VIRUS REMOVAL AND INACTIVATION IN A PHOTOCATALYTIC MEMBRANE REACTOR: DISINFECTION MECHANISMS AND EFFECT OF WATER QUALITY By Bin Guo A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Environmental Engineering Œ Doctor of Philosophy 2016 ABSTRACT VIRUS REMOVAL AND INACTIVATION IN A PHOTOCATALYTIC MEMBRANE REACTOR: DISINFECTION MECHANISMS AND EFFECT OF WATER QUALITY By Bin Guo Waterborne diseases pose great health threat to humans and result in huge economic losses. One of the effective way to avoid the infections by waterborne microorganisms is water disinfection. Conventional disinfection methods include chlorination, chloramination and ozonation. However, the inevitable production of disinfection by-products (DBPs) and the inability to inactivate certain resistant microbial species are drawbacks of the conventional disinfection methods. In addition, with the transition to lower quality water sources and an increasing role of water reuse, conventional disinfection methods may no longer be sufficient. Alternative treatment methods with higher efficiency and smaller energy demand are urgently required. Numerous studies have been conducted to explore the application of photocatalytic membrane reactors (PMRs) in water treatment. Most of these studies have focused on the removal of chemicals, often employing dyes as model pollutants. PMRs applications to water disinfection, however, are very limited. Only five studies employed concurrent filtration and photocatalytic disinfection. In four of the five publications, the same type of bacterium was used as the bacterial model. In the present work, a novel hybrid photocatalytic UV-membrane filtration system was designed and applied for water disinfection. To the best of our knowledge, this is the first application of a PMR for virus removal and inactivation in water. Two types of viruses and two types of waters were used to test the performance of the hybrid system. The hybrid system is shown to retain the advantages of photocatalytic UV disinfection and membrane filtration and to synergistically mitigate drawbacks of each of these two processes. In addition, batch experiments were also conducted to understand the mechanism of photocatalytic inactivation of viruses in water and to examine the effect of water quality on the photocatalytic inactivation of viruses. Water quality affects the kinetics of photocatalytic inactivation, which fits Collins-Selleck model in DI water and a first-order reaction in pre-filtered surface water. Copyright by BIN GUO 2016 v This thesis is dedicated to my family. Thank you for always loving and supporting me. vi ACKNOWLEDGEMENTS First of all, I would like to express my sincere thanks to my advisor, Dr. Volodymyr Tarabara, for his patience and guidance throughout my graduate study. I am also grateful to my dissertation committee members: Dr. Irene Xagoraraki, Dr. Thomas W. Hamann and Dr. Kristin N. Parent, for their time and valuable input throughout the entire project. In addition, I would also like to thank Lori Larner, Margaret Conner, Laura Taylor, Mary Mroz, Laura Post, Joseph Nguyen, Craig Burck and Yanlyang Pan, for their administrative and technical support during my graduate study. Furthermore, my appreciation also extended to my former and current group members for the inspiring discussions and their positive attitudes in and outside the lab, which bring me a very pleasant time at Michigan State University. The collaboration with many of them is really an enjoyable experience for me, and all their great contribution and generous sharing of experiences are highly appreciated. Finally, I would like to express my gratitude to my beloved family members, especially my mother, my aunts and my husband, Dr. Yunyi Jia. Without their continuous support and encouragement I would not be where I am. And all your love and understanding will always be my strongest backing. vii TABLE OF CONTENTS LIST OF TABLES ....................................................................................................................... ix LIST OF FIGURES ...................................................................................................................... x KEY TO ABBREVIATIONS..................................................................................................... xii CHAPTER 1. INTRODUCTION ................................................................................................ 1 1.1 Importance and challenges of current water disinfection technologies ................................ 1 1.2 Implication of photocatalysis in disinfection: mechanisms and applications ....................... 3 1.2.1 Mechanisms ................................................................................................................... 3 1.2.2 Factors influencing photocatalytic activity .................................................................... 4 1.2.2.1 Loading of photocatalysts ....................................................................................... 5 1.2.2.2 Initial concentration of the substance ..................................................................... 5 1.2.2.3 Characteristics of UV lamp ..................................................................................... 5 1.2.2.4 The components of the solution .............................................................................. 5 1.2.3 Applications: photocatalytic antimicrobial activity ....................................................... 6 1.3 Photocatalytic membrane reactors (PMRs): configurations and applications ...................... 8 1.3.1 PMRs configurations ..................................................................................................... 9 1.3.1.1 PMRs with suspended catalysts ............................................................................ 10 1.3.1.2 PMRs with immobilized catalysts ........................................................................ 11 1.3.2 Applications of PMRs in water treatment .................................................................... 13 1.3.2.1 Application of PMRs for chemical removal ......................................................... 13 1.3.2.2 Application of PMRs for water disinfection ......................................................... 13 1.4 Dissertation overview ......................................................................................................... 14 REFERENCES ............................................................................................................................ 16 CHAPTER 2. VIRUS REMOVAL AND INACTIVATION IN A HYBRID MICROFILTRATION-UV PROCESS WITH A PHOTOCATALYTIC MEMBRANE.... 29 2.1 Introduction ......................................................................................................................... 29 2.2 Experimental ....................................................................................................................... 32 2.2.1 Reagents ....................................................................................................................... 32 2.2.2 Bacteriophage propagation and preparation of feed suspension .................................. 32 2.2.3 Membranes and deposition of photocatalytic coating on their surface ........................ 33 2.2.4 Membrane cleaning ...................................................................................................... 33 2.2.5 Hybrid UV-membrane filtration unit: Design and operation ....................................... 34 2.2.6 Sample collection and storage ..................................................................................... 36 2.2.7 UV dose quantification ................................................................................................ 38 2.2.8 Quantifying the efficacy of disinfection by direct UV only ........................................ 39 2.2.9 Electron microscopy of the membrane surface ............................................................ 40 2.2.10 Quantification of the viable bacteriophage ................................................................ 40 2.2.11 Quantification of the total bacteriophage ................................................................... 41 2.3 Results and Discussion ....................................................................................................... 43 viii 2.3.1 Hybrid membrane filtration-UV process: The concept and a brief rationale .............. 43 2.3.2 Efficacy of disinfection by direct UV irradiation ........................................................ 45 2.3.3 Characterization of the tubular ceramic membrane ..................................................... 47 2.3.4 Removal and inactivation of bacteriophage P22 ......................................................... 52 2.3.5 Potential applications in water treatment ..................................................................... 55 2.4 Conclusions ......................................................................................................................... 57 APPENDICES ............................................................................................................................. 58 Appendix A: Supporting information ....................................................................................... 59 Appendix B. Photocatalytic coating on borosilicate glass slides .............................................. 62 Appendix C. Photodegradation test of TiO2 coated borosilicate glass slides .......................... 67 REFERENCES ............................................................................................................................ 70 CHAPTER 3. PHOTOCATALYTIC INACTIVATION OF HUMAN ADENOVIRUS 40 IN NATURAL SURFACE WATER: EFFECT OF WATER QUALITY .................................. 78 3.1 Introduction ......................................................................................................................... 78 3.2 Experimental ....................................................................................................................... 81 3.2.1 Reagents ....................................................................................................................... 81 3.2.2 A549 cell line and HAdV40 propagation .................................................................... 81 3.2.3 Batch UV photoreactor ................................................................................................ 82 3.2.4 Photocatalytic membrane and membrane reactor ........................................................ 83 3.2.5 Sample collection and storage ..................................................................................... 84 3.2.6 Lake water pre-treatment ............................................................................................. 84 3.2.7 UV dose quantification ................................................................................................ 85 3.2.8 Photochemical characterization ................................................................................... 85 3.2.9 Total virus quantification with qPCR .......................................................................... 85 3.2.10 Quantification of culturable virus: Cell culture assays and most probable number (MPN) calculation ................................................................................................................. 86 3.3 Results and discussion ........................................................................................................ 87 3.3.1 OHŁ radical production and quenching: Effect of water quality ................................. 87 3.3.2 HAdV40 removal and inactivation in a batch UV reactor ........................................... 91 3.3.3 Virus removal and inactivation in photocatalytic membrane reactor .......................... 96 3.4 Conclusions ....................................................................................................................... 101 APPENDIX ................................................................................................................................ 102 REFERENCES .......................................................................................................................... 110 CHAPTER 4. CONCLUSIONS AND FUTURE WORK ..................................................... 116 4.1 Conclusions ....................................................................................................................... 116 4.2 Future research work......................................................................................................... 117 ix LIST OF TABLES Table 1.1 Disadvantages of selected advanced oxidation processes .............................................. 2 Table 1.2 Selected representative studies of microbial disinfection by UV/TiO2 ......................... 7 Table 2.1 Rationale for the proposed UV-microfiltration hybrid process ................................... 45 Table A.1 Sequences of Primers and Taqman probe ................................................................... 59 Table A.2 Log removal of viable P22 in different treatment processes as determined by plaque assay analysis ................................................................................................................................ 60 Table B.1 Summary of dip coating parameters from selected representative references ............ 64 Table S.1 Composition of tissue culture medium ..................................................................... 104 Table S.2 Media formulations of Basal Medium Eagle ............................................................. 104 Table 4.1 Knowledge gaps of the novel hybrid photocatalytic UV-membrane filtration system..................................................................................................................................................... 118 x LIST OF FIGURES Figure 1.1 Two types of configurations of photocatalytic ceramic membranes .......................... 12 Figure 2.1 Schematic diagram of the hybrid membrane filtration-UV treatment system ............ 35 Figure 2.2 Design of custom-made parabolic UV light reflectors and the membrane housing unit........................................................................................................................................................ 37 Figure 2.3 Conceptual illustration of the hybrid membrane filtration-UV disinfection process . 43 Figure 2.4 Log removal of viable P22 bacteriophage as a function of UV fluence. ................... 47 Figure 2.5 Scanning electron micrographs of the tubular ceramic membrane ............................ 48 Figure 2.6 Specific permeate flux of ultrapure water .................................................................. 50 Figure 2.7 Inactivation and/or removal of viable P22 bacteriophage .......................................... 53 Figure 2.8 Log concentration of viable and total bacteriophage P22 in the feed solution and in the permeate 30 min into the filtration process. .................................................................................. 54 Figure A.1 UV fluence as a function of the exposure time .......................................................... 61 Figure B.1 SEM images of different coating layers on borosilicate glass slides under various magnifications ............................................................................................................................... 65 Figure C.1 Photodegradation of MB using borosilicate glass slides with different coating layers....................................................................................................................................................... 68 Figure C.2 The change of degradation rate with the increased amount of TiO2 catalysts ........... 