Author: Benjamin Tan | Major: Biology
My name is Benjamin Tan and I am a junior majoring in Biology in the Fulbright College of Arts and Sciences. During the fall of 2021 and the spring of 2022, I worked in Dr. Mack Ivey’s lab culturing and analyzing certain bacterial species for my thesis project. The core idea of my thesis is to explore the carbon requirements of certain bacterial species that hold the potential to colonize other planets. My advisor and I chose this topic due to its tangential relationship to his own work with the same organisms. This information is very relevant today as interest in astrobiology grows and technology becomes increasingly available to allow us to explore other planets. There is a risk of contamination from Earth-originating bacteria to a different planet, and knowing what environments potential colonizers could grow in is crucial for distinguishing them from potential otherworldly flora.
The two species my advisor and I collectively decided on testing were Desulfotalea psychrophila and Desulfovibrio arcticus. Both species are anaerobic sulfate reducers and cannot grow at room temperature, with D. psychrophila growing at around -5° C and D. arcticus growing around 42° C. Over the course of this semester, I spent most of my time in the lab growing the bacteria and cleaning up the RNA from each trial.
Although it sounds simple, growing these bacteria proved more difficult than I initially expected. Because of their anaerobic nature, the media had to be gassed to be rid of oxygen, the bacteria had to be grown in sealed bottles, and all the media serving and inoculation had to take place in an anaerobic hood. When I set out to do this, I quickly found out how challenging it was going to be. The first two times that we (me and our lab’s graduate student, Sergio) attempted to grow D. psychrophila, it would not grow. The first time, it was due to the contents of the medium. Consequently, we had to adjust it to normalize the pH across all carbon sources. The second time, there was oxygen contamination within the samples due to a faulty catalyzer in the anaerobic hood that has since been replaced. On this third attempt, D. psychrophila is growing well.
We decided on testing four carbon sources (glycerol, dextrose, isobutyric acid, and sodium acetate) each with five different concentrations (0.5%, 1%, 2%, 3%, and 4%). In addition, each carbon source had a positive and negative control. In total, that was 68 bottles per organism tested. I used two media for this experiment, with D. psychrophila growing best in DSMZ 141 and D. arcticus growing best in DSMZ 1040. However, each media was modified to be rid of any carbon sources that they should have been prepared with. Instead, these carbonless media were gassed with nitrogen and carbon dioxide to rid the oxygen and served into beakers containing each salt inside the anaerobic hood before being sealed. After regulating the pH of each medium, the bacteria were inoculated into the beakers inside of the anaerobic hood. D. psychrophila was grown in a freezer while D. arcticus was grown in a hot water bath set to its optimum growth temperature. Both sets need one month to double, after which the RNA in each have to be extracted and cleaned.
That leaves me where I find myself today. I have extracted all of the RNA belonging to my D. arcticus samples and done the cleanups using kits, but I am waiting on D. psychrophila because it is still growing. There is still much work to be done on the project before I will be finished. In the coming days, D. psychrophila will be finished growing and I will need to do RNA extraction and cleanups for all those samples. I also need to run qPCR on each of my samples to quantify each species’ response to each carbon source and their varying concentrations.
Throughout this whole process, I was assisted by Dr. Ivey and our lab’s graduate student, Sergio Mosquera, who has been a major help due to his vast experience with these bacteria and the process for culturing them. He has given me guidance at every turn, and I have learned much from him in lab. Dr. Ivey has also given me much guidance in regard to the direction of my project and has been key in guiding my project when I felt unclear about where my project should go.
The Honors College Research Grant has been crucial to my project, allowing me to purchase kits and supplies that are invaluable to the research I conduct. Because of it, I have learned so much about this topic and have expanded my knowledge on lab techniques and procedures. This knowledge will be helpful in my pursuit of medical school and very applicable to my future career as a doctor. Overall, I am proud of the work that I have accomplished this semester and look forward to what I have in store for me.