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Production of Butyric Acid and Hydrogen by Metabolically Engineered Mutants of Clostridium Tyrobutyricum

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Production of Butyric Acid and Hydrogen by Metabolically Engineered Mutants of Clostridium Tyrobutyricum PRODUCTION OF BUTYRIC ACID AND HYDROGEN BY METABOLICALLY ENGINEERED MUTANTS OF CLOSTRIDIUM TYROBUTYRICUM DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Xiaoguang Liu, M.S. ***** The Ohio State University 2005 Dissertation committee: Approved by Professor Shang-Tian Yang, Adviser Professor Barbara E Wyslouzil Adviser Professor Hua Wang Department of Chemical Engineering ABSTRACT Butyric acid has many applications in chemical, food and pharmaceutical industries. The production of butyric acid by fermentation has become an increasingly attractive alternative to current petroleum-based chemical synthesis. Clostridium tyrobutyricum is an anaerobic bacterium producing butyric acid, acetic acid, hydrogen and carbon dioxide as its main products. Hydrogen, as an energy byproduct, can add value to the fermentation process. The goal of this project was to develop novel bioprocess to produce butyric acid and hydrogen economically by Clostridial mutants. Conventional fermentation technologies for butyric acid and hydrogen production are limited by low reactor productivity, product concentration and yield. In this project, novel engineered mutants of C. tyrobutyricum were created by gene manipulation and cell adaptation. Fermentation process was also optimized using immobilizing cells in the fibrous-bed bioreactor (FBB) to enhance butyric acid and hydrogen production. First, metabolic engineered mutants with knocked-out acetate formation pathway were created and characterized. Gene inactivation technology was used to delete the genes of phosphotransacetylase (PTA) and acetate kinase (AK), two key enzymes in the acetate-producing pathway of C. tyrobutyricum, through homologous recombination. The metabolic engineered mutants were characterized by Southern hybridization, enzyme assay, protein expression and metabolites production. The enzyme assays showed that ii PTA and AK activities in the pta-deleted mutant (PPTA-Em) were reduced by 44% and 91%, respectively, whereas AK activity in the ack-deleted mutant (PAK-Em) decreased by 50%. Meanwhile, the activity of BK in PPTA-Em increased by 44%, and hydrogenase activity in PAK-Em increased by 40%. The SDS-PAGE showed that the expression of the proteins around 32 kDa and 70 kDa had significant changes in the mutants. Two dimensional protein electrophoresis gels showed that both PTA and AK were deleted from mutants. Butyric acid tolerances were improved significantly in the mutants, indicating high butyric acid productivity. The free cell fermentation by PPTA-Em produced 40 g/L of butyric acid using glucose with lower specific cell growth rate, lower acetate yield (0.058 g/g), higher butyric acid yield (0.38 g/g), butyrate productivity (0.63 g/L·h), and consequent higher selectivity of butyrate over acetate as compared with that of wild type. In order to improve butyric acid and hydrogen production, a novel fibrous-bed bioreactor (FBB) was applied to immobilize the metabolically engineered mutants. The immobilization fermentation using PPTA-Em showed that butyric acid concentration was improved to 50 g/L with higher butyric acid yield compared with that of free cell fermentation. The effect of sugar sources and cell adaptation on the fermentation was also studied. As compared with wild type, the specific growth rate of PAK-Em from glucose decreased by 50% (from 0.24 h-1 to 0.14 h-1) because of the impaired PTA-AK pathway. Meanwhile, butyric acid production by the mutant was improved greatly, with higher butyric acid yield (0.42 g/g vs. 0.34 g/g) and final concentration (42 g/L vs. 20 g/L), Also, hydrogen production by PAK-Em mutant increased significantly, with higher hydrogen yield (2.61 vs. 1.35 mol/mol glucose) and H2/CO2 ratio (1.44 vs. 1.04). Free cell iii fermentation using various sugar sources, including glucose, xylose and fructose, were carried out to evaluate their abilities to produce butyric acid and hydrogen. Repeated fed- batch immobilization fermentations using FBB were optimized to improve butyric acid and hydrogen production further. Through adaptation in the FBB fibrous matrix, a high butyric acid concentration of 81 g/L was obtained at pH 6.3 with PAK-Em. This concentration was the highest ever attained in butyric acid fermentation to date. The butyrate yield was also increased to ~0.45 g/g due to the reduced cell growth in the immobilized-cell fermentation. A new adaptation mutant that produced even more hydrogen with a H2/CO2 ratio of 2.69 and with very fast growth rate was discovered from the FBB matrix. In order to better understand the metabolic mechanism of butyrate production by the metabolically engineered mutant of C. tyrobutyricum, metabolic flux analysis was applied to quantitatively describe the global cellular metabolism. Different pH values, from pH 5.0 to pH 7.0, were applied with the FBB fermentation by PAK-Em using glucose and xylose to evaluate the metabolic changes. The stoichiometric analysis indicated the PTA-AK metabolic pathway to produce acetic acid was blocked completely in the mutant. The novel metabolic engineered mutants and the FBB application are important to the development of an economical bioprocess for butyric acid and hydrogen production from biomass by C. tyrobutyricm. iv Dedicated to my parents and my husband Lufang Zhou v ACKNOWLEDGMENTS My great appreciation goes to my advisor, Dr. Shang-Tian Yang, for his enormous help both academically and financially throughout my Ph.D. study. I am eternally grateful for his inspiring advice, warm support and encouragement, and the flexibility he gave me in conducting my research. I gained a lot from his insights and I have greatly enjoyed my staying here as his student. I would also like to acknowledge with sincere gratitude to the members of my dissertation committee, Dr. Hua Wang and Dr. Barbara Wyslouzil. I am grateful for their helpful suggestions and advice offered on a variety of topics. I would like to thank Dr. Ying Zhu and Ms. Yali Zhang for their help in the work reported in Chapters 3 and 8, respectively. The technical assistance provided by Dr. Ying Zhu at the initial stage of my research is gratefully acknowledged. My colleagues within my research group in the Department of Chemical Engineering offered many kinds of support over the last four years. I have been fortunate in working in such a good team. Financial support from the Department of Energy-STTR Program, the Consortium for Plant Biotechnology Research, and the U.S. Department of Agriculture-NRI Program to the various phases of this work is acknowledged. Special thanks go to my husband Lufang Zhou and my parents for their sustained support over the last four years. vi VITA November 3, 1975 ...……………………………….Born – Shouguang, China July, 1997…...………………………………………B.S. Chemical Engineering Shandong (Tech) University Shandong, China April, 2000 …...………………………………..……M.S. Biochemical Engineering Tianjin University Tianjin, China August, 2000 – July, 2001...………………………...University Research Assistant University of Connecticut September, 2001 – August, 2002...…………………University Fellow The Ohio State University September, 2002 – September, 2003..……………... Graduate Research Assistant Biochemical Engineering The Ohio State University October, 2003 – September, 2004 ………………….CPBR Research Fellow The Ohio State University October, 2004 – August, 2005..……………............. Graduate Research Assistant Biochemical Engineering The Ohio State University June, 2005..……………………………...…............. Alumni Grants for Graduate Research and Scholarship (AGGRS) The Ohio State University vii PUBLICATION 1. Liu, X., Zhu, Y., Yang, S.T. 2005. Butyric acid and hydrogen production by Clostridium tyrobutyricum ATCC 25755 and mtants. Enzyme Microbial Technolo. In press. 2. Zhu, Y., Liu, X., Yang, S.T. 2005. Construction and characterization of pta gene deleted mutant of Clostridium tyrobutyricm for enhanced butyric acid fermentation. Biotechnol. Bioeng. 90, 154-166. 3. Dong, X., Wang, Y., Liu, X., Y. Sun. 2001. Kinetic model of lysozyme renaturation with the molecular chaperone GroEL. Biotechnol. Lett. 23, 1165-1169. 4. Dong, X., Bai, S., Liu, X., Sun, Y. 2001. Kinetics of lysozyme refolding facilitated by molecular chaperone GroEL. Huagong Xuebao. 52, 1049-1053. 5. Liu, X., Dong, X. 2000. Molecular chaperone and protein renaturation. Chem. Ind. Eng. 17, 120-124. 6. Liu, X., Dong, X., Zhou, L., Wang, Y., Zeng, K., Sun, Y. 2000. Kinetics of lysozyme refolding assisted by chaperonin GroEL. Proceeding paper at 10th National Conference on Chemical Engineering. FIELD OF STUDY Major Field: Chemical Engineering Specialty: Biochemical Engineering viii TABLE OF CONTENTS Page ABSTRACT ....…...……………….……………………………………….…….….……ii DEDICATION .…..…...………………………..……………………………...…….…...v ACKNOWLEDGEMENT ....….……………………………………………….…….....vi VITA ...………………...…………………………………….…………………….........vii LIST OF TABLES ….…………….……………….……….…………...………….…viii LIST OF FIGURES …....…………………………………..…………………………..xiv Chapters: 1. Introduction ...………….…………………………………………………..…….1 2. Literature Review………………………………………………………..……..13 2.1 Metabolic Engineering and Mutant Development ….……...……………13 2.1.1 Gene Manipulation..…………...….………………………….....…..…14 2.1.2 Other Methods...…………………….….…………………….....…..…19 2.1.3 Mutant Characterization..….………..….…………………….....…..…22 2.2 Butyric Acid and Hydrogen Production from Fermentation ..……...…23 2.2.1 Butyric acid and Hydrogen…….……….…………………….....…..…23 2.2.2 Microorganisms for Fermentation…..………………………….....…..…25
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