Virus Dynamics and their Interactions with Microbial Communities and Ecosystem Functions in Engineered Systems. A thesis submitted to Newcastle University in partial fulfilment of the requirements for the degree of Doctor of Philosophy in the Faculty of Science, Agriculture and Engineering. Author: Mathew Robert Brown Supervisor: Professor Tom Curtis Co-Supervisor: Dr Russell Davenport 2019 Declaration I hereby certify that this work is my own, except where otherwise acknowledged, and that it has not been submitted for a degree at this, or any other, institution. Mathew Robert Brown Abstract Climate change, population growth and increasingly strict environmental regulation means the global water industry is currently facing an unprecedented coincidence of challenges (Palmer, 2010). Better microbial ecology could significantly contribute, since explicitly engineering and maintaining efficient and functionally stable microbial communities would allow existing assets to be optimised and their robustness improved. Given its role in natural systems viral infection could be an important, yet overlooked, factor. Here we attempt to address this lacuna, particularly within activated sludge systems. To facilitate this process we developed, optimised and validated a flow cytometry method, allowing rapid (relative to other methods), accurate and highly reproducible quantification of total free viruses in activated sludge samples (mixed liquor (ML)). Its use spatially identified viruses are highly abundant, with concentrations ranging from 0.59 - 5.14 × 109 viruses mL-1 across 25 activated sludge plants. Subsequently we applied this method to ML collected from one full- and twelve replicate lab-scale activated sludge systems respectively. At both scales viruses in the ML were shown to be both abundant and temporally/spatiotemporally dynamic, thus ever present across activated sludge systems. Through statistical inference they were shown to be associated (positively) with total host (bacterial) abundance, with microbial community structure and with a systems function (the + removal of COD and NH4 -N from influent wastewaters), whilst exogenous factors, particularly those involved in adsorption processes, played an important role in their dynamics. Evidence of predator-prey dynamics between a subset of measured viruses and a key functional group (ammonia oxidising bacteria (AOB)) within the full-scale system is also presented, whilst a detailed examination of all garnered abundances highlights the relative abundance of viruses, as reported in marine systems, declined with increasing host density. Finally preliminary metagenomic data shows wastewater viromes are largely phylogenetically and functionally uncharacterised, yet relative abundances of known viruses vary throughout the wastewater treatment stream. Considering the evidence presented viruses appear to play a more central role in the dynamics of activated sludge systems than hitherto realised and thus should be considered more frequently when assessing the key factors governing bacterial abundance, community composition and functional stability. Table of Contents CHAPTER 1 1 INTRODUCTION 1 1.1. Background 3 1.2 Viruses in Wastewater Treatment Processes 7 1.2.1. The Role of Bacteriophages in Wastewater Treatment: Interactions with Microbial Community composition 8 1.2.2 The Role of Bacteriophages in Wastewater Treatment: Interactions with food web processes and biogeochemical cycles 12 1.2.3 The Role of Bacteriophages in Wastewater Treatment: Can they influence process performance? 13 1.3 Insights from Natural Systems 15 1.3.1 Expanding the Spatial and Temporal Resolution of Viral Studies 15 1.3.2 Identifying True Viral Diversity 16 1.3.3 Elucidating who infects whom 17 1.4 Aims and Objectives 17 CHAPTER 2 19 FLOW CYTOMETRIC QUANTIFICATION OF VIRUSES IN ACTIVATED SLUDGE 19 2.1. Introduction 21 2.2. Materials and Methods 22 2.2.1. Protocol Optimisation 22 2.2.2. Fluorescent Staining and FCM Analysis 24 2.2.3. Virus Recovery Efficiency 24 2.2.4. Virus abundance at a suite of AS WWTP’s 25 2.2.5. Comparison of FCM and TEM counts 25 2.2.6. Statistical Analyses 26 2.3. Results 26 2.3.1. Optimisation of Protocol for AS Virus Enumeration by FCM 26 2.3.2. Virus Recovery and Enumeration Efficiency 28 2.3.3. Virus Abundance in Full Scale Activated Sludge WWTP’s 28 2.3.4. FCM vs. TEM 30 2.4. Discussion 31 2.5. Conclusions 34 2.6. Acknowledgments 34 CHAPTER 3 35 COUPLED VIRUS-BACTERIA INTERACTIONS AND ECOSYSTEM FUNCTION IN AN ENGINEERED MICROBIAL SYSTEM 35 I 3.1. Introduction 37 3.2. Materials and Methods 39 3.2.1. Sample Collection 39 3.2.2. Analytical Methods 39 3.2.3. Molecular Methods 39 3.2.4. Statistical Analysis 41 3.3. Results 44 3.3.1. Bioreactor Performance and Abiotic Conditions 44 3.3.2. Temporal Abundance Dynamics of Viruses and Bacteria 45 3.3.3. Virus Interactions with Biotic and Abiotic Conditions 