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The Microbiology and Metal Attenuation in a Natural Wetland Impacted by Acid Mine Drainage

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The Microbiology and Metal Attenuation in a Natural Wetland Impacted by Acid Mine Drainage The microbiology and metal attenuation in a natural wetland impacted by acid mine drainage A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Science and Engineering 2018 Oscar E. Aguinaga Vargas School of Earth and Environmental Sciences TABLE OF CONTENTS ABSTRACT ................................................................................ 6 DECLARATION ........................................................................... 7 COPYRIGHT STATEMENT ............................................................. 8 ACKNOWLEDGMENTS ................................................................. 9 Chapter 1: GENERAL INTRODUCTION ..................................... 10 1.1 ACID MINE DRAINAGE ..................................................... 10 1.2 ENVIRONMENTAL IMPLICATIONS OF ACID MINE DRAINAGE ... 13 1.2.1 Presence of trace metals .............................................. 13 1.2.2 Environmental impact .................................................. 14 1.3 ACID MINE DRAINAGE REMEDIATION .................................. 18 1.3.1 Acid mine drainage remediation by wetlands ................... 19 1.3.1.1 Physicochemical processes ...................................... 19 1.3.1.2 Role of plants ........................................................ 21 1.3.1.3 Role of microorganisms ........................................... 23 1.4 CASE STUDIES OF ACID MINE DRAINAGE REMEDIATION BY WETLANDS ............................................................................ 24 1.4.1 Constructed wetlands ................................................... 24 1.4.2 Natural wetlands ......................................................... 28 1.5 PARYS MOUNTAIN ............................................................. 30 1.6 HYPOTHESES, AIMS AND OBJETIVES OF THE THESIS ............ 39 Chapter 2: CHARACTERIZATION OF ACID MINE DRAINAGE POLLUTION AND MICROBIAL COMMUNITY SHIFTS IN THE SOUTHERN AND NORTHERN AFON GOCH ............................... 42 2.1 ABSTRACT ....................................................................... 43 2.2 INTRODUCTION ................................................................ 44 2.3 MATERIALS AND METHODS ................................................ 46 2.3.1 Study site and sample collection .................................... 46 2.3.2 Water and sediments quality analysis ............................. 48 2.3.3 DNA extraction ............................................................ 52 2.3.4 16S rRNA gene sequencing ........................................... 52 2.3.5 Statistical analysis of environmental data ....................... 53 2 2.3.6 Sequence data analysis ................................................ 54 2.4 RESULTS ......................................................................... 56 2.4.1 Environmental characterization of the SAG and NAG ........ 56 2.4.2 Microbial community structure from MiSeq data .............. 62 2.4.3 Relative abundance of known bacteria related to S and Fe metabolism ......................................................................... 69 2.4.4 Prediction of microbial taxonomic-derived metabolism ...... 75 2.5 DISCUSSION .................................................................... 76 2.5.1 Microbial structure related to AMD pollution levels ........... 76 2.5.2 Prediction of metabolic potential .................................... 86 2.6 CONCLUDING REMARKS .................................................... 87 Chapter 3: ANALYSIS OF BACTERIA ACTIVITY AND METAL DISTRIBUTION ALONG THE SOUTHERN AFON GOCH WETLAND .............................................................................................. 89 3.1. ABSTRACT ...................................................................... 90 3.2 INTRODUCTION ................................................................ 91 3.3 MATERIALS AND METHODS ................................................ 94 3.3.1 Field site locations and sampling ................................... 94 3.3.2 In situ water analysis and total aqueous metal ................ 96 3.3.3 Metal analysis in sediments........................................... 96 3.3.4 Measurement of C and N along core depths .................... 97 3.3.5 DNA and RNA extraction ............................................... 98 3.3.6 Construction of 16S rRNA libraries ................................. 98 3.3.7 Quantification of RNA transcripts ................................... 99 3.3.8 Bioinformatic analysis ................................................ 100 3.3.9 Statistical analysis ..................................................... 