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Quantifying the threshold of biogenic detection in evaporites: constraining potential Martian biomarker preservation

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Quantifying the threshold of biogenic detection in evaporites: constraining potential Martian biomarker preservation

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Content Quantifying the Threshold of Biogenic Detection in Evaporites: Constraining Potential Martian Biomarker Preservation By Scott M. Perl A Dissertation Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY in GEOLOGICAL SCIENCES December 2019 Perl, S.M. Biogenic Detection in Evaporites 1 Table of Contents Acknowledgements……………………………………………………………………………….8 Chapter 1. Introduction………………………………………………………………………..11 Chapter 2. Evaporite Minerals as a Record of Biomarker Preservation in Closed Basin Lake Systems and Implications for the Martian Subsurface..................................................19 Abstract…………………………………………………………………………………………..19 Introduction………………………………………………………………………………………20 Field Sites………………………………………………………………………………………..22 Discussion………………………………………………………………………………………..26 Conclusions………………………………………………………………………………………28 Acknowledgements………………………………………………………………………………28 References………………………………………………………………………………………..30 Figures……………………………………………………………………………………………50 Chapter 3. Laboratory and in-situ features of β-carotene and Mineral Pigments due to terrestrial Evaporite Preservation and Longevity in the Mineral record on Mars…...……55 Abstract………………………………………………….……………………………………….55 Introduction……………………………………………………………………………………....56 Methods…………………………………………………………………………………………..57 Results………………………………………………………………………………………...….60 Discussion…………………………………………………………………………………….….63 Conclusions…………………………………………………………………………………...….67 Acknowledgements……………………………………………………………………………....69 References…………………………………………..……………………………………………70 Figures……………………………………………………………………………………………79 Perl, S.M. Biogenic Detection in Evaporites 2 Chapter 4. Evaporitic Preservation of Modern Carotenoid Biomarkers and Halophilic Microorganisms in Martian Analogue Hypersaline Environments…………………...…….91 Abstract…………………………………………………………………………………………..91 Introduction……………………………………………………………………………………....92 Methods…………………………………………………………………………………………..96 Results………………………………………………………………………………………........98 Discussion………………………………………………………………………………….…...101 Conclusions…………………………………………………………………………………......103 Acknowledgements……………………………………………………………………………..104 References………………………………………………………………………………………105 Figures………………………………………………………………………..…………………115 Chapter 5. Investigation of Pigment Preservation in Pleistocene and Permian Evaporites with Implications to Biotic Entombment over Geologic Time………………….….……….125 Abstract…………………………………………………………………………………………125 Introduction……………………………………………………………………………………..126 Methods……………………………………………………………………………………..…..127 Results…………………………………………………………………………………………..129 Discussion………………………………………………………………………………………130 Conclusions……………………………………………………………………………………..132 Acknowledgements……………………………………………………………………………..134 References………………………………………………………………………………………135 Figures…………………………………………………………………………………………..142 Chapter 6. Recommendations and Nomenclature for Astrobiological and Geobiological in- situ Analyses of Martian Samples and Returned Sample Analyses……………….……….148 Perl, S.M. Biogenic Detection in Evaporites 3 Abstract…………………………………………………………………………………………148 Introduction……………………………………………………………………………………..149 Methods……………………………………………………………………………………..…..150 Results…………………………………………………………………………………………..155 Discussion………………………………………………………………………………………158 Conclusions……………………………………………………………………………………..159 Acknowledgements……………………………………………………………………………..162 References………………………………………………………………………………………163 Figures…………………………………………………………………………………………..172 Appendix……………………………………………………………………………………….183 Perl, S.M. Biogenic Detection in Evaporites 4 List of Figures Chapter 2. Evaporite Mineralogy as a Record of Biomarker Preservation in Closed Basin Lake Systems and Implications for the Martian Subsurface Figure 1. Biogenic Preservation and Biotic Information over Modern and Geologic Time Figure 2. Jezero Crater, Mars. Figure 3. Boxwork structures of sulfate mineral veins (likely gypsum) nicknamed “Garden City” within Gale Crater (from April 2015). Figure 4. Adopted from Ehlmann and Edwards (2014), this global map and icons shows confirmed CRISM detections of hydrated minerals. Figure 5. Hypersaline brine geochemical pathways and notable locations (Adopted from Warren, 2010). Chapter 3. Laboratory and in-situ features of β-carotene and Mineral Pigments due to terrestrial Evaporite Preservation and Longevity in the Mineral record on Mars Figure 1. Structures of Bacterioruberin C50H76O4 and β-Carotene C40H56. Figure 2. Optical observations at 10x and 100x showing original halite hopper crystal and microbial movement within fluid inclusions entombed within the crystal structure. Figure 3. Raman map of β-carotene at 1152cm-1 superimposed over the 40x visible light microscopy image of entombed fluids within a halite crystal face Figure 4. Abiotic laboratory precipitated NaCl with varying amounts of entombed β-carotene. Figure 5. Abiotic laboratory precipitated NaCl with varying amounts of entombed β-carotene under UV-C conditions. Figure 6. β-carotene entombment process without biology. Figure 7. Raman observations of β-carotene from fluids in Figs 7a-7c (within DNA-free water) and 7d-7f (precipitated NaCl). Figure 8. Raman observations of β-carotene embedded within laboratory-precipitated NaCl after 120 hours of UV-C exposure. Figure 9. Raman observations of β-carotene embedded within laboratory-precipitated NaCl within greater salinities than in-situ Searles Lake brine. Perl, S.M. Biogenic Detection in Evaporites 5 Figure 10. Evaporated β-carotene brine solutions showing preservation pathways into fluid inclusions. Figure 11. Optical image and Raman map of β-carotene concentrated within fluid inclusions during brine Evaporation. Figure 12. Raman analyses of several β-carotene-entombed fluid inclusions during the complete brine evaporation process. Chapter 4. Evaporitic Preservation of Modern Carotenoid Biomarkers and Halophilic Microorganisms in Martian Analogue Hypersaline Environments Figure 1. Overview of the Great Salt Lake (GSL) Figure 2. Brine and Evaporite Mineral comparisons between Great Salt Lake (Rozel Bay, UT, USA) and Searles Lake (Mojave Desert, CA, USA). Figure 3. Overview of the modern preserved evaporite mineralogy from Great Salt Lake and Searles Lake for halite and gypsum, the source lake settings, and the environmental controls. Figure 4. Optical imagery of evaporite halite minerals Figure 5. Rozel Bay sampling campaigns. Figure 6. Searles Lake location between the Argus and Slate mountain ranges (from Smith, 1979, Figure 1, p. 3). Figure 7. Raman scan representative of unfiltered brine waters from Great Salt Lake and from the dried Searles Lake Figure 8. Raman scan representative of fluid inclusions from Great Salt Lake and from the dried Searles Lake Figure 9. Raman scans of mineral salt structures from Great Salt Lake (blue) and from Searles Lake site (red) Figure 10. Raman spectra of Halobacteria sp. NRC-1 Chapter 5. Investigation of Pigment Preservation in Pleistocene and Permian Evaporites with Implications to Biotic Entombment over Geologic Time Figure 1. Sedimentology of salt and mud layers of Searles Lake Figure 2. Searles Lake (Mojave Desert, CA, USA) modern to Pleistocene field site Perl, S.M. Biogenic Detection in Evaporites 6 Figure 3. Overview of the Permian Boulby salt mine Figure 4. The three major Permian evaporite mineral assemblages from the Zechstein Formation Boulby salt mine. Figure 5. Raman analyses of Pleistocene Searles Lake trona, hanksite, mirabilite, halite, and Thenardite. Figure 6. Raman analyses of Permian Boulby Mine mine NaCl, KCl, and Polyhalite Chapter 6. Recommendations and Nomenclature for Astrobiological and Geobiological in-situ Analyses of Martian Samples and Returned Sample Analyses Figure 1. Comparisons of the origins of life on Earth and history of hydrated mineral and sedimentary features on Mars. Figure 2. Mineral signature of the Fe-oxide hematite in Meridiani Planum. Figure 3. Approximate location and stratigraphic column positions for rocks abraded by the RAT in the Karatepe section of Endurance crater. Figure 4. The Karatepe section of Burns Cliff within Endurance crater, part of the Burns fm. set of rocks. Figure 5. Scanning Electron Micrograph (SEM) image of the Alan Hills meteorite (ALH84001). Figure 6. “Pendulum” diagram showing examples and definitions of the proposed astrobiology nomenclature for a NaCl hopper crystal. Figure 7. The classical interpretation of habitability for planetary systems. Figure 8. A proposed update to the classical perspective of habitability for planetary systems. Figure 9. The evolution of the interpretation of microbial ecology. Figure 10. Bayesian framework for exoplanet spectral bio-indicator assessment (from Catling et al. 2017) Figure 11. Electrochemical impedance relationships between living microbes and dead cells. Perl, S.M. Biogenic Detection in Evaporites 7 Appendix Appendix Figure 1. Evaporite salt inhibition validation study results showing selected field samples spiked with ZymoBIOMICS DNA community standards (MMC) Appendix Figure 2. Total DNA (ng) as a function of evaporite sample (g) with environmental controls Appendix Table 1. Data table of Great Salt Lake evaporite samples used in 16S rRNA gene sequencing Appendix Figure 3. Operational Taxonomic Units (OTUs) derived from 16S rRNA gene sequences: Cumulative Appendix Figure 4. Operational Taxonomic Units (OTUs) derived from 16S rRNA gene sequences: By Site Appendix Figure 5. Operational Taxonomic Units (OTUs) derived from 16S rRNA gene sequences: Halite Only, By Site Appendix Figure 6. Operational Taxonomic Units (OTUs) derived from 16S rRNA gene sequences: Halite Only, By Category Appendix Figure 7. Operational Taxonomic Units (OTUs) derived from 16S rRNA gene sequences: Gypsum Only, By Category Appendix Figure 8. Operational Taxonomic Units (OTUs) derived from 16S rRNA gene sequences: Halite vs. Gypsum: Cumulative by Type Appendix Figure 9. Validation of an uncontaminated DNA extraction using three-dimensional Bray-Curtis PCoA for both evaporite categories observed in the Great Salt Lake Perl, S.M. Biogenic Detection in Evaporites 8 Acknowledgements I would like to thank my fellow classmates of the University of Southern California Earth Sciences. Specifically the newly minted (or soon to be) Drs. Pieter-Ewald Share, Olivia Piazza, Rob Zinke, Dylan Wilmeth, Erin McParland, Joyce Yager, Jeff Thompson, Kristin Washington, Paulina Pinedo, Marshall Rogers-Martinez, Max Dahlquist, Sylvia Dee, Mark Peaple, Cooper Harris, Reena Joubert, and Casey Barr. From the beginning new graduate student field trip in August 2013 to our individual departing events now and in the recent months, your friendships were and are just as much a positive impact on my time at USC as my research investigations were. From pizza and beer specials at Rosso’s to rooftop gatherings to tailgating to everything in- between, those times truly kept me grounded while trying to navigate my way between the “real world” and academia. I would like to thank Cindy Waite for being the positive cornerstone of so many doctoral student milestones and events. From Cindy’s nighttime reminder phone calls to register for classes, management of my credit timeline and comparing my course spreadsheet pre and post quals, to qualifying exam room schedules, or her devotion to graduate student life and well-being, Cindy has been such as asset to the Earth Sciences department and to the students. Being able to manage academic life and JPL work was difficult on its own and Cindy was vital with her support and helpfulness. I would like to thank my advisor and committee Frank Corsetti, Will Berelson, Dave Bottjer, Dave Caron, and Aaron Celestian. Having the research perspectives and personalities of this group was very important for my investigations and research avenues. From the beginning meetings between Frank, Will, and I before starting as a Ph.D. student in 2012 and their common advice on separating mission data from my own research contributions to their ongoing support Perl, S.M. Biogenic Detection in Evaporites 9 throughout my doctorate I cannot thank them enough. I’d like to thank Dave Bottjer for his significant interest and insight into my work and for his continued curiosity in taking this work into Precambrian timeframes and those implications for similar preserved features from ancient Mars and the potentially newly circulated ice crusts on Europa. To Dave Caron for his opinions and perspectives during my qualifying exams and being able to discuss the microbiological components of my evaporite investigations. Moreover, to Aaron Celestian for his continued collaboration with our funded projects and to our future ideas involving mineral-microbial interactions, the new OHL Geobiology Analysis Suite set of instruments (and the learning curve we have ahead of us!), and for the full academic circle coming from students at SUNY Stony Brook to colleagues together at the LA-NHM. Perl, S.M. Biogenic Detection in Evaporites 10 “But there are no distant relatives, no humans, and apparently no life waiting for us on those other worlds. No letters conveyed by recent émigrés help us to understand the new land – only digital data transmitted at the speed of light by unfeeling, precise robot emissaries. They tell us that these new worlds are not much like home. But we continue to search for inhabitants. We can’t help it. Life looks for life.” -Carl Sagan Wanderers: An Introduction (page xvii) in a Pale Blue Dot Perl, S.M. Biogenic Detection in Evaporites 11 Chapter 1 Introduction Evaporite minerals can capture and entomb organic matter within their intercrystalline and intracrystalline structure because they precipitate relatively quickly (nomenclature adopted from Schopf et al. 2012). Thus, evaporite minerals constitute a target for biosignature investigation on Earth and Mars, where evaporitic deposits are known to exist. However, little is known about the process of organic preservation and detection in evaporites, or the stability of such molecules when exposed to significant UV radiation (as would be present on the surface of Mars). Halite and gypsum have been chosen for investigation because they have been detected on Mars by the MRO Compact Reconnaissance Imaging Spectrometer for Mars (CRISM) instrument (Ehlmann et al. 2014; Murchie et al. 2009; Viviano ‐Beck et al. 2014), their physical transparency and short-term precipitation timescales, which are amenable to experimentation. Halite and other sulfate salts have additionally been observed within the shallow subsurface of Mars in proximity to ancient aqueous settings either via groundwater or evaporated lake beds, and thus these minerals are relevant to Mars investigations (Barbieri & Stivaletta, 2011; Ehlmann et al. 2014 ). Moreover, the current in-situ observations on mineral veins on Mars that show these hydrated features from ancient surface and groundwaters are of significant height and size, being able to survive physically over geologic time. Moreover these meter-scale regions can provide ample sample size for future Mars Sample Return analyses from due to these proven sites containing hydrated mineralogy and from direct exposure to ancient fluids and water-mineral interactions. Perl, S.M. Biogenic Detection in Evaporites 12 Motivating Questions Given the presence of evaporites on Mars and the paucity of data with respect to organic matter preservation in evaporite minerals, it is important to investigate the potential for evaporite minerals to serve as targets for organic matter preservation. Several overarching questions motivated this research: 1. “How much, if any, of the biological material is transferred/captured from the original in- situ waters to the evaporite mineral precipitated nearby?” Do the molecules reside predominantly in fluid inclusions or within the crystal lattice? a. Significance: knowledge of the efficiency of transfer from the environment to the evaporite minerals can inform future missions with respect to instrument detection limit, lithological search parameters. b. Research Plan: This question will be investigated first using laboratory experiments on known, composed samples and then via modern field sites (e.g., Great Salt Lake, Searles Lake). 2. If entombed in evaporites, what is the potential for preservation on geologic time scales? Do these chemical biomarkers from life need the biotic activity of life to be alive in order for the measurement to be valid? a. Significance: This defines the geobiological categories between extinct and extant life, the conditions in which chemical biomarkers are the most detectable, and being able to observationally link the presence of specific biosignatures to time duration and timing of active biology. Perl, S.M. Biogenic Detection in Evaporites 13 b. Research Plan: this question will be investigated by examining evaporite samples from modern, Pleistocene (~1.1 Myr years ago), and Permian (~255 Myr years ago) deposits using the techniques developed during this study. 3. How might the strong UV flux affect preservation of organic matter in evaporites? Can the natural precipitation processes of evaporite minerals mitigate destruction of organics by UV-C? Does the protection by pigmented mineralogy for halophilic microorganisms need the organisms to continually produce carotenoid biomarkers? a. Significance: Mars has an intense UV flux at the surface which has the potential to destroy organic material. How much, if any, protection from UV destruction might evaporite minerals impart to the organic matter? How robust are the pigmented after long durations of UV-C? b. Research Plan: this question will be investigated via laboratory experiments that emulate days and weeks of Martian UV-C in order to examine how robust set quantities of the carotenoid pigments in question are pre- and post-UV-C exposure. Raman spectra and optical images of samples will be compared and investigation strategies for how to look for these from a rover’s perspective will be generated. Specific Methods and Accompanying Targets of Study for Organic Matter Preservation Raman Spectroscopy: On Earth, there are many techniques with which to study how biological materials may be preserved during evaporite precipitation, and how long the biologic materials might last in the geologic record once entombed. However, only a certain subset of techniques would be available in situ on Mars given the nature of which instruments can engineered to operate on the Martian surface. Here, we focus on Raman spectroscopic Perl, S.M. Biogenic Detection in Evaporites 14 investigation because it is likely to be on the next planetary rover mission and if successful, may be part of future astrobiological payloads. Raman spectroscopy works by exciting covalent molecular bonds at specific frequencies based on the wavelength of the laser. The excitement of those molecules vibrates at specific frequencies. The difference between non-excitement and the excited state is then translated (and read by the detector) as a spectra in which the peak magnitude corresponds to the volume of the content being measured and the width of the peak as the contributions of other components that share that excitement/vibrational range. Substances with purely ionic bonds do not have a Raman signal (e.g., Halite / NaCl, see below). Carotenoid Pigments: Carotenoids compose one of the conspicuous biologic components of most modern hypersaline environments. They are involved in many biological processes and are produced by halophilic microorganisms as a sunscreen of sorts that blocks out the wavelengths of terrestrial UV-A and B. As a pigment, they are visible with the naked eye and could potentially be visible to a rover on Mars, and as relatively complex organic molecules, they have a specific chemical structure that is highly amenable to analyses via Raman spectroscopy. Although other organic molecules will be presented, a major focus is placed on the measurement and preservation of carotenoids, including β-Carotene C40H56 and Bacterioruberin C50H76O4. Other molecules will be examined (e.g., DNA in the Appendix), but the focus will be on the incorporation, measurement, and preservation in the face of UV flux on carotenoid molecules (Mathews-Roth et al. 1970; Jehlička et al. 2014; Fendrihan et al. 2003; Jones and Baxter, 2017; Baxter et al. 2007; Sankaranarayanan et al. 2014; Winters et al. 2013) Perl, S.M. Biogenic Detection in Evaporites 15 Mars Relevant Minerals There are a wide range of spectrum from evaporite minerals that form as marine or lacustrine evaporates. Halite (NaCl) and gypsum (CaSO₄·2H₂O) are two of the most common on Earth, and both have been found on Mars. Reconnaissance Imaging Spectrometer for Mars (CRISM) instrument (Ehlmann et al. 2014; Murchie et al. 2009; Viviano ‐Beck et al. 2014; Knoll et al. 2005), their physical transparency, and short-term precipitation timescales. Halite and other sulfate salts have additionally been observed within the shallow subsurface of Mars in proximity to ancient aqueous settings either via groundwater or evaporated lake beds, and thus these minerals are relevant to Mars investigations. Furthermore, Halite, because of its purely ionic bonding, is transparent with respect to Raman analyses, and thus presents a good target via a Raman-equipped rover on Mars (that is, the halite mineral matrix or substrate does not interfere with the Raman spectra of whatever may be trapped/entombed inside of it). Roadmap Here, I will address the overarching questions outlined above via laboratory and field experiments, focusing on the preservation of carotenoids in gypsum and halite using Raman spectroscopy. In addition to modern samples, ancient samples will be analyzed to address the longevity of the target molecules in the evaporite minerals. Chapter 2 will introduce the field sites, evaporite mineralogy in terrestrial closed basin lake systems, and implications for these evaporite assemblages on Mars. Chapter 3 will investigate the Raman signature and preservation potential of carotenoids via carefully composed experiments in the lab, in order to understand detection limits and better interpret samples collected from the field. In addition, UV-C measurements of abiotic laboratory- Perl, S.M. Biogenic Detection in Evaporites 16 precipitated NaCl with entombed β-carotene has been used to assess the UV shielding capabilities of NaCl. Chapter 4 will investigate the entombment of pigments and preserved carotenoids in the modern evaporites from the Great Salt Lake and Searles Lake. Chapter 5 will investigate ancient Pleistocene and Permian in-situ evaporites from the buried 1.1 Myr old Searles Lake and the ~255 Myr old Zechstein Formation evaporites in the Boulby salt mine in order to help address the longevity of pigments over geologic time. Chapter 6 will take into account the previous chapters conclusions and discuss the nomenclature needed for astrobiological measurements and interpretations, recommendations for proper life detection taking into account life “as we know it” to help or hinder the decisions to search for life “as we don’t know it” in on Mars. References Barbieri, R. and Stivaletta, N. (2011), Continental evaporites and the search for evidence of life on Mars. Geol. J., 46: 513–524. doi:10.1002/gj.1326 Baxter, B.K., Eddington, B., Riddle, M.R., Webster, T.N. and Avery, B.J. Great Salt Lake Halophilic Microorganisms as Models for Astrobiology: Evidence for Desiccation Tolerance and Ultraviolet Radiation Resistance. In: Hoover, R.B., Levin, G.V., Rozanov, A.Y., and Davies, P. C.W. (eds.) Instruments, Methods, and Missions for Astrobiology X, 6694:669415. SPIE, Bellingham, WA, 2007. Perl, S.M. Biogenic Detection in Evaporites 17 Ehlmann, B.L., J.F. Mustard, G.A. Swayze, R.N. Clark, J.L. Bishop, F. Poulet, D. Des Marais, L.H. Roach, R.E. Milliken, J. Wray, O. Barnouin-Jha S.L. Murchie. Identification of hydrated silicate minerals on Mars using MRO-CRISM: geologic context near Nili Fossae and implications for aqueous alteration, J. Geophys. Res, E00D08, doi:10.10292009 JE003339 Fendrihan S, Musso M, Stan-Lotter H. Raman spectroscopy as a potential method for the detection of extremely halophilic archaea embedded in halite in terrestrial and possibly extraterrestrial samples. Journal of Raman spectroscopy : JRS. 2009;40(12):1996-2003. doi:10.1002/jrs.2357. Jehlička J., Edwards H.G., Oren A. (2014) Raman spectroscopy of microbial pigments. Appl Environ Microbiol. 2014 Jun;80(11):3286-95. doi: 10.1128/AEM.00699-14. Epub 2014 Mar 28. Jones, D.L. and Baxter, B.K. (2017) DNA Repair and Photoprotection: Mechanisms of Overcoming Environmental Ultraviolet Radiation Exposure in Halophilic Archaea. Front. Microbiol., 29 September 2017 | https://doi.org/10.3389/fmicb.2017.01882 Knoll, A. H., Carr, M., Clark, B., Des Marais, D. J., Farmer, J. D., Fischer, W. W., Grotzinger, J. P., McLennan, S. M., Malin, M., Schröder, C., Squyres, S. W., Tosca, N. J., Wdowiak, T. (2005) An astrobiological perspective on Meridiani Planum. Earth and Planetary Science Letters v240, 1, 30 November 2005, 179-189 Perl, S.M. Biogenic Detection in Evaporites 18 Lowenstein, T.K., Lauren A.C. Dolginko, Javier García-Veigas; Influence of magmatic hydrothermal activity on brine evolution in closed basins: Searles Lake, California. GSA Bulletin 128 (9-10): 1555–1568. doi: https://doi.org/10.1130/B31398.1 Mathews-Roth, M. M., and Krinsky, N. I. (1970): Studies on the protective function of the carotenoid pigments of Sarcina lutea. Photochem. Photobiol. 11:419–428. Murchie S.L., Mustard J.F., Ehlmann B.L., Milliken R.E., Bishop J.L., McKeown N.K., Dobrea E.N, Seelos F.P., Buczkowski D.L.,Wiseman S.M., Arvidson R.E., Wray J.J., Swayze G., Clark R.N., Marais D.J.D., McEwen A.S., Bibring J.-P. (2009). A synthesis of Martian aqueous mineralogy after 1 Mars year of observations from the Mars Reconnaissance Orbiter. Journal of Geophysical Research 114, doi: 10.1029/2009JE003342 Sankaranarayanan K., Lowenstein T.K., Timofeeff M.N., Schubert B.A., Koji, L.J.. Astrobiology. July 2014, 14(7): 553-560. Viviano-Beck, C. E., et al. (2014), Revised CRISM spectral parameters and summary products based on the currently detected mineral diversity on Mars, J. Geophys. Res. Planets, 119, 1403– 1431, doi:10.1002/2014JE004627. Winters, Y.D., Lowenstein, T.K. & Timofeeff, M.N., (2013). Identification of Carotenoids in Ancient Salt from Death Valley, Saline Valley, and Searles Lake, California, Using Laser Raman Spectroscopy. Astrobiology, 13(11), pp.1065-1080. Perl, S.M. Biogenic Detection in Evaporites 19 Chapter 2 Evaporite Mineralogy as a Record of Biomarker Preservation in Closed Basin Lake Systems and Implications for the Martian Subsurface Abstract Evaporite minerals can precipitate on Earth and other planets following the evaporation of hypersaline fluids (e.g., closed basin lakes). Evaporite minerals are diagnostic of past aqueous activity and constitute potential sites for biogenic entombment of organic matter that may have populated the lake system. Furthermore, evaporites may have the potential to shield organic matter from the heavy UV load that the Martian surface experiences. Thus, evaporites constitute an excellent target for the search for biomarkers on Mars, where sulfate and chloride minerals have been identified from orbit and on the surface. The sites explored in this chapter and throughout this dissertation are Rozel Bay in the northern Great Salt Lake, Utah, USA, Searles Lake in the Mojave Desert, USA and the Boulby Salt Mine near Whitby, UK. These three sites have recorded modern surface water, modern surface to buried Pleistocene lake drying events, and enclosed Permian waters from ancient seas. The Great Salt Lake (GSL) is a remnant from the Pleistocene Lake Bonneville. The dried Searles Lake was a Pleistocene lake that has had several complete evaporation events followed by at least four fluidic/active periods. The Boulby evaporite salt mine was precipitated from ponded cliffside waters from the Zechstein Sea leading to the Zechstein Formation, stretching from northeastern England southward through parts of western and central Europe. Extreme life on our own planet in hypersaline settings gives us a wealth of knowledge for potentially extreme cellular life elsewhere in the solar system. Perl, S.M. Biogenic Detection in Evaporites 20 Introduction Microorganisms that are tolerant to hypersaline settings, known as halophilic microorganisms (or sometimes termed generally as “halophiles”), can reside in closed basin lake systems where salinity is typically ten times that of average seawater salinity. These hypersaline settings also readily precipitate evaporite minerals. Evaporite minerals allow for organic matter to be captured and entombed within their intercrystalline and intracrystalline structure because they precipitate relatively quickly (nomenclature adopted from Schopf et al. 2012). Thus, evaporite minerals constitute a target for biosignature investigation on Earth and Mars, where evaporitic paleoenvironments are known to have existed. Halite and gypsum have been chosen for investigation due to their confirmed detection by the MRO Compact Reconnaissance Imaging Spectrometer for Mars (CRISM) instrument (Ehlmann et al. 2014; Murchie et al. 2009; Viviano ‐ Beck et al. 2014), their physical transparency, and short-term precipitation timescales. Halite and other sulfate salts have additionally been observed within the shallow subsurface of Mars in proximity to ancient aqueous settings either via groundwater or evaporated lake beds, and thus these minerals are relevant to Mars investigations. The chosen field sites are the active closed-basin lake represented by the Rozel Bay site in north arm of the Great Salt Lake, Utah, USA, the dried evaporite lakebed of Searles Lake (Mojave Desert), California, USA, and the Permian Boulby salt mine formed from the Zechstein Sea (near the town of Whitby, UK. These three sites all serve as analogue environments across time due to the similar evaporitic settings interpreted to have existed on ancient Mars. These three analogue sites all have or had evaporite deposition and contain similar evaporite minerals to those found on Mars (Viviano ‐Beck et al. 2014). The evaporite minerals found in all of the analogue field sites provide the proper Martian analogue settings since they have similar geochemical and fluvial Perl, S.M. Biogenic Detection in Evaporites 21 settings as the ancient sites that Opportunity