Author: Sylvia Nupp                     Majors: Biology and Chemistry

Preparing Samples in the Lab

This past semester, I studied the metabolism of four different methanogens under Dr. Kral in the Biological Sciences department. My experiment tested if any of the four methanogens, Methanothermobacter wolfeii, Methanosarcina barkeri, Methanobacterium formicicum, or Methanococcus maripaludis, could use either zero-valent iron or basalt as a hydrogen source in solution. These organisms typically use H₂ gas as their energy source and CO₂ as their carbon source. Our lab studies these organisms in the context of astrobiological research, as methanogens are a likely candidate for life on Mars. They have several traits that may make them able to survive Martian conditions, such as being aerobic, using inorganic energy and carbon sources, and being able to survive the conditions present on Mars. Additionally, methane gas has been found in Mars’ atmosphere and has yet to be explained, and on Earth the majority of methane is produced by methanogens, meaning that this could be a potential source for the methane on Mars as well. H₂ is not known to be plentiful on Mars, but if these organisms could use a different source of hydrogen, this would add more evidence that methanogens could survive and grow on Mars.

For zero-valent iron, a stainless-steel nail was added to the experimental groups, and for basalt, a granular form was added. This semester I restarted my stock solutions, which took some time to start growing but were eventually usable for my experiment. The four organisms were grown in specialized liquid media prepared anoxically: the organisms are killed by the presence of oxygen, so several steps are taken to eliminate oxygen from the media tubes before they are inoculated. Typically, the media is prepared and left in an anoxic chamber for 24 hours to remove any oxygen. The chamber’s gas mixture contains hydrogen, so for my experiment, I had to utilize a different method for preparing the media. I bubbled CO₂ through each of the tubes to remove any oxygen in the solution and then the tubes were quickly stoppered to avoid contact with oxygen in the air. Sodium sulfide is then added to the solution to bind any residual oxygen. Since the organisms are grown in ideal conditions, I also needed to remove hydrogen from the stock solutions. This was done by separating the cells from the media and then resuspending the cells in hydrogen-free media. This process can expose the cells to some amount of oxygen if I am not quick enough in transferring them, which occurred this semester and forced me to start over. However, I was eventually able to get cells from my stock solutions transferred to begin the experiment. I took readings of the percent methane in the headspace every two weeks as a proximal measure of growth.

Although my experiment was cut short in the spring semester, the samples maintain levels of methane for some time after the organisms die. This allowed me to measure percent methane in the samples from the previous semester to give some indication of the growth that had occurred, and to compare to once I achieved results from this experiment. The headspace measurements indicated that M. wolfeii and M. maripaludis were both growing in the samples with the only zero-valent iron present, and as I gathered more data, I achieved the same results. In fact, M. maripaludis showed significant amounts of growth, with percent methane levels that I was not expecting to see without its primary hydrogen source. None of the organisms produced methane with only basalt present, which may occur because not enough hydrogen is produced from basalt reactions. This semester I was able to complete my experiment and learn throughout the entirety of the experiment, and I was able to collect enough data to analyze to support my conclusions.

As I am nearing the end of my undergraduate degree and looking towards graduate school this experiment allowed me to develop my skills in studying methanogens, maintaining growth, and overall working with organisms that are specialized and difficult to maintain. As I hope to continue working with methanogens or other microorganisms that are hard to culture, these skills are incredibly applicable and something I would not have been able to do in any other setting. Additionally, I learned a lot about the experimental methods and presenting data in a format that is useful, which will be valuable in my future endeavors as a scientist. This project demonstrates not only my proficiency in performing an experiment but my ability to cope with changing conditions and adapt through an experiment, which may or may not go according to plan. I learned how to refine my experimental design, how to evaluate a situation when it does not go as planned, and how to articulate my research findings in a precise and understandable manner. I am incredibly proud of my project, and my experiences with it will allow me to pursue even greater projects in astrobiology.