South Dakota State University Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange Electronic Theses and Dissertations 2018 Diversity of Free – Living Nitrogen – Fixing Bacteria in Soil of Sioux Prairie of South Dakota Nabilah Ali Alshibli South Dakota State University Follow this and additional works at: https://openprairie.sdstate.edu/etd Part of the Microbiology Commons Recommended Citation Alshibli, Nabilah Ali, "Diversity of Free – Living Nitrogen – Fixing Bacteria in Soil of Sioux Prairie of South Dakota" (2018). Electronic Theses and Dissertations. 2959. https://openprairie.sdstate.edu/etd/2959 This Thesis - Open Access is brought to you for free and open access by Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange. For more information, please contact [email protected]. DIVERSITY OF FREE – LIVING NITROGEN – FIXING BACTERIA IN SOIL OF SIOUX PRAIRIE OF SOUTH DAKOTA BY NABILAH ALSHIBLI A thesis submitted in partial fulfillment of the requirements for the Master of Science Major in Biological Sciences Specialization in Biology South Dakota State University 2018 iii ACKNOWLEDGEMENTS To my life-coach, my Dad Ali Alshibli: because I owe it all to you. Many Thanks! I would like to express my profuse thanks and heartfelt appreciation to my advisor Dr. Volker Brӧzel for helping me and guiding me throughout the entire research. I am thankful for his encouragement, guidance, and support throughout my research. This research would not be successful without his guidance, help, and inspiration. Without your knowledge and supporting, I cannot stand where I do today. Thanks to your support I can now see what I am capable of; I know that if I pushed hard enough I have ability to do whatever I set my mind too. My eternal cheerleader, Gita Kafle: I miss our interesting and long-lasting chats. She was standing with me step by step. She was always after her graduation keen to know what I was doing and how I was proceeding, I will miss your screams of joy whenever a significant momentous was reached. I am grateful to my mother, husband, and siblings, who have provided me through moral and emotional support in my life. I am also grateful to my other family members and friends who have supported me along the way. My most grateful acknowledgment goes to my friends Arwa and Zynab who were very supportive and encouraging, although it is likely that they have never grasped what it was all about! I would like to thank all the faculties and staffs of Department of Biology and Microbiology who have directly or indirectly supported me to accomplish this goal. A very special gratitude and grateful thanking to my lab mates Amrit, Bikram, and Dakota. iv With a special mention goes out to the funding source South Dakota Agricultural Experiment Station for its funding for the work. And finally, last but by no means least, also to everyone in the impact hub. It was great sharing laboratory with all of you during last two years. Thanks for all your encouragement! v CONTENTS ABBREVIATIONS ......................................................................................................... viii LIST OF FIGURES ............................................................................................................ x LIST OF TABLES ........................................................................................................... xiii ABSTRACT ..................................................................................................................... xiv Chapter 1: Literature Review .............................................................................................. 1 1. Diversity of Free-Living Nitrogen-Fixing Bacteria in Soil of diverse Ecosystems ................. 1 1.1. Nitrogen is Important for All Forms of Life.............................................................................. 1 1.2. Nitrogen cycle .......................................................................................................................... 2 1.3. Biological Nitrogen Fixation ..................................................................................................... 5 1.3.1. Biochemistry of Nitrogen Fixation ...................................................................................... 6 1.4. Diversity of Nitrogen fixing Bacteria ...................................................................................... 16 1.4.1. Origins and Evolution of Nitrogenase ............................................................................... 16 1.4.2. Life Style of Nitrogen Fixers .............................................................................................. 17 1.5. Nitrogen in diverse ecosystems: ............................................................................................ 23 1.6. Methods for studying of Biological Nitrogen Fixation: .......................................................... 26 1.6.1. Culture Dependent methods: ........................................................................................... 26 1.6.2. Culture Independent Methods ......................................................................................... 28 1.6.3. Biochemical determination of nitrogen fixation .............................................................. 35 1.7. The objective of this study:.................................................................................................... 36 Chapter 2 ........................................................................................................................... 37 2. Diversity of Free-living Nitrogen Fixing bacteria in Soil of Native South Dakota Prairie... 37 2.1. Introduction ........................................................................................................................... 37 2.2. Materials and Methods ......................................................................................................... 40 2.2.1. Sample Source................................................................................................................... 40 2.2.2. Isolation of free-living putative diazotrophs from soil: .................................................... 41 2.2.3. Purification on R2A, Solid NFM, and Liquid NFM ............................................................. 43 vi 2.2.4. DNA Extraction and PCR Amplification of 16S rRNA from pure cultures ......................... 43 2.2.5. Confirming nitrogen fixation in an ammonia free atmosphere ....................................... 44 2.2.6. Amplification of nifH by PCR ............................................................................................. 45 2.2.7. Validation of pure culture by 16S rRNA amplification ...................................................... 47 2.3. Results .................................................................................................................................... 48 2.3.1. Diversity of free-living putative nitrogen fixing bacteria .................................................. 49 2.3.2. Growth in liquid NFM in nitrogen depleted atmosphere: ................................................ 54 2.3.3. nifH gene amplification: .................................................................................................... 54 2.4. Discussion: ............................................................................................................................. 67 Chapter 3 ........................................................................................................................... 73 3. Bacterial nitrogen-fixing cultures from prairie soil comprised of two-species interactions ...............................................................................................................................................73 3.1. Introduction ........................................................................................................................... 73 3.2. Materials and Methods ......................................................................................................... 74 3.2.1. nifH gene amplification ..................................................................................................... 75 3.2.2. Acetylene Reduction Assay ............................................................................................... 77 3.2.3. Incorporation of 15N2 isotope in pure cultures ................................................................ 77 3.2.4. Complete Genome Sequence of Streptomyces sp ........................................................... 78 3.2.5. Multilocus sequences typing of selected Streptomyces .................................................. 79 3.2.6. Obtaining strains into single culture ................................................................................. 80 3.2.7. Transformation of Bacillus with GFP................................................................................. 82 3.2.8. The separation of Streptomyces from Bacillus using Chloramphenicol and Ampicillin ... 84 3.3. Results .................................................................................................................................... 85 3.3.1. nifH amplifications of Streptomyces and their pattern of growth