and Curiosity have explored (Squyres et al. 2005; Grotzinger et al. 2005, 2015; McLennan et al. 2005, 2014; Ming et al. 2006, 2014; Clark and Knoll, 2006; Bish et al. 2013). The benefit of having active modern study sites is being able to investigate mineral precipitation reactions in real time These modern records also provide a proper initial “stopwatch” for the monitoring of preserved biological processes and mineral modification solely due to the added presence of microbial life (Figure 1). Modern setting (Great Salt Lake): This setting represents a closed basin lake system that is inherently “active” with respect to modern biology and provides the highest volume of biological material for transfer into evaporite mineralogy. The geologically modern features of this site (flooding, rain and snowfall, etc.) also allows for the evaporites to be the most susceptible to mineral dissolution should lake levels rise above the current lake-shoreline boundary where the majority of the NaCl terraces are located. If (and only if) lake level does not rise to cause shoreline dissolution, then those evaporites (at the time of the last precipitation) and the halophilic microorganisms potentially preserved in them would be a proper “t0” for preservation potential for extant biology at its “highest” magnitude. Modern to Pleistocene setting (Searles Lake surface to buried evaporites): Searles Lake does not have persistent standing water like the Great Salt Lake, and thus provides another test case for modern environments where evaporites form. In addition, the system has been cored and buried salt deposits (at least 4-5) from modern to Pleistocene in age are available. The modern site (the surface) has significant pigmented evaporites whereas the buried salts and evaporites have different pigment variations in the mineral and regolith layers. The highly pigmented surface salts Perl, S.M. Biogenic Detection in Evaporites 22 show deep red and pink colors whereas the buried (older) layers show pigments of green, yellow, light orange, and light pink. The main hypothesis here is that these geologically older soil and salt sediments have degraded versions of the surface pigments since the β-carotene and bacterioruberin pigments being generated are from halophilic microorganism response to solar flux. Smith (1970) found at least 4-5 evaporite layers and in-situ fieldwork confirms that number and potentially reveals more minor salt layers in-between major layers. Permian setting (Boulby Mine): Ponded Zechstein Sea waters led to the precipitation of the Zechstein Formation salt layers ~255 Myr ago. The timeline of ponded seawater into precipitated evaporite minerals would have started off slow (due to seawater having an average salinity of 3.5%). However as evident from the Great Salt Lake and the Searles Lake surface brines (which have salinities of 23-27% and ~19-25%, respectively) closed basin lake systems can have salinities that are ten-fold greater than seawater. So the ponding process would have greater salinities as time progressed and the frequency and volume of evaporite layers (NaCl, KCl, polyhalite, and trace others). Organic matter preservation in this site occurred in the salt layers due to there being no soil or non-hydrated mineralogy observed in the Permian layers of the Boulby mine. Atop the salt layers are shale and non-permeable bedrock that made surface water permeability very unlikely. Closed aquafers have been observed throughout the Permian layers and likely contain Permian brines that did not evaporate but still have mineral-fluid interaction in some cases. Field sites Modern evaporitic preservation in the Great Salt Lake Great Salt Lake occupies the Bonneville Basin, which is one of the largest depressions in the Great Basin, the largest contiguous inland watershed in North America (Cohenour and Perl, S.M. Biogenic Detection in Evaporites 23 Thompson, 1966). Over time, the Bonneville Basin primarily held shallow lakes such as the modern terminal lake, however, it was home to several deep lakes over the last 780 ka, including late Pleistocene Lake Bonneville, 30-12 ka (Oviatt et al. 1999; Shroder et al. 2016). The north arm of modern Great Salt Lake, isolated by a railroad causeway, is an ideal modern field site for this investigation due to the active and receding nature of the lakebed system and isolation from new fluid inputs from the south arm (Chapter 4, Figure 1) (Madison, 1970; Cannon & Cannon 2002; Baxter et al., 2005). This separation is visually evident as the northern waters have a characteristic pink hue due to the presence of halophilic microorganisms (Baxter, 2018). The pigmentation, in particular of halophilic archaea, is critical for their survival due to the solar flux at this site (Jones and Baxter, 2017). The combined closed-basin nature of the north arm and representation of the entire Great Salt Lake as a fraction of the larger Pleistocene Lake Bonneville is similar to the dried river valleys and deltaic deposits found extensively in ancient Mars (see Figure 2 for the upcoming rover landing site launching in the Spring of 2020). Primarily the hypersaline waters of the enclosed north arm of Great Salt Lake are comparable to the fluids that were the solvent within the Burns Formation set of rocks in Endurance, Erebus, and Victoria craters in Meridiani Planum (Knoll et al. 2005, McLennan et al. 2005, Grotzinger and McLennan, 2008, Herkenhoff et al. 2005, Andrews-Hanna et al. 2010). The lack of plate tectonics on Mars has allowed the preservation of ancient aqueous settings, and the original geomorphological features (Figure 3). Therefore, the spectral and visual orbiter data can be used to directly interrogate the original mineralogical/sedimentological depositional paleoenvironment. Halite and gypsum evaporites are found extensively at the shoreline in layered terraces (Spencer et al. 1985) in west Rozel Bay. Halite precipitation is observed in a layered fashion at Perl, S.M. Biogenic Detection in Evaporites 24 the shoreline-lake boundary and record several rapid evaporation events if lake levels continue to recede seasonally. The non-permeable nature of most of the terrace spans the entire length of the north arm and can withstand mineral dissolution due to rain and snowfall. Faults in the north arm may allow for groundwater seeping where microbialites have been observed (Lindsay et al. 2017) but appear not to disturb halite terraces from the shoreline-lake boundary to significant distances in Rozel Bay. As lake waters withdraw and remaining fluids evaporate, the formation of layered halite has the ability to capture organic material and enclose it within the layers. Gypsum (CaSO4.2H2O) crystals are abundant in the north arm sediment. Measuring 4-13 cm, these crystals are found in the halite-rich clay (Eardley and Stringham, 1952). The genesis of these crystals is from the same lake waters as the halite but may be from groundwater-derived evaporation and not surface lake water evaporation. Nevertheless, if the primary gypsum is from groundwater evaporation or from rapid surface water evaporation the original source is still from the high saline lake waters. These gypsum crystals precipitate only within polygonal ripple megastructures and from visual inspection have entombed sediment and clays within its crystal structure. These ripple megastructures may be from groundwater upwelling and evaporation and if so, would utilize the nearby north arm Great Salt Lake groundwaters as its source. Modern aged evaporitic preservation in the shallow subsurface salt crusts of Searles Lake The dried salt crusts of Searles Lake have modern-aged evaporites in addition to Pleistocene-aged buried sections both consisting of thenardite (Na2SO4), hanksite (Na22K(SO4)9(CO3)2Cl), halite (NaCl), gypsum (CaSO4·2H2O), mirabilite (Na2SO4·10H2O), and trona (Na3HCO3(CO3)•2H2O). The deepest salts at Searles Lake have been dated by the USGS at approximately 200 ka years old (Smith, 2009; Smith et al. 1979) and have not been diagenetically Perl, S.M. Biogenic Detection in Evaporites 25 altered by modern surface processes. While ongoing studies continue to examine the deeper Pleistocene-aged evaporite material, this first investigation is focusing on the modern shallow subsurface salts that contain the youngest/newest biogenic preservation within the suite of salt mineralogy. Permian aged halite, potash, and polyhalite from the Zechstein Formation. in the Boulby Salt Mine Located ~1.2km below the surface, the Boulby salt mine was formed from the ponding of portions of the Zechstein Sea (Woods, 1979) leading to evaporation and evaporite deposition. The mine is currently partially located underneath the northwestern North Sea boarding the northeastern shoreline near Whitby, United Kingdom. After the isolation of the Permian waters several hundred meters of NaCl, KCl (“potash”), and polyhalite were precipitated in-situ with eventual burial by hundreds of meters of separate, perhaps interbedded at the boundary layers, shale and sandstone layers. Access to the mine was provided by this author’s participation in the MINAR and BASALT programs (see Cockell, Perl, and 48 others (2018) for details on MINAR V). Comparison of Terrestrial analogues with Martian environments/mineralogy Because of the similar salt mineral assemblages on Mars and their widespread diversity of chlorides, phyllosilicate clays, sulfate salts, and other hydrated minerals it is likely that many of the ancient lake systems had higher than nominal salt concentrations (Figs 3,4). On Earth, ocean salinity is ~3.5% whereas in closed-basin lake systems salinities can range ten-fold from this value. The GSL salinity ranges between 23-27% depending on the season while other terrestrial closed basins can be even higher (e.g., Dead Sea at 35-38% (Goetz, 1986) or Don Juan Pond at ~41% Perl, S.M. Biogenic Detection in Evaporites 26 (Hammer, 1986)). The widespread evaporite mineralogy and closed basin nature of many of the regions on Mars observed by digital elevation models from HiRISE and CRISM onboard the Mars Reconnaissance Orbiter (MRO) (Viviano-Beck et al. 2014; Ehlmann and Edwards, 2014; Weitz & Bishop, 2018; Jolliff et al. 2019; Ye and Glotch, 2018) shows how craters and other closed-basin hydrogeological settings are host to these hydrated minerals. Regions where depressions (from craters or other similar landmass) and hydrated minerals are found together show the frequency of potentially hundreds of ancient salt lake sites on the Martians surface. On Earth, previous studies (Perl et al. 2019) have utilized the north arm of the GSL as a site where halophilic microbial life, living in the water columns of the lake, has allowed for a transfer of biological/cellular material from lake to the evaporitic minerals. This process uses the hypersaline waters as the source for both the mineral precipitation and the transference mechanism (Figure 5). On Mars, these minerals from late-Noachian waters would have remained behind as the atmosphere dissipated over millions of years (Mancinelli et al., 2004). In addition, the halite and gypsum deposits on Mars are favorable to radiation protection that supports photosynthesis (Cockell and Raven, 2004). If life (as we don’t know it) were ever present on Mars during a time where water was stable on the surface and that cellular life would likely be halophilic and could tolerate the hypersaline waters. These microorganisms would have had the slow-changing geologic time to manage the osmotic stress as their once aqueous ecosystem dried up. Discussion Great Salt Lake as an Astrobiological Analogue for Halophilic Life The halophilic microorganisms, or “halophiles,” that thrive in the conditions described above teach us about the limits of life on Earth, and these lessons may be applied elsewhere in the Universe (Rothchild, 1990; Baxter et al. 2013, 2007). From environmental molecular biologic Perl, S.M. Biogenic Detection in Evaporites 27 studies, we know that the microbial communities in GSL are composed predominantly of halophilic archaea and bacteria (Baxter et al., 2005; Weimer et al. 2009; Parnell et al. 2011, Meuser et al. 2013; Tazi et al., 2014; Almeida-Dalmet et al., 2015; Boogaerts, 2015; Perl et al. 2019). Phototrophs power the system (Stephens, 1974; Lindsay et al., 2017), anaerobic activities are prevalent (Boyd et al., 2017), and methanogenesis has been detected (Baxter et al., 2005). The metabolism of these microbial communities, living at salt saturation, is complex, but such reactions occur more slowly than at lower salinity levels (Ward and Brock, 1978; Post and Stube, 1988; Stube et al., 1976; Fendrich and Schink, 1988). The pink color of the salt-saturated GSL north arm waters reveals carotenoid-containing microorganisms in abundance (Perl et al. 2019). Although there are bacterial and algal species that have these pigments, the consortia here are largely dominated by halophilic archaea (Almeida-Dalmet et al., 2015). These pigments underlie important strategies for overcoming the challenges of an extreme environment and also give us clues to potential biosignatures even after DNA has been lost to time. These same detection strategies can be critical for future astrobiology and planetary landed campaigns to determine not only the survivability of organics, which can be found with no relationship to biological processes, but to future life “as we don’t know it” mission concepts that since the Viking missions, we have not done yet. GSL halophilic microorganisms can survive high doses of ultraviolet (UV) light, desiccation of their environment, and osmotic challenges. These poly-extremophile microorganisms may be excellent life forms to study when considering a search for potential current or extant life in a Martian evaporite formation. Considering that the timescales of geological changes are magnitudes longer than the adaptation of halophilic and other extreme life, Perl, S.M. Biogenic Detection in Evaporites 28 survivability of biological evidence in ever-changing hypersaline settings can be both physical and chemical. Physical biosignatures in this case (e.g., pigments, organic layering, and fossilization) have the ability to be seen with the naked eye or a visual color image. Chemical biomarkers (e.g., highly variable and concentrated amino acids, hopanes, fatty acids, other long-chained macromolecules from life) however cannot be detected visually and their sole existence is not diagnostic of life but are significant alongside other validated evidence of a biological process, extinct or extant. Conclusions The global distribution of hydrated minerals, including Gyr-old sulfate veins, large gypsum deposits, and mm-scale evidence of soluble salts on Mars that are in close proximity to ancient aqueous settings from the late Noachian/early Hesperian could have been host to in-situ microbial processes/organic matter should it have ever existed within the water column of a Martian lake or evaporative setting. Ongoing experiments have taken these diagnostic Raman peaks from carotenoid biomarkers and their representative measurements to follow the preservation processes we’ve established in this investigation to determine the likelihood of biogenic preservation over geologic time such that assessments can be made for the modern Martian surface environment and the extent of biogenic degradation within evaporites detected on the shallow subsurface of Mars today. Acknowledgements I would like to thank Bonnie Baxter for her ongoing collaboration and research efforts for the Great Salt Lake and using it as a modern astrobiological analogue for ancient closed basin lake Perl, S.M. Biogenic Detection in Evaporites 29 systems on Mars. Bonnie’s initial introduction to the Spiral Jetty field site through colleague Olivia (Piazza) Paradis’s ooid research allowed for the initial sample collection and proof of concepts in 2014-2015 that verified that the GSL lake waters and the life residing in them can be transferred and protected inside the crystalline matrices. I would also like to thank Jaimi Butler for her huge assistance in in-situ collection during the 2 nd sampling trip. Jaimi has been a constant advocate for the Great Salt Lake and her work with Bonnie in the Great Salt Lake Institute has allowed for significant research collaborations that will be fruitful in the coming years. I want to thank Olivia Paradis for her help in my first GSL sampling trip in 2014 and her guidance regarding how (and why) the gypsum megastructures exist in such few spots in Rozel Bay. Over the years our overlapping field site locations have led to great conversations that helped shaped the interpretations of the local fluvial and geochemical environments. I would like to thank Arman Seuylemezian for his initial lab assistance when he was a JPL intern and now a planetary protection scientist. Arman was my co-investigator on the early proposals I led the initial proof of concept work into understanding salt inhibition and its quantitative differences just due to the Na ions present as part the evaporite mineral matrix. From an intern to being a full-time PP scientist in a short time, I have watched Arman grow and continue to use his scientific integrity, microbiological perspectives, and honest personality in the dynamic JPL environment with success through determination and forward thinking. I would like to thank Parag Vaishampayan for his insight into the initial aspects of this work during 2013-2014 when his guidance helped me build the SOPs for working around salt inhibition and the use of the PP laboratory space. I would like to thank my first official intern Preston Tasoff for his quick learning, determination, and perseverance in learning how to process 16S metadata for expanded analyses Perl, S.M. Biogenic Detection in Evaporites 30 from the GSL, the (purposefully) contaminated Boulby brine, and the (purposefully) contaminated Mono Lake samples. Preston’s curiosity drives his ability to fold new ideas into historically separate disciplines, which has led to his early academic career as both a microbiologist and a future geobiologist. 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High-level investigation space using terrestrial life (“as we know it”) and the loss of biotic information within biogenic preserved settings with respect to time. Perl, S.M. Biogenic Detection in Evaporites 51 Figure 2. Jezero Crater, Mars. One of the key sites where CRISM detections of sulfate salts, phyllosilicate clays, and other hydrated band (VNIR and IR wavelengths) minerals. Multiple orbiter spectrometer observations were utilized to generate this map on the backdrop of a HiRISE digital elevation map. Green and blue colors here show likely clay minerals while red-orange colored areas show sulfate salt mineralogy. CRISM scans of gypsum are shown to have triple peaks at wavelengths of 1.4nm, 1.9nm, and 2.4nm and separate IR laboratory scans of GSL gypsum minerals matched these peaks (from Vivano-Beck et al. 2014). In late 2018 this site was chosen as the landing site for the Mars 2020 rover mission landing site and primary science waypoint for their primary mission. Perl, S.M. Biogenic Detection in Evaporites 52 Figure 3. Boxwork structures of sulfate mineral veins (likely gypsum) nicknamed “Garden City” within Gale Crater (from April 2015). Calcium sulfate mineral veins located in Gale Crater, Mars. The boxwork structure, along with vein thickness may imply multiple late-diagenetic fluidic flow. Some of the lamination and dark textures suggest that at least some mineralization via fracture fills. Linear patterns occur broadly perpendicular to fracture walls, and are interpreted to represent epitaxial crystal growth, suggesting lower flow rates and fluid flow (Kronyak et al. 2015 AGU) Perl, S.M. Biogenic Detection in Evaporites 53 Figure 4. Adopted from Ehlmann and Edwards (2014), this global map and icons shows confirmed CRISM detections of hydrated minerals. While sulfate salts are of the most interesting for the investigations discussed in these dissertation chapters the suite of hydrated minerals include phyllosilicate clay minerals, chlorides, carbonates, and silica minerals. Perl, S.M. Biogenic Detection in Evaporites 54 Figure 5. Hypersaline brine geochemical pathways and notable locations (Adopted from Warren, 2010). An overview of the potential pathways brine chemistry can take based on salinity and chemistry. Searles Lake has a Ca and Mg-poor chemistry with Mg being much higher than Ca leading to Na-CO 3 - SO 4 -Cl brine chemistry. Conversely, the Lake Bonneville waters that led to the GSL were rich in Mg, Ca with also being greater than Ca leading to halite and gypsum precipitation from Na-Mg-Cl brine chemistry. Both of the modern field sites in this dissertation follow the Path III pathway. Great Salt Lake salt mineralogy follows the purple dashed arrows while Searles Lake follows the green arrows. Perl, S.M. Biogenic Detection in Evaporites 55 Chapter 3 Investigation of Experimental Incorporation of β-carotene in halite via Raman spectroscopy before and after UV-C exposure: Implications for Preservation of Biomarkers in Evaporites on Mars Abstract Features from mineral-microbe interactions in modern and former hypersaline settings can simultaneously act as both a marker for aqueous environments as well as signs of halophilic extant or extinct life. Evaporite minerals can capture and entomb organic matter within their intercrystalline and intracrystalline structure because they precipitate relatively quickly (nomenclature adopted from Schopf et al. 2012) and can further preserve metabolic processes within its fluidic structures. Thus, evaporite minerals constitute a target for biosignature investigation on Earth and Mars, where evaporitic deposits are known to exist. However, little is known about the process of organic preservation and detection in evaporites, or the stability of such molecules when exposed to significant UV radiation (as would be present on the surface of Mars). Here, we investigate the incorporation of β-carotene into halite via Raman spectroscopy by growing halite in the lab in the presence of know concentrations of beta carotene and examining the resultant crystals and fluid inclusions via Raman spectroscopy. Following evaporation, the experimental beta carotene-containing halite was exposed to UV-C to simulate conditions on the Martian surface. Results reveal that β-carotene (Figure 1) has a strong Raman signature that remains strong even when entombed in halite. In particular, fluid inclusions within the halite displayed particularly strong β-carotene Raman signatures. Little change was observed even after several weeks of UV-C delivery. Our results reveal that complex organic molecules like β-carotene should Perl, S.M. Biogenic Detection in Evaporites 56 be preserved well in halite (especially in fluid inclusions) and that halite does provide some protection from organic matter degradation via UV-C. Thus, evaporites constitute a good target for the search for biomarkers on Mars (Figure. 2). These findings will allow for proper criteria for the discovery of any potential physical biosignature and chemical biomarker that would be on active ocean worlds (Europa, Enceladus) and for future Mars subsurface roving or drilling missions. Introduction Terrestrial evaporite minerals like halite and gypsum can preserve biological products over geologic time and can provide micron-scale ecosystem for life to continue post-preservation (Perl et al. 2019). Halite and gypsum precipitation can incorporate fluid inclusions within the crystalline structures (Figure. 3) where biological matter can be preserved, potentially offering solar UV protection, desiccation protection from the outside environment, and shielding from potentially damaging enzymatic processes both chemical and physical. While we have studied these fluid inclusions in previous work (Perl et al. in-prep), their importance is also noted by others (e.g., Jehlicka et al. 2008; Roedder, 1984; Van den Kerkhof & Hein, 2001; Fendrihan et al., 2009; Winters et al. 2010). Isolated pockets of brine trapped in halite crystalline structures have been used to study the ancient environments of where fluids originated as well as microorganisms from ancient waters (Satterfield et al., 2005; Benison, 2006; Lowenstein et al. 2011; Perl, Cockell, et al. in-prep). The purpose of the work accomplished here are to put constraints on the robustness of the β-carotene carotenoid in the modern Martian UV-C environments. Should these validated signs of biological activity (and life) be observed in the Martian subsurface from mineral veins or buried Perl, S.M. Biogenic Detection in Evaporites 57 evaporite minerals, the threshold of detection will need to be understood. These experiments show how the Raman signal from abiotic carotenoid biomarkers can be detectable after significant and direct UV-C upon the carotenoid surface and while entombed within NaCl minerals. Moreover, these experiments will show where to look for these UV-C bleached physical biosignatures and how to decipher their presence while entombed within Martian-observed evaporite minerals. Methods All experiments and Raman analyses were conducted in the Mineral Sciences lab at the Los Angeles Natural History Museum (LA-NHM) β-carotene was mixed in with 500mL of UltraPure DNA-free water in three different volumes (Figure. 6). The molecular weight of β- carotene is 536.8726 g/mol. The “small” volume had 0.02684g of β-carotene powder (0.1mM), the “medium” volume had 0.07.068g of β-carotene powder (0.263 mM), and finally the “large” concentration had 0.16995g β-carotene powder (0.633mM). These three lab analogue samples were then distributed into four different solutions of DI water and the salinity was increased to speed up time for NaCl precipitation. The high salinity is meant to emulate Searles Lake (salinity is 39-41%, from Smith, 1979) and Great Salt Lake (salinity is 23-27%, from Baxter et al. 2007) and to go beyond the measured in-situ salinities of the investigation sites. After substantial NaCl precipitation at room temperature and separately at 70°C oven temperature, eight Petri dishes of laboratory salts were generated. Raman analyses were performed on all the samples prior to and after UV-C exposure to have the proper non-UV exposed baselines for comparison (Figures 7,8). After baselines were measured, the UV-C LED was placed in direct above a portion of the lab NaCl samples for 24 hours and for ~120 hours. The NaCl with the highest concentration of β- carotene was used for the UV-C studies to ensure the sample had a higher probability of being Perl, S.M. Biogenic Detection in Evaporites 58 modified by the UV diode. The other concentrations would have equal probability of being altered due to the UV-C but the highest concentration was used to ensure we had enough entombed β- carotene for the UV exposure observation timescales. Higher salinities (e.g., >44% salinity) than the natural waters were investigated and as discussed subsequently, resulted in breaking the carotenoid bonds, indicating that there is an upper limit for the preservation of β-carotene with respect to salinity (Figure 9). Moreover, and likely just as important, is that halite likely occurred on Mars in regions where water activity was at the highest. First, we will produce a baseline NRC- 1 halobacteria. Secondly, use these samples as a baseline itself for UV-C exposure experiments to quantify the breakdown of carotenoids emulating the modern Mars UV-C exposure that is currently occurring on the surface and though most of the Amazonian. To simulate the UV conditions on modern/present-day Mars the separate use of a UV-C LED and a UV hood were utilized for direct exposure of UV-C light onto lab precipitated NaCl samples containing different mM amounts of β-carotene. Bacterioruberin was not found to be available commercially so only β-carotene was used. β-carotene and bacterioruberin share very close major Raman peaks and only differ by 0.5cm -1 in some cases (Jehlicka et al. 2008; Winters et al. 2010). Prior to any UV-C exposure, understanding the entombment of the β-carotene carotenoid into the evaporite minerals in the first place is critical for linking the total preserved biomarker quantity to the mineral at the time of mineral precipitation. An “entombment volume” in this case is equal to the timing of mineral precipitation and source material (e.g., the carotenoid). Figure 10 shows the counter-clockwise evaporation pathways of NaCl brines that were in solution with three different mM concentrations (mentioned at the beginning of this Methods section). These were evaporated at room temperature and micron-scale trail of precipitated material leaves behind a ring of salts with increasing precipitated NaCl minerals along the evaporation pathway. Focusing on Perl, S.M. Biogenic Detection in Evaporites 59 the largest concentration, Figure 11 shows the heat map of a single Raman peak linked to the β- carotene carotenoid. The left side of figure 11 was taken from an optical image (Figure 11, left) and a concentration map overlaid on the 3-4 NaCl crystals (seen as black cubes in the optical image). The sections of the heat map in green correspond to the parts of the β-carotene carotenoid that are within the crystal. Red dots are β-carotene sections that are outside of the NaCl crystal (Figure 11, right). Figure 12 shows the 532nm Raman spectra that contributed to the heat map. Evaporation Studies of Abiotic Entombed β-carotene With respect to mineral precipitation and the evaporation of closed-basin lake systems on Earth and Mars it is imperative to show if there are preferences of β-carotene for eventual placement/movement into the fluid inclusions upon generation. It’s already observed that the carotenoid biomarkers are present in the mineral matrices where no fluid inclusions are present in addition to within the inclusions themselves. The pathways in which carotenoids can become entombed within the inclusions, and the preference for their placement in the inclusions over the solid evaporite mineral matrices is critical for understanding the highest probability of discovering in-situ biomarkers within evaporites on Mars using Earth’s terrestrial evaporite record. Abiogenic β-carotene was mixed within three brine solutions at different concentrations and the evaporation of the brines was studied optically and with Raman mapping set to the major carotenoid peaks that have been observed (Figure 7). Since there were no microorganisms as part of this portion of the study, all Raman peaks observed had to originate from the abiogenic β- carotene. The three different concentrations were evaporated and it was clear that the counter- clockwise (with respect to the figure) pathway