University of Arkansas, Fayetteville ScholarWorks@UARK Theses and Dissertations 8-2017 Growth and Survivability of Microorganisms at Martian Temperatures and Pressures Rebecca Lynne Mickol University of Arkansas, Fayetteville Follow this and additional works at: http://scholarworks.uark.edu/etd Part of the Microbiology Commons, and the The unS and the Solar System Commons Recommended Citation Mickol, Rebecca Lynne, "Growth and Survivability of Microorganisms at Martian Temperatures and Pressures" (2017). Theses and Dissertations. 2453. http://scholarworks.uark.edu/etd/2453 This Dissertation is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of ScholarWorks@UARK. For more information, please contact [email protected], [email protected]. Growth and Survivability of Microorganisms at Martian Temperatures and Pressures A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Space and Planetary Sciences by Rebecca Lynne Mickol University of Colorado Bachelor of Arts in Astrophysics, 2010 University of Colorado Bachelor of Arts in Ecology and Evolutionary Biology, 2010 August 2017 University of Arkansas This dissertation is approved for recommendation to the Graduate Council. __________________________________ Dr. Timothy Kral Dissertation Director __________________________________ __________________________________ Dr. John Dixon Dr. Dan Kennefick Committee Member Committee Member ___________________________________ ___________________________________ Dr. Dan Lessner Dr. Andrew Schuerger Committee Member Ex-officio Member Abstract The discovery of methane in the martian atmosphere via numerous ground- and space- based sources has prompted the study of methanogens as models for life on Mars. Methanogens are microorganisms within the domain Archaea, many of which utilize carbon dioxide (CO2) and hydrogen to produce methane. The non-photosynthetic nature of methanogens indicates that they could exist in sub-surface environments, protected from harmful UV and ionizing radiation on the surface of Mars. These organisms also do not require organics, which are sparse on the planet. Additionally, the wide variety of environments we find life in on Earth, as well as evidence for liquid brines on the surface of Mars, suggest that habitable environments may still exist on the planet. However, there are a variety of conditions that any extant life on Mars would need to endure, including wide variations in temperature over one sol, a low-pressure atmosphere, and a limited availability of liquid water, among others. This dissertation encompasses various experiments that examined the ability of four species of methanogens (Methanosarcina barkeri, Methanobacterium formicicum, Methanococcus maripaludis, and Methanothermobacter wolfeii) to survive and/or grow under 1) low-pressure conditions and 2) freeze/thaw cycles. Low pressure studies include both survival and active growth experiments conducted between 7 mbar (the average surface pressure on Mars) and 143 mbar. Freeze/thaw experiments utilized short- and long-term cycles varying in temperature between the organisms’ growth temperatures (22 °C, M. maripaludis; 37 °C, M. barkeri and M. formicicum; 55 °C, M. wolfeii) and -80 °C, encompassing Mars-relevant temperature changes. As a comparison to methanogen growth and survivability, additional experiments were conducted using a non-spore-forming bacterium, Serratia liquefaciens, previously shown capable of growth at 7 mbar, 0 °C and within an anoxic CO2 atmosphere. The experiments described here assessed the survivability of S. liquefaciens exposed to martian UV irradiation within liquid brines and ices. The experiments discussed here demonstrate the ability of Earth microorganisms to withstand certain extreme conditions on Mars and suggest that the planet may contain relatively habitable microenvironments within the near subsurface. ©2017 by Rebecca Lynne Mickol All Rights Reserved Acknowledgements Portions of this work were funded by the National Aeronautics and Space Administration (NASA) Astrobiology: Exobiology and Evolutionary Biology Program grant #NNX12AD90G and by grants from the Arkansas Space Grant Consortium. Additionally, some portions of this research were conducted at the Space Life Sciences Lab, Kennedy Space Center under grant #NNX12AJ84G from NASA’s Planetary Protection Office. I thank all of the undergraduates who conducted research with me during my graduate school career, notably, Sarah Laird and Yuta Takagi, for their assistance in parts of the research discussed here. I’d also like to acknowledge Coleman McFerrin for his unending moral support and companionship in the lab and the cities of Chicago and Boston. Thank you to my committee members, Dr. Daniel Lessner, Dr. Daniel Kennefick and Dr. John Dixon for your support and guidance throughout my graduate career. Thank you to Walter Graupner for technical assistance with the Pegasus Planetary Simulation Chamber and construction and design of the specialized puncture device. Thank you to Dr. Andrew Schuerger for the opportunity to conduct research with you and for your guidance through the process and successful completion of a manuscript. Your assistance regarding research, data analysis, and manuscript preparation has been greatly appreciated and often, necessary, and these skills will remain with me throughout my scientific career. To all my family and friends, near and far, thank you for the laughter and the commiseration. I love you all dearly. To Dr. Cassie Marnocha and Dr. Erika Kohler, there aren’t words to describe how amazing you two are, so I’ll just put it in an email. Let’s meet at the Drury and have Outback for dinner. Finally, to Dr. Timothy Kral, you’re welcome. I mean, thank you for being a great mentor and advisor. I greatly enjoyed my time in your lab and leaving will be bittersweet. Never a dull moment. Love, Spike. Dedication In loving memory of Lynne Marie Rinaldis. Mom, I love you always and forever, and you will be with me always. I wouldn’t be who I am today without your endless love and devotion. May your hard work, determination, and spirit live on. Ain’t no mountain high enough. To Nick, thank you for being the best brother anyone could have and for your continuous love and support. Table of Contents Introduction ................................................................................................................1 Methane on Earth – Biological and Geological Sources .....................................................2 Methane on Mars .................................................................................................................8 Methanogens as Candidates for Life on Mars ...................................................................13 1.3.1 Methanosarcinales..................................................................................................17 1.3.1.1 Methanosarcina barkeri ..........................................................................17 1.3.1.2 Methanochondroitin ................................................................................18 1.3.2 Archaeal surface layer (S-layer) ............................................................................19 1.3.3 Methanobacteriales ................................................................................................20 1.3.3.1 Methanobacterium formicicum ...............................................................20 1.3.3.2 Methanothermobacter wolfeii .................................................................20 1.3.3.3 Pseudomurein ..........................................................................................21 1.3.4 Methanococcales ....................................................................................................22 1.3.4.1 Methanococcus maripaludis ....................................................................22 Dissertation Goals and Significance ..................................................................................22 Dissertation Outline ...........................................................................................................25 References ..........................................................................................................................26 Survival of Non-psychrophilic Methanogens Exposed to Extreme Temperature Changes. ...........................................................................................................37 Abstract ..............................................................................................................................38 Introduction ........................................................................................................................38 Materials and Methods .......................................................................................................45 2.3.1 Microbial Procedures .............................................................................................45 2.3.2 Experiment 1: Growth at 4 °C and 22 °C ..............................................................46 2.3.3 Experiment 2: 5 g sand, 10 mL medium ................................................................46 2.3.4 Experiment 3: 10 g sand, 5 mL medium ................................................................49 2.3.5 Experiment 4: 5 mL medium .................................................................................54