69 Figure 3.1 Schematic diagram of the batch UV reactor ............................................................... 83 Figure 3.2 UV absorptivity (254 nm) and TOC of water samples used in photocatalytic tests .. 88 Figure 3.3 Degradation of pCBA normalized by UV254 fluence for different water types with and without TiO2 (0.83 mg/L) and MEM (1%) in the solution ........................................................... 90 Figure 3.4 Log removal of total HAdV40 (as measured by qPCR) in photocatalytic UV tests with different waters ............................................................................................................................. 92 Figure 3.5 Comparison of total HAdV40 removal (as determined by qPCR) by direct UV (--, --) and by photocatalytic UV (--, --) in batch inactivation tests ......................................... 93 xi Figure 3.6 Photocatalytic inactivation of culturable HAdV in different waters .......................... 94 Figure 3.7 Comparison of culturable HAdV40 inactivation with and without catalysts ............. 96 Figure 3.8 Removal of total HAdV40 by (1) microfiltration only (2) non-photocatalytic hybrid MFŒUV process and (3) photocatalytic hybrid MFŒUV process ................................................. 97 Figure 3.9 Inactivation and/or removal of culturable HAdV40 by microfiltration only, in a sequential MFŒUV process, and in a photocatalytic MF membrane reactor ................................ 98 Figure 3.10 Concentration ratio of culturable and total HAdV40 in feed and permeates ........... 99 Figure S.1 UV fluence required for 99.9% reduction of representative human enteric viruses 103 Figure S.2 Schematic diagram of concentrate system ............................................................... 105 Figure S.3 UV absorbances for several experimental conditions .............................................. 106 Figure S.4 pCBA degradation over time for various water sources .......................................... 107 Figure S.5 Inactivation of culturable HAdV in lake water pre-filtered through 0.03 µm membrane..................................................................................................................................................... 108 Figure S.6 Normalized permeability of microfiltration membranes to deionized water ........... 109 xii KEY TO ABBREVIATIONS AOP Advanced oxidation process BDF Buffered demand-free CPE Cytopathic effects DBPs Disinfection by-products DDI distilled deionized water DNA Deoxyribonucleic acid DOC Dissolved organic carbon DOM Dissolved organic matter DI Deionized water EDTA Ethylenediaminetetraacetic acid FBS Fetal bovine serum HAdV Adenoviruses HAdV40 Human adenovirus serotype 40 H2O2 Hydrogen peroxide H3PO4 Phosphoric acid KI Potassium iodide KIO3 Potassium iodate LBL Layer-by-layer LRV The log removal MB Methylene blue MEM Minimum essential medium xiii MF Microfiltration MPN Most probable number NaOH Sodium hydroxide NEAA Non-essential amino acid NF Nanofiltration NOM Natural organic matter O3 Ozone OH· Hydroxyl radicals P22 Enterobacteria phage P22 PBS Phosphate buffered saline PBW Phosphate buffered water pCBA para-Chlorobenzoic Acid PDADMAC Polydiallyldimethylammonium chloride PFU Plaque forming unit qPCR Real-time polymerase chain reaction RNA Ribonucleic acid RO reverse osmosis ROS Reactive oxygen species SDW Sterile distilled water SEM Scanning electron microscopy SEW Steriie estuarine water SUVA Specific UV absorbance TiO2 Titanium dioxide xiv TOC Total organic carbon TSA Trypticase soy agar TSB Trypticase soy broth UF Ultrafiltration 1 CHAPTER 1. INTRODUCTION 1.1 Importance and challenges of current water disinfection technologies Waterborne diseases, which are caused by pathogenic microorganisms and/or chemicals transmitted in the contaminated water, pose a major threat to human health world-wide. According to the report by World Health Organization (WHO) [1, 2], as of 2013, 700 million people still lack access to an improved drinking water sources and ~ 1.5 million people die from waterborne disease (e.g. diarrhea) annually, mostly children in developing countries. Every year huge amounts of financial and human resources are spent to prevent and reduce the risk of waterborne infectious. Water disinfection has been considered as a promising technology to prevent or decrease the deaths from waterborne diseases caused by pathogens. Conventional disinfection methods include chlorination, chloramination and ozonation. By adding strong oxidants, harmful pathogens (e.g. viruses, bacteria and protozoa) are inactivated in the treatment, storage and distribution systems. However, the traditional technologies have their drawbacks. These include: 1) formation of disinfection by-products (DBPs) that are carcinogenic [3, 4]; 2) some pathogens, such as Legionella, Cryptosporidium, Giardia lamblia cysts, have been proved to be resistant to disinfection by chlorine [5, 6]. A suite of alternative treatment processes Š advanced oxidation process (AOP) Œ that utilize the strong oxidizing property of hydroxyl radicals (·OH) has been developed. Although proven to be a powerful treatment alternative, AOPs is not free of its own drawbacks (Table 1.1) [7-9]. With the increasing awareness of persistent microbial pathogens detected in treated wastewater and drinking water sources worldwide, a sufficient, economical and environment-friendly treatment technology is necessary and urgent. 2 Table 1.1 Disadvantages of selected advanced oxidation processes AOPs Disadvantages UV/H2O2 Treatment effectiveness is greatly affected by the water quality (e.g. alkalinity, turbidity) Excess peroxide can limit the effectiveness The production of ·OH is limited by the small molar extinction coefficient of H2O2 O3/H2O2 Treatment performance depends on pH and water quality O3 production is expensive Control of O3/H2O2 dosage ratio is difficult Additional treatment of excess H2O2 and O3 O3/UV Treatment effectiveness is dependent on pH High amount of O3 and energy consumption The presence of UV absorbing compounds is problematic May require O3 off-gas treatment UV/H2O2 Treatment effectiveness is dependent on pH Turbidity and UV absorbing compounds is problematic Less stoichiometrically efficient at generating ·OH O3/H2O2/UV Turbidity and UV absorbing compounds is problematic May require O3 off-gas treatment Control of O3/H2O2 dosage High energy and cost consumption 3 1.2 Implication of photocatalysis in disinfection: mechanisms and applications Photocatalysis is classified as an AOP because the oxidation in this process occurs primarily though reactions with hydroxyl radicals (·OH) which are non-selective and potent oxidizers for organic matter in water [10, 11]. The term photocatalyst refers to a semiconductor that is able to convert light energy to the chemical energy of electron-hole pairs. Although many semiconductor materials such as TiO2, ZnO, Fe2O3, WO3, BiOBr, Bi3O4Br and CuS have been investigated as photocatalysts [12-18], titanium dioxide (TiO2) is still the most popular catalyst because of its high photoactivity, chemical stability, commercial availability, no demonstrable toxicity and low cost [19, 20]. 1.2.1 Mechanisms There are three polymorphs of TiO2: anatase (tetragonal minerals), rutile and brookite (a rare orthorhombic mineral). With the different crystalline structures, these three types of TiO2 exhibit different properties. Many studies show that the rutile is the most stable form but less active, whereas anatase is metastable but the most effective photocatalyst [21-23]. It was believed that the photoactivity is associated with the energy structure, recombination rate of electron-hole pairs [24, 25] as well as the surface physical/chemical properties [26]. Moreover, nanosized TiO2 usually show higher photoactivity due to the quantum size effect and the larger surface area. Miyagi et al. [25] also pointed out that the mixture of anatase and rutile was more effective in photocatalytic process than the pure anatase form of TiO2 . Thus, the commercial available Degussa P25 TiO2, which contains ~ 80% anatase and 20% rutile, is the catalyst most frequently used in the fundamental studies of microbial disinfection. 4 Because of the relatively wide band gap (3.2 eV for anatase), non-modified TiO2 can be excited only under UV irradiation with a wavelength less than 400 nm [19, 27]. When a photon with energy that is equal to or greater than the band gap energy is absorbed by TiO2, an electron in the valence band may be excited to the conduction band, resulting in the formation of electron-hole pairs in femtoseconds [7, 28]: + (1) With the formed hole and the presence of appropriate scavengers (e.g. H2O and/or OH-), ·OH can be produced [29]: + + · (2) + · (3) Meanwhile, the electron may react with dissolved oxygen to produce other reactive oxygen species (ROS) like superoxide radical ion (·) and hydrogen peroxide (), which ultimately lead to the production of ·OH [30]: + · (4) ·+ · (5) ·+ + (6) + · + (7) + · · + + (8) 1.2.2 Factors influencing photocatalytic activity The efficiency of photocatalytic activity depends upon many factors, including loading of the photocatalyst, initial concentration of the substrate, characteristics of UV lamp and the components of the solution. 5 1.2.2.1 Loading of photocatalysts Generally, the reaction rate is proportional to the mass of catalysts in the initial step due to the higher number of available sites on the catalyst [31]. However, when the mass of catalysts is above the optimized amount, the reaction rate is independent of catalysts. The rate may remain constant or even decrease because of the increased solution opacity and agglomeration of catalyst particles [32-35]. 1.2.2.2 Initial concentration of the substance The degradation rate increases with an increase in the initial concentration of the substrate till a certain level. A further increase in the concentration leads to the decreased degradation rate [35]. It has been reported by Molinari et al. [26] that the increased substrate concentration may cause light scattering, thus reducing the generation of ·OH. 1.2.2.3 Characteristics of UV lamp Several studies have reported that the wavelength and the intensity of the light source affect the photocatalytic activity [36-38]. However, the influence decreases with an increase in light intensity: the dependence changes from a linear relationship at low light intensity (<20 mW/cm2) to saturation at high light intensity (>25 mW/cm2) [35, 39]. 1.2.2.4 The components of the solution The photocatalytic activity is strongly affected by the pH of the solution. The agglomeration of TiO2 particles was reported under acidic condition [40], thus alkaline solution is preferred for photocatalytic activity. In addition, it is commonly considered that ·OH is easier to be produced in 6 alkaline solution due to the presence of sufficient hydroxide ions (eq. (3)). However, decreased photocatalytic activity was observed by Molinari et.al. [26], and the possible reason may be the repulsion force between the negatively charged TiO2 surface and the hydroxide ions. Besides, the photocatalytic activity is also related to the presence of oxygen and inorganic ions (e.g. ClŠ, NO3Š, CO3Š, etc.). As an electron scavenger (eq. (4)) and a strong oxidant, oxygen is known to promote photocatalytic reactions [26]. The presence of inorganic species may have positive or negative effects depending on the reaction mechanism [34, 35, 41]. 1.2.3 Applications: photocatalytic antimicrobial activity The use of TiO2 for the photoinactivation of Lactobacillus acidophilus, Saccharomyces cerevisiae, and Escherichia coli was first reported by Matsunaga et al. in 1985 [42]. Later on many studies showed TiO2 was an effective catalyst for the inactivation of a great range of microorganisms especially bacteria [43-46] and viruses [43, 47-50] (Table 1.2). The mechanism of bacterial inactivation by photocatalysis was initially proposed as depleting coenzyme A by dimerization and therefore inhibiting respiration [43, 51]. However, with the development of analysis technology, more evidences show that the lethal action is due to the damage to the membrane of bacterial cells. For example, a rapid leakage of potassium ions (K+) followed by a slow release of cellular components, such as RNA and protein, were observed from treated Streptococcus sobrinus AHT cell by Satio et al. [52]. Later, Sunada et al. [53] reported the destruction of endotoxin indicating that TiO2 photocatalysts destroy the outer cell membrane of Gram-negative bacteria E. coli. It was suggested that the ROS generated on the TiO2 surface may attack the polyunsaturated phospholipids in the bacterial cell membrane, resulting in breakdown 7 Table 1.2 Selected representative studies of microbial disinfection by UV/TiO2 Immobilization Quantity of catalysts UV wavelength Microorganism Removal efficiency References TiO2 layer - 300-400 nm Bacteriophage kNM1149 99.6% after 6h Belhacova, L., et al. [54] TiO2 suspension 1.0 or 2.0 g/L, 50 mL 300-420 nm Escherichia coli 99% after 2h Cho, M., et al. [55] TiO2 suspension 1.0 g/L, 50 mL <300 nm Escherichia coli, Bacteriophage MS-2 ~90% after 2h >99% after 2h Cho, M., et al. [56] TiO2-coated glass 400nm thick 300-400 nm Bacteriophage T4, Escherichia coli 100% after 3h Ditta, I.B., et al. [57] TiO2 suspension - 100-280 nm Escherichia coli K12 PHL849, Escherichia coli K12 PHL1273 ~100% after 5h Guillard, C., et al. [58] TiO2 layer - 254 nm Coliphage 98~100% after 89-104s Guimaraes, J.R., et al. [59] TiO2 100nm thick 300Œ400 nm Lactobacillus casei phage PL-1 99.9% after 24h Kashige, N., et al. [60] TiO2 layer - - Bacterio >99% after 1h Lee, S., et al. [61] 8 of cell membrane and the further damage of the cytoplasmic membrane and intracellular components [53, 62]. The cytoplasmic membrane contains the necessary enzymes which are closely associated with the synthesis, assembly, and transport functions of viable cells. Therefore, any disruption to the cell membrane will threaten to cell survival [63]. In addition, DNA damage was reported in many studies when microorganisms are subjected to treatment by TiO2 photocatalysis [64-69]. Although DNA damage was considered as an event that follows destruction of cell membrane, it is still responsible for the cell death. Moreover, following the cell death, complete mineralization of bacteria in water has also been reported [70-72]. It is well known that ROS generated on the TiO2 surface is responsible for the microorganism inactivation [62]. Nevertheless, ·OH among all types of ROS is proposed as the most important component [55, 56, 73-76]. For example, Ogino et al. [77] and Takashima et al. [78] showed that the inactivation of bacteria E. coli in closely correlated with the concentration of ·OH. Another study by Cho et al. [76] also indicated that ·OH to be a major contributor to the inactivation of Cryptosporidium parvum. However, it is noteworthy that photocatalytic inactivation of microorganism is very complex and may vary from case to case. Thus the contribution of other ROS, such as H2O2 and · to the overall performance should not be neglected. 