Imperial College London Soil Microbial Interactions
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IMPERIAL COLLEGE LONDON SOIL MICROBIAL INTERACTIONS AFFECTING ENTERIC PATHOGEN SURVIVAL IN SEWAGE SLUDGE-AMENDED AGRICULTURAL SOIL PhD Thesis by Felipe Perez Viana February 2010 Supervisor Stephen R. Smith Department of Civil and Environmental Engineering Abstract The natural inactivation of enteric pathogens in soil is a critical component of the multi-barrier approach to prevent infectious disease in humans by enteric microorganisms when biosolids are used as a fertiliser and soil conditioner on agricultural land. The addition of biosolids to agricultural soil modifies the soil microbial community and ecological interactions. Ecological processes, especially the activities of predatory protozoa, may have a critical role in reducing the survival of enteric pathogenic bacteria when biosolids are applied to agricultural soil. To test this hypothesis a series of field experiments were established on two soils of contrasting organic matter content and fertility status, amended with different sludge types, to examine the interactions between the soil microbial biomass, total protozoa numbers, environmental and soil factors and their effects on the decay of the enteric indicator bacteria, Escherichia coli , in biosolids-amended soil. Soil microbial biomass carbon (SMBC) concentrations were influenced by soil physico-chemical properties and, in particular, larger background biomass concentrations were measured in unamended control soil containing the largest amount of organic matter. The microbiological content and substrate availability of the supplied materials also influenced the extent of the increases in SMBC. Soil protozoa numbers consistently -1 increased in both experimental field soils from background values of 3-3.5 log 10 g -1 ds to 4-4.5 log 10 g ds after sludge application. The extent of the increase was consistent with the effect of the organic amendments on SMBC. Laboratory investigations indicated the direct involvement of bacteriophagous protozoa activity in the soil ecological processes responsible for E. coli inactivation in biosolids- amended agricultural soil. This was linked to the addition of an active protozoa population to the soil in sludge, as well as to the stimulation of protozoa indigenous to the soil due to inputs of substrates and microbial biomass in sludge. Consequently, the survival of enteric organisms is a self-limiting process, due to the stimulation of microbial predatory activity in amended soil. Overall, the results provide assurance that assumptions relating to soil decay during waiting periods stipulated for agricultural use of sludge are highly conservative. They also confirm that the cropping/harvesting restrictions prescribed in legislation and guidance controlling the application of biosolids on farmland allow the natural attenuation of pathogens to protect human health with a significant margin of safety. 2 Declaration of own work I declare that this thesis: `Soil Microbial Interactions Affecting Enteric Pathogens Survival in Biosolids-Amended Agricultural Soil’, is my own work and that the work of others is fully cited and referenced, and/or with appropriate acknowledgment given. 3 Acknowledgments This thesis was completed as part of EPSRC grant: Enteric pathogen decay kinetics in biosolids amended agricultural soils. The financial support of DEFRA, NFU, UKWIR, Anglian Water, Scottish Water, Thames Water and Yorkshire Water is also gratefully acknowledged. My sincere and especial thanks to my supervisor Professor Stephen R. Smith for all his support, guidance, patience and encouragement. Thank-you to my research team, Dr James Cass, Dr Hannah Rigby and Dr Michael Rogers with whom I established the field trials and collaborated with in sampling and trial maintenance. Thank-you to Jeremy Hall for help with field work, and to Carol Edwards and Dr Geoff Fowler for help in the laboratory. To all my family and friends, Thank-you for your unconditional support. 