Physiological Characteristics and Genomic Properties of Nitrosomonas mobilis Isolated from Nitrifying Granule of Wastewater Treatment Bioreactor December 2016 Soe Myat Thandar ソー ミャット サンダー Physiological Characteristics and Genomic Properties of Nitrosomonas mobilis Isolated from Nitrifying Granule of Wastewater Treatment Bioreactor December 2016 Waseda University Graduate School of Advanced Science and Engineering Department of Life Science and Medical Bioscience Research on Environmental Biotechnology Soe Myat Thandar ソー ミャット サンダー Contents Abbreviations ................................................................................................................... i Chapter 1-General introduction .................................................................................... 1 1.1. Nitrification and wastewater treatment system .......................................................... 3 1.2. Important of Nitrosomonas mobilis ........................................................................... 8 1.3. Objectives and outlines of this study ....................................................................... 12 1.4. Reference.................................................................................................................. 12 Chapter 2- Physiological characteristics of Nitrosomonas mobilis Ms1 ................... 17 2.1. Introduction .............................................................................................................. 19 2.2. Material and methods ............................................................................................... 22 2.2.1. Cell cultures and media ......................................................................................... 22 2.2.2. Fluorescence microscopy ...................................................................................... 23 2.2.3. Measurement of ammonium and nitrite concentrations ........................................ 24 2.2.4. Growth temperature .............................................................................................. 24 2.2.5. Growth pH ............................................................................................................. 25 2.2.6. Salt tolerance ......................................................................................................... 25 2.2.7. Substrate inhibition ............................................................................................... 26 2.2.8. Nitrite tolerance of Nitrosomonas mobilisMs1 ..................................................... 27 2.2.9. Maximum specific growth rate (μmax) of Nitrosomonas mobilisMs1 ................... 29 2.2.10. Activity kinetics of Nitrosomonas mobilis Ms1 and Nitrosomonas europaea ... 29 2.3. Results ...................................................................................................................... 31 2.3.1. Growth temperature and pH .................................................................................. 31 2.3.2. Salt tolerance of Nitrosomonas mobilisMs1 ......................................................... 34 2.3.3. Substrate inhibition on Nitrosomonas mobilisMs1 ............................................... 35 2.3.4. Nitrite tolerance of Nitrosomonas mobilisMs1 ..................................................... 37 2.3.5. Growth and activity kinetics of Nitrosomonas mobilisMs1 .................................. 39 2.4. Discussion ................................................................................................................ 44 2.5. Conclusion ............................................................................................................... 51 2.6. References ................................................................................................................ 53 Chapter 3- Genomic properties of Nitrosomonas mobilis Ms1 .................................. 58 3.1. Introduction .............................................................................................................. 60 3.2. Material and methods ............................................................................................... 63 3.2.1. Cultivation ............................................................................................................. 63 3.2.2. Sequencing and Assembly of Nitrosomonas mobilis Ms1 .................................... 63 3.2.3. Average nucleotide identity between bacterial genomes ...................................... 64 3.2.4. Annotation and identification of the genes ........................................................... 65 3.2.5. PCR amplification of the gene fbp encoding fructose 1, 6-bisphosphatase .......... 65 3.2.6. Quantification of the gene expression levels under the high ammonia and nitrite concentration ........................................................................................................... 66 3.3. Results and Discussion ............................................................................................. 70 3.3.1. Genome overview of Nitrosomonas mobilis Ms1 ................................................. 70 3.3.2. Energy metabolism of Nitrosomonas mobilis Ms1 ............................................... 72 3.3.2.1. Nitrogen metabolism .......................................................................................... 72 3.3.2.2. Sulfur metabolism .............................................................................................. 82 3.3.2.3. Hydrogen oxidation ............................................................................................ 84 3.3.2.4. Haem-copper oxidase gene cluster..................................................................... 84 3.3.3. Central carbon metabolism of Nitrosomonas mobilis Ms1 ................................... 85 3.3.4. Other functional genes .......................................................................................... 92 3.3.5. Quantification of the gene expression levels under the high ammonia and nitrite concentration ........................................................................................................... 95 3.4. Conclusion ............................................................................................................. 100 3.5. References .............................................................................................................. 101 Chapter 4 ..................................................................................................................... 107 General conclusion and future perspectives .................................................................. 108 Appendix ...................................................................................................................... 115 Supplementary information-1- Physiological characteristics of Nitrosomonas mobilis Ms1 ........................................................................................................................ 116 1.1. Fluorescence microscopy ....................................................................................... 118 1.2. Inhibition of ammonia concentrations on N. mobilisMs1 ...................................... 120 1.3. Inhibition of nitrite concentrations on N. mobilisMs1 ........................................... 125 Supplementary information-2- Genomic properties of Nitrosomonas mobilis Ms1 ..... 128 2.1. Circular view of the genome of Nitrosomonas mobilis Ms1 ................................. 130 2.2. Sulfur metabolism .................................................................................................. 131 2.3. Carbon fixation pathways ...................................................................................... 132 2.4. Glycolysis and gluconeogenesis ............................................................................ 133 2.5. Tricarboxylic acid (TCA) cycle ............................................................................. 134 2.6. Pentose phosphate pathway ................................................................................... 135 2.7. PCR amplification of the gene fbp encoding fructose 1, 6-bisphosphatase ........... 136 2.8. Quantification of the gene expression levels under the high ammonia and nitrite concentration .......................................................................................................... 139 Synopsis of the thesis .................................................................................................... 140 Publication lists ............................................................................................................. 149 Acknowledgements ....................................................................................................... 150 Abbreviations List of Abbreviations AOA Ammonia-oxidizing archaea AOB Ammonia -oxidizing bacteria AMO Ammonia monooxygenase CBB Calvin–Benson–Bassham cycle CDS Coding DNA sequences g Generation time HAO Hydroxylamine oxydoreductase HCl Hydrochloric acid KEGG Kyoto Encyclopedia of Genes and Genomes 퐾 + m(NH4 ) Half saturation constant for ammonium uptake 퐾m(O2) Half saturation constant for oxygen uptake NaHCO3 Sodium bicarbonate NaOH Sodium hydroxide NaNO2 Sodium nitrite NH3 Ammonia + NH4 Ammonium NH4Cl Ammonium chloride i Abbreviations (NH4)2SO4 Ammonium sulfate NOB Nitrite-oxidizing bacteria - NO2 Nitrite - NO3 Nitrate NO Nitric oxide N2O Nitrous oxide TCA Tricarboxylic acid cycle μmax Maximum specific growth rate 푉 + max (NH4