1.3 Photocatalytic membrane reactors (PMRs): configurations and applications Since its first industrial application in 1950s, membrane filtration has been considered as a highly automated, operationally simple and efficient technology [79]. With the increasing attention on microorganisms in water and the higher removal requirement from regulatory agencies, membrane filtration is widely applied in water industry. However, several challenges still limit its practical application: a) membrane fouling b) trade-off between the permeability and selectivity c) lack of 9 degradation capacity. Therefore, additional processes for membrane cleaning and concentrate treatment are required. Photocatalyzed oxidation, as an effective chemical process, was first used to degrade cyanide in water [80]; since then its application in water purification has been extensively studied [28, 81-85] and a number of studies have verified its applicability to water disinfection [47, 86-91]. Photocatalytic membranes combine photocatalysis with membrane separation. The hybrid technology is a potential alternative that may overcome the obstacles associated with conventional membrane filtration and photocatalytic oxidation. In addition to size exclusion, photocatalytic membranes reactively degrade organic pollutants [92-95], disinfect water [96-101], and may possess self-cleaning properties with respect to common membrane foulants [102-106]. Typically, fiphotocatalytic membrane reactorsfl (PMRs) refers to the hybridization of photocatalysis with membrane process. Compared to the conventional photoreactors, PMRs have many advantages: 1) membrane serves as a barrier to retain the catalysts; 2) PMR enables the control of the residence time of the pollutants in the reactor; 3) PMR enables simultaneous photocatalysis and product separation; 4) PMR avoids the additional treatment processes used for the separation of catalysts, lowering the consumption of energy. 1.3.1 PMRs configurations According to the state of the catalysts, PMRs can be simply divided into two groups: 1) reactors with suspended catalysts 2) reactors with catalysts immobilized in/on the membrane. In the first case, the active surface is large and the system has been found to be more efficient [107-110]. 10 However, the recovery of the catalysts and the membrane fouling may be the problems [111]. In contrast, the separation of catalysts is easier in the immobilized system, but the fixation of catalysts results in the limited mass transfer and a potential loss of available active surface [112]. 1.3.1.1 PMRs with suspended catalysts Most studies describe PMRs that utilize as the driving force for separation; the corresponding membrane processes include microfiltration (MF) [113-117], ultrafiltration (UF) [109, 118-121] and nanofiltration (NF) [122-125]. The use of reverse osmosis (RO) [126, 127] is rare, because usually RO is not applied for the feed water containing suspended solids. However, photocatalysis combined with RO for water treatment were still investigated in some studies. For example, Lehr et al. [126] observed less membrane fouling by applying RO with suspended catalysts. Additionally, Tay et al. [127] reported that no fouling occurred using photocatalytic RO membrane for the pretreatment of water containing humic acid. Since the catalyst is suspended and not attached to the membrane, the location of the light source is more flexible than that in PMRs with immobilized catalyst. The most commonly used configurations are: 1) light irradiation above the feed tank 2) light irradiation above the membrane module 3) light irradiation above the additional reservoir located between feed tank and the membrane module. To choose the configuration in terms of the light source, many factors (e.g. properties of feed solution, treatment target, cost of installation, etc.) need to be considered. Besides the position of light source, other parameters that affect the performance of PMRs with suspended catalysts are: driving force, characteristics of the membrane module (e.g. type of membrane, hydraulic properties in the membrane module), operational mode (e.g. pressurized or 11 depressurized), composition of feed solution, efficacy of photocatalytic degradation (impact factors are discussed in section 1.2.1), etc. [109, 111, 113-119, 121-123, 128]. 1.3.1.2 PMRs with immobilized catalysts In the PMRs with immobilized catalysts, the membrane has a dual function: a support for the photocatalysts and a barrier for the target compounds. Depending on the different membrane structures, the photocatalytic activity may occur on the membrane surface or within the membrane pores. Therefore, in the PMRs with immobilized catalysts, membrane itself is the only component that needs to be irradiated. There are various methods of fabricating photocatalytic membranes. Membranes may be coated with catalyst material or membrane material itself can be catalytic. A variety of materials, including inorganic, organic and metallic, have been investigated as the support where the catalysts can be deposited or imbedded. In particular, for TiO2 catalysts, polymer and ceramic membranes are the most often used supports [99, 129-131]. Taking ceramic membranes as an example, two asymmetric configurations are widely used. In the first case (Fig. 1.1a), the photocatalytic layer is on the same side with the membrane separation layer. The major advantage of this configuration is that the organic contaminants can be decomposed, therefore, the membrane fouling can be relieved. However, since the light source is on the feed side, low turbidity of the feed solution is necessary to maintain the sufficient irradiation. In the second case (Fig. 1.1b), the photocatalytic layer is separate from the separation layer. Although membrane fouling and the production of concentrated stream are observed in this configuration, this configuration can be used for the turbid water treatment. Also, any organic contaminants transported through the membrane may be decomposed 12 by the ROS in the permeate; thus this type of photocatalytic membrane can be applied to purify turbid water. Some photocatalytic membranes are made of the pure photocatalyst materials. One classical example is the development of TiO2 nanofibers, nanowire or nanotubes [98, 100, 132, 133]. Zhang et al. [134] reported that photocatalytic TiO2 nanowire is more effective in mitigating membrane fouling. Moreover, an increased permeate flux was observed with the UV irradiation enhanced by TiO2 nanotubes [135]. Figure 1.1 Two types of configurations of photocatalytic ceramic membranes 13 1.3.2 Applications of PMRs in water treatment 1.3.2.1 Application of PMRs for chemical removal Numerous studies have shown that the PMR is a promising technology for water treatment and has been widely applied for removal of various chemical pollutants including pharmaceuticals [117, 123, 124, 136], humic acid [119, 128, 134, 135, 137], dyes [97, 100, 113, 118, 125, 129, 131, 138], bisphenol A [115, 116], 2,4-dichlorophenol [139], phenol [140], etc. Mutiple studies have shown that the removal efficiency of organic pollutants is higher than 90%. For example, Zhang et al. [134] found that the TiO2 nanowires could remove 100% humic acid and 93.6% total organic carbon (TOC) in a continuous operation mode. The same in the continuous flow reactor, Romanos et al. [141] indicated that 90% methyl orange (MO) was removed by TiO2/Al2O3 membrane in 10 hours. In another study, 95%-100% removal of MO was reported [142] by using TiO2-Al2O3-ZrO2 nanofiber membrane. More than 99% removal of Reactive Black 5 in 60 min was obtained by Damodar et al. [102]. 1.3.2.2 Application of PMRs for water disinfection The application of PMRs to water disinfection is very limited. Some studies showed that the photocatalytic membranes, such as TiO2 film [97], TiO2 deposited thin-film-composite [130], TiO2 entrapped PVDF membrane [102], silver decorated carbon nanofibers [143], have antibacterial properties. In these studies the physical (e.g. permeability) and chemical (e.g. photocatalytic disinfection) properties were quantified in the separation tests. For example, Kim et al. [130] reported less flux decline and higher salt rejection with UV irradiated photocatalytic membrane using deionized water (DI) with the addition of sodium chloride, while a complete disinfection of 14 Escherichia coli (E. coli) was observed by pipetting E. coli cell dilution onto the membranes and illuminated by a UV lamp for up to 4 hours. To the best of our knowledge, there have been only five reports on the concurrent filtration and photocatalytic disinfection of microorganism [98, 100, 144-146]. In four of these studies [98-100, 144], gram-negative bacterium E. coli was used as the model pathogen, and three of the four also used silver as a component of the photocatalytic membranes. Only one study of the five investigated the photocatalytic disinfection of viruses. Although bacteriophage f2, a single-stranded RNA virus, is used as a representative in this study, the system configuration in this case is a PMR with suspended catalysts which is quite different from the previous four studies which have immobilized catalysts. Therefore, the application of PMRs with immobilized catalysts for water disinfection needs to be explored. 1.4 Dissertation overview The objective of this study is to develop an effective PMR for water disinfection and to understand the mechanisms and impacts of water quality on the photocatalytic inactivation of virus in the PMR. In chapter 2, we report on the design of a hybrid photocatalytic UV-membrane filtration system and its application to the treatment of DI water seeded with a bacteriophage. To our knowledge, this work is the first application of a PMR with immobilized catalysts to virus disinfection. 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Dionysiou, V. Likodimos, P. Falaras, Double-side active TiO2-modified nanofiltration membranes in continuous flow photocatalytic reactors for effective water purification, Journal of Hazardous Materials, 211 (2012) 304-316. [142] H.-J. Hong, S.K. Sarkar, B.-T. Lee, Formation of TiO2 nano fibers on a micro-channeled Al2O3-ZrO2/TiO2 porous composite membrane for photocatalytic filtration, Journal of the European Ceramic Society, 32 (2012) 657-663. [143] A.A. Taha, Direct synthesis of mesostructured carbon nanofibers decorated with silver-nanoparticles as a multifunctional membrane for water treatment, Advances in Natural Sciences-Nanoscience and Nanotechnology, 6 (2015). [144] R. Goei, T.-T. Lim, Ag-decorated TiO2 photocatalytic membrane with hierarchical architecture: Photocatalytic and antibacterial activities, Water Res., 59 (2014) 207-218. [145] N. Ma, X. Fan, X. Quan, Y. Zhang, AgŒTiO2/HAP/Al2O3 bioceramic composite membrane: Fabrication, characterization and bactericidal activity, J. Membr. Sci., 336 (2009) 109-117. [146] X. Zheng, Q. Wang, L. Chen, J. Wang, R. Cheng, Photocatalytic membrane reactor (PMR) for virus removal in water: Performance and mechanisms, Chemical Engineering Journal, 277 (2015) 124-129. 29 CHAPTER 2. VIRUS REMOVAL AND INACTIVATION IN A HYBRID MICROFILTRATION-UV PROCESS WITH A PHOTOCATALYTIC MEMBRANE 2.1 Introduction Photocatalytic membrane reactors (PMRs) combine membrane separation and photocatalysis in one hybrid process [1, 2]. PMR is a highly versatile technology due to the range of engineering designs it affords and the realm of possible applications. Water treatment is a salient example of such application area. Since 1985 when Matsunaga et al. [3] used Pt-loaded TiO2 for catalytic inactivation of three types of bacteria, applications of photocatalysis to water disinfection have been growing [4]. Indeed, there is a large body of literature on the use of photocatalysis for inactivating microorganisms in water [5, 6]. Notably, photocatalytic treatment can be highly effective with respect to viruses (e.g. [7-10]). Most of the PMR-based water treatment work, however, has focused on chemical pollutants. To our knowledge, there have been only five reports on the application of photocatalytic membranes to water disinfection [11-15]. All five studies were on E. Coli control and three of the five [13-15] also used silver, a known bactericide, as a component of photocatalytic membranes. Several studies (e.g. [16]) also explored how an added photocatalytic function can help improve membrane™s resistance to biofouling. Various PMRs have been implemented that employ different types of light sources and membranes. UV lamps have been the most common choice of the source of photons although new visible light catalyst materials make using visible light possible [17]. Because of the ability to support oxidation reaction, ceramic membranes have been used much more commonly than their polymeric and nanocomposite counterparts [18]. Many different photocatalytic materials have been explored as well, with TiO2 being by far the most studied and applied photocatalyst [17, 19]. 30 PMRs can be categorized into two major groups: a) PMRs with the catalyst materials suspended in the bulk of the feed solution and b) reactors where the catalyst is immobilized on the membrane surface. In the latter case, typically it is the feed surface of the membrane that supports the catalyst. By illuminating such surface with UV light, photocatalysis occurs in the immediate vicinity of the separation layer bringing about potential additional advantages of fouling control and retentate disinfection. This PMR configuration, however, couples separation and catalytic properties of the membrane making their optimization more challenging. An alternative configuration is when it is the membrane support layer (i.e. permeate side of the membrane) that is photocatalytic. To our knowledge, the only study that explored such configuration is the work by Bosc et al. [20]. One major benefits of such approach is the possibility of an independent control of the separation and photocatalytic functions. Another benefit is an extension of the first: by regulating what materials are retained by the membrane, one can control the make-up of the permeate solution to improve the photocatalytic function. For example, catalyst poisons or particulates capable of shielding UV light may be removed at the feed-membrane interface to make photocatalysis on the permeate side more efficient. The choice of PMRs configuration has implications for the design of the catalytic layer. Because most practicable membranes are asymmetric, the feed and permeate faces of membranes have dramatically different morphologies. The membrane fiskinfl (i.e. the feed side) has much smaller pores and typically a much smoother surface than the permeate side. This implies that different coating strategies might be needed to form photocatalytic layers on these supports. The goal of this work is to extend the PMR design concept proposed by Bosc et al. [20] to tubular 31 membranes and to apply such PMR to photocatalytic disinfection of viruses. We employ P22 bacteriophage as a model virus and compare the performance of the proposed PMR against that of its constituent processes Œ UV disinfection and microfiltration. To our knowledge, this is the first application of photocatalytic membranes to virus removal or inactivation. 