Investigating the Variability of Stable Isotope Fractionation (C, Cl) During Microbial Reductive Dehalogenation of Chlorinated Ethenes

Abstract

Chlorinated ethenes, such as tetrachloroethene (PCE) and trichloroethene (TCE), are common groundwater contaminants. Bioremediation harnessing microbial reductive dehalogenation provides a valuable in situ clean-up strategy. Key microorganisms for successful remediation are organohalide-respiring bacteria (OHRB) which catalyze reductive dehalogenation by reductive dehalogenases enzymes (RdhAs). Compound specific isotope analysis (CSIA) allows to track and even quantify in situ contaminant transformation. In addition, CSIA can be used to identify the underlying reaction mechanism. Application of CSIA to assess in situ biotransformation of chlorinated ethenes is impeded by highly variable kinetic isotope fractionation (expressed as ε) during microbial dehalogenation of these compounds. Recent studies with chemical model systems showed that dual isotope analysis of carbon and chlorine (expressed as ΛC/Cl ) is a promising tool to elucidate the causes (e.g. different reaction mechanisms) that lead to variable isotope fractionation of chlorinated ethenes. This work investigated the underlying factors responsible for the variability of chlorinated ethene isotope fractionation reported for pure cultures of OHRB. Furthermore, potential causes for variable isotope fractionation of dehalogenating enrichment cultures (i.e. microbial communities) were assessed using a simplifying mixed culture approach. To this end, dual isotope analysis was combined with microbial dehalogenation experiments using strains of the Desulfitobacterium genus as model organisms and PCE/TCE as model compounds. Previous studies with chemical model systems proved that different reaction mechanisms of PCE dehalogenation resulted in significant different ε values. In the first part of this study, it was investigated whether different reaction mechanisms of PCE-transforming enzymes (PCE-RdhAs) cause the variable PCE ε values of different pure cultures of OHRB. Dual isotope fractionation (εC, εCl, ΛC/Cl ) was determined for PCE dehalogenation by several Gram-positive Desulfitobacterium strains differing in their PCE-RdhA. Although ε values varied considerably, similar ΛC/Cl values allowed to assign the same reaction mechanism to the different PCE-RdhAs. Due to similar cell envelope properties of the strains, PCE mass transfer limitations to the enzymes were excluded to cause varying ε values. The results thus revealed that different rate-limiting steps (e.g. substrate channel diffusion) in the enzymatic multistep reactions of individual PCE-RdhAs rather than different reaction mechanisms determine the extent of PCE isotope fractionation in the Desulfitobacterium genus. Variable isotope fractionation was reported for single pure cultures of OHRB carrying multiple RdhAs. Thus, the effect of different RdhA expression patterns of an OHRB on isotope fractionation was evaluated in the second part of this study. Carbon and chlorine isotope fractionation of PCE was determined for living cells of Desulfitobacterium dehalogenans strain PCE1 differing in their expressed RdhA prior to PCE transformation (i.e. enzymatic phenotype). Similar dual isotope fractionation (εC , εCl , ΛC/Cl ) and significant lag-phases were observed during PCE dehalogenation for all enzymatic phenotypes. This suggested that in all experiments, PCE dehalogenation was predominantly catalyzed by the specialized PCE-RdhA after de novo synthesis. The results indicate that OHRB catalyze chlorinated ethene dehalogenation predominantly by the specialized RdhA for the individual transformation step despite the presence of other RdhA. Thus, differences in RdhA expression patterns are presumably not responsible for variable isotope fractionation of single pure cultures of OHRB. Multiple OHRB are typically present in microbial communities at contaminated sites and their simultaneous contribution to transformation of a single chlorinated ethene is likely to occur. In the third part, it was investigated how changing dehalogenation activities of strains that simultaneously transform the same chlorinated ethene affect isotope fractionation of microbial communities. To this end, PCE isotope fractionation and strain cell numbers were monitored in binary mixed cultures of two Desulfitobacterium strains, which differ in their strain intrinsic εC value for PCE. Highly variable PCE εC values were obtained for different initial strain abundances and under changing cultivation conditions (e.g. prior provision of TCE). This was attributed to different strain specific contributions to PCE transformation (i.e. dehalogenation activities) which were calculated from measured isotope effects. Dehalogenation activities of the strains did not correlate with strain cell numbers. This emphasizes the need for conclusive biomarkers to assess dehalogenation activities of OHRB in microbial communities. The dehalogenation activity of the single strains changed during the deployed cultivation conditions. This indicates that at contaminated sites the activity of single OHRB depends on environmental conditions. Thus, specific ε values for contaminated sites should be determined in laboratory experiments and re-measured on a regular basis or under different cultivation conditions. This would allow to estimate potential changes in observable isotope fractionation of the present microbial community. This work provides evidence that variable PCE ε values of OHRB do not result from different reaction mechanisms but different rate-limiting steps in the reaction sequence. Furthermore, similar PCE isotope fractionation of different enzymatic phenotypes of an OHRB suggests that different RdhA expression patterns do not cause variable ε values of OHRB carrying multiple RdhAs. This needs to be substantiated in further studies with OHRB which simultaneously express multiple RdhAs during PCE dehalogenation. Mixed cultures of two OHRB showed that chlorinated ethene isotope fractionation in microbial communities can be highly variable depending on dehalogenation activities of single strains. Based on this work, future studies can be designed with more complex mixed cultures or enrichment cultures to further assess the variability of chlorinated ethene isotope fractionation in microbial communities.