Substrate Hydrophobicity and Cell Composition Influence the Extent of Rate Limitation and Masking of Isotope Fractionation during Microbial Reductive Dehalogenation of Chlorinated Ethenes

This study investigated the effect of intracellular microstale mass transfer on microbial carbon isotope fractionation of tetrachloroethene (PCE) and trichloroethene (TCE). Significantly stronger isotope fractionation was observed for crude extracts vs intact cells Of Sulfurospirillum multivorans, Geobacter lovleyi, Desulfuromorias michiganensis, Desulfitobacterium hafniense strain PCE-S, and Dehalobacter restnctus. Furthermore, carbon stable isotope fractionation was stronger for microorganisms with a Gram-positive cell envelope compared to those -with a Gramnegative cell envelope. Significant differences were observed between model organisms in cellular sorption capacity for PCE In(Ct/Co) (S. multivorans-Kd-PCE = 0.42-0.51 L g(-1); D. hafniense-Kd-PCE = 6.13 L g '), as well as in envelope hydrophobicity (S. niultivorans 33.0 degrees to 72.2 degrees; D. hafniense 59.1 degrees to 60.8 degrees) when previously cultivated with fumarate or PCE as electron acceptor, but not for TCE. Cell envelope properties and the tetrachloroethene reductive dehalogenase (PceA-RDase) localization did not result in significant effects on observed isotope fractionation of TCE. For PCE, however, systematic masking of isotope effects as a result of microscale mass transfer limitation at microbial membranes was observed, with carbon isotope enrichment factors of -2.2 parts per thousand, 1.5 to 1.6 parts per thousand, and 1.0%0 (CI95% < +/- 0.2 parts per thousand) for no membrane, hydrophilic outer membrane, and outer + cytoplasmic membrane, respectively. Conclusively, rate-limiting mass transfer barriers were (a) the outer membrane or cell wall and (b) the cytoplasmic membrane in case of a cytoplasmic location of the RDase enzyme. Overall, our results indicate that masking of isotope fractionation is determined by (1) hydrophobicity of the degraded compound, (2) properties of the cell envelope, and (3) the localization of the reacting enzyme.