Provided by the author(s) and NUI Galway in accordance with publisher policies. Please cite the published version when available. Microbial ecology of E.coli removal mechanisms and drinking Title water production in slow sand filters exposed to emerging contaminants Author(s) Domingo, Octavi Publication Date 2019-04-19 Publisher NUI Galway Item record http://hdl.handle.net/10379/15110 Downloaded 2021-09-27T09:20:16Z Some rights reserved. For more information, please see the item record link above. MICROBIAL ECOLOGY OF E. COLI REMOVAL MECHANISMS AND DRINKING WATER PRODUCTION IN SLOW SAND FILTERS EXPOSED TO EMERGING CONTAMINANTS Octavi Domingo Fernandez A thesis submitted to the National University of Ireland Galway for the degree of: Doctor of Phylosophy Faculty of Science, Department of Microbiology, School of Natural Sciences, National University of Ireland Galway Supervisor: Co-Supervisor: Dr. Gavin Collins Dr. Eoghan Clifford Head of Department: Head of School: Dr. Gerard Wall Prof. Ciaran Morrison April 2019 ACKNOWLEDGEMENTS I would like to thank to all the people that, in many ways, have made this thesis possible. First, to my supervisor, Dr. Gavin Collins, for giving me the opportunity to do this PhD and for his guidance during these last few years. To Dr. Florence Abram, Dr. Conor O’Byrne and Dr. Eoghan Clifford, members of my GRC, for the advice and support provided during the project. To all the people from the Microbial Communities Lab. Este, Sean, Joe, Sarah, Kittie, Simon, Victor, Anna… for being always good colleagues and providing support in times of stress. To Maurice, Ann, Mike, Kathy, Caroline, from the Microbiology Department, for keeping this department running day after day. To all the people from this Department who I have had the pleasure of meeting during all these years. To all my Galway friends Jorge, Oscar, Encarni, Mona, Enrico, Juan, Paolo, Fabiana, Natalia, Camila, Paul, Daniel, Jaume and the rest of amazing people I have met during this time. For all the good memories we gathered together during these years. To the Masqueperros, for the great get aways when I needed them most. To my friends in Barcelona, for always making me feel like home when I was there. To Ama, for being by my side at all times, the Apache’s pizza’s nights, the trips… For everything. To my family, specially my parents, Angel and Elvira, to whom I will never be able to compensate everything they have done for me. To my sister, Alexandra, and my nephews Mario and Gala, who have been born during this period and who I have not seen as much as I would have liked. To my grandmothers, Teresa and Isabel, for all the pampering and being there for me. To my grandfathers, Toni and Angel, who helped me to get here and being the person I am, sure they would had loved to see me being successful in this adventure. Contents List of Figures …………………………………………………………...……… V List of Tables …………………………………………………………………... XI Abbreviations ……………………………………………………...…………. XIII Chapter 1: Introduction ........................................................................................ 1 1.1 Scope and Organization of the Thesis ........................................................... 3 Chapter 2: Literature Review ............................................................................... 5 2.1 Drinking Water Production with Slow Sand Filters ...................................... 5 2.1.1 Drinking Water Production Systems ................................................. 5 2.1.2 Slow Sand Filters .............................................................................. 7 2.2 E. coli Environmental Strains ...................................................................... 12 2.3 Emerging Contaminants .............................................................................. 14 2.4 Molecular Techniques in Microbial Ecology Studies ................................. 17 2.5 General Questions Addressed In the Thesis ................................................ 23 Chapter 3: Impact of Sporadic Faecal Contamination in the Microbial Community Structure of Lab-Scale Slow Sand Filters .................................... 25 3.1 Introduction.................................................................................................. 25 3.2 Materials and Methods ................................................................................ 28 3.2.1 Construction and Operation of the Lab-Scale SSFs ........................ 28 3.2.2 Faecal Contamination Simulations ................................................. 29 3.2.3 Water Sampling and Quality Assessment ....................................... 31 3.2.4 Schmutzdecke Sampling ................................................................. 32 3.2.5 DNA Extractions ............................................................................. 33 I 3.2.6 Quantitative Polymerase Chain Reactions (qPCRs) ....................... 33 3.2.7 High-throughput Sequencing .......................................................... 36 3.2.8 Statistical analysis ........................................................................... 37 3.3 Results ......................................................................................................... 37 3.3.1 Optical Density / E. coli Concentration Standard Curves ............... 37 3.3.2 Water Quality of Influent and Effluent ........................................... 38 3.3.3 Concentration of 16S rRNA, 18S rRNA and rodA genes in SMD samples 42 3.3.4 Bacterial Community Structure of the SSFs ................................... 44 3.3.5 Eukaryotic Community Structure of the SSFs ................................ 53 3.3.6 Protozoa Population Dynamics ....................................................... 63 3.4 Discussion .................................................................................................... 65 3.5 Conclusions ................................................................................................. 69 Chapter 4: Impact of Estrogens and Pharmaceuticals on the Microbial Ecology and total coliforms and E. coli removal in Slow Sand Filters ........... 72 4.1 Introduction.................................................................................................. 72 4.2 Materials and Methods ................................................................................ 75 4.2.1 Filters Operation and ECs Dosing .................................................. 75 4.2.2 Water and Schmutzdecke Sampling................................................ 77 4.2.3 Quantification of Total Coliforms and E. coli ................................ 78 4.2.4 Quantification of Nutrients ............................................................. 79 4.2.5 Quantification of Estrogens and Pharmaceuticals........................... 79 4.2.6 DNA Extraction and Amplicon Sequencing ................................... 80 4.2.7 Quality Trimming and Taxonomical Classification of the Sequences 81 4.2.8 Statistical Analysis .......................................................................... 81 4.3 Results ......................................................................................................... 81 II 4.3.1 Removal of Total Coliforms and E. coli ......................................... 81 4.3.2 Concentrations, and Removal, of Nutrients .................................... 85 4.3.3 Concentrations, and Removal, of ECs ............................................ 88 4.3.4 Impact of Estrogen and Pharmaceuticals on the Prokaryotic Community Structure of SSFs ...................................................................... 94 4.3.5 Impact of Estrogen and Pharmaceuticals on the Eukaryotic Community Structure of SSFs .................................................................... 105 4.4 Discussion .................................................................................................. 116 4.5 Conclusions ............................................................................................... 120 Chapter 5: Stable Isotope Probing to Unravel E. coli Removal Mechanisms in SSFs Exposed to Estrogen and Pharmaceuticals ............................................ 122 5.1 Introduction................................................................................................ 122 5.2 Material and Methods ................................................................................ 124 5.2.1 Lab-scale SSFs Used for the SIP Experiment ............................... 124 5.2.2 E. coli labelling with 13C ............................................................... 125 5.2.3 Filters Spike, and SMD and Effluent Sampling ............................ 125 5.2.4 DNA Extraction ............................................................................ 126 5.2.5 Separation of 12C and 13C DNA Fractions .................................... 126 5.2.6 Illumina MiSeq Amplicon Sequencing ......................................... 127 5.2.7 Statistical analysis ......................................................................... 128 5.3 Results ....................................................................................................... 128 5.3.1 13C-labelled E. coli Mass Balance ................................................. 128 5.3.2 12C and 13C DNA Fractions Separation ......................................... 130 5.3.3 Biological E. coli Removal Mechanisms ...................................... 130 5.3.4 Differences in E. coli Removal Mechanisms Produced by Exposure to Estrogen