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Community and Whole Genome Analysis of Wastewater Bacteria

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Community and Whole Genome Analysis of Wastewater Bacteria Community and Whole Genome Analysis of Wastewater Bacteria By Daniel Rice BSc(Hons) A thesis submitted in total fulfilment of the requirements for the degree of Master of Science School of Life Sciences College of Science, Health and Engineering La Trobe University Victoria Australia February 2020 1 TABLE OF CONTENTS TABLE OF CONTENTS ................................................................................................................ i LIST OF FIGURES ..................................................................................................................... iii LIST OF TABLES ....................................................................................................................... iv LIST OF ABBREVIATIONS .......................................................................................................... v STATEMENT OF AUTHORSHIP ................................................................................................vii ACKNOWLEDGEMENTS ......................................................................................................... viii SUMMARY ..............................................................................................................................ix SECTION 1: INTRODUCTION 1.1 Activated sludge process ................................................................................................... 2 1.2 Community profiling of activated sludge bacterial communities ....................................... 7 1.2.1 Phosphorous removal ........................................................................................... 8 1.2.2 Nitrogen removal ................................................................................................. 9 1.2.3 Flocs and filamentous bacteria ........................................................................... 11 1.2.3.1 Foaming ......................................................................................................... 12 1.2.3.2 Bulking ........................................................................................................... 13 1.3 Isolation and genomic characterisation of filamentous bacteria ...................................... 15 1.4 Aims ................................................................................................................................ 15 SECTION 2: MATERIALS AND METHODS 2.1 General protocols ............................................................................................................ 18 2.1.1 Media, buffers and solution ................................................................................ 18 2.1.2 Bacterial strains .................................................................................................. 18 2.1.3 Wastewater treatment plants and sampling ....................................................... 19 2.2 Microbiology procedures ................................................................................................ 20 2.2.1 Micromanipulation ............................................................................................. 20 2.2.2 Gram staining ..................................................................................................... 20 2.3 Genetic procedures ......................................................................................................... 20 2.3.1 DNA extraction ................................................................................................... 22 2.3.1.1 Bacterial genomic DNA extraction .................................................................. 22 2.3.1.2 Genomic DNA extraction of whole community wastewater samples .............. 23 2.3.2 Determination of DNA concentration ................................................................. 23 2.3.3 Polymerase chain reaction procedure and purification of products .................... 24 2.3.4 Bead clean-up of DNA ......................................................................................... 24 2.3.5 Agarose gel electrophoresis ................................................................................ 25 2.3.6 Sequencing ......................................................................................................... 25 2.3.6.1 Amplicon sequencing library prep .................................................................. 25 i 2.3.6.1.1 PCR amplification of amplicon libraries .............................................. 25 2.3.6.1.2 Library pooling and denaturation ...................................................... 26 2.3.6.2 Whole genome library prep and sequencing .................................................. 26 2.3.6.2.1 Illumina library prep and sequencing ................................................. 26 2.3.6.2.2 Oxford nanopore library prep and sequencing................................... 27 2.3.6.3 Computational DNA sequence analysis .......................................................... 27 2.3.6.3.1 Analysis of amplicons ........................................................................ 27 2.3.6.3.2 Analysis of whole genome ................................................................. 28 SECTION 3: CHANGES IN THE COMMUNITY COMPOSITIONS OF THREE NITROGEN REMOVAL WASTEWATER TREATMENT PLANTS OF DIFFERENT CONFIGURATIONS IN VICTORIA, AUSTRALIA OVER A 12-MONTH OPERATIONAL PERIOD 3.1 Introduction ................................................................................................................... 30 3.2 Results ............................................................................................................................ 32 3.2.1 General comments ............................................................................................. 32 3.2.2 Bacterial communities in the Woodend wastewater treatment plant ................. 42 3.2.3 Bacterial communities in the Gisborne wastewater treatment plant .................. 46 3.2.4 Bacterial communities in the Melton wastewater treatment plant ..................... 51 3.3 Discussion ....................................................................................................................... 55 SECTION 4: ISOLATION AND WHOLE GENOME SEQUENCE ANALYSIS OF FILAMENTOUS BACTERIA ISOLATED FROM ACTIVATED SLUDGE 4.1 Introduction .................................................................................................................... 58 4.2 Acidovorax ...................................................................................................................... 60 4.2.1 Acidovorax genome ............................................................................................ 60 4.2.1.1 Central pathways ........................................................................................... 61 4.2.1.2 Carbon storage material................................................................................. 63 4.2.1.3 Acidovorax metabolism ................................................................................. 63 4.2.1.4 Defence systems identified in the genome sequence ..................................... 66 4.2.1.5 Genes involved in cell formation and morphology ......................................... 67 4.2.2 Genomic variations between Acidovorax spp. Strains ......................................... 69 4.3 Significance in activated sludge ....................................................................................... 71 References ............................................................................................................................ 73 Appendix 1 – Supplemental data for amplicon analysis ......................................................... 80 Appendix 2 – Operational data .............................................................................................. 94 Appendix 3 – Scripts used for amplicon analysis.................................................................. 198 ii LIST OF FIGURES Figure 1.1 Schematic diagram of the activated sludge process ......................................... 3 Figure 1.2 Stages of the nitrogen cycle ........................................................................... 11 Figure 3.1 PCoA ordination of bacterial communities ..................................................... 34 Figure 3.2 Box plot showing average abundance of bacterial phyla ................................ 35 Figure 3.3 Heatmaps of filament populations ............................................................ 38-40 Figure 3.4 Gram stains of foam samples ......................................................................... 41 Figure 3.5 Line graph of SVI operational data ................................................................. 42 Figure 3.6 PCoA ordination of the Woodend communities ............................................. 45 Figure 3.7 Heat map of the Woodend community over a twelve-month period ............. 46 Figure 3.8 PCoA ordination of the Gisborne community ................................................. 49 Figure 3.9 Heat map of the Gisborne community over a twelve-month period .............. 50 Figure 3.10 Heat map of known nitrifying bacteria ..................................................... 51-53 Figure 3.11 PCoA ordination of the Melton communities at monthly intervals ................. 57 Figure 3.12 Heat map of the Melton community over a twelve-month period ................. 58 Figure 4.1 Gram stain of putative Acidovorax isolate ...................................................... 60 Figure
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