of evaporation also brought large β-carotene and µ- scale precipitated NaCl crystals along with the pathway. Perl, S.M. Biogenic Detection in Evaporites 60 Search Strategies for Carotenoid Pigments and Spectral Interpretation In total, ~50 sections of pigmented β-carotene were optically observed and ~30 spectra were taken of those regions. To avoid photobleaching by the 532nm laser, lower power modes were used at times (0.1%, 1.0%, 3.2%, 10%) depending on pre and post visual comparison of the pigmented sections. Figure 5 shows the β-carotene powder prior to any UV-C interference and after exposure to UV-C for 24 and 120 hours. It was trivial to find a section of the β-carotene pigment prior to the UV-C phase of the experiment. Figure 5 also shows what it was like to find pigmented sections after UV-C has physically bleached the β-carotene powder. During the brine evaporation / NaCl precipitation phase of the experiments, the β-carotene powder that ended up entombed within fluid inclusions yielded the same magnitude peaks as when the powder was alone in the previous powder-only experiment. Results Raman analyses of the laboratory analogue β-carotene entombed NaCl samples matched the same peaks as the pigmented samples (collected in-situ) but without any visible pigment in the NaCl matrix. Measurements were taken in fluid inclusions from the laboratory precipitated salt crystals and observed in 10x, 40x, and 100x. The 100x also showed features of β-carotene taking up/filling a significant portion of the fluid inclusions when entombed. These features were the targets of the UV-C exposure to test how much the layers of the NaCl protected the signature β- carotene Raman peaks at 532nm. Figure 2 show the before and after images of the UV-C exposure on a section of carotenoid preserved in the laboratory salts. β-carotene is detectable in a fluid form and within varied concentrations, can still physically modify its fluid medium. Raman detection Perl, S.M. Biogenic Detection in Evaporites 61 peaks are able to be reproduced as the pigmented fluids, having lost much of its mm-scale red color after UV-C exposure. During the timeframe of the UV-C exposure, pigments can remain in the fluid inclusions formed as the lab-precipitated halite grows or can be concentrated into the inclusions and take up much of the space there. The salts at this point retain trace amounts of pigment but nothing compared to the host/master mixtures of β-carotene + DNA-free water. Once part of the evaporite matrix, these samples were then exposed to the aforementioned UV-C for 24 and ~120 hours. After what would be more than a full Earth day of constant and direct Martian sunlight with no atmospheric opacity (low tau value) a longer period of time was met with no chemical change to the remaining visual pigment spots, about 120 hours of the same UV-C was exposed on the same NaCl samples. After 24 and ~120 hours of direct UV-C exposure, green (at 532nm) Raman analysis was conducted on the specific red spots shown in Figure 2 and on sections that were now clearly “bleached” by the UV-C. It was clear that the direct UV-C diode had visually bleached most of the β-carotene in the original powder-only experiments (24 and ~120 hours) and in the lab- precipitated NaCl trails (24 and ~120 hours). However even though the pure red powder and light red fluid inclusions were bleached the 532nm Raman scans showed the same peaks of β-carotene in the NaCl samples as were in the in-situ samples from Great Salt Lake and Searles Lake (as discussed in Chapter 4). The amount of UV-C that was directly received by the β-carotene samples, both in powder form (without any crystal substrate) and in NaCl form, was higher than what is occurring presently on modern Mars. The UV-C diode does not emulate atmospheric opacity (tau), differences in solar UV-C based on time of day on Mars (sol), or for dust coverage all over the surface. The fact that the β-carotene peaks are the same in the red sections of the bleached NaCl samples as what was observed from the in-situ samples (see Figure 3 for comparison) raises the Perl, S.M. Biogenic Detection in Evaporites 62 bar for carotenoids as a highly important chemical biomarker and physical biomarker even in high solar radiation environments. High saline brines were created to speed up the process of precipitating salt crystals but to also show the same salinities of the Great Salt Lake (23-27%), Searles Lake (39-41%), and Boulby Mine brine aquafers (25-36%, so far from 12 brine pools visited in 2017 and 2018). The highest saline brine composed was ~44% salinity and that was used for the first β-carotene entombed samples. Upon Raman inspection, the typical β-carotene peaks that were expected were not present and what appeared to be peaks from the pigmented carotenoid were shifted to the left with a significant energy drop-off and a large peak toward the right of the wavelength spectrum. It was deduced after an extensive literature review that the high salinity from the ~44% brine actually broke the long-chained β-carotene bonds and what was being observed was their broken chains in solution. Jaramillo-Flores et al. (2006) note this in their study of carrot roots that carotenoid microstructures can easily break with high sodium chloride content. While their study was in food science and not in halobacteria production of β-carotene or laboratory precipitated salts and brines, the fact that this was only occurring in the highest saline content brine generated (without any biology producing any β-carotene in these samples) there was no biological response to the high saline solution. The data in Figure 3 also supports the survivability of β-carotene and pigmented salt minerals in hypersaline sites at ~44% or above (e.g., Dead Sea, Israel; Lake Vanda, Antarctica, Salar de Atacama, Chile). Having the features of life adapting to such high saline settings and producing β-carotene chains that would be able to withstand the stress to the carbohydrate matrix structure. Figure 10 shows the three different concentrations pathways (in the counter-clockwise direction). It should be noted that the highest β-carotene concentration had more opportunity for Perl, S.M. Biogenic Detection in Evaporites 63 entombment within the fluid inclusions whereas the lowest concentration had the smallest amount of organic material to utilize. Figure 11 shows a comparison between a set of NaCl hopper crystals under normal optics and a Raman map of that same region. The green dots represent the presence of the β-carotene peaks and the red shows areas where there are no carotenoids. This relationship between the inclusions and the dynamically entombed β-carotene is clear evidence that as the evaporites are precipitated from brine and/or closed basin lake system, should there be any organic particles that are produced from in-situ microorganisms. Their entombment is highly likely and that the highest probability of their preservation would be in the trapped fluid inclusions within the mineral (intercrystalline preservation) evaporite matrix. Figure 11 shows the map data (from Figure 12) in spectral form over several dozen spectral points as the crystals were formed and the brines were evaporating. The map data alongside the spectral count data shows the direct relationship between the carotenoid preservation and the entombed fluid inclusions during evaporite precipitation. Discussion The future targets for in-situ Martian evaporite and sulfate mineral vein analyses need to focus on signs of fluid both on the vein surface (e.g., vertical sediment features aligned with ancient fluids) as an indication of where within the evaporite or mineral vein (that contains evaporitic material). Within those features mm-abrasions of said vein could reveal microtextures that were formed from fluids. It is in these features that fluid inclusions should be the primary target for Mars Sample Return (MSR), in-situ analyses, and biogeochemical analysis. Current MSR plans do not call for the preservation of microtextures and, in turn, fluid inclusions from inside those textures. However, the next rover will have a Raman on its payload and hopefully it will be utilized in this manner. Perl, S.M. Biogenic Detection in Evaporites 64 Molecules from ancient preserved fluids can provide clues about extinct life on early Earth or potentially on other space bodies. Which biological molecules can be preserved and for how long? Modern GSL studies in halite and gypsum will help us identify the parameters for preservation in minerals (Perl et al., 2016, 2019). Consideration should be made for the stability of each type of molecule and the environmental solar radiation exposure over time (Kminek et al., 2003; Fendrihan et al., 2009). There has been much controversy over the study ancient biomolecules and many former works into the ancient DNA of halophilic bacteria. This type of scrutiny is justified due to the high probability of contamination from the natural environment (i.e., the minerals not providing a true enclosed/protected environment, leading to “younger” bacteria and archaea contaminating geologically older minerals) and cross-contamination between samples (i.e., the spread of the dominant OTUs to other features in the micron-scale settings observed in the crystal structure). Of note are the technical problems including contamination have been noted (Pääbo et al., 2004; Hebsgaard et al., 2005). Of particular concern are analyses of environmental samples utilizing amplification methods such as the polymerase chain reaction (PCR) (e.g. Fish et al., 2002) or microbial cultivation techniques (e.g. Vreeland et al., 2000), which have the caveat of contamination possibilities. Significant issues here also relate to the preservation of extinct vs. extant life and the issues behind cultivation of “ancient DNA” in ancient mineralogy. Other studies may avoid this with extensive surface sterilization (Sankaranarayanan et al., 2011) or by employing more direct methods like electron microscopy of the fluid from the inclusions, coupled with biochemical assays to identify the molecules (Griffith et al., 2008). Perl, S.M. Biogenic Detection in Evaporites 65 Raman Detections of β-carotene within precipitated evaporites, and UV-C doped salt minerals Throughout the course of these investigations the validation of biogenic preservation has been the centerpiece of the motivation. The purpose of the work described here has isolated the carotenoid pigments of β-carotene into fluids, precipitated NaCl crystals, and exposed these crystals to the modern Mars equivalent of several days of UV-C to try to visually and chemically modify the evaporite structure. These laboratory-precipitated crystals have been partially stained by the β-carotene pigments without the “biogenic factory” of preserved halophilic microorganisms generating these carotenoids, thus creating a more tractable experimental system from which to investigate biosignature entombment. Figure 2 highlights the visual changes on the µ-scale to the entombed β-carotene within the fluid inclusions and to the matrix itself. Bleaching was observed to the majority of the former red pigment, however there are still tiny dot-sized amounts of pigment that does not become visually altered. This UV-C dosage was after the equivalent of ~two weeks of direct Martian sunlight with no cloud cover and atmosphere, albeit thin on Mars, as interference to limit the UV-C exposure. The difference between the visual alteration (the loss of the majority of the physical biosignature to the naked eye) and the robustness of the chemical biomarker here is extremely significant for in-situ sample analyses of surface evaporite minerals and vein mineral boxwork structures on Mars. The key feature here is that even after several days of UV-C, these bleached features still show small sections of unaltered β-carotene that remain intact with respect to intensity/counts as observed by Raman. In addition, while these are albeit smaller than from the original prior to any UV-C, the diagnostic pigment regions that yield the same wavelengths as the in-situ samples with biology entombed within. Figures 3-5 show the process of the laboratory precipitated salts and their ability, along with the carotenoid pigments, to remain robust after significant UV-C bleaching to the powder, then to the NaCl matrix containing the entombed Perl, S.M. Biogenic Detection in Evaporites 66 pigmented waters. Should any independent Martian life have evolved to the point to be able to react and photoprotect itself via biomodification of evaporites, these would be the turning point in biogenic preservation and discovery as well as designing future deep-subsurface missions to Mars around these evaporite preservation features. Given the near constant, global dust covering on Mars, pigments would be more visual/physical away from the hostile surface of the planet in the subsurface. The key feature of the evaporite minerals is the presence of evaporating late Noachian/early Hesperian fluids where via groundwater downwelling may have provided life (as we don’t know it) the benefit of a natural Petri dish for photoreactive processes as the global climate and atmosphere of Mars was slowly changing. As discussed in the next and last chapter of this dissertation, biological processes, given the proper environment, climate, energy, and fluid availability, act much faster than geological processes. The former Earth-like setting of Mars ~3.5 Gyr and overlap of the aforementioned conditions could have yielded a proper starting point for independent life on Mars that had the chance to migrate into the shallow-to-deep subsurface as the planet lost its surface water stability. The pigments generated by life in these terrestrial hypersaline closed basin lake systems would still have the opportunity to evaporate and perhaps modify the salts and regolith on Mars in similar fashion to the Searles Lake Pleistocene field site. If this were the case, a Martian deep subsurface mission to ancient buried fluvial sites would be imperative for the future of Mars astrobiology. Evaporite mineral changes that are biogenic (pigments) vs. abiotic (precipitation) are observed together but are assessed independently. Due to halite (NaCl) being Raman transparent due to the Rule of Mutual Exclusion, sodium chloride is an ideal mineral for these experiments. Perl, S.M. Biogenic Detection in Evaporites 67 Conclusions In conclusion, the physical preference of the β-carotene carotenoid to become entombed within the sodium chloride mineral structure yields a significant motivation for these chemical structures to not only be a part of the evaporite post-ancient Martian lacustrine evaporation, but to be preserved even after large doses of UV-C. Targeting of these carotenoids within salt structures further preserves these typical biomarkers over geologic time periods. The clearly visible pigments that can be seen with the naked eye (as well as a rover’s camera after surface dust removal). This is the first step for both terrestrial discovery in the field and for Martian astrobiology after care has been taken to remove surface dust. While on the surface, and likely, after the ancient Martian river deltas started to evaporate in the late Noachian, these minerals would have precipitated out. Should Martian life have existed during this timeframe and should it have the biological adaptation abilities for UV protection, it could have created the same type of solar flux protection we see on Earth. If these three factors were in place when surface waters were stable on Mars then the starting source preservation would have had a high likelihood of modifying the evaporite mineral matrix to preserve a carotenoid-like structure and the pigments generated therein. The fact that the β- carotene carotenoids can withstand directed UV-C both in the raw form (the powder) and the entombed form (within NaCl crystals) shows that these biomarkers are robust enough to be geologic, and more accurately, geobiological markers over geologic time. The clear pathways for β-carotene carotenoids to be preserved in the fluid inclusions now provides an optimal in-situ structure in which to search for validated biomarkers on Mars. The aforementioned controversial studies all have one thing in common – they are trying to prove the absolute age of the biomass as a function of the age of the mineral substrate. While this endeavor is important, it subtracts from the preservation attributes of the evaporite minerals and constitutes Perl, S.M. Biogenic Detection in Evaporites 68 a temporal roadblock that may not exist on Mars. Should future robotic in-situ subsurface missions be able to extract buried salts and evaporite veins that are far from the surface UV-C (or if the evaporite vein is large enough where it hinders the damaging UV-C from any potential Martian biomass), their largest concern will be proving that it is not terrestrial contamination. After and only after the proper burden of proof is established for showing that the Martian organics (or biomass / biological matter) then can the analysis of the relative or absolute age of the discovered in-situ biomass take place. The unnecessary portion of this type of work is due to the dynamic properties of life and biological processes. The failure of the previous studies was that they did not take into account that the source of the biology and its age is the higher priority rather than proving that it was geologically old. The example comes from the 1.2km buried Boulby brines that have been observed. Should those saline fluids have been buried and untouched by mankind since the formation of the Zechstein Formation ~255 Myr ago then the microorganisms since that time would have evolved differently than their surface counterparts, differently from closed aquafers that are buried far away from other aquafers (potentially having different evaporite chemistry and kinetics from the NaCl vs. the KCl vs. the polyhalite). The ecological differences in those separate evolutions would have the largest delta compared to surface microorganisms ponded at the same time as the formation of the Zechstein evaporites. The different m-to-km-scale layers here illustrate the different chemistries, reduction and oxidization pathways that biology could have taken. It is interesting to be able to relatively date these but it is more interesting to be able to prove the evolution differences here all coming from the original Zechstein Sea ecology and how the common ancestor here spawned likely hundreds (if not more) evolutionary pathways. Showing those differences via molecular clock analyses and how those could be preserved in the evaporite matrixes would intern provide a set of temporal constraints to indeed prove that these are not only Perl, S.M. Biogenic Detection in Evaporites 69 different but they are product of their buried environment and ecology. This type of comparative molecular analysis can be done on Martian subsurface regolith and evaporite veins should the future robotic sample return mission yield enough of a distribution of sample depth and geological time but in the future with human-led missions to Mars the prospect of drilling down into ancient Noachian and Hesperian crusts and evaporites where surface waters have seeped into the subsurface would provide an even higher probability of organic and biological entombment and the prospects of discovering potentially extant life on the planet would increase significantly, if a Martian Last Universal Common Ancestor (LUCA) were present. Acknowledgements I would like to thank Aaron Celestian for his continued collaborations, partnership in research since his arrival at the Los Angeles Natural History Museum, and expertise in mineralogy and crystallography. Having met Aaron while I was an undergraduate at SUNY Stony Brook and he a doctoral student in the Department of Geosciences, we were able to quickly start up our research together based on our overlapping interests in geobiology and microbial pigments and preservation. Aaron has been critical in the evaporite analyses and with him and his laboratory as the epicenter for many of the scientific contributions that are discussed in this dissertation and accompanying manuscripts to Astrobiology and other related journals. As this work and its spin- offs continue, Aaron and I will proceed with our exciting upcoming proposals and work efforts from new field sites and updated research questions. In addition, I will try to not leave my salt samples and brine dishes all over the NHM lab! Although it does make for good conversations with visitors and a reminder of work still left to do. Perl, S.M. Biogenic Detection in Evaporites 70 References Bada, J.L. (2001). 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Carotenoids are usually classified between carotenes or carotenoid hydrocarbons. β- Carotene is a carotene carotenoid and, along with Bacterioruberin, can protect cells from oxidative damage, fatal UV irradiation injury to the cell structure, DNA damage resulting from radiography, and high doses (5 kGy) of gamma irradiation (Rodrigo-Baños et al. 2015). Bacterioruberin is one of the most abundant C50 chain in halophilic microorganisms. β-Carotene pigments in biomass can be used to measure density of halophilic communities due to their formation when solar flux is high (i.e., summer months or desert environments). (A) (B) Perl, S.M. Biogenic Detection in Evaporites 80 Figure 2. Optical observations at 10x and 100x showing original halite hopper crystal and microbial movement within fluid inclusions entombed within the crystal structure. (A) Entire halite cubic crystal showing rectangular fluid inclusions on all four faces with an air bubble on the left face. Scale bar is at 100μm. (B) Enhanced image of (A) showing fluid inclusion patterns within cubic halite habits. Scale bar is at 100μm. (C) 100x image highlighting a fluid inclusion within a single crystal. Red arrows show microbial movement. Green targeting dot highlights an entombed biomass. Scale bar is 2μm. (D) Another 100x image showing a larger fluid inclusion (when compared to (C)) with increased microbial movement within the fluid inclusion. Scale bar is 2μm for 2c,d. The fluid inclusion is within the green dashed line in the red arrow example but can also be observed on several “z” planes above and below the current fluid inclusion in focus. (C) (D) (A) (B) Perl, S.M. Biogenic Detection in Evaporites 81 Figure 3. (A) Raman map of β-carotene at 1152cm -1 superimposed over the 40x visible light microscopy image of entombed fluids within a halite crystal face. Warmer colors represent higher volumes of carotenoid compounds at this single wavelength. Scale bar is 20μm. (B) The same halite crystal with a β-carotene “heat map” at the 1152cm -1 wavelength showing where the carotenoids are concentrated the most near the fluid-crystal interface. Scale bar is 5μm. The warmer colors (red, orange) show the highest of concentrations of β-carotene with the black background showing none of these features. Scale bar is 5μm. (B) (A) Perl, S.M. Biogenic Detection in Evaporites 82 Figure 4. Abiotic laboratory precipitated NaCl with varying amounts of entombed β- carotene. (Top-L) Fluid inclusions as viewed from the 40x objective showing small dark red sections of β-carotene entombment from the “large” carotenoid sample (0.633 mM). (Top-R) Fluid inclusions as viewed from 40x showing almost no β-carotene from the “small” carotenoid sample (0.1 mM). (Botom-L) 100x objective perspective of an inclusion with no visible carotenoid. (Bottom-R) 100x objective from the same crystal as the image on the left but with entombed β-carotene. Scale bars for the Top-L and Top-R are 20µm. Bottom-L and Bottom-R scale bars are 2µm. Perl, S.M. Biogenic Detection in Evaporites 83 Figure 5. Abiotic laboratory precipitated NaCl with varying amounts of entombed β- carotene under UV-C conditions. (Top-L) Reference β-carotene prior to any UV-C exposure. (Top-R) β-carotene after 24 hours of UV-C exposure. Scale bars are 100µm. (Middle-L) “Bleached” β-carotene in solution. (Middle-R) Fluid inclusion after 24hrs of direct UV-C exposure. Raman observations in the following figure were conducted on the remaining red- colored sections. Scale bars are 2µm. (Bottom-L,R) Two sections of β-carotene after ~120hrs of UV-C exposure. Scale bars are 2µm. Perl, S.M. Biogenic Detection in Evaporites 84 Figure 6. β-carotene entombment process without biology. (Top Row, Figs. 6a-c) Mixtures of β- carotene (molecular weight = 536.8726 g/mol) in three different “small”, “medium”, and “large” concentrations as listed in the Methods section. The small volume had 0.02684g of β-carotene powder (0.1 mM), the medium concentration 0.07068g of β- carotene powder (0.263mM) and the large a concentration of 0.16995g (0.633mM). All β- carotene was mixed into 500mL of DNA-free water to ensure no biology or biological processes took place. (Bottom Figs 6d-f) The precipitated NaCl crystals of the three concentrations of β-carotene. Small is 6d from 6a; medium is 6e from 6b; large is 6f from 3c. Figure 6g is the control with no β-carotene powder. (6a) (6b) (6c) (6d) (6e) (6f) (6g) Perl, S.M. Biogenic Detection in Evaporites 85 Figure 7. Raman observations of β-carotene from fluids in Figs 6a-6c (within DNA-free water) and 6d-6f (precipitated NaCl). Green Raman (532nm) observations of β-carotene mixed within DNA-free water are shown above as the light blue (Fig 6a, large concentration), green (Fig 6b, medium concentration), and black (Fig 6c, small concentration). Using these same pigmented fluids and the addition of sterile NaCl from Sigma-Aldrich halite crystals were precipitated and re-analyzed with 532nm Raman (see Figs 6d-6f). The large concentration β-carotene NaCl is shown as the purple spectra (Figure 6f), the medium concentration β-carotene NaCl is shown as the gold spectra (Figure 6e), followed by the small concentration β-carotene NaCl shown in pale blue (Figure 6d). It should be noted that these ~7 major peaks are the same for the in-situ Great Salt and Searles Lake samples that have biogenic preservation where pink and red pigments have resulted from photobiological responses to the solar UV-A and UV-B and preserved in the evaporite NaCl crystal matrix. Note that the peaks >2000cm -1 are from a harmonic reflection that occurs when the vibrational magnitude of the “real” peaks are high enough to cause a self-reflection in the detector. = β-carotene (0.02684g / 0.1 mM concentration) = β-carotene (0.07068g / 0.263mM concentration) = β-carotene (0.16995g / 0.633mM concentration) = β-carotene entombed halite (0.02684g / 0.1 mM) = β-carotene entombed halite (0.07068g / 0.263mM) = β-carotene entombed halite (0.16995g / 0.633mM) Perl, S.M. Biogenic Detection in Evaporites 86 Figure 8. Raman observations of β-carotene embedded within laboratory-precipitated NaCl after 120 hours of UV-C exposure. The seven major spectral peaks are after direct UV-C exposure from a LED onto the surface of entombed β-carotene. These spectral peaks shown above match the seven quantified peaks (1001.2, 1145.3, 1502.9, 2149.1, 2289.8, 2504.0, 2641.6) from the highly pigmented Searles Lake samples with the first three being the strongest and most visible ones from Great Salt Lake (1000.2, 1150.1, 1507.0) with the latter four being barely noticeable at first (2149.9, 2296.2, 2506.5, 2649.0). Note that the peaks >2000cm -1 are from a harmonic reflection that occurs when the vibrational magnitude of the “real” peaks are high enough to cause a self-reflection in the detector. = β-carotene after ~120 hours of UV-C (small conc.) = β-carotene + NaCl after ~120 hours UV-C (medium conc.) = β-carotene after ~120 hours of UV-C (medium conc.) = β-carotene +NaCl after ~120 hours of UV-C (large conc.) = β-carotene after ~120 hours of UV-C (large conc.) Perl, S.M. Biogenic Detection in Evaporites 87 Figure 9. Raman observations of β-carotene embedded within laboratory-precipitated NaCl within greater salinities than in-situ Searles Lake brine. In generating the lab salts in the previous figures the abiotic brine that was made was supersaturated with NaCl and made saline amounts higher than Great Salt (23-27%) and Searles Lakes (~39%). This brine had salinity of ~44% and caused the long-chained β-carotene macromolecules to break (black spectra). The original β-carotene peaks for GSL is shown here in blue. This provides an ecological and in-situ salinity control on how β-carotene can exist in the environment. Moreover, the broken β-carotene chains are still Raman active and still are able to vibrate at the same 532nm wavelength that have been used for quantification previously. = β-carotene + NaCl = β-carotene + NaCl from 44% salinity brine Perl, S.M. Biogenic Detection in Evaporites 88 Figure 10. Evaporated β-carotene brine solutions showing preservation pathways into fluid inclusions. Large (6a), medium (6b), and small (6c) β-carotene concentrations from evaporated brines showing that evaporation occurs in a counter-clockwise fashion with higher β-carotene particles (in fluid inclusions and in free brine) getting push all into one direction. (7a) (7b) (7c) (7a) Perl, S.M. Biogenic Detection in Evaporites 89 Figure 11. Optical image and Raman map of β-carotene concentrated within fluid inclusions during brine Evaporation. While brine solutions were evaporating (see Fig 7a) a section of the brine droplet was focused on for the precipitation of µ-scale NaCl crystals and then for fluid inclusions (left image). During the evaporation process it was observed that β- carotene was contained solely entombed within the fluid inclusions (green dots, right image) via a Raman map at the three peaks that β-carotene has been observed throughout this investigation. Perl, S.M. Biogenic Detection in Evaporites 90 Figure 12. Raman analyses of several β-carotene-entombed fluid inclusions during the complete brine evaporation process. During the large, medium, and small β-carotene brine evaporation experiments, several map spectra were taken to monitor any changes to the magnitudes of the β-carotene measurements. This was to ensure that we were monitoring the β-carotene from the initial brine deposition all the way to the complete evaporation where β- carotene particles (within fluid inclusions and in free fluid) ended up at the end of the process (Figure 7). Perl, S.M. Biogenic Detection in Evaporites 91 Chapter 4 Evaporitic Preservation of Modern Carotenoid Biomarkers and Halophilic Microorganisms in Martian Analogue Hypersaline Environments Abstract Modern closed basin lake systems produce evaporite minerals that form from lake waters during evaporation. Since hypersaline waters can host microbial life, it is possible that biomass from the lake could