4 Table of contents Abstract 2 Declaration of own work 3 Acknowledgments 4 Table of contents 5 List of Figures 10 List of Tables 15 Abbreviations 18 1 Introduction 20 2 Aims and Objectives 24 3 Literature review 26 3.1 Application of Biosolids to Agricultural Land 26 3.2 Legislation Related to Application of Biosolids to Agricultural Land 27 3.3 Sewage Sludge Generated from Wastewater Treatment 33 3.4 Pathogens Present in Sewage Sludge 36 3.5 Effect of Sewage Sludge Treatment Processes on Pathogen Removal 36 3.5.1 Pasteurisation 39 3.5.2 Lime Treatment 39 3.5.3 Mesophilic Anaerobic Digestion (MAD) 40 3.5.4 Thermophilic Aerobic Digestion (TAD) 40 3.5.5 Composting 41 3.6 Fate of Pathogens in Agricultural Soil Amended with Biosolids 42 3.6.1 Introduction 42 3.6.2 Temperature 44 3.6.3 Soil Moisture Content 45 3.6.4 Soil Matrix 46 3.6.5 Soil pH 47 3.6.6 Interactions Between Organisms 47 3.6.6.1 Competition 47 3.6.6.2 Parasitism 48 3.6.6.3 Predation 49 3.7 Effect of Biosolids on Soil properties 49 3.7.1 Biosolids as a Fertiliser 50 5 3.7.2 Effect of Biosolids Application on Soil Physical Properties 52 3.7.3 Effect of Biosolids Application on Soil Biological Properties 53 3.8 Soil Microbial Biomass 54 3.8.1 Environmental Factors Affecting SMBC 55 3.8.1.1 Temperature and Moisture Content 55 3.8.1.2 Soil Matrix 56 3.8.1.3 Soil Management 57 3.8.1.4 pH 57 3.8.2 Effect of Biosolids Application on the Soil Microbial Biomass 57 3.9 Soil Protozoa 59 3.9.1 Introduction 59 3.9.2 Soil Protozoan Classification 60 3.9.2.1 Ciliates 59 3.9.2.2 Heterotrophic Flagellates 61 3.9.2.3 Naked Amoebae 61 3.9.2.4 Testate Amoebae 61 3.9.3 Life Cycle 61 3.9.4 Ecological Role of Protozoa 62 3.9.4.1 Effect of Protozoan Predation on Bacterial Populations 63 3.9.4.2 Effect of Protozoa in Waste Water Treatment Plants 66 3.9.4.3 Effect of Protozoa on Nutrient Cycle 67 3.9.5 Factors Affecting Activity of Protozoa in Soil 69 3.9.5.1 pH 69 3.9.5.2 Temperature 69 3.9.5.3 Moisture Content 70 3.9.5.4 Soil Texture and Structure 70 3.9.5.5 Biocides 71 3.9.6 Method for the Enumeration of Soil Protozoa 71 3.9.6.1 Dilution Culture Methods. 72 3.9.6.2 Direct Counting Method 73 4 General Materials and Methods 75 4.1 Introduction 75 4.2 Site Location and Soil Characteristics 75 4.2.1 Soil Characteristics 76 6 4.2.2 Soil Classification 78 4.3 Biosolids, Livestock Wastes and Cattle Slurry Characteristics 79 4.3.1 Source, Collection and Processing 79 4.3.2 Physicochemical Characteristics 81 4.4 Experimental Field Trials 81 4.4.1 Design and Establishment 81 4.4.2 FT1 85 4.4.3 FT2 86 4.4.4 FT3 86 4.4.5 FT4 88 4.5 Soil Sampling and Analysis 88 4.5.1 Sample Collection, Transport and Storage 88 4.6 Soil Microbial Biomass C Analysis 88 4.7 Most Probable Number (MPN) Technique for Enumeration of soil Protozoa 90 4.8 Environmental Data 93 4.9 Statistical Analysis 93 5 Effect of Organic Residual Soil Amendments on the Microbial Biomass C Concentration in Two Contrasting Agricultural Soils 97 5.1 Introduction 97 5.2 Materials and Methods 99 5.3 Results 99 5.3.1 Effect of Soil Type and Season on SMBC 99 5.3.2 Effect of Organic Amendments 100 5.3.2.1 DMAD and DRAW Amendments 100 5.3.2.2 Other Sludge Amendments 111 5.3.2.3 Livestock Manures and Slurries 112 5.3.3 Effect of environmental factors on SMBC 114 5.4 Discussion 120 5.5 Conclusions 126 6 Influence of Biosolids Type and Application on Soil Protozoa Population Dynamics in Two Contrasting Agricultural Field Soils and Under Different Temperate Seasonal Conditions 128 7 6.1 Introduction 128 6.2 Materials and Methods 129 6.3 Results 130 6.3.1 General Factors Influencing Soil Protozoa Populations 130 6.3.2 Unamended Control Soil 131 6.3.3 Organic Amendments 133 6.3.3.1 General Response 133 6.3.3.2 Dewatered Mesophilic Digested Biosolids (DMAD) 136 6.3.3.3 Dewatered Raw Sludge (DRAW) 142 6.3.3.4 Enhanced Treated Biosolids 147 6.3.4 Protozoa inhibitor 149 6.3.5 Effect of Environmental Variables on Soil Protozoa Populations 150 6.3.6 Influence of Soil Protozoa on the Decay of Enteric Bacteria 151 6.4 Discussion 154 6.5 Conclusions 161 7 Influence of Soil and Sludge Microbiological Properties on Protozoa Populations in Biosolids-amended Soil and Implications for Survival of Enteric Bacteria 163 7.1 Introduction 163 7.2 Materials and Methods 164 7.2.1 Soil and Biosolids Characteristics 164 7.2.2 Soil Protozoa 165 7.2.3 Enteric Bacteria 166 7.2.4 Design and Establishment of the Laboratory Incubation 167 7.3 Results 169 7.3.1 Sterile Soil 169 7.3.1.1 Protozoa Dynamics in Control and Inoculated Soil 169 7.3.1.2 Effect of DMAD Application in Protozoa Dynamics in Sterile Soil 170 7.3.2 Unsterile Soil 173 7.3.2.1 Protozoa Dynamics in Unamended Control and Inoculated Soil 174 7.3.2.2 Effect of DMAD Application on Protozoa Dynamics in Unsterile Soil 174 7.3.2.3 Effect of Sterile DMAD Application in Protozoa Dynamics in Unsterile Soil 176 7.3.3 Interactions Between Protozoa and Bacteria 178 8 7.3.3.1 Effect of Protozoa and Sludge Microbiology on Inoculated E. coli Populations in Sterile Soil 178 7.3.3.2 Effect of Protozoa and Bacteria in E.