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Interaction of Selected Actinides (U, Cm) with Bacteria Relevant to Nuclear

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Interaction of Selected Actinides (U, Cm) with Bacteria Relevant to Nuclear Interaction of Selected Actinides (U, Cm) with Bacteria Relevant to Nuclear Waste Disposal DISSERTATION zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) vorgelegt der Fakultät Mathematik und Naturwissenschaften der Technischen Universität Dresden von Diplom-Chemikerin Laura Lütke geboren am 27.04.1984 in Dresden Eingereicht am 21.02.2013............. Die Dissertation wurde in der Zeit von August 2009 bis Januar 2013 im Institut für Ressourcenökologie des Helmholtz-Zentrums Dresden-Rossendorf angefertigt. Gutachter: Prof. Dr. rer. nat. habil. Gert Bernhard Prof. Dr. rer. nat. habil. Jörg Steinbach Prof. Dr. rer. nat. habil. Petra Panak Datum der Disputation: 23.04.2013 Table of Contents I Table of Contents List of Abbreviations and Symbols Abstract 1 MOTIVATION & AIMS.................................................................................................. 1 2 INTRODUCTION ............................................................................................................ 5 2.1 Aqueous Chemistry of Actinides ............................................................................ 5 2.2 Bacteria an Introduction and Their Diversity at Äspö and Mont Terri ................. 11 2.3 Bacterial Isolates of Interest .................................................................................. 15 2.3.1 The Äspö Strain Pseudomonas fluorescens ............................................ 15 2.3.2 The Mont Terri Opalinus Clay Isolate Paenibacillus sp. MT-2.2 .......... 16 2.4 Impact of Bacteria on Actinide Speciation............................................................ 17 3 MATERIAL & METHODS ........................................................................................... 23 3.1 Cultivation of Strains............................................................................................. 23 3.1.1 P. fluorescens .......................................................................................... 23 3.1.2 Paenibacillus sp. MT-2.2........................................................................ 23 3.1.3 Paenibacillus wynnii ............................................................................... 24 3.2 Molecular Biological Analyses ............................................................................. 24 3.2.1 In situ Polymerase Chain Reaction ......................................................... 24 3.2.2 Restriction Fragment Length Polymorphism .......................................... 25 3.2.3 Sequencing and Phylogenetic Analysis................................................... 25 3.3 Microbiological Characterization.......................................................................... 26 3.4 Cell Surface Characterization by Potentiometry................................................... 27 3.5 Actinide Binding Experiments .............................................................................. 28 3.5.1 P. fluorescens and U(VI)......................................................................... 28 3.5.2 Paenibacillus sp. and U(VI).................................................................... 29 3.5.3 Paenibacillus sp. and Cm(III) ................................................................. 29 3.6 Techniques to Study Bacterial Actinide Species................................................... 30 3.6.1 Potentiometric Titration .......................................................................... 30 3.6.2 Time-resolved Laser-induced Fluorescence Spectroscopy..................... 30 3.6.3 X-Ray Absorption Spectroscopy............................................................. 31 3.6.4 Transmission Electron Microscopy......................................................... 33 4 RESULTS & DISCUSSION .......................................................................................... 35 4.1 Proof of Culture Purity.......................................................................................... 35 4.1.1 P. fluorescens .......................................................................................... 35 4.1.2 Paenibacillus Species.............................................................................. 36 4.2 Morphology & Physiology.................................................................................... 37 Dissertation Laura Lütke II Table of Contents 4.3 Characterization of Bacterial Surface Functionalities........................................... 41 4.4 Uranium Biosorption and Bacterial Response ...................................................... 44 4.4.1 P. fluorescens and U(VI)......................................................................... 