The bioremediation potential of an aquifer contaminated with tetrachloroethene (PCE) was assessed by combining hydrogeochemical data of the site, microcosm studies, metabolites concentrations, compound specific-stable carbon isotope analysis and the identification of selected reductive dechlorination biomarker genes. The characterization of the site through 10 monitoring wells evidenced that leaked PCE was transformed to TCE and cis-DCE via hydrogenolysis. Carbon isotopic mass balance of chlorinated ethenes pointed to two distinct sources of contamination and discarded relevant alternate degradation pathways in the aquifer. Application of specific-genus primers targeting Dehalococcoides mccartyi species and the vinyl chloride-to-ethene reductive dehalogenase vcrA indicated the presence of autochthonous bacteria capable of the complete dechlorination of PCE. The observed cis-DCE stall was consistent with the aquifer geochemistry (positive redox potentials; presence of dissolved oxygen, nitrate, and sulphate; absence of ferrous iron), which was thermodynamically favourable to dechlorinate highly chlorinated ethenes but required lower redox potentials to evolve beyond cis-DCE to the innocuous end product ethene. Accordingly, the addition of lactate or a mixture of ethanol plus methanol as electron donor sources in parallel field-derived anoxic microcosms accelerated dechlorination of PCE and passed cis-DCE up to ethene, unlike the controls (without amendments, representative of field natural attenuation). Lactate fermentation produced acetate at near-stoichiometric amounts. The array of techniques used in this study provided complementary lines of evidence to suggest that enhanced anaerobic bioremediation using lactate as electron donor source is a feasible strategy to successfully decontaminate this site. Capsule The combination of complementary diagnostic techniques provides different lines of evidence for in situ bioremediation potential of a tetrachloroethene-contaminated aquifer.

Many aquifers around the world are impacted by toxic chlorinated methanes derived from industrial processes due to accidental spills. Frequently, these contaminants co-occur with chlorinated ethenes and/or chlorinated benzenes in groundwater, forming complex mixtures that become very difficult to remediate. In this study, a multi-method approach was used to provide lines of evidence of natural attenuation processes and potential setbacks in the implementation of bioremediation strategies in multi-contaminated aquifers. First, this study determined i) the carbon and chlorine isotopic compositions (δ 13 C, δ 37 Cl) of several commercial pure phase chlorinated compounds, and ii) the chlorine isotopic fractionation (εCl =-5.2 ± 0.6‰) and the dual C-Cl isotope correlation (Λ C/Cl = 5.9 ± 0.3) during dichloromethane (DCM) degradation by a Dehalobacteriumcontaining culture. Such data provide valuable information for practitioners to support the interpretation of stable isotope analyses derived from polluted sites. Second, the bioremediation potential of two industrial sites contaminated with a mixture of organic pollutants (mainly DCM, chloroform (CF), trichloroethene (TCE), and monochlorobenzene (MCB)) was evaluated. Hydrochemistry, dual (C-Cl) isotope analyses, laboratory microcosms, and microbiological data were used to investigate the origin, fate and biodegradation potential of chlorinated methanes. At Site 1, δ 13 C and δ 37 Cl compositions from field samples were consistent with laboratory microcosms, which showed complete degradation of CF, DCM and TCE, while MCB remained.

Based on the previously observed intrinsic bioremediation potential of a site originally contaminated with perchloroethene (PCE), field-derived lactate-amended microcosms were performed to test different lactate isomers and concentrations, and find clearer isotopic and molecular parameters proving the feasibility of an in-situ enhanced reductive dechlorination (ERD) from PCE-to-ethene (ETH). According to these laboratory results, which confirmed the presence of Dehalococcoides sp. and the vcrA gene, an in-situ ERD pilot test consisting of a single injection of lactate in a monitoring well was performed and monitored for 190 days. The parameters used to follow the performance of the ERD comprised the analysis of i) hydrochemistry, including redox potential (Eh), and the concentrations of redox sensitive species, chlorinated ethenes (CEs), lactate, and acetate; ii) stable isotope composition of carbon of CEs, and sulphur and oxygen of sulphate; and iii) 16S rRNA gene sequencing from groundwater samples. Thus, it was proved that the injection of lactate promoted sulphate-reducing conditions, with the subsequent decrease in Eh, which allowed for the full reductive dechlorination of PCE to ETH in the injection well. The biodegradation of CEs was also confirmed by the enrichment in 13 C and carbon isotopic mass balances. The metagenomic results evidenced the shift in the composition of the microbial population towards the predominance of fermentative bacteria. Given the success of the in-situ pilot test, a full-scale ERD with lactate was then implemented at the site. After one year of treatment, PCE and trichloroethene were mostly depleted, whereas vinyl chloride (VC) and ETH were the predominant metabolites. Most importantly, the shift of the carbon isotopic mass balances towards more positive values confirmed the complete reductive dechlorination, including the VC-to-ETH reaction step. The combination of techniques used here provides complementary lines of evidence for the diagnosis of the intrinsic biodegradation potential of a polluted site, but also to monitor 3 the progress, identify potential difficulties, and evaluate the success of ERD at the field scale.

We used C-Cl dual isotope analysis and microcosm studies for elucidating the origin and fate of the common groundwater pollutant dichloromethane (DCM) in two different multi-contaminant field sites in Catalonia, Spain; where DCM contamination could be the result of direct solvent releases and/or chloroform (CF) transformation. Known commercial solvents isotopic compositions as well as characteristic C-Cl dual isotope slopes from our anaerobic enrichment culture containing Dehalobacterium sp., capable of fermenting DCM, and other bacteria from the literature were used for field data interpretation.