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Characterization of the Microbial Communities Responsible for Ammonia Oxidation in Activated Sludge Systems of Municipal Wastewater Treatment Plants

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Characterization of the Microbial Communities Responsible for Ammonia Oxidation in Activated Sludge Systems of Municipal Wastewater Treatment Plants CHARACTERIZATION OF THE MICROBIAL COMMUNITIES RESPONSIBLE FOR AMMONIA OXIDATION IN ACTIVATED SLUDGE SYSTEMS OF MUNICIPAL WASTEWATER TREATMENT PLANTS by GM Itheshamul Islam Ryerson University, 2012 Master of Science, Molecular Science Ryerson University, 2010 Bachelor of Science, Biology A dissertation presented to Ryerson University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the program of Molecular Science Toronto, Ontario, Canada, 2018 ©GM Itheshamul Islam, 2018 AUTHOR’S DECLARATION FOR ELECTRONIC SUBMISSION OF A DISSERTATION I hereby declare that I, GM Itheshamul Islam am the sole author of this dissertation. This is a true copy of the dissertation, including any final revisions as accepted by my examiners. I hereby authorize the University of Ryerson to lend this dissertation to other institutions, organizations and individuals for the purpose of scholarly research. I further authorize Ryerson University to reproduce this dissertation by photocopying or by other means, in total or in part, at the request of other institutions or individuals for the purpose of scholarly research. I understand that my dissertation may be made electronically available to the public. ii CHARACTERIZATION OF THE MICROBIAL COMMUNITIES RESPONSIBLE FOR AMMONIA OXIDATION IN ACTIVATED SLUDGE SYSTEMS OF MUNICIPAL WASTEWATER TREATMENT PLANTS GM Itheshamul Islam Doctor of Philosophy, Molecular Science, Ryerson University, 2018 ABSTRACT Nitrification is an essential microbial process in the global nitrogen cycle. The first step of nitrification is ammonia oxidation which is achieved by bacteria and archaea and is crucial in decreasing ammonia concentrations that are persistently high in wastewater. This study examined the composition, abundance and identity of the microbial community in activated sludge with a focus on characterizing ammonia oxidizing bacteria and archaea in a full-scale municipal wastewater treatment plant (MWTP). Specifically, two pharmaceutical compounds Tetracycline and Ibuprofen, and their effects on the community composition of bacteria and protozoa in activated sludge was investigated using PCR coupled with denaturing gradient gel electrophoresis (DGGE). In addition, the composition, abundance and activity of the ammonia oxidizing bacteria (AOB) and ammonia oxidizing archaea (AOA) were analyzed from aerobic activated sludge, recycled sludge and anaerobic digesters of the Humber MWTP using molecular techniques such as PCR, Quantitative PCR, Reverse Transcription- PCR and DGGE. The findings demonstrated that Tetracycline did not appear to alter community composition of bacteria in the activated sludge, rather, the operational parameters of the sequencing batch reactors such as feeding rates and SRT have shown to alter the richness of bacterial communities. However, Ibuprofen affected some members in the protozoan community in activated sludge. In the full-scale Humber MWTP using the conventional activated sludge system, the aeration tanks contained 1.8 × 105 copies of the AOB amoA gene per 100 ng of DNA. In contrast, the anaerobic digester tanks contained 7.3 × 102 copies of the AOA amoA gene per 100ng of DNA. This study also found that AOB were dominant in activated sludge samples, regardless of the operational parameters. The quantification of cDNA transcripts of the amoA gene also indicated that AOB may be more active than AOA in the activated sludge system. Overall, it appears that AOA are very niche specific and thrive in very low oxygenated environments, while AOB proliferate and play a major role in aerobic ammonia oxidation occurring in MWTPs. iii ACKNOWLEDGEMENTS Just as the farmer toils day and night for the seeds he plants to blossom and bear fruit, my parents have toiled day and night for me to achieve success in whatever I have desired. Thank you Abbu (Dad), for being the rock that has kept me from withering away by providing shelter, support, protection and discipline. Your support as a father will always live on for me and for the next generations to come. To my mother, Ammu (Mom), the greatest gift of my life has been you. You taught me never to say NO when anybody asks something of you, it may seem like a weakness to many, but it is my greatest strength as it leads me to be always helpful, kind and generous. I count the blessings from high above to be more than enough for me because I get to see you smile everyday. To my wife, you are my present and future. The way you have shown me understanding and patience and love, nobody in the world can match. I can only promise you that in my heart, I will always appreciate the sacrifices you make for us. To my family, thank you for being the source of peace for me through good times and bad. My son Zaid and my nephew Rayan (lalu), I love you. To my supervisor, Dr. Kimberley Gilbride, I do not think I will ever meet a better role model, who taught me how to tackle challenges with patience, perseverance and confidence. You always found a way to raise my spirit and motivated me to continue striving, and I will be forever indebted to your support and guidance. To my friend Adil Syed, no matter where the road leads us, I know one of us will wait at the destination and be eager to meet and laugh about our childhood and the things we have seen and learned as we grew older. I would like to thank my lab mates, Simon, Farhan and everybody that I had the pleasure to meet and interact with throughout my whole project. My friends Amir, Krishna, Micheal, Christian, Tracy and Farhana. Thanks for being there. A special thanks to Dr. Hausner, Dr. Wolfaardt, Dr. Botelho, Dr. McCarthy and all of the faculty members and staff here at Ryerson who made my education here a pleasant experience. I also would like to thank my committee members, Dr. Andrew Laursen, Dr. Robert Delatolla, Dr. Mehrab Mehrvar and Dr. Marthinus Kroukamp. iv QUOTE “Verily After Hardship, Comes Ease” - Surah Al Inshirah v TABLE OF CONTENTS AUTHOR’S DECLARATION FOR ELECTRONIC SUBMISSION OF A DISSERTATION .......... ii ABSTRACT ................................................................................................................................................ iii ACKNOWLEDGEMENTS ...................................................................................................................... iv QUOTE ........................................................................................................................................................ v LIST OF TABLES ...................................................................................................................................... x LIST OF FIGURES ................................................................................................................................... xi LIST OF ABBREVIATIONS .................................................................................................................. xii CHAPTER 1: INTRODUCTION .............................................................................................................. 1 1.1. Introduction ...................................................................................................................................... 2 1.2. Purpose.............................................................................................................................................. 8 1.3. Hypothesis and Objectives .............................................................................................................. 9 CHAPTER 2: BACKGROUND .............................................................................................................. 10 2.1. The Importance of Wastewater Treatment ................................................................................. 11 2.2. The Wastewater Treatment Process ............................................................................................ 15 2.3. Wastewater in Toronto .................................................................................................................. 18 2.3.1. Humber Wastewater Treatment Plant………………………………….………………19 2.4. Different Types of Biological Wastewater Treatment ................................................................ 20 2.4.1. Activated Sludge System .................................................................................................... 21 2.4.2. Anaerobic Digestion ........................................................................................................... 23 2.5. Activated Sludge Microbial Community ..................................................................................... 24 2.6. Methods to Study the Composition of the Microbial Community............................................. 26 2.6.1. Polymerase Chain Reaction (PCR) .................................................................................. 27 2.6.2. Denaturing Gradient Gel Electrophoresis ....................................................................... 28 2.7. Structure and Composition of Microbial Communities in Activated Sludge ........................... 29 2.8. Factors Affecting the Composition and Function of Activated Sludge ..................................... 33 2.9. Types of Contaminants in Activated Sludge ................................................................................ 35 2.10. Antibiotics ..................................................................................................................................... 36 2.11. Non-Steroidal Anti-Inflammatory
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