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The biotransformation of hexachlorocyclohexane isomers (HCH) by two Dehalococcoides mccartyi strains (195 and BTF08) and an enrichment culture was investigated and compared to conversion by the obligate anaerobic strain Clostridium pasteurianum strain DSMZ 525. The D. mccartyi strains preferentially transformed γ-HCH over α-HCH and δ-HCH isomers while β-HCH biotransformation was not significant. In case of the enrichment culture, γ-HCH was preferentially transformed over the δ-HCH, β-HCH and α-HCH isomers. Major observed metabolites in both cases were tetrachlorocyclohexene and as end products monochlorobenzene (MCB) and benzene. Dechlorination of the γ-HCH isomer was linked to an increase in cell numbers for strain 195. γ-HCH transformation was linked to considerable carbon stable isotope fractionation with the enrichment factor εc = − 5.5 ± 0.8‰ for D. mccartyi strain 195, εc = − 3.1 ± 0.4‰ for the enrichment culture and εc = − 4.1 ± 0.6‰ for co-metabolic transformation by C. pasteurianum. [ABSTRACT FROM AUTHOR]

In anaerobic environments microorganisms exchange electrons with community members and with soil and groundwater compounds. Interspecies electron transfer (IET) occurs by several mechanisms: diffusion of redox compounds or direct contact between cells. This latter mechanism may be facilitated by the presence of conductive nanoparticles (NP), possibly serving as electron conduits among microorganisms. Our study examined the effect of magnetite (Fe 3 O 4 ) NP on the dechlorination of 1,2-dichloroethane (1,2-DCA) by a mixed-culture. The addition of NP (170 mg L − 1 total Fe) enhanced the acetate-driven reductive dechlorination of 1,2-DCA to harmless ethene (via reductive dihaloelimination) up to 3.3-times (2.3 μeq L − 1 d − 1 vs. 0.7 μeq L − 1 d − 1 ), while decreasing the lag time by 0.8 times (23 days) when compared to unamended (magnetite-free) microcosms. Dechlorination activity was correlated with the abundance of Dehalococcoides mccartyi , which accounted up to 50% of total bacteria as quantified by CARD-FISH analysis, pointing to a key role of this microorganism in the process. Given the widespread abundance of conductive minerals in the environment, the results of this study may provide new insights into the fate of 1,2-DCA and suggest new tools for its remediation by linking biogeochemical mechanisms. [ABSTRACT FROM AUTHOR]

The remediation of chlorinated solvent contaminated sites frequently involves bioaugmentation with mixed cultures containing Dehalococcoides mccartyi . Their activity is then examined by quantifying reductive dehalogenase (RDase) genes. Recently, we described a rapid, low cost approach, based on loop mediated isothermal amplification (LAMP), which allowed for the visual detection of RDase genes from groundwater. In that study, samples were concentrated (without DNA extraction), incubated in a water bath (avoiding the use of a thermal cycler) and amplification was visualized by the addition of SYBR green (post incubation). Despite having a detection limit less than the threshold recommended for effective remediation, the application of the assay was limited because of the semi-quantitative nature of the data. Moreover, the assay was prone to false positives due to the aerosolization of amplicons. In this study, deoxyuridine triphosphate (dUTP) and uracil DNA glycosylase (UNG) were incorporated into the assay to reduce the probability of false positives. Optimization experiments revealed a UNG concentration of 0.2 units per reaction was adequate for degrading trace levels of AUGC based contamination (~ 1.4 × 10 4 gene copies/reaction) without significant changes to the detection limit (~ 100 gene copies/reaction). Additionally, the optimized assay was used with the most probable number (MPN) method to quantify RDase genes ( vcrA and tceA ) in multiple groundwater samples from a chlorinated solvent contaminated site. Using this approach, gene concentrations were significantly correlated to concentrations obtained using traditional methods (qPCR and DNA templates). Although the assay underestimated RDase genes concentrations, a strong correlation (R 2 = 0.78 and 0.94) was observed between the two data sets. The regression equations obtained will be valuable to determine gene copies in groundwater using the newly developed, low cost and time saving method. [ABSTRACT FROM AUTHOR]