be transferred to the minerals, making evaporite minerals a target for the search for life elsewhere. On Mars, sulfate salts and other evaporitic minerals has been observed globally across the planet where late Noachian/early Hesperian surface waters once were stable and present for significant durations over Martian geologic time. Here, we investigate evaporite minerals and the waters from which they precipitated to assess their ability to trap and potentially preserve biomass from the hypersaline lake environment. On Earth, these hypersaline systems can be host to halophilic microorganisms where overall microbial diversity is low but salt tolerance is high. Minerals precipitated from these regions can transfer organic and biological material from the lake system (e.g, the water column and microbes from the photic zone). Should these terrestrial systems start to dry up, leftover minerals from previous waters can be a record of past halophilic life and, after preservation occurs, fluid inclusions within these evaporites can maintain the metabolic processes once carried on in the lake system. Hydrated minerals on Mars are found globally with their locations between the northern lowlands and the southern highlands. These mineral signatures, when found within depressions from craters or other ancient sign of aqueous activity (e.g., deltas, alluvial fans), are ideal for in- Perl, S.M. Biogenic Detection in Evaporites 92 situ analysis of former mineral-water interactions and will be targets for future life detection missions. Introduction The preservation of biomarkers and biosignatures depend on the source biological matter and the ability for the host evaporite mineral to maintain the original biogenic environmental settings, a solvent (for these samples this is saline and hypersaline water) which act as the media for biological processes, and emulation of their original environment. These features are found in both Great Salt Lake (Figs 1,2) and Searles Lake (Figure 3) such that the chemical biomarkers (e.g., carotenoids) as well as their associated physical biosignatures (e.g., visible pigments) match features found in their source lake/water column settings. Furthermore, the subset of these biogenic features, if found after the original aqueous setting has fully evaporated, can act as a record of bacteria and/or archaea that were once prevalent in that setting. What were the quantifiable metrics of biogenicity between the ancient lake settings and the minerals? Could these features be used to assess in-situ physical biomarker(s) that were a product of microbial activity? How long-lasting are these features in their current environment and would these sustainable over geologic time? Would these features survive from the late Noachian/early Hesperian timeframe onwards to modern day Mars? On Mars the limited availability of groundwater after evaporite precipitation near present- day dried lakebeds could allow for stability of highly soluble evaporites to remain physically intact. Secondary mineral precipitation can also play a role if post-evaporation fluids are at low enough volumes as not to dissolve the primary mineralogy likely in close proximity to evaporating lakebeds. The volume of water, water chemistry, and salinity are the major controls on the type Perl, S.M. Biogenic Detection in Evaporites 93 and size of evaporite minerals precipitated. Evaporite minerals are uniquely suited for both informing the former (ancient surface waters) and modern aqueous (closed basin lake systems) activity of a region and for entombing sediment, clays, and cellular life in its crystal substrates via fluid inclusions (Figure 4) providing latter with enough energy to maintain homeostasis and nutrient cycling. As Mars lost the ability to have water remain stable on the surface the original sulfate salts and phyllosilicate clays that were formed by fluid evaporation and water activity, respectively, would have not been modified by later aqueous or groundwater events. This investigation shows in terrestrial closed basin lake systems that the mineral-microbe relationships can show that the evaporite mineralogy are an excellent preserver of biological matter due to the commonality of microbial-rich lake settings and those same fluids being utilized for evaporite precipitation (i.e., the same fluids that are evaporating to precipitate the mineralogy also contain halophilic microbial communities inhabiting the water column). Of course, this is not to say that all evaporite minerals contain microorganisms, but having preserved records of ancient water activity in rover-accessible settings would be vital for future sample return mission decisions and in-situ analysis. This also intuitively shows the direct relationship between the medium of preservation and the ability to “protect” its entombed organic contents for an extended period. Summons et al. (2011) and the Biosignature Preservation Working Group assessed several early environments in the context of ancient Mars to determine which settings had the likelihood of supporting organic matter formation both abiotically and biotically, concentration/preservation of those organics, and the probability of in-situ discovery within geomorphic, mineralogical, and stratigraphic contexts. This assessment ranks lacustrine settings “high” for relative preservation ability, pointing to the significance of our study sites. Perl, S.M. Biogenic Detection in Evaporites 94 The preservation of chemical biomarkers in the form of carotenoids sourced from the activity of halophilic microbes constitutes the target of investigation for this study. Carotenoid pigments have been analyzed via Raman spectroscopy and compared to reference microorganisms and literature and show overlaps to biogenic pigments that have been preserved in evaporite minerals in the field sites discussed here. Links between overlapping Raman spectra from vastly different field sites tell equal parts of a story between mineral preservation and microbial survivability over time. Moreover, should evaporite precipitation occur over longer periods of time, trace mineralogy and other minor minerals can also be entombed or formed within the same evaporitic substrate. Mars contains a global record of these hydrated minerals including phyllosilicate clays, carbonates, sulfate salts, and a lack of plate tectonics, making these in-situ minerals more than likely in the very same regions where ancient late Noachian / early Hesperian waters once flowed creating massive river deltas, closed basin systems, modifying primary minerals via physical and chemical weathering. More significantly, the loss of stable surface waters provided several sweet spots where primary evaporite minerals were formed and later not dissolved or modified further. The loss a water cycle on Mars on a global scale could have provided these preservation features across the planet in the best possible spots to search for ancient life. Evaporite Pigments as Both Physical Biosignatures and Chemical Biomarkers Carotenoids are usually classified between carotenes or carotenoid hydrocarbons. Two of these, β-Carotene (C40H56) and Bacterioruberin (C50H76O4) can protect cells from oxidative damage, fatal UV irradiation injury to the cell structure, DNA damage resulting from radiography, and high doses of gamma irradiation (Rodrigo-Baños et al. 2015). Bacterioruberin is one of the most abundant C50 chain in halophilic microorganisms. β-Carotene (C40) pigments in biomass can Perl, S.M. Biogenic Detection in Evaporites 95 be used to measure density of halophilic communities due to their formation when solar flux is high (i.e., summer months or desert environments). These carotenoids yield a diagnostic Raman signature under 532nm (green) such that they can be distinguished from any mineral background. Halite, because of the principle of mutual exclusion, is transparent (not Raman active) and allows for direct detection of these carotenoid and their pigments. Moreover, extracted DNA from the lake water can be used to show the source of these carotenoid signatures. Methodology Roadmap The fact that halophilic microorganisms produce carotenoids in hypersaline systems and they are preserved in evaporite minerals is the main motivation for this investigation. In the hypersaline systems, we can explore the amount of organic matter, with carotenoids as a proxy, is transferred and preserved into the evaporite mineral systems after hypersaline brine evaporation. The following sections will discuss: • Sample sites and reasoning for their section (e.g., mineralogy, ecosystem) • The types of in-situ and laboratory analyses conducted for investigation of entombment of halophilic microorganisms (optical interpretations, Raman observations of evaporite features, cell motility). • Microbial community preservation observations (DNA extraction and salt inhibition workarounds, and verification of extraction protocols). Features that are physical biosignatures (e.g., visible pigments, cell motility) are assessed alone and together from chemical biomarkers (e.g., carotenoid abundance, DNA). Perl, S.M. Biogenic Detection in Evaporites 96 Methods Modern Evaporite Sample Site – the Great Salt Lake (Rozel Bay, Utah, USA) The Rozel Bay section of the north arm of the Great Salt Lake (GSL) contains halite terraces and vertically-precipitated gypsum crystals that are found southwest of Spiral Jetty art structure and eastward, away from the lake-shoreline boundary (Figure 5). A total of 58 samples were collected from seven sites along the shoreline where halite precipitation had occurred and was preserved (e.g., not subsequently dissolved during lake level change). Emergent halite hopper crystals, halite hopper crystals submerged in the ponded fluids in the terrace, fluids from underneath the terrace, and a soil beneath the fluids were collected at each site. Samples were collected using sterile techniques and samples were placed in 50ml Falcon® tubes with the larger lake water samples being collected in Nalgene © 1000ml bottles. All tubes and bottles were autoclaved prior to usage and gloves were worn and changed when needed during in-situ collection. Raman Analyses for lake waters and minerals Raman spectroscopy was conducted at the Los Angeles Natural History Museum on micron-scale features within selected sections of the evaporite minerals and fluids to determine baseline mineral peaks via green (532 nm) and near infrared (785 nm) Raman laser scattering and photoprotective responses as a function of pigment. Halite is a Raman inactive mineral, and thus observations provide us with a clear window into the biogenic preservation within. Water samples, mineral fluid inclusions, and mineral lacking fluid inclusions were analyzed from Great Salt Lake (Figures 1,2, and 5) and Searles Lake. Perl, S.M. Biogenic Detection in Evaporites 97 In addition, we observed Raman spectra matching β-carotene and bacterioruberin in Halobacterium sp. NRC-1 as a single-source halophilic microorganism (Figure 10) and references that have used this same microorganism (Fendrihan et al. 2009; Jehlička et al. 2014; Winters et al. 2013). Three Raman peaks were observed in all fluid inclusions from the Great Salt Lake and Searles Lake samples. The magnitude (e.g., Raman intensity) of these peaks denote the volume of the carotenoids that are being preserved in the inclusion. The first three peaks (~1000.2, ~1150.1, and ~1507.0) are the diagnostic peaks for β-carotene and bacterioruberin as tested from NRC-1 per the above references. Jehlička et al. (2014) goes into detail with a table of organics and their Raman intensity (weak “w”, moderate “m”, and strong “s”) and provides reference for both β- carotene and bacterioruberin. However, these carotenoids also share many of the Raman bands peaks (1000 cm -1 vs. 1001 cm -1 , 1155 cm -1 vs. 1152 cm -1 , and 1505 cm -1 vs. 1524 cm -1 ). More of the third pair of peaks correspond to the β-carotene’s 1505 cm -1 value but in some samples the ambiguity between 19 cm -1 leads to the conclusion that these preserved carotenoids are both contributing to the Raman spectra. Moreover, bacterioruberin tends to be more common among halophilic bacteria but both β-carotene and bacterioruberin are responsible for the pink pigment gradients in the field site studies – lighter pink in the Great Salt Lake samples and dark pink to dark red in the Searles Lake samples. These include the NaCl samples and the bodies of water. Sample Location Description and Tested Evaporite Features Great Salt Lake halite and brine samples were collected in varying wind flux and lake level environments from May 2014 to February 2016. DNA extraction was performed on solid samples of halite (pigmented and non-pigmented), gypsum (containing clays and clays removed), lake waters, and environmental controls for both halite and gypsum evaporite sets. Average weights for the pigmented and non-pigmented halite were ~50g, gypsum without clays were ~20g, gypsum Perl, S.M. Biogenic Detection in Evaporites 98 containing clays were ~9.4g. Lake water samples were 250ml while halite terrace fluids (trapped in-between crystal hopper macrotextures) was 100ml. Finally, halite and gypsum environmental controls (soils beneath the evaporite terraces and megastructures, respectively) was ~0.5g each. All of the DNA descriptions and data can be found in the Appendix and Appendix Figures. Results Halobacterium sp. NRC-1 To ensure we were able to isolate the β-carotene and bacterioruberin carotenoids we employed the use of the Halobacterium sp. NRC-1 (Figure 10) also known as strain ATCC- 700922, as a single-source halophilic microorganism for reference (Fendrihan et al. 2009; Jehlicka et al. 2014; Winters et al. 2013). NRC-1’s Raman spectra has all the major peaks that the Great Salt Lake samples have < 2000 cm -1 in addition to the Searles Lake samples that have all the major peaks spanning from ~1000 cm -1 to ~2700 cm -1 that contains the span of all observed carotenoid peaks thus far. Winters et al. (2013) and the three aforementioned manuscripts in this paragraph have been utilized to show Raman spectra of carotenoids up to ~2000 cm -1 but do not report values greater than that wavelength. Raman Spectroscopy - Lake Water The modern Great Salt Lake samples contained strong peaks at 1000cm -1 , 1150cm -1 , and 1507cm -1 correspond to the β-carotene and bacterioruberin carotenoids (Figure 7). The peaks from carotenoids have a high degree of peak overlap therefore making it difficult to distinguish if both are present in the same in-situ spectra (Figs 7-9). These peaks were observed in strong-to-moderate bands along with pigmented sections and observed in all halite samples across all seven sites at the Spiral Jetty (in Rozel Bay) in the northern Great Salt Lake. Weaker versions of these peaks Perl, S.M. Biogenic Detection in Evaporites 99 were found in the lake waters in addition to apparently non-pigmented halite. To ensure proper sample comparisons, fluid inclusions were largely targeted since that was where the majority of microbial cell movement and pigment was concentrated. Deeper pink pigments (visually) were found in Searles Lake and associated higher intensities of the carotenoid peaks were found in all analyzed samples. Raman Spectroscopy - Carotenoids in Fluid Inclusions Figure 8 shows two types of Raman peaks. The first category are the real Raman vibrations that are showing the carotenoid peaks from halophilic microorganisms. The second category are harmonic overtones that are generated due to the high volume of the original / real vibrations. The mathematical reasoning for these overtones is explained in the following sections. The Raman peaks from biogenic preservation are shown as 1000, 1150, and 1507 cm -1 for both the Great Salt Lake and Searles Lake samples. In addition, the OH stretch for water is shown at ~3340 cm -1 . Raman Spectroscopy - Carotenoids in Evaporites Figure 9 shows the Raman observations from the halite minerals from the in-situ samples from the Great Salt Lake and Searles Lake. The NaCl minerals from Searles Lake are a deeper purple and pink compared to the light pinks and oranges from the Great Salt Lake. The harmonic overtones mentioned earlier (and mathematically explained in the next section) are found with moderate peaks in Searles but weak in Great Salt Lake. This distinction is important due the desert environment of the Mohave Desert where Searles Lake is located. Raman Resonant Peaks Perl, S.M. Biogenic Detection in Evaporites 100 There are at least four peaks that are beyond the 2000 cm -1 that were identified for the first time during this study due to their relationship to the overall volume of carotenoid preservation. The peaks at 2149.1 cm -1 , 2289.8 cm -1 , 2504.0 cm -1 , and 2641.6 cm -1 , beyond the previously known peaks for β-carotene and bacterioruberin, are caused by a series of combination bands, overtones, and resonance. The peaks greater than 2000 cm -1 are a result of how the fundamental bands interact with molecule to create other resonant vibrations. Similar to harmonics, the new peaks have a multiplicative relationship to their source Raman vibrations (e.g., the direct β- carotene and bacterioruberin carotenoid measurements). Using the first two prominent peaks in the region 2149 cm -1 is a summation combination of the 960 cm -1 and 1188 cm -1 fundamental carotenoid peaks. In addition, the 2289 cm -1 peak is the 1145 cm -1 1 st order overtone (i.e., the 1145 cm -1 peak multiplied twice). While these secondary harmonic peaks are not the direct carotenoid measurements, they only exist if the magnitude of the original/fundamental three peaks of 1000.2 cm -1 , 1150.1 cm -1 , 1507.0 cm -1 are present in a high enough magnitude such that there would be the vibration summation. Thus, the presence and magnitude of the peaks beyond 2000 cm-1 may have some utility in determining the abundance of the carotenoid biomarkers. Discussion Significance of the resonant (harmonic overtone) peaks: The presence of the harmonic overtones in the Searles Lake samples (moderate peaks) and the weak harmonic overtones in the Great Salt Lake samples is significant due to the visually deeper pigments in Searles and the desert environment that these samples come from. The higher solar flux that these salt minerals receive may correlate to the blocked wavelengths of UV-A and UV-B that occurs in this setting. The higher the solar flux would mean that the halophilic Perl, S.M. Biogenic Detection in Evaporites 101 microorganisms have to produce more of the β-carotene and other carotenoid pigments in order to shield themselves from solar radiation. The higher volumes of the carotenoids in Searles is so great that the vibrations of those harmonic overtones (2149.1, 2289.8, 2504.0, and 2641.6 cm -1 ) are actually a reflection of the real peaks at 1000.2, 1150.1, and 1507 cm -1 . This is both a secondary measurement of the highest volume of preserved carotenoid biomarkers and a measurement that can be quantified back to the source halophilic microorganisms and their production of the β- carotene carotenoid. Comparison of Lake Water, Fluid Inclusions, and Minerals: The Raman spectra of carotenoids are highest in the fluid inclusions vs. the minerals and lake water due to the harmonic overtones being both present and highest in the inclusion spectra. Due to these acting as a reflection as a function of initial volume of the real spectra they can be used as a metric for volume or quantity of the β-carotene carotenoid. Originally the overtones in the Great Salt Lake samples were disregarded since they were weak peaks. However, after analysis of the overlapping overtones from both hypersaline sites proved that not only were the weak overtones legitimate but that they also can be compared to high and very high volumes of the β- carotene carotenoid. Searles Lake biogenic pigments (Desert environment / high solar flux) By observing halite samples in it is clear that the pigmented halite samples are due to the preservation of carotenoid-containing bacteria and archaea that give the salts and brine of the north arm of Great Salt Lake the characteristic pinkish hue. Moreover, in Searles Lake these pigments are a darker pink that grade into purple, both in the in-situ brines and the accompanying sulfate Perl, S.M. Biogenic Detection in Evaporites 102 salts in the region. These pigments and their color gradients are a product of photobiology during the adaption of the halophilic bacteria that are preserved in the salts. The microorganisms produce carotenoids of these different pigments to help shield themselves from different amounts of solar flux (Truscott, 1990; Mathews-Roth & Krinsky, 1970; Hirabayashi et al. 2004). Searles Lake is in the Mojave Desert and has a higher frequency of sunlight whereas the Great Salt Lake has more of a seasonal climate where sunlight is not as constant and direct. The dried Searles Lake samples were analyzed using the same methodology with a focus on trapped fluids and pigment gradients. As mentioned earlier, the solar flux of the desert environment of Searles Lake vs. the seasonal environment of Great Salt Lake have a direct impact on the pigment and biogenic entombment. The modern Searles Lake evaporite suite consisting of halite, with minor phases of hanksite, thenardite, gypsum, mirabilite, and trona, and contained the same Raman frequency peaks as the Great Salt Lake evaporites but in higher magnitudes, 2-3 times greater, than the modern Great Salt Lake salts. In addition, at least four major peaks were detected > 2000 cm -1 at 2149 cm -1 , 2290 cm -1 , 2504 cm -1 , and 2642 cm -1 . Re-inspection of the weakest peaks after 2000 cm -1 in the Great Salt Lake evaporites show these frequencies present but at magnitudes but at much lower intensity. Due to the biogenic activity occurring in the Searles Lake evaporite samples, the pigments that have stained the internal structure of the hydrated minerals from the fluid inclusions, and the carotenoid differences based on environmental responses from the entombed halophiles, we have treated the suite of carotenoids as a chemical biomarker whereas the mineral pigments from halophilic microorganisms as a physical biosignature. Perl, S.M. Biogenic Detection in Evaporites 103 Conclusions In conclusion the highest volume of biogenic preservation in the form of carotenoid pigments, carotenoid chemistry, and the Raman vibrational observations of both genuine peaks and harmonic overtones as a diagnostic feature of higher than normal volumes are found within fluid inclusions of halite evaporite minerals. The longevity of these pigments as a chemical biomarker over geologic time can be measured from the modern preservation that takes place in these in-situ sites where evaporite entombment is taking place and new biological material and microorganisms can be added to the minerals as the surface lake beds and brines dry up and new minerals are precipitated. Pigments in the studied surface evaporite minerals from Great Salt Lake appear from light pink a pale pink whereas surface and shallow subsurface pigments from Searles Lake appear as a dark pink to red and dark orange in some cases. These carotenoid pigments (specifically β-carotene and bacterioruberin that has been measured in the GSL and SL samples) are the most significant form of biogenic validation since these simultaneously meet the requirements for being a chemical biomarker and a physical biosignature that could only exist if life was present in the evaporite mineral system. Perl, S.M. Biogenic Detection in Evaporites 104 Acknowledgements I would like to thank Bonnie Baxter and Jaimi Butler for their ongoing collaborations and work since the initial Great Salt Lake investigations in 2013. Part of our collaboration has gone into a book chapter that will be featured in the first Great Salt Lake book that has our section as the first astrobiology-driven chapter strictly devoted to the lake*. I look forward to our continued collaboration and our upcoming exploration Delaware Basin salt in WIPP during the Mars Extant Life workshop this winter. *Perl, S.M. & Baxter, B.K. (2020) Great Salt Lake as an astrobiology analogue for ancient Martian hypersaline aqueous systems. (in “Great Salt Lake: biology of a terminal lake in the age of change”). Springer. Perl, S.M. Biogenic Detection in Evaporites 105 References Almeida-Dalmet, S., Sikaroodi, M., Gillevet, P.M., Litchfield, C.D. & Baxter, B.K. (2015). 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(2005) New evidence for 250 Ma age of halotolerant bacterium from a Permian salt crystal. Geology 33, 265-268. Perl, S.M. Biogenic Detection in Evaporites 113 Schopf, J & Farmer, J. & S Foster, Ian & B Kudryavtsev, Anatoliy & Gallardo, Victor & Espinoza, Carola. (2012). Gypsum-Permineralized Microfossils and Their Relevance to the Search for Life on Mars. Astrobiology. 12. 619-33. 10.1089/ast.2012.0827. Shroder JF, Cornwell K, Oviatt CG & Lowndes TC (2016) Landslides, alluvial fans, and dam failure at Red Rock Pass: the outlet of Lake Bonneville. In: Oviatt CG & Shroder JF (eds) Lake Bonneville a Scientific Update, 1st edn. Elsevier, Netherlands, pp 75-85 Smith, G.I. (1979). Subsurface stratigraphy and geochemistry of late Quaternary evaporites, Searles Lake, California, U.S. Geol. Surv. 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Truscott, G.T. (1990) New trends in photobiology: The photophysics and photochemistry of the carotenoids. Journal of Photochemistry and Photobiology B: Biology. Volume 6, Issue 4, August 1990, Pages 359-371. https://doi.org/10.1016/1011-1344(90)85110-I Viviano-Beck, C. E., et al. (2014), Revised CRISM spectral parameters and summary products based on the currently detected mineral diversity on Mars, J. Geophys. Res. Planets, 119, 1403– 1431, doi:10.1002/2014JE004627. Winters, Y.D., Lowenstein, T.K. & Timofeeff, M.N., (2013). Identification of Carotenoids in Ancient Salt from Death Valley, Saline Valley, and Searles Lake, California, Using Laser Raman Spectroscopy. Astrobiology, 13(11), pp.1065-1080. Perl, S.M. Biogenic Detection in Evaporites 115 Figures Figure 1. Overview of the Great Salt Lake (GSL). Highlighted is the Rozel Bay (Spiral Jetty) field site containing in-situ pigmented halite terraces, gypsum crystals. (Source: https://commons.wikimedia.org/wiki/File:Great_Salt_Lake_ISS_2003.jpg) Perl, S.M. Biogenic Detection in Evaporites 116 (A) (B) (C) (D) Figure 2. Brine and Evaporite Mineral comparisons between Great Salt Lake (Rozel Bay, UT, USA) and Searles Lake (Mojave Desert, CA, USA). Taken during the field campaigns in April 2014, October 2014, February 2015, and June 2015 showing where samples were collected. (2a) Halites from GSL are from the lake-shoreline boundary showing pink pigments just below a non-pigmented top terrace layer (red dashed line). (2b) Gypsum crystals (red arrows) precipitating vertically from “ripple fan” megastructures west of the Spiral Jetty. (2c) Surface brines from Searles Lake near Cement Plant Rd. showing deeply pigmented waters and modified salt mineral structures. (2d) Unnamed site on the southern section of the dried Searles Lake where shallow subsurface samples were collected for brine and evaporite sets including hanksite, trona, mirabilite, thenardite, and halite. Perl, S.M. Biogenic Detection in Evaporites 117 Figure 3. Overview of the modern preserved evaporite mineralogy from Great Salt Lake and Searles Lake for halite and gypsum, the source lake settings, and the environmental controls. (A) Fluid inclusions with halite crystals (B) usually range from 2µm to 8µm. Not all halite crystals have fluid inclusions and if present, not all fluid inclusions contain halophilic microorganisms. (C) Gypsum crystals showing entombed clays. Source fluid environments of the Great Salt Lake (D) and Searles Lake (E) where the fluid originated that eventually precipitated the evaporite mineralogy. (F) Environmental controls for halite (subsurface muds containing no hopper crystals) and (G) gypsum (subsurface muds containing no gypsum crystals). (A) (B) (C) (D) (E) (F) (G) Perl, S.M. Biogenic Detection in Evaporites 118 Figure 4. Optical imagery of evaporite halite minerals from Great Salt Lake (A,C,E) and Searles Lake (B,D,F). Figs 4A, 4B show the NaCl crystalline structure with potential traces of epsomite in close proximity to the crystal edges of the Searles halite. Figs 4C, 4D show entombed fluid inclusions within the crystal structure. Figs 4E, 4F show where carotenoids were later observed. While carotenoids are mostly found within fluid inclusions, they have been observed in trace amounts in the crystal structures. (A) (B) (C) (D) (F) (E) Perl, S.M. Biogenic Detection in Evaporites 119 “A”, “B”, “C” “E” “D” Gypsum evaporate crystals “F” Dot Sample Tube Fluids under halite terraces A Halite crystals with fluids B Halite crystals without fluid C Gypsum crystals D Unfiltered lake water E Environmental control F Figure 5. Rozel Bay sampling campaigns. The modern pigmented NaCl and gypsum crystals were the primary focus of the original sampling trips in 2013-2015. Note the Spiral Jetty artwork in the center of the figure above and to the left (south of the Jetty) are the only sections of the area that contained gypsum crystals in the ripple fan megastructures. Halite sites 1-7 were labeled from left-to-right above and the same category of sample was collected per site. All samples had environmental controls collected at the same time as the in-situ samples so a reference preservation metric could be established. For halite the environmental control were the wet soils below the massive salt terrace. For gypsum the environmental control was the ripple fan sediment below the vertical precipitating crystal. Scale bar on top right corner is ~40 meters. N Perl, S.M. Biogenic Detection in Evaporites 120 Figure 6. Searles Lake location between the Argus and Slate mountain ranges (from Smith, 1979, Figure 1, p. 3). The modern component of the now dried Searles Lake is on the surface where mild rains have ponded above the hypersaline crusts. The modern-to-Pleistocene stratigraphic column (shown in Figure 3) and excavated buried salt and mud layers (shown in Figure 4) are all taken from field campaigns done by the author (during Feb 2016, “EX 1337”). Perl, S.M. Biogenic Detection in Evaporites 121 Figure 7. Raman scan representative of unfiltered brine waters from Great Salt Lake and from the dried Searles Lake. Both field site samples show a large OH band (green dashed line) at ~3400cm - 1 indicating bound water within the mineralogy. Since the halite cubic structure is Raman transparent (absent of impurities), these detections are found within the hopper crystal itself. Perl, S.M. Biogenic Detection in Evaporites 122 Figure 8. Raman scan