The Geomicrobiology of Cementitious Radioactive Waste A thesis submitted to The University of Manchester for the degree of Doctor of Philosophy in the Faculty of Engineering and Physical Sciences 2014 Adam John Williamson School of Earth, Atmospheric and Environmental Sciences Table of Contents Table of Contents .............................................................................................................. 2 List of Figures ................................................................................................................... 8 List of Tables................................................................................................................... 13 Abbreviations .................................................................................................................. 16 Abstract ........................................................................................................................... 18 Declaration ...................................................................................................................... 19 Copyright Statement ....................................................................................................... 20 Acknowledgments .......................................................................................................... 21 The Author ..................................................................................................................... 22 1. Introduction and Thesis Content ............................................................................ 23 1.1. Project relevance ................................................................................................ 24 1.1. Research objectives ............................................................................................ 25 1.2. Thesis structure .................................................................................................. 25 1.3. Paper status and collaborator contributions .................................................. 27 1.4. References .......................................................................................................... 28 2. A Review of Neutral and Alkaline Biogeochemistry, Relevant to Radioactive Waste Disposal .............................................................................................................. 30 2.1. The nuclear legacy ............................................................................................. 31 2.1.1. The nuclear fuel cycle .................................................................................... 31 2.1.2. Radioactive waste legacy ............................................................................... 32 2.1.3. Geological disposal ........................................................................................ 33 2.1.4. Natural analogues .......................................................................................... 34 2.2. Background to the elements in this study ........................................................ 35 2.3. Stable element biogeochemistry ....................................................................... 36 2.3.1. Iron biogeochemistry ..................................................................................... 37 2.3.2. Metal reduction mechanisms ......................................................................... 39 2.4. Alkaline biogeochemistry .................................................................................. 43 2.4.1. Physiology ..................................................................................................... 43 2.4.2. Environments ................................................................................................. 44 2 2.4.3. Phylogenetic diversity and metal reduction ................................................... 45 2.5. Radionuclide biogeochemistry ......................................................................... 47 2.5.1. Uranium biogeochemistry ............................................................................. 47 2.5.2. Technetium biogeochemistry......................................................................... 49 2.5.3. Neptunium biogeochemistry .......................................................................... 50 2.6. Summary and site overview ............................................................................... 51 2.6.1. Summary ........................................................................................................ 51 2.6.2. Overview of the Buxton site ......................................................................... 51 2.7. References .......................................................................................................... 53 3. Materials and Methods ............................................................................................ 66 3.1. Geochemical analyses ........................................................................................ 67 3.1.1. pH .................................................................................................................. 67 3.1.2. Eh (the reduction potential) ........................................................................... 67 3.2. Spectrophotometry (UV-Vis ) ............................................................................ 67 3.2.1. Determination of Fe(II): the ferrozine method ............................................. 69 3.2.2. Determination of U(VI): the bromo-PADAP method .................................. 69 3.3. Ion chromatography (IC) ................................................................................. 70 3.4. Inductively coupled plasma (ICP) ..................................................................... 72 3.4.1. Inductively coupled plasma-atomic emission spectroscopy (ICP-AES) ....... 72 3.4.2. Inductively coupled plasma-mass spectroscopy (ICP-MS) ........................... 72 3.5. Electron microscopy .......................................................................................... 73 3.5.1. Transmission electron microscopy (TEM) ................................................... 73 3.5.2. Select area electron diffraction (SAED) ........................................................ 74 3.5.3. Energy dispersive X-ray spectroscopy (EDX) .............................................. 75 3.6. X-ray techniques ................................................................................................ 75 3.6.1. X-ray absorption spectroscopy (XAS)........................................................... 75 3.6.2. X-ray magnetic circular dichroism (XMCD) ................................................ 81 3.6.3. X-ray fluorescence (XRF) ............................................................................. 82 3.6.4. X-ray diffraction (XRD) ................................................................................ 84 3.7. Microbiological techniques ............................................................................... 85 3.7.1. Polymerase chain reaction (PCR) .................................................................. 85 3.7.2. 16S rRNA gene based clone libraries ........................................................... 86 3 3.7.3. Ribosomal intergenic spacer analysis (RISA) ............................................... 87 3.7.4. 16S amplicon pyrosequencing and data analysis .......................................... 87 3.7.5. Microcosm experiments ................................................................................ 88 3.8. Radiological techniques ..................................................................................... 89 3.8.1. Liquid scintillation counting (LSC) ............................................................... 89 3.8.2. Determination of pertechnetate: the TPAC method ..................................... 90 3.8.3. Gamma camera imaging ............................................................................... 90 3.9. Radiation safety ................................................................................................. 91 3.9.1. Protocols ....................................................................................................... 91 238 99 99m 237 3.9.2. Radioactive characteristics of U, Tc, Tc and Np ........................... 91 3.10. References ........................................................................................................ 93 4. Microbial Reduction of Fe(III) under Alkaline Conditions Relevant to Geological Disposal ...................................................................................................... 97 Abstract ................................................................................................................. 3320 Introduction .......................................................................................................... 3320 Materials and methods ......................................................................................... 3321 Microcosm experiments....................................................................................... 3321 Geochemical analyses .......................................................................................... 3321 Mineralogical characterization X-ray (powder) diffraction ................................ 3321 X-ray absorption spectroscopy (XAS) and X-ray magnetic circular dichroism (XMCD) ..............................................................................................................