Amino Acid-Fermenting Bacteria from the Rumen of Dairy Cattle Enrichment, Isolation, Characterization, and Interaction with Entodinium caudatum THESIS Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Jacqueline M. Gano Graduate Program in Animal Sciences The Ohio State University 2013 Master's Examination Committee: Dr. Zhongtang Yu, Advisor Dr. Jeffrey Firkins Dr. Macdonald Wick Copyrighted by Jacqueline M. Gano 2013 i ABSTRACT Excess ammonia emissions are a major concern for the dairy industry due to the detrimental impact ammonia emissions have on the environment and wastage of dietary nitrogen. Hyper-ammonia-producing bacteria (HAB) and protozoa in the rumen are the major contributors of excessive ammonia excretions from cattle. Besides Clostridium aminophilum, C. sticklandii, and Peptostreptococcus anaerobius, little is known about the HAB present in the rumen. In addition, rumen protozoa prey on bacteria and other microbes, excreting considerable amounts of amino acids and/or peptides that could promote the growth of HAB. In addition, inhibition of HAB by plant secondary metabolites may ultimately reduce ammonia production by HAB, thereby lowering excess nitrogen emissions. The studies presented in this thesis investigate HAB characterizations and interactions. In the first study, mixed microbes were obtained from the rumen of three fistulated dairy cows and further enriched and isolated for amino acid-fermenting bacteria. As a result, new isolates displayed high rates of ammonia production, ranging from 0.87 to 2.45 mg N/dL, and identified through 16S rRNA gene sequencing as a Bacillus sp. and Proteus mirabilis. In the second study, HAB enrichment cultures were co-cultured with an Entodinium caudatum culture. The co- culturing experiment was conducted with or without a feed substrate for E. caudatum ii and Micrococcus luteus to assess the impact of feeding the protozoan. Ammonia concentrations were higher in the E. caudatum alone treatments, both with and without the addition of the feed substrate compared with HAB alone or co-culture of HAB and E. caudatum, with 20.5 ± 0.8, 18.5 ± 0.2, 23.9 ± 0.3, and 23.2 ± 1.1 mg ammonia N/dL in treatment groups E. caudatum with feed substrate, E. caudatum without feed substrate, E. caudatum with feed substrate and with M. luteus, and E. caudatum without feed substrate but with M. luteus, respectively. Ammonia concentration was also significantly (P < 0.05) increased by M. luteus addition in the E. caudatum alone treatment group without feed substrate but with M. luteus. In the third study, HAB in enrichment cultures were examined for inhibition by varying plant secondary metabolites in terms of ammonia concentration. Results indicate that carvacrol, origanum oil, clove oil, and vanillin all successfully (P < 0.01) reduced ammonia concentration 24 h post incubation both in the presence and absence of a feed substrate. Overall, results indicate the presence of additional amino acid-fermenting bacteria in the rumen, capable of rapid ammonia production. These amino acid- fermenting bacteria, when grown in co-cultures with E. caudatum may interact with protozoa present within the rumen. These amino acid-fermenting bacteria are also sensitive to certain plant secondary metabolites, therefore decrease in ruminal ammonia concentration and shifts in ruminal microbe populations may be achieved through dietary supplementation with some of these plant secondary metabolites. iii ACKNOWLEDGEMENTS I would like to thank everyone who has helped me over the last year. I would like to thank Dr. Yu for helping me and pushing me to succeed; Jill Stiverson, for teaching me how to use everything in the lab as well as putting up with my late night texts when something went wrong in the beginning; Josie Plank, for helping me figure out how to work with protozoa and not kill them; Lingling Wang for helping me with sequencing; Deng Pan for helping me with SAS; and everyone else in the lab for dealing with my questions and helping me along the way. And, lastly, I would also like to thank my parents for standing beside me no matter what. iv VITA June 2007………………………………….Wilmington High School, Wilmington, Ohio June 2011………………………………….B. S., Animal Sciences, The Ohio State University 2011-2013…………………………………Graduate Research Assistant, Department of Animal Sciences, The Ohio State University Field of Study Major Field: Animal Sciences v TABLE OF CONTENTS Abstract …………………………………………………………………………………………………………….…………..ii Acknowledgments …………………………………………………………………………………………………….…..iv Vita…………………………………………………………………………………………………………………………………v List of Tables ………………………………………………………………………………………………………….….....ix List of Figures ………………………………………………………………………………………………………...........x CHAPTER 1. Review of Literature ……………………………….