representative of fluid inclusions from Great Salt Lake and from the dried Searles Lake. Both field sites show a large OH band at ~3400cm -1 indicating bound water within the mineralogy. Both samples display notable Raman peaks around 1000, 1150, and 1507 cm -1 . The Searles Lake sample shows additional peaks atfor the Great Salt Lake inclusions; and 1001.2, 1145.3, 1502.9, 2149, 2289, 2504, and 2641 cm -1 , but only trace peaks at these values for the Great Salt Lake inclusions. These additional peaks from Searles Lake inclusions correspond to the carotenoid β-carotene when compared to the Halobacteria sp. NRC-1 (see Figure 10 for NRC-1 Raman spectra) and from Fendrihan et al. 2009 and Jehlicka et al. 2014. Note that there are trace peaks at these wavelengths in the Great Salt Lake samples when compared to Searles Lake. Water from the fluid inclusion is shown as the OH band by the green dashed line. Perl, S.M. Biogenic Detection in Evaporites 123 Figure 9. Raman scans of mineral salt structures from Great Salt Lake (blue) and from Searles Lake site (red). These sections of halite and trace sulfate salts (hanksite, trona, mirabilite, others found in Searles Valley region) have no bound water as shown from the absent OH peak. Great Salt Lake Raman peaks are prominent at around 1000.2, 1150.1, and 1507 with very minor peaks after 2000cm -1 whereas Searles shows similar peaks as Great Salt Lake but include major peaks at 2149.1, 2289.8, 2504.0, and 2641.6cm -1 . The Halobacteria sp. NRC-1 (see Figure 10) shares these later peaks also correspond to carotenoids β-carotene and Bacterioruberin (Fendrihan et al. 2009; Jehlicka et al. 2014). Perl, S.M. Biogenic Detection in Evaporites 124 Figure 10. Raman spectra of Halobacteria sp. NRC-1 showing prominent peaks at 1000.2 cm -1 , 1150.1 cm -1 , and 1507 cm -1 with very minor peaks after 2000 cm -1 whereas Searles shows similar peaks as Great Salt Lake but include major peaks at 2149.1 cm -1 , 2289.8 cm - 1 , 2504.0 cm -1 , and 2641.6 cm -1 due to the high volume of β-carotene and Bacterioruberin. The dry Searles Lake halite are the green and light blue spectra while the Great Salt Lake halite spectra is purple and the fluid inclusion is in dark blue. The baseline Halobacteria sp. NRC-1 is in the black spectra. = Halobacteria sp. NRC-1 = Searles Lake halite 1 = Great Salt Lake halite 1 = Searles Lake halite 2 = Great Salt Lake fluid inclusion Perl, S.M. Biogenic Detection in Evaporites 125 Chapter 5 Investigation of Pigment Preservation in Pleistocene and Permian Evaporites with Implications to Biotic Entombment over Geologic Time Abstract Biogenic preservation is a function of volume of biotic material and the preservation medium. The issue of preservation of organic matter over geologic presents a two-fold issue. Primarily, how long do the specific chemical biomarkers and physical biosignatures last in the mineral matrix? Are these biotic features diagnostic for life or can they be replicated abiotically? On Earth, terrestrial preservation over geologic time usually relies on organic chemical species that are linked to a bulk volumetric amount and cannot provide taxonomic unit information. Which leads into the secondary knowledge gap with respect to extant vs. extinct life. Should diagnostic signs of life be present at the information level (e.g., DNA, hydrocarbons, amino acids, fatty acids) how long can these features last over geologic time before that biogenic information is lost? Moreover, how can one distinguish signs of ancient/extinct life long after DNA has been lost to time separately from false positives? Finally, how can extant life be geobiologically separated from younger contamination? The purpose of this paper is to examine Pleistocene and Permian evaporite minerals that have signs of biogenic preservation in the form of organic biomarkers detected via Raman analyses and pigment-centric mineral-microbe interaction during and directly after evaporite precipitation. Pleistocene samples in question are from the deepest sections of an at least four-pronged set of wet-dry cycles whereas the Permian samples examined here are from the ponding of a land-driven marine region that has precipitated the same lithology and provide a single Permian age set of evaporites with similar pigments but buried 1.2km below the surface. Perl, S.M. Biogenic Detection in Evaporites 126 Introduction The search for life in our solar system, and more specifically on Mars, Europa, and Enceladus, all share a common element that has been deemed necessary for life: water as a solvent. Without getting into the topic of the origins of life on our own planet and the search for life as we don’t know it outside of Earth, several temporal and evolutionary constraints eventually come up. The jump from the “standard” planetary geology mindset of searching for water as a proxy for habitable environment to truly understand what to specifically search for using a single data point of Earth is a conundrum in it of itself. How do we search for life as we don’t know it using life as we do know it? Using geobiology, a more complete question becomes: How do we search for life as we don’t know it as a function of where an organism is in its evolutionary timeline and do the capabilities of that organism allow for a measurable biomarker or biosignature to be generated as a normal or adaptative biotic function? While this may be long-winded it does touch upon several aspects of these investigations. The latter part of this question: the generation of biotic evidence is a given in the modern geologic timeframe. Modern evaporite minerals can preserve modern lake water columns and transfer biogenic evidence into a preserved mineral environment. But what happens after life becomes extinct? Do the lines of evidence also decay without its biological source? How can we determine extant life that has evolved potentially separately and isolated from a reference taxonomic outgroup? How can we decipher between modern/younger contamination and extant microorganisms? More specifically how can we use hypersaline mineralogy that provides this preservation in the modern periods to tell us where to look for life (both as we don’t know it and terrestrial)? Since modern evaporites provide not only preservation of biotic material and biogenic modifications from cellular activity the answer to the aforementioned question for terrestrial life needs to Perl, S.M. Biogenic Detection in Evaporites 127 simultaneously preserve chemical biomarkers over geologic time and remain physically robust to geologic changes. Due to these constraints, two distinct evaporite mineral sites have been chosen that perform these actions in-situ due to burial and due to the fact that their original in-situ fluids both were contained in enclosed settings such that no further physical destructive fluids, post- precipitation, dissolved these highly soluble minerals (Barbieri & Stivaletta, 2001). The Pleistocene salt layers of Searles Lake (approximately ~1.1 Myr, see Smith (1970) and Figs. 1,2) and the 1.2km buried Permian evaporite layers of the Zechstein Formation in the Boulby salt mine (approximately ~255 Myr, see Cockell, Perl, and 48 others (2018), Glennie et al.(1998), and Figs 3,4) will be examined to evaluate the preservation of potentially organic Raman peaks that may be degraded versions of carotenoid biomarkers, and present results that show the robustness of this potentially biotically-generated chemistry over geologic time. Methods Sample collection for the Pleistocene Searles Lake salts and muds was done during February 2017 as part of the 1336EX Searles Valley Mining (SVM) expedition where buried evaporite and regolith layers were exhumed and brought to the surface using a pressure-gradient rotating coring mechanism. Due to the typical geochemical analyses SVM does for every expedition, the rotating cores were cleaned and decontaminated from their previous use. While not a true core in the sense of a cylinder-type of material, the “cored” sediment piles were collected upon being unearthed (Figure 2). The Pleistocene muds here were also collected but not part of this study and will be used for future analyses (Perl & Sessions, in-prep). The Permian samples collected were from the MINAR V expedition (see Cockell, Perl, and 48 others (2018)) and the MINAR VI expedition (Perl & Cockell, in-prep) where sites were Perl, S.M. Biogenic Detection in Evaporites 128 preselected that had recently discovered aquafers that had prevented further mining operations in the Boulby potash salt mine (Figure 3). Further sites explored showed thermally-induced (from tectonics in this case) polygonal structures in the evaporitic layers, typical NaCl, KCl, and polyhalite samples with the latter having a chemical composition (Figure 4) only found in this section of the world (Cockell et al. 2018). Pleistocene Sample Site – the (dry) Searles Lake (Mojave Desert, California, USA) The Searles Lake site contained highly pigmented NaCl crystals and brines with minor to major amounts of thenardite, hanksite, trona, mirabilite, and gypsum (Figure 2). Pigmented brines and evaporite minerals were collected from near Cement Plant Road with permission and authorization to the author from Searles Valley Minerals. Sample collection took place in and around the brine pools where evaporite minerals were fairly homogenous. The same aforementioned sterile sample collection techniques were used, and samples were stored in both 50ml Falcon tubes as well as brines in 1000ml Nalgene bottles. Due to the varied evaporite mineral diversity here samples were analyzed using XRF to see the typical composition of the in-situ salts. Solid evaporite minerals were dissolved and concentrated as the previous GSL samples were, but these samples took far longer than the GSL salts due to even higher salinity. Brines and dissolved salts were concentrated using the Innovaprep Concentrating Pipette using the UltraFine filter and the 0.82µm filter when the salinity proved too much for the vacuum pump. Permian Sample Site – the 1.2km Buried Boulby Salt Mine (near Whitby, UK) Due to low biomass concerns, Permian evaporite samples were collected using individual sealed gamma-treated Nalgene bottles, such that any background organics or biology could not interfere with in-situ measurements, future QuBit fluorometer (version 4) observations, and Perl, S.M. Biogenic Detection in Evaporites 129 Maxwell DNA extractions for eventual 16S OTU analyses. About 20 brine samples from six different aquafers with some duplicates due to accessibility issues or unique pigmented at the aquafer-evaporite wall boundary. In total for MINARs V and VI ~86kg and ~129kg of samples were collected per expedition, respectively and were used in these analyses. Raman analyses of Preserved Organics and Potentially Carotenoid Biomarkers Green Raman (532nm) spectroscopic analyses were conducted on Searles Lake mineral lithologies that were present in each of the evaporite layers (Figure 5). Fluid inclusions and distinctive organic peaks were present alongside trace hydrated mineralogy. Raman analyses were conducted on the Permian NaCl, Permian KCl, Permian polyhalite, and aquafer brine samples. It is unknown how old the age of the brines are but due to non-permeable regions of shale and other rock layers above the evaporite sections of the mine it is very possible that these closed aquafers were/are the original Permian fluids ponded from the Zechstein sea to the Zechstein formation. Fluid inclusions were also present in all Permian evaporite samples (Figure 6) and magnitude analyses of the OH stretches ~3340 show these fluids are still indeed present and in very high volumes and at many times comparable with modern Great Salt Lake NaCl fluid inclusions. Results Both the Pleistocene and Permian NaCl samples (~1.1 Myr and ~255 Myr, respectively) contained significant amounts of trapped fluids (H2O shown as an OH stretch ~3340cm -1 ). This was measured using the aforementioned green Raman at 532nm. Raman peak magnitudes of the preserved fluids can be inferred by the peak heights and trace fluids (shown as a lower OH stretch relative to the higher peaks) were also observed in some of the NaCl matrix sections. Figure 5 shows ~20 spectra from buried Searles Lake sodium chloride lithologies from 128, 135, and 153 Perl, S.M. Biogenic Detection in Evaporites 130 meter depths. While the peak at ~1000cm -1 may be trace mineral (potentially serperite or another hydrated sulfate salt due to common peaks at this point) all of the moderate peaks prior to and after this point do not match to any mineral database (RRUFF, BioRad, or relevant literature) and due to the waveform pattern and wide base it is likely a suite of organics that are vibrating due to the 532nm absorption. Water content preserved in these samples were fairly low but fluid inclusions were observed, albeit with lower volumes. While not a part of this study, the Pleistocene muds that were extracted from the 1336EX drilling campaign at these depths came out with light green, light yellow, and light orange pigments, as did the salts above and below these features. Figure 6 shows the same type of analysis for the Permian Boulby NaCl evaporites. Keeping the lithologies the same between the two geologic ages allows for a proper comparison of Raman peaks and spectral patterns. It was apparent that the NaCl samples from Boulby (specifically the hopper crystals extracted from the evaporite walls near the “New West B” brine pool) not only contained a greater number of fluid inclusions, but the volumes of their water content were higher than the buried Searles Lake samples. As with the earlier Pleistocene samples, the peaks and energy drop-off ~320cm -1 is not diagnostic of any mineral assemblage and since halite (NaCl) is Raman transparent, we are seeing directly into the crystal structure without any mineral substrate interference. The wide range of peaks from ~1500cm -1 to ~3100cm -1 do not match mineralogy or elemental chemistry and are likely the remains of ancient organic molecules left behind from halophilic microorganisms. Pigments in the Permian samples are all a light to moderate orange with some samples containing a yellow hue that appears in a rare set of samples (Hirabayashi et al. 2004). Perl, S.M. Biogenic Detection in Evaporites 131 Discussion The degradation of carotenoid biomarkers over geologic time is related to the preservation medium (e.g., the evaporite minerals they are produced in by halophilic microorganisms), the original source biology, and the lack of mineral dissolution or transformation such that both the chemical biomarkers from life and any physical biosignature pigments are well preserved. Should these features become buried their chances for remaining more intact over geological periods of time significantly increases (Sankaranarayanan et al. 2014; Lowenstein et al. 2016; Cockell et al. 2014). Moreover, carotenoid pigments by themselves can be measured over long periods of time with established organic detection techniques such as Raman, ICP-MS, and GC-MS to name the major instruments (Mathews-Roth et al. 1970; Perl, Cockell, Celestian, et al. in-prep; Truscott, 1970). The utility of carotenoids in the modern evaporitic preservation environments is the same in the geologically older settings but due to the extended periods of preserved time and loss of biological inputs (i.e., more carotenoid structures being added to the evaporites by halophilic microorganisms) the longevity and robustness of these pigments can now be established in the mineral record from two ancient aqueous environments. The major and obvious difference between the level of biotic information one can obtain from validated carotenoid/pigment observation in hydrated salts and minerals that contain extant life in the form of halophilic microorganisms (i.e., motile cells preserved in fluid inclusions) is that you would only be able to infer the solar flux that the halophilies had to adapt to in order to maintain their metabolic processes. In the modern evaporitic setting it has been shown that biogenic preservation of specific OTUs and bulk DNA preservation is directly related to evaporite category (e.g., type of salt mineral) and feature (i.e., presence of pigments and clays). This bodes Perl, S.M. Biogenic Detection in Evaporites 132 well for future sites of interest on Mars due to the presence of clay minerals and widespread hydrated mineralogy (Ehlmann et al. 2009; Bridges et al. 2015; Murchie et al. 2009). Due to the high potential of subsurface waters far away from the current UV-C hostility on the Martian surface, signs of life in buried evaporites and salt layers from current or ancient groundwaters would provide the highest probability of in-situ discovery of extinct cellular life that may have utilized these ancient waters (Michalski et al. 2015). Moreover, should current deep subsurface groundwater even exist on modern Mars today, it would be contain features emulated in the Pleistocene Searles Lake deep evaporite-regolith layers or if groundwaters were mostly done, the Permian Zechstein formation evaporites of the Boulby Mine could provide an ideal analogue for the near total loss (excluding observed aquafers) of water in the deep subsurface. Conclusions While the search for ancient signs of life on Earth has direct applications to our astrobiological exploration strategies for searching for life in our solar system, the major hurtle to these types of endeavors is that, when applied to a planetary context, it ignores the similarities of cellular organisms over their evolutionary timelines (Maughan et al. 2002). Major misinterpretation of the age of halotolerant bacterium from the biological perspective can reside in the age of the mineral substrate and completely ignore contamination and extant life possibilities (Vreeland et al. 2000; Satterfield et al. 2005). While preserved microfossils can be rare (Schopf et al. 2012), their diagnostic features would be a problem on Mars or other icy body due to the inability of any current or near future sample extraction capability to preserve microtextures. Not even the future Mars Sample Return (MSR) mission suite will be able to retain physical textures Perl, S.M. Biogenic Detection in Evaporites 133 in mineral veins or rocks due to the sample being drilled in-situ by the next rover. With these technologies not aligning with proper sample (and potentially non-sterile) collection techniques. We know from the highly successful Opportunity rover mission to Meridiani Planum that surface waters on Mars were stable prior to Noachian/early Hesperian time frame and long enough such that groundwater movement significantly modified pore spaces in sedimentary rocks there (McLennan et al. 2005). Should these ancient fluids have been the solvent for life as we don’t know it, deep subsurface precipitated evaporite minerals would not only have been the ideal preservation medium for extant life, but their total physical timeline would have never interacted with the eventual changing surface UV-C and atmospheric weathering. It is ironic that due to permeability and secondary porosity we are aware of this groundwater movement to the degree that it was these same source fluids that formed the hematitic spherules (e.g., the Martian “blueberries” observed in the Burns Cliff section of the Burns Formation), but that it’s also this same exact pore space that would allow for damaging surface features of Amazonian Mars to percolate downwards towards potentially untouched deep surface hydrated mineralogy. Of course, the same arguments for the potential ambiguity of the age of the Permian Boulby Mine brine pools take place for Martian subsurface aquafers. If ancient waters can survive and remain stable and fluidic over geologic time, they can be a record of ancient fluid chemistry and can act as the Petri dish of sorts for metabolic processes for life as we know it. Biology is more dynamic than geology and any changes to the mineral record due to water would be slow enough for life to adapt and evolve much like the ability for halophilic microorganisms to evolve to produce the ability to generate the carotenoid pigments in the first place as a survival strategy for continued nutrient cycling. If life were to have started on Mars and evolved separately (e.g., a “Martian LUCA” and separate pathway(s) of that life) perhaps the slow changes of water-mineral and water-rock Perl, S.M. Biogenic Detection in Evaporites 134 interaction would have given that life enough time to adapt to the changes that would befall the planet. We know at least four groundwater “recharge” cycles took place in Meridiani Planum (Squyres et al. 2005; McLennan and Grotzinger, 2008; Knoll et al. 2005) and that water had to move somewhere if it didn’t all become unstable on the surface. The SHARAD shallow radar onboard the Mars Reconnaissance Orbiter has shown buried polar ice (more equivalent to terrestrial dry ice, see Nerozzi & Holt, 2019) near the Martian poles and given the sheer amount of hydrated minerals globally across the planet (Murchie et al. 2009), it would stand to reason that a significant amount of this once stable surface water made it’s way through permeable sedimentary rocks down to shallow-to-deep surface regions. If so, it would continue to precipitate salt mineralogy and, should a Martian LUCA have existed ~3.5 to 3.8 Gyr ago, would be the optimal habitat where we would find chemical biomarkers, potentially in the form of carotenoids or other chemical structure that provides the same utility (e.g., “carotenoid-like”) of solar flux protection. Most of this fluidic timeline already occurs on Earth largely with the assistance of our terrestrial water cycle. The groundwater hydrogeology is simple enough with the most difficult part being able to access these deep subsurface evaporite layers. Acknowledgments I would like to thank Charles Cockell from the University of Edinburgh for inviting me to my first MINAR program in late 2017 where I first was able to explore the Boulby salt mine for Permian evaporites and closed aquafer hypersaline brines. I’ve been able to return to the Boulby mine each year since to expand my fieldwork there and bring back “new” Permian salts for further laboratory analyses. These samples have led to spinoff work where in conjunction with extracting DNA from Perl, S.M. Biogenic Detection in Evaporites 135 known cross-contaminated saline brines (contaminated by “younger” brine sections in the mine) I am now looking at hopanes and lipid biomarkers in the Permian salts, contaminated brines, and Permian brines. The initial results have led to two co-author papers in the International Journal of Astrobiology in 2018 and 2019. I would also like to thank Sean Pailing, Tom Edwards, Emma Meehan, Louise Yeoman, and Paul Scovell for their immense assistance during the MINAR field campaigns (V and VI), assistance in getting my 97kg and 114kg Pelican crates back to JPL from 1.2km below the Earth’s surface. Without them, I would have no Permian samples for analyses and would not have been able to maintain my continued research initiatives into the Zechstein formation. References Barbieri, R. and Stivaletta, N. (2011), Continental evaporites and the search for evidence of life on Mars. Geol. J., 46: 513–524. doi:10.1002/gj.1326 Bridges, J. C., S. P. Schwenzer, R. Leveille, F. Westall, R. C. Wiens, N. Mangold, T. Bristow, P. Edwards, and G. Berger (2015), Diagenesis and clay mineral formation at Gale Crater, Mars, J. Geophys. Res. Planets, 120, 1–19, doi:10.1002/2014JE004757. Cockell, C. S. (2014). 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Provenance and diagenesis of the evaporite-bearing Burns Formation, Meridiani Planum, Mars. Earth and Planetary Science Letters 240:95–121. Michalski, J. R. Cuadros, J., Niles, P.B., Parnell, J., Rogers, A.D., Wright, S.P. (2013). Groundwater activity on Mars and implications for a deep biosphere. Nature Geoscience. 6 (2): 133-138. Murchie S.L., Mustard J.F., Ehlmann B.L., Milliken R.E., Bishop J.L., McKeown N.K., Dobrea E.N, Seelos F.P., Buczkowski D.L.,Wiseman S.M., Arvidson R.E., Wray J.J., Swayze G., Clark R.N., Marais D.J.D., McEwen A.S., Bibring J.-P. (2009). A synthesis of Martian aqueous mineralogy after 1 Mars year of observations from the Mars Reconnaissance Orbiter. Journal of Geophysical Research 114, doi: 10.1029/2009JE003342 Nerozzi, S., & Holt, J. W. ( 2019). Buried ice and sand caps at the north pole of Mars: Revealing a record of climate change in the cavi unit with SHARAD. 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Subsurface stratigraphy and geochemistry of late Quaternary evaporites, Searles Lake, California, U.S. Geol. Surv. Prof. Pap. 1043, 130 pp. Smith, G.I., 2009, Late Cenozoic geology and lacustrine history of Searles Valley, Inyo and San Bernardino Counties, California: U.S. Geological Survey Professional Paper 1727, 115 p., 4 plates. Squyres, S. W., and A. H. Knoll (2005), Sedimentary rocks at Meridiani Planum: Origin, diagenesis, and implications for life on Mars, Earth Planet. Sci. Lett., 240, 1 – 10, doi:10.1016/j.epsl.2005.09.038. Perl, S.M. Biogenic Detection in Evaporites 141 Summons, R.E., Amend, J.P., Bish, DL, Buick, R., Cody, G.D., Des Marais, D.J., Dromart, G., Eigenbrode, J.L., Knoll, A.H., and Sumner, D.Y.(2011), Preservation of Martian organic and environmental records: Final report of the Mars Biosignature Working Group. Astrobiology, 11, 157-181. Truscott, G.T. (1990) New trends in photobiology: The photophysics and photochemistry of the carotenoids. Journal of Photochemistry and Photobiology B: Biology. Volume 6, Issue 4, August 1990, Pages 359-371. https://doi.org/10.1016/1011-1344(90)85110-I Vreeland, R. & Rosenzweig, W. & Powers, D. (2000). Isolation of a 250 million year-old halotolerant bacterium from a primary salt crystal. Nature. 407. 897-900. 10.1038/35038060. Viviano-Beck, C. E., et al. (2014), Revised CRISM spectral parameters and summary products based on the currently detected mineral diversity on Mars, J. Geophys. Res. Planets, 119, 1403– 1431, doi:10.1002/2014JE004627. Winters, Y.D., Lowenstein, T.K. & Timofeeff, M.N., (2013). Identification of Carotenoids in Ancient Salt from Death Valley, Saline Valley, and Searles Lake, California, Using Laser Raman Spectroscopy. Astrobiology, 13(11), pp.1065-1080. Perl, S.M. Biogenic Detection in Evaporites 142 Figures Figure 1. Sedimentology of salt and mud layers of Searles Lake from Smith, 1979. This sedimentary column shows 3-4 salt layers from 3-4 lake evaporation events. In conjunction with the next figure (Figure 4) samples excavated from this column start at the surface (overburden muds) and extend down in depth to ~150 meters / 496 feet (halite and trona laters with some interbedded brown muds containing gaylussite). Note that many of the mud layers have light pigment green, yellow, pink, and orange pigments (see next page and figure). Letters A-F correspond to Smith (1979) Table 2 and pgs. 14-15. Perl, S.M. Biogenic Detection in Evaporites 143 Figure 2. Searles Lake (Mojave Desert, CA, USA) modern to Pleistocene field site. Taken during the February 2015 campaign showing salt and mud samples were excavated using an brine pressure gradient “drill” (no actual coring or drilling). Using the previous figure (Figure 3) as a sedimentary map, these samples show, via depth, the different evaporation events (salt layers) and active lake periods (mud layers). It should be noted that the difference between each pile is 10 feet (~3 meters) with each red flag denoting every ten piles (every 100 feet / ~30 meters). The first salt layer starts at ~34 feet (10 meters) which matches Smith, 1979 (Figure 3). It should be noted that after excavation, it was observed that the mud layers had light pigments that ranged from pink to yellow to green to orange in that order. Perl, S.M. Biogenic Detection in Evaporites 144 Figure 3. Overview of the Permian Boulby salt mine. (A) Modified from Cockell, Perl, et al. (2018) showing the relative sampling locations (not to horizontal scale) of brine pools, halite sites, potash (KCl) sites, and polyhalite sections. (B) Images of the author using a EtOH-sterilized coring tool to extract Permian halite cores. Most sample had a mixture of solid salts as shown in the middle image and ground sample near the cored diameter. (C) Map adopted from Figure 1. In Payler et al. 2016. International Journal of Astrobiology, doi:10.1017/S1473550416000045). (D) View of the “polygon region” where samples were extracted from. (A) (C) (D) (B) Perl, S.M. Biogenic Detection in Evaporites 145 Permian halite hopper crystals NaCl Polyhalite K2Ca2Mg(SO4)4·2H2O “Potash” KCl Closed Permian Aquifers Figure 4. The three major Permian evaporite mineral assemblages from the Zechstein Formation Boulby salt mine. (A) Halite hopper crystals are typically darker due to trace amounts of graphite in their mineral matrix. (B) Fluid inclusions for Permian halite. (C) Potassium chlorite evaporite (KCl) that have formed along with trace amounts of Fe in their matrix. (D) Fluid inclusions for the potash/KCl/potassium chlorite. (E) Polyhalite that is only found in this section of the Zechstein Formation and the only source of this evaporite mineral on Earth. These have been observed with the lowest volume of (F) fluid inclusions. (G) Hypersaline brine aquifer that has been uncovered by the mining company here. After aquifers are discovered, the area is closed off and either left alone or later drained if access is needed. (H) Rehydrated Permian salts along with younger precipitated salt stalactites. (A) (B) (H) (F) (E) (G) (D) (C) Perl, S.M. Biogenic Detection in Evaporites 146 Figure 5. Raman analyses of Pleistocene Searles Lake halite minerals. (Top) Unprocessed Raman data of ~20 sites in three Pleistocene evaporite mienrals. (Bottom) Baseline corrected spectra for “D_34” spectra. RRUFF and other mineralogical databases point to the major peak at ~1000cm -1 as serperite, afganite, or devilline. Based on the elemental composition it is more likely serperite since that is the only mineral of the three that has Na. However, the first β-carotene peak is also at 1000.2cm -1 and very close to the above datasets. It is likely not β-carotene since the diagnostic other peaks are not present. All other peaks do not match mineral or elemental peaks and are likely moderate organic peaks. Perl, S.M. Biogenic Detection in Evaporites 147 Figure 6. Raman analyses of Permian Boulby Mine NaCl. Raman analyses of trapped Permian fluid inclusions with NaCl hopper crystals. The OH stretch at ~3340cm -1 is diagnostic of liquid water and the magnitude of that peak shows the abundance of that water molecule. Note the delta between the red and purple peaks. Both are water but the