MICROBIAL HABITATS IN HIGH-ALTITUDE LAVA TUBES. Kimberly B. Teehera Department of Chemistry University of Hawai`i at Mānoa Honolulu, HI ABSTRACT

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1 MICROBIAL HABITATS IN HIGH-ALTITUDE LAVA TUBES Kimberly B. Teehera Department of Chemistry University of Hawai`i at Mānoa Honolulu, HI ABSTRACT Ice-filled lava tubes on Mauna Loa can serve as analogs to lava tubes on Mars. The purpose of this project is to characterize the microbes found in the caves and determine how secondary minerals are linked to microbial life. Mineral samples collected from the cave were analyzed with scanning electron microscopy and energy-dispersive spectroscopy. For the microbial samples, genetic analyses were conducted by matching the DNA to relatives, producing phylogenetic trees to display relatedness, and researching the related microbes on GenBank. The minerals were identified as calcite, gypsum, and, largely, SiO2. These minerals may support microbial life in that calcite may be precipitated by the microbes, accelerate weathering of SiO2, and utilize heavy metal ions for metabolism. The Mauna Loa microbes are closely related to microbes found in similar environments. Any Martian microbes are likely to be chemolithoautotrophs, and past evidence of this could be preserved in ice in the subterranean lava tubes. INTRODUCTION The ice caves on Mauna Loa are valuable analogs to similar subterranean Martian lava tubes, as they are high-altitude caves formed from lava tubes. Two ice caves are of interest, the Mauna Loa Ice Cave (MLIC) and Arsia Ice Cave. In one hall, the continuous white secondary mineral deposit running along the side of cave walls indicate previous ice levels. Previously, the secondary minerals had been identified using Raman spectroscopy as calcite, CaCO3, and gypsum, CaSO4 2H2O (Teehera, 2016). Additionally, the area around these caves is devoid of vegetation, and therefore little to no organic carbon (the energy source of heterotrophs) can be expected in the caves. The earliest life forms on Earth may have been chemolithoautotrophs, consistent with the lack of sunlight inside of caves; photosynthesis is a relatively advanced evolutionary mechanism. Thus, studying the microbes in the caves can allow the prediction of microbes in Martian lava tubes as well. DNA sequences obtained after DNA extraction of a mineral and water sample were analyzed to identify any genetic relatives. The goal of this project was the characterize the microbes both by processing their sequenced DNA and by determining the ties between the geochemistry of the mineral analysis to the potential microbes. METHODS & MATERIALS Scanning electron microscopy (SEM) and energy-dispersive spectroscopy (EDS) were used to analyze five mineral samples (A6, A8, M3-2, M2-1, and M1) to determine if there are any biogenic mineral signature or traces of microbial life. These mineral samples were crushed, and a 81

2 few grains or flecks were placed onto an adhesive before being carbon-coated, which allows for a conductive surface. In the previous semester, DNA from Samples M1 (a mineral sample) and A7 (a water sample) were extracted and sequenced using the 16s rrna gene, a section of a gene common to all bacteria and some archaea, which allows for matching of gene sequences (Teehera, 2016). This semester, they were sorted in order of abundance from the resulting DNA files. The ten most abundant sequences, sorted using TextWrangler, were put through BLAST to obtain the two closest genetic relatives. These relatives DNA sequences were downloaded and aligned (that is, matched by nucleotides as closely as possible) using a DNA aligning program (Dereeper et al., 2008) to generate a phylogenetic tree. Finally, these relatives were characterized via their GenBank accession number to reveal any information about the microbes found in MLIC and Arsia Ice Cave. SEM/EDS RESULTS Mineral samples contained micron-sized calcite crystals, which take on a thin, needle-like appearance (Figure 1). The calcite is also superposed on gypsum, which has a surface that appears smoother. The electron beam produced cracks on its surface, indicative of hydration. Figure 1. (a) SEM image of Sample M2-1, showing calcite on gypsum. The calcite crystals are on the scale of 1 μm, and are hence cryptocrystalline. (b) The larger gypsum crystal has cracked from the laser beam, evidence for former hydration. (c) Sample A6 showing calcite on gypsum. (d) gypsum blades in the center. Crystal identities are based on EDS points. The EDS analyses of the mineral samples contain high peaks of C, Ca, and O, and/or S, which are indicative of calcite and gypsum. The appearance of these minerals is not uniform across 82

