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Characterization of Aerobic Methane Oxidizing Bacteria in Oil Sands Tailings Ponds University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2014-01-29 Characterization of Aerobic Methane Oxidizing Bacteria in Oil Sands Tailings Ponds Saidi-Mehrabad, Alireza Saidi-Mehrabad, A. (2014). Characterization of Aerobic Methane Oxidizing Bacteria in Oil Sands Tailings Ponds (Unpublished master's thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/25266 http://hdl.handle.net/11023/1313 master thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca UNIVERSITY OF CALGARY Characterization of Aerobic Methane Oxidizing Bacteria in Oil Sands Tailings Ponds by Alireza Saidi-Mehrabad A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF BIOLOGICAL SCIENCES CALGARY, ALBERTA January, 2014 Alireza Saidi-Mehrabad 2014 Abstract The current study for the first time investigated the potential methane oxidation capacity of the surface layers of oil sands tailing ponds in Alberta, which are anthropogenic methane sources. This research by the aid of recently developed molecular analysis tools such as 16S rRNA gene pyrotag sequencing, Stable Isotope Probing, 16S rRNA and pmoA gene sequence analysis highlighted the presence of methanotrophic bacteria (mainly Gammaproteobacteria), in these - tailings ponds, with potential methane oxidation activity ranging from 75.6 – 135.6 nmol CH4 ml 1 d-1. 16S rRNA pyrotag sequencing and construction of phylogenetic trees detected OTUs affiliated to the genus Methylocaldum present in all sampling sites and times (July/2011- December/2011). Later investigations resulted in the isolation of a novel strain dubbed “Methylobacter oleiharenae”, which is able to thrive at optimum pH of 8 and contains 729 specific genes not detected in closely related reference methanotroph strains. ii Preface Parts of this work has been published with the following title “Saidi-Mehrabad A, He Z, Tamas I, Sharp CE, Brady AL, Rochman FF, Bodrossy L, Abell G CJ, Penner T, Dong X, Sensen CW, Dunfield PF. (2012). Methanotrophic bacteria in oilsands tailings ponds of northern Alberta. International Society of Microbial Ecology Journal. 7:908-92.” The contribution of the thesis author in the mentioned publication is as follows; Alireza Saidi-Mehrabad: Processing the tailing samples (from the samples provided between July/2011- December/2011) Microbial community analysis of the tailing ponds surface layers (from the samples provided between July/2011-December/2011) via DNA extraction, analysis of the data produced from the16S rRNA gene based pyrotag sequencing, constructing 16S rRNA gene and pmoA gene phylogenetic trees, DNA-based stable isotope probing, analysis of the data resulted from the pyrotag sequencing of the heavy SIP fractions (16S rRNA gene based) Measuring the potential methane (CH4) oxidation rates via gas chromatography (GC/FID) (from the samples provided between July/2011-December/2011) Constructed Table 1 (Summary of methane oxidation rates and methanotrophic communities detected in tailings ponds and other sites at multiple sampling dates), Figure 1 (Phylogenetic tree based on 16S rRNA gene sequences of methanotrophic iii Proteobacteria), Figure 2 (Phylogenetic tree based on partial-derived PmoA sequences of Proteobacteria), Supplementary Figure 1 (Methane oxidation rate of pond B sampled in August, 2011), Supplementary Table 2A-D, 2B, 2C, 2D (The top 25 OTUs of each sample site based on % of total reads in 16S rRNA gene pyrotag sequencing analysis). And has been presented in the following presentations and posters: Presented at departmental microbiology seminars. Topic of the presentations: “Methanotrophic Bacteria Residing in Oil Sands Tailings Waste. (Canada. 2011-2012) Presented in a poster titled “Methane Oxidizing Bacteria in Surface Layers of Oil Sands Tailings Ponds” at Canadian Society of Microbiologists Conference (Canada, June 2012) Presented in a poster titled “Methanotrophic Bacteria in Oil Sands Tailings Ponds of Northern Alberta” at 13th International Society of Microbial Ecology Conference (Denmark, August 2012). Presented at Hydrocarbon Metagenomics 2012 Conference, Topic of the presentation: “Methane Oxidizing Activity in Oil Sands Tailings Ponds”. (Canada, October 2012) Presented at Genome Canada/Genome Alberta 2013 Conference. Topic of the presentation: “Methylobacter ferrophilum, sp . nov. an aerobic bacteria isolated from an oil sands tailings pond”. (Canada 2013) The final section of this study and the extracted data are planned to be published with the following