Abstract PHYSIOLOGICAL STUDIES OF ALKALIPHILIC ANAEROBIC ORGANOTROPHS IN A SERPENTINIZING SUBSURFACE HABITAT By Crystal R. George October 27, 2014 Director: Dr. Jeffrey McKinnon Department: Biology Microbial habitats in serpentinizing ultramafic rocks represent one of the largest, yet least understood portions of the biosphere, with potentially major consequences for global biogeochemical cycles. Serpentinization is a process where ultramafic rock is uplifted to Earth’s surface through tectonic activity, where it reacts with water to create a highly reducing environment rich in hydrogen, methane, and small abiogenic organic compounds. These compounds may serve as important sources of nutrients and energy to sustain microbial metabolism. While serpentinites can provide the energy to sustain microbial communities, the conditions created by serpentinization can be harsh and challenge the limits of microbial physiology in terms of extreme pH (>11), and availability of terminal electron acceptors. Ongoing culture-independent studies by our research group have identified bacterial taxa related to the order Clostridiales as important components of the highest pH, most reducing subsurface habitats. The goal of this project was to systematically analyze the nutrients necessary to sustain life in a serpentinizing subsurface habitat located on McLaughlin Natural Reserve near Lower Lake, CA. The research consisted of creating microcosm enrichment cultures to evaluate the distribution and environmental controls on Clostridiales species within a subsurface microbial observatory in serpentinizing rocks. Subsequently, the cellular abundance of a novel isolate from these experiments was measured in relation to organic carbon utilization, and utilization of iron and sulfur as terminal electron acceptors. We have also conducted a cursory analysis of annotated biochemical pathways in the genomes of two isolates in relation to respiration and metabolism of carbohydrates. From these experiments we have shown that Clostridiales may play important roles in the biogeochemistry of carbon, iron, and sulfur in serpentinite environments. The research also provides further insight into habitability in these extreme environments and sources of nutrients and energy. Knowledge on the subsurface metabolic capabilities gained through this research has implications for the development of future biotechnologies related to bioremediation and microbial fuel cells. PHYSIOLOGICAL STUDIES OF ALKALIPHILIC ANAEROBIC ORGANOTROPHS IN A SERPENTINIZING SUBSURFACE HABITAT A Thesis Presented To The Faculty of The Department of Biology East Carolina University In Partial Fulfillment of the Requirements for the Degree Master of Science Molecular Biology and Biotechnology By Crystal R George October 27, 2014 © Copyright 2014 Crystal R. George All Rights Reserved PHYSIOLOGICAL STUDIES OF ALKALIPHILIC ANAEROBIC ORGANOTROPHS IN A SERPENTINIZING SUBSURFACE HABITAT by Crystal R. George APPROVED BY: DIRECTOR OF THESIS:___________________________________________ Matthew Schrenk, PhD COMMITTEE MEMBER:__________________________________________________ Anthony Overton, PhD COMMITTEE MEMBER:__________________________________________________ Mary Farwell, PhD COMMITTEE MEMBER:__________________________________________________ Adriana Heimann-Rios, PhD CHAIR OF THE DEPARTMENT OF Biology: ________________________________________________ Jeffrey McKinnon, PhD DEAN OF THE GRADUATE SCHOOL: ________________________________________________ Paul J. Gemperline, PhD ACKNOWLEDGEMENTS First and foremost, I would like to thank Dr. Matthew Schrenk for me giving me the opportunity to conduct research in his lab and meet collaborators through travel to the Coast Range Ophiolite to further conduct hands on research for my project. Allowing me that opportunity gave me a once-in-a-lifetime experience of which I am thankful for. I would also like to thank Dr. Farwell, Dr. Overton and Dr. Heimann-Rios for providing their expertise and guidance with my writing and experimental design. I also extend my gratitude to two of my senior labmates, Melitza Crespo-Medina, and Katrina Twing for providing moral support and guidance over the duration of my research, as well as providing environmental samples and showing me how to collect my own samples during our research trip to CROMO. Secondly, I would like to thank Denise Mayer, Manager of the Genomics Core Facility at East Carolina University for her help with sequencing. In addition, I would like to thank Alyssa Kloysuntia for her help with this project and contributions that she made to our lab. Additionally, I would like to thank Dr. William Brazelton who provided his expertise with several molecular biology methodologies along with structural guidance to several of my experimental designs. Lastly, I would like to thank my family and friends for their encouragement during this program. Funding for this project was provided to Dr. Schrenk from the Division of Research and Graduate Studies at East Carolina University, the NASA Astrobiology Institute through the Carnegie Institution for Science, the Deep Carbon Observatory, and the Alfred P. Sloan Foundation TABLE OF CONTENTS Title Page………………………………………………………………………...... i Copyright…………………………………………………………………………. ii Signature Page……………………………………………………………………. iii Acknowledgements……………………………………………………………..