3 all samples and spots; they occur in different spots on different minerals (Table 1). However, the calcite and gypsum occurs in lower abundance; much of the bulk secondary mineral deposit is silicon dioxide (SiO2) or an amorphous silicate (SiO4 - ). EDS analysis contained high peaks or signals of Si and O, most times in conjunction with calcite and gypsum. In some samples, the elements of calcite only registered if an integrated area was analyzed in EDS (Figure 2). A8 is a sample with a soft, clay-like texture. EDS identified it as gypsum. However, other grains with different texture and morphology show that it consists of high amounts of Al-rich silicate. Sample Name & Type Appearance Raman Identification A6, wall white coating (veneer) Gypsum A8, deposit white, clay-like, soft Gypsum M1, deposit fine white powder Calcite M2-1, wall white coating (veneer) Calcite, Gypsum EDS Identification & Abundant Ions Gypsum, SiO2, Calcite (Si, S, O, Ca, C) Al-rich silicate (Al, Ca, Si, O, Na) SiO2, Calcite (O, Si, C, Ca) SiO2, Gypsum (Si, S, O, Ca, Mg) M2-3, wall white coating (veneer) Calcite, Gypsum - M2-5, floor white coating (veneer) Calcite, Gypsum - M3-1, wall white coating (veneer) Calcite, Gypsum - M3-2, deposit chalky whitish powder Calcite SiO2 (Si, O, Mg) Table 1. Mineral samples and their identities. Samples with 'M' are from Mauna Loa Ice Cave; samples with 'A' are from Arsia Cave. Abundant elements detected using EDS are also listed. Multiple carbon-rich fibers were found in sample M3-2 (Figure 3). SiO2 is deposited between the striations of some fibers. These fibers are not uniform in appearance. Additionally, rod-shaped depressions were found on the surface of one sample, M3-2 (Figure 4). However, EDS analysis determined that the elemental composition of these prints did not differ from that of the surrounding mineral. Figure 2. Left: SEM image of surface of sample M1 with SiO2 and calcite. Right: EDS spectrum for box 1 (orange). 83

4 Figure 3. SEM images of carbon-rich fibers. Figure 4. Sample M3-2 (powder) SEM images showing repeating rod-shaped depressions on the surface. Microbial Characterization Most of the related microbes were bacteria, mostly actinobacteria. The closest relatives match between 95 to 99% of the DNA, but no 100% DNA matches were found. Phylogenetic trees were constructed to determine the genetic relatedness of the microbial relatives to the MLIC and Arsia microbes (Figure 5). Most of the related microbes are uncultured; they have not been successfully cultured, or grown, in laboratories. Thus, not much is known about them. But, while most of the related microbes do not have species-specific information published about them, others did yield more information and provide clues for the microbes of MLIC and Arsia Cave. 84

5 Related microbes are able to adjust their metabolism to adapt to energy sources (or lack thereof), often utilizing heavy metal ions. One such microbe that is cultured is the genus Conexibacter, and there are many others that are purported to be chemolithotrophs, based on analysis of the environments from which they were collected. Another closely related proteobacteria can undergo aerobic anoxygenic mechanisms (de la Torre et al., 2003). Such characteristics of the related microbes suggests that some of the MLIC and Arsia microbes are chemolithoautotrophs. Also, related microbes live in dry, desiccant environments such as the Antarctic, and are able to survive in the insulation of porous rocks (Friedmann & Ocampo- Friedmann, 1984). Actinobacteria, specifically the genus Nocardioides, reside in carbonate stones and are able to precipitate CaCO3 (Jroundi et al., 2015). Furthermore, those exposed to radiation are adapted to cope with higher mutation rates if exposed to radiation, such as the related microbe belonging to the order Deinococcales (Ragon et al., 2011). Figure 5. Phylogenetic trees that show related microbes to those found in Sample A7 (left) and M1b (right). Red values correspond to the probability that the configured relation is correct, and the bar scale at the bottom left represents the horizontal relative difference in DNA sequences between branches. 85

6 DISCUSSION The EDS results match the results from Raman analysis in the previous semesters. SEM imaging also confirms the theoretical expectation obtained in an earlier semester that gypsum forms first, and then calcite, which is why the calcite was often superposed on the gypsum. Also the calcite and gypsum crystals are visible only on the micrometer scale; this cryptocrystallinity supports that the secondary minerals are cryogenic. EDS analysis detected SiO2, which was not identified by Raman spectroscopy. Since no strong Raman signals were found, the SiO2 is likely amorphous. The source of the SiO2 is from the host basalt, which consists of about 50% SiO2. SiO2, like the other minerals, is leached out by water; however, SiO2 is not very water-soluble, so it remains in solution longer. Once the carbonate ions are precipitated as calcite, the ph of the water decreases, thus causing the silicate to precipitate. Furthermore, clay minerals originate from weathering processes, which is often very slow in silicates (Hill & Forti, 1997). Weathering may be accelerated by microbes that live on the secondary calcite (Bennett et al., 2001). One of the samples, A8, contained an Al-rich silicate, which is anomalous because it was found in only one isolated deposit sample, whereas the other SiO2-containing samples were not Al-rich. Furthermore, Al is one of the ions that remains in solution longest, as it is not very soluble. Its appearance and elemental composition from EDS strongly suggest it is the clay silicate mineral, kaolinite, Al2Si2O5(OH)4. Systematically, there was a presence of Mg 2+ (or sometimes Na + ) ions recurring in spots that contained SiO2. This could be the previously unidentified locations of these cations (that were identified in the source water, but not in the Raman spectra); they are likely included in the structure of the SiO2. Otherwise, the Mg 2+ could replace Ca 2+ in calcium carbonate since they are similar cations. Moreover, the sample M3-2 contains many fibers. From EDS, the fibrous material is high in carbon (Figure 3). Due to its texture, appearance, and high carbon content, the fibers are likely organic. The source of these fibers is unknown, and, in lack of another explanation, they may have been introduced into the cave by humans or bats. The fibers were only found in one localized sample, although visually similar deposits are seen at other locations in the cave as well. SEM images also reveal rod-like depressions on the mineral (Figure 4). These imprints may be indicative of past microbes living in or on this mineral. However, because the SEM uses vacuum and the samples are no longer fresh, any microbes present would have been killed or would have disappeared by the time we looked at them with SEM as they were taken out from their native habitat. In addition, other heavy metal ions were detected in the secondary mineral, such as aluminum, iron, and chromium. These metals were detected in random parts of the mineral samples; they were not present in significant portions of the sample. These have been found to support microbial life in extreme environments that are devoid of organic matter. In lieu of oxygen, other ions such as Fe, NO3 -, or SO4 2- may also serve as the final electron acceptor in the electron 86

