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Molecular and Phenetic Characterization of the Bacterial MOLECULAR AND PHENETIC CHARACTERIZATION OF THE BACTERIAL ASSEMBLAGE OF BASQUE LAKE, BC, AN ENVIRONMENT WITH HIGH CONCENTRATIONS OF MAGNESIUM SULPHATE, AND ITS RELEVANCE TO MARS A Thesis by James Deven Crisler Bachelor of General Studies, Wichita State University, 2011 Bachelor of Science, Wichita State University, 2010 Submitted to the Department of Biological Sciences and the faculty of the Graduate School of Wichita State University in partial fulfillment of the requirements for the degree of Master of Science May 2017 © Copyright 2017 by James Crisler All Rights Reserved MOLECULAR AND PHENETIC CHARACTERIZATION OF THE BACTERIAL ASSEMBALAGE OF BASQUE LAKE, BC, AN ENVIRONMENT WITH HIGH CONCENTRATIONS OF MAGNESIUM SULFATE AND ITS RELEVANCE TO MARS The following faculty members have examined the final copy of this thesis for form and content, and recommend that it be accepted in partial fulfillment of the requirement for the degree of Master of Science with a major in Biological Sciences. ______________________________________ Mark Schneegurt, Committee Chair ______________________________________ Mary Liz Jameson, Committee Member ______________________________________ Janet Twomey, Committee Member iii ACKNOWLEDGMENTS First, I would like to extend my deepest gratitude to Dr. Mark Schneegurt for his eternal patience and continuous guidance and support over the years. I would like to thank Tim Eberl, Casper Fredsgaard and Amer Alsoudi for all their help and friendship over the years. I would also like to thank Justin Campbell and Jonathan Wilks for all their help in the lab and the endless support through trying times. Finally, I would like to especially thank my Mom for all her love, support and encouragement throughout my life regardless of the circumstances. I am truly blessed to have had all of you in my life. iv Success is not final, failure is not fatal: it is the courage to continue that counts. -Winston Churchill v ABSTRACT Halotolerant bacteria favor environments containing high concentrations of salts. While there are a multitude of hypersaline environments containing various salts on Earth, those heavily dominated with sodium chloride (NaCl) have been of academic rigor. This thesis pertains to environments with high concentrations of magnesium sulphate (MgSO4), which presents ample opportunity for discovery. Basque Lake, BC is one such environment that is dominated by magnesium sulphate. Basque Lake is an ephemeral lake containing near-saturated levels of magnesium sulphate that precipitates as epsomite (MgSO4●7H2O). Natural environments containing high concentrations of magnesium sulphate are rare and previous microbiological effort is limited. For a microbe to persist in Basque Lake it must withstand extreme conditions similar to those present on the Martian surface including salinity, aridity, and temperature. Any microbe isolated from Basque Lake could give astrobiologists key details on what traits a Martian life-form may have and to limit potential forward contamination. Approximately 65 bacterial isolates were obtained through repetitive streak-plating in high salt media. The bacterial isolates were characterized phenotypically and subjected to 16S rRNA sequencing and phylogenetic analyses. Gram-positive bacteria dominated the culture collection including members of Virgibacillus, Marinococcus, and Staphylococcus. Members of the Gram-negative genera Halomonas and Salinivibrio were represented in the culture collection as well. Results indicate that microbes isolated from epsom-rich environments such as Basque Lake present a potential risk of forward contamination. This research was supported by NASA ROSES Planetary Protection (PPR), KANSAS NASA EPSCoR, and KINBRE. vi TABLE OF CONTENTS Chapter Page 1. INTRODUCTION .................................................................................................. 1 1.1 Extremophiles ............................................................................................ 1 1.2 Halotolerance ............................................................................................. 2 1.2.1 Hypersaline Environments .................................................... 2 1.3 Mars ........................................................................................................... 4 1.3.1 MgSO4 ......................................................................................................................... 4 1.3.2 Epsotolerance ...................................................................... 5 1.3.3 Challenges ........................................................................... 5 1.4 Basque Lake .............................................................................................. 7 1.4.1 Martian Analog ..................................................................... 8 1.5 Salinotolerance .......................................................................................... 9 1.6 Growth in Varying MgSO4 and NaCl Concentrations ................................. 9 1.7 Effects of Water Activity upon Growth...................................................... 10 1.8 Chaotropicity, Kosmotropicity, and Ionic Strength ................................... 11 1.9 Phylogeny of Epsom Rich Environments ................................................. 12 2. RATIONALE AND HYPOTHESIS ....................................................................... 16 2.1 Rationale .................................................................................................. 16 2.2 Hypothesis ............................................................................................... 17 2.3 Objectives ................................................................................................ 18 3. METHODOLOGY ............................................................................................... 19 3.1 Sample Collection .................................................................................... 19 3.2 Enrichment and Isolation ......................................................................... 20 3.3 Morphological, Physiological and Biochemical Tests ............................... 22 3.3.1 Gram Stain ......................................................................... 22 3.3.2 Endospore Stain ................................................................. 23 3.3.3 Acid-Fast Stain ................................................................... 23 3.3.4 SIM Test ............................................................................. 23 3.3.5 Catalase Test ..................................................................... 23 3.3.6 Oxidase Test ...................................................................... 23 3.3.7 Urease Test ........................................................................ 24 3.3.8 Glucose Test ...................................................................... 24 3.3.9 Lactose Test ....................................................................... 24 3.3.10 Sucrose Test ...................................................................... 24 3.3.11 Starch Hydrolysis ............................................................... 25 3.4 Epsotolerance and Halotolerance ............................................................ 25 vii TABLE OF CONTENTS (continued) Chapter Page 3.5 Temperature Tolerance ........................................................................... 25 3.6 pH Tolerance ........................................................................................... 26 3.7 DNA Extraction ........................................................................................ 26 3.7.1 Isolate Extract ..................................................................... 26 3.8 PCR Amplification .................................................................................... 27 3.9 Sequencing and Phylogenetic Analysis ................................................... 28 4. RESULTS ........................................................................................................... 29 4.1 Assemblage and Identification of Bacterial Isolates ................................. 29 4.2 Phenotypic Analysis of Bacterial Isolates ................................................. 30 4.2.1 Stains ................................................................................. 30 4.2.2 Morphological, Physiological and Biochemical Tests .................................................................................. 30 4.3 Epsotolerance and Halotolerance ............................................................ 30 4.4 Temperature Tolerance ........................................................................... 31 4.5 pH Tolerance ........................................................................................... 32 5. DISCUSSION ..................................................................................................... 33 5.1 Epsotolerance and Halotolerance ............................................................ 33 5.2 Temperature Tolerance ........................................................................... 34 5.3 Basque Lake Phylogeny .......................................................................... 35 6. CONCLUSION .................................................................................................... 37 7. FUTURE EFFORTS ........................................................................................... 38 REFERENCES .............................................................................................................
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