UNIVERSITY OF OKLAHOMA GRADUATE COLLEGE ANAEROBACULUM HYDROGENIFORMANS: INSIGHTS INTO THE NOVEL BIOCHEMICAL AND PHYSIOLOGICAL ABILITY OF A THERMOPHILIC BIOCATALYST THAT CAN UTILIZE BOTH DEFINED AND UNDEFINED SUBSTRATES FOR THE PRODUCTION OF RENEWABLE HYDROGEN ENERGY A DISSERTATION SUBMITTED TO THE GRADUATE FACULTY in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY By Matthew William Maune Norman, Oklahoma 2011 ANAEROBACULUM HYDROGENIFORMANS: INSIGHTS INTO THE NOVEL BIOCHEMICAL AND PHYSIOLOGICAL ABILITY OF A THERMOPHILIC BIOCATALYST THAT CAN UTILIZE BOTH DEFINED AND UNDEFINED SUBSTRATES FOR THE PRODUCTION OF RENEWABLE HYDROGEN ENERGY A DISSERTATION APPROVED FOR THE DEPARTMENT OF BOTANY AND MICROBIOLOGY By Dr. Ralph S. Tanner, Chair Dr. Paul A. Lawson Dr. Michael J. McInerney Dr. Kenneth M. Nicholas Dr. Bradley S. Stevenson © Copyright by Matthew W. Maune 2011 All Rights Reserved. ACKNOWLEDGEMENTS First and foremost, I would like to thank my wife, Jennifer. Without you inspiring me to achieve my potential and seeing something in me that most only overlooked, I could not have completed this work, nor would I be the person I am today. Thank you so much for being there for me and getting me though this. I couldn’t have done it without you. Secondly, I would like to thank my daughter Averee for pointing out the significance of the little things in life. Thirdly, I would like to thank my major professor Dr. Ralph Tanner for allowing me to come into his lab and taking me under his wing. You have showed me that anything is possible as long as you pay attention to the “DETAILS, DETAILS, DETAILS”. I have to give special thanks to Dr. McInerney for allowing me to be a part of the biohydrogen grant that supported me throughout the later portion of my doctoral research and helping me with the pathway portion of my research. I can’t believe that I had the opportunity to do the things that he taught me in his Physiology of Microorganisms class, which turned me on to microbiology in the first place. I can’t forget Neil Wofford. Man without you I would have been stranded to get this work going. You are the best. I would also like to thank the members of my advisory committee, Dr. Stevenson, Dr. Lawson and Dr. Nicholas. Thank you for being there and fielding all of my questions. I would also like to thank the Department of Botany and Microbiology here at OU for providing me with the resources to conduct this iv research. I need to thank the other scientists that made my research possible, such as, Dr. Toby Allen, Dr. Matthew Caldwell, Dr. Jyotisna Saxena, Dr. Joseph Suflita, Dr. Victoria Parisi, Dr. Mark Nanny, Dr. Paul Philp, Dr. Rob Gunsalus and Dr. Margret Eastman. I have listed my specific acknowledgements at the end of each chapter to thank you for your contribution to this work. I would like to thank my lab mates in the Tanner Lab, both past and present for helping me when I was stuck and picking up the slack when I was busy. I need to especially thank Catherine “Cat” Isom for her help with the feedstock set of experiments and keeping me sane while getting through it all. Finally, I would like to thank my parents and extended family for seeing me though this journey and believing in me throughout this process. No matter how long it took and what happened along the way you were the best, and I can’t thank you enough. v Table of Contents TABLE OF FIGURES ................................................................................................................... XII TABLE OF TABLES ................................................................................................................ XVIII ABSTRACT ..................................................................................................................................................... XXI CHAPTER 1 ANAEROBACULUM HYDROGENIFORMANS SP. NOV., A NOVEL ANAEROBE THAT PRODUCES HYDROGEN FROM GLUCOSE ........................................... 1 INTRODUCTION ............................................................................................................................................... 2 MATERIALS AND METHODS .......................................................................................................................... 5 Enrichment and isolation ...................................................................................................................... 5 Phenotypic characterization ............................................................................................................... 5 pH range and optima .............................................................................................................................. 6 Alternate electron acceptor analysis ............................................................................................... 6 Types strains used for comparison .................................................................................................... 7 Chemotaxonomic analysis .................................................................................................................... 7 Genomic analysis ....................................................................................................................................... 7 Pure and co-culture growth assays .................................................................................................. 8 RESULTS AND DISCUSSION ......................................................................................................................... 10 Phenotypic analysis ............................................................................................................................... 10 Genotypic analysis .................................................................................................................................. 10 Biochemical characterization ........................................................................................................... 11 Chemotaxonomic data .......................................................................................................................... 12 Pure and co-culture growth data .................................................................................................... 13 vi CONCLUSIONS ............................................................................................................................................... 22 Emended description of the genus Anaerobaculum Menes & Muxi, 2002 ..................... 24 Description of Anaerobaculum hydrogeniformans sp. nov. ................................................. 25 ACKNOWLEDGEMENTS: .............................................................................................................................. 27 CHAPTER 2 BIOHYDROGEN FROM WASTE FEEDSTOCKS BY ANAEROBACULUM HYDROGENIFORMANS .............................................................................................................. 28 ABSTRACT ..................................................................................................................................................... 29 INTRODUCTION ............................................................................................................................................ 30 MATERIALS AND METHODS ....................................................................................................................... 32 Culture and growth conditions: ........................................................................................................ 32 Feedstocks: ................................................................................................................................................. 32 Optimization of growth parameters: ............................................................................................. 33 Batch fermentations: ............................................................................................................................ 34 Analytics: .................................................................................................................................................... 34 RESULTS AND DISCUSSION ......................................................................................................................... 37 Feedstock optimization: ...................................................................................................................... 37 Batch fermentations: ............................................................................................................................ 40 GlucosE vs. brEwEry wastEwatEr: .............................................................................................................................. 40 Sweet sorghum juice: ...................................................................................................................................................... 41 CheEsE whey wastEwatEr: ............................................................................................................................................ 45 DomEstic wastEwatEr/raw sEwagE: ......................................................................................................................... 47 AnaErobic digEstor sludgE: ........................................................................................................................................... 47 CONCLUSIONS ..............................................................................................................................................