47 3.3.4. Virus Interactions with Bacteria Community Structure 48 3.3.5. Virus interactions with community function 49 3.4. Discussion 50 3.5. Acknowledgements 54 CHAPTER 4 55 VIRUS - BACTERIA INTERACTIONS, SYNCHRONICITY AND ECOSYSTEM FUNCTION IN REPLICATE ENGINEERED MICROBIAL SYSTEMS 55 4.1. Introduction 57 4.2. Materials and Methods 58 4.2.1. Reactor Set Up 58 4.2.2. CSTR Operational Conditions 59 4.2.3. Sample Collection and Storage 59 4.2.4. Analytical Methods 59 4.2.5. Molecular Methods 60 4.2.6. Statistical Analysis 62 4.3. Results 66 4.3.1. Functional performance and Abiotic Conditions 66 4.3.2. Spatiotemporal Dynamics of Virus, Bacterial and AOB Abundances 67 4.3.3. Spatiotemporal Interactions 68 4.3.4. Similarity and Synchronicity of CSTR’s (day 62 - 204) 72 4.4. Discussion 75 4.5. Acknowledgements 78 CHAPTER 5 79 EVIDENCE OF PREDATOR-PREY DYNAMICS BETWEEN BACTERIOPHAGE AND AMMONIA OXIDISING BACTERIA IN AN ENGINEERED MICROBIAL SYSTEM 79 5.1. Introduction 81 II 5.2. Materials and Methods 82 5.2.1. Sample Collection 82 5.2.2. Flow Cytometry Analysis 82 5.2.3. DNA Extraction qPCR 82 5.2.4. Statistical Analysis 83 5.2.5. Theory 83 5.3. Results and Discussion 84 5.4. Acknowledgements 86 CHAPTER 6 89 A WASTEWATER PERSPECTIVE ON VIRAL AND MICROBIAL ABUNDANCES AND VIRUS-MICROBE RATIOS 89 6.1 Introduction 91 6.2 Materials and Methods 92 6.2.1 Sample Collection 92 6.2.2 Flow Cytometry Analysis 92 6.2.3 DNA Extraction qPCR 92 6.2.4 Statistical Analysis 93 6.3 Results and Discussion 93 6.4 Acknowledgements 96 CHAPTER 7 97 PRELIMINEARY METAGENOMIC CHARACTERISATION OF WASTEWATER VIRUSES 97 7.1 Introduction 99 7.2 Materials and Methods 100 7.2.1 Sample Collection 100 7.2.2 Sample Processing 100 7.2.3 Prophage Induction 101 7.2.4 Molecular Methods 101 7.2.5 Bioinformatics 101 7.3 Results and Discussion 103 7.3.1 General Characteristics of the Wastewater Viromes 103 7.3.2 Phylogenetic and Functional Profiles of Nucleotide Sequences 103 7.3.3 Phylogenetic Profiles of Assembled Protein Sequences 105 7.3.4 Mined Viral-like Genomes 106 7.3.5 Comparison of Wastewater Viromes 106 7.4 Preliminary Conclusions and Recommendations 107 7.5 Acknowledgements 108 III CHAPTER 8 109 GENERAL DISCUSSION AND CONCLUDING REMARKS 109 8.1 Rationale 111 8.2 Synopsis of results 111 8.3 Caveats 114 8.4 Conclusions 115 CHAPTER 9 117 FUTURE WORK 117 CHAPTER 10 125 REFERENCES 125 APPENDIX I 163 AN INTRODUCTION TO WASTEWATER TREATMENT 163 I.1. Introduction 165 I.2. Biological Secondary Treatment 165 I.3. The Activated Sludge Process 166 APPENDIX II 169 AN INTRODUCTION TO BACTERIOPHAGES 169 II.1. What is a phage?! 171 II.2. The Phage Infection Cycle 171 II.3. Phage Life Cycles 173 APPENDIX III 175 SUPPLEMENTARY INFORMATION - COUPLED VIRUS-BACTERIA INTERACTIONS AND ECOSYSTEM FUNCTION IN AN ENGINEERED MICROBIAL SYSTEM 175 III.1. Supplementary Methods 177 III.2. Supplementary Results 178 III.2.1. Bioreactor performance and abiotic conditions 178 III.2.2. Virus interactions with biotic and abiotic conditions 182 III.2.3. Virus interactions with bacteria community structure 185 APPENDIX IV 187 SUPPLEMENTARY INFORMATION - VIRUS - BACTERIA INTERACTIONS, SYNCHRONICITY AND ECOSYSTEM FUNCTION IN REPLICATE ENGINEERED MICROBIAL SYSTEMS 187 IV.1. Supplementary Results 189 APPENDIX V 203 SUPPLEMENTARY INFORMATION - EVIDENCE OF PREDATOR-PREY DYNAMICS BETWEEN BACTERIOPHAGE AND AMMONIA OXIDISING BACTERIA IN AN ENGINEERED MICROBIAL SYSTEM 203 V.1. Supplementary Theory 205 IV V.1.1. Extension of LV Equations 205 V.1.2. More complex models 205 V.1.3. AOB Mortality and Growth 206 APPENDIX VI 207 SUPPLEMENTARY INFORMATION - A WASTEWATER PERSPECTIVE ON VIRAL AND MICROBIAL ABUNDANCES AND VIRUS-MICROBE RATIOS 207 VI.1. Supplementary Results 209 APPENDIX VII 211 SUPPLEMENTARY INFORMATION - PRELIMINARY METAGENOMIC CHARACTERISATION OF WASTEWATER VIRUSES 211 VII.1. Supplementary Methods 213 VII.2. Supplementary Results 214 V VI List of Figures CHAPTER 1 Figure 1. 1. Possible scenarios for temporal changes in host abundance as a consequence of viral infection, (A – D) described in the text. 9 CHAPTER 2 Figure 2. 1. Effect of dispersants (A) and sonication time (B) on FCM virus abundance. Main bars indicate mean virus abundance across triplicates, whilst error bars indicate standard deviation across triplicates. * Significantly different from controls at the 0.05 level. 27 Figure 2. 2. Effect of DNase treatment (A), stain type and dilution (B) and incubation temperature (C) on FCM virus abundance. Main bars indicate mean virus abundance across triplicates, whilst error bars indicate standard deviation across triplicates. * Significantly different from other treatments at the 0.05 level. 27 Figure 2. 3. FCM density plots (A – C) and histograms (D – F) of AS samples taken from Tudhoe Mill WWTP stained with SG I (A and D), SG (B and E) and SG II (C and F), all at a 0.5 × 10-4 dilution of commercial stock.