Final Report BSCI223-0101

Madison Crossley, Taylor Castaneda and Valerie Bell BSCI223-0101 Introduction While working with NASA and supporting their mission to Mars through DuPunt, a company specializing in polyurethane derivatives, the polyurethane protective coating on the robot, extraterrestrial landing vehicle integrated sample (ELVIS), has degraded significantly and has reduced to a slimy residue. The original goal of the ELVIS mission and the scientists launching the ELVIS probe was to find any signs of life on Mars by collecting samples of soil for analysis. The sample return vehicle (SRV) was able to successfully obtain this soil although the polyurethane suit did not survive. To investigate the disaster further, a main project hypothesis was formed: “Samples from the ELVIS probe contain bacteria native to Mars, and these bacteria cannot be identified as earthly bacteria.” However, as employees of DuPunt, a separate hypothesis was also formed: “The degradation of polyurethane products was caused by a microorganism or microorganisms present in the soil samples collected by ELVIS.” These statements can either support or destroy DuPunt’s reputation as a company if there is no proof that organisms from Mars are responsible for destroying ELVIS. Additionally, these hypotheses serve as a guide into our study of the ELVIS mission and to identify if our isolated organisms are either Earthly or Martian organisms through our phylogenetic and phenotypic analyses. As we continue our studies, four background questions were asked: (1) are there organisms that can degrade polyurethane, (2) could life have originated on Mars and been transferred to Earth, (3) could life exist on Mars, and (4) could Earth organisms survive on Mars. It is reported that microbial degradation of polyester PUR [polyurethane] by microorganisms 10 is possible. To analyze this, biodegradation needs to be split into polyester type or polyether type as PUR degradability is largely influenced by the chemical structure of the polyol segment. 10 Both gram-positive and gram-negative bacteria have been reported as PUR degraders 10 with polyester polyurethane, as PUR degradation was caused mainly by the hydrolysis of ester bonds. 10 On the other hand, polyether PUR is relatively resistant 10 to degradation. If there were to be degradation present, studies tested that some kinds of polyether PUR were degraded by Staphylococcus epidermidis , 10 but the degradation happened very slowly. 10 Studies have considered the possibility that life from Mars was carried to Earth and that it is possible that life from Earth could have similarly been carried to Mars. 11 This suggestion came to be from the fact that many of the meteorites found on Earth have come from Mars and studies of the magnetic domains within one of these meteorites have shown that interior temperatures never exceed the survival limits of microorganisms. 11 However, it is emphasized that specialized biochemical tools for detecting life are ineffective when studying which organic material from Mars is of biological origin but does not have the biomolecules associated with Earth life. 11

