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The Response of Planktonic and Aggregated Bacterial Cells in Aqueous Media to Photodisinfection Techniques

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The Response of Planktonic and Aggregated Bacterial Cells in Aqueous Media to Photodisinfection Techniques The response of planktonic and aggregated bacterial cells in aqueous media to photodisinfection techniques A thesis submitted to Dublin City University in fulfilment of the requirements for the award of the degree of Doctor of Philosophy By Ana Carolina Maganha de Almeida, B.Sc., Lic., M.Sc. School Of Biotechnology, Dublin City University Dublin 9, Ireland Research Supervisor: Dr. Bríd Quilty July 2013 I hereby certify that this material, which I now submit for assessment on the programme of study leading to the award of PhD is entirely my own work, that I have exercised reasonable care to ensure that the work is original, and does not to the best of my knowledge breach any law of copyright, and has not been taken from the work of others save and to the extent that such work has been cited and acknowledged within the text of my work. Signed: __________________ (Candidate) ID No.: _______________ Date: ____________________ It was fair, it was honourable, and it was worthy. Dedicated to the lovely memory of Elvira Lunardelli Maganha and Lídia Tumollo de Almeida ACKNOWLEDGMENTS I would like to thank the Environmental Protection Agency (EPA) of Ireland for providing the scholarship and the financial support to carry out this research. I would like to offer my gratitude to my supervisor Dr. Bríd Quilty, School of Biotechnology, Dublin City University. Her patience, encouragement and valuable guidance were crucial to the accomplishment of this research. I would like to acknowledge the support of all the members of the School of Biotechnology. Particular thanks to Dr. Ciarán Fagán, Head of School, Dr. Michael Parkinson for guidance with statistical analysis, Dr. Enrico Marsili for reading my work and adding valuable discussion about the biofilms and photocatalysis sections and the technical staff for their constant presence. I am indebted to the collaborators on this project, Prof. Michael Oelgemöller and Dr. Kevin McGuigan. In particular Prof. Oelgemöller, James Cook University, Australia and formerly School of Chemical Sciences, Dublin City University, for his time and attention during the selective process which resulted in my enrollement as a PhD student in Dublin City University and for his guidance in photochemistry. I am very grateful for the help of Dr. Mary Pryce, School of Chemical Sciences, Dublin City University, for manufacturing the LED reactor. Also to Dr. Kieran Nolan and Dr. Alexander Yavorsky, School of Chemical Science, Dublin City University, for the discussion regarding fluence-rate measurements. I kindly thank Dr. Sharon Murphy and Dr. Oksanna Shvydkiv for their time and help with the UV reactors. Thanks to Kieran Joyce for the discussions about photosensitisers, and to Ciprian and Brendan for helping me with the light measurements. I would like to offer my sincere gratitude to my friends Prof. Dr. Sandra Mara Martins Franchetti, for the discussions about Chemistry and Photochemistry and to the kind support of my previous supervisor of my MSc. thesis, Dr. Carlos Renato Corso. I would like to express my love and gratitude to my family for all their faith on my work and choices. To my parents, Regina and Jorge, I express my gratitude for all their encouragement and the love to handle we had to be apart from each other. They are the best parents, good-tempered, full of peace and so kind to me and to the others. I wish I could become as kind and wonderful as they are. They put all the efforts on my studies, they believed on me all the time, even during the most challenging moments. They never let me give up, never accepted my excuses to not follow on my dreams, they challenged me so much to be better, to go further. They never let me forget how much I strived for my studies in Brazil to then get to Ireland. I express my love and gratitude to Kamal, who made my path full of love, patience and courage. Special thanks to Dr. Moïra Schuler for her support, friendship, discussions and kind and positive encouragement. Thanks to my friends Bianca, Cecilia, Claudia, Monika, Ross, Sahar and Tanja. We had such a good time together! Thanks to Layla, Renata and Gustavo, Camila and Karine for never stopping believing in me. Special thanks to my lab mates Aida and Markella, for their kindness and friendship! My sincere gratitude goes to the reviewers Camila, Cecilia, Ernane, Grace, Karine, Kamal, Lauren and Tanja. I could go on with saying thanks, as I did have so much help indeed, but I cannot fit here the names of everyone. But I am deeply grateful for all of you who made this work possible. You will be always in my heart, and prayers. I thank God who gave me strength and helped me to carry on through my PhD. ABSTRACT..................................................................................................... I ABBREVIATIONS......................................................................................... II LIST OF TABLES........................................................................................ III LIST OF FIGURES .......................................................................................VI 1 INTRODUCTION.................................................................................... 2 1.1 UV disinfection and Photocatalysis ...................................................... 2 1.1.1 Reactors ............................................................................................. 7 1.1.2 Light sources and measurement......................................................... 11 1.2 Photodynamic inactivation (PDI) ....................................................... 15 1.2.1 Reactors, light sources and measurement ........................................... 22 1.3 Microbes in the environment.............................................................. 24 1.3.1 Production of safe water ................................................................... 31 1.4 Effects of photodisinfection on microbial cells ................................... 33 1.4.1 Mechanism of photocatalytic disinfection.......................................... 35 1.4.2 Dark repair in UV and photocatalytic disinfection.............................. 38 1.4.3 Mechanism of photodynamic inactivation.......................................... 40 1.5 Aim and objectives of the project ....................................................... 43 1.6 Outline of experimental design........................................................... 44 2 MATERIALS AND METHODS............................................................ 46 2.1 Materials ............................................................................................ 46 2.1.1 Bacterial cultures .............................................................................. 46 2.1.2 Maintenance of cultures .................................................................... 46 2.1.3 Source of chemicals.......................................................................... 46 2.1.3.1 Titanium Dioxide ...................................................................... 46 2.1.3.2 Rose Bengal.............................................................................. 47 2.1.4 Media............................................................................................... 47 2.1.4.1 Pseudomonas Minimal Medium................................................. 47 2.1.4.2 Aqueous media used in photodisinfection studies ....................... 48 2.1.4.3 Sodium phosphate buffer........................................................... 49 2.2 Methods .............................................................................................. 49 2.2.1 Determination of bacterial numbers................................................... 49 2.2.1.1 Pour-plate method ..................................................................... 49 2.2.1.2 Miles-Misra (drop-plate) method ............................................... 49 2.2.1.3 Spread-plate method ................................................................. 50 2.2.1.4 Measurement of turbidity .......................................................... 50 2.2.1.5 Evaluation of dark repair ........................................................... 50 2.2.1.6 Determination of number of cells in aggregates of P. putida CP1 51 2.2.2 Determination of viability ................................................................. 51 2.2.3 Photochemical reactors ..................................................................... 53 2.2.3.1 UV-C disinfection ..................................................................... 53 2.2.3.2 UV-A/B disinfection and photocatalysis .................................... 56 2.2.3.3 Photodynamic inactivation ........................................................ 59 2.2.4 Inoculum preparation........................................................................ 60 2.2.4.1 Free-swimming cells (E. coli and P. putida CP1) ....................... 60 2.2.4.2 Aggregated cells (P. putida CP1)............................................... 61 2.2.5 Reactor set up................................................................................... 61 2.2.5.1 UV-C disinfection ..................................................................... 61 2.2.5.2 UV-A/B disinfection and photocatalysis .................................... 62 2.2.5.3 Photodynamic inactivation (PDI) ............................................... 63 2.2.6 Determination of aggregates size....................................................... 64 2.2.7 Dry-weight of aggregates .................................................................
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