Ph.D. thesis in Bioscience engineering Valérie Van Eesbeeck Microbial dynamics in the aquatic environments of a nuclear reactor PhD dissertation committee Supervisors: Dr. Natalie Leys (SCK CEN – Microbiology Unit) Prof. Jacques Mahillon (UCL – Earth and Life Institute) Reviewers: Prof. Claude Bragard (UCL – Earth and Life Institute) Dr. Pieter Monsieurs (ITG – protozoan pathogen units) Dr. Corinne Rivasseau (CEA – laboratory of plant cellular physiology) Prof. Jonathan Lloyd (Earth and Environmental Sciences – University of Manchester) President: Prof. Emmanuel Hanert (UCL – Earth and Life Institute) Acknowledgments This PhD has been an incredible journey, throughout which I was fortunate enough to be supported by a wonderful group of people. I can truly say that without them, this endeavor would not have been successful. First of all, I would like to express my deepest gratitude to my SCK CEN mentors Natalie Leys and Pieter Monsieurs, whose support and advice have been unwavering throughout this entire PhD. Thank you for your valuable scientific insights and suggestions, and most of all thank you for encouraging me when times were hard and never giving up on me! Thank you for allowing me the space I needed to bring this project to fruition. I would also like to immensely thank my UCL supervisor Prof. Jacques Mahillon, whose warm guidance and support kept me on track during difficult times. Thank you for the interesting discussions and for always keeping an open door for me. I will always cherish your light-hearted and humorous touch. Next, I would like to thank Prof. Claude Bragard, Dr. Corinne Rivasseau, Prof. Jonathan Lloyd and Prof. Emmanuel Hanert for accepting to be part of my thesis jury. Thank you for accepting to read this thesis and sharing your expertise on the subject. I am also deeply grateful to all the people at BR2 who have always been enthusiastic to help me when needed! I would like to especially thank Hans Ooms, Dirk Meynen and everyone at the radiation control department for providing valuable information on the BR2 and guiding me through the meanders of the reactor ;-). Thank you as well to all the people in the BIS lab who have helped me to carry out my experiments safely. I would like to warmly thank all the staff members of the microbiology unit for their support. I am especially grateful to Hugo, Rob, Kristel and Ann for always being ready to help me and giving me some much needed advice. And of course thank you to all my fellow PhD students Charlotte, Laurens, Tom and Shari for the fun times spent in the lab together! And thank you Mohamed and Gleb for helping me out with the bioinformatics analyses, I have learned a lot thanks to you! Special thanks as well to my friends and former office mates Ali, Claude, Raghda and Anu, we’ve been through a lot together and I still cherish our amazing discussions!! i Thank you to all my friends who have supported me throughout this rollercoaster, it has been one hell of a ride! Special thanks to Louise for your contribution to the thesis! ;-) I am immensely grateful to my beloved parents, Joëlle and Jean. You have no idea how much your love and support have meant to me during some of the difficult times. Thank you for always standing behind me and encouraging me to persevere towards my goals. If I am where I am today, it is mostly thanks to you. Thank you as well to my dear brothers Laurent and Denis for the support and for just being you! Finally, I want to express my profoundest gratitude to you, dear Hans. You have been my rock throughout this entire journey, I cannot put into words how grateful I am for your unconditional support. Thank you for always having believed in me and stood by my side. Thank you all! Valérie ii Abstract Nuclear reactors contain various watery environments, such as spent nuclear fuel pools for the intermediate storage of spent nuclear fuel underwater, the primary and secondary cooling circuits for the cooling of nuclear fuel in the reactor vessel and different ultrapure water tanks for the replenishing of evaporated water. These water systems are maintained at high purity levels through constant filtration and deionization in purification circuits, resulting in ultrapure waters with low conductivities and nutrient levels, occasionally exposed to high levels of radiation. Despite the extremely challenging conditions in these waters, microorganisms such as bacteria, fungi and microalgae have been previously detected. While most of the previous studies were performed on spent nuclear fuel pools, the aim of this work was to investigate the bacterial communities in different watery environments of the BR2 nuclear research reactor at SCK CEN in Mol, Belgium, with a particular focus on an open basin surrounding the reactor