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ABSTRACT ZURAWSKI, JEFFREY VANCE. Microbiological And ABSTRACT ZURAWSKI, JEFFREY VANCE. Microbiological and Engineering Studies of Extremely Thermophilic Caldicellulosiruptor Species for Lignocellulose Deconstruction and Conversion (Under the direction of Dr. Robert Kelly). The genus Caldicellulosiruptor contains extremely thermophilic, lignocellulose- degrading bacteria, capable of optimal growth at temperatures of up to 78˚C. Characteristic of these bacteria are hydrolytic thermostable enzymes that are active towards all plant polysaccharides and utilization of both pentose and hexose degradation products in fermentation pathways to produce primarily acetate, lactate, H2 and CO2. These traits make members of the genus poised for utilization in lignocellulose-based bioprocesses. Currently, the intrinsic recalcitrance of lignocellulose is the major economic hurdle to developing these bioprocesses. Lignin, a major component of plant cell walls, is a primary contributor to recalcitrance. This work describes the comparison and assessment of Caldicellulosiruptor species for deconstruction of untreated lignocellulosic feedstocks and the evaluation of genetically engineered switchgrass for recalcitrance both with and without hydrothermal pretreatment. The ability to degrade untreated lignocellulosic substrates was evaluated for three strongly cellulolytic Caldicellulosiruptor species (C. bescii, C. kronotskyensis and C. saccharolyticus). Solubilization of biomass from natural poplar variants correlated inversely with lignin levels for the three species with C. saccharolyticus affected the most. The extent of switchgrass degradation by C. bescii and C. kronotskyensis was comparable, while degradation by C. saccharolyticus was much lower and only slightly more than abiotic thermal mechanisms. The lack of two cellulases containing glycoside family 48 domains in the genome of C. saccharolyticus is believed to be responsible for its lower levels of solubilization. Fermentation product profiles indicated acetate as the primary metabolic product for all species, although lactate production was variable. Transcriptomes for growth of each species comparing cellulose to switchgrass showed differential regulation of many carbohydrate ABC transporters and multi-domain extracellular glycoside hydrolases, reflecting the heterogeneity of lignocellulose. Expression of many of these elements was high on both substrates indicating of a role in cellulose deconstruction. However, there were significant differences in transcription levels for genes conserved among the three species, indicating unexpectedly diverse regulatory strategies for deconstruction for these closely related bacteria. Next, the recalcitrance to two transgenic switchgrass lines, COMT3 and MYB, modified in lignin content and structure, were analyzed for deconstruction by C. bescii. Transgenic modification of the lignin biosynthesis pathway increased carbohydrate conversion of untreated COMT3 and MYB by 1.5- and 2.3-fold, respectively. Overall carbohydrate conversion for the transgenic COMT3 and MYB lines was 36% and 15%, respectively, compared to a naturally selected switchgrass, Cave in Rock (36% carbohydrate conversion). C. bescii planktonic growth was indicative of differences in carbohydrate conversion between switchgrass lines. Hydrothermal pretreatment significantly increased carbohydrate conversion by 15% for all switchgrass lines, indicating transgenic modification did not enhance hydrothermal release of insoluble carbohydrates. Time-course sampling of pH-controlled fermentations found transgenic switchgrass lines to have not only decreased recalcitrance but also increased solubilization and acetate production rates. Combining multiple C. bescii microbial deconstructions and hydrothermal treatments achieved over 70% carbohydrate solubilization for transgenic COMT3 and Cave in Rock switchgrass lines. Characterization of untreated and hydrothermally treated switchgrass showed while accessibility increased as a result of hemicellulose removal and cellulose disruption by C. bescii enzymes, lignin contributed significantly to residual recalcitrance. The results from this study showed cellulolytic capacity varies within the Caldicellulosiruptor genus as indicated in the genome. Likewise, the extent of carbohydrate conversion is highly dependent on lignocellulosic substrate composition and structural characteristics. From an engineering perspective, coupling genetically manipulated Caldicellulosiruptor strains and plants with mild pretreatment technologies offers opportunity to overcome lignocellulosic recalcitrance. © Copyright 2016 by Jeffrey Vance Zurawski All Rights Reserved Microbiological and Engineering Studies of Extremely Thermophilic