Study of Macromolecule-Mineral Interactions on Nuclear Related Materials
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Study of macromolecule-mineral interactions on nuclear related materials by Lygia Eleftheriou Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Supervised by: Prof John Harding Dr Maria Romero-González The University of Sheffield Faculty of Engineering Department of Materials Science and Engineering September 2016 Declaration The work described within this thesis has been completed under the supervision of Prof J. Harding and Dr M. Romero-González at the University of Sheffield between September 2012 and September 2016. This thesis along with the work described here has been completed by the author with some exceptions indicated clearly at the relevant chapters. These include: (1) the construction of ceria models for the computational work that was completed by Dr Colin Freeman and Dr Shaun Hall (described in chapter 5), (2) the purification of peptidoglycan completed by Dr Stephane Mesnage (described in chapter 4) and (3) the electron microscopy analysis completed by Dr Mohamed Merroun (described in chapter 2). Lygia Eleftheriou September 2016 Acknowledgements I would like to express my sincere gratitude to my supervisors Dr Maria Romero González and Prof John Harding for all their support during the past four years. This work would not have been possible without their endless encouragement, guidance and advice. I would also like to thank Dr Colin Freeman, Dr Shaun Hall and Riccardo Innocenti Malini for all the hours they spent trying to make things work and all their help with the computational part of this project. In addition, I would like to thank Dr Simon Thorpe, Dr Stephane Mesnage and Mr Robert Hanson for their help with the analytical methods of this project. Special thanks should go to Debbie Hill, Emma Wharfe and Gordon Brown for being my first contact for basically everything, from outreach activities to student support. Lastly, I would like to thank my family and friends. My parents were the ones to support me the most during this journey. I could never thank them enough. My dear friends, Gifty Tetteh and Joe Hufton who in one way or another shared their support and of course my partner-in- crime, Panagiotis Koulouris who has been next to me during the endless nights of analysing, correcting, writing, re-writing and so on. Thank you! The research was sponsored financially by the EPSRC Doctoral Training Centre (DTC) in Nuclear Fission Research Science and Technology and the EPSRC consortium 'Hard-soft matter interfaces: from understanding to engineering' (EP/I001514/1). Abstract Microbes have been identified close to contaminated sites such as nuclear-waste repositories indicating their ability to interact with radionuclides. One of the main mechanisms bacteria use involves the reduction of highly mobile radionuclides to less mobile minerals, often in the form of oxides. The precipitated minerals are found located at the cell wall or close to the external components of the bacterial cell suggesting a possible interaction of cell wall components or external components of the tested bacteria with the precipitated particles. Tests on ceria, urania, thoria and europium oxide confirmed that several biomolecules are responsible for the observed localisation. The first experiments used lipopolysaccharides (LPS), found at the outer site of Gram negative bacterial cells and resulted in successful sorption of LPS on all four minerals. The Purpald assay was used to quantify LPS before and after the interaction and confirmed the attachment of the biopolymers to the oxides with ΔG values in the range of physisorption (-3 to -25 kJ/mol) for all systems. ATR-FTIR, zeta potential analysis and electron microscopy also confirmed the attachment of the biomolecules on the minerals. Mycolic acids and peptidoglycans, components of Gram positive bacteria, were also assessed for their ability to interact with ceria and europium oxide. Mycolic acids showed successful sorption profiles with ΔG values in the physisorption range (-10 to -17 kJ/mol) for all systems, highly dependent on the experimental conditions. In addition, Molecular Dynamics simulations were used to examine the interaction of mycolic acid with ceria under natural conditions (pH7) in the presence of Na+ and the calculated binding energy (-11.598 kJ/mol) agreed well with the experimental results for the corresponding system (-16.485 kJ/mol). Additionally, preliminary tests on peptidoglycan interactions resulted in successful sorption of the biomolecule on both ceria and europium oxide with ΔG values in the physisorption range (-8 to -10 kJ/mol). ATR- FTIR and zeta potential analysis confirmed the attachment of mycolic acid and peptidoglycan on the minerals tested. These results suggest that the observed localisation of mineral within the bacterial cell walls are related to lipopolysaccharide and peptidoglycan-mediated sorption and have potential uses in treatment of nuclear waste and biomining processes. Contents Page Chapter 1 ........................................................................................................................................................ 15 Introduction ................................................................................................................................................... 15 Background ................................................................................................................................................ 16 Objectives ................................................................................................................................................... 17 Literature review ....................................................................................................................................... 18 1.1 Nuclear Materials: Existence and Usage.................................................................................... 18 1.2 The issue .................................................................................................................................... 19 1.3 Bacterial interactions with nuclear-related materials ................................................................. 19 1.3.1 Bacterial cell structure ............................................................................................................... 22 1.3.1.1 Gram negative and Gram positive bacteria ........................................................................ 22 1.3.1.2 The Mycolata family .......................................................................................................... 23 1.4. Interactions of cell wall bio-components with minerals .................................................................. 24 1.4.1 Lipopolysaccharides .................................................................................................................. 24 1.4.2 Peptidoglycans ........................................................................................................................... 25 1.4.3 Mycolic acids ................................................................................................................................. 25 1.5. Importance and applications of interactions between bacterial components and nuclear-related minerals ................................................................................................................................................... 26 1.6 Experimental techniques to evaluate macromolecule-mineral interactions ...................................... 28 1.6.1 Spectrophotometric Techniques ................................................................................................ 28 1.6.2 Spectroscopic techniques-FTIR ..................................................................................................... 30 1.6.3 Computational Methods............................................................................................................. 31 1.6.3.1 Molecular Dynamics .......................................................................................................... 33 Chapter 2 ........................................................................................................................................................ 45 Lipopolysaccharide Interactions with Nuclear Related Minerals ............................................................. 45 2.1 Introduction ......................................................................................................................................... 46 2.1.1 Functions of LPS ............................................................................................................................ 48 2.1.1.1 Interactions of LPS with surfaces ........................................................................................... 49 2.1.2 Objectives ...................................................................................................................................... 51 2.2 Materials and Methods ....................................................................................................................... 52 2.2.1 Chemicals ....................................................................................................................................... 52 2.2.2 Macromolecules ............................................................................................................................. 52 2.2.2.1 Molecular Weight Determination of LPS ..............................................................................