Brigham Young University BYU ScholarsArchive Theses and Dissertations 2010-03-18 Syngas Fermentation: Quantification of Assay Techniques, Reaction Kinetics, and Pressure Dependencies of the Clostridial P11 Hydrogenase Bradley E. Skidmore Brigham Young University - Provo Follow this and additional works at: https://scholarsarchive.byu.edu/etd Part of the Chemical Engineering Commons BYU ScholarsArchive Citation Skidmore, Bradley E., "Syngas Fermentation: Quantification of Assay Techniques, Reaction Kinetics, and Pressure Dependencies of the Clostridial P11 Hydrogenase" (2010). Theses and Dissertations. 2098. https://scholarsarchive.byu.edu/etd/2098 This Thesis is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. Syngas Fermentation: Quantification of Assay Techniques, Reaction Kinetics, and Pressure Dependencies of the Clostridial P11 Hydrogenase Bradley Skidmore A thesis submitted to the faculty of Brigham Young University in partial fulfillment of the requirements for the degree of Master of Science Randy S. Lewis, Chair Steven R. Herron Thomas A. Knotts Department of Chemical Engineering Brigham Young University April 2010 Copyright © 2010 Bradley Skidmore All Rights Reserved ABSTRACT Syngas Fermentation: Quantification of Assay Techniques, Reaction Kinetics, and Pressure Dependencies of the Clostridial P11 Hydrogenase Bradley Skidmore Department of Chemical Engineering Master of Science Ethanol usage as a transportation fuel is rapidly increasing in the United States. Production of ethanol from cellulose feedstocks via gasification followed by syngas fermentation offers an environmentally friendly approach that mitigates many of the adverse effects associated with production from corn. In the syngas fermentation process, the hydrogenase enzyme of the fermentation bacterium, Clostridium P11 for this work, supplies electrons to the metabolic pathway, facilitating ethanol production. In this thesis, an assay for P11 hydrogenase activity was developed. It was determined that 1) less than 4 minutes of sparging with 50 sccm H2 is needed to reduce O2 levels to below 1 ppm in a 3 mL aqueous solution, while less than 1 minute of purging at the same rate is needed to fill an air-filled 3.5 mL cuvette to 99.9999% H2, 2) 12.5 mM DTT included in the reaction mixture at pH 6 helps scavenge O2, 3) H2 diffusion is slow compared to enzymatic reaction rates, 4) CO2 lowers media pH, 5) 0.084 atm CO causes 90% inhibition of P11 hydrogenase, 6) prolonged Triton X-100 exposure diminishes hydrogenase activity, and 7) variations in H2 pressure and electron acceptor identity and concentration affect measured hydrogenase activities. The assay developed for P11 hydrogenase activity was used to perform kinetic studies. The Okura rapid-equilibrium rate law best described this activity. A constant that regulates the effect of H2 pressure on hydrogenase activity, KH2, was determined to be independent of electron acceptor and to have a value of 0.31 atm, implying that H2 must be supplied to the syngas fermentation at ~3 atm to maximize hydrogenase activity. KBV and KMV, constants that regulate the effect of benzyl viologen and methyl viologen on hydrogenase activity, were determined to be 1.7-2.4 mM and 10.6 mM, respectively. Additionally, hydrogenase activity was temporally correlated with ethanol production in batch cultures of P11 and strongly dependent on pH. The intracellular pH of P11 was determined to be approximately 5.5. Keywords: ethanol, hydrogenase, syngas, fermentation, Clostridium, P11, assay ACKNOWLEDGMENTS I am most indebted to my advisor, Randy Lewis, and to the four undergraduate students with whom I worked to perform this research: Doug Tree, Jason Bray, Dila Banjade, and Ryan Baker. Their insights, hard work, and encouragement have been invaluable. I am grateful for the other professors and graduate students who have contributed their wisdom, time, ideas, and facilities to the furtherance of this work. Additionally, I am grateful to the staffs of both Brigham Young University and Los Alamos National Laboratory who have made it possible to carry this work through to completion. Finally, I am grateful to my wife, Jessica, and to my parents, Cary and Wendy, whose love and support has strengthened me at each step along the journey. TABLE OF CONTENTS LIST OF TABLES ....................................................................................................................... ix LIST OF FIGURES ..................................................................................................................... xi 1 Introduction ........................................................................................................................... 1 1.1 Current and Projected Production of Fuel Ethanol ......................................................... 1 1.2 Methods of Ethanol Production ...................................................................................... 2 1.3 Metabolic Pathway of Acetogenic Syngas Fermentation ............................................... 3 1.4 Pressure Dependence of Enzymatic Reaction Rates ....................................................... 5 1.5 Research Objectives ........................................................................................................ 8 1.5.1 Objective 1- Assay Development ............................................................................... 8 1.5.2 Objective 2- Kinetic Modeling ................................................................................... 9 1.5.3 Objective 3- Physiological Insights ............................................................................ 9 2 Literature Review ............................................................................................................... 11 2.1 Hydrogenase Assay Procedures .................................................................................... 11 2.1.1 Manometric Assay .................................................................................................... 12 2.1.2 Ferredoxin Assay ...................................................................................................... 13 2.1.3 Artificial Dye Assays ................................................................................................ 13 2.1.4 Electrochemical Assays ............................................................................................ 14 2.2 Complications with Performing Hydrogenase Assays ................................................. 14 2.2.1 Anaerobic Conditions ............................................................................................... 15 2.2.2 Gas Solubility and Diffusion .................................................................................... 16 2.2.3 Enzyme Inhibition ..................................................................................................... 17 2.2.4 Whole Cell vs Purified Enzymes .............................................................................. 17 2.2.5 Rate Discrepancies with Electron Acceptors ............................................................ 18 v 2.3 Kinetic Modeling of Hydrogenase ................................................................................ 19 2.3.1 Nickel-Iron Hydrogenase .......................................................................................... 21 2.3.2 Iron-Iron Hydrogenase .............................................................................................. 23 2.4 Physiological Role of Hydrogenase .............................................................................. 25 2.4.1 Regulation of Redox Potential .................................................................................. 25 2.4.2 Hydrogenase Activity as a Function of Extracellular Environment ......................... 26 2.4.3 Hydrogenase Activity Linked to Ethanol Production ............................................... 27 2.4.4 Hydrogenase Activity as a Function of Internal and External pH ............................ 28 2.5 Chapter Summary ......................................................................................................... 30 3 Optimizing P11’s Hydrogenase Assay .............................................................................. 33 3.1 Current Hydrogenase Assay and Data Analysis ........................................................... 34 3.1.1 Optimized Hydrogenase Assay ................................................................................. 34 3.1.2 Assay Data Analysis ................................................................................................. 39 3.2 Anaerobic Conditions ................................................................................................... 41 3.2.1 Gas Purging ............................................................................................................... 42 3.2.2 Use of O2-scavenging Agent ..................................................................................... 44 3.3 Gas Solubility and Diffusion Modeling ........................................................................ 49 3.3.1 H2 Diffusion Modeling ............................................................................................. 50 3.3.2 CO2 Solubility and pH Effects .................................................................................