Deciphering stable isotope fractionation (d13C; d37Cl) during reductive dehalogenation of chlorinated ethenes: Effects of bacterial growth physiology and expression of key enzymes

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Compound specific isotope analysis (CSIA) is regarded as a powerful approach to assess in situ biotransformation of chlorinated organic compounds as based on kinetic isotope fractionation due to biodegradation. Although reported carbon (d13C) isotope fractionation factors for microbial reductive dehalogenation of chlorinated ethenes vary considerably even for the same bacterial strain and thus hamper the interpretation and applicability of isotope data in complex environmental settings, systematic experimental investigations on factors and mechanisms influencing observed isotope fractionation are scarce. The major objective of our proposed research is to identify and evaluate what and how physiologic and enzymatic factors control isotope fractionation during microbial reductive dechlorination of chlorinated ethenes. Generally, the extent of stable isotope fractionation may be determined largely by the reaction mechanism at the respective enzyme, but can be attenuated by uptake and transport processes of the substrate. More specifically, controlling factors may include concentrations and type of electron donor/acceptor, availability of growth supplements (vitamins/trace elements), growth phase, cell density or expression and abundance of reductive dehalogenase genes. The majority of the known and genetically characterized organohalide reducing bacteria (OHRBs) carry multiple homologous genes encoding putative reductive dehalogenases. In order to directly link observed dehalogenation activity and isotope fractionation to gene expression levels we propose Desulfitobacterium hafniense strain Y51 as model organism for our study because it possesses only two key enzymes that are both well characterized (PCE and chlorophenol reductive dehalogenases). This model organism will thus facilitate a systematic evaluation and discrimination of various potential effects of selected growth conditions on isotope fraction in conjunction with molecular analyses of specific gene expression and protein content. Our recently developed expertise in compound specific chlorine (d37Cl) isotope analysis enables us to determine also chlorine isotope fractionation factors for microbial dehalogenation. Thus, through improved mechanistic understanding of the controlling factors of microbial isotope fractionation the proposed research will enhance the applicability and practical value of 2-dimensional carbon/chlorine isotope approaches to assess in situ biodegradation of organochlorine compounds in the field and will provide an experimental basis for linking compound specific isotope data with reactive transport models.