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The Probiotics of Biofuel: a Metagenomic Study of Microalgae Grown for Fuel Production

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The Probiotics of Biofuel: a Metagenomic Study of Microalgae Grown for Fuel Production ABSTRACT Title of Thesis: THE PROBIOTICS OF BIOFUEL: A METAGENOMIC STUDY OF MICROALGAE GROWN FOR FUEL PRODUCTION Samuel Russell Major, Master of Science, 2018 Thesis Directed By: Dr. Russell T. Hill, Professor, Institute of Marine and Environmental Technology, University of Maryland Center for Environmental Science Ponds in Frederick, MD were fertilized with chicken manure to increase the nutrient load in the water and stimulate microalgal growth. Nutrient analyses indicate that fertilization results in significant increases in the DOC, TDN, and TDP. The bacterial and eukaryotic microalgal communities were analyzed using 16S and 18S rRNA gene sequencing, respectively. Communities were analyzed pre-fertilization and for 15 days following fertilization. Molecular data reveals a decrease in diversity as microalgal blooms form. The microalgal density increased following fertilization, with enrichment for the Chlamydomonadales order. Prior to fertilization the bacterial communities were dominated by five phyla: Actinobacteria, Bacteroidetes, Cyanobacteria, Proteobacteria, and Verrucomicrobia. Dominant bacterial genera post-fertilization included Flavobacterium, Limnohabitans, and Polynucleobacter. Bacteria isolated from the ponds were screened for effects on Scenedesmus sp. HTB1 to identify bacteria that either enhance or inhibit microalgal growth. The growth- promoting bacteria were closely related to bacteria found to be enriched during microalgal bloom formation. THE PROBIOTICS OF BIOFUEL: A METAGENOMIC STUDY OF MICROALGAE GROWN FOR FUEL PRODUCTION by Samuel Russell Major Thesis submitted to the Faculty of the Graduate School of the University of Maryland, College Park, in partial fulfillment of the requirements for the degree of Master of Science 2018 Advisory Committee: Professor Russell T. Hill, Chair Professor Feng Chen Associate Professor Yantao Li © Copyright by Samuel Russell Major 2018 Dedication I would like to dedicate this work to my mother and father for their unwavering support for all my endeavors. To the beautiful Carolyn for her dedication, sacrifice, and keeping me filled with donuts and cookies during my research. And to my brother and his family; hopefully the world will be a little bit better and cleaner when Chase and Olivia can read this. ii Acknowledgements I would like to thank my advisor Dr. Russell Hill for taking a chance and granting me the opportunity to become a better scientist. His patience and advice has pushed me to learn new skills I never imagined I would learn. Thank you to my committee members Dr. Feng Chen and Dr. Yantao Li for their guidance through the microalgal world. My kindest regards go out to Dr. Ryan Powell and all of Manta Biofuel for allowing us to perform the research. A giant thanks to all my interns Ema Pagliaroli, Deanna Stephens, Lily Xiao, and Bryanna Sanders for their aid in executing experiments and tolerating my teaching style as I tried to teach them the intricacies of molecular biology. Also, I would like to acknowledge the patience and assistance of IMET’s all-star bioinformaticians: Tsetso Bachvaroff, Ryan McDonald, and Amanda Maggio. The entire Hill lab, Hanzhi Lin, Daniela Tizabi, Lauren Jonas, Taylor Carter, Billie Beckley, and Jan Vincente, all deserve an extra slice of cake as well for listening to my terrible jokes, even worse singing, and I’m sorry if you ever caught me dancing. iii Table of Contents Dedication ..................................................................................................................... ii Acknowledgements ...................................................................................................... iii Table of Contents ......................................................................................................... iv List of Tables .............................................................................................................. vii List of Figures ............................................................................................................ viii List of Abbreviations ................................................................................................... xi Chapter 1: Microalgae: from their evolution to an energy revolution .......................... 1 1.1 Algae evolution, diversity, and current technological uses ................................ 1 1.2 Algae as a biofuel ............................................................................................... 5 1.3 Biofuel production .............................................................................................. 8 1.4 Bacteria to improve algae-derived biofuels ...................................................... 10 Chapter 2: Diversity of bacterial communities associated with microalgae grown for biofuel production in man-made ponds following artificial eutrophication ............... 13 2.1 Abstract ............................................................................................................. 13 2.2 Introduction ....................................................................................................... 14 2.3 Methods ............................................................................................................ 17 2.3.1 Sample collection and processing .............................................................. 17 2.3.2 DNA extraction, sequencing, and data processing .................................... 19 2.3.3 Diversity and statistical analyses ............................................................... 22 2.4 Results ............................................................................................................... 24 2.4.1 Sequence processing and control communities ......................................... 24 iv 2.4.2 Chicken manure bacterial communities ..................................................... 25 2.4.3 Nutrient analysis ........................................................................................ 25 2.4.4 Taxonomy and diversity analyses .............................................................. 29 2.4.5 Statistical similarities ................................................................................. 36 2.5 Discussion ......................................................................................................... 41 2.6 Conclusions ....................................................................................................... 52 Chapter 3: Microalgal 18S rRNA gene community dynamics in agricultural ponds fertilized to stimulate blooms for biofuel production ................................................. 53 3.1 Abstract ............................................................................................................. 53 3.2 Introduction ....................................................................................................... 54 3.3 Methods ............................................................................................................ 58 3.3.1 Sample collection and nutrient analysis ..................................................... 58 3.3.2 DNA extraction, sequencing, and data processing .................................... 59 3.3.3 Diversity, statistical, and similarity analyses ............................................. 61 3.4 Results ............................................................................................................... 63 3.4.1 Sequence processing and control communities ......................................... 63 3.4.2 Algal cell counts ........................................................................................ 64 3.4.3 Nutrient analyses ........................................................................................ 64 3.4.4 Taxonomy and diversity analyses .............................................................. 66 3.4.5 Community Similarities ............................................................................. 75 3.5 Discussion ......................................................................................................... 80 3.6 Conclusions ....................................................................................................... 88 v Chapter 4: Bacterial-algal interactions and isolation of axenic Desmodesmus cultures from artificial ponds using physical isolation and antibiotic treatment. ..................... 90 4.1 Abstract ............................................................................................................. 90 4.2 Introduction ....................................................................................................... 90 4.3 Methods ............................................................................................................ 93 4.3.1 Algal and bacterial culturing and isolation ................................................ 93 4.3.2 Antibiotic treatment ................................................................................... 94 4.3.3 Bacterial:algal ratios and algal identification ............................................ 95 4.3.4 Screen for algal-bacterial interactions ....................................................... 96 4.4 Results ............................................................................................................... 96 4.4.1 Algal culturing and treatment .................................................................... 96 4.4.2 Screen for algal-bacterial interactions ..................................................... 101 4.5
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