CHARACTERIZATION OF ORGANISMS IN VOSTOK (ANTARCTICA) GLACIAL, BASAL, AND ACCRETION ICE Colby J. Gura A Thesis Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE December 2019 Committee: Scott O. Rogers, Advisor Helen Michaels Paul Morris © 2019 Colby Gura All Rights Reserved iii ABSTRACT Scott O. Rogers, Advisor Chapter 1: Lake Vostok is named for the nearby Vostok Station located at 78°28’S, 106°48’E and at an elevation of 3,488 m. The lake is covered by a glacier that is approximately 4 km thick and comprised of 4 different types of ice: meteoric, basal, type 1 accretion ice, and type 2 accretion ice. Six samples were derived from the glacial, basal, and accretion ice of the 5G ice core (depths of 2,149 m; 3,501 m; 3,520 m; 3,540 m; 3,569 m; and 3,585 m) and prepared through several processes. The RNA and DNA were extracted from ultracentrifugally concentrated meltwater samples. From the extracted RNA, cDNA was synthesized so the samples could be further manipulated. Both the cDNA and the DNA were amplified through polymerase chain reaction. Ion Torrent primers were attached to the DNA and cDNA and then prepared to be sequenced. Following sequencing the sequences were analyzed using BLAST. Python and Biopython were then used to collect more data and organize the data for manual curation and analysis. Chapter 2: As a result of the glacier and its geographic location, Lake Vostok is an extreme and unique environment that is often compared to Jupiter’s ice-covered moon, Europa. Lake Vostok was originally thought to be sterile, but multiple studies have suggested that not only is there a variety of bacterial and eukaryotic organisms living in the lake, but it may contain a complex ecosystem. The results of this analysis yielded metagenomic and metatranscriptomic data that aligned with a wide variety of organisms from 30 different phyla. The associated organisms were capable of many metabolic pathways, such as the nitrogen cycle and carbon fixation, as well as oxidation and/or reduction pathways for sulfur, iron, arsenic, hydrogen, iv hydrocarbon, phosphorous, uranium, and chromium compounds. The number of organisms unique to each sample was quite high for all samples except the layered meteoric ice sample, which only contained sequences similar to one organism. These results, combined with previous research, indicates that Lake Vostok is a transitory repository of DNA and organisms from the glacier, and also contains a much larger dynamic community and ecosystem. v TABLE OF CONTENTS Page CHAPTER 1: INTRODUCTORY REVIEW ........................................................................ 1 1.1 Lake Vostok ....................................................................................................... 1 1.2 Glacial Ice ........................................................................................................... 3 1.3 Ice Core Drilling ................................................................................................. 7 1.4 RNA Extraction .................................................................................................. 9 1.5 DNA Extraction .................................................................................................. 10 1.6 cDNA Synthesis ................................................................................................. 11 1.7 Polymerase Chain Reaction (PCR) .................................................................... 13 1.8 Ion Torrent Sequencing ...................................................................................... 17 1.9 Basic Local Alignment Search Tool .................................................................. 19 1.10 Python and Biopython ....................................................................................... 21 1.11 Chapter 1 References ......................................................................................... 23 CHAPTER 2: INFLUX OF ORGANISMS INTO SUBGLACIAL LAKE VOSTOK FROM GLACIAL AND BASAL ICE…………. .............................................................................. 29 2.1 Abstract ............................................................................................................ 29 2.2 Introduction ........................................................................................................ 30 2.2.1 Lake Vostok ........................................................................................ 30 2.2.2 Glacial Ice Conditions ......................................................................... 31 2.2.3 Previous Research ............................................................................... 35 2.2.4 Purpose of Research ............................................................................ 37 2.3 Materials and Methods ......................................................................................... 38 vi 2.3.1 Sample Preparation .............................................................................. 38 2.3.2 cDNA Synthesis .................................................................................. 39 2.3.3 Adapter Ligation .................................................................................. 40 2.3.4 Fractionation ........................................................................................ 41 2.3.5 DNA Rehydration ................................................................................ 41 2.3.6 Polymerase Chain Reaction ................................................................. 42 2.3.7 EcoRI MID Primer Polymerization and Purification .......................... 42 2.3.8 Sample Purification ............................................................................. 45 2.3.9 Ion Torrent Primer Polymerization ..................................................... 46 2.3.10 Ion Torrent Sample Preparation ......................................................... 47 2.3.11 Sequence Clean Up ............................................................................ 47 2.3.12 Organism Determinations ................................................................... 47 2.3.13 Ecology and Physiology ..................................................................... 48 2.3.14 Comparison of Data ............................................................................ 49 2.4 Results and Discussion ........................................................................................ 49 2.4.1 Sequence Data – Overall ..................................................................... 49 2.4.2 Glacial Ice (2,149 m) ........................................................................... 50 2.4.2.1 Results Summary ................................................................... 50 2.4.2.2 Organism Overlap .................................................................. 50 2.4.3 Basal Ice (3,501 m + 3,520 m) ............................................................ 51 2.4.3.1 Results Summary ................................................................... 51 2.4.3.2 Organism Overlap .................................................................. 58 2.4.3.3 Unshared Organism Summary ............................................... 72 vii 2.4.4 Shallow Embayment Type 1 Accretion Ice (3,540 m + 3,569 m) ....... 77 2.4.4.1 Results Summary ................................................................... 77 2.4.4.2 Organism Overlap .................................................................. 83 2.4.4.3 Unshared Organism Summary ............................................... 88 2.4.5 Shallow Embayment Type 2 Accretion Ice (3,585 m) ........................ 91 2.4.5.1 Results Summary ................................................................... 91 2.4.5.2 Organism Overlap .................................................................. 101 2.4.5.3 Unshared Organism Summary ............................................... 104 2.4.6 Summary ............................................................................................. 110 2.4.7 Extremophiles ...................................................................................... 112 2.4.7.1 Glacial Ice (2,149 m) ............................................................. 112 2.4.7.2 Basal Ice (3,501 m + 3,520 m) ............................................... 112 2.4.7.3 Type 1 Accretion Ice (3,540 m + 3,569 m)............................ 114 2.4.7.4 Type 2 Accretion Ice (3,585 m) ............................................. 115 2.4.8 Metabolic Analysis .............................................................................. 117 2.4.8.1 Glacial Ice (2,149 m) ............................................................. 117 2.4.8.2 Basal Ice (3,501 m + 3,520 m) ............................................... 117 2.4.8.3 Type 1 Accretion Ice (3,540 m + 3,569 m)............................ 121 2.4.8.4 Type 2 Accretion Ice (3,585 m) ............................................. 124 2.5 Conclusions of Findings ...................................................................................... 126 2.5.1 Meteoric Ice (2,149 m) ........................................................................ 126 2.5.2 Basal Ice (3,501 m + 3,520 m) ............................................................ 127 2.5.2.1 Organism Overlap .................................................................. 127 viii 2.5.2.2 Organism Contents................................................................