Characterization of a Thermophilic, Cellulolytic Microbial Culture DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Sarah Marie Carver Graduate Program in Microbiology The Ohio State University 2011 Dissertation Committee: Dr. Olli H. Tuovinen, advisor Dr. Zhongtang Yu Dr. Ann D. Christy Dr. Hua Wang Abstract Microorganisms have evolved to degrade and hydrolyze complex matrixes and extreme environmental conditions such as higher temperature, salinity, or pH. With the appropriate inoculum and selective conditions, organisms can be enriched using selective conditions and in order to efficiently degrade a compound of interest. Cellulosic biomass is a renewable resource explored as a feedstock for bioenergy and the microbial mechanisms to hydrolyze the material are discussed in Chapter 1. This research focuses on cellulose and elevated temperatures (52 - 60 °C) as a way to select for a microbial consortium able to degrade many plant polymers and generate products of interest, including biohydrogen. The versatility of the consortium, henceforth called TC52 or TC60 (different by their enrichment temperature) was analyzed by observing growth and metabolic profiles with a variety of substrates. The first portion of the research focused on utilizing the consortium in a microbial fuel cell, Chapter 4. Unfortunately, MFC designs are not sustainable at elevated temperatures so a new design was developed and tested. TC60 was unstable at 60 °C with cellulose as a substrate but produced 375 mW/m2 when fed glucose. ii The second portion of this research monitored the ability of TC52 and TC60 to adapt and degrade a variety of substrates. In order to monitor short chain fatty acids , a solid phase extraction method (Chapter 2) was developed in order to clean culture samples prior to metabolite analysis with high-performance liquid chromatography. Chapter 3 describes a study where TC60 was enriched on a combination of microalgal biomass and cellulose in order to increase hydrogen yields. Dunaniella tertiolecta and Chlorella vulgaris, two microalgae species, were tested with several ratios of cellulose. Cultures fed a 1:2 ratio of D. tertiolecta and cellulose generated higher hydrogen yields due to lysed microalgae cells. Chapters 7 and 8 show the difference in TC52 metabolite production when grown on commercial paper samples and polysaccharides. Pyrosequencing results indicate that Lutispora thermophilia, Clostridium thermocellum, and Clostridium stercorarium were the dominant microorganisms in many conditions. It must be noted that enrichment was not done on individual substrates, rather, immediate reactions were monitored. The third portion of this research concerned the effect of the type of substrate, concentration of substrate, and temperature on the metabolism of TC60. Initial substrate concentration (2, 4, 8, 12, 16, 20 g/l), temperature (50, 55, 60 °C), and cellulosic substrates (microcrystalline cellulose Sigmacell Type 20 and 50, long fibrous cellulose, and 5 x 5 mm pieces of filter paper) were tested in all possible combinations. Data analyses showed that each individual effect can have a significant affect on metabolite production rates and yields. Also, combined iii environmental factors can have a combined effect. Statistical analyses were able to reveal which factors played a significant affect on production rates and yields of H2, CO2, ethanol, and acetate. The research and results are outlined and described in Chapters 5 and 6. This research explored the ability of a consortium to quickly adapt to a change in substrate and the affect this had on metabolite production. This study showed that it is feasible to enrich for a consortium able to generate different forms of bioenergy by changing environmental conditions. iv Dedication To all those who put up with my eccentric nature And encouraged it v Acknowledgments There are so many wonderful co-workers and friends I must acknowledge. First of all, family and friends, because the two are very similar. My mother and father for supporting me unconditionally and for knocking me off my ‘humble post’ sometimes. My brother for reminding me that science isn’t everyone’s truth. Friends, especially Srujana Samhita Yadavalli, Liang-Chun Liu, and Kiley Dare: thank you for the support through the stress, fun breaks to take my mind off of work, and for listening when I needed to vent. Secondly, I want to thank my awesome advisor, Dr. Olli H. Tuovinen. Not only have I learned microbiology, I’ve learned how to think in a whole new light. You gave me a wonderful opportunity by going to Finland to work and I will always appreciate it. Finland is a second home to me; I cannot wait to visit again. Thank you for putting up with my weird sense of humor and my rambling at times. I