BUSCADOR RECOLECTA

A surface flow constructed wetland (CW) was set in the Lerma gully to decrease nitrate (NO3 -) pollution from agricultural runoff water. The water flow rate and NO3 - concentration were monitored at the inlet and the outlet, and sampling campaigns were performed which consisted of collecting six water samples along the CW flow line. After two years of operation, the NO3 - attenuation was limited at a flow rate of ~2.5 L/s and became negligible at ~5.5 L/s. The present work aimed to assess the feasibility of using rural waste products (wheat hay, corn stubble, and animal compost) to induce denitrification in the CW, to assess the effect of temperature on this process, and to trace the efficiency of the treatment by using isotopic tools. In the first stage, microcosm experiments were performed. Afterwards, the selected waste material was applied in the CW, and the treatment efficiency was evaluated by means of a chemical and isotopic characterization and using the isotopic fractionation (e) values calculated from laboratory experiments to avoid field-scale interference. The microcosms results showed that the stubble was the most appropriate material for application in the CW, but the denitrification rate was found to decrease with temperature. In the CW, biostimulation in autumn-winter promoted NO3 - attenuation between two weeks and one month (a reduction in NO3 - between 1.2 and 1.5 mM was achieved). After the biostimulation in spring-summer, the attenuation was maintained for approximately three months (NO3 - reduction between 0.1 and 1.5 mM). The e15NNO3/N2 and e18ONO3/N2 values obtained from the laboratory experiments allowed to estimate the induced denitrification percentage. At an approximate average flow rate of 16 L/s, at least 60% of NO3 - attenuation was achieved in the CW. The field samples exhibited a slope of 1.0 for d18O-NO3 - versus d15N-NO3 -, similar to those of the laboratory experiments (0.9–1.2). Plant uptake seemed to play a minor role in NO3 - attenuation in the CW. Hence, the application of stubble in the CW allowed the removal of large amounts of NO3 - from the Lerma gully, especially when applied during the warm months, but its efficacy was limited to a short time period (up to three months). © 2019 Elsevier B.V.

A surface flow constructed wetland (CW) was set in the Lerma gully to decrease nitrate (NO3-) pollution from agricultural runoff water. The water flow rate and NO3
- concentration were monitored at the inlet and the outlet, and sampling campaigns were performed which consisted of collecting six water samples along the CW flow line. After two years of operation, the NO3 -attenuation was limited at a flow rate of ~2.5 L/s and became negligible at ~5.5 L/s. The present work aimed to assess the feasibility of using rural waste products (wheat hay, corn stubble, and animal compost) to induce denitrification in the CW, to assess the effect of temperature on this process, and to trace the efficiency of the treatment by using isotopic tools. In the first stage, microcosm experiments were performed. Afterwards, the selected waste material was applied in
the CW, and the treatment efficiency was evaluated by means of a chemical and isotopic characterization and using the isotopic fractionation (ε) values calculated from laboratory experiments to avoid field-scale interference. The microcosms results showed that the stubble was the most appropriate material for application in the CW, but the denitrification rate was found to decrease with temperature. In the CW, biostimulation in autumn-winter promoted NO3-attenuation between two weeks and one month (a reduction in NO3- between 1.2 and 1.5 mM was achieved). After the biostimulation in spring-summer, the attenuation was maintained for approximately three months (NO3 - reduction between 0.1 and 1.5 mM). The ε15NNO3/N2 and ε18ONO3/N2 values obtained from the laboratory experiments allowed to estimate the induced
denitrification percentage. At an approximate average flow rate of 16 L/s, at least 60 % of NO3 -attenuation was achieved in the CW. The field samples exhibited a slope of 1.0 for δ18O-NO3 -versus δ15N-NO3 -, similar to those of the laboratory experiments (0.9-1.2). Plant uptake seemed to play a minor role in NO3 - attenuation in the CW. Hence, the application of stubble in the CW allowed the removal of large amounts of NO3 - from the Lerma gully, especially when applied during the warm months, but its efficacy was limited to a short time period (up to three months)., This work was financed by the projects REMEDIATION (CGL2014-57215-C4), AGRO-SOS (CGL2015-66016-R) and PACE-ISOTEC (CGL2017-87216-C4-1-R) financed by the Spanish Government and AEI/FEDER financed by the European Union, and by MAG (2017 SGR 1733) financed by the Catalan Government. R. Margalef-Marti thanks the Spanish Government for the Ph.D. grant BES-2015-072882. D. Merchán was supported by the “Juan de la Cierva – Formación” program (FJCI-2018-24920).

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.

Chlorinated ethanes are frequent groundwater contaminants but compound specific isotope analysis (CSIA) has been scarcely applied to investigate their degradation pathways. In this study, dual carbon and chlorine isotope fractionation was used to investigate for the first time the anoxic biodegradation of 1,1,2-trichloroethane (1,1,2-TCA) using a Dehalogenimonas-containing culture. The isotopic fractionation values obtained for the biodegradation of 1,1,2-TCA were ɛC = −6.9 ± 0.4‰ and ɛCl = −2.7 ± 0.3‰. The detection of vinyl chloride (VC) as unique byproduct and a closed carbon isotopic mass balance corroborated that dichloroelimination was the degradation pathway used by this strain. Combining the values of δ13C and δ37Cl resulted in a dual element C-Cl isotope slope of Λ = 2.5 ± 0.2‰. Investigation of the apparent kinetic isotope effects (AKIEs) expected for cleavage of a CCl bond showed an important masking of the intrinsic isotope fractionation. Theoretical calculation of Λ suggested that dichloroelimination of 1,1,2-TCA was taking place via simultaneous cleavage of two CCl bonds (concerted reaction mechanism). The isotope data obtained in this study can be useful to monitor natural attenuation of 1,1,2-TCA via dichloroelimination and provide insights into the source and fate of VC in contaminated groundwaters.