7 transport chain (a metabolic process that generates energy for the organism). Thus, the MLIC microbes may also adjust their metabolism according to the available inorganic substances in the cave, and the calcite may be a precipitated biogenic byproduct, which is also supported by the lower abundance of calcite relative to the SiO2. The related microbes are found in similar environments, such as in cold, dry, desiccant environments like Antarctica, or locations with volcanic activity. The microbial group most closely related to the group of MLIC microbes is from Kasatochi Island, Alaska. While most the microbe relatives are found in similar environments, they are also quite diverse. Some are found in prairie soils, others in warmer climates. The water sample yielded relatives also from aquatic environments such as lakes and ponds. Primarily, the microbes were actinobacteria. Actinobacteria are characterized for their high guanosine-cytidine nucleotide content (which makes for stronger, mutation-resistant DNA), gram positive membranes (thicker and more protected), and have sporing capabilities. Together, these traits make for a radiation- and desiccation-resistant group of microbes. Also, the related microbes are capable of altered metabolism to utilize the dissolved heavy metal ions. CONCLUSION The composition of the secondary mineral samples are cryogenic calcite and gypsum, and predominantly SiO2. SEM images show that calcite forms on top of gypsum, and are both cryptocrystalline, indicative of cryogenic formation. Al-rich silicate was found in one localized deposit in Arsia Ice Cave. These minerals can be expected to be found in Martian lava tubes as well. Because DNA was extracted from the powdery sample made of SiO2 and calcite, microbes reside in the secondary mineral. The SiO2 may serve as a habitat for the microbes, and thus may be weathered more quickly by microbial biological processes. Since calcite was found in lower amounts than was SiO2, perhaps calcite formation is a byproduct of and is accelerated by microbial metabolism, while the rest of the dissolved Ca 2+ remains in equilibrium in water. Based on the results, it can be expected that the microbes are radiation- and desiccationresistant chemolithoautotrophs that utilize the dissolved metal ions for metabolism. ACKNOWLEDGMENTS Many special thanks to Norbert Schorghofer for his guidance and support throughout a total of five semesters of my research fellowship. Also, thank you to the many contributors including my co-mentors Eric Hellebrand, Sean Jungbluth, and Myriam Telus. Thank you also to the Raman Lab for training me and allowing me to use the micro-raman spectrometer. Last, but not least, I also thank the Hawai`i Space Grant Consortium and NASA for affording me the opportunity to research as an undergraduate and learn all the things I have through this invaluable experience. 87

8 REFERENCES Bennett, P.C., Rogers, J.R., & Choi, W.J. (2001). Silicates, silicate weathering, and microbial ecology. Geomicrobiology Journal, 18, De la Torre, J. R., Goebel, B. M., Friedmann, E. I., & Pace, N. R. (2003). Microbial diversity of cryptoendolithic communities from the McMurdo Dry Valleys, Antarctica. Applied and Environmental Microbiology, 69(7), Dereeper A., Guignon V., Blanc G., Audic S., Buffet S., Chevenet F., Dufayard J.F., Guindon S., Lefort V., Lescot M., Claverie J.M., Gascuel O. Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res Jul 1;36(Web Server issue):w Epub 2008 Apr 19. Friedmann, E. & Ocampo-Friedmann, R. (1984). The antarctic cryptoendolithic ecosystem: relevance to exobiology. Origins of Life and Evolution of Biospheres, 14(1-4), Hill, C.A., and Forti, P. (1997). Cave Minerals of the World. 2nd ed., Huntsville, Ala.: National Speleological Society. Jroundi, F., Gonzalez-Muñoz, M.T., Sterflinger, K., & Piñar, G. (2015). Molecular tools for monitoring the ecological sustainability of a stone bio-consolidation treatment at the Royal Chapel, Granada. PLoS ONE, 10(7), e Ragon, M., Restoux, G., Moreira, D., Møller, A. P., & López-García, P. (2011). Sunlight-exposed biofilm microbial communities are naturally resistant to Chernobyl ionizing-radiation levels. PLoS ONE, 6(7), e Teehera, K. (2016). Secondary Minerals in High Altitude Lava Tubes on Mauna Loa. Undergraduate Fellowship Reports: Fall Hawai`i Space Grant Consortium, Honolulu. 88