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Universität Bonn Physikalisches Institut DE13F7563 Universität Bonn Physikalisches Institut X-Ray Absorption Spectroscopy in Biological Systems: Opportunities and Limitations von Gudrun Lisa Bovenkamp X-ray absorption spectroscopy has become more important for applications in the material sciences, geology, environmental science and biology, specifically in the field of molecular biology. The scope of this thesis is to add more experimental evidence in order to show how applicable X-ray absorption near edge structure (XANES) is to biology. Two biological systems were investigated, at the molecular level, lead uptake in plants and the effect of silver on bacteria. This investigation also included an analysis of the sensitivity of Pb L3- and Ag L3-XANES spectra with regard to their chemical environment. It was shown that Pb L3- and Ag L3 -XANES spectra are sensitive to an environment with at least differences in the second coordination shell. The non-destructive and element specific properties of XANES are the key advantages that were very important for this investigation. However, in both projects the adequate selection of reference compounds, which required in some cases a chemical synthesis, was the critical factor to determine the chemical speciation and, finally, possible uptake and storage mechanisms for plants and antibacterial mechanisms of silver. The chemical environment of Pb in roots and leaves of plants from four different plant families and a lichen from a former lead mining site in the Eifel mountains in Germany was determined using both solid compounds and aqueous solutions of different ionic strength, which simulate the plant environment. The results can be interpreted in such a way that lead is sorbed on the surface of cell walls. Silver bonding as reaction with Staphylococcus aureus, Listeria monocytogenes, and Escherichia coli bacteria was determined using inorganic silver compounds and synthesized silver amino acids. Silver binds to sulfur, amine and carboxyl groups in amino acids. Post address: BONN-IR-2013-07 Nussallee 12 Bonn University 53115 Bonn Mai 2013 Germany ISSN 0172-8741 Universität Bonn Physikalisches Institut X-Ray Absorption Spectroscopy in Biological Systems: Opportunities and Limitations von Gudrun Lisa Bovenkamp aus Dinslaken Dieser Forschungsbericht wurde als Dissertation von der Mathematisch-Naturwissenschaftlichen Fakultät der Universität Bonn angenommen. Angenommen am: 24.5.2013 1. Gutachter: Prof. Dr. Josef Hormes 2. Gutachter: Prof. Dr. Karl Maier Dedicated to my parents, Brigitte Bovenkamp and Armin Bovenkamp Acknowledgements Primarily, I would like to express my deepest gratitude to Prof. Dr. Josef Hormes, my advisor. He gave me this once in a life time chance of becoming a doctor of physics and spending major parts of my PhD time at CAMD, Baton Rouge, LA. It is so much fan to work with him! I am very grateful for all the interesting discussions we had, his patience, his support, and his guidance. I would like to thank Prof. Dr. Karl Maier and Prof. Dr. Marcel Drees for their participation in my PhD committee. I am very grateful to Prof. Dr. Dr. Alexander Prange, our expert in microbiology, for being the fourth member of my PhD committee, for the exciting discussions, and the invaluable experience of working together. I would like to thank the (previous and current) members of the Bonn Synchrotron Radiation (Syli) group: Hubert Blank, Dr. Timna-Joshua Kühn, Martin Niestroj, Mathias Fischer, and Dr. Henning Lichtenberg for their friendship and the discussions over long distances. I would like to thank the members of the INE beamline at ANKA, Dr. Melissa Denecke, Dr. Kathy Dardenne, Dr. Tonya Vitova, Dr. Boris Brendebach, Dr. Jörg Rothe; Dr. Stefan Mangold from the XAS beamline, and Dr. Jörg Göttlicher and Dr. Ralf Steininger from the SUL-X beamline at ANKA; for their great support at all times and the great atmosphere. I would like to thank Dr. Challa Kumar and Dr. Sai Krishna Katla for their discussions and help with the syntheses of the additional compounds and chemical experiments. I am very grateful for the patience and help of Dr. Joshua J. Kas from the FEFF team during our long discussions on the theory of XAS and FEFF calculations. I would like to thank the CAMD family for welcoming me as part of the family, especially Craig Stevens and Dr. John Scott. I would like to thank Dr. Amitava Roy, Dr. Kyungmin Ham, and Gregory Merchan from the X-ray group at CAMD. It is a pleasure working with you! I would like to express my gratitude to Rose and Dr. John Howe for their invaluable help with editing and correcting. Finally, I would like to thank my family and my friends for their love and their unwavering support, especially Brigitte Bovenkamp and John D. Langlois. Contents Chapter 1 - Introduction 1 Chapter 2 - XAS - Theory and Experiment 5 2.1 The Physics of X-ray Absorption Spectroscopy............................................................................ 5 2.1.1 Basics of XAS.......................................................................................................................... 5 2.1.2 XA N ES................................................................................................................................... 6 2.2 Theoretical Aspects of XAS and the FEFF9 c o d e .......................................................................... 7 2.3 Synchrotron Radiation .................................................................................................................. 11 2.4 The XAS experiment....................................................................................................................... 12 2.4.1 Experimental setup and Instrumentation.......................................................................... 12 2.4.2 Data an alysis....................................................................................................................... 15 2.5 Materials and Sample Preparation .............................................................................................. 17 2.5.1 Powder materials and solution experiments for Pb experiments .................................... 17 2.5.2 Powder materials and solution experiments for Ag experim ents.................................... 17 2.5.3 Plant preparation ................................................................................................................ 18 2.5.4 Bacterial strains, media and cultivation ............................................................................ 18 2.6 Other experimental methods . ...................................................................................................... 19 2.6.1 ICP-AES and AAS analysis ................................................................................................... 19 2.6.2 X-ray Tomography experiment and param eters................................................................ 20 2.6.3 XRF experiments .................................................................................................................. 20 2.6.4 XRD experiments.................................................................................................................. 20 2.6.5 FTIR experiments.................................................................................................................. 20 Chapter 3 - Lead uptake in plants 21 3.1 Sensitivity of XANES spectra regarding the chemical environment............................................ 24 3.1.1 Determination of the limit of uncertainty for the sensitivity analysis of XANES spectra .. 25 3.1.2 Determination of the level of sensitivity of XANES spectra................................................ 28 3.1.3 Sensitivity analysis of Pb L3-edge XANES spectra............................................................... 30 3.1.4 Sensitivity analysis of Pb Lr edge XANES spectra............................................................... 32 3.1.5 Sensitivity analysis of Pb M5-edge XANES spectra ............................................................. 33 3.2 Quantitative Analysis using linear combination fittin g ................................................................ 36 3.3 Simulating the biological environment - adequate reference compounds................................. 40 3.3.1 Biological functional groups................................................................................................ 40 3.3.2 Lead in solutions.................................................................................................................. 41 3.4 The uptake of lead in plants........................................................................................................... 43 3.4.1 Pb L3-XANES measurements .............................................................................................. 43 3.4.2 Other techniques................................................................................................................... 50 3.5 FEFF Simulations at Pb L3-edge...................................................................................................... 53 3.5.1 DOS calculations for Pb compounds.................................................................................... 54 i 3.5.2 Simulation of the plant model.............................................................................................. 56 Chapter 4 - Silver speciation in bacteria 59 4.1 Sensitivity of Ag L3-XANES spectra regarding the chemical environment................................... 60 4.1.1 Determination of the
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