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BUTANOL PRODUCTION from GLYCEROL by Clostridium Pasteurianum in DEFINED CULTURE MEDIA- a PHENOTYPIC APPROACH

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BUTANOL PRODUCTION from GLYCEROL by Clostridium Pasteurianum in DEFINED CULTURE MEDIA- a PHENOTYPIC APPROACH ABSTRACT Title of Document: BUTANOL PRODUCTION FROM GLYCEROL BY Clostridium pasteurianum IN DEFINED CULTURE MEDIA- A PHENOTYPIC APPROACH. David Leonardo Ramos Sanchez, Master of Science, 2009 Directed By: Associate Professor Nam Sun Wang Department of Chemical and Biomolecular Engineering ABSTRACT The fluctuations in oil prices have stimulated the production of renewable biofuels, in particular the production of bioethanol and biodiesel. The production of biodiesel has expanded almost six fold in the past years. The ten wt% of the biodiesel process results in crude glycerol. Once a valuable product, nowadays glycerol is considered a waste and a surplus material. Its current low price makes it an attractive substrate for a fermentation process. Molecular genetics have unveiled new insights about solvent production in Clostridia. It has been recognized that endospore development and solvent formation share a regulatory mechanism. The solvent production, particularly the butanol fermentation of glycerol by Clostridium pasteurianum was studied. Taking advantage of the characteristics of the sporulation phenotype, the study of the butanol fermentation was approached. A relation between spore formation and butanol production was found in C. pasteurianum by applying molecular genetics concepts. BUTANOL PRODUCTION FROM GLYCEROL BY Clostridium pasteurianum IN DEFINED CULTURE MEDIA- A PHENOTYPIC APPROACH By David Leonardo Ramos Sanchez Thesis submitted to the Faculty of the Graduate School of the University of Maryland, College Park, in partial fulfillment of the requirements for the degree of Master of Science 2009 Advisory Committee: Professor Nam Sun Wang, Chair Ganesh Sriram John Fisher © Copyright by David Leonardo Ramos Sanchez 2009 Dedication To Jesus Christ who never abandoned me and answered my prayers. He is worthy to receive praise. ii Acknowledgements To Catalina for all her patience, support and love showed during all this process. To my mother and sister and family members who encouraged me during the hard times. To my professors and friends who gave me wise advise. To the Fulbright commission, LASPAU, Sucromiles S.A, and Colciencias for sponsoring my studies.. iii Table of Contents Dedication..................................................................................................................... ii Acknowledgements......................................................................................................iii Table of Contents......................................................................................................... iv List of Tables ............................................................................................................... vi List of Figures............................................................................................................. vii Chapter 1: Introduction and Overview ......................................................................... 1 1.1. Problem description and motivation ............................................................. 1 2. Chapter 2: Background: glycerol, Clostridium pasteurianum and ABE fermentation process ..................................................................................................... 4 2.1. Glycerol from Biodiesel: A premier .......................................................... 4 2.1.1. Biodiesel Feedstocks............................................................................. 4 2.1.2. Transesterification reaction................................................................... 5 2.2. Lessons from the ABE process..................................................................... 6 2.2.1. Brief history of the industrial process................................................... 6 2.2.2. Previous Work Reviewed ..................................................................... 9 2.3. Clostridium pasteurianum and the sporulation process .............................. 10 2.3.1. Class Clostridia and relevant aspects to C. pasteurianum .................. 10 3. Chapter 3: Materials and Methods...................................................................... 12 3.1. Strain utilized.............................................................................................. 12 3.2. Growth Medium and fermentation conditions............................................ 12 3.3. Glycerol Quantification .............................................................................. 16 3.3.1. Spectophotometric method by Biodioli .............................................. 16 3.3.2. Description of Bondioli Method ......................................................... 17 3.3.3. Glycerol Quantification using AOCS method .................................... 19 3.4. Fermentation Products ................................................................................ 21 3.4.1. Quantitative analysis........................................................................... 22 3.4.2. Internal standard method..................................................................... 22 3.4.3. Gas chromatograph method ................................................................ 24 4. Chapter 4: Media Development.......................................................................... 26 4.1. Introduction................................................................................................. 26 4.2. Defined media Formulation ........................................................................ 27 4.2.1. Materials and Methods........................................................................ 31 4.2.2. Results................................................................................................. 34 4.2.3. Discussion........................................................................................... 37 5. Chapter 5: Fermentation Parameters................................................................ 39 5.1. Introduction................................................................................................. 39 5.2. Kinetic Fermentation Parameters................................................................ 40 5.2.1. Specific growth rate ............................................................................ 40 5.3. Determining Yield coefficients................................................................... 43 5.3.1. Growth Yield ...................................................................................... 44 5.4. Product Formation Kinetics ........................................................................ 47 iv 5.4.1. Type of microbial product .................................................................. 47 6. Chapter 6: Sporulation and Solvent Production............................................... 51 6.1. Introduction................................................................................................. 51 6.2. Clostridial Sporulation................................................................................ 52 6.3. Methods and rationale behind the experiments .......................................... 54 6.3.1. Selection of heat resistant spores ........................................................ 54 6.3.2. Experimental procedures .................................................................... 55 6.4. Results......................................................................................................... 56 6.4.1. Selection of heat resistant cells........................................................... 59 6.4.2. Heat resistant individuals and the carbon source................................ 60 6.4.3. Number of heat resistant cells and solvent production ....................... 61 6.5. Discussion................................................................................................... 62 Chapter 7: Study of the effects of hydrosulfite during the growth and the effects in the solvent production....................................................................................................... 65 7.1. Introduction...................................................................................................... 65 7.1.1.Selecting the reducing Agent..................................................................... 65 7.1.2.Dissimilative Sulfate Reduction ................................................................ 67 7.2.Methods............................................................................................................. 69 7.2.1. Experimental methods .............................................................................. 70 7.4.Discussion......................................................................................................... 76 8. Chapter 8: Conclusions........................................................................................... 78 9. Chapter 9: Future Work .......................................................................................... 80 Bibliography ............................................................................................................... 81 v List of Tables Table 2-1 ABE fermentation parameters at industrial scale. ........................................ 8 Table 2-2 Relevant results in Biebl’s work (Biebl 2001) ........................................... 10 Table 4-1 Macronutrients and micronutrients required for most organisms. ............. 28 Table 4-2 Media formulations .................................................................................... 31 Table
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