32 2.2 Experimental 2.2.1 Reagents Aeroxide TiO2 P25 powder was provided by Evonik Industries. Lysozyme (from chicken egg white), ethylenediaminetetraacetic acid (EDTA) and phosphoric acid were purchased from Sigma-Aldrich. Trypticase soy broth (TSB), trypticase soy agar (TSA) and Bacto agar were purchased from Becton, Dickinson and Co. Glycerol and sodium hydroxide were obtained from Avantor Performance Materials. KI/KIO3 solution was a mixture of 0.6 M potassium iodide (Jade Scientific) and 0.1 M iodate (EM Industries) in 0.01 M borate buffer (Sigma-Aldrich). Ultrapure water (~ 17 -pure water purification system (Thermo Fisher Scientific). The bacteriophage was propagated by inoculating 25 mL trypticase soy broth with Salmonella enterica serovar Typhimurium LT2 and allowing for growth at 37°C. 2.2.2 Bacteriophage propagation and preparation of feed suspension To evaluate virus removal and inactivation efficiency of the hybrid photocatalytic UV-MF process, bacteriophage P22 was used as a model virus. P22 is a dsDNA virus [21-25] that has been used as a surrogate for human viruses to study their attenuation [25] and their fate in sewage [26]. The bacteriophage was propagated by inoculating 25 mL trypticase soy broth with Salmonella enterica serovar Typhimurium LT2 and allowing for growth at 37 °C. After overnight incubation, 0.1 mL of lysozyme (50 mg/mL) and 0.75 mL of 0.5 M EDTA were added to lyse host bacterial cells. The culture was then centrifuged at 4000 rpm for 10 min and the supernatant was filtered through a concentration of 5·109 PFU/mL and was maintained at 4 °C. The P22 bacteriophage feed 33 suspension used in all disinfection and filtration experiments was prepared by diluting 300 µL of P22 stock in 3 L of ultrapure water and thus had P22 concentration of ~ 5·105 PFU/mL. 2.2.3 Membranes and deposition of photocatalytic coating on their surface Membranes used in all tests were TiO2 tubular ceramic microfilters (TAMI Industries) with a cm. The permeate side of the membrane was coated with commercial Aeroxide TiO2 P25 powder (50 ± 15 m2/g, 78-85% anatase and 14-17% rutile [27], mean particle size of 21 nm) by dip-coating the membrane with a 10 wt% TiO2 solution prepared following the procedure described by Wang et al. [28]. Prior to its use in the dip-coating procedure, the TiO2 suspension was stirred and sonicated for 24 h. The tubular ceramic membrane with both ends sealed by Parafilm (to avoid coating the internals walls of the membrane channel) was vertically dipped into the TiO2 suspension, maintained submerged for 30 s, and then withdrawn at a constant speed of 4.7 cm/min. The dip-coating instrument was constructed in-house using a syringe pump (55-2219, Harvard apparatus). The entire procedure included 10 coating cycles with 5 min drying at 80°C after the deposition of each coat. After the tenth coating cycle, the membrane was dried at 80°C for 24 h, and then calcined in a furnace (RHF 15/3, Carbolite Ltd). The furnace temperature was programmed to increase to 500 °C with a ramp rate of 4.0 °C/min, stay constant for 45 min, and finally decrease to the room temperature at a rate of 4.0 °C/min. 2.2.4 Membrane cleaning Prior to each filtration experiment, the membrane was cleaned by following the procedure recommended by the membrane supplier: the membrane was first soaked in 20 g/L NaOH at 85 34 °C for 30 min, rinsed with ultrapure water to bring pH to 7, soaked in 75% H3PO4 at 50 °C for 15 min, and then again rinsed with DI water to bring pH to 7. The efficacy of cleaning was verified by performing a pure water flux test and comparing membrane resistances before and after cleaning. 2.2.5 Hybrid UV-membrane filtration unit: Design and operation Figure 2.1 shows the schematic of the hybrid MF-UV disinfection unit used in all filtration experiments. The membrane and the UV lamp were placed in the foci of two alumina parabolic reflectors (Fig. 2.2) positioned to face each other at a distance that could be adjusted to regulate UV fluence on the membrane surface. The parabolic design ensured that the outer surface of the membrane was evenly irradiated by the UV light. UV-C irradiation was generated by a preheated germicidal UV lamp (16 W, model GPH330T5L/4, Atlantic Ultraviolet Corp.) The crossflow was provided by a peristaltic pump (model 621 CC, Watson-Marlow) equipped with a pulsation dampener (AD-10 PS, Yamada America). Transmembrane pressure was measured by pressure gauges (0 to 15 psi range, Ashcroft) installed on the feed and retentate sides of the membrane unit. The crossflow flux was measured using a flowmeter (model 101-8, McMillan). Permeate was collected on an electronic mass balance (Adventurer Pro AV8101C, Ohaus) interfaced with a data acquisition system (model NI PCI-6221, National Instruments). All filtration tests were performed in a constant pressure mode with the average transmembrane pressure of 2.8 ± 0.2 psi (19.4 ± 1.5 psi). Average pressure values in filtration tests of different types were 2.67 ± 0.14 psi, 2.83 ± 0.14 psi, and 2.95 ± 0.30 psi in experiments on MF only, UV +MF with non-catalytic membranes, and UV + MF photocatalytic membrane, correspondingly 35 (see Appendix A, Table A1). The membrane, which was operated in an inside-out flow geometry, was housed in a quartz sleeve (160 mm in length, 20.5 mm in outer diameter) to allow for both illumination of the permeate side of the membrane by UV light and permeate collection. At the membrane ends, the space between the membrane and the quartz sleeve was sealed using two silicone stoppers (Fig. 2.2). The permeate was allowed to leave the quartz sleeve through a syringe needle into the permeate collection tube and the permeate mass flow rate was recorded at 1 s intervals. The average crossflow rate was 1.06 ± 0.09 L/min translating into the average crossflow velocity of 0.62 ± 0.05 m/s. Average crossflow rate values in filtration tests of different types were 1.1 ± 0.0 L/min, 1.0 ± 0.1 L/min, and 1.1 ± 0.0 L/min in experiments on MF only, UV +MF with non-catalytic membranes, and UV + MF photocatalytic membrane, correspondingly (see Appendix, Table A1). Figure 2.1 Schematic diagram of the hybrid membrane filtration-UV treatment system 36 Temperature of the permeate as a function of filtration time and UV exposure was measured in real time in a separate crossflow test with the membrane tilted at an angle to fasten permeate collection and minimize heat loss prior to the measurement. The samples of permeate were collected in a 2 mL vial (2 mL) periodically for 2 h. The temperature of the solution was measured with a digital thermometer (model S407993; Fisher Scientific: accuracy: ±1°C). 2.2.6 Sample collection and storage Samples of the feed solution were withdrawn from the feed tank before and after each filtration experiment. In each filtration test, permeate samples were collected immediately after the start of filtration as well as 10, 20, 30, 45 and 60 min into the experiment. Each sample was divided into two aliquots. One aliquot was placed in a glass vial with a plastic cap, wrapped in foil, and stored at 4 °C. The second aliquot was frozen in a 5mL cryogenic vial at -80°C as a backup. The cryoprotectant (20% glycerol) to sample volume ratio was 1:1. 37 Figure 2.2 Design of custom-made parabolic UV light reflectors and the membrane housing unit Note: The membrane housing unit is drawn not to scale. Membrane™s length and outer diameter are 0.25 m and 0.01 m, respectively. 38 2.2.7 UV dose quantification Chemical actinometry [29-31] was used to measure UV fluence by determining the UV absorbance of KI/KIO3 solution. The exposure of the KI/KIO3 solution to UV light results in the formation of triiodide, the concentration of which can be determined spectrophotometrically at 352 nm (MultiSpec 1501, Shimadzu). For each measurement, the absorbance of the KI/KIO3 solution in dark was used as a baseline. To determine the quantum yield (mole of product formed per mole of photons absorbed), the concentration of KI was first measured by recording KI absorbance at 300 nm and applying the Beer-Lambert law: = , where and =1.061 M-1 cm-1 are the absorbance of KI at 300 nm and the extinction coefficient of KI at 300 nm, respectively [32], and = 1 cm is the optical path length of the spectrophotometer cell. With known, was computed as: =0.75(1+(20.7))(1+(0.577)), where is the solution temperature in oC. Fluence (mJ/cm2) is given by [32] = (1) where = 4.72·105 (J·E-1) is a conversion factor for 254 nm wavelength, = 27,600 M-1cm-1 is the extinction coefficient of triiodide at 352 nm [33], (mL) is the total volume of solution in the quartz sleeve, and = 96.6 cm2 is the surface area of the quartz sleeve exposed to UV light. 39 2.2.8 Quantifying the efficacy of disinfection by direct UV only The disinfection efficacy of UV light in the crossflow system in the absence of photocatalyst could not be measured because the possibility of a photocatalytic effect could not be eliminated. Instead, to quantify virus inactivation due to UV only we employed the following multistep procedure: Step 1: Measuring the permeate retention time in the quartz sleeve. Based on the measured values of the permeate mass flow rate, , and the mass of the residual permeate solution, , in the quartz sleeve, the retention time of permeate solution was calculated as t= , where is the length of the quartz sleeve ( = 15 cm); = is the flux of permeate solution in quartz sleeve, is the density of permeate solution and = is the cross-sectional area of the residual permeate solution in the sleeve. Step 2: Relating UV fluence to permeate retention time. This was done by placing the KI/KIO3 indicator solution in the permeate chamber (the quartz sleeve) of the hybrid UV- MF unit, without applying any pressure or crossflow and exposing the solution to UV. Because the permeate retention time values calculated at Step 1 did not exceed 30 s in any of the experiments, the KI/KIO3 solution was exposed to UV irradiation for 5, 10, 20 and 30 s. Based on these measurements, the dependence of UV fluence on retention time was established. Step 3: Determining P22 inactivation as a function of UV fluence. To determine the dependence of P22 inactivation efficiency on UV fluence, P22 suspensions were exposed to UV for 5, 10, 15, 20 and 30 s in a sequence of separate tests. The obtained values of P22 inactivation (see section 2.10) were related to UV fluence using the relationship established at step 2. 40 Following the above three steps, the P22 removal efficacy was related to the mass flow rate. Thus, in each test of the hybrid microfiltration-UV process, the contribution of direct UV to virus inactivation could be determined. 2.2.9 Electron microscopy of the membrane surface Scanning electron microscopy (SEM) images of the tubular membrane surface as well as the membrane™s cross-section were recorded (JEOL 6610LV SEM) under magnifications of ×700 and ×1000. Membrane samples for SEM imaging were obtained by breaking the membrane, mounting a piece with membrane™s cross-section exposed onto an aluminum stub and coating the mounted sample with ~ 20 nm thick layer of gold (Emscope Sputter Coater, model SC 500, Quorum Technologies). 2.2.10 Quantification of the viable bacteriophage The concentration of viable P22 bacteriophage was quantified by plaque assaying. TSA plates (1.5%) and 1% top agar tubes were prepared according to the standard method [34]. On the same day as the experiment, the Salmonella enterica serovar Typhimurium LT2 stock was removed from -80 ºC and defrosted. One milliliter of the defrosted stock was introduced into 10 mL TSB media under sterile conditions and placed in a 37 °C incubator. After overnight incubation, 1 mL Salmonella enterica serovar Typhimurium LT2 culture was transferred to 30 mL TSB at 37 °C for 3 h to reach the log phase of growth. The concentrations of viable P22 in feed solution and filtrate samples were determined by the double agar layer method [34]. First, top agar tubes were boiled and then placed in a 45-48 °C water bath. A series of dilutions (101 to 104) was prepared for each sample and each diluted sample was analyzed in triplicate. Second, one top agar tube was removed 41 from the water bath, 0.3 mL of log phase Salmonella enterica serovar Typhimurium LT2 culture and 1 mL of sample were sequentially added. Then, the mixture was gently agitated and poured on a 1.5% TSA bottom agar plate. Slight shaking and swirling was applied to distribute the agar evenly on the plate. After the top agar hardened at room temperature, the plates were inverted and incubated for 16 to 18 h at 37 °C. Finally, the number of circular clear spots in each lawn of host bacteria was counted to determine plaque-forming units (PFU/mL) for each sample. 2.2.11 Quantification of the total bacteriophage The total P22 bacteriophage count, which includes both the viable (infective) and non-viable (non-infective) virus, was determined by qPCR. Within 24 h of the filtration experiment, DNA of the bacteriophage was extracted using a MagNA Pure automatic extraction machine and MagNA Pure added to prevent DNA adsorption on the surfaces of the extraction kit. The nucleic acid eluents Each eluate was analyzed by real-time qPCR in triplicate following the procedure described by Masago et al. [25] (also see Appendix; Table A2). Each sample that was PCR-grade water (Qiagen). This study used same sequences of primers and probe as in Masago et al. [25]. The qPCR analysis started with 95°C for 15 min then followed by 45 amplification cycles at 95 °C for 10 s, 60 °C for 20 s and 72 °C for 10 s and finally cooling at 40°C for 30 s. To 42 relate the crossing-point () values to the numbers of P22 DNA copies, a standard curve developed in our laboratory was used. 43 2.3 Results and Discussion 2.3.1 Hybrid membrane filtration-UV process: The concept and a brief rationale UV disinfection is effective against a broad range of microorganisms and has unique advantages over other disinfection processes. As a unit operation, UV disinfection does not involve addition of chemicals and does not generate harmful disinfection by-products typical for chemical disinfection unit processes such as chlorination and ozonation. Figure 2.3 Conceptual illustration of the hybrid membrane filtration-UV disinfection process UV light is also effective for inactivating chlorine-resistant pathogens such as Cryptosporidium and Giardia protozoa (e.g. [35, 36]). A fundamental limitation of UV disinfection is that RNA-based microorganisms Œ a group that includes many EPA-regulated viruses such as enteroviruses, hepatitis A virus, and caliciviruses Œ are resistant to UV. Furthermore, some pathogens can repair 44 UV-induced damage to their DNA. Another challenge is presented by water turbidity. Turbidity, when present at high levels, limits UV light access to microorganisms and is known to diminish the efficacy of UV disinfection [37, 38]. The proposed novel approach combines microfiltration and UV disinfection into a hybrid photocatalytic process (Fig. 2.3) to overcome the above two challenges. The ultra- or microfiltration membrane operated in an inside-our geometry removes turbidity so that the UV irradiation is applied to a relatively turbidity-free permeate stream. The degree of turbidity removal is controlled by an appropriate choice of the membrane pore size. At the same time, the membrane serves as a support for photocatalytic nanoparticles immobilized on the outer (i.e. permeate) membrane surface exposed to the UV light. The catalytic enhancement of UV disinfection is due to non-specific chemical oxidation by reactive oxygen species (ROS) catalytically generated at the membrane surface. The oxidation complements direct UV to pose a fidual threatfl to pathogens with direct UV targeting microorganism™s DNA and ROS damaging cellular membrane (in cases of bacteria and protozoa) or viral capsid (in case of viruses). These and several other advantages of the proposed hybrid process are summarized in Table 2.1. 45 Table 2.1 Rationale for the proposed UV-microfiltration hybrid process Technology and its benefits Challenges How the challenge is addressed in a hybrid process Photocatalytic UV disinfection Photocatalytic UV is effective against a wide range of microbial pathogens. Chemicals demand and harm to receiving waters are minimal UV disinfection is catalytically enhanced Some pathogens are resistant to UV or can repair UV-induced damage Catalytic oxidation at the membrane surface by ROS complements the physical effect of the direct UV. Catalyst needs to be recovered Membrane-supported catalyst is immobilized and does not need to be recovered. Efficiency is limited when turbidity is present Turbidity is removed by the microfilter fiupstreamfl from the UV reactor Membrane filtration Membranes provide absolute barrier to pathogens Lower pore size for removal of smaller pathogens results in lower permeate fluxes Redundancy introduced with catalytic UV disinfection enables trade-offs in pore sizes and product water fluxes 2.3.2 Efficacy of disinfection by direct UV irradiation First, batch experiments were conducted to determine UV fluence as a function of UV exposure time by using KI/KIO3 solution as an indicator. Following the procedure described in section 2.7 (see eq. (1)), values of UV fluence were calculated (see Appendix, Fig. A1). The exposure time was considered to be equal to the retention time of permeate solution in the quartz sleeve. The 46 results show that fluence increased linearly with exposure time (see SI). Second, the log removal (LRV) of viable P22 was measured as a function of UV fluence LRV is defined as ()= (2) where and are P22 concentrations in the batch reactor at time 0 and time into the reaction, respectively. The kinetics of P22 inactivation by UV light could be approximated (Fig. 2.4) by the Collins-Selleck model [39, 40]: =[()ln ()] (3) where is Collins-Selleck coefficient of specific lethality and is the lag coefficient. Based on the fit of experimental data to eq. (3), the following values of these two coefficients were determined: = 1.972; = 0.376 mW·s/cm2. With () and () dependencies determined, the dependence of the efficacy of disinfection (expressed in terms of ) by direct UV irradiation on the UV exposure time was established. The small negative filagfl described by (i.e. non-zero extrapolated value of P22 inactivation based on the fit given by eq. (3)) is attributed to an experimental error. The decelerating kinetics described by the Collins-Selleck model could be a consequence of P22™s being shielded from the UV light by residual components of the virus growth media. 