PRODUCTION OF ALCOHOLS VIA SYNGAS FERMENTATION USING ALKALIBACULUM BACCHI MONOCULTURE AND A MIXED CULTURE By KAN LIU Bachelor of Science in Pharmaceutical Engineering Tianjin University of Science & Technology Tianjin, China 2006 Master of Science in Fermentation Engineering Tianjin University of Science & Technology Tianjin, China 2009 Submitted to the Faculty of the Graduate College of the Oklahoma State University in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY December, 2013 PRODUCTION OF ALCOHOLS VIA SYNGAS FERMENTATION USING ALKALIBACULUM BACCHI MONOCULTURE AND A MIXED CULTURE Dissertation Approved: Dr. Hasan K. Atiyeh Dissertation Adviser Dr. Babu Z. Fathepure Dr. Raymond L. Huhnke Dr. Mark R. Wilkins ii ACKNOWLEDGEMENTS I appreciate my advisor Dr. Hasan Atiyeh giving me the opportunity to study in the United States and obtain my Ph.D. at Oklahoma State University. During the four years study, he gave me a lot of encouragements and guidance on my Ph.D. project, making me grow up quickly and become more professional in the way to think, to perform and to present. The experience and knowledge I learned from him will be a precious treasure for my future career. I also acknowledge the financial support for this project from USDA-NIFA 2010-34447-20772, Oklahoma NSF EPSCoR, Oklahoma Bioenergy Center and the Oklahoma Agricultural Experiment Station. I also appreciate Dr. Raymond Huhnke for guidance on my Ph.D. project, presentations and papers. Also, I am very grateful to Dr. Mark Wilkins’s help and support for designing my experiments, reviewing my papers, presentations and providing valuable suggestions, especially giving me a precious opportunity to visit Washington D.C. to present the Biowinol project at 2012 National Sustainable Design Expo, which greatly increased my experience to explain the technology to different people. In addition, I thank my committee member Dr. Babu Fathepure who provided great support and encouragement for my whole education at OSU. I specially thank Dr. Ralph Tanner who provided the Alkalibaculum bacchi strains and gave valuable suggestions for my research. Also, I appreciate Dr. Ralph Tanner and iii Acknowledgements reflect the views of the author and are not endorsed by committee members or Oklahoma State University. Dr. Bradley Stevenson who helped me to identify the strains in the mixed culture used in this research via the 16s rRNA test and provided a solid information for my paper and dissertation. I am very lucky to have wonderful lab colleagues during the Ph.D. life. Firstly, I would like to thank Prasanth Maddipati who trained me all research related equipment and techniques from scratch. Also, I thank Dr. Marthah De Lorme and Dr. Jennine Terrill for their help and training when I was a new comer to the lab. I show my great gratitude to the Research Engineer, Dr. John Randy Philips, for his help when I had research problems and gave me valuable advice on my project. I also thank Jie Gao and Research Engineer Oscar Pardo Planas’s help. I specially thank lab manager, Mark Gilstrap, who was always helpful to resolve issues with GC, HPLC, lab operation and safety. For the four years lab work and life at OSU, I highly appreciate my colleagues and friends Naveen Pessani, Karthikeyan Ramachandriya, Michael Muller, Mamatha Devarapalli. You gave me a great time not only in academics but also in my life. I enjoyed the every moment with you at OSU. Finally, I want to show my deepest appreciation to my parents’ wholehearted support, love and expectation. iv Acknowledgements reflect the views of the author and are not endorsed by committee members or Oklahoma State University. Name: KAN LIU Date of Degree: DECEMBER, 2013 Title of Study: PRODUCTION OF ALCOHOLS VIA SYNGAS FERMENTATION USING ALKALIBACULUM BACCHI MONOCULTURE AND A MIXED CULTURE Major Field: BIOSYSTEMS AND AGRICULTURAL ENGINEERING Abstract: Gasification-syngas fermentation is a hybrid conversion technology. In this process, feedstocks such as biomass or municipal solid waste are gasified to syngas (CO, H2 and CO2), which is then converted into biofuels and chemicals using biocatalysts. Recently isolated Alkalibaculum bacchi strains CP11T, CP13 and CP15 were found to convert CO and H2 into ethanol and acetic acid at initial pH 8.0. Bottle fermentations showed that CP15 was the most promising strain for ethanol production because of its higher growth and ethanol production rates and yield than CP11T and CP13. The cost of CP15 medium was reduced by 27% by removing TAPS buffer and replacing yeast extract (YE), minerals and vitamins with corn steep liquor (CSL). The use of CSL resulted in a twofold increase in ethanol production in bottle fermentations. Fermentations were scaled up to 3-L and 7-L fermentors