101 3.4 RESULTS ....................................................................... 103 3.4.1 Phylogenetic analysis of 16S rRNA libraries from W2 surface sediments ......................................................................... 103 3.4.2 Measurement of pH in water samples ........................... 107 3.4.3 Sulphur content and speciation in water samples ........... 108 3.4.4 Iron content and speciation in wetland water samples .... 108 3.4.5 Trace metal levels in water samples along the wetland ... 111 3.4.6 Metal distribution along sediment depths ...................... 111 3.4.7 Expression of dsrA and soxB genes .............................. 118 3 3.4.8 Expression of 16S rRNA genes from Fe oxidizing bacteria118 3.4.9 Expression of soxB and F. myxofaciens genes in different plant stand in W2 sediment ................................................. 121 3.4.10 ITRAX analysis ........................................................ 122 3.4.11 Total and dissolved organic carbon and nitrogen concentrations along core depths ......................................... 127 3.5 DISCUSSION .................................................................. 130 3.5.1 Surface sediment bacteria and Fe and S speciation along the wetland ............................................................................ 130 3.5.2 Metal distribution along sediment depths ...................... 131 3.5.3 Expression of S metabolic genes .................................. 134 3.5.4 Expression of Fe bacteria ribosomal genes .................... 135 3.6 CONCLUDING REMARKS .................................................. 137 3.7 SUPLEMENTARY DATA ..................................................... 139 Chapter 4: EFFECT OF OXYGEN AND ORGANIC SUBSTRATES ON BACTERIAL ACTIVITY AND METAL ATTENUATION IN INCUBATED WETLAND SEDIMENTS ...................................... 143 4.1 ABSTRACT ..................................................................... 144 4.2 INTRODUCTION .............................................................. 145 4.3 MATERIALS AND METHODS .............................................. 148 4.3.1 Sediment sampling .................................................... 148 4.3.2 Experimental design .................................................. 149 4.3.3 Measurement of environmental variables ...................... 151 4.3.4 DNA extraction and shotgun metagenomic sequencing ... 152 4.3.5 Bioinformatic analysis ................................................ 153 4.3.6 Statistical analysis ..................................................... 154 4.4 RESULTS ....................................................................... 154 4.4.1 Changes in water chemistry during microcosm incubations ....................................................................................... 154 4.4.2 General characteristics of the sediment metagenomes ... 177 4.4.3 Comparison of taxonomy profiles ................................. 177 4.4.4 Comparison of functional profiles ................................. 184 4.4.5 Sulphate metabolism analysis ..................................... 187 4.5 DISCUSSION .................................................................. 198 4.5.1 Changes in S and metal chemistry ............................... 198 4 4.5.2 Differences in bacterial taxonomy and metabolism ......... 201 4.6 CONCLUDING REMARKS ................................................ 207 Chapter 5: GENERAL DISCUSSION AND CONCLUSIONS ........ 210 5.1 SUMMARY AND KEY FINDINGS ......................................... 210 5.2 RESEARCH IMPLICATIONS AND RELEVANCE ....................... 216 5.3 FUTURE DIRECTIONS ...................................................... 223 6 REFERENCES ..................................................................... 225 Word count: 44,049 5 ABSTRACT A natural wetland that has historically received acid mine drainage (AMD) from the abandoned metal mine at Parys Mountain (Anglesey, UK) has proved efficient in the removal of Fe and other trace metals. This study aims to evaluate the microbial and physicochemical mechanisms involved in the metal attenuation process observed along the wetland by assessing the role of the microbial communities and microbial – derived changes in metal chemistry that leads to an improvement in the water quality. A combination of molecular microbiology approaches, metal analytical techniques and microcosm experiments show that the wetland retains the diversity and metabolic structure of sediment bacteria communities despite the high acidity and metal concentration. In the middle of the wetland, increased bacterial activity related to Fe and S oxidation coincided with a removal of sulphate and metals from the water column. Incubation experiments of wetland sediments showed that the presence of bacteria generated higher levels of sulphide and particulate metals
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