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ABSTRACT WHITHAM, JASON MICHAEL. Identifying Genetic Targets and Growth Conditions for Higher Ethanol Production by the Synthesis Gas Fermenting Bacterium Clostridium ljungdahlii (Under direction of Dr. Amy M. Grunden and Dr. Joel J. Pawlak). The United States has increasing volume mandates for cellulosic biofuels production to reduce our dependence on foreign oil for the sake of transportation fuel security. For the past few years, mandated volumes have not been met by industry though for a variety of reasons. Clostridium ljungdahlii, an established cellulosic ethanol- producing industrial biocatalyst can produce ethanol from synthesis gas; however, relatively low concentrations of ethanol compared to traditional yeast fermentations of sugar are costly to separate from the fermentation medium, and C. ljungdahlii’s growth and productivity can be inhibited by contaminants in syngas which are expensive to remove. Synthesis gas is predominantly composed of carbon monoxide, carbon dioxide, and hydrogen, but also can contain a variety of contaminants (e.g. hydrogen cyanide, mono-nitrogen oxides, ammonia, sulfur dioxide, carbonyl sulfide, hydrogen sulfide, oxygen) depending on the carbonaceous feedstock from which it is produced (e.g. biomass, agricultural waste, municipal waste, industrial waste). The purpose of this work was, therefore, to identify genetic targets and growth conditions for higher ethanol production. To accomplish this, the metabolic and physiological response of C. ljungdahlii PETC to oxygen was investigated and a lab-derived hyper ethanol-producing strain of C. ljungdahlii (designated OTA1) was characterized. Preliminary investigation showed that C. ljungdahlii PETC has some tolerance to oxygen when cultured in a rich mixotrophic medium. Batch cultures not only continued to grow and consume H2, CO, and fructose after 8% O2 exposure, but fermentation product analysis revealed an increase in ethanol yield and decreased acetate yield compared to non-oxygen exposed cultures. Acetate reductase activity by a more highly expressed predicted aldehyde oxidoreductase (encoded by CLJU_c24130) and hydrogen peroxidase activity by a rubrerythrin (encoded by CLJU_c39340) were identified as important contributions to higher ethanol production and oxygen detoxification, respectively. Higher ethanol production and oxygen/ROS detoxification were also found to be linked by cofactor management as well as substrate and energy metabolism. The C. ljungdahlii OTA1 strain was previously shown to produce twice as much ethanol as the C. ljungdahlii PETC strain when cultured in mixotrophic medium containing fructose and syngas. This work identified four single nucleotide polymorphisms (SNPs) unique to its genome. SNPs in acsA (CLJU_c37670) and/or hemL (CLJU_c04490) likely cause the OTA1 strain to have an inoperable Wood-Ljungdahl pathway. OTA1 also has a mutation in the ADI transcriptional regulator gene (CLJU_c09320) which was found to deregulate expression of arginine catabolism genes while a mutation in the DeoR transcriptional regulator (CLJU_c18110) deregulated expression of 2-deoxy-D-ribose catabolism genes. Fermentation studies, differential expression analysis, and enzyme assays showed that the mutation(s) which resulted in OTA1 having an inoperable Wood-Ljungdahl pathway had the greatest effect on growth and fermentation products, and the higher ethanol to acetate fermentation product ratio could be explained by those mutations alone. The mutations in the transcriptional regulators and the increased expression of their respective operons seemed to have more minor effects on growth and fermentation products based on fermentation supplementation experiments. Besides providing genetic targets and growth conditions for higher ethanol production, this work also contributes new knowledge about C. ljungdahlii’s metabolism and physiology. © Copyright 2015 by Jason Michael Whitham All Rights Reserved Identifying Genetic Targets and Growth Conditions for Higher Ethanol Production by the Synthesis Gas Fermenting Bacterium Clostridium ljungdahlii by Jason Michael Whitham A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the dual Degree of Doctor of Philosophy Microbiology and Forest Biomaterials Raleigh, North Carolina 2015 APPROVED BY: ________________________________ ________________________________ Dr. Amy M. Grunden Dr. Joel J. Pawlak Committee Co-Chair Committee Co-Chair ________________________________ ________________________________ Dr. José M. Bruno-Bárcena Dr. David C. Tilota ________________________________ Dr. Mari S. Chinn DEDICATION I dedicate these first fruits and all works here after to the Father, Son and Holy Spirt. In God’s great grace and to His glory, He has provided