Probing molecular tracers in geobiological systems using imaging mass spectrometry
von Tim Leefmann
Datum der mündl. Prüfung:2013-04-11
Betreuer:Prof. Dr. Volker Thiel
Gutachter:Prof. Dr. Volker Thiel
Gutachter:Prof. Dr. Joachim Reitner
EnglischIn this thesis ToF-SIMS was applied to the study of geobiological samples. Except for a few pioneering studies, ToF-SIMS has rarely been used in the field of geobiology and thus only a limited number of SIMS reference spectra for biogeochemical relevant compounds has as yet been published. One major goal of this thesis was to extend the spectral library by compounds particularly used as molecular markers in the analysis of geobiological systems. Consequently in the first study reference spectra of eight different glycerolipids functioning as important membrane constituents in eukaryotes, bacteria, and archaea, were presented and their detection in an extract and cryosections of microbial mats demonstrated. The analyses of the standard compounds showed that the detection of fragment ions, in particular from headgroups and alkyl chains, is particularly important for the reliable identification of glycerolipids in complex, environmental samples. As a follow-up study, ten cyclic lipid standards were analyzed by ToF-SIMS. These cyclic lipids are known as important membrane constituents or fulfill a protective function from oxidative stress in eukaryotes, bacteria, or archea. The results obtained showed that most of the compounds can reliably be analyzed in only one of the two polarity modes. In addition the study demonstrated, that not all fragment ions detected in the analyses of the standard compounds can be expected to be detectable in environmental samples containing this compound in complex mixtures with other organic molecules. The knowledge gained in the analysis of standard compounds was used to study a phototrophic microbial mat in detail for their lipid biomarker content by a combining ToF-SIMS, optical microscopy and GC-MS analyses. ToF-SIMS analyses of a cryosection of the microbial mat showed, that a wide range of lipids and pigments, e.g. acylglycerols, carotenoids and chlorophyll a, were present in the microbial mat. Testing the limits of the lateral resolution of the ToF-SIMS instrument, the burst alignment mode was used, to probe single cells for their biomarker content. By subsequent optical microscopy these cells were identified as the diatom species Planothidium lanceolatum and as a major source of lipids and pigments in the microbial mat. The results of the Tof-SIMS analyses were consistent with the diatom specific fatty acid pattern obtained from GC-MS analyses of the bulk microbial mat. This study demonstrated a potential future application of ToF-SIMS in biomarker related research, namely as a tool for the rapid, clear-cut assignment of biomarkers to specific microbial sources in complex environmental samples. This technique could be particularly useful for biomarker studies on the majority of microorganisms, which can as yet not be grown in pure cultures. However, it should be noted that most prokaryotic cells are by an order of magnitude smaller than the diatom cells analyzed in this study and thus more effort has to be spent on increasing the useful lateral resolution of ToF-SIMS imaging. In the last study ToF-SIMS was applied in combination with SEM to the analysis of thin organic films, i.e. conditioning films, forming on solid surfaces exposed to aquifer water prior to the attachment of microorganisms. The results showed that the initial conditioning film forming after a few minutes of exposure to aquifer water was inhomogenous and composed of amino acids, carbohydrates, and fatty acids. With increasing time, the film became more homogenous and microorganism had attached to the surface after a few hours. After 90 days biofilm like accumulations of cells enclosed in extracellular polymeric substances had formed on the wafer surface. This study gave an insight into the chemistry and growth rates of conditioning films and biofilms forming in aquifers in the subsurface and demonstrated the applicability of ToF-SIMS for the analysis of very low amounts of organic material in environmental samples. The studies comprised in this thesis showed that ToF-SIMS can offer unique analytical capabilities for the analyses of large organic ions in environmental samples.
Keywords: ToF-SIMS; biomarkers; lipids; hopanoids; steroids; pigments; algae; biofilms