Hydrogen Stress and Syntrophy of Hyperthermophilic Heterotrophs and Methanogens
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University of Massachusetts Amherst ScholarWorks@UMass Amherst Doctoral Dissertations Dissertations and Theses July 2018 HYDROGEN STRESS AND SYNTROPHY OF HYPERTHERMOPHILIC HETEROTROPHS AND METHANOGENS Begum Topcuoglu Follow this and additional works at: https://scholarworks.umass.edu/dissertations_2 Part of the Environmental Microbiology and Microbial Ecology Commons, and the Microbial Physiology Commons Recommended Citation Topcuoglu, Begum, "HYDROGEN STRESS AND SYNTROPHY OF HYPERTHERMOPHILIC HETEROTROPHS AND METHANOGENS" (2018). Doctoral Dissertations. 1299. https://scholarworks.umass.edu/dissertations_2/1299 This Open Access Dissertation is brought to you for free and open access by the Dissertations and Theses at ScholarWorks@UMass Amherst. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of ScholarWorks@UMass Amherst. For more information, please contact [email protected]. HYDROGEN STRESS AND SYNTROPHY OF HYPERTHERMOPHILIC HETEROTROPHS AND METHANOGENS A Dissertation Presented by BEGÜM D. TOPÇUOĞLU Submitted to the Graduate School of the University of Massachusetts Amherst in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY May 2018 Microbiology © Copyright by Begüm Topçuoğlu 2018 All Rights Reserved HYDROGEN STRESS AND SYNTROPHY OF HYPERTHERMOPHILIC HETEROTROPHS AND METHANOGENS A Dissertation Presented by BEGÜM D. TOPÇUOĞLU Approved as to style and content by: ____________________________________ James F. Holden, Chair ____________________________________ Kristen DeAngelis, Member ____________________________________ Klaus Nüsslein, Member ____________________________________ Steven Petsch, Member __________________________________ Steven Sandler Microbiology Department Head DEDICATION This dissertation is dedicated to my parents Eser and Ersin for being the source of my perseverance. And to my best friend Başak who always has my back. ACKNOWLEDGMENTS I would like to thank my advisor Jim Holden for his unwavering support and invaluable mentorship throughout the years. His dedication to his work and to his students inspire me every day. I would like to acknowledge my dissertation committee – Klaus Nüsslein, Kristen DeAngelis and Steve Petsch for their support. I would also like to thank the Microbiology Deparment at UMass for being my home for the past six years. Within our lab, I would like to thank my fellow graduate students – Lucy Stewart, Jenn Lin, Srishti Kashyap, Emily Moreira, Sarah Hensley, and James Llewellyn. I would also like to thank Roberto Orellana and Cem Meydan for their mentorship. I would like to thank our fieldwork collaborators for this research – Julie Huber, Caroline Fortunato, Joe Vallino, Chris Algar, Dave Butterfield, Ben Larson and the crews of the R/V Thomas G. Thompson, the R/V Ronald H. Brown, as well as the operating teams of ROV Jason II. I am grateful for my chosen family, scattered around the world but always a phone call away, especially Duygu Ula who has been my rock. Your love and support made this possible. I am also grateful for the many friends I had in Amherst who made these six years a joyful time. I would like to thank my family for their encouragement and support that made this transatlantic journey possible. Finally, I would like to thank my grandmother Aysel Deniz for being my first teacher. Thank you for giving me the love of reading. v ABSTRACT HYDROGEN STRESS AND SYNTROPHY OF HYPERTHERMOPHILIC HETEROTROPHS AND METHANOGENS MAY 2018 BEGÜM D. TOPÇUOĞLU B.S., SABANCI UNIVERSITY Ph.D., UNIVERSITY OF MASSACHUSETTS AMHERST Directed by: Dr. James F. Holden 15 Approximately 1 giga ton (Gt, 10 g) of CH4 is formed globally per year from H2, CO2, and acetate through methanogenesis, largely by methanogens growing in syntrophic association with anaerobic microbes that hydrolyze and ferment biopolymers. However, our understanding of methanogenesis in hydrothermal regions of the subseafloor and potential syntrophic methanogenesis at thermophilic temperatures is nascent. This dissertation shows that thermophilic H2 syntrophy can support methanogenesis within natural microbial assemblages at hydrothermal vents and that it can be an important alternative energy source for thermophilic autotrophs in marine geothermal environments. This dissertation also elucidates H2 stress survival strategies of the H2 producing heterotrophs and the H2 consuming methanogens as well as their cooperation with one another for survival. The growth of natural assemblages of thermophilic methanogens from Axial Seamount was primarily limited by H2 availability. Heterotrophs supported thermophilic methanogenesis by H2 syntrophy in microcosm incubations of hydrothermal fluids at 80°C supplemented