In recent decades, concepts involving compound-specific stable isotope analysis have evolved which allow the assessment of organohalide (bio)transformation in situ as well as to evaluate complex (bio)chemical reactions. However, isotope analysis of halogenated compounds is still hampered by the difficulty of measurements of hydrogen (H) and chlorine (Cl) by available standard methods. This research project was focused on the development of alternative methods for H and Cl isotope analysis and the application of the compound-specific stable isotope analysis (CSIA) for characterization of microbial reductive dehalogenation reaction. The development of a novel method for isotope analysis of H and Cl was realized by constructing an analytical set-up with a simultaneously operating dual-detection system, including ion trap mass spectrometry (MS) and isotope ratio mass spectrometry (IRMS). Application of two simultaneously operating detector systems offered the opportunity for a characterization of the conversion process (via MS) prior to isotope analysis (via IRMS) in great detail. On-line Cl- isotope analysis was established by coupling gas chromatography (GC) to a high-temperature conversion (HTC) at 1450 – 1500 °C and subsequent isotope ratio mass spectrometry (IRMS) as GC-HTC-IRMS system. This approach was successfully applied for Cl- isotope analysis for different compound classes, including chloroethenes, chloroethanes, chloromethane, hexachlorocyclohexane and chloroacetic acids. The on-line H- isotope analysis could be significantly improved with a novel chromium-based reactor system operating in at 1100 – 1500 °C. The improvement of H- isotope analysis via hot chromium reduction was extended to various heteroatom (N, Cl, S) containing compound classes and was able to demonstrate its accuracy and precision. The characterization of reductive dehalogenation by compound-specific stable isotope analysis was applied for abiotic and enzymatic reactions. The bottlenecks of single-element (carbon) isotope analysis for microbial systems could be identified and mainly addressed to limitation in micro-scale mass transfer though the membranes, as well as at the reductive dehalogenase enzyme itself. In contrast to the single-element approach, dual-element isotope analysis of carbon and chlorine was able to elucidate reductive dehalogenation reaction mechanism in much more detail. Conclusively, compound-specific stable isotope analysis C and Cl supported a similar reaction mechanism of reductive dehalogenation in abiotic (mediated by corrinoids) and enzymatic (mediated by RDases) system. Furthermore, the effect of different corrinoid cofactors on the reaction mechanism of RDase enzyme could be excluded.
|School:||Technische Universitaet Berlin (Germany)|
|Source:||DAI-C 81/1(E), Dissertation Abstracts International|
|Subjects:||Bioengineering, Chemical engineering|
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