Investigating Mechanisms of Reductive Chlorinated Hydrocarbon Degradation with Compound-Specific Isotope Analysis

Abstract

Chlorinated hydrocarbons, such as chlorinated methanes and ethenes, are large-scale industrial products and frequently detected in the environment as toxic contaminants. As remediation approach the reductive dechlorination through bacteria or reducing agents is commonly applied. However, for organisms it is often observed that the degradation stops at toxic intermediates and no full dechlorination is achieved. The reason why degradation stops is not understood so far and even the elucidation with mechanistic experiments and interpretation were ultimately not successful because until today multiple reaction mechanisms are plausible candidates for the degradation. Therefore, compound specific isotope analysis, a promising tool for identifying reaction mechanisms was combined with chemical model systems to identify the reductive dechlorination mechanisms in this thesis. Chlorine isotope measurement for chlorinated ethenes (CEs) became accessible in 2006 and is frequently used to follow the fate of CEs in groundwater and to identify their reaction mechanisms. However, an established and verified method for chlorine isotope measurement of chlorinated methanes was not available. Therefore, in first instance, a method for chlorine isotope analysis of tetrachloromethane and trichloromethane was realized for Gas Chromatography - quadropol Mass Spectrometry (GC-qMS) and Gas Chromatography - Isotope Ratio Mass Spectrometry (GC-IRMS) and both methods were compared in points of precision and trueness. Thus, for the first time it became possible to combine information from carbon and chlorine isotope values to form a dual element isotope plot for reductive trichloromethane degradation. Direct investigation of the reaction mechanism of chlorinated ethene degradation by organisms is no simple task, because processes like diffusion through the cell membrane or binding at the enzyme complicate the interpretation of the mechanism. Therefore, often simplified model systems such as degradation of chlorinated ethenes by pure enzymes or the enzymatic cofactor vitamin B12 are used. The enzymatic cofactor vitamin B12 (which lies at the heart of practically all reductive dehalogenases identified to date) was chosen as model system for this thesis. . Despite numerous studies which investigated this model system, the prevailing mechanism still remains elusive. Based on these results and the suggested reaction mechanisms for this system (1. Outer-Sphere or Inner-Sphere Single Electron Transfer 2. Nucleophilic Substitution 3. Nucleophilic Addition), first the isotope fractionation of the Outer-Sphere Single Electron Transfer (OS-SET) mechanism was investigated for tetra-, tri and cis-dichloroethene. The OS-SET reaction was simulated using different single electron IV transfer reagents (e.g. CO2 radical anions) in water and organic solvent. Through the absence of chlorine isotope effects the OS-SET reaction for CEs by organism and vitamin B12 could be ruled out and furthermore it was indicated that the OS-SET reaction does not prevail for reductive dechlorination of CEs by zero valent iron (ZVI). In last instance, carbon and chlorine isotope effects revealed a mechanistic shift for PCE and cis-DCE reaction with vitamin B12 and a pH dependent shift for TCE. Based on carbon-, chlorine- and hydrogen isotope effects, pH-dependent shifts in reaction rates and TCE product distribution the existence of two possible pathways (1. addition-elimination, 2. addition-protonation) was narrowed down. Reversible and irreversible cobalamin-substrate association was detected by mass balance deficits, whereas possible structures of chloroalkyl and chlorovinyl cobalamin complexes could be analyzed by high-resolution mass spectrometry. By combining experimental evidence it was revealed that initial electron transfer or alkyl or vinyl complexes as crossroads of both pathways are not consistent with experimental observations. In contrast, the formation of cobalamin chlorocarbanions is supported as key intermediates, where chloride elimination produces vinyl complexes (explaining rates and products of TCE at high pH) and protonation generates less reactive alkyl complexes (explaining rates and products of TCE at low pH). Furthermore, circumstantial evidence indicates that PCE reacts only via addition-elimination and cis-DCE only via addition-protonation. Transferring these results into biosystems implies that enzymes which react via the addition-elimination cannot degrade cis-DCE. Finally this fact can provide a possible answer why degradation often stops at the step of cis-DCE.