47 Figure 2.4 Log removal of viable P22 bacteriophage as a function of UV fluence Note: Each data point is based on a triplicate measurement. Error bars represent standard deviations (n=3). 2.3.3 Characterization of the tubular ceramic membrane SEM images of the as-received tubular ceramic membrane and the same membrane coated with TiO2 P25 nanocatalyst are presented in Fig. 2.5. The separation layer of this 0.8 µm nominal pore size membrane is on the inner wall of the membrane channel making the membrane suitable for use in the inside-out flow geometry only. 48 Figure 2.5 Scanning electron micrographs of the tubular ceramic membrane Note: A) planar view of the inner surface, B) planar view of the uncoated outer surface, C) planar view of the TiO2-coated outer surface, and D) the cross-sectional view of the coated outer surface of the tubular ceramic membrane. Accordingly, the inner (feed) surface of the membrane has a finer pore structure (Fig. 2.5A) than the more porous and rough outer (permeate) surface composed of larger TiO2 grains (Fig. 2.5B). The coating-induced morphological changes of the outer membrane surface could be clearly observed: the 10-layer coating covered the outer surface of the membrane with a layer of TiO2 P25 that is relatively smooth but cracked (Fig. 2.5C). The cracking might be due to the high roughness of the underlying membrane surface, which could lead to an uneven tensile stress in the coating 49 [41, 42]. The coating was not homogeneous over the entire membrane surface with some portions of the membrane coated with a denser catalyst layer. The reasons were not clear and an additional study would be required to optimize the coating process. Coating the permeate surface with a TiO2 layer led to ~ 40% decline in the permeability of the membrane. Figure 2.6A illustrates how the specific permeate flux, , of uncoated and coated membranes changed with the time of filtration of ultrapure water first in the absence of UV and then after exposed to UV irradiation. In the absence of UV (i.e. during the first 60 min of the filtration test) the specific permeate flux through an uncoated membrane declined by ~ 17.5%. A declining trend for pure water permeate flux for ceramic membranes has been reported in the past [43-45] and attributed by Mendret et al [45] to the very slow hydration of the membrane surface. However, given the very large nominal pore size (0.8 µm) of the membrane employed in our study, hydration shell should be much thinner than the pore size so that hydration can be eliminated as the reason for flux decline. A part of the reason for this flux behavior is the change in water temperature (Fig. 2.6B) as it decreased throughout the first 60 min of the test from its initial value (23 0C) towards the lower temperature of the ambient air (20.3 ºC). When the temperature induced changes were factored out by normalizing values of the specific permeate flux by viscosity, the resulting time dependence of membrane permeability still showed a 16.5% decline (Fig. 2.6C). Tentatively, we attribute the observed flux behavior to the re-arrangement of loosely affixed TiO2 particles due to permeate flow. 50 A) B) C) Figure 2.6 Specific permeate flux of ultrapure water Note: (A) temperature of ambient air, feed water and permeate water, (B) and membrane permeability, (C) as functions of filtration time for uncoated and TiO2 P25-coated membranes. 51 When the outer membrane surface was illuminated by the UV light the permeate flux (Fig. 2.6A) started to increase. We attribute this increase to higher temperature (Fig. 2.6B) and resulting lower viscosity of the permeating water due to membrane heating by the UV light. The temperature effect could not fully explain away the increase in flux - the permeability still increased with time (Fig. 2.6C) in the presence of UV. We attribute this fact to errors in temperature measurements: collecting a sample for temperature measurements takes time during which the water in the sample can cool down. Mendret et al [45] reported similar UV-induced increases in permeate flux and explained them as stemming from photoinduced hydrophilicity [46, 47]. In our case though it was the porous permeate side of the membrane, and not the permeability-controlling separation layer, that was exposed to UV irradiation (Fig. 2.3). Because the membrane is not transparent to UV, only a thin sublayer of the membrane on its permeate side could have experienced photoinduced changes in surface hydrophilicity. The resulting improved wettability of this part of porous structure could not have been responsible for the observed increase in the overall permeability of the membrane. Notably, the coated membrane did not show a similar dependence on filtration time on the absence of UV light nor did it show a response to the UV irradiation. This behavior can be rationalized by posing that a) loose particles in the membrane are stabilized during the coating and sintering procedures, and b) energy of the UV irradiation is absorbed by the coating and not dissipated as heat that can increase the temperature of the permeating solution. 52 2.3.4 Removal and inactivation of bacteriophage P22 Separate experiments were performed on the removal and inactivation of P22 bacteriophage in three treatment processes: 1) MF only, 2) hybrid UV-MF process with an uncoated membrane, and 3) hybrid UV-MF process with a TiO2-coated membrane. Figure 2.7 summarizes LRV data for viable P22 by the four processes for six different times into the filtration process (also see Appendix, Table A3): 1) Among all processes tested, microfiltration, applied alone, was the least effective in removing viable P22 (LRV = 0.5 ± 0.5). The low removal rate was due to the large nominal pore size of the microfilter (0.8 µm) relative to the hydrodynamic diameter of P22 bacteriophage ( = 68.8 nm) [48]. The overall removal of viable P22 by the membrane can be attributed to a combination of adsorption, size exclusion, and inactivation upon contact with the membrane surface. Despite the mismatch between the pore size and virus diameter, size exclusion may still be possible because the membrane pore size distribution is of finite width and may include very small pores. 2) The estimated (see section 2.8) inactivation by direct UV was very stable throughout the entire 60 min of filtration with an average LRV of 1.6 ± 0.1. The contribution of the UV process to bacteriophage inactivation is due to UV™s germicidal effect, which reduces the number of infective viruses but not the total number of viral particles. 3) Averaged over filtration time, the LRV removal of viable P22 by the hybrid UV-MF process with an uncoated MF membrane (2.3 ± 0.2) was not statistically different from the arithmetic sum 53 of LRVs achieved by the two constituent processes, UV and MF with uncoated membrane - applied separately. 4) By contrast, the hybrid UV-MF process with a membrane coated with TiO2 photocatalyst resulted in an average LRV of 5.0 ± 0.7, which was more than two times higher than the corresponding value for the hybrid UV-MF test with uncoated membranes. Figure 2.7 Inactivation and/or removal of viable P22 bacteriophage Note: 1) direct UV only 2) microfiltration only, 3) non-photocatalytic hybrid UV-MF process, and 4) photocatalytic hybrid UV-MF process. Error bars represent standard deviations (n=3). 54 Figure 2.8 Log concentration of viable and total bacteriophage P22 in the feed solution and in the permeate 30 min into the filtration process The synergistic effect was due to the membrane-based photocatalysis, wherein reactive oxygen species (ROS) generated at the surface of TiO2 nanoparticles of the coating provide non-specific oxidation that complemented the effect of direct UV. While direct UV inactivates viruses by dimerizing their DNA [49], ROS contribute to disinfection by oxidizing the protein capsid of viruses. As mentioned earlier, this contribution of photocatalysis to the overall removal of viable viruses is especially important because, in contrast to direct UV, it can inactivate RNA viruses. Figure 2.8 provides absolute values of the total concentration of P22 and the concentration of viable P22 in the effluent after 30 min of operation of each of the three treatment processes. The total virus count (i.e. viable and non-infective fractions together) in feed and permeate samples was estimated based on DNA copy counts measured by qPCR. The data show that the 55 photocatalytic UV-MF process is effective in inactivating viable P22 even though the reduction of the total virus is not significant. The large value of the LRV recorded for the hybrid process points to the possibility of employing membranes with even larger pore size to enable higher flow rates. 2.3.5 Potential applications in water treatment The proposed hybrid process can mitigate two salient disadvantages of UV disinfection: resistance of certain environmentally important pathogens to UV disinfection and low efficacy of UV light when applied to highly turbid waters. Coupling microfiltration with photocatalytic UV process can make disinfection of highly turbid or large flow rate streams (e.g. ballast and storm water) more efficient in terms of the required UV dose for a given level of disinfection. This improvement, however, would likely come at the expense of membrane fouling and, therefore, a higher cost of membrane operation. A possible application for the proposed hybrid process and an important environmentally-relevant example of a high flow rate operation requiring disinfection is ballast water treatment. Recent International Marine Organization D2 regulations impose limits on the concentration of microbes in ballast water. According to these regulations, all vessels built after January 1, 2016 must comply with the U.S. Coast Guard Discharge Standards Phase 2 that require that no more than 103 bacteria and 104 viruses are present in 100 mL of treated ballast water. Given the very small size of microorganisms and the large flow rates typical for ballast water treatment, complete physical removal of bacteria and especially viruses is unlikely, which makes disinfection a critical second barrier. It is now recognized that no single process efficiently removes the wide range of potential invasive species in ballast water and that a combination of technologies must be considered [50]. 56 A combination of filtration and subsequent disinfection has been identified as the best available treatment [51]. To date, UV disinfection has been applied as a stand-alone unit [52-56] and in combination with physical separation methods that use filters [57, 58] and hydrocyclones [59, 60]. The prior history of adoption of these technologies by the shipping industry bodes well for the application of new hybrid technologies that combine filtration and UV light Innovative reflector designs can further facilitate field applications of the proposed photocatalytic membrane reactor. For example, we envision a reflector with a parabolic profile of corrugation as a large surface area lens that focuses incident light on tubular filters positioned in foci of the parabolas. Finally, membranes can support a broad range of photocatalytic materials including high efficiency UV and visible-light photocatalysts that tap into solar energy and may enable low cost disinfection. 57 2.4 Conclusions We report on the first application of photocatalytic membranes for virus removal and inactivation. In the proposed hybrid technology, UV light is focused on a TiO2-coated outer surface of a tubular ceramic membrane operated in an inside-out geometry. The hybrid process is evaluated with respect to removal and inactivation of P22 bacteriophage, a model virus. The kinetics of P22 inactivation by direct UV was first evaluated in a separate set of tests in a batch UV reactor and found to fit Collins-Selleck model. To gauge the performance of the hybrid UV-microfiltration process, a number of crossflow filtration tests were performed with and without UV light as well as with and without photocatalytic coating on the membrane. Compared to stand-alone microfiltration, stand-alone UV disinfection and UV-microfiltration with a non-photocatalytic membrane, the hybrid photocatalytic UV-microfiltration process was considerably more effective in inactivating the virus. Average values of log removal of viable P22 by these four processes were 0.5 ± 0.5, 1.6 ± 0.1, 2.3 ± 0.2, and 5.0 ± 0.7, respectively. The proposed hybrid process can mitigate two salient disadvantages of disinfection by direct UV: resistance of certain environmentally important pathogens to UV and low efficacy of UV disinfection when applied to highly turbid waters. Virus removal and inactivation can be regulated by the choice of the membrane pore size, design of the photocatalytic coating, and by controlling UV fluence applied to the permeate stream. Potential applications of the hybrid UV-microfiltration technology include treatment of turbid, high fouling potential and high flow rate streams that cannot be cost-effectively disinfected by other means. 58 APPENDICES 59 Appendix A: Supporting information Table A.1 Sequences of Primers and Taqman probe Primers/probe Sequence Reverse CTT AAC AAG CTC TGA CTG CTC ATC A Forward CCA TCG CCT GTG ACT CGA T Taqman Probe FAM-TCG CAA CGA TGC AGA ACG ACT CG-TAMRA Note: Reference [29]. 