Geological Carbon Sequestration and its Influence on Subsurface Microbial Diversity and Metabolic Carbon Cycling Mariana Erasmus Geological Carbon Sequestration and its Influence on Subsurface Microbial Diversity and Metabolic Carbon Cycling By Mariana Erasmus Submitted in fulfillment of the requirements for the degree PHILOSOPHIAE DOCTOR In the Department of Microbial, Biochemical and Food Biotechnology Faculty of Natural and Agricultural Sciences University of the Free State Bloemfontein Republic of South Africa August 2015 Promotor: Prof. E. van Heerden Co-promotors: Prof. D. Litthauer : Prof. T.C. Onstott : Prof. T.J. Phelps : Dr. T. Surridge I dedicate this thesis to my godchild, Hayley Shardelow. “Words of wisdom are spoken by children at least as often as scientists.” ~ James Newman “The more I study science, the more I believe in God.” ~ Albert Einstein “If at first, the idea is not absurd, then there is no hope for it.” ~ Albert Einstein “The value of a college education is not the learning of many facts, but the training of the mind to think.” ~ Albert Einstein “It isn't what you learn that makes you successful, but what you use of what you learn.” ~ Percy H Whiting CONTENTS CONTENTS .....................................................................................................................i ACKNOWLEDGEMENTS .............................................................................................. vii LIST OF SYMBOLS AND ABBREVIATIONS ...................................................................x LIST OF FIGURES ....................................................................................................... xvi LIST OF TABLES ...................................................................................................... xxviii CHAPTER 1: LITERATURE REVIEW ................................................................................ 1 1.1 GLOBAL CLIMATE CHANGE AND THE GREENHOUSE EFFECT ................. 1 1.2 CARBON SEQUESTRATION .......................................................................... 5 1.2.1 Geological Sequestration ............................................................................ 6 1.2.2 Ocean Sequestration .................................................................................. 8 1.2.3 Terrestrial Sequestration ............................................................................. 9 1.2.4 Mineral Sequestration ............................................................................... 10 1.3 CARBON SEQUESTRATION IN SOUTH AFRICA ......................................... 10 1.4 THE CARBON CYCLE ................................................................................... 12 1.5 THE DEEP SUBSURFACE AND CARBON CYCLING ................................... 13 1.6 CONCLUSIONS ............................................................................................. 17 1.7 REFERENCES............................................................................................... 19 CHAPTER 2: INTRODUCTION TO PRESENT STUDY ................................................... 28 2.1 ABSTRACT .................................................................................................... 28 2.2 POSSIBLE OCCURENCES DURING CARBON SEQUESTRATION ............. 29 2.3 REFERENCES............................................................................................... 31 Contents i CHAPTER 3 - SAMPLING, CHARACTERIZATION AND MICROBIAL DIVERSITY OF THE SITE DESIGNATED TO MIMIC CARBON SEQUESTRATION CONDITIONS ................. 35 3.1 INTRODUCTION ............................................................................................ 35 3.1.1 Geological Sequestration Sedimentary Rock ............................................ 37 3.2 AIMS OF THIS CHAPTER ............................................................................. 38 3.3 MATERIALS AND METHODS ........................................................................ 39 3.3.1 Sampling Requirements ............................................................................ 39 3.3.2 The Study Site .......................................................................................... 40 3.3.3 The Borehole ............................................................................................ 41 3.3.4 Fissure Water and Gas Sampling and Analyses ....................................... 42 3.3.4.1 On-Site Physicochemical Analyses ..................................................... 43 3.3.4.2 Collection of Fissure Water for Geochemical Analyses ........................ 45 3.3.4.3 Collection of Gas and Fissure Water for Gas Chemistry and Isotope Analyses .............................................................................................. 47 3.3.5 Sandstone Sampling and Analyses........................................................... 50 3.3.5.1 Crushing and Sterilizing ....................................................................... 50 3.3.5.2 X-Ray Fluorescence Analyses ............................................................. 51 3.3.6 Diversity Analyses of the Deep Subsurface Microbial Biome .................... 51 3.3.6.1 Cornelius® Canister Sampling ............................................................. 52 3.3.6.2 SterivexTM Filter Sampling ................................................................... 54 3.3.6.3 Tangential Flow Filtration .................................................................... 54 3.3.6.4 Massive Filter Sampling ...................................................................... 55 3.3.6.5 Polyvinyl Chloride Cartridge Sampling ................................................. 58 3.3.6.6 Microbial Diversity Assessments using Denaturing Gradient Gel Electrophoresis .................................................................................... 59 3.3.6.6.1 Genomic DNA Isolation ................................................................. 59 3.3.6.6.2 Polymerase Chain Reaction .......................................................... 59 3.3.6.6.3 Nested Polymerase Chain Reaction.............................................. 61 3.3.6.6.4 Denaturing Gradient Gel Electrophoresis ...................................... 62 3.3.6.6.5 Gel Extraction, Re-amplification and Purification ........................... 62 3.3.6.6.6 Sanger Sequencing and Analyses ................................................ 63 3.3.6.7 Microbial Diversity Assessments using Targeted 16S and 18S rRNA Gene Sequencing ................................................................................ 65 3.3.6.7.1 Genomic DNA Isolation ................................................................. 65 3.3.6.7.2 MiSeq Illumina® Sequencing and Analyses ................................... 66 Contents ii 3.3.6.7.3 Roche GS Junior Sequencing and Analyses ................................. 66 3.3.6.7.4 Ion Torrent PGMTM Sequencing and Analyses .............................. 67 3.3.6.8 Microbial Diversity Assessments of the Fissure Water using Visual Methods .............................................................................................. 67 3.3.6.8.1 Negative Stain .............................................................................. 68 3.3.6.8.2 Live/Dead Stain ............................................................................ 68 3.3.6.8.3 Gram Stain.................................................................................... 69 3.3.6.8.4 Scanning Electron Microscopy ...................................................... 69 3.3.7 Summary of all Samplings and Collaborators ........................................... 71 3.4 RESULTS AND DISCUSSIONS ..................................................................... 73 3.4.1 Fissure Water and Gas Analyses .............................................................. 73 3.4.1.1 On-Site Physicochemical Analyses ..................................................... 73 3.4.1.2 Geochemical Analyses ........................................................................ 74 3.4.1.3 Gas Chemistry and Isotope Analyses .................................................. 76 3.4.2 Sandstone X-Ray Fluorescence Analyses ................................................ 77 3.4.3 Characterization and Diversity of the Deep Subsurface Microbial Biome .. 78 3.4.3.1 Microbial Diversity Assessments using Denaturing Gradient Gel Electrophoresis .................................................................................... 78 3.4.3.1.1 Genomic DNA Isolation and Polymerase Chain Reaction ............. 78 3.4.3.1.2 Nested Polymerase Chain Reaction.............................................. 79 3.4.3.1.3 Denaturing Gradient Gel Electrophoresis, Sanger Sequencing and Analyses ....................................................................................... 80 3.4.3.2 Microbial Diversity Assessments using Targeted 16S and 18S rRNA Gene Sequencing ................................................................................ 82 3.4.3.3 Microbial Diversity Assessments of the Fissure Water using Visual Methods .............................................................................................. 88 3.4.3.3.1 Staining ......................................................................................... 88 3.4.3.3.2 Scanning Electron Microscopy ...................................................... 92 3.5 CONCLUSIONS ............................................................................................. 94 3.6 REFERENCES..............................................................................................