with tryptone only. Based on 16S rRNA gene sequencing, H2 producing vi heterotrophs, Thermococcus, and H2-consuming methanogens, Methanocaldococcus were abundant in 80°C tryptone microcosms from an Axial Seamount hydrothermal vent. In order to model the impact of H2 syntrophy at hyperthemophilic temperatures, a coculture was established consisting of a H2-producing hyperthermophilic heterotroph and a H2- consuming hyperthermophilic methanogen. The model organisms, hyperthermophilic heterotroph Thermococcus paralvinellae and the hydrogenotrophic methanogen Methanocaldococcus jannaschii, were examined in monocultures and cocultures for their H2 stress and syntrophy strategies. In monocultures, H2 inhibition changed the growth kinetics and the transcriptome of T. paralvinellae. A significant decrease in batch phase growth rates and steady state cell concentrations was observed with high H2 background. Metabolite production measurements, RNA-seq analyses of differentially expressed genes, and in silico experiments performed with a constraint-based metabolic network model showed that T. paralvinellae produces formate by a formate hydrogenlyase to survive H2 inhibition. H2 limitation on M. jannaschii caused a significant decrease in batch phase growth rates and CH4 production rates but also caused a significant increase in cell yields. In cocultures, H2 syntrophy relieved H2 stress for T. paralvinellae but not for M. jannaschii. T. paralvinellae only produced formate when grown in monoculture while no formate was detected during growth in coculture. While M. jannaschii was capable of growth and methanogenesis solely on the H2 produced by T. paralvinellae, the growth and CH4 production rates of M. jannaschii decreased in coculture compared to when M. jannaschii was grown in monoculture. vii TABLE OF CONTENTS Page ACKNOWLEDGMENTS ...................................................................................................v ABSTRACT ....................................................................................................................... vi LIST OF TABLES ............................................................................................................. xi LIST OF FIGURES ......................................................................................................... xiii CHAPTER 1. INTRODUCTION ...................................................................................................1 1.1 Objectives ...........................................................................................................1 1.2 Life in the Hot Subsurface Biosphere ................................................................3 1.2.1 Marine Hydrothermal Vents ...............................................................5 1.2.2 Terrestrial Hot Springs ........................................................................8 1.2.3 Marine Sediments and Hydrocarbon Reservoirs ................................9 1.2.4 Microbiology of Hot Biosphere Habitats ..........................................11 1.3 Syntrophy Among Thermopiles .......................................................................24 1.4 Research Approach and Significance ..............................................................25 2. HYDROGEN LIMITATION AND SYNTROPHIC GROWTH AMONG NATURAL ASSEMBLAGES OF THERMOPHILIC METHANOGENS AT DEEP-SEA HYDROTHERMAL VENTS ......................................................27 2.1 Abstract ............................................................................................................27 2.2 Introduction ......................................................................................................28 2.3 Methods............................................................................................................30 2.3.1 Field sampling ...................................................................................30 2.3.2 Microcosm incubations .....................................................................33 2.3.3 DNA extraction and 16S rRNA amplicon sequencing .....................35 2.3.4 Growth Media ...................................................................................36 2.3.5 Most Probable Number (MPN) cell estimates ..................................37 2.3.6 Pure and coculture growth conditions ...............................................37 2.4 Results ..............................................................................................................40 2.4.1 MPN cell estimates in hydrothermal fluids .......................................40 viii + 2.4.2 Growth in microcosms on H2, CO2 and NH4 ...................................45 2.4.3 H2 syntrophy in microcosms .............................................................49 2.5 Discussion