The aim of this work was to study the effects of the presence of oxygen on the dechlorination of trichloroethene by a microbial consortium containing D. mccartyi . The 16S rRNA and reductive dechlorination genes of the functional bacteria and the non-dechlorinators were monitored. Exposing the consortium to oxygen altered the overall biotransformation rate of the dechlorination process, biotransformation processes prolonged with oxygen concentrations changing from 0 to 7.2 mg/L, however, trichloroethylene was eventually dechlorinated to ethene. The qPCR analyses revealed that the D. mccartyi strains containing the tceA gene were less sensitive to exposure to oxygen than were the D. mccartyi strains containing the vcrA gene. High-throughput sequencing by Illumina MiSeq indicated that the non-dechlorinating organisms were probably crucial to scavenge the oxygen to protect D. mccartyi from being damaged. [ABSTRACT FROM AUTHOR]

Dehalococcoides mccartyi exhibits versatile capabilities to respire halogenated compounds under anaerobic conditions. In this study, we report the assembly and annotation of the complete genome of a chloroethene dechlorinating D. mccartyi strain 11a5. Bearing a 1461 973 base-pair chromosome, strain 11a5 distinguishes itself from other D. mccartyi strains by possessing a 5940 base-pair circular extrachromosomal genetic element which contains a reductive dehalogenase homolog. The whole genome of strain 11a5 harbors 31 putative reductive dehalogenase genes. Through transcriptional and proteomic analyses, we identified a new tetrachloroethene (PCE) reductive dehalogenase, PteA (11a5 1355), which catalyzes reductive dechlorination from PCE to trichloroethene (TCE) and shares only 38% similarity in amino acid sequence with its closest relative PceA in D. mccartyi strain 195. The acquisition of the genome of strain 11a5 enlarged the database of D. mccartyi and enriched our understanding of this unique species, among which, the identification of a new PCE reductive dehalogenase can assist in understanding PCE dechlorination process. In addition, the discovery of the circular extrachromosomal genetic element in strain 11a5 may provide insights to investigate how reductive dehalogenase homologous genes are transferred and carried by organohalide respiring bacteria. [ABSTRACT FROM AUTHOR]

The present study evaluates the PCB-dehalorespiring capabilities and dynamics of indigenous Dehalococcoides mccartyi population in a PCB contaminated marine sediment. Specialized PCB-dechlorinase genes pcbA1 , pcbA4 and pcbA5 previously characterized in pure cultures of D. mccartyi , were here found for the first time in environmental samples. Reductive dechlorination was stimulated by spiking Aroclor1254 to the sediment and by imposing strictly anaerobic conditions both with and without bioaugmentation with a Dehalococcoides mccartyi enrichment culture. In line with the contaminant dechlorination kinetics, Dehalococcoides population increased during the entire incubation period showing growth yields of 4.94E + 07 Dehalococcoides per μmol Cl − 1 and 7.30E + 05 Dehalococcoides per μmol Cl − 1 in the marine sediment with and without bioaugmentation respectively. The pcbA4 and pcbA5 dechlorinase genes, and to a lesser extent pcbA1 gene, were enriched during the anaerobic incubation suggesting their role in Aroclor1254 dechlorination under salinity conditions. [ABSTRACT FROM AUTHOR]

The application of bioelectrochemical systems (BES) for the treatment of chloroethanes has been so far limited, in spite of the high frequency that these contaminants are detected at contaminated sites. This work studied the biodegradation of 1,2-dichloroethane (1,2-DCA) in a lab-scale BES, inoculated with a municipal activated sludge and operated under a range of conditions, spanning from oxidative to reductive, both in the presence and in the absence of the humic acid analogue anthraquinone-2,6-disulfonate (AQDS) as a redox mediator. The results showed stable dechlorination of 1,2-DCA to ethene (up to 65 ± 5 μmol/L d), when the BES was operated at a set potential of −300 mV vs. SHE, in the presence of AQDS. Sustained filled-and-draw operation resulted in the enrichment of Dehalococcoides mccartyi . The results of this work provide new insights into the applicability of BES for groundwater remediation and the potential interaction between biogeochemistry and 1,2-DCA in humics-rich contaminated aquifers. [ABSTRACT FROM AUTHOR]