vessel. In a first study, we investigated the viable microbial population in a range of interlinked watery environments using a cultivation-based approach. This yielded an extensive strain collection of 33 distinct bacterial species, which is the largest catalogue of isolates described so far in a single study. Furthermore, we attempted to characterize the radiation susceptibility of some of the isolated strains, which resulted in the identification of Sphingomonas melonis as the most radio-resistant species, as it survived an acute irradiation dose of 2.1 kGy. As the BR2 reactor runs in successive cycles of operation and shutdown, this generates highly dynamic conditions in the basin surrounding the reactor core, with periodically shifting physico-chemical parameters such as temperature, radiation and flow rate. In the second part of our work, we characterized the long-term microbial community dynamics in this basin through 16S rRNA amplicon sequencing. Two sampling campaigns spanning several months were performed, which resulted in the characterization of a diverse bacterial population displaying clear shifts in community profiles: cycles were mostly dominated by an unclassified Gammaproteobacterium and Pelomonas, whereas Methylobacterium prevailed during shutdowns. iii Finally, in order to dig deeper into the taxonomic and functional characteristics of the microbial community in the basin and characterize its dynamics more in depth, we adopted a shotgun metagenomics sequencing approach. To this aim, we designed a specialized filtration system in order to be able to collect a sufficient amount of cell material. With regard to the functional characterization of the community, several pathways believed to play a role in cell function recovery after irradiation were more highly represented during shutdowns. Furthermore, we managed to almost entirely reconstruct two MAGs from the metagenome, corresponding to Bradyrhizobium sp. BTAi1 and Methylobacterium sp. UNC378MF. These strains harbored significant adaptations in their genome allowing them to cope with the extremely challenging conditions prevailing in the basin. In conclusion, we managed to uncover a large microbial diversity in the various watery environments of the BR2, which were shown to be mostly dominated by bacteria. Members of the community were believed to harbor significant evolutionary adaptations allowing them to survive in these extremely challenging environments. iv Included manuscripts Published research papers: • Petit, P. C. M., Pible, O., Van Eesbeeck, V., Alban, C., Steinmetz, G., Mysara, M., Monsieurs, P., Armengaud, J. and Rivasseau, C. (2020). Direct Meta-Analyses Reveal Unexpected Microbial Life in the Highly Radioactive Water of an Operating Nuclear Reactor Core. Microorganisms, 8. To be submitted research papers: • Van Eesbeeck, V., Props, R., Mysara, M., Petit, P. C. M., Rivasseau, C., Armengaud, J., Monsieurs, P., Mahillon, J. and Leys, N. (2021). Cyclical patterns affect microbial dynamics in the water basin of a nuclear research reactor. • Van Eesbeeck, V., Mysara, M., Goussarov, G., Monsieurs, P., Mahillon, J. and Leys, N. (2021). Microbial dynamics in the water basin surrounding a nuclear reactor in operation: a metagenomic approach. v List of abbreviations AA Aluminum Alloy ALARA As Low As Reasonably Achievable ANI Average Nucleotide Identity ASV Amplicon sequence Variants ATP Adenosine Triphosphate BLAST Basic Local Alignment Search Tool bp base pair Bq Becquerel BR2 Belgian Reactor 2 ca. circa CAHB Culturable Aerobic Heterotrophic Bacteria CEA Atomic Energy Commission CFU Colony-Forming Unit CMF Core Mock-up Facility D10 Decimal reduction dose DAPI 4′,6-diamidino-2-phenylindole DGGE Denaturing Gradient Gel Electrophoresis DOC Dissolved Organic Carbon DSB Double-Strand Break dsDNA double-stranded DNA DW Demineralized Water e.g. example given ECCS Emergency Core Cooling System eDNA Extracellular DNA ENA European Nucleotide Archive EPS Extracellular Polymeric Substance ESDSA Extended Synthesis Dependent Strand Annealing FBTR Fast Breeder Test Reactor FDA Food and Drug Administration FGMSP First Generation Magnox Storage Pond FTC Fuel Transfer Channel FT-IR Fourier Transform Infrared Spectroscopy GFP Green Fluorescent Protein GIF Gamma Irradiation Facility vii Gy Gray HEU Highly Enriched Uranium HR Homologous Recombination i.e. id est IAEA International Atomic Energy Agency ILL Institut Laue Langevin INTEC Idaho Nuclear Technology Center IOB Iron-Oxidizing Bacteria IRB Iron Reducing Bacteria ITS Internal Transcribed Spacer kb kilobase Km Michaelis-Menten constant KO Kegg Orthology LB Luria-Bertani medium LET Linear Energy Transfer LMW Low Molecular Weight LPS Lipopolysaccharide MAG Metagenome-Assembled Genome MAPS Madras Atomic Power