Caldicellulosiruptor Species for Lignocellulose Deconstruction and Conversion by Jeffrey Vance Zurawski A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Chemical and Engineering Raleigh, North Carolina 2016 APPROVED BY: _______________________________ ______________________________ Dr. Robert Kelly Dr. Jason Haugh Committee Chair ________________________________ ________________________________ Dr. Chase Beisel Dr. Amy Grunden BIOGRAPHY Jeffrey Zurawski was born in Albuquerque, New Mexico to his parents Dwight and Loretta Zurawski. He has one younger brother, Daniel Zurawski. During the first sixteen years of life in Albuquerque, he developed a love for bicycling, skiing and the outdoors, this passion still endures. At age sixteen, he moved to Golden, Colorado where he graduated from Arvada West high school. With his high school diploma, he moved one-hour north to Fort Collins, Colorado to attend Colorado State University. He graduated in 2009 with a Bachelor of Science majoring in Chemical and Biological Engineering. With a passion for learning and a knack for problem solving, he began his graduate studies in the Department of Chemical and Biomolecular Engineering at North Carolina State University in the fall of 2010. He began his studies on extremophilic bacteria under the advisement of Dr. Robert Kelly. After completion of his graduate studies in May 2016, Jeffrey will move on to pursue a career in the biotechnology industry. ii ACKNOWLEDGMENTS My academic advisor, Dr. Robert Kelly, has been an excellent mentor and leader. The Kelly method for scientific research and presentation kept me on a trajectory for success throughout my graduate studies. The lessons I have learned from Dr. Kelly about leadership, collaboration and science will be instrumental in my career. The members of the hyperthermophile lab have always made science fun and been available for useful discussion. I am forever grateful for the constructive environment they fostered. Dr. Sara Blumer- Schuette’s knowledge and encouragement was instrumental in my early research. The BioEnergy Science Center (BESC) provided me with financial suppor and many opportunities for professional development. The knowledge, experience and comradery gained at BESC and in the hyperthermophile lab has been vital to my graduate experience. Finally, I am grateful for the relationships I have outside of the laboratory. First, I would like to thank my parents, Dwight and Loretta, for their unwavering support and guidance. My brother, Daniel for always being ready to go to the mountains. My graduate school class of 2010: Mike, Andrew, Dan, Garrett, Collin, Steven, and Dennis. They kept my spirits high, supported me through the perils of graduate school, and always reminded me to work hard, play hard. My friends from Colorado State University: Pat, Doss, Guerin, Val, Brenda, and Sheehan are always ready to take a vacation and available for a good laugh. Last but certainly not least, I am extremely thankful for my best friend and partner, Elizabeth Yount. She has always been there reminding me to not take life too seriously and with a piece of yoga philosophy to ease my mind. I am forever grateful for her enthusiasm to go on adventures with me, whether it is a bike ride, hike or traveling to a new place. I am excited for our next chapter and journey through life. iii TABLE OF CONTENTS LIST OF TABLES ................................................................................................................... vi LIST OF FIGURES ................................................................................................................ vii CHAPTER 1 The Extremely Thermophilic Genus Caldicellulosiruptor: Physiological and Genomic Characteristics for Complex Carbohydrate Conversion to Molecular Hydrogen .............................................................................................1 Abstract ..................................................................................................................................... 2 General Caldicellulosiruptor Background ................................................................................ 3 Extracellular Deconstruction of Lignocellulosic Biomass ....................................................... 5 Lignocellulose Composition and Recalcitrance .................................................................5 Enzymatic Lignocellulose Deconstruction .........................................................................6 Core Caldicellulosiruptor Hydrolytic Enzymes ...............................................................10 Cellulolytic Caldicellulosiruptor Enzymes.......................................................................11 Carbohydrate
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