Fate of antibiotic resistance in carcass landfill leachate : biological and physicochemical treatment

다국어 초록 (Multilingual Abstract)

This study aimed to investigate and understand the behavior of leachate and the fate of its antibiotic resistance. A synthetic leachate was formulated and monitored for more than 2 years. This is done to simulate the fate of antibiotic resistance in leachate from anaerobic carcass landfill site. Three anaerobic reactors were set-up. Initially, Escherichia coli DH5α with tetracycline resistance pB10 plasmid was inoculated in nutrient rich anaerobic reactors. The fate of tetracycline-resistant bacteria (TRB) was tracked by analysis using culture-based method, EC50 (half maximal effective concentration), and quantitative polymerase chain reaction (qPCR). Though the EC50 profile looks maintained, values during the second year shows lower standard deviation. Interestingly, pB10 plasmid has the same pattern. While the two parameters agreed with each other, the TRB time profile decreased after 300 days or almost one year. At the end of experiment, the tetracycline resistance microbial communities were investigated by 16s RNA gene-based pyro sequencing. The results of this study indicated that leachate with high organic strength in anaerobic conditions could be an antibiotic resistant point source in several year periods.
To simulate the treatment of leachate, the effects of the solids retention time (SRT), using sequencing batch reactors (SBRs), was investigated. This is done to see the fate of antibiotic resistance in the leachate sample. SRTs were varied to see its influence on antibiotic resistance. Escherichia coli DH5a was used as the representative tetracycline-resistant bacteria with multiple antibiotic-resistant genes encoded in plasmid pB10. SRT contributed to an increase in antibiotic resistance in SBR C (SRT 25 days) with TRB values up to 1.25 x 107 CFU mL-1 which is one log higher than the influent. Microbial community analysis of the DNA samples from effluent of SBR C showed four major phyla: Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria under which are ecologically-important microbial species. It was shown that antibiotic resistance genes cannot be eliminated during treatment of synthetic carcass leachate in a lab-scale sequencing batch reactor.
Lastly, the fate of antibiotic resistance in an effluent after ozone disinfection was investigated by comparing the ARB and ARG removal efficiencies of ozonation using pure culture in buffer and using E. coli pB10 in wastewater samples. Based on the results, ozonation on E. coli K-12, as a representative of non-ARB, showed higher removal efficiencies than the samples inoculated with antibiotic-resistant E. coli. Another aspect of this study is monitoring the fate of antibiotic resistance in the wastewater effluent. It was found out that ARB and ARGs still perpetuated in the last stage of effluent disinfection showing that treatment in WWTPs is not effective in eliminating the antibiotic resistance.

목차 (Table of Contents)

• ACKNOWLEDGEMENT i
• DEDICATION ii
• ABSTRACT i
• LIST OF ABBREVIATIONS xii
• CHAPTER 1 Background 1
• 1.1 Introduction 1
• 1.1.1 Statement of the problem 2
• 1.1.2 Research objectives 2
• 1.1.3 Hypotheses 2
• 1.1.4 Significance 4
• 1.1.5 Organization of the dissertation 5
• 1.2 Background of the research 5
• 1.2.1 Carcass landfill 5
• 1.2.2 Procedure 8
• 1.2.3 Status 11
• 1.2.3.1 In foreign countries 11
• 1.2.3.2 In South Korea 11
• 1.2.4 Leachate treatment 12
• 1.2.4.1 Biological treatment 13
• 1.2.4.2 Physicochemical treatment 16
• 1.2.4.2.1 Chlorination 18
• 1.2.4.2.2 Ozone 19
• 1.3 Antibiotic resistance in environment 20
• 1.3.1 Definition 20
• 1.3.2 Mechanisms 21
• 1.3.3 Antibiotic resistance significance 23
• 1.3.3.1 In foreign countries 24
• 1.3.3.2 In Korea 26
• 1.3.4 Antibiotic resistance treatment in environment 26
• 1.3.4.1 Biological treatment for ARB and ARGs 26
• 1.3.4.2 Physicochemical treatment for ARB and ARGs 27
• 1.4 This study 29
• 1.5 Experimental approach in hypothesis testing 29
• CHAPTER 2 Fate of tetracycline resistance in synthetic livestock carcass leachate for two years 48
• 2.1 Introduction 48
• 2.2 Materials and methods 51
• 2.2.1 Preparation of microorganisms 51
• 2.2.2 Synthetic carcass landfill leachate 52
• 2.2.2.1 Physicochemical characteristics of the synthetic leachate. 52
• 2.2.2.2 Synthetic carcass landfill leachate model set-up. 54
• 2.2.3 Physicochemical tests 56
• 2.2.4 Determination of the fate of TRB and TR plasmid 57
• 2.2.4.1 Analysis of the behavior of TRB 57
• 2.2.4.2 Analysis of the behavior of TRGs. 58
• 2.2.4.3. Microbial biodiversity analyses. 59
• 2.2.5 Statistical analyses 59
• 2.3 Results and discussion 59
• 2.3.1 Nutrient content monitoring and comparison 59
• 2.3.2 Monitoring of TRB and TR plasmid abundance 61
• 2.3.3. Microbial community analysis 68
• 2.5 Conclusions 71
• 2.6 References 72
• CHAPTER 3 Effects of solids retention time on the fate of tetracycline resistance in SBRs for the treatment of carcass leachate 83
• 3.1 Introduction 83
• 3.2 Materials and methods 85
• 3.2.1 Sequencing batch reactors (SBRs) design and operation 85
• 3.2.1.1 SBR performance monitoring 87
• 3.2.2 Evaluation of antibiotic resistance in the SBR system 88
• 3.2.2.1 Quantification of tetracycline-resistant bacteria (TRB) and determination of EC50. 88
• 3.2.2.2 Quantification of tetracycline resistance genes 89
• 3.2.2.3 Statistical analysis 90
• 3.3 Results and discussion 90
• 3.3.1 SBR operations 90
• 3.3.2 Influence of SRT on tetracycline-resistant bacteria 91
• 3.3.3 Fate of tetracycline resistance genes 95
• 3.3.4 Bacterial community analysis 97
• 3.4 Conclusions 99
• 3.5 References 100
• CHAPTER 4 Comparison of antibiotic resistance removal efficiencies using ozone disinfection under different pH and suspended solids and dissolved organic matter concentrations 107
• 4.2. Materials and methods 110
• 4.2.1 Bacterial culture preparation 110
• 4.2.2 Disinfection experiments 112
• 4.2.2.1 Free Chlorination 112
• 4.2.2.2 Ozonation 112
• 4.2.3. Effects of pH, SS and humic acids 114
• 4.2.4. Antibiotic resistance 114
• 4.2.4.1 Measurement of ARB concentrations 115
• 4.2.4.2 pB10 plasmid transfer efficiency analysis 115
• 4.2.4.3 pB10 plasmid measurement 116
• 4.2.5 3-D fluorescence EEM spectrophotometry 117
• 4.2.6 Data analysis 118
• 4.2.7 Statistical analysis 119
• 4.3. Results and discussion 119
• 4.3.1 Efficacy of disinfection processes for controlling ARB and plasmid 119
• 4.3.2 Factors affecting the efficiency of removal of ARB, pB10 plasmid transfer, and pB10 plasmids by ozonation 129
• 4.3.2.1 pH 129
• 4.3.2.2 Effect of SS and humic acids 131
• 4.4 References 141
• CHAPTER 5 Final conclusions 152