University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 12-2014 Toward Understanding the Physiological Determinants of Microbial Competitiveness in Methanogenic Processes Si Chen University of Tennessee - Knoxville, [email protected] Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Environmental Engineering Commons Recommended Citation Chen, Si, "Toward Understanding the Physiological Determinants of Microbial Competitiveness in Methanogenic Processes. " PhD diss., University of Tennessee, 2014. https://trace.tennessee.edu/utk_graddiss/3191 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Si Chen entitled "Toward Understanding the Physiological Determinants of Microbial Competitiveness in Methanogenic Processes." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the equirr ements for the degree of Doctor of Philosophy, with a major in Civil Engineering. Qiang He, Major Professor We have read this dissertation and recommend its acceptance: Chris Cox, Terry Hazen, Gary Sayler Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) Toward Understanding the Physiological Determinants of Microbial Competitiveness in Methanogenic Processes A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Si Chen December 2014 Copyright © 2014 by Si Chen All rights reserved ii DEDICATION This dissertation is dedicated in loving memory to my maternal grandfather. iii ACKNOWLEDGEMENTS I would like to express the highest level of gratitude to my advisor, Dr. Qiang He, for all the knowledge and wisdom he imparted on me over the last six years. I would also like to express my gratitude to my committee numbers, Dr. Chris Cox, Dr. Gary Sayler and Dr. Terry Hazen who have given their advice and perspective as I have pursued my Ph.D. Their time and efforts are greatly appreciated. Many thanks are due to my colleagues, Dr. Yan Zhang, Dr. Zhenwei Zhu, Esteban Zamudio Cañas, and Kristen Wyckoff for their friendship and assistance during my doctoral study. I would also like to express my gratitude to my cooperators, Dr. Jun Yan, Yi Yang, Steve Higgins from Dr Loffler’s Lab; Jiang Liu, Dominique Joyner from Dr. Hazen’s Lab; Dr. Alice Layton from the Center for Environmental Biotechnology (CEB) and Dr. Karen Lloyd from the Department of Microbiology for their generous help and precious ideas in my research. Last but not least, I would like to thank my parents for their encouragement, love and support. iv ABSTRACT Methanogenesis is of great significance in both natural and engineered processes. Anaerobic digestion technology represents the engineering-scale implementation of methanogenesis in waste treatment processes. Despite the broad application of anaerobic digestion as a common waste treatment option, much remains to be learned on the anaerobic food web underlying methanogenesis for more effective process modeling and control. Following an initial screening of various substrates, six continuous lab-scale anaerobic digesters were developed with animal waste as the substrate. The linkage between microbial community composition and process performance was studied by initiating process imbalance with organic overloading. As a result, accumulation of short chain fatty acids, particularly acetate, occurred in response to the onset of process imbalance. Populations related to Methanosaeta vastly outnumbered those of Methanosarcina as the dominant acetoclastic methanogen throughout the study period, even at elevated acetate concentrations during process imbalance, which is unexpected as it is considered that Methanosaeta can out-compete Methanosarcina only at low acetate concentrations, typical for balanced anaerobic food webs. Subsequent methanogenic batch cultures enriched with high concentrations of acetate further confirmed the dominance of Methanosaeta over Methanosarcina. Given the significance of acetoclastic methanogenesis in the anaerobic food web and global carbon cycling, it is evident that the interactions between Methanosaeta and Methanosarcina need to be better defined. Other novel microbial populations were also identified as abundant constituents of the microbial communities in the anaerobic digesters, including populations related to Crenarchaeota. It is critical to understand the roles these abundant but poorly-described populations play in the anaerobic food web. v TABLE OF CONTENTS Chapter I Introduction......................................................................................................... 1 Introduction..................................................................................................................... 2 Research Outline............................................................................................................. 8 References..................................................................................................................... 10 Chapter II Optimizing Methane Production from Organic Wastes................................... 14 Improving Biogas Production from Dilute Dairy Waste by Anaerobic Co-digestion with Food Waste............................................................................................................ 15 Abstract..................................................................................................................... 15 Introduction............................................................................................................... 15 Materials and methods .............................................................................................. 16 Results and discussion .............................................................................................. 18 Conclusion ................................................................................................................ 27 References................................................................................................................. 28 Chapter III Linkage between Microbial Populations and Process Conditions in Anaerobic Digestion........................................................................................................................... 30 Part I: Impact of Substrate Overloading on Archaeal Populations in Anaerobic Digestion of Animal Waste ........................................................................................... 31 Abstract......................................................................................................................... 31 Introduction............................................................................................................... 31 Materials and Methods.............................................................................................. 33 Anaerobic digester set-up ......................................................................................... 33 Chemical analysis ..................................................................................................... 33 Clone library analysis of archaeal microbial populations......................................... 34 Statistical Data Analysis ........................................................................................... 34 Results........................................................................................................................... 35 Anaerobic co-digester operation and performance................................................... 35 Archaeal populations in pre and post-overloading co-digesters by clone library analysis...................................................................................................................... 37 Discussion..................................................................................................................... 40 References..................................................................................................................... 43 Part II: Dynamics of Two Dominant Bacterial Phyla, Bacteroidetes and Firmicutes in Anaerobic Digestion Process with Organic Loading Perturbations ............................. 46 Abstract......................................................................................................................... 46 Introduction................................................................................................................... 46 Materials and Methods.................................................................................................. 48 Anaerobic co-digester setting and performance monitoring..................................... 48 Sample collection and DNA extraction..................................................................... 48 454 pyrosequencing and sequence analysis.............................................................. 48 Community diversity analysis .................................................................................. 49 Statistical