The Adaptation Mechanisms of Shwanella oneidensis MR-1 Against on Imidazolium-Based Ionic Liquid

The Adaptation Mechanisms of Shwanella oneidensis MR-1 Against on Imidazolium-Based Ionic Liquid
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Ionic liquids (ILs) are known as “green solvents”. However, it is necessary to determine the potential toxic effects of ILs before they can be released into the environment. In this study, the relative toxicity and adaptation consequences of the selected Imidazolium-based ILs using Shewanella oneidensis MR-1, an important environmental model organism which possess a diverse respiratory capabilities. Adaptation of MR-1 in the presence of ILs was investigated in different aspects such as, SEM for morphological adaptation, fatty acid analysis for the membrane adaptation, FT-IR analysis for the effects of ILs on major biochemical components of MR-1 as well as for the accumulation of ILs in the cells, and last but not the least is the adaptation of MR-1 at the proteomic level using 2D-Electrophoresis and the identification of the most significant up-regulated spots caused by the treatment of sublethal dose of ILs. Scanning Electron Microscope (SEM) was used for the analysis of the morphological changes caused by the ILs. The increased in cellular size of MR-1 after exposure to ILs was observed in some samples indicating a morphological adaptation by using the principle of surface-to-volume ratio. Also, intact cell membranes of pretreated MR-1 were observed. To further investigate the adaptation mechanism of Shewanella oneidensis MR-1 based from the observed changes in cellular morphology, fatty acid composition was investigated to analyze the membrane adaptation during the treatment of ILs using GC-MS. Significant changes in membrane fatty acid composition were observed in response to sublethal-dose pretreatment of ILs as compared to non-pretreated cells. The changes comprised the alterations in the percentage of selected groups of fatty acids: saturated, branched-chain (iso and anteiso), unsaturated (cis and trans) fatty acids. It may suggest that ‘homeoviscous adaptation’ had occurred by incorporating increasing proportions of saturated and branched-chain fatty acids
1. Introduction 1 1.1 Ionic Liquids 1 1.1.1 Novel Application of Ionic Liquids 2 1.2 Shewanella oneidensi MR-1 2 1.3 Toxicity of Ionic Liquids 3 1.3.1 Different Levels of (eco) Toxilogical Test Battery 5 Toxicity Effects of ILs on Aquatic Systems 5 Toxicity Effects of ILs on Terrestrial Systems 10 Toxicty Effects of ILs on Mamalian Cellular Test Systems 11 1.3.2 Substructure of ILs that Influences Their Biological Effects 14 Effects of Side Change Length 15 Effects of Anion Moiety 17 Effect of Cation "head group" 18 1.4 Adaptation of Shewanella oneidensis MR-1 in ILs 20 1.4.1 Adaptation of the Cell Membrane 22 Membrane Fatty Acids 22 1.4.2 Fourier-Transdorm Infrared (FT-IR) Spectroscopy 23 1.4.3 Proteins 24 2. Objectives 26 3. Material and Methods 27 3.1 Microorganisms and Culture Conditions 27 3.2 Adaptation and Challenge 27 3.3 Fatty Acid Extraction and Analysis 28 3.4 Scanning Electron Microscopy (SEM) 29 3.5 FT-IR Analysis 29 3.5.1 Sample Preparation 30 3.5.2 Subcellular Fractionation 30 3.6 Protein Analysis 31 3.6.1 Protein Extraction 31 3.6.2 Subcellular Protein Fractionation 31 3.6.3 Protein Quantification Analysis 32 3.6.4 Two-Dimensional Electrophoresis 32 Protein Sample Preparation 33 2D-PAGE 33 Image Analysis 34 Peptide Mass Fingerprinting (PMF) 34 4. Results and Discussion 36 4.1 EC50 Values and Minimum Inhibitory Concentration (MIC) 36 4.2 Adaptation Tes 39 4.2.1 Fatty Acid Analysis 40 4.2.2 Morphological Analysis 45 4.2.3 Fourier Transform Infrared Spectroscopy Analysis 47 4.2.4 Total Protein Analysis 58 2D-E Analysis of Protein 58 Identification of Protein with Altered Expression in Response Stress 62 4.3 Mechanisms of ILs Toxicity 68 5. Conclusion 71 6. References 74
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College of Natural Science(자연과학대학) > Ocean Sciences (해양과학) > Theses(해양과학 석박사 학위논문)
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