Similarly, the presence of fluvial features provides evidence that liquid water was once present on the martian surface. 12 By analogy with Earth, life may have originated on Mars early in its history, possibly during the end of the late heavy bombardment. 12 Additionally, spacecraft exploration of Mars has shown that the essential resources necessary for life support are present on the martian surface. 13 Finally, studies reported by NASA have shown that microbes, as the simplest and most ancient organisms on Earth, could survive the extremely thin air of Mars. 14 Researchers found that four species of methanogens — Methanothermobacter wolfeii, Methanosarcina barkeri, Methanobacterium formicicum, and Methanococcus maripaludis — all survived exposure of lengths varying from 3 to 21 days at pressures down to roughly six-thousandths of Earth’s surface pressure. 14 Since methanogens neither require oxygen nor photosynthesis means that they could live just beneath the martian surface, shielded from harsh levels of ultraviolet radiation on the Red Planet. 14 Results/Data While completing our phylogenetic reports, we all matched our sequences to different organisms and each ended up with a 100% match. Organism A matched 100% with Escherichia coli , Organism B was matched 100% with Serratia marcescens and Organism C was 100% matched with Mammaliicoccus/Staphylococcus sciuri . This indicates that our sequences were matched perfectly with a specific type of bacteria which makes them a 100% match. Based on the Phylogenetic report, we were able to distinguish our bacterias from one another. To begin, we performed a Gram-stain test and determined that Phylogenetic Organism A, E. coli , was purple bacillus meaning Gram-negative, 7 Phylogenetic Organism B, S. marcescens, was also purple bacillus meaning Gram-negative, 8 and Phylogenetic Organism C, S. sciuri, was pink cocci meaning Gram-positive. 9 The next test we looked at was what kind of growth was present on MacConkey’s medium. Phylogenetic Organism A, E. coli , grew in the MacConkey’s medium and created pink colonies which represent the ability to ferment lactose. 1 Phylogenetic Organism B, S. marcescens, also grew in the MacConkey’s medium but produced white colonies which represent the inability to ferment lactose. 2 Finally for Phylogenetic Organism C, S. sciuri , had no growth on MacConkey’s agar but not necessarily meaning it did or did not ferment lactose. 3 Finally, we observed the motility of our different bacterias and compared these. We determined that Phylogenetic Organism A, E. coli , is motile, 4 Phylogenetic Organism B, S. marcescens, was also motile but, 5 Phylogenetic Organism C, S. sciuri, was non-motile. 6 All of these results represent 3 different types of bacteria which further supports our conclusion that our original sample contained different bacterias. We also analyzed three different TSA plates where our different bacterias were purely isolated and grown. Phenotypic Organism A (Figure 1), is rather opaque and pale yellow/off-white in color with an irregular/circular form that has undulate/lobate margins. The bacteria presented as rather flat with very small elevation in the plate. The bacteria dilutes into very small individual colonies. Phenotypic Organism B (Figure 2), has a darker, more tannish

Streaked and Incubated at 37ºC for 24 hours Figure 3: Phenotypic Organism C : TSA Plate Streaked and Incubated at 37ºC for 24 hours Based on our phenotypic reports, we were able to differentiate our organisms in the lab through different types of experimental tests and the results. To begin, we were able to perform a Gram-stain experiment to distinguish the shape and Gram-stain of our bacteria. The Gram-stain test helps identify whether a bacteria is Gram-positive or Gram–negative. The difference between these two types of bacteria is mainly the presence of an outer-membrane on the Gram-negative bacteria. This extra membrane helps the Gram-negative bacteria resist the crystal violet stain from sticking to the cell and this is why Gram-negative bacteria will stain pink. The crystal violet is washed off with alcohol and the cell is stained pink with Safranin in order to add contrast to the Gram-negative bacteria since the crystal violet will not stick. I personally worked with Organism C which had pink bacteria representing Gram-positive. Organisms A and B however, were presenting purple which indicates Gram-positive. This test also helped distinguish the shape of our bacterias. For organism A and B, we determined that the shape of the bacteria were rod-shaped, bacillus and for organism C, the shape of the bacteria was cocci. This confirmed that Phenotypic Organism A and B were Gram-negative bacillus and Organism C was Gram-positive cocci. This means that E. coli and S. marcescens which are Gram-negative bacillus must be either Phenotypic Organism A or B and S. sciuri is most likely Phenotypic Organism C.

Another test we performed to confirm our organism matches was streaking our samples on a MacConkey’s medium. The MacConkey’s medium is used for the identification of lactose fermenting from lactose non-fermenting bacteria and it is selective for Gram-negative bacteria. If the petri dish grows pink colonies, this means it ferments lactose whereas the formation of white colonies represents non-lactose fermenters. I personally worked on Organism C which had no growth which indicated that the organism was Gram-positive because the MacConkey medium selects for Gram-negative bacteria. Organism A however, had white growth which indicates that the bacteria is Gram-negative but unable to ferment lactose. Organism B had pink growth which indicated that the bacteria is Gram-negative and can ferment lactose. This further clarifies which bacterias match with which because E. coli grows on MacConkey’s medium and creates pink colonies which match Organism B. S. marcescens had growth on the MacConkey’s agar but produced white colonies which matched Organism A. Finally, S. sciuri had no growth on MacConkey’s medium which matches with Organism C. Our final test that confirmed our organism matches was motility. In order to determine the motility of our bacteria, we performed two different tests, one being observing our sample under a wet mount and the other was a stab test. The wet mount allowed for the bacteria to be swimming in a liquid, so the bacteria could be motile if they performed that action. We observed these mounts under a microscope where we were able to determine if the bacteria was moving. Another test to determine whether the bacteria is motile was a stab test. We essentially stabbed a sterilized needle into an agar in a tube and incubed them in order to see if there was growth. I personally worked on Organism C which presented immobile in both the stab test and the wet mount meaning no movement and this was the same results for Organism A. For Organism B, there is motility present in both the wet mount and the stab test, in the wet mount there were clear individual organisms that were swimming around and the stab test had shown that the bacteria moved around the agar rather than staying where the stab was. For Phenotypic Organism A however, these results did not match with the results for Organism B, S. marcescens, which is the organism we matched with A. We assume that there must have been an error made while performing either tests. The stab test could have been contaminated and something else was motile in the agar and the wet mount could have been incorrectly prepared or the microscope could have not been focused on the true bacteria sample. These results confirmed our matched organisms where Phylogenetic Organism A is E. coli, Phylogenetic Organism B is S. marcescens and Phylogenetic Organism C is S. sciuri.