purple peak is a fluid inclusion with greater water content than the fluid inclusion from the red peak. Moreover the weak-moderate peaks between ~1000 and ~2800cm -1 all do not align with mineralogy or elemental chemistry and are deemed to be organic in nature. Finally the energy dropoff ~320cm -1 is present in the NaCl, the KCl, and the polyhalite minerals while the peaks before that region are found in just the NaCl samples. Perl, S.M. Biogenic Detection in Evaporites 148 Chapter 6 Recommendations and Nomenclature for Astrobiological and Geobiological in-situ Analyses of Martian Samples and Returned Sample Analyses Abstract As the exploration of Mars and other worlds for signs of life have increased the need for a common nomenclature and consensus has become significantly important for proper identification for non-terrestrial/non-Earth biology, biogenic structures, and chemistry generated from biological processes. The fact that Earth is our single datapoint for all life, diversity, and evolution there is an inherent bias towards life as we know it through our own planets’ history. The search for life as “we don’t know it” then brings this bias forward to decision making regarding mission instruments and payloads. Understandably, this leads to several top-level scientific, theoretical, and philosophical questions regarding the definition of life and what it truly means for future life detection missions. How can we decide on how and where to detect known and unknown signs of life with single biased data point? The purpose of this paper is to generate a proper nomenclature for terrestrial features that have mineral-microbial interactions within structures and to confirm which features can only exist from life (biotic), features that are modified by biological processes (biogenic), features that would not change due to life (abiotic), and properties that can exist or not regardless of the presence of biology (abiogenic). These four categories are critical in understanding and deciphering future returned samples from Mars and in-situ analyses from Europa to distinguish and separate what physical structures and chemical patterns are due to life and which are not. Perl, S.M. Biogenic Detection in Evaporites 149 Introduction As the discipline of astrobiology is increasing in its parameters and practice for planetary missions the definitions and usage of terminology that allows for proper differentiation of features from life or modified from life do not yet exist. The broad term of “biosignatures” has been increasingly applied to features on Earth that can indeed exist without terrestrial life or the existence of specific patterns and/or textures is far too easily given reasoning of biological interaction with, at times, little thought into nomenclature. Clarity for this field is critical for proper identification of what is and isn’t due to biological processes and taking into account if life even could, over a geologic period of time, provide a robust sign or signs of life that we would be able to decipher from a non-biological feature, mineral, or sedimentary outcrop. Most robotic planetary missions are focused on signs of ancient or present aqueous activity on planetary bodies or moons (Figure 1). Mars exploration since Pathfinder in the late 1990s was a proof-of-concept that a rover could indeed land safety on the planet. It wasn’t until the Gamma Ray Spectrometer (GRS) and the Thermal Emission Spectrometer (TES) onboard Mars Odyssey detected the Fe-oxide hematite (Fe2O3) in the plains of Meridiani Planum (Figure 2) that the Mars Exploration Rover (MER) Opportunity was sent to that site in 2004 (McLennan et al. 2005, 2012). It was during the first six weeks of that mission that layered outcrop was observed in-situ in Endurance crater (Figs. 3,4). A year after Opportunity made this discovery, the Mars Reconnaissance Orbiter achieved orbit the Compact Reconnaissance Imaging Spectrometer for Mars (CRISM) started its global campaign to observe planet-wide signs of ancient aqueous environment to see signs of habitability from those late Noachian/early Hesperian waters and the minerals precipitated or modified by in-situ fluids. How does evidence of ancient water tell us that a site is habitable? How can we quantify habitability on another planet other than Earth without Perl, S.M. Biogenic Detection in Evaporites 150 knowing how life would have evolved in a planetary ecosystem? If a crater on Mars had more water than another does that means it is more habitable? These questions have led the planetary geology community down pathways of directly associating habitability with current and ancient signs of water on Mars. Is this association always correct? Life as we know it on Earth can survive in all known climates and extreme ecosystems albeit the highest and lowest temperature and pressure endmembers. The key here is that life persists in settings that it can adapt to and eventually, over geologic time, can thrive in. Astrobiology nomenclature needs to take into account the availability of the one single data point of life and evolutionary processes that we know of. Earth. This final dissertation chapter will discuss the shortcomings of life detection and provide a framework for how to define terrestrial life with direct associations to the evaporite minerals and series of experiments conducted for this doctoral research. The purpose of this last chapter is to formulate and discuss the proper definitions for biogenic and abiogenic processes that can lead to better assessments of planetary habitability. This proposed nomenclature will reflect life as we know it and leave enough room for life as we have yet to discover it. Methods The definition of life from a textbook will vary depending on the interpretation of the discipline. In biology if something is alive then it can respond to their current environment, has some sort of cellular composition, it is able to gain energy from chemical reactions (metabolism), it has the potential for growth, it can replicate itself, it can maintain homeostasis, and it can inherit properties or traits from previous versions of itself. On Earth the diversity of life has proven these properties of life over and over again and it is what is used for the definitions of life elsewhere outside of our own planet. The nomenclature for detecting these features (Figure 7) however does Perl, S.M. Biogenic Detection in Evaporites 151 not capture temporal and evolutionary features in the general “biosignatures” terminology that many in the planetary science communities use for advocating for mission landing sites and future instrument payloads (Figure 8). Over the course of the research investigations for this doctorate, two high-level definitions have aided in categorizing features that have been formed from or modified by halophilic microorganisms. In order for the evidence of life as we know it to be significant, both of the below criteria need to be proven in multiple parts of a sample. Using the fluid inclusion environment setting as described in Chapters 2-5 as an example, the types of validation necessary would need to be observed in multiple inclusions in one single crystal and be able to be observed in several crystals from the same aqueous environment and local sedimentology. • Chemical Biomarkers: quantified only by instrumentation and unable to be seen with the naked eye these macromolecules are from the chemistry of biological processes and interactions with minerals. These biomarkers are usually not arranged in any well-ordered set with respect to volume, (retention) time, and are only observable if active biology is present or if the preservation of extinct biology has remain intact since the point of mineral precipitation. Examples from terrestrial life include carotenoids, DNA, hopanes, cellulose, and unique sets of lipids and proteins. (Note: the chemistry that forms carotenoid form pigments that are visible to the naked eye and are simultaneously chemical in their formation but presented also as physical features). • Physical Biosignatures: quantified by images in the form of pigments, non-Brownian and independent motion, unique patterns, layering, and microfossils (although these can be the least significant when dealing with cellular life). These visible features can be seen with Perl, S.M. Biogenic Detection in Evaporites 152 the naked eye, µ-scale to mm-scale images, and do not require instrumentation other than images or video. For extinct biology, these physical signs of life can be evident if the preservation medium has been maintained and if no physical or chemical modification occurred after the last instance of biology or a biological process took place. For extant life these features should be able to respond to forms of chemotaxis or pigment generation. These two overarching definitions need widely different lines of evidence before a burden of proof is established. If either the chemical biomarker or the physical biosignature in question in an unknown sample can be established and not both, then the evidence will likely fall short. We have witnessed this before where scientists have quickly asserted that signs of extraterrestrial life have been observed based on a single data point, image, or sample. McKay et al. (1996) claimed that tube-like structures observed in a photomicrograph in the Alan Hills meteorite (ALH84001) were physical fossils from that were preserved in the Martian meteorite (Figure 5). Taking into account no biological evidence to support such a claim and in the dynamic set of events following McKay’s paper, led to informing the United States presidential administration at that time of this highly controversial line of evidence. Were McKay and co- authors wrong in making this claim based on their 1996 manuscript evidence? Did they have enough evidence to make the claim in the first place? How did such conclusions about something so significant pass through peer review, through NASA-HQ and their subject matter experts, and made its way to national and international news? While these hypothetical questions surely can exist for McKay’s work and others the proper methodology questions need to take into account life as we don’t know it using terrestrial microbial ecology and evolution as we know it (Figure Perl, S.M. Biogenic Detection in Evaporites 153 9). To start this effort the terminology of biogenic, biotic, abiotic, and abiogenic, have been utilized throughout this dissertation and the upcoming peer-reviewed manuscripts. This is to assist the planetary geology community in the nomenclature that we can understand and apply in-situ to fieldwork; sample analyses on Earth in addition to Martian rover campaigns into microtextures laid down and modified by ~3.5 Gyr old closed basin lake systems. Moreover, these terms can also be applied to the ocean worlds of Europa and Enceladus where fluid action has made its way from the deep icy crusts and into the vacuum of space in the form of giant frozen plume material imaged by the Hubble Space Telescope (HST). Using the quantified chemical biomarkers and physical biosignatures of evaporite minerals as the main example from these dissertation investigations the pigments observed in the modern Great Salt and Searles Lakes are the photobiological output from halophilic organisms due to their reactivity to high solar UV-A and UV-B in the desert environment compared to the seasonal environments of Rozel Bay. In order for the carotenoid pigments to be maintained over geologic periods of time the evaporite NaCl minerals need to precipitate and the halophilic organisms need to be preserved in the fluid inclusions. If one of these events does not occur, then the entire act of natural in-situ preservation of the carotenoid pigments cannot transpire. The deep purple and red pigments can remain over geologic time and the evaporite minerals can remain intact if no further waters remain in close proximity after precipitation to re-dissolve the parent mineral matrix. The pigments act as the physical biosignature, easily seen with the naked eye and potentially from rover and lander cameras on Mars (albeit Martian dust will definitely obstruct mm-scale features). Furthermore, the fluid inclusions can act as safe harbors for cellular life where chemical biomarkers can reside in and be a robust set of macromolecules for evidence of extinct or extant life. Perl, S.M. Biogenic Detection in Evaporites 154 Should these physical and chemical features occur simultaneously and remain preserved over geologic time then the interpretation of geologically old evaporites, their visual analysis, and chemical interpretation, should fall into the following four categories (Figure 6): • Biotic – a feature or measurement that would only exist if biology generated it or it was undoubtedly modified by life. Without the processes from life this measurement or feature would not exist (e.g., carotenoid pigments). • Biogenic – a feature or measurement that is found with relationships to biological processes but may exist without the influence of life. This feature would look different depending on the type of biological processes involved. • Abiogenic – a feature or measurement that is found equally with and without associations to biological processes with its existence not contributing to any biomarker or biosignature. • Abiotic – a feature or measurement that has no relationship to life at all and whose existence would be identical with or without the presence of biology or biological processes and has undeniably no relationship to biology past or present These categories can be heavily dependent on the terrestrial in-situ setting that life and its habitats would utilize for nutrient cycling (i.e., the Fe mineralogy used by Fe-reducing bacteria, or the elemental carbon or sulfur in fluid inclusions for entombed halobacteria, are some examples). For planetary exploration on Mars into outcrops and features laid down by flowing ancient waters these features could have contributed to extant life on the planet (if it existed in the first place) but by themselves are not enough evidence that it is even biotic. For example, as stated in Chapter 3, you can measure ancient fluid inclusions that have not been modified since mineral formation but that does not mean you will find microbial life in these inclusions. Furthermore, just because there has been widespread evaporite mineralogy seen globally across Mars by CRISM does not conclude that each and every pixel of hydrated mineralogy observed from orbit contain fluid inclusions nor would it ever be dateable from such a distance and resolution. Perl, S.M. Biogenic Detection in Evaporites 155 Results The proposed nomenclature and its application to geobiology would yield proper definitions for what is and is not due to the biological processes and the presence of life (or lack thereof). While not put into practice yet, these definitions will be used in the forthcoming publications that will stem from this dissertation. As stated in the previous section, the usage of these definitions will vary based on the field site or sample analysis (in-situ or laboratory), or resolution of life detection. The following examples are the results of how this nomenclature is already being utilized (Figure 7) and why the necessary updates for astrobiology nomenclature are timely and necessary (Figure 8). Potential Active Hypersaline Brines on Modern Mars Recent evidence of potentially seasonally flowing brine fluids have been observed by CRISM in the form of the Recurring Slope Lineae (RSL) (Ojha et al. 2015). These dark slope streaks seem to become elongated over several Martian years on crater walls of steep angles of repose leading into the possibility of these streaks being sourced by a highly viscous brine that is extends during the warmer Martian seasons and remains at its previous length during the colder months. The survivability and somewhat stable nature of potential surface brines bodes well for subsurface fluidic flow where more recent evaporite mineralogy may be precipitated on modern Mars. In a saturated brine water molecules that interact with ions are less available to support life (as we know it) and some have theorized that life cannot tolerate the saturated acidic Martian brines (Tosca et al. 2008) however there is ample evidence from several hypersaline environments where halophilic organisms that are able to maintain homeostasis and thrive just as non-extreme microorganisms can despite the lower aw and acidic saline lakes (Fendrihan et al. 2012; Benison et al. 2008). Significant comparisons between ancient saline lake deposits on Earth with those of the late Noachian/early Hesperian lakes on Mars (Benison, 2006). Perl, S.M. Biogenic Detection in Evaporites 156 Terrestrial salt lakes include more neutral or basic fluids (such as GSL) and those remain strong analogues to understanding how life can thrive in high salt settings with many of these overlapping features occurring in the Burns Formation fluids when surface waters were stable (Figure 8). Bio-indicators for Exoplanet Habitable Worlds and Life Detection In recent years with the ability for far-observing space telescopes to make transit observations of planets outside our solar system, exoplanet discovery has become a disciple within astrobiology all its own. Datasets have used the “habitable zone” set of characteristics to measure an exoplanet’s atmosphere and star type to determine how habitable an exoplanet is. Moreover the increased ability to measure gas content in an exoplanet atmosphere has led to the term “bio-indicators” where triplet peaks of CH3, O2, and O3 are directly measured from an exoplanet VNIR and IR spectrometer across vast distances of space (Figure 10). The term bioindictator is used freely in exoplanet science and publications who use the links between methane, oxygen, and ozone on our own planet to claim that these readings may have an exoplanet biological source or that life could likely live on such a planet. There are many caveats in exoplanet bioindictators that are loosely explained and features that would separate non-Earth biological atmospheric peaks would look the same as abiogenic gas sources even if the atmospheric sample were in our solar system. This is also the case for positive detections of methane on Mars having a abiotic source due to seasonal and yearly amounts being fairly consistent to each other (Yung et al. 2018). If biological CH3 and O2 atmospheric gases would look the same as abiogenic peaks in VNIR and IR spectroscopy then the claims for biogenic detections, especially from such distances, fall short of the proper burden of proof. While they are a step in the right direction far too easily is terminology created to meet the shortcomings of data interpretation and an overall lack of a proper sample size. Perl, S.M. Biogenic Detection in Evaporites 157 To assist exoplanet efforts such as this and to link true biogenic observations for valid geobiological sample analyses on Earth work is under way to make gas output measurements from bacteria that are grown within closed bottle experiments to replicate small scale columnar atmospheres (Kataria and Perl, 2019) in which biogenic gas outputs are measured with VNIR and IR spectroscopy. While the resulting peaks may be similar to abiotic gases, the magnitude of gas from microorganisms will be modeled to the sizes of what would be in an atmosphere to determine how much source biology would be needed to create a measureable atmospheric set of peaks as a function of biogenic metabolism vs. abiogenic atmospheric compositions. Organic vs. Biological Feedback from Electrochemical Impedance Spectroscopy (EIS) Life detection instruments will play a large role in upcoming missions after the Mars 2020 rover mission launching next year. The interpretation of data from these payloads will be the deciding factors in understanding which direction and location future missions are directed to and what they will measure. One of the main results that the 2011 Mars Science Laboratory has concluded that there is evidence of carbon, hydrogen, nitrogen, oxygen, phosphorus, and sulfur on Mars at present and that the likely timeframe of their formation came from when there were stable surface waters and the early Noachian atmosphere was able to help retain that stability (Figure 1). While the discovery of these organic molecules is critical for understanding the planetary evolution of Mars it does not conclusive evidence that life was ever present on the planet. The idea of organic detections being a stepping stone to life detection would only yield positive results on a planet that we know is already inhabited by life. To assist in separating organic detections from biological ones the use of electrochemical impedance spectroscopy (EIS) is being used to show how the measured conductance of cellular life differs from organic macromolecules (Figure 11). Specifically amino acids glycine and alanine by themselves Perl, S.M. Biogenic Detection in Evaporites 158 compared to bacteria that have these amino acids as part of their cellular composition. Cole et al. (2015) shows that lysed & dead cells along with solid particles allow electrochemical impedance to permeate through their structures while intact cells entrap ions which polarize them. The “distance” of this EIS non-permeability is being investigated to show how separations between organic molecules alone and intact cellular structures can be differentiated in soils and ice mediums (Chin, Perl, et al. 2019) and how unknown macromolecules can be deciphered using a database of planetary analogue materials and samples. Discussion The hybrid nature of geobiology has allowed for interpretations into mineral-microbial interactions from the perspectives of terrestrial microbial ecology and evolution to be studied as a reference for life as we don’t know it. Up until now the lack of a utilized nomenclature has led many abiotic and abiogenic features to be misclassified as potentially being modified from life, younger contamiated features into ancient mineralogy (Vreeland et al. 2000), or unitless measurements of habitability that do not take into account evolution and adaptation (Figs. 7,8). On Earth and yet to be discovered planets that harbor life, it would be a valid account to state that biology, acting faster than geology, can adapt to planetary changes that would occur over geologic time (Figure 9). Should those changes continue to propagate over geologic periods and globally those differences in habitability not changing too significantly, life as we don’t know it should be observable on a larger scale than the approach that planetary missions have taken in the last five decades. While the official statement from Lourens Baas Becking may have been lost to time, they are loosely quoted (Witt & Bouvier, 2006) as saying, “Everything is everywhere, but the environment selects”. While this was largely meant for terrestrial biology the application of this to our solar system and to the universe as a whole provides an interesting Perl, S.M. Biogenic Detection in Evaporites 159 paradox for life detection and for our perspectives for search for life as we don’t know it. It is this author’s viewpoint that terrestrial microbial evolution at present is not a sufficient metric for life elsewhere. Stated differently, we as a science community should never seek macromolecules such as DNA outside of our own terrestrial biomes. DNA is a product of Earth evolution and the eventual output original Last Universal Common Ancestor (LUCA) on our own world. The more specific and close to our present day evolutionary markers we get, the more distant we would be from a separate evolutionary pathway or separate tree of life whose evolution would be different both from the standpoint of a non-Earth LUCA and from different temporal, biogeochemical, and planet-wide trajectories. In short life has evolved on Earth due to the geological, chemical, and environmental histories that our planet experienced. During these events, early cellular life as we know it, evolved and responded to changes to our planet over time and led to the genetic makeups and ecological systems as we know them today. To try to search for those same systems outside of Earth makes the incorrect assumption that other planets and moons shared the same LUCA, the same planetary evolution, and the same microbial response over geologic time. Conclusions The need for nomenclature to study astrobiological features potentially generated or modified by non-Earth biological processes is paramount for the proper interpretation needed for future data analysis of unknown sample being returned from Mars withn the next decade. While no concrete plans have been set for Stages 2 and 3 of Mars Sample Return (MSR), the first stage of this MSR mission will be when samples are collected by the Mars 2020 rover. Stage 2 involves retrieving the samples from the Martian surface and sending them into Mars orbit. Stage 3 is then tasked with sending those samples from Mars orbit back to Earth for analysis. These samples will be small in size and at the time of this writing will be ~15x10x5cm in volume. It is still unknown how these samples will be analyzed Perl, S.M. Biogenic Detection in Evaporites 160 if they are able to make it back to Earth intact and what types of geobiological and microbiological laboratory work will be conducted on them to determine if life ever was present on Mars. While the types of analyses that should be done would be very similar to what we currently do for that may not yield the results that we would expect. Our modern day laboratories are geared toward life as we know it. Prior to any Earth analyses on Martian samples these soil (and hopefully evaporite mineral) samples should be inspected non-destructively and visually for any physical biosignatures that may have modified the mineral or sediment. Only after visual inspection and µ-scale assessment has been made should destructive chemical biomarker analysis take place. This can include liquid chromatography for any lipids preservation as well as potential metagenomics to see if we have contaminated the samples with any terrestrial byproducts of the initial sample capture on Mars. Would non-terrestrial life have the same common elemental chemistry as we know of on Earth? Is something as simple as organic carbon in an unique looking microstructure enough to prove that a feature is indeed biogenic and not just organic? If we go back to the Baas-Becking hypothesis we would conclude that we would not have to look very hard if we had a hand sample from another planet that was teaming with life. We should see its unique properties all over the sample much like algae atop a pond or worms underneath in soil underneath a rock. Does this mean that since see nothing alive on the surface of Mars that there is no life there now or nothing biological ever present? The lack of evidence on the surface of Mars does not infer anything for the shallow subsurface or even deeper subsurface regions km below the Martian crust. We do know that groundwaters at pH ranges from ~2.3 to near neutral levels in Meridiani Planum and Gale Crater, respectively had the ability and vertical range to make their way through permeable sedimentary rock and breach the crust. We also know that some of the sources of these fluids were closed basin lake systems where salinity was likely tenfold higher than Earth marine waters are now. If life were ever present on Mars and it resided in Perl, S.M. Biogenic Detection in Evaporites 161 these closed basin systems then the eventual downward movement of these ancient waters after the loss of the Martian atmosphere would have provided a safe haven from the impending UV-C and global desiccation that would eventually occur over the next ~3.5 Gyr into the Amazonian. This aqueous downwelling and likely hypersaline values in these waters would have led to significant precipitation of sub-crustal evaporite layers where water became stagnant and even deeper still. If cellular life utilized the current categories of habitability (Figure 7), and these categories overlapped each other (Figure 8) then that would yield a high probability that preservation could have occurred within subsurface evaporite mineralogy away from the hostile surface. The best way forward for astrobiology is to integrate planetary geology with terrestrial microbiology in order for the communities to understand how each discipline formulates their research questions for the joint in-situ sample analysis and continued global observations of Mars. Should we ever find something in-situ that fulfills both the requirements for a physical biosignature and a chemical biomarker the very next question will involve identification and classification. In turn, this will lead to noteworthy questions involving Martian cellular life, metabolisms in the deep subsurface of Mars, and evolution outside of Earth. At this point, we would finally have a second data set to life not on our own planet, but in our solar system. Perl, S.M. Biogenic Detection in Evaporites 162 Acknowledgements I would like to thank Frank Corsetti personally for his perspective, inputs, and critical questions during my time in USC Geobiology as a doctoral student and candidate. When looking for an advisor when I was only a few months into starting my tenure at JPL I wanted someone who I could I work with, discuss, and debate my scientific questions with in a constructive and insightful manner. Someone who would take the intellectual time to deliberate and give proper thought to my scientific questions and curiosities. In 2012 when I met Frank I was naïve to how I would balance my work life at JPL and my academic life at USC. I had 2-3 large scale ideas and methods for how I would go about my doctorate work. The research questions remained the same but evolved (as I know now) to match the data, application, and contribution of the work. The separate mindsets of JPL and academia became clearer as the months and years progressed and Frank’s personality and adaptability to my work commitments allowed for my own self-reflectance to grow and solve unique problems that came about. Thankfully, as my separate worlds began to merge to the point where my academic work now became what I was, and am now, actually funded to do, Frank was a constant throughout. I am extremely grateful for that. Perl, S.M. Biogenic Detection in Evaporites 163 References Andrews-Hanna, J.C., M.T. Zuber, R.E. Arvidson, and S.M. Wiseman (2010), "Early Mars Hydrology: Meridiani playa deposits and the sedimentary record of Arabia Terra", J. Geophys. Res., 115, E06002, doi:10.1029/2009JE003485. Aubrey, A.D., Cleaves, H.J. & Bada, J.L. 