Effect of climate change on permafrost microbiome at the Pleistocene-Holocene and Holocene- Anthropocene transitions by Alireza Saidi-Mehrabad A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology and Biotechnology Department of Biological Sciences University of Alberta © Alireza Saidi-Mehrabad, 2021 Abstract Understanding past and future responses of Arctic soil microbes to climate change is critical to an understanding of ecosystem function and climate change feedbacks; the goal of my thesis was to obtain a clearer understanding of these feedbacks. The remnants of the Pleistocene and early Holocene biota, including microorganisms, are preserved in the permafrost of Eastern Beringia, which could potentially show how soil microbes were shaped in Eastern Beringia by the ancient climate change that occurred at the end of the last ice age. However, contamination, low biomass, and physical complexity of permafrost samples create difficulties for molecular analyses. My results show that none of the widely used permafrost decontamination methods fully removed intentionally added biological tracer; hence, I introduced a novel method based on bleaching and scraping. Further, I showed that while permafrost chemical parameters and age did not affect decontamination, they influenced DNA extraction efficiency. I tested multiple widely used DNA extraction kits and modified one to acquire sufficient DNA from difficult permafrost samples. Using this optimized sampling and extraction protocol, I examined whether relict permafrost microbial communities provide a window into past soil microbial communities or not. Microbial community composition and soil chemistry at the Pleistocene-Holocene transition indicated that these parameters were stable until the climate system crossed a threshold, after which there was an abrupt shift to a new steady state. These findings may provide insights into possible future shifts in modern soils. I also examined the response of Arctic methanotrophs (microbes that use CH4 as their sole source of energy) to increases in temperature and active layer disturbance, which both parameters are going to be wide spread in Anthropocene. Soil disturbance had a more significant effect on methanotroph community structure than temperature. There was a strong linear relationship between CH4 oxidation rate and temperature; ii furthermore, CH4 oxidation rates significantly increased in disturbed soils. The active methanotrophs shifted in response to temperature in undisturbed samples, but not in disturbed samples. The results of my thesis complement the previous studies regarding the carbon cycle in the fragile Arctic, and could lead to generation of effective strategies for better management of novel fragile ecosystems in a warmer world. iii Preface All the figures and tables in this thesis are designed by Alireza Saidi-Mehrabad, except Figures 1.1-1.5 of the chapter 1 and Figure 2.1 of the chapter 2. Figures obtained from other publications have been cited and acknowledged. Chapter 2 of this thesis has been published in the journal “Scientific Reports” as “Saidi- Mehrabad, A., Neuberger, P., Cavaco, M., Froese, D., Lanoil, B. (2020). Optimization of subsampling, decontamination, and DNA extraction of difficult peat and silt permafrost samples. Scientific Reports. 10:14295”. Chapter 3 of this thesis has been published in the journal of “Frontiers in Environmental Science” as “Saidi-Mehrabad, A., Neuberger, P., Hajihosseini, M., Froese, D., Lanoil, B. (2020). Permafrost microbial community structure changes across the Pleistocene-Holocene boundary. Frontiers in Environmental Science. 8:133”. Chapter 4 of this thesis is in preparation for publication. No specific journal has been selected yet. The potential title for the publication is going to be “Saidi-Mehrabad, A., Strilets, T., Forster, M., Froese, D., Lanoil, B. (2021). A major shift in activity and composition of methanotrophs in permafrost active layer soil in response to soil disturbance and temperature changes”. In all these chapters, I was responsible for conceptualizing the projects, sample collection, performing the experiments, data analysis, designing of the figures and tables (except Figures iv 1.1-1.5 and 2.1), article review, and lead authorship of the manuscripts. Lanoil and Froese obtained the funding for all the projects, conceptualized the projects, and aided with the formulation of the manuscripts. Neuberger, Cavaco, Strilets, and Forster, assisted with the experiments and data analysis. Hajihosseini aided with the statistical tests. This project was made possible with the generous support of University of Alberta (UANRA grant, obtained by Alireza Saidi-Mehrabad), the Polar Knowledge Canada (NSTP grant, obtained by