“Deciphering Chlorohydrocarbon Transformation Mechanisms by Advancing δ37Cl/δ13C Compound-Specific Isotope Analysis”
Contamination of groundwater by chlorinated organic compounds originating from industry (e.g. chlorinated ethenes) or agriculture (e.g. the herbicide atrazine) is a prominent problem of our times. Investigating and understanding their environmental fate is, therefore, of major importance to develop successful strategies for bioremediation of contaminated sites. A promising approach for studying the transformation pathways of these contaminants is compound-specific stable isotope analysis (CSIA). By analyzing changes in isotope ratios (e.g. 13C/12C, 15N/14N, 37Cl/35Cl) at their low natural abundance, CSIA can detect degradation processes and provide information about the underlying chemical reaction mechanisms.
The first part of the thesis emphasized the importance of referencing and substance-specific working standards that should bracket a wide range of isotope values to guarantee accurate chlorine isotope analysis. However, almost all international referencing standards have a very similar value. Furthermore, for many organic compounds, substance-specific working standards are not available. Therefore, synthesis routes for the generation of an in-house referencing standard (CT16) depleted in 37Cl/35Cl and substance-specific working standards of the chlorinated herbicides S-metolachlor and acetochlor (Metola2/Aceto2) enriched in 37Cl/35Cl were identified. Subsequently, the standards were characterized via gas chromatography - isotope ratio mass spectrometry (GC-IRMS) and gas chromatography - multicollector inductively coupled plasma mass spectrometry (GC-MC-ICPMS). Thus, it was demonstrated that isotope effects of dehalogenation reactions can be used for the generation of much-needed standards for CSIA of chlorine.
The second part investigated why microbial reductive dehalogenation of tetrachloroethene (PCE) and trichloroethene (TCE) often stops at toxic cis-1,2-dichloroethene (cis-DCE) or vinyl chloride (VC). A model study using vitamin B12 as model reactant for reductive dehalogenase (RDase) activity recently identified two different mechanisms for reductive dehalogenation (addition-protonation vs. addition-elimination) by analyzing dual element isotope plots of carbon and chlorine. Dual element isotope plots associated with microbial reductive dehalogenation revealed that the same mechanisms are at work in bacteria. Furthermore, it was observed that precultivation conditions have an influence on the TCE dechlorination mechanism indicating that some RDases may be tailored to the dechlorination of PCE and TCE, but are not able to degrade cis-DCE or VC. This could offer a possible explanation for the question why bioremediation often stops at cis-DCE or VC.
In the third part chlorine, carbon and nitrogen isotope effects during microbial hydrolysis and microbial oxidative dealkylation of atrazine were investigated. Carbon and nitrogen isotope effects confirmed that the degradation pathways followed previously proposed mechanisms. In contrast to nitrogen and carbon, chlorine isotope effects of atrazine are not diluted by non-reacting atoms, which could turn chlorine isotope fractionation into a sensitive indicator for natural transformation. Microbial hydrolysis of atrazine resulted in unexpected small chlorine isotope effects indicating that C-Cl bond cleavage is not the rate-determining step. On the other hand, unexpected pronounced chlorine isotope effects during microbial oxidative dealkylation suggested that enzymatic interactions may have an influence on chlorine isotope fractionation. These unexpected results demonstrated that a complete understanding of underlying chemical reaction mechanisms is necessary before applying such a new approach to the field.