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 (δ¹³C, δ³⁷Cl) of several commercial pure phase chlorinated compounds, and ii) the chlorine isotopic fractionation (εCl = −5.2 ± 0.6‰) and the dual CCl isotope correlation (ΛC/Cl = 5.9 ± 0.3) during dichloromethane (DCM) degradation by a Dehalobacterium-containing 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 mono-chlorobenzene (MCB)) was evaluated. Hydrochemistry, dual (CCl) isotope analyses, laboratory microcosms, and microbiological data were used to investigate the origin, fate and biodegradation potential of chlorinated methanes. At Site 1, δ¹³C and δ³⁷Cl compositions from field samples were consistent with laboratory microcosms, which showed complete degradation of CF, DCM and TCE, while MCB remained. Identification of Dehalobacter sp. in CF-enriched microcosms further supported the biodegradation capability of the aquifer to remediate chlorinated methanes. At Site 2, hydrochemistry and δ¹³C and δ³⁷Cl compositions from field samples suggested little DCM, CF and TCE transformation; however, laboratory microcosms evidenced that their degradation was severely inhibited, probably by co-contamination. A dual CCl isotopic assessment using results from this study and reference values from the literature allowed to determine the extent of degradation and elucidated the origin of chlorinated methanes.

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 13C 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 the progress, identify potential difficulties, and evaluate the success of ERD at the field scale.

Trichloromethane (TCM) is a pollutant frequently detected in contaminated aquifers, and only four bacterial strains are known to respire it. Here, we obtained a novel Dehalobacter strain capable of transforming TCM to dichloromethane, which was denominated Dehalobacter sp. strain 8M. Besides TCM, strain 8M also completely transformed 1,1,2-trichloroethane to vinyl chloride and 1,2-dichloroethane. Quantitative PCR analysis for the 16S rRNA genes confirmed growth of Dehalobacter with TCM and 1,1,2-trichloroethane as electron acceptors. Carbon and chlorine isotope fractionation during TCM transformation was studied in cultured cells and in enzymatic assays with cell suspensions and crude protein extracts. TCM transformation in the three studied systems resulted in small but significant carbon (εC = −2.7 ± 0.1‰ for respiring cells, −3.1 ± 0.1‰ for cell suspensions, and − 4.1 ± 0.5‰ for crude protein extracts) and chlorine (εCl = −0.9 ± 0.1‰, −1.1 ± 0.1‰, and − 1.2 ± 0.2‰, respectively) isotope fractionation. A characteristic and consistent dual Csingle bondCl isotope fractionation pattern was observed for the three systems (combined ΛC/Cl = 2.8 ± 0.3). This ΛC/Cl differed significantly from previously reported values for anaerobic dechlorination of TCM by the corrinoid cofactor vitamin B12 and other Dehalobacter strains. These findings widen our knowledge on the existence of different enzyme binding mechanisms underlying TCM-dechlorination within the genus Dehalobacter and demonstrates that dual isotope analysis could be a feasible tool to differentiate TCM degraders at field studies.

1,2-dichloropropane (1,2-DCP) and 1,2,3-trichloropropane (1,2,3-TCP) are hazardous chemicals frequently detected in groundwater near agricultural zones due to their historical use in chlorinated fumigant formulations. In this study, we show that the organohalide-respiring bacterium Dehalogenimonas alkenigignens strain BRE15 M can grow during the dihaloelimination of 1,2-DCP and 1,2,3-TCP to propene and allyl chloride, respectively. Our work also provides the first application of dual isotope approach to investigate the anaerobic reductive dechlorination of 1,2-DCP and 1,2,3-TCP. Stable carbon and chlorine isotope fractionation values for 1,2-DCP (ƐC = −13.6 ± 1.4 ‰ and ƐCl = −27.4 ± 5.2 ‰) and 1,2,3-TCP (ƐC = −3.8 ± 0.6 ‰ and ƐCl = −0.8 ± 0.5 ‰) were obtained resulting in distinct dual isotope slopes (Λ12DCP = 0.5 ± 0.1, Λ123TCP = 4 ± 2). However direct comparison of ΛC-Cl among different substrates is not possible and investigation of the C and Cl apparent kinetic isotope effects lead to the hypothesis that concerted dichloroelimination mechanism is more likely for both compounds. In fact, whole cell activity assays using cells suspensions of the Dehalogenimonas-containing culture grown with 1,2-DCP and methyl viologen as electron donor suggest that the same set of reductive dehalogenases was involved in the transformation of 1,2-DCP and 1,2,3-TCP. This study opens the door to the application of isotope techniques for evaluating biodegradation of 1,2-DCP and 1,2,3-TCP, which often co-occur in groundwaters near agricultural fields.