60 Table A.2 Log removal of viable P22 in different treatment processes as determined by plaque assay analysis Treatment process Filtration time, min 0 10 20 30 45 60 UV 1.54 ± 0.07 1.51 ± 0.07 1.51 ± 0.08 1.52 ± 0.09 1.56 ± 0.08 1.58 ± 0.09 MF (uncoated membrane) 0.54 ± 0.40 0.88 ± 1.05 0.67 ± 0.74 0.67 ± 0.70 0.53 ± 0.47 0.44 ± 0.51 UV + MF 2.08 ± 0.40 2.39 ± 1.05 2.18 ± 0.74 2.19 ± 0.70 2.10 ± 0.48 2.03 ± 0.52 Hybrid UV-MF process (uncoated membrane) 1.91 ± 0.13 2.25 ± 0.07 2.38 ± 0.15 2.33 ± 0.22 2.29 ± 0.18 2.38 ± 0.19 Hybrid UV-MF process Hybrid UV-MF process (coated membrane)ne)MF process (coated membrane) 4.57 ± 0.80 4.43 ± 0.61 4.67 ± 0.39 4.49 ± 0.38 4.75 ± 0.71 4.99 ± 0.70 61 Figure A.1 UV fluence as a function of the exposure time Note: Each data point is based on a triplicate measurement. Error bars correspond to standard deviations. y = 0.2616xR² = 0.99120123 456 789010203040Fluence (mJ/cm2)Exposure time (s)62 Appendix B. Photocatalytic coating on borosilicate glass slides Borosilicate glass slides with the dimension of 24 mm×60 mm were purchased form VWR (catalog number 16004-096) and used to optimize the number of TiO2 coating layers for the preparation of a photocatalytic ceramic membrane. Borosilicate glasses are known to have a very low coefficient of thermal expansion (~3 × 106 K1 at 20 °C). Therefore they show very good thermal resistance which allows for the use of temperatures up to 500 º C or even 550 º C for a short period of time [1]. Moreover, borosilicate glasses also possess excellent chemical resistance and flatness. In addition, the borosilicate glasses do not fluoresce under UV light [2]. B.1 Borosilicate glass slides cleaning procedure Prior to coating, new borosilicate glass slides were carefully cleaned to eliminate the potential alkaline nature of new glass products and the interference of grease and/or organic matter [3]. First, borosilicate glass slides were soaked in 1% hydrochloric acid for 2 to 3 hours and rinsed with DI water [3]. Second, borosilicate glass slides were ultrasonically cleaned with detergent for 15 min and again rinsed with DI water. Third, borosilicate glass slides were ultrasonicated in acetone and ethanol solution for 15 min, respectively [4, 5]. Finally, borosilicate glass slides were rinsed with DI water and dried at ~30°C overnight prior to use. B.2 Borosilicate glass slides coated with TiO2 catalysts 63 Borosilicate glass slides were coated with TiO2 using the dip-coating method. Suspension of TiO2 particles with the concentration of 10 wt% was prepared by adding 100 g of P-25 TiO2 powder into 900 g of distilled deionized water (DDI) with the addition of 0.02 g of dioctyl sulfosuccinate as a dispersant [6]. Then, the suspension was stirred for 24 h and ultrasonicated for a24 h before use. At the same time, all the glass containers were cleaned using 10% hydrochloric acid (detergent or 70% ethanol if needed) and rinsed with DI water. To evaluate the amount of TiO2 deposited on glass slides during coating, each glass slide was weighed before and after the coating procedure. Table B.1 shows major parameters of the dip-coating method. In all referenced studies TiO2 was used as the only catalyst and glass was used as the substrate. Based on the analysis and comparison of the references, we developed our own coating procedure, which includes the following steps: 1. Cleaned borosilicate glass slide was immersed into TiO2 solution at the speed of 4.7 cm/min. 2. After 30 s of immersion, the glass slide was withdrawn at the same speed of 4.7 cm/min. 3. TiO2 coated glass slide was dried in oven at 80°C for 3-5 min after each coating. The desired number of coating layers was achieved by repeating the above steps. 4. After the last coating layer is deposited, the glass slide was dried in an oven at 80°C overnight and then sintered at 773K (~500°C) for 45 min. In total, seven different borosilicate glass slides were coated with 1, 2, 5, 8, 10, 15 and 20 TiO2 coating layers, respectively. Table B.2 shows the scanning electron microscope (SEM) images of the seven TiO2 coated glass slides under different magnifications. 64 Table B.1 Summary of dip coating parameters from selected representative references Substrate Components Concentration Speed Coating layers Drying process Sintering TiO2 amount Sources Glass TTIP in i-prOH 0.5 mol/L 20.4cm/min Several dried at 150 °C during 1 h 450 °C 3 mg/cm2 Guillard et. al. [7] Conducting glass TTIP in acetic acid solution in an ice/water 10mL/100mL acid solution - 1 - 450 °C for 1 h 0.5 mg/cm2 Yang et. al. [8] Glass Degussa P25 particles 4 g/l - 4 dried at 100 °C for 1 h 450 °C 0.28 mg/cm2 Alinsafi et. al. [9] Glass beads TTIP in i-prOH and DEA Degussa P-25 TiO2 powder 0.5 M 12.8 cm/min Several 125 °C for 24 h 100 °C for 1 h, then 600 °C for 1 h. - Balasubramanian et. al. [10] Borosilicate Petri dish TTIP and nitric acid in deionized water 15mL/150mL water Evenly applied 1 75 °C for 24 h 400 °C for 2 h ~0.11 mg/cm2 Ao et. al. [11] Borosilicate glass TTIP in ethanol and HCl (37%) 10mL/50mL ethanol 11.5 cm/min Several to get tens of nm dried at 70 °C during 5 min 80 °C for 12 h and then calcinated at 450 °C in air during 2 h - Ghazzal et. al. [12] Note: TTIPŠtitanium isopropoxide; i-prOHŠ isopropanol; DEAŠ diethanolamine; HClŠhydrogen chloride. 65 Layers Overall view With magnification of ×1,500 With magnification of ×10,000 1 5 8 Figure B.1 SEM images of different coating layers on borosilicate glass slides under various magnifications 66 Figure B.1 (cont™d) 10 15 20 67 Appendix C. Photodegradation test of TiO2 coated borosilicate glass slides To evaluate photocatalytic properties performance of the catalyst-coated borosilicate glass slides, methylene blue (MB) dye was adopted as an indicator compound and used in all photodegradation tests. All tests were conducted in batch. For each test, 200 mL MB solution with the concentration of 2 mg/L was prepared in a 250 mL glass beaker. After sufficient mixing, the coated glass slide was immersed in the beaker for 30 min to reach the adsorption and desorption balance prior to the photodegradation test. Meanwhile, UV lamp was preheated. Uncoated glass slides were used in control experiments. The first sample was collected before the MB solution was exposed to UV and this sample was used to calculate the initial concentration of the MB in the solution. Then with the UV irradiation, 1 mL of the MB solution was sampled from the beaker at 10 min intervals during the first 1 h; samples were collected every 30 min during the 2nd hour of the test. The whole experiment lasted for 2 h and the total of nine samples were collected. The MB concentration in each sample was measured using UV spectrophotometer. According to Beer-Lambert law, the concentration of MB is directly proportional to its absorbance. Thus, the degradation efficiency of the TiO2 coated glass slide, which is usually expressed as a ratio of degraded concentration to initial concentration, can be calculated from the values of MB solution absorbance. Figure C.1 shows the photodegradation performance of different coating layers. For coatings with fewer than five TiO2 layers, MB degradation performance increased with an increase in the number of coats/layers. However, when coating included more than eight layers, the MB degradation 68 efficiencies were similar and the maximum degradation efficiencies were observed in tests with 10 layers. Thus 10 layers coating was selected as optimal. Figure C.2 demonstrates the change of degradation rate with the increased amount of catalyst. At the initial stage, degradation rate improved with the increased amount of catalyst. Nevertheless, for the tests with the glass slides of more than 10 layers coatings, although more catalysts were deposited on the glass slides, the degradation efficiency remained stable. This trend indicated that the increased amount of catalysts contributed to the increase of the coating thickness but not the available area for photoactivity. In summary, according to the analysis on the photodegradation of MB in batch, 10 layers coating was selected. Figure C.1 Photodegradation of MB using borosilicate glass slides with different coating layers y = -0.0033x + 1R² = 0.9871y = -0.0033x + 1R² = 0.99660.500.55 0.60 0.650.700.750.80 0.85 0.90 0.951.00020406080100120MBt/MB0Time, minControl1 layer2 layers5 layers8 layers10 layers15 layers20 layers69 Figure C.2 The change of degradation rate with the increased amount of TiO2 catalysts -0.0035-0.003-0.0025-0.002-0.0015-0.001-0.0005000.0050.010.0150.020.025Degradation rate, 1/minIncreased weight, g70 REFERENCES 71 REFERENCES [1] A. Zakersalehi, H. Choi, J. Andersen, D.D. Dionysiou, Photocatalytic ceramic membranes, in: E.M.V. Hoek, V.V. Tarabara (Eds.) Encyclopedia of Membrane Science and Technology, John Wiley & Sons, 2013. [2] S. Leong, A. Razmjou, K. Wang, K. Hapgood, X. Zhang, H. Wang, TiO2 based photocatalytic membranes: A review, J. Membr. Sci., 472 (2014) 167-184. [3] T. Matsunaga, R. Tomoda, T. Nakajima, H. 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Ku, Photocatalytic degradation of Acid Red 4 using a titanium dioxide membrane supported on a porous ceramic tube. Water Research, 2008. 42(19): p. 4725-4732. [67] Guillard, C., et al., Influence of chemical structure of dyes, of pH and of inorganic salts on their photocatalytic degradation by TiO2 comparison of the efficiency of powder and supported TiO2. Journal of Photochemistry and Photobiology a-Chemistry, 2003. 158(1): p. 27-36. [68] Yang, J., et al., Effects of hydroxyl radicals and oxygen species on the 4-chlorophenol degradation by photoelectrocatalytic reactions with TiO2-film electrodes. Journal of Photochemistry and Photobiology a-Chemistry, 2009. 208(1): p. 66-77. [69] Alinsafi, A., et al., Treatment of textile industry wastewater by supported photocatalysis. Dyes and Pigments, 2007. 74(2): p. 439-445. 77 [70] Balasubramanian, G., et al., Evaluating the activities of immobilized TiO2 powder films for the photocatalytic degradation of organic contaminants in water. Applied Catalysis B-Environmental, 2004. 47(2): p. 73-84. [71] Ao, C.H., et al., Photocatalytic decolorization of anthraquinonic dye by TiO2 thin film under UVA and visible-light irradiation. Chemical Engineering Journal, 2007. 129(1-3): p. 153-159. [72] Ghazzal, N.M., et al., A simple procedure to quantitatively assess the photoactivity of titanium dioxide films. Journal of Photochemistry and Photobiology a-Chemistry, 2010. 215(1): p. 11-16. 78 CHAPTER 3. PHOTOCATALYTIC INACTIVATION OF HUMAN ADENOVIRUS 40 IN NATURAL SURFACE WATER: EFFECT OF WATER QUALITY 3.1 Introduction Adenoviruses, members of the family Adenoviridae, are non-enveloped viruses ranging from 70 to 90 nm in diameter and are icosahedral in shape [1]. Represented by 7 species (A through G) and 51 serotypes [1], adenoviruses are double-stranded DNA viruses, ubiquitous in the aqueous environment . Adenoviruses have been shown to be the etiologic agents of various types of diseases, including respiratory infections, conjunctivitis, gastroenteritis and pneumonia [2]. The viruses can be transmitted through direct contact, fecal-oral transmission or occasionally waterborne transmission [2]. The most common symptoms of HAdV infection are fever and coughing; however, life-threatening multi-organ diseases can also be caused by HAdV infection in extreme cases [3]. Adenoviruses have been on the Drinking Water Contaminant Candidate List of the U.S. Environmental Protection Agency (EPA) since 1998 [4]. Numerous studies have shown that adenoviruses can be stable for days and even months in water and on dry surfaces [5, 6]. Moreover, adenoviruses have been recognized as the most UV-resistant virus known to date, requiring a much higher dose for disinfection than other EPA regulated viruses [7] (see Appendix, Fig. S.1). This property is associated with the double-stranded DNA: it has been demonstrated that the viruses can utilize the host cell's DNA repairing capability [8, 9]. Since both DNA strands can serve as a template for replication, when only one of them is damaged by external factors (e.g. UV light irradiation), the other strand can still be used to repair the damaged sites [9]. The U.S. EPA has specified a UV dose of 186 mJ/cm2 for achieving a 4-log inactivation of all viruses (99.99% removal) [10]. However, human adenovirus serotype 40 79 (HAdV40) requires a UV dose of up to 226 mW/cm2 to achieve a 4-log reduction [11]. HAdV40 has been identified as the most UV resistant serotype of the adenoviruses [12]. Species F of HAdV40 has been recognized as a very important pathogenic agent associated with gastroenteritis, primarily in children [13-16]. Every year, millions of deaths are caused by adenoviruses, costing billions of dollars worldwide [17]. The concerns over the health risks of adenoviruses prompt further research on the disinfection of these microorganisms. Effective against a variety of waterborne pathogens [18-20], photocatalytic disinfection offers an alternative to traditional disinfection by chlorine. However, much of the reported studies on photocatalytic disinfection focus only on bacteria [21-23], particularly using E. coli as an indicator organism, which may not be representative of viruses [24-30]. The studies that have examined the inactivation of viruses with photochemical methods have often used bacteriophages, such as MS2 or P22, as their target agents [31-34]. Human viruses, such as adenoviruses, have received much less attention, due the more challenging culturing protocols. To our knowledge, there have been only three published reports on the photocatalytic disinfection of adenoviruses [35-37]. All three studies used UV as the light source. Gorvel et al. [35] and Li et al. [36] coupled UV irradiation with titanium dioxide for inactivation of replication-deficient recombinant adenoviruses and serotype 5 adenoviruses. Bounty et al. [37] used H2O2 in the UV treatment of serotype 2 adenoviruses. The photocatalytic inactivation of human adenovirus serotype 40 has not been explored. This knowledge is needed, given the essential role of HAdV40 as an etiologic agent of waterborne disease and it is resistance to UV disinfection. 80 An important challenge to photocatalytic disinfection is the influence of natural organic matter, which can quench the ROS produced as disinfectants. Recent work has pointed to the importance of NOM interactions with photocatalytic processes and noted a distinction between surface-based and bulk quenching of radicals [38]. None of the aforementioned studies on adenovirus disinfection investigated the role of NOM in the photocatalytic process [39]. The nature of these interactions is important when considering strategies to mitigate the quenching. Membrane filtration may provide a route to eliminate key fractions of NOM that quench ROS. The goal of this study is twofold. First, we investigate the kinetics of photocatalytic disinfection of HAdV40 in DI water and filtered surface water of different quality. Second we evaluate a photocatalytic membrane reactor as a method for high throughput inactivation and removal of HAdV40. 81 3.2 Experimental 3.2.1 Reagents Aeroxide TiO2 P25 powder was provided by Evonik Industries. Minimum essential medium (MEM), sodium pyruvate solution (100 mM), trypsin 1×, modified eagle™s medium eagle non-essential amino acid solution (NEAA), HEPES buffer (1 M), 1×PBS with W/EDTA (pH=7.4), antibiotic-antimycotic solution, and kanamycin sulfate solution (5,000 µg/mL) were purchased from VWR International. Cell culture Tris-buffered saline solution (10×) was purchased from Fisher Scientific International, Inc. Fetal bovine serum (FBS) was purchased from Atlanta -pure water purification system (Thermo Fisher Scientific). Polyelectrolytes used for the LbL deposition of catalyst included reagent grade polydiallyldimethylammonium chloride (PDADMAC, Aldrich, MW 100,000 -200,000 Da), and polyacrylic acid (Aldrich, MW 1,800 Da). Commercially available titanium dioxide (Evonik P25) was used as a catalyst in all coatings. 3.2.2 A549 cell line and HAdV40 propagation HAdV40 was obtained from the American Type Culture Collection (ATCC) and propagated in A549 cell lines (human carcinoma cells). A549 cell lines were propagated in growth medium (containing 10% FBS, see Appendix, Table S.1, for the complete composition of the growth medium) until the confluence reached 80%; after that growth medium was replaced with maintenance medium (containing 2% FBS) to retain cell activity. Typically, maintenance medium was renewed every three days. 82 The HAdV40 propagation process was conducted as follows: when the monolayer cell line was determined to reach confluence of at least 80%, the old medium was emptied from the 150 cm2 tissue culture flask and the monolayer cell line was rinsed with sterile, Tris-buffered saline. Afterwards, 4 mL of HAdV40 stock were added into each flask, which were then incubated at 37 °C and vigorously shaken every 15 min for 1 to 1.5 h. After the incubation, 46 mL of maintenance medium were added to each flask sequentially and then all flasks were incubated for 3 to 4 days until 90% of the cell monolayer was destroyed. The flasks were frozen and thawed three times, then the entire mixture was transferred to 50 mL centrifuge tubes and centrifuged at 12,000g at 4°C for 10 min [37]. After centrifugation, the supernatant was filtered using 0.22 µm in filtration experiments. 