AN ABSTRACT OF THE DISSERTATION OF Amy Renee Smith for the degree of Doctor of Philosophy in Ocean, Earth, and Atmospheric Sciences presented on November 22, 2017. Title: Impact of Igneous Mineralogy on the Composition and Metabolic Function of Microbial Biofilms in a Thermal Suboceanic Crustal Aquifer. Abstract approved: _____________________________________________________ Frederick S. Colwell Martin R. Fisk Igneous oceanic crust encompasses ~60% of Earth’s surface and is composed of basalt glass and mafic, ultramafic, and felsic minerals. A vast marine aquifer lies within the crust, exchanging geochemically altered fluids with seawater from the overlying ocean at ridge crests, flanks, seamounts, and outcrops where permeable crust is exposed. Correlation studies of crustal surface rocks have shown that mineralogy is linked to microbiology; however, the influence of individual mineral phases or their compositions on microbial communities has yet to be empirically demonstrated. In addition, the habitable zone of oceanic crust can extend to depths of several kilometers and communities deeper within this zone may be more representative of the whole suboceanic aquifer ecosystem than those communities found just at the surface where the environment is influenced by infiltration of cold, oxic seawater. The focus of this work was to explore how deep subsurface biofilm communities in the suboceanic aquifer of the Juan de Fuca Ridge (JdFR) are influenced by igneous mineral phases and their compositions. We expect that crustal mineralogy will affect the microbial community structure and metabolism of these aquifer communities. Exploring the metabolisms of these communities will also lead to a greater understanding of the functioning of the suboceanic aquifer ecosystem and its role in the global carbon cycle. Microbial communities that colonized a variety of in situ-incubated igneous minerals and glasses were investigated. We used International Ocean Drilling Program (IODP) borehole 1301A as a subseafloor observatory to incubate these mineral substrates for a four-year period. After retrieval, we found that taxa related to thermophilic and hyperthermophilic chemolithotrophs and heterotrophs were present. Archaeal taxa included three genera of Archaeoglobaceae, including members of the sulfate-reducing genus Archaeoglobus. Bacterial taxa were overwhelmingly dominated by Clostridia and other deep-branching Bacteria. Most taxa were not closely related to known organisms so their metabolic capabilities could not be predicted by taxonomic association. Microbial communities were also influenced by the mineralogical properties of attachment surfaces; particularly with respect to iron- rich phases. Communities attached to rocks, minerals, and glasses in this environment were more similar to each other than they were to aquifer fluid communities, bottom seawater, and other marine or deep crustal communities, and thus represented a distinct mineral-colonizing “attached” community. Metabolic reconstruction of metagenome-derived genomes from olivine also showed that sulfate reduction, carbon fixation, and hydrogenotrophic pathways dominated that community. These results suggest there is a potential for hydrogen-based chemolithoautotrophy in the deep oceanic crust, that these microbial communities do not fully rely on photosynthetically-derived organic carbon for energy and carbon, and that the suboceanic aquifer biosphere may play a role in global carbon cycling and productivity. ©Copyright by Amy Renee Smith November 22, 2017 All Rights Reserved Impact of Igneous Mineralogy on the Composition and Metabolic Function of Microbial Biofilms in a Thermal Suboceanic Crustal Aquifer by Amy Renee Smith A DISSERTATION submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Presented November 22, 2017 Commencement June 2018 Doctor of Philosophy dissertation of Amy Renee Smith presented on November 22, 2017. APPROVED: Co-Major Professor, representing Ocean, Earth, and Atmospheric Sciences Co-Major Professor, representing Ocean, Earth, and Atmospheric Sciences Dean of the College of Earth, Ocean, and Atmospheric Sciences Dean of the Graduate School I understand that my dissertation will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my dissertation to any reader upon request. Amy Renee Smith, Author ACKNOWLEDGEMENTS I wish to thank my committee members for their patience and support though the years. Drs. Frederick S. Colwell and Martin R. Fisk have been instrumental in helping me amalgamate my work into cohesive, influential manuscripts, and provided advice for funding applications, exams, classwork, presentations, and more. I am forever in your debt for helping me become a better scientist. Thank you to Drs. Staci Simonich, Adam Schultz, and Ryan Mueller for agreeing to serve on my committee. A special thank you goes to Dr. Mueller for providing significant support in bioinformatics which was crucial to the success of this dissertation and for our deep discussions about metabolism. Finally, the Geomicrobiology Group has provided insight and advice for presentations, support during my time here, and invaluable feedback in a warm and supportive environment. I would also like to acknowledge collaborators who have contributed to my work and offered insight when needed. For their contribution to my ocean crust research, I would like to thank Dr. Mark Nielsen, Dr. C Geoffrey Wheat, Dr. Andrew Fisher, Dr. Hans Jannasch, Dr. Keir Becker, and Dr. Stefan Sievert. Thank you also to the crews of the submersible Alvin and the RV Atlantis and JOIDES Resolution. Many people provided instruction on writing and scientific analyses throughout my time at OSU. Dr. Andrew Thurber provided instruction and guidance on community analysis with PRIMER, Teresa Sawyer trained me in the use of the Environmental Scanning Electron Microscope, and Dr. Olivia Mason introduced me to MG-RAST and STAMP. Funding for the flow cell construction was provided by a small grant from the Ocean Drilling Program. The idea for the flow cells used in this project was developed at a workshop in Bergen Norway in 2002 and was made possible by support from the University of Bergen and the Ocean Drilling Program. Unimin Co. provided the Fo90 olivine. This work was also funded by grants from the National Aeronautics and Space Administration (NASA), the National Science Foundation’s Center for Deep Energy Biosphere Investigations (C-DEBI), and the Census of Deep Life which is funded by the Sloan Foundation as part of the Deep Carbon Observatory. I would like to give a very special thanks to the folks involved with C-DEBI and the Deep Carbon Observatory. In particular, I would like to thank Rosalynn Sylvan and Katie Pratt for providing incredible support and positivity. Those involved with the sequencing of Census of Deep Life projects at the Marine Biological Laboratory provided stellar pyrotag and metagenome sequencing results and other related support that provided the basis for all the work in this dissertation. I can’t thank you all enough! Finally, I would like to acknowledge my family and friends who provided so much love and support throughout graduate school. My husband Shane and daughters Stella and Zephyr have loaded me up with enough hugs and smiles to get me through the moments when I have felt discouraged. Stella and Zephyr, you are my inspiration and I do this as much for you as I do for myself! I hope you are proud of your Mommy and know that you can do anything you set your mind to. My Mom Cynthia has been incredibly helpful by flying to conferences with me to babysit the girls while I present my work and being there at the other end of the phone line when I needed encouragement. I certainly could not have done this without you, Mom, and I appreciate everything you have done for me! I love you. CONTRIBUTION OF AUTHORS For Chapters 1 and 5, Drs. Martin R. Fisk and Frederick S. Colwell provided edits, suggested revisions, and direction. For Chapter 2, Drs. Martin R. Fisk and Frederick S. Colwell provided writing and conceptual advice, Dr. Andrew Thurber and Gilberto E. Flores helped guide the analyses of pyrotag data, and Dr. Olivia U. Mason provided technical and writing advice. Radu Popa provided support during the writing phase and was instrumental in procuring funding for pyrotag sequencing from C-DEBI and the Census of Deep Life. For Chapter 3, Ryan Mueller provided instruction and guidance on bioinformatics principles and lent his technical expertise to help me solve problems during the metagenome analysis. Olivia U. Mason aided in project design, funding, and initial analyses. Radu Popa provided support during the writing phase. Brandon Kieft analyzed genome bin purity and completeness using CheckM and produced Supplemental Figure 3.1. Frederick S. Colwell and Martin R. Fisk provided guidance and support throughout the data collection, analysis, and writing phases. For Chapter 4, again Ryan Mueller provided instruction and guidance on bioinformatics and metagenome analysis. Frederick S. Colwell and Martin R. Fisk provided guidance and support throughout the data collection, analysis, and writing phases. TABLE OF CONTENTS Page 1. INTRODUCTION………….……….……………………………………………..1 Oceanic Crust……………………………………………………….………….. …1 Structure………………………………………………………………….…… 1 The Suboceanic Aquifer…………………………………………………...…. 3 The Habitable Zone…………………………………………….………….......4