Differential tolerance of biofilms and planktonic cells of Deinococcus geothermalis to desiccation and to simulated space and Mars conditions DISSERTATION zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften – Dr. rer. nat. – vorgelegt von JAN FRÖSLER geboren in Wesel Biofilm Centre Aquatische Mikrobiologie der Universität Duisburg-Essen 2015 Die vorliegende Arbeit wurde im Zeitraum von Mai 2011 bis Dezember 2015 im Arbeitskreis von Prof. Dr. Hans-Curt Flemming/ Prof. Dr. Rainer U. Meckenstock am Biofilm Centre der Universität Duisburg-Essen durchgeführt. Tag der Disputation: 18.03.2016 Gutachter: Prof. Dr. Hans-Curt Flemming Prof. Dr. Christian Mayer Vorsitzender: Prof. Dr. Mathias Ulbricht „Forward ever – backward never“ (Dennis Alcapone) I am deeply grateful to Prof. Dr. Hans-Curt Flemming for giving me the opportunity to do research in the fascinating field of astrobiology. Hans-Curt, thank you for your guidance and inspiration, and for the countless helpful discussions you have provided throughout my studies. I am indebted to Prof. Dr. Christian Mayer for kindly accepting the task of being the co- examiner of my dissertation. I would like to express my gratitude to Prof. Dr. Rainer Meckenstock for giving me the chance to continue my work in his group. My sincerest thanks to Dr. Jost Wingender for his constant support and advice, and for always be willing to share his expertise on microbiology and chemistry with me. Many thanks to Dr. Petra Rettberg for coordinating the joint project BOSS this study was a part of, to Dr. Elke Rabbow for her efforts in conducting the space and Mars simulation experiments, and to Dr. Corinna Panitz for the kind co-operation and exchange of ideas throughout the project. This study would not have been possible without the funding provided by the German Ministry for Economic Affairs and Energy (BMWi; 50WB1154). I owe a great deal of gratitude to all my former and current colleagues at the Biofilm Centre for providing such a pleasant working atmosphere with lots of laughs, mutual support and help. Special thanks go to Anika for her excellent and much appreciated assistance, to Astrid for her guidance in the lab, to Witold for sharing his knowledge about EPS isolation and all sorts of laser-related stuff, to Silke for her advice on EPS analytics and TLC, to Jens for showing me some tricks in CLSM imaging, to Agathe for her secretarial help, to Janine, Zenyta, and Giacomo for being amazing office mates, and to Friederike, Felix, Christian, and Natascha for their friendship, enjoyable lunch breaks, and lots of cake and coffee. My warmest thanks to Elli, my personal typography expert, for helping me with the layout of this work. I want to thank my fellow ‘Easy Snappers’ Anil, Chris, Mareike, Mario, Marko, MK, and Robert, who – probably without noticing it – had an important role in sustaining my work-life balance during these last few years. Thank you for the good vibes, the fun, and the music. I further wish to thank Chris and Giacomo, who have always supported me and blessed me with their invaluable friendship. Thank you that I could always count on you. I will be forever thankful to my dear parents Wilfried and Brigitte and to my awesome brothers Philip and Luca for always being there and believing in me. I feel fortunate to have you as my family, and I wish to dedicate this thesis to you. Acknowledgments ...................................................................................................................... I Table of contents ...................................................................................................................... II List of figures ........................................................................................................................... VI List of tables .............................................................................................................................. X Abbreviations ......................................................................................................................... XII Abstract ................................................................................................................................. XIV 1. Introduction ...................................................................................................................... 1 1.1 Lithopanspermia ................................................................................................................... 1 1.1.1 The stages of lithopanspermia .................................................................................................................. 2 1.1.2 Experimental support of the lithopanspermia hypothesis ................................................................... 4 1.1.3 Lithopanspermia and Mars ....................................................................................................................... 5 1.2 The BOSS experiment .......................................................................................................... 6 1.2.1 Hypothesis ................................................................................................................................................... 6 1.2.2 EXPOSE-R2 ................................................................................................................................................ 7 1.2.3 Ground-based simulation experiments .................................................................................................. 8 1.2.4 Test organisms used in BOSS ................................................................................................................... 9 1.3 The test organism: Deinococcus geothermalis DSM 11300 ............................................ 9 1.3.1 Stress resistance of the Deinococcaceae ................................................................................................ 10 1.3.2 Physiology and metabolism of D. geothermalis .................................................................................. 13 1.3.3 Deinococcal biofilms ............................................................................................................................... 14 1.4 Effects of extraterrestrial conditions on microbial viability .......................................... 16 1.4.1 Desiccation-induced damage ................................................................................................................. 16 1.4.2 UV radiation-induced damage ............................................................................................................... 18 1.4.3 Microbial adaptation to desiccation and irradiation........................................................................... 19 1.5 Biofilms ................................................................................................................................ 23 1.5.1 Biofilm formation..................................................................................................................................... 23 1.5.2 The biofilm matrix ................................................................................................................................... 25 1.5.3 Biofilm analysis ......................................................................................................................................... 26 1.6 Survival of microbes ........................................................................................................... 28 1.6.1 The viable but non-culturable state ....................................................................................................... 29 1.6.2 Detecting microbial viability .................................................................................................................. 29 1.7 Aims of this study ............................................................................................................... 31 2. Materials and methods .................................................................................................... 32 2.1 Materials ............................................................................................................................... 32 2.1.1 Bacterial strains ........................................................................................................................................ 32 2.1.2 Nutrient media ......................................................................................................................................... 32 2.1.3 Chemicals .................................................................................................................................................. 33 2.1.4 Enzymes ..................................................................................................................................................... 37 2.1.5 Probes ........................................................................................................................................................ 37 2.1.6 Buffers and solutions ............................................................................................................................... 37 2.1.7 Commercial kits ....................................................................................................................................... 42 2.1.8 Consumables ............................................................................................................................................