Detoxification of Lignocellulose-derived Microbial Inhibitory Compounds by Clostridium beijerinckii NCIMB 8052 during Acetone-Butanol-Ethanol Fermentation DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Yan Zhang Graduate Program in Animal Sciences The Ohio State University 2013 Dissertation Committee: Thaddeus C. Ezeji, Advisor Steven C. Loerch Sandra G. Velleman Zhongtang Yu Venkat Gopalan Copyrighted by Yan Zhang 2013 Abstract Pretreatment and hydrolysis of lignocellulosic biomass to fermentable sugars generate a complex mixture of microbial inhibitors such as furan aldehydes (e.g., furfural), which at sublethal concentration in the fermentation medium can be tolerated or detoxified by acetone butanol ethanol (ABE)-producing Clostridium beijerinckii NCIMB 8052. The response of C. beijerinckii to furfural at the molecular level, however, has not been directly studied. Therefore, this study was to elucidate mechanism employed by C. beijerinckii to detoxify lignocellulose-derived microbial inhibitors and use this information to develop inhibitor-tolerant C. beijerinckii. Towards the long-term goal of developing inhibitor-tolerant Clostridium strains, the first objective was to evaluate ABE fermentation by C. beijerinckii using different proportions of Miscanthus giganteus hydrolysates as carbon source. Compared to the growth of C. beijerinckii in control medium, C. beijerinckii experienced different degrees of inhibition. The degree of inhibition was dose-dependent, and C. beijerinckii did not grow in P2 medium with greater than 25% (v/v) Miscanthus giganteus hydrolysates. To improve tolerance of C. beijerinckii to inhibitors, supplementation of P2 medium with undiluted (100%) Miscanthus giganteus hydrolysates with 4 g/L CaCO3 resulted in successful growth of and ABE production by C. beijerinckii. Spectrophotometric and HPLC analyses revealed that C. beijerinckii transformed lignocellulose-derived furan ii aldehydes such as furfural and hydroxymethylfurfural to furfuryl alcohol and 2, 5-bis- hydroxymethylfuran, respectively and at a rate of 0.15 and 0.08 g/L/h, respectively. The next study aimed to compare differential gene expressions between C. beijerinckii cultures grown in P2 medium supplemented with and without furfural during acidogenic and solventogenic growth phases. The genomic microarray was used to comprehensively evaluate the inhibitory effects of furfural on C. beijerinckii, and potential adaptation mechanisms to furfural stress. Functional gene group analysis showed that increased expression of genes related to redox balancing may be responsible for the reduction of toxic effects of furfural and alleviation of furfural induced oxidative stress in C. beijerinckii during acidogenic growth phase. However, ABE accumulation, redox balance perturbations, and repression of phosphotransferase system may have caused the termination of the growth of C. beijerinckii following furfural challenge at the solventogenic growth phase. The last objective was to accelerate biotransformation of furfural to furfuryl alcohol by overexpression of furfural-reducting enzymes in C. beijerinckii. Based on results obtained from the transcriptomic analysis of C. beijerinckii, two candidate genes, aldo/keto reductase (AKR) and short-chain dehydrogenase/reductase (SDR) encoded by Cbei_3974 and Cbei_3904 respectively, were selected, cloned and expressed in Escherichia coli to generate polyhistidine-tagged proteins and confirm the role of these enzymes in furfural reduction. Those (His)6-tagged proteins were purified by immobilized metal affinity chromatography. AKR and SDR reduced furfural to furfuryl alcohol using NADPH as cofactor, and they showed catalytic activities over a broad range of temperature, pH, and substrate specificity. Subsequently, AKR and SDR were iii overexpressed in C. beijerinckii, which resulted in the development of inhibitor-tolerant strains, C. beijerinckii AKR+ and C. beijerinckii SDR+. This integrated study enhanced our understanding of inhibitory effects of lignocellulose-derived aldehydes, and discussed poteintial strategies to engineer clostridia with high tolerance to lignocellulose hydrolysates. iv Dedication This document is dedicated to my Dearest Mother. v Acknowledgments I would like to take this opportunity to thank all the people who generously supported my research and helped me accomplish this dissertation. First of all, I would like to express my deepest gratitude to my advisor, Dr. Thaddeus Ezeji, for providing me the opportunity to one of my favorite fields, for his careful and thoughtful guidance on each of my experiment, and for all the scientific training he gave me to help me become a scientist. I would also like to thank all