me with more than I have ever asked for, including this opportunity. At times, the work seemed unbearable, but His comforting words, timely provisions, and the encouraging prayerful loving community of His people have brought me successfully through the hardest thing I have done thus far in my life. He is good. I also dedicated this work to my parents who have always encouraged me to pursue higher education. ii BIOGRAPHY Jason Michael Whitham was raised in East Granby, CT by his mother, Gwen E. Whitham. He had no brothers or sisters, but close relationships with a few boys and girls in the neighborhood. For a variety of reasons including the desire for novelty, he decided to move and go to college in North Carolina where his dad, Scott J. Whitham, and step mom, Brooke Baldridge, lived. While he was saving up to go to North Carolina State University, he was called by name and saved by the Lord. After four years of studying biochemistry, Jason wanted to go to China as a missionary, but God closed that door and opened an opportunity for him to get a dual Ph.D. in Plant and Microbial Biology and Forest Biomaterials. At the same time God enabled him to serve fellow graduate students, professors, neighbors, and homeless people in the city of Raleigh. He looks forward to what the Lord has for him next. iii ACKNOWLEDGEMENTS I would like to thank my thesis committee members – Dr. Mari S. Chinn, Dr. José M. Bruno-Bárcena, Dr. David C. Tilotta and especially my thesis advisor Dr. Amy M. Grunden and co-advisor Dr. Joel J. Pawlak for challenges, support, and knowledge. I would also like to more specifically thank Dr. Grunden for encouraging me in my faith and making my continued efforts in homeless ministry a condition of taking me on. I am grateful for the spiritual and emotional support of my best friend Robert Davis, my community group (especially Brian Shea, Matt Munse, Erica Eshe Elesthe, Bekah Charlier and Kim Vo), my spiritual brother and roommate Parham Tayeb, my spiritual brother and co-worker Kai Lee, my spiritual brother and co-worker Mark Schulte, my spiritual sister and favorite lunch buddy Mikyoung Ji, my spiritual salty brother Yonathan, and the body of believers at Vintage Church in downtown Raleigh. While there were many others who contributed to my success, these people prayed for me regularly, they were there for me during my nasal surgery, they sacrificed sleep for me, they picked up my slack when I was too busy to do house or lab chores, they worshipped with me regularly, broke bread with me regularly, listened and cared about my science stuff (at least pretended), they celebrated with me and they cried with me…they lived their lives with me. Love you all very much. I am so thankful for the patience of Dr. Jimmy Gosse, Mark Shulte, and Dr. Oscar Tirado-Acevedo who taught me most of the anaerobic techniques that I currently know and how to use and troubleshoot the gas chromatography machine in Dr. Michael C. Flickinger’s lab. Thank you Dr. Flickinger for allowing me to use your machine. Thank iv you Dr. Sarwat Khattak for being incredibly helpful in allowing me access to Nova BioProfile 400 Analyzer, even late at night. Thank you Dr. Heike Winter-Sederoff for allowing me to use your computer for transcriptome analysis, and thank you so much Naresh Vasani and Dr. Hemant Kelkar for helping me with lots of command line questions. Thank you Dr. Benjamin Bobay for your help with generating protein homology models. Thank you Dr. Chase Beisel and Ryan Leenay for your collaboration in generating a CRISPR-Cas-based point mutation technology for C. ljungdahlii. Thank you Dr. Alice Lee for seeing potential in me as an undergraduate student in your microbiology lab class and helping me get an interview with Dr. Grunden so many years ago. Last but not least, thank you Dr. Sherry Tove for your review and commentary on my manuscripts. v TABLE OF CONTENTS LIST OF TABLES..............................................................................................................x LIST OF FIGURES..........................................................................................................xi PREFACE.......................................................................................................................xiii CHAPTER 1 Clostridium ljungdahlii: A Review of the Development of an Industrial Biocatalyst...........................................................................................................................1 ABSTRACT............................................................................................................2 1.1 Introduction......................................................................................................3 1.2 History
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