The Effect of Nitrate, Live Yeast Culture or their interaction on Methane Mitigation and Nitrate Reduction in vitro THESIS Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Caitlyn M. Massie, BS Graduate Program in Animal Sciences The Ohio State University 2015 Master's Examination Committee: Dr. Jeffrey L. Firkins, Advisor Dr. Kelly C. Wrighton Dr. Zhongtang Yu Copyrighted by Caitlyn M. Massie 2015 Abstract Nitrates have been fed to dairy cows for years to decrease methane emissions. In the nitrate assimilatory pathway, bacteria reduce nitrate to nitrite to ammonia. The second step is rate-limiting leading to nitrite accumulation causing methemoglobinemia, which can hinder the adoption of nitrate feeding to compete with methanogens for hydrogen while assimilating the nitrogen from nitrate into microbial protein. The yeast Saccharomyces cerevisiae has the potential to anaerobically respire nitrite through its cytochrome c oxidase; it also can stimulate populations of bacteria that express nitrate and nitrite reductases. In the first trial, a 3 x 4 factorial within in vitro tubes included no yeast, an active live yeast culture (S. cerevisiae strain 1026), or autoclaved yeast dosed with nitrate, nitrite, ammonium, or a control to determine if yeast altered the nitrate reduction pathway. Differences were seen at h 0 (P < 0.01) for nitrogen treatments, that correlated with dosed metabolites. For the second project, 4 continuous cultures were used in 5 periods of 10 days. Treatments were arranged in a 2 x 2 factorial with sodium nitrate (NO3 at 1.5% of diet DM) or urea as an isonitrogenous control and without or with live yeast culture fed at a recommended 0.010 g/d (scaled from cattle to fermenter intakes). The second study showed a significant increase in NDF digestibility for the main effect of live yeast culture. Total VFA production (averaging 146 mmol/d; SEM = 15) and acetate: propionate (averaging 3.37, SEM = 0.10) did not differ (P > 0.15) among ii treatments. The main effect of nitrate decreased (P < 0.05) methane emissions compared to the urea control (29.5 vs 21.0 mmol/d). There was no difference (P > 0.15) for hydrogen emission for nitrate or live yeast culture (averaging 0.148 ± 0.053 mmol/d), but the main effect mean of nitrate decreased (P < 0.01) aqueous hydrogen concentration compared with the main effect mean of urea (2.51 vs 3.66 ± 1.36 mM). Live yeast culture did not alter nitrite metabolism as hypothesized; however, nitrite inhibited yeast growth in batch culture. The last experiment was a yeast growth in a YPD media control combined with a 3 by 3 factorial arrangement of nitrogen sources (nitrate, nitrite, ammonium salts) replacing peptone at 25, 50 , 75% at one of three gas phases (aerobic, transition from aerobic to anaerobic, anaerobic). No interactions were detected, but the dosed nitrite inhibited yeast growth regardless of the gas phase. Overall, nitrate decreased methane production, but live yeast did not significantly influence methane production. Live yeast culture did not alter nitrite reduction, although nitrite did inhibit yeast growth. iii Dedicated to my Parents Steve and Brenda, Fiancé Jacob, Best Friend Sara and Sister Ashley without whose support I could not have succeeded. iv Acknowledgments First, I would like to thank my advisor, Dr. Jeffrey L. Firkins, for his guidance, patience, and for sharing his wisdom with me. I am very lucky to have had the opportunity to learn from and work alongside you. Thank you for all of your assistance over my graduate experience. I would also like to thank my other committee members, Dr. Zhongtang Yu and Dr. Kelly C. Wrighton for all of their time and knowledge they shared with me. Without their lab managers I would have been lost; a special thank you goes out to Johanna Plank of the Ruminant Nutrition lab, Jill Stiverson recently of the Animal Science Microbiology lab and Rebecca Daly of the Microbiology lab. My thanks also goes out to Dr. Normand St. Pierre for his guidance and Amanda M. Gehman and Alltech, Inc. for their gracious funding of my research and graduate education. To the Ruminant Nutrition lab, I could never say how grateful I am to all of you for your assistance, time and guidance over the years. Thank you especially to Benjamin Wenner and Brooklyn Wagner my fellow Fermenter Fanatics, as well as the other numerous interns, farm personal and graduate students of the Ohio State University Animal Science Department: Nicole Danszczak, Tasha Henderson, Natalia Jurcak, Logan Morris, Sarah Waits, Jessica Pempek, Danni Ye, Bethany Keyser, Yaniris Alvarado Luna, Thiago Bompadre and Danielle Coleman. A huge thank you goes out to Lorraine v English without whom I might have lost my sanity. Also without Rebekah Meller I could not have survived my first year of graduate school, thank you for being there in the wee morning to learn with me. Lastly I would like to again thank my family and friends for all of your encouragement and support throughout my life and most importantly my graduate career. Thank you Dad for opening my eyes to the possibilities in the Dairy industry, Mom for my love of learning, Jacob and Sara for your positivity and love and lastly Ashley, Megan and Hannah for your support. vi Vita 2009................................................................Graham High School, St. Paris, OH 2013................................................................BS Biology Life Science, Wilmington College, Wilmington, OH 2013 to present ..............................................Graduate Research Associate, Department of Animal Science, The Ohio State University, Columbus, OH Fields of Study Major Field: Animal Sciences vii Table of Contents Abstract ............................................................................................................................... ii Acknowledgments............................................................................................................... v Vita .................................................................................................................................... vii List of Tables ...................................................................................................................... x List of Figures .................................................................................................................... xi Chapter 1: Introduction ...................................................................................................... 1 Methanogenesis ............................................................................................................... 3 Mitigation Strategies ................................................................................................... 4 Nitrate .............................................................................................................................. 7 Thermodynamics ......................................................................................................... 8 Methemoglobinemia .................................................................................................... 8 Nitrate Reduction Pathways ........................................................................................ 9 Live Yeast ..................................................................................................................... 12 Nitrite Reduction ....................................................................................................... 13 Digestibility ............................................................................................................... 13 Chapter 2: The Effect of Live Yeast Culture on Nitrate and Nitrite Disappearance in vitro ........................................................................................................................................... 15 viii Introduction ................................................................................................................... 15 Methods ......................................................................................................................... 15 Statisticsal Analysis ................................................................................................... 16 Results and Discussion ..................................................................................................... 17 Chapter 3: The Effect of Live Yeast Culture and Nitrate on Methane Mitigation and Nitrate Reduction in a Continuous Culture System .......................................................... 20 Introduction ................................................................................................................... 20 Methods ......................................................................................................................... 20 Statisticsal Analysis ................................................................................................... 23 Results and Discussion ..................................................................................................... 23 Chapter 4: The Effect on Live Yeast Culture Growth by Replacement of 25%, 50%, or 75% of Peptone within YPD Media with Nitrate, Nitrite, or Ammonium at aerobic, transition between aerobic and anaerobic, or anaerobic respiration phases ..................... 48 Introduction ..................................................................................................................