Distribution of Dehalococcoidia in marine sediments and strategies for their enrichment vorgelegt von Master of Science Camelia Algora geb. in Madrid, Spanien von der Fakultät III – Prozesswissenschaften der Technischen Universität Berlin zur Erlangung des akademischen Grades Doktor der Naturwissenschaften / Doctor rerum naturalium -Dr. rer. nat.- genehmigte Dissertation Promotionsausschuss: Vorsitzender: Prof. Dr. Juri Rappsilber Gutachter: Prof. Dr. Peter Neubauer Gutachter: PD Dr. Lorenz Adrian Gutachter: Prof. Dr. Ricardo Amils Tag der wissenschaftlichen Aussprache: 11. Juli 2016 Berlin 2016 I II “Life on earth is such a good story you cannot afford to miss the beginning. Beneath our superficial differences we are all of us walking communities of bacteria. The world shimmers a pointillist landscape made of tiny living beings” Lynn Margulis Dedicado a mis tíos, Dr. José Luis Gallardo y Dra. María del Carmen López, por plantar la semilla del interés por la Ciencia en mi adolescencia, por su ejemplo y por todo su apoyo III IV DECLARATION OF INDEPENDENT WORK Erklärung zur Dissertation Ehrenwörtliche Erklärung zu meiner Dissertation mit dem Titel: ‘Distribution of Dehalococcoidia in marine sediments and strategies for their enrichment’ hiermit erkläre ich, dass ich die beigefügte Dissertation selbstständig verfasst und keine anderen als die angegebenen Hilfsmittel genutzt habe. Alle wörtlich oder inhaltlich übernommenen Stellen habe ich als solche gekennzeichnet. Wörtlich oder inhaltlich Stellen, die aus meinen persönlichen Publikationen stammen, wurden nicht zusätzlich gekennzeichnet. Ich versichere, dass ich die beigefügte Dissertation nur in diesem und keinem anderen Promotionsverfahren eingereicht habe und dass diesem Promotionsverfahren keine endgültig gescheiterten Promotionsverfahren vorausgegangen sind. _____________________ ______________________________ Ort, Datum Unterschrift V ACKNOWLEDGEMENTS The present work was carried out within the research group of PD Dr. Lorenz Adrian at the Department of Isotope Biogeochemistry (ISOBIO) at the Helmholtz Centre for Environmental Research–UFZ in Leipzig, Germany, from the beginning of November 2008 to end of February 2012, except for a period of 10 weeks on board the Polarstern, during the expedition ARKXXV/3. The PhD work was done under the research project MICROFLEX (Proposal Nr. 202903), which was funded by the European Research Council. In the first instance, I would like to deeply thank my supervisor, Dr. Lorenz Adrian, for giving me the great opportunity to accomplish this PhD project, for supporting me throughout its development, for innumerable ideas, and contagious enthusiasm. I thank him for sharing his knowledge with numerous BBCs, inspiring discussions, and for providing excellent advice on publications and this thesis. I would like to thank Prof. Peter Neubauer for external supervision and review of my thesis work, and Prof. Ricardo Amils for reviewing my dissertation work and for being a deeply inspirational lecturer during my time at UAM (Universidad Autónoma de Madrid). I thank Prof. Dr. Juri Rappsilber for being the head of the dissertation committee. I am honoured to have worked with Dr. Kenneth Wasmund, whom I profoundly thank for immense support, brilliant ideas, fabulous scientific discussions, and great friendship. This work could not have been done without his help. I am immensely grateful to Dr. Martin Krüger, for the opportunity to join the research expedition on the Polarstern and for fruitful collaboration. Thanks to Friederike Gründger for sharing field work and time on board the Polarstern and further collaboration in the BGR. Thanks to all the colleagues at the expedition ARK XXV/3 and especially to Dr. Volkmar Damm and Dr Thomas Pletsch. I would like to thank Dr. Timothy G. Ferdelman and Dr. Casey Hubert, from the Max Planck Institute for Marine Microbiology in Bremen, for kindly suppling sediment samples. It has been an immense pleasure to me to collaborate with Dr. Sotirios Vasileiadis and Dr. Edoardo Puglisi. I especially would like to thank them for their dedication and a fantastic working atmosphere. My most sincere gratitude goes to Dr. Paula Martínez, Dr. Petra Bombach, Dr. Anke Wagner, and Dr. Kelly E. Fletcher, for their great scientific advice, suggestions, enthusiasm, support, and friendship. VI It has been a pleasure to me to work with an international lab group, full of lovely and talented microbiologists from all over the world. I especially want to thank Dr. Ernest Marco-Urrea, Anja Kublik, Josefine Müller, Dr. Chang Ding, Katja Seidel, and Chao Yang, among many others. I particularly would like to thank Myriel Cooper for helpful discussions and splendid collaboration within our MICROFLEX project, and for her