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Biogenic Detection in Evaporites 172 Figures Mars Earth Time (Gyr) 4.0 Noachian Pre-Noachian? Early Mid. Late Hesperian Early Late Amazonian Early Middle Late 3.0 2.0 1.0 Hadean Archean Proterozoic Phanerozoic Eoarchean Paleoarchean Mesoarchean Neoarchean Paleoproterozoic Mesoproterozoic Neoproterozoic Paleozoic Mesozoic Ceno- zoic Complex life Eukaryotes Photosynthesis Prokaryotes Deep Alteration ------------------------------------------? -Late Heavy Bombardment--- ------Layered clays-----------------------------? -Late Heavy Bombardment--- ?------Layered sulfates---------------------?--------------------------------?-------------------------? ?-----Polar sulfates Burns Formation (Meridiani Planum) ALH84001 Nakhlites Shergottites Columbia Hills (Inner Basin) Gusev Plains -------------------------------------------------------Anhydrous ferric oxides------------------------------------------------------ Figure 1. Comparisons of the origins of life on Earth and history of hydrated mineral and sedimentary features on Mars. Relationship between habitable periods on Earth and Mars. During the periods when the first life evolved on Earth, the Martian surface was warmer, wetter, and habitable. The age of sedimentary rocks from the Opportunity rover landing site are ~3.5 Gyr (late Noachian/early Hesperian). It is also in this time period, where the surface of Mars likely had water stable on the surface (Squyres et al. 2005, McLennan et al. 2005) in liquid form versus recent discoveries of surface brine fluids on present-day slopes (Ohja et al. 2015). Perl, S.M. Biogenic Detection in Evaporites 173 Figure 2. Mineral signature of the Fe-oxide hematite in Meridiani Planum. The TES spectral signature of Fe2O3 that led the MER rover Opportunity to land in what would be later labeled the Burns Formation set of rocks in Eagle and Endurance craters on Mars. The justification for this landing site was due to hematite forming on Earth with relationships to hot springs and standing bodies of water. Perl, S.M. Biogenic Detection in Evaporites 174 Figure 3. Approximate location and stratigraphic column positions for rocks abraded by the RAT in the Karatepe section of Endurance crater. (a) Partial panorama of Endurance Crater with rock target locations shown with their designations on the lower left. (b) Single filter Pancam image of the Karatepe section showing all of the rocks in the Karatepe portion of the Burns Formation. (c) Magnified portions of the panorama to show the location of the Wharenhui rock that contains similar pore modification as seen around the Whatanga contact, southwest of Burns Cliff (see McLennan & Grotzinger, 2008 for stratigraphic unit boundaries). (a) (b (c) Perl, S.M. Biogenic Detection in Evaporites 175 Figure 4. The Karatepe section of Burns Cliff within Endurance crater, part of the Burns fm. set of rocks. Abraded rocks that make up ~7m of vertical sedimentology showing where fluidic flow in the form of groundwater recharge flowed upwards and dissolved Mg- and Fe-sulfate salts. Later modification occurred after the original secondary pore spaces were defined by the stagnant groundwater table in-between the middle and upper units of the Whatanga contact. Perl, S.M. Biogenic Detection in Evaporites 176 Figure 5. Scanning Electron Micrograph (SEM) image of the Alan Hills meteorite (ALH84001). McKay et al. (1996) let the investigation to the physical evidence of life potentially from Mars using visual evidence only. While on its own the features can appear biological in nature without further microbiological lab analyses or even organic separation technique done on this same sample, evidence was not conducive to validating that this was an extraterrestrial microbe. Perl, S.M. Biogenic Detection in Evaporites 177 Figure 6. “Pendulum” diagram showing examples and definitions of the proposed astrobiology nomenclature for a NaCl hopper crystal. The example above is for a single pigmented halite hopper crystal (biogenic) and then brought from left-to-right showing how features lose their biological validity due to the definitions proposed in the Methods section of Chapter 4. Biotic Biogenic Abiogenic Abiotic • DNA and measured preserved OTUs • Carotenoid pigments from biological processes • Gradients in elemental observed chemistry used for nutrient cycling in fluid inclusions • Elemental chemistry present in fluid inclusions • Entombed fluids that are from the original evaporite precipitation • The evaporite mineralogy and fluid inclusions, if present • Trace minerals within evaporites 0% 20% 40% 60% 80% 100% Halite and Gypsum Evaporite OTU Abundances (by site) Biology or biological processes Not from life (as we know it) (as we know it) Perl, S.M. Biogenic Detection in Evaporites 178 Figure 7. The classical interpretation of habitability for planetary systems. Proposed by DesMarais, Buz, Perl, Freissenet, Slipski, Mickol, and Shkolyar (2014) this represents a perspective for measuring how habitable a location and measurable chemistries and environmental features are. This represents a geobiological issue for astrobiology due to the lack of any temporal overlap between these four conditions and states. Figure 8 on the next page presents an updated viewpoint of this outdated venn diagram. From (https://www.hou.usra.edu/meetings/8thmars2014/presentations/8th%20Mars%20Conferen ce%20Synthesis.pdf) Perl, S.M. Biogenic Detection in Evaporites 179 Figure 8. A proposed update to the classical perspective of habitability for planetary systems. The previous Figure 7 has been used for discussions into Martian habitability and taking global and local measurements from orbiters, rovers, and landers to determine how “habitable” a location was. That former assessment did not taken into account the need for the four environmental and aqueous features to overlap in time. It would only be the timeframe of the combined overlap of all four products that life, as we know it, would have the highest probability for survival after a separate origins and last universal common ancestor should microbial evolution took place outside of Earth. “t 0 ” Presence and proximity of C,H,O,N,P ,S Relevant solvent (likely water) for medium Energy for microbial activity (photo or chemical) Environmental settings (pH, temperature, etc.) Timeframe for “ h a b i t a b i l i t y ” Perl, S.M. Biogenic Detection in Evaporites 180 Figure 9. The evolution of the interpretation of microbial ecology. (Left) Phenotype- based, 1879 showing the “Pedigree of Man” and the links between humankind and other forms of life (Top) 16S-based “Trees of Life” noted by Woese et al. (1980) showing Bacteria, Archaea, and Eucarya. (Bottom) Updated metagenomic-based tree of life from Hug et al. (2016). Perl, S.M. Biogenic Detection in Evaporites 181 Figure 10. Bayesian framework for exoplanet spectral bio-indicator assessment (from Catling et al. 2017) . Exoplanet spectral and photometric data that may contain bio- indictators are used with models to measure the probabilities of life on a planet in the habitable zone and an exoplanet that has no life. Input variables are only from exoplanet spectra and do not use any biological feedback into the model. Moreover this type of decision process or conditional probability has no measureable scope for biogenic atmospheres and ones that naturally contain CH3, O2 and O3 naturally without life. Perl, S.M. Biogenic Detection in Evaporites 182 Figure 11. Electrochemical impedance relationships between living microbes and dead cells. During EIS activity when fields are active in a substrate containing living and dead microorganisms the ions of the cell medium migrate towards the opposite charge. This polarizes the cells and cell wall whereas dead/lysed cells do not store such a charge. Perl, S.M. Biogenic Detection in Evaporites 183 Appendix DNA Extraction alongside salt inhibition After collection samples were analyzed for bulk DNA using a ThermoFisher QuBit Flourometer 4. After DNA yields were established the solid NaCl and gypsum samples were dissolved in Invitrogen UltraPure DNase/RNase-Free Distilled Water and concentrated using Ultra Fine filters within the Innovaprep Concentrating Pipette Select system. Due to the high salinity it took a significant amount of time to concentrate samples. After concentration, samples were eluted and cold stored for eventual 16S gene sequencing. Metadata files were extracted using open-source QIIME software and Operational Taxonomic Unit (OTU) bar charts were generated directly from queued .fastq files based on the research questions needed between the seven GSL sites, the evaporite minerals, features (pigments, clays, etc,) in the evaporite minerals, and cumulative values independent of site location. DNA Extraction, qPCR Verification, and Sequencing Due to recognized salt inhibition of DNA extraction (Stevens, 1977; Redon and Bonner, 2011, among others) we performed a custom saline validation experiment using ZymoBIOMICS Microbial DNA Community standard (MMC). Selected qPCR verified in-situ samples were spiked with the DNA MMC standard and compared to the original in-situ samples. It was found that the ~91% of the in-situ preserved DNA was inhibited naturally by the hypersaline salts preserving the microbial signatures of the lake DNA entombed within the crystal structure (Appendix Figs. 3-8). Furthermore, we concluded that the original qPCR measurements were significantly obscured by the saline mineralogy. Based on this, we concentrated and filtered the samples, keeping the sample type and dissolution medium (if needed) uniform throughout each process without losing potential Perl, S.M. Biogenic Detection in Evaporites 184 biogenic information due to underexposure or consequently reducing our yield from the actual process. The samples were divided based on estimated yield of DNA, site location within the north arm of the Great Salt Lake field site, and sample type (halite hopper crystals, gypsum crystals and clays, halite crystal fluids, and Great Salt Lake brine). Samples were suspended in 200 ml of sterile ultrapure water (Invitrogen) in 500 ml Corning bottles, they were subsequently dissolved under shaking conditions (200 rpm overnight), once samples were dissolved, they were filtered through UltraFine micron filters using the InnovaPrep concentrating pipette (CP Select). Concentrated solution was eluted into 300µl of elution fluid using 3 pumps. Subsequently the eluted concentrate was used to extract DNA using the MoBio Powersoil DNA extraction kit (Cat # 1288-100). DNA was quantified using the Promega Quantus fluorometer (version 4). DNA was then frozen at -80°C until it was shipped to Chun Lab LLC. The “which regions were amplified” hyper variable regions were amplified using kit primers and PCR conditions “put in conditions of PCR”. Sequencing was performed on an Illumina Mi-Seq, resulting in 58 raw reads. Reads were subsequently trimmed and adapters were removed using the CLC Genomics Workbench (put in version number). Filtered reads were analyzed using local BLAST against the SILVA LTP type strain database (put in version). Plots were generated using QIIME and Principal Components Analysis (PCA) was performed using the CLC Genomics Workbench. Mineral vs. Fluid Preservation of DNA & Optimal Biomass Preservation Due to the preservation of DNA in the mineral matrix as well as the fluid inclusions within there are accompanying Raman peaks of the carotenoid biomarkers in varying concentrations. The fluid inclusions have in general have higher concentrations of these pigmented biomarkers in a Perl, S.M. Biogenic Detection in Evaporites 185 given area but high magnitudes of the β-carotene and bacterioruberin carotenoids can be found in just the mineral matrix as well. Comparison of Preserved DNA Yields and Raman Spectra of Biomarkers The major difference between the preserved taxonomic unit OTU yields and the Raman biomarker spectral data are the distributions of the carotenoids. While Raman analysis of motile halophilic microorganisms always showed β-carotene and bacterioruberin spectra (with the higher carotenoid volumes showing a harmonic reflection in peaks >2000cm -1 ) these spectra were also found in many regions where halophilic microorganisms were not present. This implies that during the evaporite precipitation, the halophilic organisms were still producing these biomarkers and the rate in which this production occurred was faster than the precipitation kinetics of the evaporite. The fact that the pigments are observed on the mm-scale from microbial community cell concentrations ranging from 10 1 in the lowest fluid inclusions to 10 7 in the highest concentration in an inclusion that is on average from 2x4µm to 6x10µm is critical for understanding rates of biomarker production and evaporite pigment staining. This was calculated via a digital cytometer on a Olympus digital and fluorescence microscope over several hour periods of time. Preserved Halophilic Microorganism Communities Appendix Figure 3 shows the 16S rRNA gene sequencing results for the Great Salt Lake waters, gypsum crystals, extracted gypsum clays from the host crystals, non-pigmented halite crystals, pigmented halite, and extracted halite fluid inclusions. Seven sites along the north arm of Great Salt Lake were selected and DNA was extracted, qPCR was executed, and samples were sequenced. Table 1 shows all the evaporite categories by each site that samples were collected from. Gypsum had only one major site near the Spiral Jetty. Appendix Figure 4 shows the OTUs Perl, S.M. Biogenic Detection in Evaporites 186 of only site-specific halite evaporites with the halite environmental control. Appendix Figure 5 takes the previous figure data and illustrates the cumulative halite minerals by pigment or lack thereof. Appendix Figure 7 shows the cumulative gypsum evaporite minerals and illustrates gypsum with and without the Fe-rich clays that were observed. Appendix Figure 8 shows the OTUs of all halite, gypsum, (by category), lake water, and environmental controls for each evaporite set. Appendix Figure 2 along with Appendix Table 1 illustrates the differences between sample volumes and total DNA preservation. Moreover, halite fluid inclusions tend to preserve more DNA than the parent halite minerals. Sequenced DNA within these evaporites show the microbial communities preserved. Appendix Figures 6 and 7 shows Operational Taxonomic Unit (OTU) of the halite groups (including environmental control) and the OTUs for the gypsum groups (including environmental control). Appendix Figure 9 shows a three-dimensional and three component Bray-Curtis Principal Component Analysis (PCoA), respectively, of both the 16S rRNA gene sequence metadata of the cumulative taxonomic units preserved in the halite and gypsum samples without environmental controls to highlight differences in individual evaporites. Note the three-component version includes the environmental controls separately for both evaporite sample sets. Note that ~64,000 sequences were recovered and of those, 21,000 were assigned to novel halophilic bacteria and archaea species northern arm of the Great Salt Lake. As with most extreme environments, the volume of microbial communities and diversity is lower than compared to “normal” salinity environments. This is observed in the total sequences in the hypersaline environments in the Great Salt Lake samples. To ensure the microbial communities are assessed per evaporite category properly we have employed PCoA measurements both as a validation and as a calculation for differences in taxa as they are preserved in the modern environment. It should be noted that the control groups for the both evaporite categories, halite Perl, S.M. Biogenic Detection in Evaporites 187 and gypsum independently from each other, while all plotting together per evaporite group (Figure 14). Furthermore the environmental control groups are separated from the taxa of the regular in- situ samples showing that our methodology for DNA extraction and sequencing work but that it can accurately calculate the taxa differences and be able to rank the preservation potentials for the mineral intercrystalline and intracrystalline structures, clays, and evaporite matrices themselves. Conclusions (Evaporite preservation as a function of OTU) The fact that there are different volumes to the preserved bacteria and archaea in the evaporite sets speak to the fluid volumes required to precipitate these minerals in-situ. The variation in fluid volumes and mineral/chemical components in each evaporite category and the individual volumes of preserved microorganisms shows a preference on what the halobacteria and haloarchaea are using to maintain their metabolic processes. Since after mineral entombment the microorganisms are still active and motile, they will continue their processes now in the preserved settings (as opposed to when they were in the lake water column) and can continue gain nutrients from their new fluid inclusion environment. It is critical that visual assessment also includes a biological response to stimulus inputted into the fluid or sample in question, multiple observations showing potential halophilic bacteria (or other domain) over substantial time (minutes to hours) and over multiple samples from same in-situ site that were collected under sterile conditions such that no contamination or cross- contamination occurred from handling. Dormant microorganisms may not be as motile as high- velocity swimming organisms and those that are able to achieve their nutrients from the water column or fluid inclusion environment without significant motility may swum slower or not at all. Perl, S.M. Biogenic Detection in Evaporites 188 Conclusions (DNA-related only) Moreover, the changes in OTU volumes and microbial community differences speak to the potential metabolisms the bacteria are utilizing. While the robustness of these features over geologic time on Earth and Mars will be discussed in the next chapter, the initial/modern preservation of validated biogenic matter in these notable crystalline minerals gives us a real-time stopwatch for how long active biology can be maintained. Perl, S.M. Biogenic Detection in Evaporites 189 Appendix Figures Sample A* MMC- spiked (A) Sample (B) In-situ gypsum Sample B* MMC- spiked (B) Sample (C) In-situ pigmented halite hopper Sample C* MMC-spiked (C) MMC only Negative Control Sample (A) In-situ halite hopper fluids Appendix Figure 1. Evaporite salt inhibition validation study results showing selected field samples spiked with ZymoBIOMICS DNA community standards (MMC). The x-axis shows the samples vs. the mg/ml quantity of measured DNA on the y-axis. Validation samples A, B, and C are unmodified Great Salt Lake precipitated evaporites halite, trapped terrace halite fluids, and gypsum. Validation samples A*, B*, and C* are spiked samples of their originals mixed with 50ul of MMC (as shown in sample D by itself). Solid portions of the bar graph show Quantitative polymerase chain reaction (qPCR)-verified DNA whereas dashed bars show the estimated preserved DNA without any natural saline inhibitors. The red-dashed lines show the expected DNA from the ZymoBIOMICS MMC standard based on the MMC-only value. This value was added to the spiked A*, B*, C* sample values to generate the red-dashed bar value. = Expected MMC + in-situ DNA sample = Measured DNA quantity Perl, S.M. Biogenic Detection in Evaporites 190 Appendix Figure 2. Total DNA (ng) as a function of evaporite sample (g) with environmental controls. Cumulative amounts are for all samples across all north arm Great Salt Lake sites. The environmental controls for the halite are the wet soils beneath the salt terrace whereas the environmental controls for the gypsum are the regolith surrounding and beneath the precipitated gypsum crystals. The purpose of the environmental control samples are to show how much DNA is found in the open environment that is not ecologically part of the preserved halite and gypsum evaporites. 0 500 1000 1500 2000 2500 DNA (ng) / Weight of Sample Processed (grams) Perl, S.M. Biogenic Detection in Evaporites 191 Evaporite Mineral Evaporite Properties In-situ Location Extraction Method Mineral sample size used in DNA extraction (g or ml) DNA Concentration (ng/µl) Total DNA (ng) Halite Non-Pigmented Site 1 Maxwell 51.45g 63.3 6330 Halite Non-Pigmented Site 1 Maxwell 51.63g 104.2 10420 Halite Non-pigmented Site 1 Maxwell 51.29g 79.15 7915 Halite Non-pigmented Site 7 Maxwell 50.00g 89.65 8965 Halite Non-pigmented Site 7 Maxwell 47.31g 49.20 4920 Halite Non-pigmented Site 7 Maxwell 56.28g 62.9 6290 Gypsum Non-clay S. Rozel Bay Maxwell 18.6g 146.9 14690 Gypsum Non-clay S. Rozel Bay Maxwell 19g 107 10700 Gypsum Non-clay S. Rozel Bay Maxwell 22.6g 72.1 7210 Gypsum Clays S. Rozel Bay Maxwell 9.04g 23.1 2310 Gypsum Clays S. Rozel Bay Maxwell 10.27g 69.3 6930 Gypsum Clays S. Rozel Bay Maxwell 8.85g 12.6 1260 Halite Non-pigmented Sites 3,4 Maxwell 50.48g 40.6 4060 Halite Non-pigmented Sites 3,4 Maxwell 53.47g 41.7 4170 Halite Non-pigmented Sites 3,4 Maxwell 49.03g 14.4 1440 Lake water (n/a) Site 1 Maxwell 250ml 134.85 13485 Lake water (n/a) Site 4 Maxwell 250ml 155.15 15515 Lake water (n/a) Site 7 Maxwell 250ml 71.25 7125 Halite Pigmented Site 1 Maxwell 49.96g 29.8 2980 Halite Pigmented Site 1 Maxwell 48.99g 31.45 3145 Halite Pigmented Site 1 Maxwell 52.97g 42 4200 Halite Pigmented Sites 3,4 Maxwell 52.48g 4.8 480 Halite Pigmented Sites 3,4 Maxwell 44.35g 29.1 2910 Halite Pigmented Sites 3,4 Maxwell 52.91g 75.35 7535 Halite Pigmented Site 7 Maxwell 52.18g 34.6 3460 Halite Pigmented Site 7 Maxwell 46.73g 13.2 1320 Halite Pigmented Site 7 Maxwell 53.08g 16.1 1610 Halite Terrace Pigmented Site 1 Maxwell 100ml 281.9 28190 Halite Terrace Pigmented Site 4 Maxwell 100ml 161.5 16150 Halite Terrace Pigmented Site 7 Maxwell 100ml 43.1 4310 Halite Envir. Control Sites 1,3,4,7 Power Soil 0.49g 5.5g 550 Halite Envir. Control Sites 1,3,4,7 Power Soil 0.46g 5.8g 580 Halite Envir. Control Sites 1,3,4,7 Power Soil 0.58g 6.9g 690 Halite Envir. Control Sites 1,3,4,7 Power Soil 0.51g 7.3g 730 Gypsum Envir. Control S. Rozel Bay Power Soil 0.47g 10.3g 1030 Gypsum Envir. Control S. Rozel Bay Power Soil 0.57g 8.5g 850 Gypsum Envir. Control S. Rozel Bay Power Soil 0.6g 7g 700 Gypsum Envir. Control S. Rozel Bay Power Soil 0.58g 6.3g 630 Appendix Table 1. Data table of Great Salt Lake evaporite samples used in 16S rRNA gene sequencing. All Maxwell extractions used 500ml of water to dissolve solid salts except for the gypsum clay samples which used 250ml. Since environmental controls were regolith samples from beneath evaporite sections ZymoBIOMICS Power Soil Extraction was used. If preliminary yields were too low than sites in close proximity were combined (i.e., sites 3 and 4). Total DNA in nanograms was calculated taking the product of the concentration amount (ng/µl) and the volume (100 µl). Complete data table can be found in Supplemental Data 1. Perl, S.M. Biogenic Detection in Evaporites 192 Halite (cumulative) 0% 20% 40% 60% 80% 100% Halite (cumulative) Gypsum (cumulative) Lake Water (cumulative) Cumulative OTU Summary of Halite vs. Gypsum vs Lake water k__Bacteria;p__[Thermi] k__Bacteria;p__WWE1 k__Bacteria;p__WS4 k__Bacteria;p__WS3 k__Bacteria;p__WS1 k__Bacteria;p__WPS-2 k__Bacteria;p__Verrucomicrobia k__Bacteria;p__Tenericutes k__Bacteria;p__TM7 k__Bacteria;p__TM6 k__Bacteria;p__Synergistetes k__Bacteria;p__Spirochaetes k__Bacteria;p__SR1 k__Bacteria;p__Proteobacteria k__Bacteria;p__Planctomycetes k__Bacteria;p__OP8 k__Bacteria;p__OP3 k__Bacteria;p__OP1 k__Bacteria;p__OD1 k__Bacteria;p__Nitrospirae k__Bacteria;p__NKB19 k__Bacteria;p__Lentisphaerae k__Bacteria;p__LD1 k__Bacteria;p__Gemmatimonadet es Gypsum (cumulative) GSL water (cumulative) Appendix Figure 3. Operational Taxonomic Units (OTUs) derived from 16S rRNA gene sequences sourced from Great Salt Lake halite salts, pigmented salts, non-pigmented halite, gypsum without entombed clays, gypsum with entombed clays, unfiltered lake waters, and environmental controls for both evaporite mineral sets. This figure shows all cumulative sequenced samples. Cumulative describes the average of all sites and evaporite category. OTU legend colors shown have the same microbial communities in Figs. 8-13. Perl, S.M. Biogenic Detection in Evaporites 193 0% 20% 40% 60% 80% 100% Halite (cumulative) Gypsum (cumulative) Lake Water (cumulative) Cumulative OTU Summary of Halite vs. Gypsum vs Lake water k__Bacteria;p__[Thermi] k__Bacteria;p__WWE1 k__Bacteria;p__WS4 k__Bacteria;p__WS3 k__Bacteria;p__WS1 k__Bacteria;p__WPS-2 k__Bacteria;p__Verrucomicrobia k__Bacteria;p__Tenericutes k__Bacteria;p__TM7 k__Bacteria;p__TM6 k__Bacteria;p__Synergistetes k__Bacteria;p__Spirochaetes k__Bacteria;p__SR1 k__Bacteria;p__Proteobacteria k__Bacteria;p__Planctomycetes k__Bacteria;p__OP8 k__Bacteria;p__OP3 k__Bacteria;p__OP1 k__Bacteria;p__OD1 k__Bacteria;p__Nitrospirae k__Bacteria;p__NKB19 k__Bacteria;p__Lentisphaerae k__Bacteria;p__LD1 k__Bacteria;p__Gemmatimonadet es 0% 20% 40% 60% 80% 100% Halite and Gypsum Evaporite OTU Abundances (by site) Halite (by site) Gypsum (by site) GSL water (by site) Appendix Figure 4. Operational Taxonomic Units (OTUs) derived from 16S rRNA gene sequences sourced from Great Salt Lake halite salts, pigmented salts, non-pigmented halite, gypsum with and without entombed clays, unfiltered lake waters, and environmental controls for both evaporite mineral sets. This figure shows all sequenced samples categorized by evaporite type only (cumulative of all sites). Perl, S.M. Biogenic Detection in Evaporites 194 0% 20% 40% 60% 80% 100% Halite (cumulative) Gypsum (cumulative) Lake Water (cumulative) Cumulative OTU Summary of Halite vs. Gypsum vs Lake water k__Bacteria;p__[Thermi] k__Bacteria;p__WWE1 k__Bacteria;p__WS4 k__Bacteria;p__WS3 k__Bacteria;p__WS1 k__Bacteria;p__WPS-2 k__Bacteria;p__Verrucomicrobia k__Bacteria;p__Tenericutes k__Bacteria;p__TM7 k__Bacteria;p__TM6 k__Bacteria;p__Synergistetes k__Bacteria;p__Spirochaetes k__Bacteria;p__SR1 k__Bacteria;p__Proteobacteria k__Bacteria;p__Planctomycetes k__Bacteria;p__OP8 k__Bacteria;p__OP3 k__Bacteria;p__OP1 k__Bacteria;p__OD1 k__Bacteria;p__Nitrospirae k__Bacteria;p__NKB19 k__Bacteria;p__Lentisphaerae k__Bacteria;p__LD1 k__Bacteria;p__Gemmatimonadet es Halite (by site) 0% 20% 40% 60% 80% 100% Halite (non- pigment) - Site 1 Halite (non- pigment) - Site 7 halite (non- pigment) - Sites 3 and 4 Halite (pigmented) - Site 1 halite (pigmented) - Site 3 and 4 halite (pigmented) site 7 halite (terrace fluids) - Site 1 halite (terrace fluids) - Site 4 halite (terrace fluids) - Site 7 Halite Environmental Control Halite OTU Abundance (by site) Appendix Figure 5. Operational Taxonomic Units (OTUs) derived from 16S rRNA gene sequences sourced from Great Salt Lake halite salts, pigmented salts, non-pigmented halite, and halite environmental controls for the halite evaporite suite by category and by site. Perl, S.M. Biogenic Detection in Evaporites 195 Appendix Figure 6. Operational Taxonomic Units (OTUs) derived from 16S rRNA gene sequences sourced from Great Salt Lake halite salts, pigmented salts, non-pigmented halite, and halite environmental controls for the halite evaporite suite by category. 0% 20% 40% 60% 80% 100% Halite (non-pigment) Halite (pigmented) Halite (terrace fluids) Halite Environmental Control Halite OTU Abundances (by category) Halite (by category) Perl, S.M. Biogenic Detection in Evaporites 196 0% 20% 40% 60% 80% 100% Halite (cumulative) Gypsum (cumulative) Lake Water (cumulative) Cumulative OTU Summary of Halite vs. Gypsum vs Lake water k__Bacteria;p__[Thermi] k__Bacteria;p__WWE1 k__Bacteria;p__WS4 k__Bacteria;p__WS3 k__Bacteria;p__WS1 k__Bacteria;p__WPS-2 k__Bacteria;p__Verrucomicrobia k__Bacteria;p__Tenericutes k__Bacteria;p__TM7 k__Bacteria;p__TM6 k__Bacteria;p__Synergistetes k__Bacteria;p__Spirochaetes k__Bacteria;p__SR1 k__Bacteria;p__Proteobacteria k__Bacteria;p__Planctomycetes k__Bacteria;p__OP8 k__Bacteria;p__OP3 k__Bacteria;p__OP1 k__Bacteria;p__OD1 k__Bacteria;p__Nitrospirae k__Bacteria;p__NKB19 k__Bacteria;p__Lentisphaerae k__Bacteria;p__LD1 k__Bacteria;p__Gemmatimonadet es Gypsum (by category) 0% 20% 40% 60% 80% 100% Gypsum (no clays) Gypsum (with clays) Gypsum Environmental Control Gypsum OTU Abundances (by category) Appendix Figure 7. Operational Taxonomic Units (OTUs) derived from 16S rRNA gene sequences sourced from Great Salt Lake gypsum crystals with clays removed, gypsum crystals with clays still entombed, and gypsum environmental controls. Perl, S.M. Biogenic Detection in Evaporites 197 Halite (cumulative by category) 0% 20% 40% 60% 80% 100% Halite (cumulative) Gypsum (cumulative) Lake Water (cumulative) Cumulative OTU Summary of Halite vs. Gypsum vs Lake water k__Bacteria;p__[Thermi] k__Bacteria;p__WWE1 k__Bacteria;p__WS4 k__Bacteria;p__WS3 k__Bacteria;p__WS1 k__Bacteria;p__WPS-2 k__Bacteria;p__Verrucomicrobia k__Bacteria;p__Tenericutes k__Bacteria;p__TM7 k__Bacteria;p__TM6 k__Bacteria;p__Synergistetes k__Bacteria;p__Spirochaetes k__Bacteria;p__SR1 k__Bacteria;p__Proteobacteria k__Bacteria;p__Planctomycetes k__Bacteria;p__OP8 k__Bacteria;p__OP3 k__Bacteria;p__OP1 k__Bacteria;p__OD1 k__Bacteria;p__Nitrospirae k__Bacteria;p__NKB19 k__Bacteria;p__Lentisphaerae k__Bacteria;p__LD1 k__Bacteria;p__Gemmatimonadet es Gypsum (cumulative by category) GSL water (cumulative by category) 0% 20% 40% 60% 80% 100% Halite (non-pigment) Halite (pigmented) Halite (terrace fluids) Halite Environmental Control Gypsum (no clays) Gypsum (with clays) Gypsum Environmental Control Lake waters Halite vs. Gypsum OTU Abundances (cumulative by type) Appendix Figure 8. Operational Taxonomic Units (OTUs) derived from 16S rRNA gene sequences sourced from Great Salt Lake gypsum crystals with entombed clays, gypsum with clays removed, and gypsum environmental controls alongside the pigmented halite, non-pigment halite, and halite environmental controls. These are the cumulative OTU volumes by evaporite category. Perl, S.M. Biogenic Detection in Evaporites 198 Appendix Figure 9. Validation of an uncontaminated DNA extraction using three- dimensional Bray-Curtis PCoA for both evaporite categories observed in the Great Salt Lake. (A) For the halite suite: pigmented halite samples are shown as the pink dot, non- pigmented halite samples are shown as gold dots, and halite fluids are shown as blue dots. Halite environmental controls are shown as green dots. Note that the all four environmental controls (left side) fall in the same categories. (B) For the gypsum suite: gypsum crystals with clays removed are shown as red dots (note their orientation close to each other and far removed from other taxonomic sets), gypsum clays are shown as light green dots, and gypsum environmental controls are shown as purple dots.

