UNDERSTANDING PHYSIOLOGICAL ADAPTATIONS, METABOLIC POTENTIAL AND ECOLOGY IN A NOVEL PHOTOAUTOTROPHIC ALGA FOR BIOFUEL PRODUCTION by Luisa Fernanda Corredor Arias A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology MONTANA STATE UNIVERSITY Bozeman, Montana November 2019 ©COPYRIGHT by Luisa Fernanda Corredor Arias 2019 All Rights Reserved ii DEDICATION To Ben, the love of my life, partner in crime and my happy place. Your love, support, devotion and kindness are beyond measure. To our little sunshine Lily. You turned my world into a wonderful rollercoaster of love and filled my life with joy and purpose. Para mi mamá Magda, por el inmenso amor, la dedicación y la ternura que siempre me brinda. Para mi papá Germán, quién despertó en mi la pasión por la ciencia y el conocimiento. iii ACKNOWLEDGEMENTS I would like to thank the Fulbright Scholarship Program for opening my mind to the world and its endless possibilities. It truly changed my life in the most positive ways. I would like to thank my advisor, Dr. Matthew Fields, for giving me the chance to be part of his lab, the opportunities he provided for my professional development and for his support and advice for the last seven years of my life. I am very grateful to my committee members, Drs. Robin Gerlach, Mensur Dlakic and Abigail Richards for their support and always taking the time to help me, teach me and guide me through grad school and science. A special thank you to former and current members of the Fields lab for their support and friendship, coming to work was always great fun. I would also like to thank the members of the Center for Biofilm Engineering and the Microbiology and Immunology Department for all their assistance. Finally, I would like to thank my husband Ben, for always keeping me strong and motivated, for his hopeful and contagious view of life and for always believing in me, even when I don’t. My husband and my daughter Lily are my source of strength and encouragement for my personal and professional improvement every day. I would also like to thank my loving parents, Magda y Germán, for instilling in me the work ethic, commitment and perseverance that guides me through life, and my sister for her kind and loving soul. To my girls, Anali, Andrea, Erika, Dayane, Fede and Chiachi, I could not have done this without them. They are the family I found in the big sky country and will always be until we are old sipping wine and laughing without teeth. To Sandy. iv TABLE OF CONTENTS 1. INTRODUCTION ...........................................................................................................1 Factors that Influence Algal Growth and Lipid Accumulation .......................................2 Physical Factors: Temperature, Light and pH .........................................................3 Chemical Factors: Carbon and Nitrogen ..................................................................4 Nitrate Depletion and Bicarbonate Addition for Lipid Production .........................6 Algal Growth in Standardized Media versus Low Quality Water ..................................7 Coal Bed Methane Production Water as a Medium for Algal Growth ...........................8 Omics Approach to Understanding Algal Physiology ....................................................9 Ecological Approach to Algal Cultivation ....................................................................12 Research Goals and Objectives .....................................................................................14 2. IMPACT OF TEMPERATURE, NITRATE CONCENTRATION, PH AND BICARBONATE ADDITION ON BIOMASS AND LIPID ACCUMULATION OF A SPORULATING GREEN ALGA .....................................16 Contribution of Authors and Co-Authors .....................................................................16 Manuscript Information Page ........................................................................................17 Abstract .........................................................................................................................18 Introduction ...................................................................................................................19 Materials and Methods ..................................................................................................21 Results and Discussion ..................................................................................................29 Conclusions ...................................................................................................................51 Acknowledgments .........................................................................................................51 3. GENOMIC INSIGHTS INTO A SPORULATING, NON-MOTILE, OLIGOTROPHIC GREEN MICROALGA (PW95) ....................................................53 Contribution of Authors and Co-Authors .....................................................................53 Manuscript Information Page ........................................................................................54 Abstract .........................................................................................................................55 Introduction ...................................................................................................................56 Materials and Methods ..................................................................................................57 Results and Discussion ..................................................................................................64 Conclusions ...................................................................................................................80 Acknowledgments .........................................................................................................81 v TABLE OF CONTENTS CONTINUED 4. TRANSCRIPTOMIC PROFILING OF Chlamydomonas-like PW95 CULTIVATED IN COAL BED METHANE PRODUCTION WATER WITH THE NATIVE MICROBIAL COMMUNITY ...................................82 Contribution of Authors and Co-Authors .....................................................................82 Manuscript Information Page ........................................................................................83 Abstract .........................................................................................................................84 Introduction ...................................................................................................................85 Materials and Methods ..................................................................................................87 Results and Discussion ..................................................................................................92 Conclusions .................................................................................................................105 Acknowledgments .......................................................................................................108 5. ASSOCIATIONS BETWEEN SYMPATRIC BACTERIAL GROUPS AND A NOVEL GREEN ALGA CULTIVATED IN COAL BED METHANE PRODUCTION WATER .......................................................................109 Contribution of Authors and Co-Authors ...................................................................109 Manuscript Information Page ......................................................................................110 Abstract .......................................................................................................................111 Introduction .................................................................................................................112 Materials and Methods ................................................................................................115 Results .........................................................................................................................122 Discussion ...................................................................................................................141 Conclusions .................................................................................................................148 Acknowledgments .......................................................................................................149 6. EPILOGUE ..................................................................................................................151 REFERENCES ................................................................................................................163 APPENDICES .................................................................................................................179 APPENDIX A: Supplementary Materials for Chapter Two ................................180 APPENDIX B: Supplementary Materials for Chapter Three ..............................183 APPENDIX C: Supplementary Materials for Chapter Four ................................187 APPENDIX D: Supplementary Materials for Chapter Five ................................223 vi LIST OF TABLES Table Page Chapter 2 1. Comparison of growth metrics between all the treatment groups evaluated in this study ...........................................................................38 Chapter 3 1. PW95 de novo genome assembly metrics ..........................................................66 2. Chlamydomonas PW95 genome sequence annotation. .....................................68 3. Fundamental