I would like to thank my supervisors - Raul Bettencourt, Conceição Egas and Ricardo Serrão Santos - for providing valuable advice, guidance and support during the course of my studies. My appreciation for the freedom to explore new paths and the continuous encouragement which allowed me to pursue my research scope, using often the funds from their own projects. I truly appreciate the opportunity to have worked with a great committee, in and outside the laboratory. Each committee member – my co-author or colleague – brought a different perspective that enhanced my research work. At DOP, University of the Azores, Valentina Costa, Inês Martins, Inês Barros, Joana Goulart, Sílvia Lino, Lucia Bongiorni and Paola Parretti; at Next Generation Sequence Unit, BIOCANT, Diogo Pinho, Hugo Froufe and Cristina Barroso; at Norstruct, University of Tromsø, Bjørn Altermark, Miriam Grgić, Tim Mee and at GeoBioTec, University of Aveiro, Carla Candeias. I am also grateful to the chief scientists from missions DeepFun (Ana Colaço) and BioBaz (François Lallier) for allowing the access to the hydrothermal samples for this work, and Eva Martins and Cátia Cardoso for samples preservation on board. Thanks to scientific and technical crews from R/V "Thalassa”, R/V "Pourquoi Pas ?" and ROV "Victor6000". A special consideration to all the members of the Department of Oceanography and Fisheries of the University of the Azores, past and present for their cooperation. In particular to Marina Carreiro-Silva, Gui Menezes, Daphne Cuvelier, Maria Magalhães, Verónica Neves, Fernando Tempera, Íris Sampaio, Diya Das and all the “DOP chef team”, Ricardinho, Paulinha, Sandra Andrade, Sandra Silva, Norberto, Sr. Santos and many others which, in a way or another, helped me fulfilling my needs and cross boundaries of my work. I would also like to express a profound gratitude to all my friends and family. Above all, I am grateful to my parents, Fernanda and João, both brother and sister, Bernardo and João and also my godmother Inha for their love, support and encouragement. A special acknowledgment to Roman, with whom this adventure began, and to all my friends from capoeira Garcia, Fred, Isabel, Álvaro, Ana, Anaïs, Maria, Alex, Tiago, Tiagão, Djemy, José, Rodrigo, Rafa, Eneko, Clothilde, Januy, Lise, Jordi, Cleuza, Mané and everyone who pushed me to challenge myself. Finally, I would like to thank my life companion Tomás for his day- to-day love, humour, support and motivation and for all his help with thesis design. None of this would be possible without all of you. I would also like to acknowledge the funding agencies, particularly Fundo Regional para a Ciência e Tecnologia for the financial support through the fellowship M3.1.2/F/052/2011 and IMAR-Centre management unit of the University of the Azores. This is a second version of my doctoral thesis originally delivered on the 30th December, 2016. Due to recent changes in the PhD regulations of the University of the Azores that became effective on August 2017, I was given an opportunity to improve my dissertation before the public defense and submit a second version, including the proposed changes to the original version. Acknowledgments ix Contents xi Abstract xvii Resumo xix I. GENERAL INTRODUCTION 1 1. Deep-sea Hydrothermal Vents 3 1.1 Historical background of deep-sea biology 3 1.2 Distribution of Hydrothermal activity in the oceans 5 1.3 Hydrothermal systems at Mid-Ocean Ridges 6 1.4 Deep-sea hydrothermal sediments 8 1.5 Mid-Atlantic hydrothermal vents 8 1.5.1 The basalt-hosted Menez Gwen 10 1.5.2 The basalt-hosted Lucky Strike 10 1.5.3 The ultramafic-hosted Rainbow 11 2. The microbial realm in hydrothermal vents 13 2.1 Microbial chemosynthesis 13 2.2 Microbial diversity at hydrothermal vent habitats 14 2.2.1 Microbial communities in the Menez Gwen, Lucky Strike and Rainbow 17 3. Metagenomics as a tool to study the environment 21 4. Aim of the thesis 25 4.1 Thesis outline 26 II. MICROBIAL DIVERSITY IN SEDIMENTS FROM THE MENEZ GWEN VENT FIELD 29 1. Introduction 31 2. Materials and Methods 35 2.1 Study site 35 2.2 Collecting sediment samples 35 2.3 Chemical analysis 36 2.4 DNA extraction 36 2.5 Sequencing of SSU rRNA gene amplicons 37 2.6 Sequence processing 38 2.7 Statistical analysis 38 3. Results 41 3.1 Chemical composition of the sediment samples 41 3.2 SSU tag pyrosequencing results 42 3.3 DNA extraction methods 42 3.4 Bacteria and Archaea diversity analysis 43 3.5 Comparative analysis of sediment-associated microbial communities 45 3.6 Bacterial community composition 46 3.7 Archaeal community composition 49 3.8 Micro-eukaryotic community composition 50 4. Discussion 51 4.1 DNA extraction methods 51 4.2 Microbial diversity 52 4.3 Vent Chimney site-specific microbial diversity 52 4.4 Bathyal Plain site-specific microbial diversity 54 4.5 Micro-Eukaryotic community composition 55 5. Conclusion 57 Appendix II 61 III. MICROBIAL DIVERSITY IN GEOCHEMICALLY CONTRASTING SEDIMENTS 69 1. Introduction 71 2. Materials and Methods 73 2.1 Sampling Sites 73 2.2 Sample collection 74 2.3 Chemical