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Abstract Slivka, Rachel Marie ABSTRACT SLIVKA, RACHEL MARIE. Clostridium autoethanogenum Fermentation Using Sugar and Gaseous Carbon Substrates. (Under the direction of Dr. Mari S. Chinn). Clostridium autoethanogenum is an anaerobic acetogen that is capable of metabolizing both energy-rich xylose and the single carbon gases (CO and CO2+H2) found in synthesis gas (syngas). These features are desirable because they enhance the amount of carbon in lignocellulosic biomass that can be converted through hydrolysis, gasification, and fermentation technology to value-added products such as bio-fuels and bio-chemicals. While C. autoethanogenum may be among the limited number of microorganisms that can naturally metabolize xylose, its innate ability to utilize the substrate is hindered without optimization of the fermentation conditions. Assessment of the factors which impacted xylose metabolism ranged from initial xylose concentration to nutrient limitation to pH optimization studies. Once it was discovered that C. autoethanogenum preferred a growth-optimized pH (~5.8) for sugar metabolism, the bacteria was able to fully consume all fed xylose (up to 30 g/L) and yield an ideal ethanol to acetate ratio of 1:3 (mass-basis). Unlike other acetogens, C. autoethanogenum is a pH sensitive organism which co-produces both acetate and ethanol during growth rather than having a distinct growth-associated acetogenic phase followed by non-growth-associated solventogensis. Ethanol formation serves to prevent acidification of the culture by sequestering excess reductant in an electron sink. C. autoethanogenum uses the Wood-Ljungdahl pathway to metabolize CO and CO2+H2; however, due to the high reductant demand of the pathway, the bioenergetics provided by syngas as the sole substrate is limited. Preadaptation studies were conducted to determine if inoculum adapted to xylose, syngas, or a sugar-gas mixture would provide an energy boost to the bioenergetics of fermentation systems containing syngas as a sole or co-substrate. Surprisingly, the method by which the organism was acclimated to syngas, and not sugar preadaptation of the organism, had the largest effect on whether C. autoethanogenum could metabolize syngas in an effective and timely manner. Cultures which had stagnant preadaptation to syngas experienced long lags in cell growth, while cultures that used reactor agitation during preadaptation to syngas demonstrated ideal growth profiles. Since the gases present in syngas are sparingly soluble, enhanced mass transfer by means of reactor agitation was required to increase the concentrations of CO, CO2, and H2 in the liquid phase so that the cells were able to uptake the gaseous substrate. Based on physiology differences observed between C. autoethanogenum cultures grown on xylose, syngas, or a mixture of xylose-syngas, a proteomic analysis was used to illustrate how protein expression changes under each substrate condition. The main pathways which showed changes in protein regulation as a result of the carbon source available included those related to sugar transport, carbon fixation, and energy metabolism. Although future optimization work remains, this study has advanced our overall understanding of C. autoethanogenum fermentations involving xylose and syngas as substrates. © Copyright 2018 by Rachel Marie Slivka All Rights Reserved Clostridium autoethanogenum Fermentation Using Sugar and Gaseous Carbon Substrates by Rachel Marie Slivka 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 Biological and Agricultural Engineering Raleigh, North Carolina 2018 APPROVED BY: _______________________________ _______________________________ Dr. Mari Chinn Dr. Amy Grunden Committee Chair _______________________________ _______________________________ Dr. Praveen Kolar Dr. Michael Goshe ii DEDICATION This dissertation is dedicated to my three dogs: To the memory of Betsy, I asked God to let you stay with me through graduate school and bless your heart you did. You will always be my little dog. To Electra, my little red ball of fun. To Blue, my special friend. iii BIOGRAPHY Who am I? I am the granddaughter of a Polish immigrant who used every ounce of gumption she had to raise my father and to give him a better life than she had as a child. I am the great-granddaughter of an Irish milkman from Northeast Ohio. I am a proud descendant of a young couple by the name of Slivka who left their homeland in Slovakia and started a family in America in 1908. I am the sister of a veterinarian who lets me think that I am the “smart” one. I am the daughter of two incredibly gifted, intelligent parents – one is a mechanical engineer and the other is a Special Education teacher. Hello, I am Rachel. I was born in Northeast Ohio, but moved to Danville, VA with my family when I was nine-years-old. Thank goodness because the weather in the South is considerably more pleasant. After spending my school days as “the girl who always ruined the curve”, I decided to attend Virginia Tech and pursued a BS in Chemical Engineering. When I wasn’t studying during undergrad, I loved to hike the trails in the area surrounding Blacksburg, VA. My younger sister followed me to Virginia Tech, so I also spent many free hours with her in the land of Hokies. After undergrad, I moved to West Virginia for a brief period to work as a Process Engineer in the polymer industry. Although I enjoyed the hiking there, the job just wasn’t for me and I decided to return to academia for a PhD. During graduate school at NC State, I majored in Biological and Agricultural Engineering and found my calling doing fermentation research. I love to bake so the ability to mix ingredients together in a lab was fun for me. While living in Raleigh, NC during graduate school, I took up the new hobbies of Zumba and salsa dancing. I also continued my existing hobbies of “making stuff” which included knitting my first afghan and sewing Halloween costumes for several of the dogs in my family (I love dogs). iv ACKNOWLEDGMENTS I wish acknowledge first and foremost my parents, John and Laura Slivka, who raised me to be the person I am today. Without your love and guidance, I would not have had the courage and perseverance to accomplish as much as I have thus far in life. And thank you so very much for supporting me when I decided to get a PhD! Appreciation also goes to my sister, Reba, who is my best friend and has supported me in my endeavors just as much as I have supported her in her own. You are my cheerleader for life! A big thank you goes to Dr. Mari Chinn who is absolutely the best research advisor. You have been a fantastic role model and mentor and, on the days when I needed it, you treated me like a true friend. Last of all, I want to acknowledge my research family: Joscelin Diaz, Matt Whitfield, Ed Godfrey, and Ana Zuleta Correa. We have laughed together, vented about crazy things together, and gone on many a road trip to the field or conferences together. Without your help, graduate life would have been considerably more hectic. v TABLE OF CONTENTS LIST OF TABLES ........................................................................................................................ vi LIST OF FIGURES ..................................................................................................................... vii Chapter 1: Gasification and Synthesis Gas Fermentation: An Alternative Route to Biofuel Production ....................................................................................................................... 1 Summary ............................................................................................................................ 2 Introduction ........................................................................................................................ 2 Gasification ........................................................................................................................ 5 Microorganisms that can convert synthesis gas components to biofuels .......................... 8 Mechanisms of microbial synthesis gas utilization ........................................................... 9 Synthesis gas fermentation .............................................................................................. 12 Future perspective ............................................................................................................ 20 References ........................................................................................................................ 32 Chapter 2: Influence of Carbon Source Preadaptation on Clostridium autoethanogenum........................................................................................................................ 39 Abstract ............................................................................................................................ 40 Introduction ...................................................................................................................... 41 Materials and Methods ..................................................................................................... 43 Results and Discussion .................................................................................................... 48 Conclusion ....................................................................................................................... 62 References .......................................................................................................................
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