3.2.3 Batch UV photoreactor Figure 3.1 shows the schematic of the photoreactor used in the batch UV disinfection experiments. The photoreactor has two chambers connected through a circular opening 9.3 cm in diameter. In the upper chamber, a preheated germicidal UV lamp (16 W, model GPH330T5L/4, Atlantic Ultraviolet Corp.) was fixed above the center of the opening. The temperature within reactor was maintained constant by circulating air by a fan. A beaker with a solution was placed in the lower chamber and the solution was mixed with a magnetic stirrer to ensure its homogeneous irradiation by the UV light. A shutter positioned between two chambers controlled the irradiance of UV light to the solution. 83 ACopenclosemagnetic stir platebeakerUV lampfancircular opening Figure 3.1 Schematic diagram of the batch UV reactor 3.2.4 Photocatalytic membrane and membrane reactor A tubular ceramic (TiO2) membrane (TAMI Industries) with the nominal pore size of 0.8 µm was coated with TiO2 nanoparticles (100% anatase, Sigma) by the layer-by-layer method as described earlier [40]. The design of the photocatalytic membrane reactor was also described previously [41]. Crossflow rate and permeate mass flow rate were automatically logged into a computer at 1 s intervals. The crossflow rate was maintained constant in the 1.1 to 1.2 L/min range, which translates to the crossflow velocity of ~ 0.17 m/s. Transmembrane pressure was measured by two pressure gauges installed on the feed and retentate sides of the membrane unit. 84 3.2.5 Sample collection and storage Surface water was collected from Lake Lansing at the boat ramp in Lake Lansing Park-South (Haslett, MI) in November, 2015 and stored at 4 oC. All feed water samples were characterized in for UV/Vis absorbance (MultiSpec 1501 spectrophotometer, Shimadzu) and total organic carbon (TOC) content. The TOC in each water sample was measured at least in triplicate (OI Analytical model 1010 analyzer, OI Analytical, College Station, TX). In batch tests, samples were collected at 0, 1, 5, 10, 15, 20, 30, 45, and 60 min into each experiment and stored in 5 mL cryogenic vials eate solutions were collected. Feed solution was withdrawn from the feed tank prior to the start of the filtration test. Permeate samples were first collected into a foil-wrapped Erlenmeyer flask positioned on an electronic mass balance, and then after the filtration, three or four mL sample was withdrawn from the flask for qPCR analysis. The remaining permeate solution was transferred to the pressurized tank (see Appendix, Fig. S.2) for concentration using 50 kDa ultrafiltration membrane discs (PBQK06210, EMD Millipore) and then the concentrated samples were used for cell culture assays. 3.2.6 Lake water pre-treatment In batch tests, both raw lake water and pre-filtered lake water were tested. Lake water was pre-filtered through membranes of one of three different nominal pore sizes: 0.8 µm (tubular TiO2 membrane, TAMI Industries), 0.45 µm (mixed cellulose esters membrane, HAWP09000, Merck Millipore Ltd.) and 0.03 µm (PVP-treated, low non-specific binding polycarbonate track etch membranes, PCT0039030, Sterlitech Corp.) and stored in separate glass flasks at 4 oC. In all 85 crossflow filtration experiments, lake water pre-filtered through 0.45 µm membrane (HAWP09000, Merck Millipore Ltd.) was used to prepare the feed solution. 3.2.7 UV dose quantification A UVX Radiometer (UVP, LLC) was used to measure the incident light intensity (254 nm) at the surface of the reaction 2 for the initial time point, the average fluence throughout the reactor for each water source was estimated using a standard procedure described by Bolton and Linden [43]. The fluence was re-calculated iteratively after each sample aliquot withdrawn to account for the changes in reaction volume. 3.2.8 Photochemical characterization The batch photochemical reactor was used to determine steady state OHŁ concentrations in experiments with different waters. The concentrations of OHŁ was determined indirectly by measuring the degradation of a probe compound, pCBA, with a known rate constant for reaction 9 L mol-1s-1) [44]. The degradation of pCBA was measured as a function of irradiation time using a Perkin Elmers Series 200 HPLC equipped with a Waters 2487 Dual Lambda absorbance detector, at 235 nm, and a C-18 column. 3.2.9 Total virus quantification with qPCR All samples from batch UV and crossflow filtration tests were subjected to qPCR analysis. The DNA extraction process was the same as described earlier [41]. The generic primers and TaqMan probe used for quantification were described previously [45]. The qPCR analysis started with 15 min denaturation at 95 °C then followed by 45 amplification cycles at 95 °C for 10 s, 60 °C for 30 86 s and 72 °C for 12 s and finally cooling at 40 °C for 30 s. To relate the crossing-point values to the numbers of HAdV40 DNA copies, a standard curve developed earlier was used [45]. 3.2.10 Quantification of culturable virus: Cell culture assays and most probable number (MPN) calculation The culturable virus from both batch and membrane reactor samples was quantified by cell culture assays [46]. Each sample was prepared in a ten-fold series dilutions (10-1 to 10-4) and cell culture assays were conducted. First, the monolayer cell line in 25 cm2 tissue culture was checked for a confluence of at least 90%. The old medium was then emptied and the monolayer of cells was rinsed with sterile Tris-buffered saline (1×). Second, 1 mL of diluted sample was inoculated into a flask. Each diluted sample was analyzed in triplicate. Third, flasks were incubated at 37 °C and vigorously shaken every 15 min for approximately 1 to 1.5 h. After the incubation, the 1 mL sample was decanted and 8 mL of maintenance medium was added. Cytopathic effects (CPE), which indicate viral infection in the cell cultures, were monitored for up to 14 days. Maintenance media in the flasks was changed every 7 days. Positive and negative results of the samples were determined according to the U.S. EPA protocol [46]. The mean concentration of HAdV40 in each sample was estimated using MPN calculator [47]. 87 3.3 Results and discussion 3.3.1 OHŁ radical production and quenching: Effect of water quality In order to design an effective PMR, it is critical to understand how permeate quality affects the photocatalytic process. Water quality is an important factor that affects UV fluence and OHŁ lifetimes. Specifically, DOM is a potent OHŁ scavenger and typically absorbs UV light effectively. Further, suspended and dissolved organics can scatter light, which, combined with the absorption, reduces the intensity of radiation available for disinfection in the bulk of the water sample. Thus, knowledge of UV absorbance by organic matter is needed for attenuation calculations. Spectrophotometry can also provide insights into the type and quantity of organics in solution. To explore the effects of permeate water quality on photocatalysis, a photocatalytic batch reactor was used to evaluate the role of OHŁ in the inactivation of HAdV40. First, raw feed samples were filtered through membranes of different pore sizes and UV absorbance as well total organic content (TOC, Appendix Fig. S.3, Fig. 3.2) were measured. The absorptivity values for DI water containing TiO2, MEM, or both were also determined to assess the impact of these two constituents (Fig. 3.2). As expected, using membranes with smaller nominal pore sizes led to improved water quality, where improvement is defined as reduced UV absorbance in the UVC range. A notable difference in water quality was observed between samples prefiltered through membranes with an amino acid mixture, see Appendix, Table S2), was commensurate with that due to organics in the lake water. TiO2 also exerted significant, broad-band absorption of UV light. Figure 3.2 presents total organic carbon (TOC) and UV254 absorbance values in the lake water samples. The small difference in DOC 88 size range does not significantly affect DOC rejection for clean membranes. The difference in DOC rejection for the different filters could become significant with the addition of fouling layers after continued membrane use. Therefore, the effects of DOC concentration on photocatalysis are not expected to be significantly different between the prefiltered lake water samples, since clean membranes were used. DI with MEM0.03 µm prefiltered0.45 µm prefilteredTOC (mg/L)051015202530Absorbance (254 nm, cm-1)0.000.050.100.150.200.250.30TOC Absorbance Figure 3.2 UV absorptivity (254 nm) and TOC of water samples used in photocatalytic tests Note: To parallel the photocatalytic batch experiments (Figures 3.3 ~ 3.7 in sections 3.3.1 and 3.3.2 and Figures S4 and S5 in the Appendix), each water sample contained TiO2 (0.83 mg/L). In addition, DI water sample contained MEM (1% v/v). Specific UV absorbance (SUVA), a common metric for the aromaticity of organic matter, is given by UV254 absorbance normalized by dissolved organic carbon (DOC) contents of the sample. In this study we used TOC as an estimate of DOC. To maintain this assumption, the raw water and 89 -1m-1 were obtained for the pure water with 1% of chemical reactivity of DOC; disinfection byproduct formation, for example, was shown to increase with increasing SUVA values [51]. Given that SUVA is a predictor of aromaticity of DOC and that aromatic compounds are electron rich, higher SUVA values may also be predictive of higher reactivity with OHŁ. Increased reactivity of water constituents with OHŁ would directly correlate to a decrease in efficacy of photocatalytic disinfection; thus waters with high SUVA are likely to inhibit OHŁ driven disinfection more than those with lower SUVA. If this mechanism proves significant in mediating the photocatalytic efficiency, then higher photoactivity should be observed in DI water with MEM (low SUVA sample) than in the lake water samples. The observation of pCBA degradation provides a convenient method for the estimation of a pseudo steady state OHŁ concentration in a photocatalytic reaction. The steady state estimation is possible given the known reaction rate of OHŁ with pCBA. While it was expected that the presence of DOM would impact the photocatalytic production of OHŁ, the magnitude of this effect (see Appendix, Fig. S.4) was surprisingly high. The difference in pCBA degradation was not -photocatalytic) UV and photocatalytic UV. In the lake water, the NOM quenched OHŁ significantly and more rapidly than the quenching reaction with pCBA. The steady state OHŁ concentration estimation for pure water resulted in a value of 1.2×10-13 ± 7.2×10-15 M after subtracting the degradation caused by UV alone. 90 1000×ln([pCBA]/[pCBA]0)/Fluence, cm2/µW0.00.1 0.23.04.0DI Water 0.03 µm prefiltered 0.45 µm prefiltered 0.8 µm prefiltered Raw Water With MEMWith TiO2With TiO2 & MEM Figure 3.3 Degradation of pCBA normalized by UV254 fluence for different water types with and without TiO2 (0.83 mg/L) and MEM (1%) in the solution Note: Error bars correspond to 95% confidence intervals for the linear fit of the dependence of pCBA degradation on UV254 fluence. Given the difficulty with distinguishing the degradation of pCBA by direct UV from that caused by reaction with OHŁ, pCBA degradation rates normalized by fluence (Fig. 3.3) were calculated in lieu of steady state OHŁ concentrations. The data shows that there is no significant difference between any of the prefiltered lake water samples used for HAdV40 experiments. Likewise, MEM is observed to quench OHŁ to the same extent as the NOM present in the lake water. Comparing the pure water case to any other sample, it is clear that the DOM exerts a strong quenching action on OHŁ. If viruses react with OHŁ more readily than pCBA, then enhanced viral inactivation by photocatalytically produced OHŁ can be expected even in the presence of DOM. 91 3.3.2 HAdV40 removal and inactivation in a batch UV reactor Batch experiments were conducted to investigate the removal and inactivation of HAdV40 in four different waters: (1) raw lake water, (2) lake water prefiltered through 0.8 µm membrane, (3) lake water prefiltered through 0.45 µm membrane and (4) lake water prefiltered through 0.03 µm membrane. UV fluence was measured following the procedure described in section 2.6. Total numbers of HAdV40 were quantified using the qPCR method (see section 2.7). Figure 3.4 shows log removal values (LRVs) of total HAdV40 as a function of UV fluence. The combined dataset can be fit well (R = 0.9026) by a linear dependence on the UV fluence. Since the qPCR method quantifies the number of organisms by counting the number of target DNA sequences in the sample, the only direct way to reduce the qPCR count is to damage the DNA. Thus, the increased LRVs can be attributed to the UV-induced DNA damage, including dimerization and the oxidation of DNA by ROS formed in the photocatalytic UV process [19]. All experiments were conducted with identical sample time points. The fluence was highest for the experiments conducted with 0.03 µm, due to the relatively lower absorbance in that water type. In samples with higher water quality (i.e. lower TOC values), more UV-induced DNA damage was observed leading to higher LRVs of total HAdV40. The LRVs for each water type at a given fluence value were not significantly different; UV fluence was the main determinant of DNA damage. These observations suggest that water quality impacted the DNA damage pathway via fluence attenuation only. 92 Figure 3.4 Log removal of total HAdV40 (as measured by qPCR) in photocatalytic UV tests with different waters Note: Error bars represent standard deviations (n = 3). Comparison of total HAdV40 removal with and without catalysts was performed in lake water pre-filtered through 0.03 µm and 0.45 µm membranes (Fig. 3.5). In each situation, no significant differences in the removal of total virus with and without catalysts were observed. In other words, the presence of TiO2 catalyst did not cause apparent increase of total HAdV40 removal. Thus, we conclude that UV-induced damage, such as dimerization, rather than the ROS generated during photochemical process was the main mechanism for the total virus removal in high quality water. 