Date of Graduation

5-2024

Document Type

Thesis

Degree Name

Bachelor of Science in Biology

Degree Level

Undergraduate

Department

Biological Sciences

Advisor/Mentor

Kral, Timothy

Committee Member/Reader

Lessner, Daniel

Committee Member/Second Reader

Millet, Francis

Committee Member/Third Reader

Levine, Bill

Abstract

Methanogens have been studied as a model for life on Mars for 28 years now in the Kral lab. The discovery of methane in the Martian atmosphere by ground-based and orbital observations as well as Curiosity Rover (Formisano, V. et al., Krasnopolsky, V.A. et al., Mumma, M.J. et al.) has added relevance to these types of studies. Methanogens were chosen due to their ability to live in harsh environments, very similar to the Martian terrain. In addition to methane in the atmosphere, phyllosilicate clays have also been identified. One of those clays is kaolinite. Kaolinite has been found to not be toxic to methanogens. In the research reported here, the methanogens were placed in a bicarbonate buffer containing kaolinite along with molecular hydrogen as an energy source and carbon dioxide as a carbon source, to determine if the kaolinite could support the growth of the methanogens.

The methanogens tested were Methanothermobacter wolfeii, Methanosarcina barkeri, Methanobacterium formicicum and Methanococcus maripaludis. Organisms were inoculated into their respective media followed by incubation at each organism’s growth temperature. Following two weeks of growth, cells from each culture were centrifuged and washed with sterile buffer three times. Cell pellets were suspended in sterile buffer, then added to anaerobic tubes containing sterile kaolinite (0.5g per tube). Sterile buffer was added to each tube to reach a final volume of 5 mL. Each tube was pressurized with 2 atm of molecular hydrogen followed by incubation at each organism’s ideal growth temperature. Following six weeks of incubation, 0.5 mL of each culture was transferred to a fresh, sterile tube containing 0.5g kaolinite. Again, volumes were increased to 5 mL with sterile buffer. The purpose of the transfers was to dilute out any residual nutrients from the original stock cultures. Methane production, commonly used to measure methanogen growth, was measured by gas chromatography of headspace samples at regular time intervals.

Three of the four methanogens tested, M. wolfeii, M. formicicum, and M. barkeri, showed measurable methane following incubation. The amounts were far less than found in the control tubes which contained growth media, which is expected. M. maripaludis did not show any methane production, most likely because it is a halophile, and no salts were added. The methane production eventually tapered off, and the methanogens were unable to sustain methane production. It is unclear if these microorganisms could survive an extended period of time using kaolinite as a main substrate.

Keywords

methanogens; Mars; kaolinite; methanogen; astrobiology

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