Characterization of Stress Tolerance and Metabolic Capabilities of Acidophilic Iron-Sulfur-Transforming Bacteria and Their Relevance to Mars
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Characterization of stress tolerance and metabolic capabilities of acidophilic iron-sulfur-transforming bacteria and their relevance to Mars Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften – Dr. rer. nat. – vorgelegt von Anja Bauermeister aus Leipzig Im Fachbereich Chemie der Universität Duisburg-Essen 2012 Die vorliegende Arbeit wurde im Zeitraum von März 2009 bis Dezember 2012 im Arbeitskreis von Prof. Dr. Hans-Curt Flemming am Biofilm Centre (Fakultät für Chemie) der Universität Duisburg-Essen und in der Abteilung Strahlenbiologie (Institut für Luft- und Raumfahrtmedizin, Deutsches Zentrum für Luft- und Raumfahrt, Köln) durchgeführt. Tag der Einreichung: 07.12.2012 Tag der Disputation: 23.04.2013 Gutachter: Prof. Dr. H.-C. Flemming Prof. Dr. W. Sand Vorsitzender: Prof. Dr. C. Mayer Erklärung / Statement Hiermit versichere ich, dass ich die vorliegende Arbeit mit dem Titel „Characterization of stress tolerance and metabolic capabilities of acidophilic iron- sulfur-transforming bacteria and their relevance to Mars” selbst verfasst und keine außer den angegebenen Hilfsmitteln und Quellen benutzt habe, und dass die Arbeit in dieser oder ähnlicher Form noch bei keiner anderen Universität eingereicht wurde. Herewith I declare that this thesis is the result of my independent work. All sources and auxiliary materials used by me in this thesis are cited completely. Essen, 07.12.2012 Table of contents Abbreviations ........................................................................................ 0 1 Introduction .................................................................................. 1 1.1 The search for life on Mars ...................................................................... 1 1.1.1 Findings of the Viking mission ................................................................. 1 1.2 Mars in the past and present ................................................................... 4 1.2.1 The history of Mars and the possible origin of life ................................... 4 1.2.1.1 Water and environmental evolution on early Mars ........................... 4 1.2.1.2 Implication for the evolution of life .................................................... 8 1.2.2 Present Mars and potential microbial metabolisms ................................. 9 1.2.2.1 Physical and geochemical conditions on present-day Mars ............. 9 1.2.2.2 Water on Mars today? .................................................................... 11 1.2.2.3 Permafrost environment and methanogenesis ............................... 12 1.2.2.4 Potential electron donors and acceptors on Mars for chemolithotrophic metabolism ....................................................................... 14 1.2.2.5 Hypothetical microbial food web in the subsurface of Mars ............ 16 1.2.3 Rio Tinto as a Mars analog study site ................................................... 19 1.3 Acidophilic iron-sulfur bacteria ............................................................ 22 1.3.1 Acidithiobacillus ferrooxidans ................................................................ 23 1.3.2 Sulfobacillus thermosulfidooxidans ....................................................... 25 1.4 Environmental stressors and microbial resistance strategies .......... 26 1.4.1 Water stress .......................................................................................... 26 1.4.1.1 Desiccation ..................................................................................... 27 1.4.1.2 Salt stress ....................................................................................... 28 1.4.2 Low temperatures ................................................................................. 29 1.4.3 Radiation ............................................................................................... 31 1.4.3.1 Ultraviolet (UV) radiation ................................................................ 31 1.4.3.2 Ionizing radiation ............................................................................ 32 1.4.4 Life in biofilms as a survival strategy ..................................................... 33 1.5 Maintenance metabolism and methods for viability determination ... 34 1.6 Open fields .............................................................................................. 37 1.7 Objectives ............................................................................................... 38 2 Materials and Methods .............................................................. 40 2.1 Chemicals ............................................................................................... 40 2.2 Bacterial strains and cultivation ........................................................... 42 2.2.1 Bacterial strains .................................................................................... 42 2.2.2 Media, buffers, and solutions ................................................................ 43 2.2.2.1 Basal salt medium (BSM) ............................................................... 43 2.2.2.2 Trace element solution ................................................................... 43 2.2.2.3 Sporulation medium........................................................................ 44 2.2.2.4 Yeast extract stock solution (10%) ................................................. 44 2.2.2.5 Fe 2+ -stock solution (200 g/l) ........................................................... 44 2.2.2.6 Fe 3+ -stock solution (164 g/l) ........................................................... 45 2.2.2.7 R2A medium ................................................................................... 45 2.2.2.8 Phosphate-buffered saline (PBS) ................................................... 45 2.2.3 Cultivation conditions ............................................................................ 46 2.2.3.1 Stock cultures ................................................................................. 46 2.2.3.2 Cultivation of D. geothermalis ......................................................... 46 2.2.3.3 Cultivation of A. ferrooxidans and S. thermosulfidooxidans ............ 46 2.2.3.3.1 Aerobic cultivation ..................................................................... 46 2.2.3.3.2 Anaerobic or microaerobic cultivation ....................................... 47 2.2.3.4 Cultivation of biofilms...................................................................... 48 2.2.3.4.1 Sequential detachment of biofilm cells of A. ferrooxidans from membrane filters ......................................................................................... 49 2.2.4 Mars regolith simulants ......................................................................... 50 2.2.4.1 Cultivation of A. ferrooxidans with Mars regolith simulants ............. 51 2.2.4.2 Attachment of A. ferrooxidans to Mars regolith simulants............... 52 2.3 Exposure to stress conditions .............................................................. 53 2.3.1 Desiccation ........................................................................................... 53 2.3.1.1 Strains of acidophilic iron-sulfur bacteria screened for desiccation tolerance ....................................................................................................... 53 2.3.1.2 Sample preparation and analysis ................................................... 54 2.3.1.2.1 Drying planktonic cells and biofilms of A. ferrooxidans and S. thermosulfidooxidans ................................................................................. 54 2.3.1.2.2 Drying planktonic cells and biofilms of D. geothermalis ............ 56 2.3.1.2.3 Exposure to different conditions of relative humidity (RH) and oxygen .................................................................................................. 56 2.3.1.2.4 Vacuum-drying of A. ferrooxidans with compatible solutes ....... 58 2.3.2 Salt stress ............................................................................................. 58 2.3.3 UV radiation .......................................................................................... 59 2.3.3.1 Irradiation of planktonic cells ( A. ferrooxidans and D. geothermalis ) .. ....................................................................................................... 59 2.3.3.2 Irradiation of biofilms of A. ferrooxidans ......................................... 60 2.3.4 X-rays ................................................................................................... 61 2.3.5 Freezing ................................................................................................ 61 2.3.6 Mars simulation ..................................................................................... 62 2.3.6.1 Survival of dried biofilms of A. ferrooxidans and S. thermosulifdooxidans ..................................................................................... 62 2.3.6.2 Growth of A. ferrooxidans at low O 2 partial pressure ...................... 63 2.4 Determination of viable (culturable) and total cell numbers .............. 64 2.4.1 Total cell counts (TCC) ......................................................................... 64 2.4.2 Assay for colony formation ability .......................................................... 64 2.4.3 Most Probable Number (MPN) assay ................................................... 65 2.4.4 Calculation of the culturability fraction after stress exposure ...............