00.10.20.30.40.50.6 0.70100200300400500LRV of total HAdV 40UV254fluence (mJ/cm2)raw (unfiltered) surface waterpre-filtered through 0.8µmpre-filtered through 0.45µmpre-filtered through 0.03µm93 Figure 3.5 Comparison of total HAdV40 removal (as determined by qPCR) by direct UV (--, --) and by photocatalytic UV (--, --) in batch inactivation tests Note: Error bars represent standard deviations (n = 3). The data points representing photocatalytic tests (--, --) are the same as shown in Figure 3.4. Figure 3.6 shows LRVs of culturable HAdV40 in different waters. Because of the limitation of the measurement method, no culturable virus could be detected after 10 min in all experiments. Thus, only four samples (initial, 1 min, 5 min and 10 min into the experiment) were analyzed for viable virus concentration in each test. An increase in LRVs with irradiation time was observed for each water type. The LRVs of culturable HAdV40 were much higher (Fig. 3.6) than the LRVs of total 0.00.1 0.2 0.3 0.4 0.5 0.6 0.70100200300400500600Log removal of total HAdV0Fluence (mJ/cm2)pre-filtered through 0.03µm: UV onlypre-filtered through 0.03µm: photocatalytic UVpre-filtered through 0.45µm: UV onlypre-filtered through 0.45µm: photocatalytic UV94 HAdV40 under the same fluence (Fig. 3.5). This enhanced removal efficacy may be attributed to the fact that in addition to the UV-induced DNA damage, the oxidation by reactive oxygen species may also lead to the loss of viability [19]. The cell culture method is more sensitive than the qPCR technique, since it is capable of observing these losses to viability that are not detected with qPCR. Figure 3.6 Photocatalytic inactivation of culturable HAdV in different waters Note: Error bars represent standard deviations (n = 3). LRVs of culturable HAdV40 in lake water pre-filtered through 0.03 µm membrane at all sampling time were much higher than that in the other water samples (i.e. raw, prefiltered through 0.8 µm, prefiltered through 0.45 µm). The kinetics of HAdV40 inactivation in lake water pre-filtered with 0.03 µm membrane fit the Collins-Selleck model [52, 53] reasonably well with Collins-Selleck y = 3.70E-01ln(x) + 3.54E-01R² = 1.00E+0000.511.522.533.50.0020.0040.0060.0080.00LRV of infective HAdV 40Fluence (mJ/cm2)raw (unfiltered) surface waterpre-filtered through 0.8µmpre-filtered through 0.45µmpre-filtered through 0.03µm95 coefficient = 0.8513 and with the lag coefficient b = 0.6596 mJ/cm2 (see Appendix, Fig. S.5). The decelerating kinetics showed that at higher UV fluence, the inactivation of HAdV40 exhibited a fitailingfl effect, where large increases in fluence resulted in only gradual increases in LRVs. Similar fitailingfl effects were also reported in other studies on photocatalytic inactivation of viruses [54, 55]. Possible reasons for tailing include the presence of resistant subpopulation due to genetic or morphological differences [56], aggregated state of viruses [57] and the competition for adsorption sites between the remnants of inactivated viruses and infective viruses [54]. The virus inactivation due to direct UV and photocatalytic oxidation is presented in Figure 3.7. In both waters, the LRVs with UV only were similar, which indicates that water quality has little effect on the viral DNA damage due to dimerization. In water pre-filtered through a 0.03 µm membrane, photocatalytic oxidation appeared to contribute significantly to HAdV40 inactivation, where the LRV due to direct UV was approximately one log lower than that of the photocatalytic process. However, the inactivation of HAdV40 in water pre-filtered through a 0.45 µm membrane relied more on direct UV, especially at low UV fluence. This observation may be attributed to the presence of NOM in water, which consume the OHŁ produced by the photocatalytic process, thereby reducing the effects of photocatalysis 96 Figure 3.7 Comparison of culturable HAdV40 inactivation with and without catalysts Note: Error bars represent standard deviations (n = 3). 3.3.3 Virus removal and inactivation in photocatalytic membrane reactor Three different processes: (1) MF only, (2) non-photocatalytic hybrid MFŒUV process, and (3) photocatalytic hybrid MFŒUV process, were conducted using the crossflow filtration system. To minimize membrane fouling, lake water used this study was pre-filtered through 0.45 µm membrane. However, a decline in the permeate flux was still observed in all types of filtration processes (see Appendix, Fig. S.2). For one hour filtration, the permeate flux shows a 67±9.7% decline in microfiltration only process, 55±4.9% in the non-photocatalytic hybrid MF-UV process and 31±19.7% in the photocatalytic hybrid MF-UV process. Therefore, with the layer-by-layer TiO2 coating, the coated membrane shows less permeate flux decline, which indicates that the 0.000.501.001.50 2.00 2.503.003.500.0020.0040.0060.0080.00100.00Log removal of culturable HAdV0Fluence (mJ/cm2)pre-filtered through 0.03µm: UV onlypre-filtered through 0.03µm: photocatalytic UVpre-filtered through 0.45µm: UV onlypre-filtered through 0.45µm: photocatalytic UV97 coating layer may relieve membrane fouling to some extent. The fully explanation for this improvement caused by coating is unclear and further investigation is needed. The removal of total HAdV40 was quantified by qPCR for all three processes and expressed in terms of LRV (Fig. 3.8). Permeate samples were collected at 30 min and 60 min into the filtration experiment. Due to the large nominal pore size of the membrane (d = 0.8 diameter of HAdV40 (d = 90 to 100 nm) [58], the MF process was the least effective in removing HAdV40 (LRV = 0.96 ± 0.08). The non-photocatalytic hybrid MFŒUV process gave LRV of 1.37 ± 0.24 and the photocatalytic hybrid MFŒUV process gave the highest LRV of 1.58 ± 0.24. Figure 3.8 Removal of total HAdV40 by (1) microfiltration only (2) non-photocatalytic hybrid MFŒUV process and (3) photocatalytic hybrid MFŒUV process Note: Error bars represent standard deviations (n = 3). 0.00.5 1.01.52.0 2.53060LRV of total HAdV40 Time (min)MF onlyNon-photocatalytic processPhotocatalytic process98 Figure 3.9 shows the inactivation of culturable HAdV40 by each of the three processes. The MF process and the non-photocatalytic hybrid MFŒUV process have almost same LRVs (2.21 ± 0.02 and 2.09 ± 0.22). By contrast, the removal efficacy of the photocatalytic hybrid MFŒUV process is significantly larger: 3.03 ± 0.35. The difference could be a consequence of the combined effect of membrane adsorption, size exclusion (for the possible existence of very small pore size), and the UV-induced inactivation of viruses in the permeate. Figure 3.9 Inactivation and/or removal of culturable HAdV40 by microfiltration only, in a sequential MFŒUV process, and in a photocatalytic MF membrane reactor Note: Error bars represent standard deviations (n = 3). Figure 3.10 provides the ratio of culturable to total HAdV40 in the feed and in the effluent of each of the three treatment processes. Approximately 13.5% of the total virus load in the feed was culturable, and after membrane filtration the ratio decreased to 1.5%. This significant decrease 0.00.5 1.0 1.52.02.5 3.0 3.54.0MF onlyNon-photocatalyticprocessPhotocatalyticprocessLRV of cultuable HAdV40MF onlyNon-photocatalytic processPhotocatalytic process99 may due to adsorption, size exclusion and possibly damage to the virus capsid during permeation, leading to infectivity loss. The addition of UV source did not lead to a major change in the viable-to-total ratio. However, after the photocatalytic process, the ratio of culturable to total virus dropped dramatically to 0.1%. This large drop is believed to be a result of the synergistic effects of combining membrane filtration with photocatalytic UV disinfection. The ROS generated at the surface of TiO2 coating layer can oxidize the protein capsid of viruses, which complements the direct DNA damage due to the germicidal property of the UV lamp. Figure 3.10 Concentration ratio of culturable and total HAdV40 in feed and permeates Note: Error bars represent standard deviations (n = 3). 13.5%1.5%1.2%0.1%-5%0%5%10%15% 20%25%FeedMF onlyNon-photocatalyticprocessphotocatalyticprocessMPN/106copies100 In summary, the photocatalytic hybrid MFŒUV process developed in our group was successfully applied for turbid water treatment and has proven to be highly efficient in virus removal and inactivation. The combined process overcomes the limitations of UV disinfection due to turbidity and the existence of UV-resistant viruses. Moreover, it also relives the permeate flux decline. 101 3.4 Conclusions In this study, we extended the application of the photocatalytic hybrid MFŒUV process for the removal and inactivation of widespread naturally occurring virus from natural water resource. The batch experiments conducted with HAdV40 and natural surface water suggested that the inactivation kinetics may varied in different water quality, especially under lower light source. The total virus removal linearly depend on the UV fluence, and the removal mechanism mainly due to the UV-induced DNA dimerization. The removal of culturable virus in higher water quality (e.g. lake water pre-filtered through 0.03 µm membrane) had a fitailingfl effect and fit Collins-Selleck model. However, the removals in lower water quality, such as raw lake water, lake water pre-filtered through 0.45 µm and through 0.8 µm membrane, were fit a two-stage linear relationship. To investigate the performance of the photocatalytic hybrid MF-UV process, a number of experiments with filtration alone, non-photocatalytic membrane and photocatalytic membrane were performed respectively. Although challenged with the most UV resistant virus Œ HAdV40 and complex surface water, the photocatalytic hybrid process showed its superiority as a promising treatment process for water disinfection. It achieved a 96.8% removal of total virus (LRV of 1.58 ± 0.24), while more significantly, a 99.9% inactivation of infectious virus (LRV of 3.03 ± 0.35). Nevertheless, reasons for the improved permeate flux observed by adding UV and TiO2 coating are still unclear, and further studies are very necessary. 102 APPENDIX 103 APPENDIX: Supporting Information Figure S.1 UV fluence required for 99.9% reduction of representative human enteric viruses Notes: BDF-buffered demand-free; SDW-sterile distilled water; SEW-steriie estuarine water; PBS-phosphate buffered saline; PBW- phosphate buffered water. 104 Table S.1 Composition of tissue culture medium Composition Volume (mL) Growth medium Maintenance medium FBS 50 10 antibiotic-antimycotic solution 6 6 kanamycin sulfate solution 6 6 sodium pyruvate solution 6 6 NEAA 6 6 HEPES buffer 10 10 MEM 500 500 Table S.2 Media formulations of Basal Medium Eagle Note: Media purchased from Thermo Fisher Scientific Inc. (Catalog number: 21010-046) Amino Acids Molecular weight, Da Concentration, mg/L L-Arginine hydrochloride 211.0 21.0 L-Cystine 2HCl 313.0 16.0 L-Histidine 155.0 8.0 L-lsoleucine 131.0 26.0 L-Leucine 131.0 26.0 L-Lysine hydrochloride 183.0 36.47 L-Methionine 149.0 7.5 L-Phenylalanine 165.0 16.5 L-Threonine 119.0 24.0 L-Tryptophan 204.0 4.0 L-Tyrosine disodium salt dihydrate 261.0 26.0 L-Valine 117.0 23.5 105 Figure S.2 shows the concentration unit for permeate concentration after crossflow filtration. The stirred dead-end filtration cell (Model 8050, EMD Millipore) was connected to a feed tank (serial NO. 22111-041, Alloy Products Corp.) which was pressurized by compressed nitrogen at 30 psi. Biomax® 50 KDa ultrafiltration membrane discs (PBQK06210, EMD Millipore) was used to concentrate the permeate sample collected in the previous crossflow filtration. This entire process was stopped when the volume of residual solution in filtration cell was about 10 mL. Mass weights of initial permeate from crossflow filtration and final residual solution collected after concentration were measured by mass balance, which were used to calculate concentrate ratio for culturable virus quantification. filtration cellgas cylinderfeed tankoutletinletmagnetic stir plateElectronic BalancePermeate Collected Tank Figure S.2 Schematic diagram of concentrate system 106 Wavelength, nm200250300350400Absorbance, cm-10.00.2 0.40.60.81.0Raw Water 0.8 µm prefiltered 0.45 µm prefiltered0.03 µm prefilteredDI Water with MEM & TiO2 DI Water with MEM DI Water with TiO2 Figure S.3 UV absorbances for several experimental conditions Note: Lake water solutions and the DI cases with MEM contained 1% MEM to parallel HAdV40 experiments. 107 Time, minutes020406080100120140160180ln([pCBA]/[pCBA]o)-6-5-4-3-10DI Water, no TiO2 DI Water, with TiO2 Raw Water, no TiO2 Raw Water with TiO2 0.03 µm prefiltered, no TiO2 0.03 µm prefiltered, with TiO2 DI Water, with TiO2, Dark Figure S.4 pCBA degradation over time for various water sources Note: experiments. 108 Figure S.5 Inactivation of culturable HAdV40 in lake water pre-filtered through 0.03 µm membrane Note: Error bars represent standard deviations (n = 3). y = -0.8513x -0.3543R² = 0.9999-2.5-2-1.5-1-0.5000.511.52log (N/N0) for infective HAdV 40log (Fluence, mJ/cm2)surface water pre-filtered through 0.03µm109 A B C Figure S.6 Normalized permeability of microfiltration membranes to deionized water Note: A) uncoated membrane; B) the same membrane with a photocatalytic coating in the absence of UV light; C) the same membrane with a photocatalytic coating exposed to UV light. 110 REFERENCES 111 REFERENCES [1] J.H. 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The hybrid system retains the advantages of photocatalytic UV disinfection and membrane filtration while mitigating drawbacks of each of these two processes. The membrane, operated in the inside-out geometry, removes the turbidity of the water thereby enhancing the efficacy of UV disinfection. The membrane also serves as a support for immobilized catalysts to avoid the recovery and secondary separation of catalysts. The presence of catalysts complements direct UV to enhance pathogen removal. The additional photocatalytic UV disinfection applied on the permeate side of the membrane enables trade-offs in pore sizes and product water fluxes. A number of experiments were conducted to determine the optimized operational parameters, understand mechanisms of virus inactivation in complex water matrices and evaluate the performance of the hybrid photocatalytic UV-membrane filtration system. To make this investigation feasible, we started from a simple model system of bacteriophage P22 suspended in DI water (chapter 2). The hybrid process was shown to be considerably more effective in inactivating bacteriophage P22 than the constituent processes applied in series. P22 inactivation 117 by direct UV in a batch rector followed Collins-Selleck model. Similar results were observed in the second part of the study (chapter 3) that involved human adenovirus suspended in lake water pre-filtered through membranes of different porosities. The virus inactivation by the hybrid photocatalytic UV-membrane filtration process was ~1.5 times higher than that with the non-photocatalytic UV-membrane filtration process. UV disinfection experiments in a batch reactor with adenovirus suspended in pre-filtered lake water showed that the water quality has a major impact on the efficacy of virus inactivation but does not affect qPCR count. 4.2 Future research work The efficiency of photocatalysis depends upon many factors: photocatalyst loading, initial concentration of the substance, characteristics of UV lamp, and composition of the solution. Besides, the operational parameters, such as crossflow rate, distance between the photocatalytic surface of the membrane and light source as well as membrane™s pore size, also affect the performance of the hybrid system. Table 4.1 summarizes what is known and unknown knowledge of this novel hybrid photocatalytic UV-membrane filtration system. From this table, we can see that there are still many areas that lack of the comprehensive understanding. Therefore, plenty of experiments with more in-depth researches are necessary to be conducted to find out the optimal combination for this hybrid system. 118 Table 4.1 Knowledge gaps of the novel hybrid photocatalytic UV-membrane filtration system Parameters Knowns Unknowns photocatalysts loading a) The photocatalytic efficiency is proportional to the mass of catalysts on the membrane within a reasonable range. Adding catalyst in excess of the optimum does not lead to increased reactivity. b) Excessive coverage by the photocatalyst results in a dense coating layer causing significant decrease in membrane permeability. The optimal amount of catalysts. initial concentration of the substance - Whether and how the change of the initial concentration affects the treatment efficiency. characteristics of UV lamp - a) How do the wavelength and the intensity of the UV irradiation influence the treatment efficiency? b) What is the optimal intensity? components of the solution Water quality affects the treatment efficiency, especially the results of virus inactivation. a) How does the pH of the solution affect the treatment efficiency? b) What is the treatment efficiency when more than one type of microbial species exists in the solution? crossflow rate - How does the crossflow rate affect the treatment efficiency? distance between membrane and light source - The optimal distance. membrane pore size - How does the membrane pore size influence the treatment efficiency?