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Spectral Signs of Life in Ice

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Spectral Signs of Life in Ice SPECTRAL SIGNS OF LIFE IN ICE by Mitch Wade Messmer A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Chemical Engineering MONTANA STATE UNIVERSITY Bozeman, Montana April 2020 ©COPYRIGHT by Mitch Wade Messmer 2020 All Rights Reserved ii ACKNOWLEDGEMENTS I would like to thank my Principal Investigator, Dr. Christine Foreman, for all her guidance and for the incredible opportunity to be a part of the Foreman Lab. I would also like to thank my committee members, Dr. Heidi Smith and Dr. Robin Gerlach for their help and support throughout my research. I would like to express my thanks to Markus Dieser for his assistance in the lab, and for all the skills that he taught me. Additionally, I would like to thank Dr. Al Parker for his insight into the statistical analysis that laid the foundation for my project. Thank you to the National Aeronautics and Space Administration for the grant that funded this research. I extend my gratitude to all members of the Foreman Lab for both their help with my experiments and for creating a supportive environment where I could grow academically and personally. Lastly, I would like to thank my friends Jake, Carter, Esther, KaeLee, and Humberto for all the encouragement that made this whole journey possible. iii TABLE OF CONTENTS 1. INTRODUCTION ...........................................................................................................1 Background ......................................................................................................................1 Hypotheses .....................................................................................................................17 2. METHODS ....................................................................................................................18 Determining Cell Characteristics ...................................................................................18 16S rRNA Gene Sequencing .........................................................................................19 Substrate Utilization Profiles .........................................................................................20 UV/Vis Absorbance Measurements ...............................................................................21 Fourier-Transform Infrared Spectroscopy .....................................................................22 Raman Spectroscopy Analysis .......................................................................................22 2. RESULTS ......................................................................................................................26 Determining Cell Characteristics ...................................................................................26 16S rRNA Gene Analysis ..............................................................................................28 Substrate Utilization Profiles .........................................................................................30 UV/Vis Absorbance Measurements ...............................................................................31 Fourier-Transform Infrared Spectroscopy .....................................................................44 Raman Spectroscopy Analysis .......................................................................................54 2. Discussion ......................................................................................................................72 Characteristics of Bacterial Isolates ...............................................................................72 Application of UV/Vis and FTIR for Detecting Biosignatures .....................................73 Application of Raman Spectroscopy for Identifying Microbial Biosignatures .............77 REFERENCES CITED ......................................................................................................87 APPENDICES .................................................................................................................101 APPENDIX A: Tables .........................................................................................102 APPENDIX B: Additional UV/Vis Absorbance Spectrum .................................108 APPENDIX C: Microscopy Images ....................................................................110 APPENDIX D: Principal Component Analysis ...................................................127 iv LIST OF TABLES Table Page 1. Cell Morphologies and Colony Color ..............................................................103 2. Gram Staining and Catalase Tests ...................................................................104 3. Substrate Utilization Assay ..............................................................................105 4. Utilization of Individual Substrates .................................................................106 5. Degree of Lipid Saturation Table ....................................................................107 v LIST OF FIGURES Figure(s) Page(s) 1. Image of Martian Surface Taken by the Mariner 4 Spacecraft (1965) ................3 2. Image of Martian Surface Taken by the Mariner 9 Spacecraft (1971) ................3 3. Sojourner Rover ...................................................................................................5 4. Curiosity Rover ....................................................................................................9 5. Mars Cumberland Drill Site .................................................................................9 6. Perseverance Rover Schematic ..........................................................................12 7-8. Omnilog Plate Reader Instrument ..................................................................21 9-10. LabRAM HR Evolution Raman Spectrometer .............................................25 11. Microscopy Image of Polaromonas eurypsychrophilia_GRIS 1-12 .......27, 111 12. Maximum Likelihood Phylogeny Tree ............................................................29 13. 96-Well EcoPlate .............................................................................................31 14-20. UV/Vis Absorbance Spectra of Pigment Extractions ........................... 34-37 21. Janthinobacterium svalbardensis_GRIS 1-11 on a R2A Media Plate ............38 22-28. UV/Vis Absorbance Spectra of Whole Cells ...................................... 40-43 29-35. FTIR Absorbance Spectra .................................................................... 46-49 36. FTIR Absorbance Spectra PCA Scatterplot .....................................................51 37-38. FTIR Absorbance Spectra Principal Components ......................................52 39-46. Raman Spectra of Bacterial Isolates ..................................................... 56-59 47. Raman Spectra of Mesophilic Bacteria ............................................................60 vi LIST OF FIGURES CONTINUED Figure(s) Page(s) 48. Raman Spectra of Bacterial Isolates PCA Scatterplot .....................................64 49-50. Raman Spectra of Bacterial Isolates Principal Components ......................65 51. Raman Spectra of Bacterial Isolates PCA Biplot ............................................68 52. Raman Spectra of Algal Isolates ......................................................................70 53. UV/Vis Absorbance Spectrum with Peptide and DNA Absorbance Peaks ...109 54-77. Microscopy Images of Bacterial Isolates.......................................... 111-123 78-82. Microscopy Images of Algal Isolates ............................................... 124-126 83-163. PCA of Raman Spectra for Individual Bacterial Isolates ............... 128-154 vii ABSTRACT In astrobiology, new technologies are being implemented in the search for extraterrestrial life. Interpreting results from new analytical techniques requires additional information about microbial properties. A catalogue of identifying characteristics, called biosignatures was created for bacterial and algal isolates from Greenland and Antarctica by measuring substrate utilization, UV/Vis absorbance, Fourier-Transform Infrared Spectroscopy, and Raman spectroscopy. Organisms were chosen from environments analogous to Martian glacier systems. Spectral properties of these polar isolates could serve as a reference for interpreting results from NASA’s Perseverance rover. Substrate utilization was evaluated using EcoPlates on an Omnilog plate reader (Biolog, California, U.S.A.). UV/Vis absorbance spectra indicated that nine of the twenty-five bacterial isolates contained carotenoid pigments, and one contained violacein. UV/Vis analysis was effective at identifying the presence of pigments, but was insufficient for distinguishing between the types of carotenoids. FTIR analysis identified general biological features such as lipids, proteins, and carbohydrates, but did not detect pigments. Raman analysis of isolates with a 532 nm laser identified both the presence of carotenoid and violacein pigments, and the general cell features observed with FTIR. The degree of saturation of membrane lipids was evaluated for the bacterial isolates by comparing the ratio of unsaturated and saturated fatty acid peaks in the Raman spectra. Results were similar for the polar isolates and mesophiles, excluding the Bacillus subtilis spores. A principal component analysis was conducted to determine the regions of the spectra that contributed the variability between samples. The spectra of the bacterial isolates
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