1. Oxygen Tolerance in Methanogens Jill K. Jackson 1,2, Dr. Timothy Kral 2,3 1 William Jewell College Depts. of Biology and Chemistry, 2 University of Arkansas Center for Space and Planetary Sciences, 3 University of Arkansas Dept. of Biological Sciences When analyzing the possibility of life beyond Earth, an important organism to consider is the methanogen. Methanogens are highly anaerobic and can reduce CO, CO 2, formate, methanol, methylamines, or acetate to methane (Reeve, Annu. Rev. Microbiol., 1992). Mars is one location within the solar system which could potentially be conducive to this form of life, and the implications of these organisms existing on Mars would be far-reaching. However, to test the viability of methanogens under Martian conditions, it is necessary to perform thorough analyses in the laboratory. Maintenance of a highly anaerobic environment is somewhat of a hindrance to the advancement of methanogenic studies. However, researchers have shown that oxygen exposure does not necessarily have a lethal effect; rather, some strains of methanogens can endure exposure to high levels of oxygen (Kato et. al., Braz. J. Chem. Eng., 1997). Experimental results indicating high oxygen tolerance in methanogens are important in the field of exobiology. Such a discovery could lead to increased expediency in experimentation and thereby to increased understanding of methanogens and their possible habitats. Introduction Materials and Methods Objectives Results Exposure to oxygen has not thus far shown a clear effect on methanogen growth, although in many cases, cultures exposed to atmospheric O 2 in the absence of Na 2 S have recovered just as well as their counterparts receiving treatment with the reducing agent. Three trials using M. wolfeii and two using M. barkerii have been performed, with methane production being analyzed daily following O 2 exposure. Preliminary results from M. wolfeii are given in Figures 3 and 4; however, because this procedure is still being perfected and many more trials are upcoming, no conclusions have been drawn. Figure 2. MM and MS media, respectively, including control tubes and tubes exposed to O 2 in the presence and absence of Na 2 S. To analyze the recovery of three strains of methanogens, M. barkeri, M. wolfeii, and M. formicicum, upon exposure to oxygen both in the presence and absence of the commonly used reducing agent Na 2 S. To analyze whether the production of methane following oxygen exposure is due to the fact that only a few surviving organisms are present or to many surviving organisms being present but hindered by atmospheric O 2. These experiments utilize the strains of Methanogens listed in Table 1. Optical density measurements were taken prior to each experiment to determine that cultures were actively growing. Cells were centrifuged, washed and suspended in sterile buffer to remove residual Na 2 S both prior to and following oxygen exposure. This buffer solution was used to inoculate tubes of media both containing and lacking Na 2 S. All tubes except the controls were exposed to atmospheric oxygen, and a pair of tubes (one containing and one lacking Na 2 S) were removed from oxygen contact at designated time intervals. Tube contents were centrifuged and the supernatant discarded to remove any residual oxygen present in the exposed media from contact with cells. Cells were re-suspended in sterile buffer and then introduced into ideal conditions and allowed to recover. For several days following oxygen exposure, analysis using Gas Chromatography (GC) was performed to quantify methane composition of the headspace, a characteristic indicative of methanogen growth. Table 1. Ideal growth conditions for the strains used in these experiments. Acknowledgements A big thanks to Dr. Tim Kral for his expert guidance and support, and to NASA for funding the REU program. Future work The remainder of the summer will be devoted to perfecting the procedure and performing more trials with all three organisms to attain reliable, repeatable data. Figure 3. Methane Production of M. wolfeii six days following O 2 exposure in the presence of Na 2 S. Figure 4. Methane Production of M. wolfeii six days following O 2 exposure in the absence of Na 2 S. Figure 1. Dr. Tim Kral working in the anaerobic chamber in the exobiology laboratory.