my committee members for discussing on my progress updates. Dr. Sandra Velleman always inspires me with her questions and comments on my presentations and progress reports. Dr. Zhongtang Yu offered me a great chance to work and learn techniques in his laboratory when I took classes in the Columbus campus, and continued to encourage me after I moved to Wooster. Dr. Steve Loerch has taught me and warmed me more than he will ever know. I really appreciate every word he talked to me, especially when he pointed out my weaknesses and helped me to improve. Dr. Venkat Gopalan is one of the best teachers I have ever met. He is so knowledgeable and his ideas are always logical. I benefited a lot from his questions and suggestions. vi I would like to thank Dr. Bei Han for being a great colleague during my first two years of graduate study and a great friend to date. I also appreciate Dr. Victor Ujor for his help on my molecular cloning experiment. Many thanks to Christopher Abraham, Angufor Numfor, Catherine Richmond, and Jenny Orozco for the conversations and laughter in the lab which made life not that stressful. I would also like to thank Lingling Wang, Danni Ye, Shan Wei, Min Seok Kim, Jill Stiverson, Thavamathi Annamalai, and Revathi Shanmugasundaram for their friendship. Finally, I would like to thank my family. No words can express how happy and thankful I am as the daughter of my Mom and Dad. I would never have made it this far without your love and support. I am very grateful to have my sister with me to share my happiness and sadness. The most sincere thanks go to my husband, Bin You, the person who knows me best and supports me through good times and bad. vii Vita 2007................................................................B.S. Biotechnology, Shandong Normal University 2008 to present ...............................................Graduate Research Associate, Department of Animal Sciences, The Ohio State University Publications Zhang, Y., Han, B., Ezeji, T.C., 2012. Biotransformation of furfural and 5- hydroxymethyl furfural (HMF) by Clostridium acetobutylicum ATCC 824 during butanol fermentation. N. Biotechnol. 29, 345-351. Zhang, Y., Ezeji, T.C., 2013. Transcriptional analysis of Clostridium beijerinckii NCIMB 8052 to elucidate role of furfural stress during acetone butanol ethanol fermentation. Biotechnol. Biofuels. In revision. viii Fields of Study Major Field: Animal Sciences ix Table of Contents Abstract ............................................................................................................................... ii Dedication ........................................................................................................................... v Acknowledgments.............................................................................................................. vi Vita ................................................................................................................................... viii Publications ...................................................................................................................... viii Fields of Study ................................................................................................................... ix List of Tables ................................................................................................................. xviii List of Figures .................................................................................................................. xxi Chapter 1: Introduction ...................................................................................................... 1 References ....................................................................................................................... 5 Chapter 2: Literature Review .............................................................................................. 8 2.1 Acetone-Butanol-Ethanol (ABE) fermentation .................................................... 8 2.1.1 History of ABE fermentation ........................................................................ 8 2.1.2 Solventogenic Clostridium sp. and mechanisms of ABE fermentation ..... 10 x 2.2 Lignocellulosic biomass ..................................................................................... 13 2.2.1 Sources of Lignocellulosic biomass............................................................ 13 2.2.2 Pretreatment of lignocellulosic biomass ..................................................... 14 2.2.3 Enzymatic hydrolysis of lignocellulosic biomass ....................................... 16 2.2.4 Lignocellulose-derived microbial inhibitory compounds ........................... 17 2.3 Molecular