titles “Saidi-Mehrabad A, Tamas I, Kim JJ, Rijpstra IC, Damsté JS, Dunfield PF. (2013). Methylobacter oleiharenae, sp. nov., an aerobic methanotroph isolated from an oilsands tailings pond.” iv The mentioned thesis author has also contributed in the following publication; “An D, Caffrey SM, Soh J, Agrawal A, Brown D, Budwill K, Dong X, Dunfield PF, Foght J, Gieg LM, Hallam SJ, Hanson NW, He Z, Jack TR, Klassen J, Konwar KM, Kuatsjah E, Li C, Larter S, Leopatra V, Nesbø CL, Oldenburg T, Pagé AP, Ramos EP, Rochman FF, Saidi- Mehrabad A, Sensen CW, Sipahimalani P, Song YC, Wilson S, Wolbring G, Wong ML, Voordouw G. (2013). Metagenomics of Hydrocarbon Resource Environments Indicates Aerobic Taxa and Genes to be Unexpectedly Common. Environmental Science & Technology. 47:10708-10717. v Acknowledgements Foremost I would like to thank GOD almighty who led me to my advisor Dr. Peter Dunfield and his wonderful lab members. I would like to express my sincere gratitude from the bottom of my heart to him for providing me an opportunity, which comes once in life to accomplish my master study and research under his guidance by sharing his immense knowledge. I could not have imagined having a better mentor and lab mates for my master study. I would like to include my thesis committee members: Professor. Michael Hynes and Professor. Gerrit Voordouw in this acknowledgment and thank them for their leadership and insightful comments. My sincere thanks also goes to the fund providers: Institute for Sustainable Energy, Environment and Economy (ISEEE), Genome Alberta, Genome Canada, Hydrocarbon Metagenomics group and Syncrude Ltd, particularly Tara Prenner. Beside the funding groups I would like to thank all these fabulous people which made this research possible such as metagenome analysis group from University of Calgary Visual Genomics Center Xiaoli Dong and Christoph Sensen, people at Core DNA sequencing facility, Wei-Xiang Dong (TEM Expert) from microscopy and imaging facility, Wah Lai (Rebecca) Lee from preparation room and all the special people from the office of the graduate studies specially Karen Barron, Christine Goodwin, David Bininda and Sophia George. I thank my fellow lab mates in Dunfield lab, especially Dr. Joong-jae Kim who never said no to my requests and always was present whenever I needed him, Dr. Allyson Brady for her vi encouragement, Fauziah Rochman for being a good friend, Xianqing (Emily) Wang for her good and cheerful nature, Dr. Ivica Tamas, Gareth Jones, Christine Sharp, Roshan Khadka, Ilona Ruhl, and Evan Haupt. Last but not least, I would like to thank my wonderful family for their unquestionable support and love throughout my life. I would like to send my gratitude to all my friends in and out of university of Calgary. I should finish this section by thanking GOD almighty again for his spiritual support and guidance throughout the darkest and happiest day of my life that without his grace everything was impossible. vii Dedication “ Read! In the Name of your Lord, Who has created (all that exists), He has created man from a clot (a piece of thick coagulated blood) Read! And your Lord is the Most Generous, Who has taught (the writing) by the pen. He taught man that which he knew not.” (Quran 96: 1-4) I dedicate this work to my father Dr.Mohammad Saidi-Mehrabad, my mother Nasrin Amel, my bother Amir Reza Saidi-Mehrabad and Dr. Mohammad Sadek Eid (GOD bless his soul) whom have changed my life once and for all. “At about twenty-four miles from the Fork, are some bitumenous fountains; into which a pole of twenty feet long may be inserted without the least resistance. The bitumen is in a fluid state, and when mixed with gum or the resinous substance collected from the Spruce Fir, serves to gum the canoes. In its heated state it emits a smell like that of Sea Coal. The banks of the river, which are there very elevated, discover viens of the same bitumenous quality” Sir Alexander Mackenzie, 1788 Alexander Mackenzie, Voyages from Montreal Through the Continent of North America to the Frozen and Pacific Oceans in 1789 and 1793 Vol. I (1902 ed.) and Vol. II (1903 ed.) viii Table of Content Abstract……………………………………………………………………………………..ii Preface……………………………………………………………………………………...iii Acknowledgments………………………………………………………………………….vi Dedication………………………………………………………………………………….viii List of Tables………………………………………………………………………………xiv List of Figures and Illustrations……………………………………………………………xv List of Symbols, Abbreviations, Nomenclature…………………………………………...xvii CHAPTER ONE: GENERAL INTRODUCTION……………………………..1 1.1
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