Conclusion The main hypothesis indicated that samples from the ELVIS probe contain bacteria native to Mars and these bacteria cannot be identified as related to earthly bacteria. Based on the results, this hypothesis is not supported. All three organisms found in the sample were able to be distinguished as Earthly organisms. The Earthly bacteria present in the samples were E. coli, S. sciuri, and S. marcescens. When comparing the data between the rDNA sequence and its phenotypic data, each organism had mostly matches among the different test runs which led to the conclusion of what these organism samples were. Specifically, comparing the motility, Gram type, and lactose fermentation helped confirm the identity of each organism. The Gram-stain and MacConkey tests mainly helped bring all of this information together. The first organism showed it was a motile bacillus Gram-negative and was not able to ferment lactose which led to the conclusion of identifying this as S. marcescens. Meanwhile, the second organism shared similar shape and Gram type but was able to ferment lactose on the MacConkey agar which categorized this organism as E. coli. The last organism was a coccus Gram-positive bacterium with no motility that could ferment lactose which helped determine that this organism was S. sciuri. With all of these different tests conducted, it helped us determine that these were identifiable organisms from Earth therefore rejecting the initial hypothesis. There was some inconsistency in observations due to numerous reasons. S. marcescens does have motility according to the rDNA sequence data, but the observations showed otherwise. This was most likely due to inoculation error of not getting enough bacteria on the plates. This reason can be said for a couple other tests that were run in this experiment such as the glucose minimal media, and simmon citrate media. For future technique, sterilizing properly and getting enough colonies on the inoculating tool will prevent this inconsistency from happening. Another inconsistency that occurred was the oxidase test. The oxidase test had all results indicate a positive for oxidase being present, even in the negative control. This was due to the reagent disks being defective so these results were ignored when classifying the organisms. These organisms tend to be found in similar areas to Earth. They all grow around room temperature and prefer an aerobic atmosphere but can survive in anaerobic conditions. However, there are also distinguishable features to each organism that have them grow better in different conditions such as halophilic properties. A mechanism that can isolate each organism from the ELVIS probe is the aseptic technique to create a pure culture. Each organism in the mixed sample had a specific colony appearance so they were able to be isolated with an inoculating loop. The inoculating loop was sterilized with fire then the organism colony was collected and moved to a separate Petri plate in order for just that organism to be grown. This was repeated multiple times to make sure the culture was purely isolated to run tests. The DuPunt hypothesis was that the degradation of polyurethane products was being caused by microorganisms present in the soil sample. Based on the results and research done, this hypothesis can be supported. To test if the organisms isolated could live on polyurethane, each

organism can be grown on a defined minimal media with polyurethane being the only nutrient source. It is important for the media to be defined and minimal because it will give specific selection as to which organisms can use polyurethane as a nutrient source and grow. The organisms can be isolated into a pure culture onto this media and incubated overnight to see growth. Since the polyurethane on the ELVIS probe has deteriorated, the expected results would be that the organisms will grow on the media.