in semi-continuous and continuous modes with and without cell recycle. Results of the continuous syngas fermentation with cell recycle showed a maximum of 5.5 g/L cell mass concentration at a dilution rate of 0.033 h-1 in the YE medium. Cell mass and ethanol concentrations were 2.2 g/L and 6.5 g/L, respectively, at a dilution rate of 0.011 h-1. When CSL medium was used in continuous syngas fermentation, the maximum produced concentrations of ethanol, n-propanol and n- butanol were 8 g/L, 6 g/L and 1 g/L, respectively. n-Propanol and n-butanol were not typical products of strain CP15. A 16S rRNA gene-based survey revealed a mixed culture in the fermentor dominated by A. bacchi strain CP15 (56%) and Clostridium propionicum (34%). The mixed culture presents an opportunity for higher alcohols production from syngas. Semi-continuous fermentations in a 3-L fermentor with the mixed culture and CSL medium resulted in a twofold more total alcohol production than in the YE medium. The synergy between strain CP15 and C. propionicum in the mixed culture in bottle fermentations resulted in 50% higher efficiency in converting propionic acid, butyric acid and hexanoic acid to their respective alcohol. v TABLE OF CONTENTS Chapter Page CHAPTER I INTRODUCTION ......................................................................................... 1 CHAPTER II LITERATURE REVIEW ............................................................................ 9 2.1 Biofuels development ............................................................................................... 9 2.2 Routes for lignocellulosic biomass conversion to biofuels .................................... 11 2.2.1 Biochemical platform....................................................................................... 11 2.2.2 Thermochemical platform ................................................................................ 13 2.3 Conversion of syngas to advanced biofuels ............................................................ 15 2.3.1 Fischer-Tropsch process .................................................................................. 15 2.3.2 Syngas fermentation......................................................................................... 16 2.3.2.1 Comparison of syngas fermentation to FT process for biofuels production ........................................................................................................ 16 2.3.2.2 Acetogens ...................................................................................................... 17 2.3.2.3 Metabolism and bioenergetics ...................................................................... 20 2.3.2.3.1 Metabolism .......................................................................................... 20 2.3.2.3.2 Bioenergetics ....................................................................................... 23 2.3.2.4 Syngas fermentation operation...................................................................... 26 2.3.2.4.1 Mass transfer ....................................................................................... 26 2.3.2.4.2 Medium composition........................................................................... 31 2.3.2.4.3 Fermentation pH .................................................................................. 34 2.3.2.4.4 Redox potential ................................................................................... 35 2.3.2.4.5 Syngas compositions and impurities ................................................... 35 2.3.2.4.6 Substrates concentration ...................................................................... 38 2.3.2.4.7 Mixed culture fermentation ................................................................. 39 2.3.2.4.8 Fermentation mode .............................................................................. 39 2.4 Commercialization .................................................................................................. 40 2.5 References ............................................................................................................... 44 CHAPTER III OBJECTIVES ........................................................................................... 59 CHAPTER IV FERMENTATIVE PRODUCTION OF ETHANOL FROM SYNGAS USING NOVEL MODERATELY ALKALIPHILIC STRAINS OF ALKALIBACULUM BACCHI ....................................................................................... 61 4.1 Introduction ............................................................................................................. 62 4.2 Materials and methods ............................................................................................ 65 vi Chapter Page 4.2.1 Microorganisms and fermentation