capacity: 68% for the daylight blue and 75% for the glass filter (see table 2.1 and Fig 5.

Chapter 4

Characterization and microbial disinfection of GO/TiO 2 and GO/BiVO 4 composite 1) either the light that falls on the composite is not used, ultimately resulting in less ROS production, or

2) when the concentration of the nanocomposite is in excess, the light absorption increased resulting in the maximum generation of ROS that causes the suspension to be turbid. This turbidity blocks the utilization of photons, and therefore, a decline in photocatalytic efficiency is seen [86,88].

With the incident light absorption of a specific wavelength by the photocatalyst, a photoreduction phase occurred. Here, the electrons from the valence band (VB) excite towards the conduction band (CB), leaving behind the positive photo-holes (h + ). These electrons can convert the adsorbed oxygen to hydroxyl (•OH) radicals and superoxide radicals (O2•⁻). These generate highly reactive radicals and intermediates for the inactivation of the bacteria. A proposed mechanism of disinfection is depicted in Fig 4 .17. The steps involved in the disinfection mechanism of GOT composite is as follows [89,90]:

. + →

The mechanism followed for the GOB nanocomposites is below [91]:

The effect of light intensity is also counter-intuitive because the recovery was faster under the highest light intensity. These apparently striking results may mean that the externally generated ROS, eventually combined with high light intensity have enhanced the defence mechanisms of the algae against the oxidative risk more efficiently than in the absence of externally generated ROS. These mechanisms would include the activation of the enzymatic arsenal (peroxidases, superoxide dismutase, catalases) inside the cells but also outside, with the externalization of superoxide dismutase in the extracellular medium [67] (for a review, see [68]). This last possibility may be efficient enough to decrease the externally generated ROS under a level lower than under the one generated during lysis conditions. Altogether, these reasonings allow me to propose that diatoms might also be able to sense the externally generated ROS. To this end, it is worth to mention that ROS sensors located in plasma membranes for ROS detection in plant cell apoplast have been reported (for a review, see [66]).

(b) Effects of diatom density on diatom inactivation by photocatalysis

Two different concentrations of diatoms were tested in f2 media to study the effects of algal density on the photocatalytic disinfection of P. tricornutum.

General conclusions

The research work presented and discussed in this thesis aims to portray the significant water quality issues around the globe. The work details the drawbacks of currently available disinfection techniques, claiming the importance and versatility of nanoparticles and their application in heterogeneous photocatalytic disinfection. In this work specifically, we have designed a nanocomposite by embedding metal oxides, i.e., titanium dioxide (TiO2) and bismuth vanadate (BiVO4) over graphene oxide (GO)

sheets by an environmental-friendly and green methodology, one-step simple blending process, and used for the photocatalytic disinfection of microorganisms as well as microalga.

First, we have explored various nanoparticles and their synthesis methodologies to conclude the selection of materials for an efficient photocatalytic antibacterial property.

This study consisted of synthesis of TiO2 anatase nanoparticles through sol-gel method using titanium chloride and ethanol as the precursors and formation of five different graphene oxide/titanium dioxide (GO/TiO2) composites (0.5 %, 1 %, 1.5 %, 2 %, 2.5 %) using a simple method without the addition of any surfactants or protective agents. All the characterizations were conducted to confirm the formation of the GO/TiO2 (GOT) nanocomposite. The synthesized composites were checked for photocatalytic antibacterial activity, ROS effect, and disinfection kinetics. Escherichia coli K12, which is a model organism for the photocatalytic disinfection study, was used.

From the results, the best efficiency was found for the 1.5% GO/TiO2 composite with 99.9 % disinfection in 30 min for a bacterial concentration of 10 7 CFU/mL, by a dosage of 1.05 g/L. All these provide a novelty to this work to the best of our knowledge.

We have also explored water disinfection using graphene oxide/bismuth vanadate (GO/BiVO4) or GOB nanocomposite against Escherichia coli K12 (E. coli K12). The composite was developed through a facile and simple blending method at room temperature. Bismuth vanadate (BiVO4) nanoparticles synthesized through the sol-gel method with a particle size of 21.3 nm were used to decorate graphene oxide (GO)

sheets. The composites where of five different combinations, namely, 0.5 %, 1 %, 1.5 %, 2 % and 2.5% of GO/BiVO4 (wt.% of GO). The characterization confirmed the