University of Kentucky UKnowledge University of Kentucky Doctoral Dissertations Graduate School 2008 METABOLITE ANALYSIS OF CLOSTRIDIUM THERMOCELLUM USING CAPILLARY ELECTROPHORESIS BASED TECHNIQUES Anup P. Thakur University of Kentucky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Thakur, Anup P., "METABOLITE ANALYSIS OF CLOSTRIDIUM THERMOCELLUM USING CAPILLARY ELECTROPHORESIS BASED TECHNIQUES" (2008). University of Kentucky Doctoral Dissertations. 647. https://uknowledge.uky.edu/gradschool_diss/647 This Dissertation is brought to you for free and open access by the Graduate School at UKnowledge. It has been accepted for inclusion in University of Kentucky Doctoral Dissertations by an authorized administrator of UKnowledge. For more information, please contact [email protected]. ABSTRACT OF DISSERTATION Anup P. Thakur Graduate School University of Kentucky 2008 METABOLITE ANALYSIS OF CLOSTRIDIUM THERMOCELLUM USING CAPILLARY ELECTROPHORESIS BASED TECHNIQUES ABSTRACT OF DISSERTATION A dissertation submitted in partial fulfillment of degree of Doctor of Philosophy in the college of Arts and Sciences at the University of Kentucky By Anup P. Thakur Director: Dr. Bert C. Lynn Professor of Chemistry Lexington, KY 2008 Copyright © Anup P. Thakur 2008 ABSTRACT OF DISSERTATION METABOLITE ANALYSIS OF CLOSTRIDIUM THERMOCELLUM USING CAPILLARY ELECTROPHORESIS BASED TECHNIQUES Clostridium thermocellum is a thermophilic bacterium that converts biomass to ethanol directly; however, high sensitivity of this bacterium toward ethanol limits its commercial utility. To elucidate the effect of ethanol on the growth of this bacterium a metabolite analysis of C. thermocellum was performed. The hypothesis of the project was that exogenous ethanol alters the metabolite profile of C. thermocellum. For metabolite analysis, capillary electrophoresis-electrospray ionization-mass spectrometry method (CE-ESI-MS) was developed due to highly polar and charged nature of metabolites. To increase the sensitivity of CE-ESI-MS, several parameters at the ESI interface were optimized. The application of 50% isopropanol as a sheath liquid increased sensitivity for metabolite analysis dramatically. Trimethylamine acetate (pH 10) was used as background electrolyte (BGE) due to its ability to separate the structural isomers of glucose phosphate. For metabolite sample preparation, novel methods for quenching and CE compatible metabolite extraction protocols were developed. Newly developed protocols were applied to metabolite analysis of wild type (WT) and ethanol adapted (EA) strains of C. thermocellum grown in batch cultures. Significant differences were found in key intracellular metabolites such as NAD+ and pyruvic acid. Intracellular concentrations of NAD+ were low in EA cells compared to WT cells and pyruvic acid was only detected in EA cells. To further understand the effect of ethanol on metabolite fluxes, WT and EA cells were grown in increasing concentrations of ethanol and the metabolite profile for each ethanol treatment was obtained. Significant changes were found in intracellular metabolite concentrations. Metabolic data showed that the glycolysis process in WT cells was obstructed due to exogenous ethanol which was evident from accumulation of G6P. On the other hand, no such accumulation of G6P was observed in the EA strain; however pyruvate began to accumulate in EA strain. These changes in intracellular metabolite concentrations due to perturbation of exogenous ethanol supported the hypothesis. Also, this investigation revealed a correlation between ethanol and metabolite profile changes and was able to explain a possible mechanism of growth inhibition of C. thermocellum which will certainly help genetic engineers to develop superior strains of C. thermocellum for commercial cellulosic ethanol production. Keywords: C. thermocellum, metabolomics, quenching, extraction, capillary electrophoresis-electrospray ionization-mass spectrometry. Anup Thakur 2008 IV METABOLITE ANALYSIS OF CLOSTRIDIUM THERMOCELLUM USING CAPILLARY ELECTROPHORESIS BASED TECHNIQUES By Anup P. Thakur ______________________________________ Dr. Bert C. Lynn (Director of Dissertation) ______________________________________ Dr. Robert B. Grossman (Director of Graduate Studies) RULES FOR THE USE OF DISSERTATIONS Unpublished dissertations submitted for the Doctor’s degree and deposited in the University of Kentucky Library are as a rule open for inspection, but are to be used only with due regard to the rights of authors. Bibliographical references may be noted, but quotations or summaries of the parts may be published only with permission of the author, and with the usual scholarly acknowledgements. Extensive copying or publication of the dissertation in whole or in part also requires the consent of the Dean of the Graduate School of the University of Kentucky. A library that borrows this dissertation for use by its patrons is expected to secure the signature of each user. Name Date ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ____________________________________________________________________ DISSERTATION Anup P. Thakur Graduate School University of Kentucky 2008 METABOLITE ANALYSIS OF CLOSTRIDIUM THERMOCELLUM USING CAPILLARY ELECTROPHORESIS BASED TECHNIQUES ABSTRACT OF DISSERTATION A dissertation submitted in partial fulfillment of degree of Doctor of Philosophy in the college of Arts and Sciences at the University of Kentucky By Anup P. Thakur Director: Dr. Bert C. Lynn Professor of Chemistry Lexington, KY 2008 Copyright © Anup P. Thakur 2008 Dedicated to My beloved Uncle Arvind Thakur ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Bert Lynn for accepting me in his group and introducing me to the wonderful world of mass spectrometry. A million thanks to him for providing me guidance, motivation, and inspiration throughout my research work. He is really a wonderful person and great mentor and helped me to grow from a chemist to a scientist. My sincere thanks to Dr. Herbert Strobel, Professor of Animal and Food Sciences, without his guidance and help I would not have completed my research project. Constant interactions with him provided opportunities to learn basic as well as modern aspects of microbiology, especially when I didn’t have much credentials of biology. I am also thankful to Dr. Barbara Knutson, Professor of Chemical Engineering; Dr. Nokes, Professor of Biosystems and Agriculture engineering; Dr. Satyakrishna Jujjuri, Postdoctoral fellow at Department of Chemical Engineering; and all other people involved in KREC project. I also take this opportunity to thank Dr. Jack Goodman, Manager of University of Kentucky Mass Spectrometry Facility for his resourceful and kind assistance on several occasions. I really appreciate all the members and colleagues from Lynn laboratory for their fellowship and making graduate career an amazing experience. I will always relish those memories in future. I also show my gratitude toward all the graduate students and staff from the Department of Chemistry at the University of Kentucky. Lastly, I will be indebted all my life to my uncle Mr. Arvind Thakur for his constant inspiration and support during the tough time of my PhD. He helped me to make my academic journey really smooth sailing when the path was obstructed by the storms of challenges and shadows of uncertainties. I want to thank you all for helping me in fulfilling my academic goals. I TABLE OF CONTENTS ACKNOWLEDGEMENTS ............................................................................................... I TABLE OF CONTENTS.................................................................................................. II LIST OF TABLES .......................................................................................................... VI LIST OF FIGURES .......................................................................................................VII LIST OF FILES .............................................................................................................. X Chapter 1 : Introduction ..................................................................................................1 1.1 Study goal ......................................................................................................................... 2 1.2 Study hypothesis ............................................................................................................... 2 1.3 Study rationale .................................................................................................................. 2 1.4 Specific goals .................................................................................................................... 3 Chapter 2 : Background ..................................................................................................4