Genes and Microbes Impacting the Geochemistry of Arsenic Mobilised Aquifers in Bangladesh and Cambodia A thesis submitted to The University of Manchester for the degree of Doctor of Philosophy in the Faculty of Science and Engineering 2017 Edwin Thadheu Gnanaprakasam School of Earth and Environmental Sciences 0 Table of Contents List of Figures …………………………………………………………………………………………………………….. 6 List of Tables ……………………………………………………………………………………………………………… 9 List of Abbreviations………………………………………………………………………………………………….. 11 Thesis Abstract …………………………………………………………………………………………………………. 13 Declaration ……………………………………………………………………………………………………………….. 14 Copyright Statement …………………………………………………………………………………………………. 15 Acknowledgements ………………………………………………………………………………………………….. 16 About the Author ……………………………………………………………………………………………………… 18 Chapter 1 : Introduction ............................................................................................................... 19 1.1. Project context ................................................................................................................. 19 1.2. Aims and objectives ....................................................................................................... 20 1.3. Thesis structure .............................................................................................................. 22 1.4. Paper status and author contributions ......................................................................... 25 Chapter 2 : Literature Review ...................................................................................................... 27 2.1. Arsenic general introduction ......................................................................................... 27 2.2. Arsenic geochemistry .................................................................................................... 30 2.2.1. Arsenic minerals and compounds ......................................................................... 30 2.2.2. Arsenic speciation ................................................................................................... 31 2.3. Arsenic bio-geochemical cycle ...................................................................................... 33 2.4. Geomicrobiology of arsenic mobilisation..................................................................... 35 2.4.1. Extracellular interaction ........................................................................................ 35 2.4.2. Intracellular interaction ........................................................................................ 36 2.4.2.1. Methylation and demethylation ..................................................................... 36 2.4.2.2. As(III) oxidation ............................................................................................. 38 2.4.2.3. As(V) reduction............................................................................................... 38 2.5. Role of other elements in the arsenic biogeochemical cycle ....................................... 40 2.6. Metagenomics of arsenic ............................................................................................... 41 2.6.1. 16S rRNA gene based community analysis ............................................................ 42 2.6.2. Functional genes analysis (targeted amplification and sequencing) .................. 44 2.6.3. Whole genome sequencing (WGS) based community analysis and gene annotation ....................................................................................................................... 50 2.7. Metatranscriptomic analysis of arsenic mRNAs ........................................................... 51 2.8. Metaproteomic analysis of arsenic proteins ................................................................. 52 2.9. Summary ........................................................................................................................ 54 1 Chapter 3 : Methodology ............................................................................................................. 55 3.1. Introduction ................................................................................................................... 55 3.2. Sampling methods ......................................................................................................... 55 3.3. General methods involved in the molecular analysis................................................... 57 3.3.1. DNA extraction ....................................................................................................... 57 3.3.2. RNA extraction ....................................................................................................... 58 3.3.3. Protein extraction .................................................................................................. 58 3.3.4. Primer design ......................................................................................................... 59 3.3.5. Thermal cycler and polymerase chain reaction (PCR) ......................................... 59 3.3.6. Gel electrophoresis and documentation ............................................................... 59 3.3.7. DNA and RNA quantification ................................................................................ 60 3.3.8. SequalPrep™ normalisation of PCR products ....................................................... 62 3.3.9. Pooling ................................................................................................................... 62 3.3.10. Next generation sequencing ................................................................................. 62 3.3.10.1. Roche/454 (Pyrosequencing) ......................................................................... 63 3.3.10.2. MiSeq and HiSeq (Illumina) .......................................................................... 65 3.3.11. High performance computing (HPC) ................................................................... 66 3.3.12. Bioinformatics tools .............................................................................................. 66 3.4. Metagenomic methods .................................................................................................. 69 3.4.1. 16S rRNA gene based community analysis ............................................................ 70 3.4.1.1. Roche 454 pyrosequencing and QIIME pipeline ............................................ 70 3.4.1.2. MiSeq and the Amplicon analysis pipeline .................................................... 72 3.4.2. WGS based metagenomic analysis ........................................................................ 74 3.5. Metatranscriptomics methods ...................................................................................... 76 3.6. Proteomic methods ....................................................................................................... 77 3.7. Geochemical methods ................................................................................................... 78 3.7.1. pH and Eh ............................................................................................................... 78 3.7.2. Diffuse reflectance spectrum: ................................................................................ 79 3.7.3. X-Ray fluorescence ................................................................................................. 79 3.7.4. EXAFS spectra for iron and arsenic ....................................................................... 79 3.7.5. Analysis of trace elements ...................................................................................... 81 3.8. Statistical methods ........................................................................................................ 82 3.8.1. O2-PLS method (Orthogonal Projection to Latent Structure) ............................. 82 3.8.2. Spearman’s rank correlation ................................................................................. 83 2 Chapter 4 : Paper I - The microbial community structure and arsenic biogeochemistry in two arsenic impacted aquifers in Bangladesh…………………………… .............................................. 84 4.1. Introduction ................................................................................................................... 85 4.2. Materials and methods .................................................................................................. 89 4.2.1. Study site ................................................................................................................ 89 4.2.2. Sample collection ................................................................................................... 90 4.2.3. Sediment chemistry ................................................................................................ 91 4.2.3.1. X-Ray fluorescence ........................................................................................... 91 4.2.3.2. EXAFS spectra for iron and arsenic ................................................................ 92 4.2.4. Aqueous chemistry ................................................................................................ 94 4.2.5. Microbial Ecology .................................................................................................. 94 4.2.5.1. 16S rRNA Based Bacterial Community Analysis ............................................. 94 4.2.5.2. Functional Gene Analyses .............................................................................