have grown so much through graduate school and you have been a great mentor. In addition to my advisor, I must thank my doctoral committee without whom I would not be completing this document. Dr. Ann D. Christy, you have always been sweet, supportive, and great for engineering questions. Dr. vi Zhongtang Yu, thank you for keeping me on my toes and forcing me to think twice as hard as I usually do. Dr. Hua Wang, while I did not get to work with you much, you have been extremely insightful during meetings and the dissertation process. A large part of my research was completed in Finland and I cannot forget to thank the wonderful people I met there. The Department of Chemistry and Bioengineering at Tampere University of Technology was extremely supportive, financially and mentally while I carried out my studies. Of particular thanks, Dr. Jaakko Puhakka, Pertti Vuoriranta, Raghida Lepistö, and Uwe Münster for being mentors in their own way. The biohydrogen researchers, including two of my best-friends-forever ever Aino-Maija Lakaniemi and Marika Nisillä, helped me polish my communication skills and increased my interested and appreciation of bioenergy research. Aino-Maija and Marika, you two will always hold a special place for being the best officemates a crazy American like I could have. Paper samples used in this research were supplied by the paper companies UPM and E- Real (Tampere, Finland). Special thanks go to Suvi Nieminenat at UPM who facilitated the transfer. Chris Hulatt from the University of Wales for collaboration on the co-degradation study, you are the only person as crazy as me that I have yet to find in science. Good luck on all your endeavors. While researching at Ohio State University, I have collaborated and learned much from a diverse bunch of people. Of particular note is the Yu lab in the Animal Sciences Department. Mike Nelson, Jill Stephens Stiverson, and Wen vii Lv have all taught me a variety of molecular techniques necessary to carry out this research. Also, you’ve taught me patience, each in a different way. Mike, thank you for your extensive help with sequencing and analysis. Trent Bower, Alan Yost, and C.J. Morabito, thank you for assisting with the final changes to the microbial fuel cell design and Trent for the time and effort of making Figure 1 for the thermophilic microbial fuel cell manuscript. I would like to acknowledge Sandy Jones for assistance with XRD and surface area analysis. Thank you to Sasha Bai and the Statistical Consulting Service at OSU for teaching me analyses that I would have struggled with alone. Also, Dr. Sukhbir Grewal and Dr. Fredrick Michel at the OSU-Wooster extension campus for the time to teach me T-RFLP even though another method was eventually chosen. Last, but not least, thank you to the Department of Microbiology and the Graduate School for technical support throughout my doctoral studies. If there is anyone I have missed in this acknowledgement section, please track me down and I will thank you personally. viii Vita 2002................................................................Olivet High School 2006................................................................B.A. Biology, Albion College 2006-present ...................................................Graduate Teaching Associate, Department of Microbiology, The Ohio State University June 2008-December 2009 ...........................Research Associate, Department of Chemistry and Bioengineering, Tampere University of Technology, Finland Publications Carver, S.M., Vuoriranta, P., and O.H. Tuovinen. 2011. A Thermophilic Microbial Fuel Cell Design. J Power Sources 196:3757-3760. Carver, S.M., Hulatt, C.J., Thomas, D.N., and O.H. Tuovinen. 2011. Thermophilic, Anaerobic Co-Digestion of Microalgal Biomass and Cellulose for H2 Production. Biodegradation. doi:10.1007/s10532-010-9419-2 Carver, S.M., Lepistö, R., and O.H. Tuovinen. 2010. Hydrolysis and Metabolism of Cellulose by an Anaerobic, Thermophilic Consortium. Proceedings of the 3rd International Symposium on Energy from Biomass and Waste, paper no 436, p.9. Rismani-Yazdi, H., Christy, A.D., Carver, S.M., Yu, Z., Dehority, B.A., and O.H. Tuovinen. 2011. Effect of External Resistance on Bacterial Diversity and Metabolism in Microbial Fuel Cells. Bioresour Technol 102:278-283. ix Rismani-Yazdi, H., Carver, S.M., Christy, A.D., and O.H. Tuovinen. 2008. Cathodic Limitations in Microbial Fuel Cells: An Overview. J Power Sources 180: 683-694. Fields of Study Major Field: Microbiology x Table of Contents Abstract ................................................................................................................... ii Acknowledgments.................................................................................................