Understanding the microbial ecology of highly radioactive nuclear storage facilities A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Science and Engineering 2018 Lynn Foster School of Earth and Environmental Sciences 1 Contents List of Figures……………………………………………………………………..7 List of Tables……………………………………………………………………..13 List of Equations…………………………………………………………………14 List of abbreviations……………………………………………………………...15 Abstract…………………………………………………………………………..17 Declaration……………………………………………………………………….18 Copyright statement……………………………………………………………...19 Acknowledgements………………………………………………………………20 The author………………………………………………………………………...22 1 Introduction ........................................................................................ 24 1.1 Project context .................................................................................................. 24 1.2 Aims and objectives ......................................................................................... 25 1.3 Thesis structure................................................................................................. 27 1.4 Author contributions......................................................................................... 28 2 Literature review ................................................................................ 32 2.1 The nuclear fuel cycle ...................................................................................... 32 2.1.1 The fuel cycle ............................................................................................ 32 2.1.2 Spent nuclear fuel storage ponds (SNFPs) ................................................ 34 2.1.3 Magnox Reactors and the First Generation Magnox Storage Pond (FGMSP) at Sellafield ............................................................................................. 34 2.2 Microorganisms in nuclear storage facilities.................................................... 36 2.2.1 Biofilms, microbial induced corrosion (MIC) and biofouling .................. 36 2.2.2 Techniques used to study microbial communities in SNFPs. ................... 37 2.2.3 Microbes in SNFPs ................................................................................... 38 2.2.4 Microbial diversity in storage ponds at Sellafield .................................... 44 2.3 Cyanobacteria and eukaryotic algae ................................................................. 45 2.3.1 Introduction to cyanobacteria.................................................................... 45 2.3.2 Introduction to eukaryotic algae ............................................................... 47 2.3.3 Bloom formation and collapse .................................................................. 48 2.4 Radiation resistance .......................................................................................... 59 2.4.1 Ionizing radiation ...................................................................................... 59 2 2.4.2 Reactive oxygen species ........................................................................... 59 2.4.3 Radiation and microorganisms .................................................................. 64 2.4.4 Desiccation tolerance linked to radiation tolerance .................................. 65 2.4.5 Enzymes involved in ROS response ......................................................... 66 2.4.6 Non- enzymatic protection against ROS- carotenoids, melanin and micosporin-like amino acids (MAAs) ..................................................................... 69 2.5 Interactions of microorganisms and radionuclides ........................................... 71 2.5.1 Interaction between microbes from SNFPs and radionuclides ................. 74 2.5.2 Sr interactions............................................................................................ 75 2.6 References ........................................................................................................ 77 3 Methodology ....................................................................................... 92 3.1 Pond water sample collection, procedures and safety ...................................... 92 3.2 Molecular biology- DNA extraction, amplification, quantitative amplification and visualisation .......................................................................................................... 92 3.2.1 DNA extraction ......................................................................................... 92 3.2.2 Polymerase Chain Reaction (PCR) ........................................................... 95 3.2.3 Quantitative polymerase chain reaction (qPCR) ....................................... 97 3.2.4 Agarose gel electrophoresis ...................................................................... 98 3.3 Pyrosequencing ................................................................................................ 99 3.4 MiSeq, Illumina next generation sequencing ................................................. 103 3.4.1 Sample preparation.................................................................................. 103 3.4.2 Cluster generation ................................................................................... 104 3.4.3 Sequencing .............................................................................................. 106 3.4.4 Data analysis ........................................................................................... 109 3.5 Measurement of Pseudanabaena catenata growth ........................................ 110 3.5.1 Chl-a concentration analysis ................................................................... 112 3.6 X-irradiation of P. catenata ............................................................................ 112 3.7 Microscopy ..................................................................................................... 113 3.7.1 Cell counts ............................................................................................... 113 3.7.2 Calcofluor white cell staining ................................................................. 114 3.7.3 Electron microscopy................................................................................ 114 3.8 Fourier-transform infrared (FT-IR) spectroscopy .......................................... 118 3.9 Inductively-coupled plasma atomic emission spectroscopy (ICP-AES) ........ 120 3.10 X-ray absorption spectroscopy ....................................................................... 120 3.11 X-ray diffraction ............................................................................................. 122 3 3.12 References ...................................................................................................... 123 4 Research chapter 4: The microbial ecology of a high pH spent nuclear fuel pond on the Sellafield site .................................................. 128 4.1 Abstract .......................................................................................................... 128 4.2 Introduction .................................................................................................... 129 4.3 Materials and Methods ................................................................................... 131 4.3.1 Pond samples, sampling and pond description ....................................... 131 4.3.2 DNA extraction and 16S rRNA gene sequencing ................................... 133 4.3.3 qPCR analysis of DNA extracts to determine approximate total microbial numbers 136 4.4 Results ............................................................................................................ 137 4.4.1 General FGMSP pond conditions over study period .............................. 137 4.4.2 Photosynthetic pigment, residence time, and turbidity in the FGMSP during the microbial blooms ................................................................................. 138 4.4.3 Quantitative polymerase chain reaction (qPCR) ..................................... 141 4.4.4 Microbial community analysis of 16S rRNA gene of the FGMSP ......... 143 4.4.5 Microbial community analysis of 16S rRNA gene of the auxiliary pond 145 4.4.6 Microbial community analysis of 18S rRNA gene and fungal ITS2-region 147 4.5 Discussion ...................................................................................................... 148 4.6 Conclusion ...................................................................................................... 151 4.7 Acknowledgements ........................................................................................ 152 4.8 References ...................................................................................................... 153 4.9 Supplementary material:................................................................................. 160 4.9.1 18S rRNA gene community analysis of the FGMSP .............................. 168 4.9.2 Fungal ITS2 region community analysis of the FGMSP ........................ 169 4.9.3 18S rRNA gene community analysis of the auxiliary pond .................... 170 4.9.4 Fungal ITS2 region community analysis of the auxiliary pond .............. 172 5 Research Chapter 5: The effect of X-irradiation on the growth and metabolic status of Pseudanabaena catenata ......................................... 175 5.1 Abstract .........................................................................................................