Abstract (if available)

Abstract Evaporite minerals can capture and entomb biology and organic matter within their intercrystalline and intracrystalline structure because they precipitate relatively quickly. Thus, evaporite minerals constitute a target for biosignature investigation on Earth and Mars, where evaporitic deposits are known to exist. However, little is known about the process of organic preservation and detection in evaporites, or the stability of such molecules when exposed to significant UV radiation (as would be present on the surface of Mars). Halite and gypsum have been chosen for investigation because they have been detected on Mars by the MRO Compact Reconnaissance Imaging Spectrometer for Mars (CRISM) instrument, their physical transparency and short-term precipitation timescales, which are amenable to experimentation. Halite and other sulfate salts have additionally been observed within the shallow subsurface of Mars in proximity to ancient aqueous settings either via groundwater or evaporated lake beds, and thus these minerals are relevant to Mars investigations. Moreover, the current in-situ observations on mineral veins on Mars that show these hydrated features from ancient surface and groundwaters are of significant height and size, being able to survive physically over geologic time. Moreover these meter-scale regions can provide ample sample size for future Mars Sample Return analyses from due to these proven sites containing hydrated mineralogy and from direct exposure to ancient fluids and water-mineral interactions. Should cellular life have existed in ancient or modern saline waters on Mars and Europa, respectively, their preservation within evaporite minerals should be a high priority target for life detection missions and in-situ analyses.

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Asset Metadata

Creator Perl, Scott Michael (author)

Core Title Quantifying the threshold of biogenic detection in evaporites: constraining potential Martian biomarker preservation

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Geological Sciences

Publication Date 06/06/2021

Defense Date 12/06/2019

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag astrobiology,carotenoids,closed-basin lake systems,Europa,evaporites,fluid inclusions,Geobiology,groundwater,gypsum,halite,halophiles,hypersaline,life detection,Mars,OAI-PMH Harvest,Raman,salt,UV-C radiation

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Corsetti, Frank (committee chair), Berelson, William (committee member), Bottjer, Dave (committee member), Caron, Dave (committee member), Celestian, Aaron (committee member)

Creator Email scottmpe@usc.edu,scottperl@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-247726

Unique identifier UC11673601

Identifier etd-PerlScottM-8022.pdf (filename),usctheses-c89-247726 (legacy record id)

Legacy Identifier etd-PerlScottM-8022.pdf

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Document Type Dissertation

Rights Perl, Scott Michael

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Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA