Acetone-Butanol-Ethanol Fermentation by Engineered Clostridium Beijerinckii and Clostridium Tyrobutyricum
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Acetone-Butanol-Ethanol Fermentation by Engineered Clostridium beijerinckii and 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 Wei-Lun Chang Graduate Program in Food Science and Technology The Ohio State University 2010 Dissertation Committee: Professor Shang-Tian Yang, Advisor Professor Jeffrey Chalmers Professor Ahmed Yousef Copyright by Wei-Lun Chang 2010 Abstract Recently, biofuel production is a popular topic with the rising concerns about the limited supply of fossil fuel and the fluctuating price of crude oil. Biofuels can be categorized into different groups based on their characteristics and applications, including biodiesel, bioalcohols, biogas, and solid biofuels. Bioalcohols, such as methanol, ethanol, and butanol, have been considered to replace transportation fuels. Among the bioalcohols, butanol attracted more and more attentions because of its superiority over other bioalcohol candidates with its similar properties to automobile gasoline, such as octane numbers, energy content, and energy density. Other advantages for using butanol as a biofuel are its low hygroscopicity and low vapor pressure which prevent increase in moisture content in butanol and make butanol a safe and environmentally friendly biofuel. Butanol, a butyl alcohol, can be produced from sugars and starch by solvent- producing clostridia through acetone-butanol-ethanol (ABE) fermentation. ABE fermentation was once a large industrial fermentation for solvent production from starch- based substrates and molasses during the World Wars in the early twentieth century. However, this practice gradually disappeared because its products could not compete with solvents produced through petro-chemical syntheses. Another reason that butanol production from ABE fermentation declined was because it suffered from low titer, yield and productivity, which made it not economically feasible for commercialization with the ii high cost in downstream product recovery. In order to improve butanol production from ABE fermentation, extensive studies have been investigated in the aspects of metabolic engineering and process development. The objectives of this study were to develop enhanced butanol production from ABE fermentation in continuous FBB by mutants of C. beijerinckii, and to explore the possibility of producing butanol by metabolic engineered hosts. ABE fermentation has two phases, acidogenesis and solventogenesis. In acidogenesis, acetic acid and butyric acid are produced, while acetone, ethanol, and butanol are produced in solventogenesis. A stoichiometric model was developed for the analysis of the metabolic pathway in solvent producing clostridia that carry out ABE fermentation. The model predicted the maximum butanol yield based on mass and energy balance at steady state in continuous process. This helped process development without redundant experiments. In the stoichiometric analysis, the supplement of butyric acid in the medium was shown to benefit butanol production from ABE fermentation by reducing required energy to produce butanol (NADH) and increasing butyrate phosphate production which upregulates solventogenesis. Two different mutants of C. beijerinckii were cultured in continuous fermentation to study the effect of using butyric acid as a supplement in the medium under different conditions. At a high dilution rate of 1.88 h-1, asporogenic C. beijerinckii ATCC 55025 in the fibrous bed bioreactor (FBB) was able to utilize glucose and butyrate and steadily give a high butanol productivity of 17.29 g/L∙h, which was 170 folds higher than that in conventional free-cell batch fermentation. A new strain isolated from C. beijerinckii iii ATCC 55025 in repeated-batch culture in the FBB, C. beijerinckii JB200 was studied on its performance in continuous fermentation. C. beijerinckii JB200 is superior to other industrial strains with its better tolerance of solvent, acid and feeding substrate. In continuous fermentation, butanol titer was increased to 12 g/L at lower dilution rate by C. beijerinckii JB200. In the studies of continuous fermentation by two C. beijerinckii mutants, fibrous bed bioreactor (FBB) was used. It is a novel technology that can help cell immobilization at high cell density for adaptation. In continuous FBB, C. beijerinckii mutants were able to be retained at high cell density (>100 g dried weight/L) in the bioreactor and steadily produce butanol without contamination or degeneration. With incorporation of FBB, C. beijerinckii was able to effectively use both glucose and butyric acid to produce butanol. Besides solvent producing clostridia, metabolically engineered Clostridium tyrobutyricum was studied for its potential to produce butanol. The metabolic engineered mutant of C. tyrobutyricum ATCC 25755 was able to actively produce butanol over six repeated batches in the FBB using glucose and/or xylose. In batches with only xylose as the carbon source, the mutant was able to produce more butanol at 7.12 g/L with a higher yield because of the slower cell growth that made the foreign aldehyde/alcohol dehydrogenase competitive to the native enzyme for butyryl CoA. A pilot scale FBB was constructed for the evaluation of the feasibility of large scale butanol production in the FBB. Two batches of batch fermentation were operated and the results of ABE fermentation were similar to those in batch fermentation at lab scale. ABE fermentation at pilot scale was able to produce butanol at 8.7 g/L and 9.4 g/L iv with feed media containing glucose and butyric acid of 2 g/L and 5 g/L, respectively. The butyrate uptake was found better and higher ratio of butanol to total produced solvents in FBB under continuous, repeated batch, and pilot scale. In summary, continuous butanol production with improved productivity from ABE fermentation with supplement of butyric acid as additional carbon source in the FBB was demonstrated. The supplement of butyric acid was able to maintain the process in solventogenesis and produce higher ratio of butanol to total produced solvents. The FBB was able to stabilize butanol production in continuous process. More studies are necessary to improve butanol production by metabolically engineered strains. At pilot scale, the FBB was able to produce butanol with a result that was comparable to that in free cell batch reactor at lab scale. The pilot scale FBB can be further operated under different modes of fermentation for process optimization. v Dedication Dedicated to my parents and my sisters vi Acknowledgments First of all, I would like to thank my advisor, Dr. Shang-Tian Yang, for his guidance, encouragement, patience, and full support during my graduate study. I am eternally grateful for his help academically and financially throughout the completion of my Ph.D study. I have benefited a lot from his expertise in science and his great personality. I wish to thank Dr. Ahmed Yousef and Dr. Jeffrey Chalmers for their time serving on my committee and their valuable suggestions and advices to my research project. I would like to acknowledge Dr. An Zhang for his help on preliminary experiment setup and sincere thanks to all the previous and current laboratory members, especially Dr. Yali Zhang, Dr. Mingrui Yu, Ching-suei Hsu, Jianxin Sun, and Congcong Lu for their encouragement and support. Special thanks to Mr. Bingxu Song for his help on the establishment of the foundation of pilot plant, valuable discussion with Dr. Yali Zhang on the study of stoichiometric analysis, and Dr. Mingrui Yu for his construction of C. tyrobutyricum mutants. Financial supports from the Ohio Department of Development Third Frontier Advanced Energy Program, the Consortium for Plant Biotechnology Research, Inc., and EnerGenetics International during the course of this research are appreciated. vii Finally I would like to thank my parents Mr. Tien-Yang Chang and Mrs. Chao-Yu Chang Yu, my sisters, and my boyfriend for their unconditional love and support, and all of my family members and friends for their having faith in me. viii Vita June 1999 .......................................................Song-Shan Senior High 1999-2003 ......................................................B.S. Chemical Engineering, National Taiwan University 2003-2005 ......................................................Research Assistant, Academia Sinica 2005 to present ..............................................Graduate Research Associate, Department of Food Science and Technology, The Ohio State University Fields of Study Major Field: Food Science and Technology ix Table of Contents Abstract……………………………………………………………………………………ii Dedication………………………………………………………………...……………….v Acknowledgments………………………………………………………………………..vi Vita………………………………...…………………………………………………….vii Table of Contents………………………………………………………………………..viii List of Tables…………………………………………………………………………....xiii List of Figures……………………………………………………………………………xv Chapter 1: Introduction……………………………………………………………………1 1.1 Objectives and Research Tasks………………………………………………..3 1.2 Reference……………………………………………………………………7 Chapter 2: Literature Review…………………………………………………………….11 2.1 Biofuels………………………………………………………………………11 2.1.1 Bioalcohols – Bioethanol…………………….…………………….12 2.1.2 Biodiesel………………………………………………………….14 2.1.3 Biogas and solid biofuels…………………………………………..16 2.2 Production of Biobutanol…………………………………………………….18 2.2.1 Butanol……………………………………………………………18 2.2.2 ABE fermentation……………………………………………….19 2.3 Process