Deep-sea bacteria: a quest for sulfur and manganese oxidizers Ana Marta Rodrigues Lourenço Thesis to obtain the Master of Science Degree in Microbiology Supervisors: Prof. Rogério Paulo de Andrade Tenreiro Prof. Rodrigo da Silva Costa Examination Committee Chairperson: Prof. Jorge Humberto Gomes Leitão Supervisor: Prof. Rogério Paulo de Andrade Tenreiro Member of the Comittee: Dr. Tina Keller Costa November, 2018 ACKNOWLEDGMENTS This thesis is the culminate of one year of work on the Microbiology and Biotechnology Laboratory. I would like to thank the team of the Estrutura de Missão para a Extensão da Plataforma Continental (EMEPC) that collected the samples used in this work. It was not an easy year and I could have not gone through it without the precious help of my friends and family. Firstly, to my supervisor Professor Rogério Tenreiro, for the opportunity to work with him and for the help but mostly for the patience and for never giving up on me. It was so great to learn from you, even when you were mocking on my excel sheets. A real big thank you from the one and only (I hope), La Bufadora. To Professor Rodrigo Costa, for the help and availability during this work. To Professor Ana Tenreiro for the everyday smile and good mood she always brings, for the hundreds of gums and more important, for the care. To Filipa Silva and Francisca Moreira for maintaining the most organized laboratory I have ever been. To my “Lindos do Toclabs”, it is so great when you work in a place where more than colleagues you have friends. To Cláudia, for everything she taught me, always with such caring. To Pedro, for the help, advice and for bringing us Mateus. To André and Beatriz, for the patience, advice and companionship. To João, for always having a bad joke that makes you smile. To Catarina, for always having a helping hand with the greatest smile and cutest eyes. To Ana, for the endless hugs and for always having room for me in her shoulders. To Miguel, whose gratitude goes beyond this thesis, for being such an amazing person you can count on. To Inês and Mariana, thank you is such a powerless word to express how much gratitude I owe you for the past years, for always being there, for the good, the great, the bad and the worse. Thank you for helping me, for believing in me, for the strength, for all the tears dried and for making me smile. You will always be the other two vertices of my triangle. To the rest of the Bugworkers team, to Raquel, for always bringing laugh to the table. To Jéssica, for the opportunity to getting to know you after sharing a whole bachelor, for the sharing and for being such a good friend when we both moved to Lisbon. For the help and companionship throughout these years and for being the friend with such bad humor at night that I can always mock on. To my friends from Leiria, some of which I have known for the majority of my life, for everything that we shared and lived. To the more recent ones, for such good diners, sharing and for pushing me. To my girls and boys from Coimbra, for still being there; it is true when people say your college friends are for life no matter the distance. Last but definitely not least, to my parents and my sister, for all the support throughout my entire life; for being there, no matter what; for the endless calls, the cuddling, for always pushing me further but mostly, for believing in me even when I didn’t. You are amazing. Truly, thank you. ii ABSTRACT Deep-sea environments are hosts of several microorganisms, such as sulfur and manganese oxidizing bacteria (SOB and MOB). These can oxidize reduced sulfur and manganese compounds as energy sources, fixing carbon dioxide autotrophically or growing heterotrophically on carbon sources. Sixty-two deep-sea sediment samples, obtained from five frozen sub-seafloor corers collected along Azores island in 2007, were enriched in sulfur and manganese media to isolate putative sulfur and manganese oxidizers. The isolates were characterized from morphophysiological features to gene detection. Moreover, based on M13 PCR fingerprints of isolates, dendrograms were constructed and diversity was also assessed; 16S rRNA gene sequencing was also performed for representative isolates. A collection of 32 and 144 putative SOB and MOB, respectively, was obtained, being the majority of them gram-positive rods, most likely to belong to Bacillus spp.. Moreover, even though it seems that there is a great diversity throughout the collection of isolates, it was possible to see that these seem to be spread along the corers and in each corer, in terms of depth. Furthermore, through the sequencing results and the constructed phylogeny, it seems that the majority of the isolates is related to Bacillus spp., what goes into accordance with the morphophysiological characterization. Concerning the search for functional genes related to sulfur oxidation and carbon metabolism, the results were not so promising in the sense that, in fact, no gene presence could be doubtless assumed. However, the amplifications were also tested for the enrichment cultures and regarding sulfur oxidation some results seem more promising. Key words: deep-sea; sulfur-oxidizing bacteria; manganese-oxidizing bacteria; fingerprinting; gene detection. iii RESUMO O fundo do mar é um ambiente propício ao desenvolvimento de vários grupos de microrganismos, como as bactérias oxidantes de enxofre e manganésio. Estas são capazes de oxidar compostos de enxofre e manganésio reduzidos de forma a obter energia, fixando o dióxido de carbono autotroficamente ou crescendo com fontes de carbono heterotroficamente. Sessenta e duas amostras de sedimentos marinhos foram enriquecidas em meios com fontes de enxofre e manganésio com o objetivo de isolar bactérias oxidantes de enxofre e manganésio. Os isolados foram caracterizados em termos de características morfofisiológicas e de deteção de genes. Através de amplificação de M13, foram construídos dendrogramas permitindo estudar a diversidade nas amostras e nos meios; para alguns isolados foi também realizada sequenciação de rRNA 16S. Uma coleção de 32 e 144 isolados para enxofre e manganésio, respetivamente, foi obtida, sendo a maioria deles bastonetes gram-positivos, podendo pertencer ao género Bacillus. A diversidade da coleção era grande sendo que os isolados parecem estar presentes em vários corers e em várias das suas secções. No que toca aos resultados de sequenciação, a maioria dos isolados parece pertencer ao género Bacillus, o que é concordante com as características morfológicas. Relativamente à pesquisa de genes para a oxidação de enxofre e metabolismo do carbono dos isolados, não houve resultados positivos claros. No entanto, as mesmas amplificações foram testadas nos enriquecimentos e os resultados parecem mais promissores. Palavras-chave: fundo do mar; bactérias oxidantes de enxofre; bactérias oxidantes de manganésio; fingerprinting; deteção de genes. iv INDEX ACKNOWLEDGMENTS .............................................................................................................................................. ii ABSTRACT ................................................................................................................................................................ iii RESUMO .................................................................................................................................................................. iv INDEX ....................................................................................................................................................................... v LIST OF FIGURES ..................................................................................................................................................... vii LIST OF TABLES ........................................................................................................................................................ ix LIST OF ABBREVIATIONS........................................................................................................................................... x INTRODUCTION ....................................................................................................................................................... 0 1. Deep-sea: an overview ............................................................................................................................... 1 2. Sulfur Cycle and Sulfur Oxidizing Bacteria ................................................................................................. 2 2.1. Sulfur-Oxidizing Bacteria (SOB) ......................................................................................................... 3 3. Manganese Cycle and Manganese Oxidizing Bacteria ............................................................................. 11 3.1. Manganese-Oxidizing Bacteria (MOB) ............................................................................................ 12 4. Thesis goal ................................................................................................................................................ 20 MATERIALS AND METHODS .................................................................................................................................. 21 1. Sampling................................................................................................................................................... 21 2. Enrichment cultures ................................................................................................................................