……………………………………..……….1 Ammonia ….…………………………………………………………....................................1 Deposition of Ammonia and Ammonium …..……...……………………………………2 Ammonia Emissions in Animal Agriculture …..……………………………..……….…3 The Ruminant Forestomach …..…………………………………………………………..…..5 Microorganisms in the Reticulorumen …….…………………………………………..…6 Hyper-Ammonia-Producing Bacteria .……………………………………………………..9 Ammonia Assimilation ………………………………………………............................11 vi Transamination ……………………………………………………………………………….…..13 Inefficient N Retention in Cattle …………………………………………………………...14 Efficiency of Amino Acid Deamination .…………………………………………………15 Dietary Modification of Rumen Fermentation Using Plant Secondary Metabolites …………………………………………………………………………….……………16 CHAPTER 2. Enrichment, Isolation, and Characterization of Amino Acid-Fermenting Bacteria ………..…………………………………………………………………………………………………..………..18 Abstract …………………………………………………………………….…………..…………….18 Introduction ………………………………………………………………….……………..……...19 Materials and Methods ………………………………………………….………………….…23 Results and Discussion ………………………………………………………………….….…..26 CHAPTER 3. Investigation into the Interactions of a Consortium of Hyper-Ammonia- Producing Bacteria and Protozoa ….……………………………………………………………….….………..33 Abstract ………………………………………………………………………………….………..….33 Introduction ………………………………………………………………………….…...………..34 Materials and Methods ……………………………………………………….……...…….…36 Results and Discussion ……………………………………………………………………..…..39 vii CHAPTER 4. Investigation into the Inhibition of Ammonia production by a Hyper- Ammonia-Producing Bacterial Consortium by Plant Secondary Metabolites ..………..…...47 Abstract …………………………………………………………………………….………………...47 Introduction ………………………………………………………………………………….……..48 Materials and Methods …………………………………………………………………….….51 Results and Discussion …………………………………………………………………..……..53 Literature Cited ……………………………………………………………………………………………………..…….63 Appendix A: Extraneous Tables and Figures …………………………………………………………………92 viii LIST OF TABLES Table 2.1. Characterization of isolates ..………………………………………………….….…………….….28 Table 3.1. Treatment Groups ………………………………………………………………………………....…..43 Table A.1. Effects of HAB and E. caudatum co-cultures without M. luteus on pH, ammonia production, and protozoal counts in vitro ……………………..…………….125 Table A.2. Effects of HAB and E. caudatum co-cultures with M. luteus on pH, ammonia production, and protozoal counts in vitro …………………....………..……126 Table A.3. Effects of plant secondary metabolites on ammonia production and pH in vitro …………………………….……………………………………………….….……..….127 Table A.4. Effects of plant secondary metabolites with feed substrate on ammonia concentration and pH in vitro …………………………………………………………………..…128 ix LIST OF FIGURES Figure 2.1. Growth curve of HAB3 when grown on Casamino Acids, casein, xylose, glucose, maltose, cellobiose, or starch ……………………………………………29 Figure 2.2. Growth curve of HAB5 when grown on Casamino Acids, casein, xylose, glucose, maltose, cellobiose, or starch ……………………………………………30 Figure 2.3. Growth curve of HAB7 when grown on Casamino Acids, casein, xylose, glucose, maltose, cellobiose, or starch ……………………………………………31 Figure 2.4. Growth curve of HAB8 when grown on Casamino Acids, casein, xylose, glucose, maltose, cellobiose, or starch ……………………………………………32 Figure 3.1. Ammonia concentration in co-cultures 12 h post incubation ..…….………..……44 Figure 3.2. Ammonia concentration in co-cultures 24 h post incubation .…………...…….…45 Figure 3.3. E. caudatum counts in co-cultures 24 h post incubation …………….……..…..….46 Figure 4.1. Ammonia concentration in plant secondary metabolite treatments 12 h post incubation …………………………………………………………………………...…………..59 x Figure 4.2. Ammonia concentration in plant secondary metabolite treatments 24 h post incubation ……………………………………………………………………………………60 Figure 4.3. Ammonia concentration in plant secondary metabolite treatments with feed substrate 12 h post incubation …………………….………………………………61 Figure 4.4. Ammonia concentration in plant secondary metabolite treatments with feed substrate 24 h post incubation …………………….………………………………62 Figure A.1. Growth curve of HAB3 when grown on Casamino Acids ………………….…….…..93 Figure A.2. Growth curve of HAB3 when grown on Casein ……………..…………………………...94 Figure A.3. Growth curve of HAB3 when grown on Cellobiose …………………………………….95 Figure A.4. Growth curve of HAB3 when grown on Control …………..…………………………….96 Figure A.5. Growth curve of HAB3 when grown on Glucose …………..……………………………97 Figure A.6. Growth curve of HAB3 when grown on Maltose …………..……………………………98 Figure A.7. Growth curve of HAB3 when grown on Starch .……………..…………………………..99 Figure A.8. Growth curve of HAB3 when grown on Xylose ……………..…………………………100