analysis 75 2.4 DNA extraction and PCR amplicon sequencing 75 2.5 Sequence processing 76 2.6 Statistical analysis 76 3. Results and Discussion 77 3.1 Trace element composition of the sediments 77 3.2 Pyrosequencing and microbial richness 78 3.3 Comparative analysis of the microbial community associated with the three hydrothermal sediments 80 3.4 Menez Gwen associated microbial communities 84 3.5 Lucky Strike associated microbial communities 86 3.6 Rainbow associated microbial communities 91 4. Conclusion 93 Appendix III 97 IV. METAGENOMIC SIGNATURES OF MICROBIAL COMMUNITIES FROM AZORES DEEP-SEA HYDROTHERMAL VENTS 103 1. Introduction 105 2. Materials and Methods 107 2.1 Samples Collection and DNA extraction 107 2.2 Metagenomic Sequencing and Annotation 109 2.3 Comparative metagenome analyses 109 2.4 Data availability 109 3. Results and Discussion 111 3.1 Microbial community composition 112 3.2 Functional composition 116 3.2.1 Carbon fixation 116 3.2.2 Sulfur metabolism 118 3.2.3 Nitrogen metabolism 122 3.2.4 Methane metabolism 123 3.2.5 Alternative electron donors 124 3.2.6 Oxygen as electron acceptor 124 4. Conclusions 127 Appendix IV 131 V. GENERAL DISCUSSION AND CONCLUSIONS 139 1. General Discussion 141 2. Enzymes with potential biotechnological applications 145 3. Final conclusions 147 List of abbreviations 151 List of figures 153 List of tables 157 Publications 159 Bibliography 163 The world’s oceans cover two-thirds of the planet’s surface being by far the largest habitat on Earth. The marine habitats range from sunlight surface waters to ocean trenches 11,000 m deep with pressures exceeding 1000 bar. Water temperatures range from sea ice, in the polar regions, to over 300 ºC, at deep-sea hydrothermal vents. Microorganisms are able to survive and grow throughout these environments, including the subsurface and the deep-sea. Deep- sea hydrothermal systems represent an important interface between the lithosphere and the oceans, and are considered to be “windows” into the subsurface biosphere. At these locations, thermally charged hydrothermal fluids, enriched with metal compounds and dissolved gases, are ejected into the ocean, providing conditions for supporting chemosynthesis and microbial growth. Extremophile organisms have adapted to the high pressures and temperature shifts, developed novel physiological strategies to thrive in such conditions resorting to unique enzymes and proteins with interesting activities and potential biotechnological applications. Chemoautotrophic Bacteria and Archaea are the primary source of nutrition for the overall organisms dwelling around the vents. They are primary producers of organic carbon, able to establish intricate chemosynthetic symbioses with micro- and macroorganisms, and to transfer the energy up to the food web, sustaining life in the deep-sea. Because of their unique features, they are considered plausible analogues to the early microorganisms of Earth. Therefore, the study of such putative early microbes may help us understanding the origin and evolution of life, and the adaptation mechanisms to these extreme environmental conditions. Moreover, these microorganisms are potentially playing important roles in global geochemical cycling between crusts and oceans, what makes their distribution and activities in deep-sea floors interesting subjects for contemporary microbial ecologists. So far, the diversity and distribution patterns of invertebrate vent communities have been reasonably investigated, however few studies reported on the microbial ecology of free-living Archaea and Bacteria in deep-sea hydrothermal sediments, even though their remarkable activity at these ecosystems. Prior to this work, sediment-associated microbial diversity in the Menez Gwen, Lucky Strike and Rainbow hydrothermal vent fields, southwest of the Azores, was still largely unexplored. Only three microbiological surveys based on 16S clone libraries were conducted in sediments from Rainbow field. The overall goal of this thesis was therefore to characterize the microbiota associated with hydrothermal sediments from the three most visited deep-sea vent fields of the Azores region, and the elucidation of the microorganisms’ metabolic potential. The outcome of this work gives useful insights into yet undiscovered organisms with unique living conditions, molecular mechanisms, and promising enzymes for biotechnological applications. As most of the vent-associated microorganisms remain uncultured, diversity studies are usually based in culture-independent sequencing technologies. Here, the bacterial, archaeal and micro-eukaryotic taxonomic profiles were addressed by barcoded pyrosequencing of a segment of the ribosomal RNA gene. The taxonomic profiles obtained were used to compare communities associated with different hydrothermal sediments retrieved from different sites. Metagenomic approaches were then used to characterize the metabolic potential of the communities. The microbial community associated with sediments from the Menez Gwen vent system was surveyed for the first time.