Alireza Saidi-Mehrabad), Queen Elizabeth II graduate scholarship (QEII, obtained by Alireza Saidi-Mehrabad), Alberta Graduate Excellence Scholarship (obtained by Alireza Saidi-Mehrabad), Alberta Innovates Graduate Student Scholarship (obtained by Alireza Saidi- Mehrabad), Steve and Elaine Antoniuk Graduate Scholarship in Arctic research in biological sciences (obtained by Alireza Saidi-Mehrabad), NSERC Discovery Grant (obtained by Brian Lanoil and Duane Froese), NSERC Northern Research Supplements (obtained by Duane Froese) and ArcticNet project (obtained by Brian Lanoil). Some components of this project have been used in the following posters and presentations: Saidi-Mehrabad, A., Neuberger, P., Hajihosseini, M., Froese, D., and Lanoil, B. (2019). Soil chemistry, not age, is the primary driving factor correlated to difference in Pleistocene-aged and Holocene-aged permafrost microbiomes. ASM-Arctic Net, Halifax Canada (Oral and poster presentations). v Saidi-Mehrabad, A., Neuberger, P., Hajihosseini, M., Froese, D., and Lanoil, B. (2017). Microbial community structure is distinct across the Holocene-Pleistocene boundary in ancient Beringian permafrost. ASM-Arctic Net, Québec Canada (Poster presentation). Lanoil, B., Neuberger, P., Saidi-Mehrabad, A., and Froese, D. (2019). AGU conference, San Francisco USA. (Oral and poster presentations). Saidi-Mehrabad, A. (2019). ACUNS conference, Edmonton Canada. (Guest speaker). Saidi-Mehrabad, A. (2019). RENR GSA Conference, Edmonton Canada. (Guest speaker). My findings have been also featured at the University of Alberta’s scientific news under the title “A climatic crystal ball: How changes in ancient soil microbes could predict the future of the Arctic”. https://www.ualberta.ca/science/news/2020/august/arctic-soil-climate-change.html. CTV EDMONTON NEWS “Soil microbes may be ‘crystal ball’ in future climate”. (https://www.facebook.com/watch/?v=365633934429447) University of Alberta’s CJSR-FM campus radio station “Ice age microbes” (Broadcasted in Persian/Farsi language). vi توانا بود هر که دانا بود ز دانش دل پير برنا بود “Mighty is he who has knowledge; from knowledge the old heart becomes young” Ferdowsi Shahnameh (The book of kings) I dedicate this work to my family and friends. I am forever grateful for their compassion and support. vii Acknowledgment First and foremost, I would like to sincerely thank my advisor, Dr. Brian Lanoil, for his guidance, for his patience, and for all the opportunities that he provided me. Joining his lab was a dream come true. Brian, it was an honor working with you. Furthermore, I would like to thank Dr. Duane Froese for his support and guidance. Thank you so much Duane for sharing your immense knowledge with me. I would have never thought one day I can go beyond all those documentaries and posters about the ice age and mammoth and tag along with your awesome lab on a journey to wonderful Yukon for collecting samples. I thank you Brian and Duane for providing me this once in a lifetime opportunity. I would like to extend my thanks to the members of my supervisory committee: Dr. Suzanne Tank, Dr. Maya Bhatia, and Dr. Lyle Whyte. I sincerely appreciate you taking the time to get involved in my Ph.D. project and your insightful comments that helped to perfect my project. I thank my past and present fellow lab mates in Lanoil lab and Froese lab, especially Patrick Neuberger, William Kirby, Juan Camilo Santana, Angelica Aguirre, Julian Ariel Cabrera, Helena Magaldi, Joseph Young, Sasiri Bandara, Joel Pumple, and Casey Buchanan. Of course, I will never forget my amazing friends at the University of Alberta, Morteza Hajihosseini, Peyman Derik Vand, Maria Cavaco, Dillon Lee, Tania Strilets, Helen Trinh, Natalie Leung, Erin Macdonald, Sarah Shakil, Sattar Soltani, Dimitri Kits, and Philip Sun. viii The Department of Biological Sciences has been a great environment, I thank each and everyone in the graduate office, the 265 microbiology lab, the microscopy lab, and the MBSU lab for helping me with my research. Thank you University of Alberta for shaping my career and being my second home for five years. I finish this acknowledgment section by thanking my wonderful family for their unquestionable support and love throughout my life. GOD, thank you for your amazing power and work in our lives, thank you for your goodness and for your blessings over us. Thank you for your great love and care. Thank you for your mercy and grace. ix Table of content List of Tables .............................................................................................................................. xiii List of Figures ............................................................................................................................