friendship. Special thanks go to Christina Lachmann, for fabulous help with the first pioneering months setting up the lab and the first cultures and experiments. Many thanks go to our technician, Benjamin Scheer, for his support from the beginning until the end of my PhD project. My very special thanks go to my students: Christina M. Ridley, Vaida Vaitkeviciute, Sebastian Röther, and Sukwon Jang. I thank them for their hard work and dedication, their eagerness to learn and enthusiasm. I would like to thank all my colleagues at the ISOBIO department, and specially, I would like to thank Dr. Ivonne Nijenhuis, our department leader Dr. Hans-Hermann Richnow, Dr. Sara Herrero, Dr. Mònica Rosell, Dr. Felipe Bastida, Dr. Maria Luisa Feo, Marie Markantonis, Kristina Hitzfeld, Jan Birkigt, and Julian Renpenning among many others. I am honoured to have been part of the HIGRADE graduate school of the UFZ, and I would like to thank Vera Bissinger and Barbara Timmel, among the many people who run it. I very much would like to thank all the friends that have enriched my life throughout these years in the UFZ, their names are too many to be written, but their support, love and friendship was essential. Mi mayor agradecimiento se lo dedico a mi familia, por su inmenso apoyo, comprensión y amor, sin el cual hubiera sido totalmente imposible alcanzar la consecución de este trabajo de tesis doctoral. VII ABSTRACT The marine subsurface is one of the largest microbial habitats on Earth. Microbial community studies based on the 16S rRNA gene have revealed that bacteria of the class Dehalococcoidia, phylum Chloroflexi, are one of the most widespread and abundant bacteria inhabiting the marine subsurface. However, their physiology and ecological role are unknown. In this study, marine sediments were cultivated with various potential electron acceptors to explore respiration modes catalysed by Dehalococcoidia. The cultivation revealed that Dehalococcoidia can be cultivated in an anoxic minimal medium amended with hydrogen and acetate as potential electron donor and carbon source, respectively, at a temperature of 30ºC and at atmospheric pressure, and thus Dehalococcoidia are not strict piezophilic bacteria. An increase in Dehalococcoidia 16S rRNA gene copy numbers of one order of magnitude, as evidenced by quantitative PCR with newly designed primers, could be observed in a time span of months for several sediment cultures, indicating that their replication times are not in geological times, i.e., thousands to millions of years. Dehalococcoidia 16S rRNA gene copy numbers (in the range of 102–105 ml-1 culture) among the various sediment cultures inoculated with diverse sediments and amended with different potential electron acceptors suggested no specific respiratory mode to be preferred by subseafloor Dehalococcoidia. Additionally, subseafloor Dehalococcoidia were not reliant on organohalide respiration under the conditions tested, in contrast to cultivated Dehalococcoidia species such as Dehalococcoides mccartyi. Interestingly, among the tested organohalide compounds, 1,2,3-trichlorobenzene was transformed to 1,3-dichlorobenzene in sediment cultures. Inhibition studies using antibiotics against Gram-positives and microbial community analyses by 454-pyrosequencing of 16S rRNA genes, indicated members affiliated with the phylum Firmicutes were involved in the transformation of 1,2,3-trichlorobenzene. The presence, abundance, and distribution of Dehalococcoidia were investigated in sediments of the Arctic Baffin Bay. Geochemical studies showed the shelf as an area rich in organic matter, with indications for the presence of sulphate reduction (highest dsrA gene copies and a decrease in sulphate concentration in its pore-waters). On the other hand, the basin area was comparably poor in organic matter with high concentrations of iron(II) and manganese(II) in its pore-waters. Dehalococcoidia were present at all investigated sites and depths in the range of 1.1 x 103–8.4 x 105 16S rRNA gene copy numbers g-1 sediment. Dehalococcoidia copy numbers were highest and generally stable with depth in shelf sites of the Baffin Bay compared to any other area. In contrast, Dehalococcoidia copy numbers pronouncedly decreased with depth at basin sites. Dehalococcoidia accounted for the highest proportion of the total bacterial 16S rRNA genes when low bacterial copy numbers were found, mostly in samples from deeper sediment layers, indicating that subseafloor Dehalococcoidia are VIII resilient to burial. Illumina sequencing of 16S rRNA genes gave further evidence that Dehalococcoidia were mostly associated with shelf sediments. The relative abundance of Dehalococcoidia and, most specifically, the clade GIF-9 within the class Dehalococcoidia, positively correlated