44 4.4.2 Paenibacillus sp. and U(VI).................................................................... 50 4.5 Bacterial Surface Complexation Constants........................................................... 53 4.5.1 U(VI)-P. fluorescens Species.................................................................. 53 4.5.2 U(VI)-Paenibacillus sp. Species............................................................. 55 4.5.3 Eu(III)-P. fluorescens Species................................................................. 58 4.5.4 Eu(III)-Paenibacillus sp. Species............................................................ 59 4.6 Speciation Calculations and Comparison of Strains ............................................. 62 4.6.1 Bacterial Impact on U(VI) Speciation..................................................... 62 4.6.2 Baterial Impact on Eu(III) Speciation ..................................................... 67 4.7 Direct Actinide Speciation Studies in Bacterial Suspensions by TRLFS ............. 70 4.7.1 P. fluorescens and U(VI)......................................................................... 70 4.7.2 Paenibacillus sp. and U(VI).................................................................... 77 4.7.3 Paenibacillus wynnii and U(VI).............................................................. 82 4.7.4 Paenibacillus sp. and Cm(III) ................................................................. 86 4.8 Structures of U(VI) Complexes at Bacterial Cell Surfaces................................... 94 4.8.1 U(VI)-P. fluorescens Species.................................................................. 94 4.8.2 U(VI)-Paenibacillus sp. Species............................................................. 98 4.9 Cellular Localization of U(VI) ............................................................................ 101 4.9.1 P. fluorescens and U(VI)....................................................................... 101 4.9.2 Paenibacillus sp. and U(VI).................................................................. 102 5 SUMMARY & CONCLUDING REMARKS.............................................................. 103 6 REFERENCES ............................................................................................................. 109 Appendix Dissertation Laura Lütke List of Abbreviations and Symbols III List of Abbreviations and Symbols Abbreviations AAS atomic absorption spectroscopy AMP adenosine monophosphate ATP adenosine triphosphate a.u. arbitrary unit BLAST basic local alignment search tool bp base pairs CASO casein soytone DNA deoxyribonucleic acid dNTP deoxynucleotide triphosphate DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen (German collection of microorganisms and cell cultures) EDTA ethylene diamine tetraacetate EDS energy-dispersive spectroscopy EDX energy-dispersive x-ray analysis EXAFS extended x-ray absorption fine structure GC guanine cytosine Hi-Di highly deionized HLW high-level waste HPLC high performance liquid chromatography HRL hard rock laboratory ICCD intensified charge-coupled device ICP inductively coupled plasma IEC ion exchange chromatography LPS lipopolysaccharide M metal MOPS 3-(N-morpholino)propane sulfonate MS mass spectrometry or multiple scattering NB nutrient broth Dissertation Laura Lütke IV List of Abbreviations and Symbols Nd:YAG neodymium: yttrium aluminium garnet NEA nuclear energy agency OD optical density OPO optical parametrical oscillator PCR polymerase chain reaction PG peptidoglycan PT potentiometric titration RFLP restriction fragment length polymorphism rRNA ribosomal ribonucleic acid SAXS small angle x-ray scattering SS single scattering SSM standard succinate medium TBE tris-borate-ethylene diamine tetraacetate TEM transmission electron microscopy TRLFS time-resolved laser-induced fluorescence spectroscopy XANES x-ray absorption near edge structure XAS x-ray absorption spectroscopy Symbols E energy Eh redox potential F(R) Fourier transform I intensity or ionic strength k electron wave vector K complex formation constant KD distribution coefficient LIII characteristic x-ray line, e.g. of uranium N coordination number pH negative base 10 logarithm of the proton concentration pKa negative base 10 logarithm of the acid dissociation constant R bond distance 2 S0 amplitude reduction factor Dissertation Laura Lütke List of Abbreviations and Symbols V β overall stability constant σ2 Debye-Waller factor τ luminescence lifetime χ(k) EXAFS function χ2 chi to the square as parameter for goodness of fit Indices ax axial eq equatorial Dissertation Laura Lütke VI Abstract Abstract To assess the safety of a site destined for storage of nuclear waste enhanced research effort is demanded to investigate the complex interactions of released radionuclides with parts of the environment that includes indigenous microorganisms. As part of a
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