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Doctoral Dissertation Template UNIVERSITY OF OKLAHOMA GRADUATE COLLEGE THE IMPACT OF LONG-TERM ELEVATED ATMOSPHERIC CARBON DIOXIDE ON BELOWGROUND MICROBIAL COMMUNITY AT CONTRAST NITROGEN CONDITIONS A DISSERTATION SUBMITTED TO THE GRADUATE FACULTY in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY By FEIFEI LIU Norman, Oklahoma 2017 THE IMPACT OF LONG-TERM ELEVATED ATMOSPHERIC CARBON DIOXIDE ON BELOWGROUND MICROBIAL COMMUNITY AT CONTRAST NITROGEN CONDITIONS A DISSERTATION APPROVED FOR THE DEPARTMENT OF MICROBIOLOGY AND PLANT BIOLOGY BY ______________________________ Dr. Jizhong Zhou, Chair ______________________________ Dr. Elizabeth Karr ______________________________ Dr. Bradley Stevenson ______________________________ Dr. Yiqi Luo ______________________________ Dr. Michael Patten © Copyright by FEIFEI LIU 2017 All Rights Reserved. Acknowledgements This is a dissertation studying microbial community and it is really a work of a community. Many people contributed to this study and I would like to express my gratitude to all of them who encouraged me and helped me out through the past years. First, my advisor, Jizhong Zhou, was generous to take me on as a student and introduced me to the field of environmental microbiology. Dr. Zhou is an excellent, incredibly hard- working scientist. He has provided much guidance, encouragement, and patience over the years. This project would not be possible without his support. Dr. Zhili He and Dr. Kai Xue also contributed much to this project for instructing me to gather, analyze, and interpret of the large datasets. Dr. Liyou Wu spent lots of efforts to develop the high-throughput sequencing pipelines. Without their help, I could be still struggling working on generating data and making sense of it. I truly learned a lot from them in organizing massed results into logical scientific work. I also owe great thanks to my committee members—Dr. Elizabeth Karr, Dr. Yiqi Luo, Dr. Bradley Steven, and Dr. Michael Patten for serving as my committee members for this long time. As a graduate student trying to develop his own background in a multidisciplinary area, I deeply appreciated their guidance by providing valuable suggestions and assistance for every stages throughout this degree period. The different perspectives they look at questions greatly benefitted me to address problems comprehensively. Members of IEG have been very helpful, particularly Dr. Joy Van Nostrand, Dr. Daliang Ning, Dr. Renmao Tian, Dr. Arthur Escalas, Dr, Anyi Hu, Dr. Jialiang Kuang, Dr. Ye Deng, and Yujia Qin. The data analysis methods and scientific writing skills I iv learned from them would also benefit me for life. Dr. Peter Reich and Dr. Sarah Hobbie from the University of Minnesota also kindly offered perspective on potential research ideas, helped me collect samples, and generously shared data. Grants from several agencies have supported my research including the US Department of Energy and the China Scholarship Council. Thank you for the investment and the trust placed in me. My final sincerest gratitude belongs to my family, my parents, and my fiancée, for their continuous encouragements and support in my whole life. It is them who helped me to survive all the stress for years and not letting me give up. Nothing could compensate the great efforts and sacrifice they have made for my achievements. v Table of Contents Acknowledgements ......................................................................................................... iv Table of Contents ............................................................................................................ vi List of Tables ................................................................................................................... xi List of Figures ................................................................................................................. xii Abstract ......................................................................................................................... xvii Chapter 1: Introduction ..................................................................................................... 1 1.1 Atmospheric CO2: the background ....................................................................... 1 1.2 Consequences of elevated atmospheric CO2 for macroecosystems ..................... 2 1.3 Consequences of elevated atmospheric CO2 for microbial communities belowground ................................................................................................... 3 1.4 Foci of this study .................................................................................................. 7 Chapter 2: Responses of soil microbial communities to elevated carbon dioxide and nitrogen deposition differ by C3 and legume plants .......................................... 11 2.1 Abstract ............................................................................................................... 11 2.2 Introduction ........................................................................................................ 13 2.3 Materials and Methods ....................................................................................... 16 2.3.1 Site description and experimental design: ................................................. 16 2.3.2 Plant and soil property measurements ....................................................... 17 2.3.3 DNA isolation, PCR amplification of the 16S rRNA gene and Illumina sequencing .............................................................................................. 18 2.3.4 Sequence data preprocessing and statistical analysis ................................ 18 2.4 Results ................................................................................................................ 19 vi 2.4.1 Structure of soil bacterial and archaeal communities ................................ 19 2.4.2 Effect of eCO2 and eN on microbial community diversity and structure .. 21 2.4.3 Changes in community composition along eCO2 and eN ......................... 22 2.4.4 Distribution of copiotrophic-, oligotrophic-like microorganisms along treatments in C3 grass and legume plots ................................................ 25 2.4.5 Linkages between microbial composition and selected environmental properties ................................................................................................ 28 2.5 Discussion ........................................................................................................... 30 Chapter 3: Contingency in belowground microbial functional responses to rising atmospheric CO2 at contrast nitrogen conditions ............................................... 37 3.1 Abstract ............................................................................................................... 37 3.2 Introduction ........................................................................................................ 39 3.3 Materials and Methods ....................................................................................... 42 3.3.1 Site description and sampling .................................................................... 42 3.3.2 Soil DNA extraction, amplification, and labeling ..................................... 43 3.3.3 Microarray hybridization ........................................................................... 43 3.3.4 Microarray scanning and data pre-processing ........................................... 44 3.3.5 Ecosystem C flux measurement ................................................................ 45 3.3.6 Stable isotope analysis of soil C sequestration and N fixation .................. 45 3.3.7 Statistical analysis ..................................................................................... 46 3.4 Results ................................................................................................................ 47 3.4.1 Effects of eCO2 and eN on plant and soil attributes .................................. 47 3.4.2 Overall responses of soil microbial functional genes to eCO2 and eN ...... 48 vii 3.4.3 Effects of eCO2 and eN on key functional genes ...................................... 50 3.4.4 Soil and microbial heterotrophic respiration ............................................. 56 3.4.5 Stable isotope analysis of soil C sequestration and N fixation .................. 56 3.4.6 Linkages between microbial functional structure and selected environmental attributes ......................................................................... 57 3.5 Discussion ........................................................................................................... 60 3.6 Conclusions ........................................................................................................ 64 Chapter 4: Impact of Elevated CO2 and N Addition on Metabolic Diversity of C3 grass- and Legume-associated Microbial Communities ............................................... 66 4.1 Abstract ............................................................................................................... 66 4.2 Introduction ........................................................................................................ 68 4.3 Materials and Methods ....................................................................................... 69 4.3.1 Site description and sample collection ...................................................... 69 4.3.2 Sample preparation ...................................................................................
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