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CALIFORNIA STATE UNIVERSITY, NORTHRIDGE Identifying Changes in the Active, Dead, and Dormant Microbial Community Structure Across a Chronosequence of Ancient Alaskan Permafrost A thesis submitted in partial fulfillment of the requirements For the degree of Master of Science in Biology By Alexander Burkert December 2017 The thesis of Alexander Burkert is approved: ______________________________________ ______________ Dr. Kerry Cooper Date ______________________________________ ______________ Dr. Gilberto Flores Date ______________________________________ ______________ Dr. Rachel Mackelprang, Chair Date California State University, Northridge ii Acknowledgements There are many people who have contributed to the success of this work and who deserve acknowledgement and thanks. To my advisor, Dr. Rachel Mackelprang of the College of Math and Science at CSU Northridge, who took a chance on a student with very little experience in microbiology. I have been afforded more opportunities in her lab than I had ever expected from this program. From collecting my own permafrost samples in Fairbanks, Alaska to presenting my research in Nuuk, Greenland and even learning a little bit of beekeeping along the way. Her dedication to science is an inspiration. To the members of my committee: if Dr. Gilberto Flores, of the College of Math and Science at CSU Northridge, had a dollar for every question of mine that he answered, he would be a very wealthy man. I appreciate his ability to get to the heart of the problem and give me guidance without directly telling me what to do. I also thank Dr. Kerry Cooper, of the College of Math and Science at CSU Northridge, for his jovial persona and his valuable input in my thesis project. To our collaborators, thank you to Tom Douglas from the Alaska Projects Office of the Cold Regions Research and Engineering Laboratory for his assistance collecting permafrost core samples and to Mark Waldrop of the United States Geological Survey for his help performing soil chemistry analysis on our samples. To the community of brilliant scientist in my lab (The Mackelprang Gang) and in my cohort at CSU Northridge who have provided both camaraderie and a place to commiserate with others who understand the late nights and failed experiments that led to this work. Lastly to my family and friends who have been there to keep me grounded and have encouraged me at every step of the way, I thank you. Alex Burkert iii Table of Contents Signature Page .................................................................................................................... ii Acknowledgements ............................................................................................................ iii List of Figures ..................................................................................................................... v List of Tables ..................................................................................................................... ix Abstract ............................................................................................................................... x 1. Introduction ................................................................................................................. 1 2. Materials & Methods .................................................................................................. 9 Permafrost Sample Collection ...................................................................................................... 9 Permafrost Subsampling ............................................................................................................ 12 Soil Chemistry ............................................................................................................................ 12 Cell Separation for Enumeration via Microscopy ...................................................................... 12 Live/Dead Staining ..................................................................................................................... 13 DAPI Staining ............................................................................................................................ 13 Cell Enumeration........................................................................................................................ 13 Separation of Biomass from Soil Matrix.................................................................................... 14 Depletion of DNA from Dead Cells via Propidium Monoazide Treatment .............................. 15 Endospore Enrichment via Lysozyme Enzyme Treatment ........................................................ 15 DNA Extraction.......................................................................................................................... 17 PCR Amplification and Sequencing .......................................................................................... 17 Statistical Analysis ..................................................................................................................... 18 3. Results ....................................................................................................................... 20 Soil Chemistry ............................................................................................................................ 20 Cell Enumeration........................................................................................................................ 20 16S rRNA gene-based community analysis ............................................................................... 24 Depletion of DNA from Dead Cells via Propidium Monoazide Treatment .............................. 32 Endospore Enrichment via Lysozyme Enzyme Treatment ........................................................ 33 4. Discussion/Conclusion .............................................................................................. 39 Literature Cited ................................................................................................................. 46 Appendix A: Supplementary Material .............................................................................. 55 iv List of Figures Figure 1. Experimental strategy— To obtain direct counts for live, dead, and total cells, we first separated cells from soil using a Nycodenz density centrifugation and stained using either a Live/Dead differential stain or DAPI stain before counting cells via fluorescence microscopy. In addition, we separated biomass from soil using a gravity separation technique and treated with either a propidium monoazide treatment (to deplete DNA from dead organisms) or a lysozyme enzyme treatment (to enrich for endospores). We conducted 16S rRNA gene amplicon sequencing for each treatment plus an untreated control for all age categories to observe changes in alpha diversity, beta diversity, and taxa abundance in the active dead, and dormant populations. ............................................ 8 Figure 2. Map of Alaska with the marked location of the United States Cold Regions Research and Engineering Laboratory (CRREL) Permafrost Tunnel Research Facility. The tunnel is located 16 km north of Fairbanks, Alaska. (Figure from Cyzewski et al 2010). ................................................................................................................................ 10 Figure 3. Photograph taken from the entrance to the CRREL Permafrost tunnel. ........... 10 Figure 4. Schematic drawing of the CRREL Permafrost tunnel. The age of exposed permafrost increases with distance from the tunnel portal. (Figure from Mackelprang et al 2017). ................................................................................................................................ 11 Figure 5. Diagram showing the core sampling strategy used in the CRREL Permafrost Tunnel. The cores were dug into the wall at three depths. Microbiological studies were conducted with the second and third depth cores which were interior to the wall and less likely to suffer from contamination or thaw. The outermost core was used for soil chemistry analysis. (Figure from Mackelprang et al 2017). ............................................. 11 Figure 6. Direct cell counts as determined by fluorescent microscopy using SYTO 9 (B), propidium iodide (C), and DAPI (D). Total counts were highest in the intermediate aged category for the live counts (B, Kruskal Wallis, X2(2) = 46.25, p < 0.001, Dunn’s post hoc test, 19K – 27K p < 0.01 27K – 33K p < 0.01), dead counts (C, Kruskal Wallis, X2(2) = 53.16, p < 0.001, Dunn’s post hoc test, 19K – 27K p < 0.01 27K – 33K p < 0.01), and total counts gdw-1 (D, Kruskal Wallis, X2(2) = 53.58, p < 0.001, Dunn’s post hoc test, 19K – 27K p < 0.01 27K – 33K p < 0.01). The total counts gdw-1 were lowest for the oldest samples (D, Dunn’s post hoc test, p < 0.05). (** = p < 0.01, * = p < 0.05). Values show averages of five replicates and error bars show standard error of the mean. .......... 22 Figure 7. Direct cell counts determined by fluorescent microscopy showed that the proportion of live cells increased with increasing age. The intermediate aged samples had a significantly higher proportion of live cells than the youngest samples (Kruskal Wallis, X2(2) = 9.84, p < 0.01, Dunn’s post hoc test, ** = p < 0.01). Values show averages of five replicates and error bars show standard error of the mean. ....................................... 23 Figure 8. Alpha diversity estimates based on 16S rRNA gene sequences