… iv List of Tables............................................................................................................ vii List of Figures.......................................................................................................... viii Chapter 1: Review of Literature………..…..…………………………………….... 1 Biogeochemistry of Serpentinization in Ophiolites…………………...……. 1 Study Site: Coast Range Ophiolite, Lower Lake, California………....…...... 4 Molecular Biology Analysis of Microbial Communities at CROMO…...…. 4 Importance of Dethiobacter alkaliphilus…………....………….…..... 7 Metabolic Capabilities of Alkaliphilic Anaerobes ……………………....…. 8 Iron……………………………………………......…………….……. 8 Sulfur……………………………………………………………..…... 12 Assessment of Metabolic Capabilities at an Isolate Level………………...... 13 Relevance of Experiment…………......………………………………….…. 14 Chapter 2: Research Objectives…………………………………………….....…... 16 Systematically Enrich for Anaerobic Microbial Communities………....…... 1 6 Assess the Diversity of Microcosm Experiments Through TRFLP.....…..…. 17 Partially Characterize a Microorganism from Microcosm Experiment ….... 17 Discover Genes Relevant to Iron, Sulfur and Carbon Metabolic Pathways through Genomic Analyses............................................................................. 17 Chapter 3: Central Hypotheses…………………………………………………...... 18 Chapter 4: Methods……………………………………………………………....... 20 Sample Collection…………………………..………………………………. 20 Culturing and Enrichments………………………………………………….. 22 Cellular Abundance………………………………………………………..... 23 Dilution to Extinction………………………………………………....…….. 24 DNA Extraction……………………………………………......................…. 25 TRFLP Analyses……………………………………............………………. 25 Polymerase Chain Reaction………………………….....…………………… 27 16S rRNA Sequencing…………………………………..............………….. 28 Characterization of an Isolate.......................................................................... 28 Genomics Analyses and Annotation in RAST……………………......…….. 29 Chapter 5: Results………………………………………………………....………. 30 Electron Acceptor Microcosm Treatments…………………………....…….. 30 March Microcosm Experiment………....................................................…… 33 August Microcosm Experiment……….………….................................……. 36 Assessment of Genetic Diversity at CROMO through TRFLP Utilization.... 40 Sugar Utilization by Anaerobic Culture WB…….................…..................... 44 Genomics Comparison of AHT1 and WB and WB1……………………….. 45 Chapter 6: Discussion……………………………………………....……………… 49 References................................................................................................................ 55 APPENDIX A: TRFLP Cluster Dendrograms (Bray-Curtis Similarity)…….. 59 LIST OF TABLES 1. CROMO Wells, Depth, pH and Eh……………………………….………........ 21 2. Restriction Enzymes used in TRFLP…………………………….…………..... 26 3. Beginning and Ending Cellular Abundance – March 2013……………….…... 31 4. Beginning and Ending Cellular Abundance – August 2013…………….…...... 32 5. Normalized (n) values for microcosms March 2013…………………………... 33 6. Statistical correlation between (n) and environmental parameters, March 2013……………………..................................................................................... 35 7. Normalized (n) values for microcosms August 2013…………..……………… 37 8. Statistical correlation between (n) and environmental parameters, August 2013..................................................................................................................... 38 9. Electron Acceptor (n) values for isolate WB....................……………….……... 44 10. Sugar Utilization (n) values for isolate WB…....................…………….………. 45 11. Genes related to respiration in WB, WB1 and AHT1………………..………... 46 12. Genes related to carbohydrate utilization in WB, WB1 and AHT1……..…….. 47 13. Genes related to carbohydrate utilization in WB, WB1 and AHT1..................... 48 LIST OF FIGURES 1. Biogeochemical Processes in a Serpentinite Habitat……………………... 3 2. 16S rRNA sequences mapped to Clostridiales phylogeny……………....... 5 3. Pyrotag sequences from March 2013……………………………………... 6 4. Collection of environmental samples from CROMO…………………….