Polychlorobiphenyl (PCB) biodegradation was followed for 1 year in microcosms containing marine sediments collected from Mar Piccolo (Taranto, Italy) chronically contaminated by this class of hazardous compounds. The microcosms were performed under strictly anaerobic conditions with or without the addition of Dehalococcoides mccartyi, the main microorganism known to degrade PCBs through the anaerobic reductive dechlorination process. Thirty PCB congeners were monitored during the experiments revealing that the biodegradation occurred in all microcosms with a decrease in hepta-, hexa-, and penta-chlorobiphenyls (CBs) and a parallel increase in low chlorinated PCBs (tri-CBs and tetra-CBs). The concentrations of the most representative congeners detected in the original sediment, such as 245-245-CB and 2345-245-CB, and of the mixture 2356-34-CB+234-245-CB, decreased by 32.5, 23.8, and 46.7 %, respectively, after only 70 days of anaerobic incubation without any bioaugmentation treatment. Additionally, the structure and population dynamics of the microbial key players involved in the biodegradative process and of the entire mixed microbial community were accurately defined by Catalyzed Reporter Deposition Fluorescence In Situ Hybridization (CARD-FISH) in both the original sediment and during the operation of the microcosm. The reductive dehalogenase genes of D. mccartyi, specifically involved in PCB dechlorination, were also quantified using real-time PCR (qPCR). Our results demonstrated that the autochthonous microbial community living in the marine sediment, including D. mccartyi (6.32E+06 16S rRNA gene copy numbers g sediment), was able to efficiently sustain the biodegradation of PCBs when controlled anaerobic conditions were imposed. [ABSTRACT FROM AUTHOR]

Effective treatment of sites contaminated with dense non-aqueous phase liquids (DNAPLs) requires detailed understanding of the microbial community responses to changes in source zone strength and architecture. Changes in the spatial and temporal distributions of the organohalide-respiring Dehalococcoides mccartyi ( Dhc ) strains and Geobacter lovleyi strain SZ ( Geo SZ) were examined in a heterogeneous tetrachloroethene- (PCE-) DNAPL source zone within a two-dimensional laboratory-scale aquifer flow cell. As part of a combined remedy approach, flushing with 2.3 pore volumes (PVs) of 4% (w/w) solution of the nonionic, biodegradable surfactant Tween® 80 removed 55% of the initial contaminant mass, and resulted in a PCE–DNAPL distribution that contained 51% discrete ganglia and 49% pools (ganglia-to-pool ratio of 1.06). Subsequent bioaugmentation with the PCE-to-ethene-dechlorinating consortium BDI-SZ resulted in cis -1,2-dichloroethene ( cis -DCE) formation after 1 PV (ca. 7 days), while vinyl chloride (VC) and ethene were detected 10 PVs after bioaugmentation. Maximum ethene yields (ca. 90 μM) within DNAPL pool and ganglia regions coincided with the detection of the vcrA reductive dehalogenase (RDase) gene that exceeded the Dhc 16S rRNA genes by 2.0 ± 1.3 and 4.0 ± 1.7 fold in the pool and ganglia regions, respectively. Dhc and Geo SZ cell abundance increased by up to 4 orders-of-magnitude after 28 PVs of steady-state operation, with 1 to 2 orders-of-magnitude increases observed in close proximity to residual PCE–DNAPL. These observations suggest the involvement of these dechlorinators the in observed PCE dissolution enhancements of up to 2.3 and 6.0-fold within pool and ganglia regions, respectively. Analysis of the solid and aqueous samples at the conclusion of the experiment revealed that the highest VC (≥ 155 μM) and ethene (≥ 65 μM) concentrations were measured in zones where Dhc and Geo SZ were predominately attached to the solids. These findings demonstrate dynamic responses of organohalide-respiring bacteria in a heterogeneous DNAPL source zone, and emphasize the influence of source zone architecture on bioremediation performance. [ABSTRACT FROM AUTHOR]

Due to the direct involvement in the biodegradation of chlorinated solvents, reductive dehalogenase genes (RDase) are considered biomarkers of the metabolic potential of different strains of Dehalococcoides mccartyi ( Dhc ). This is known to be the only microbe able to completely reduce toxic chlorinated solvents to harmless ethene. In the last years, several Molecular Biological Tools (MBTs) have been developed to optimize the detectability of Dhc cells and/or the RDase genes, with particular attention to the most important indicators of ethene formation, namely tceA and vcrA genes. Despite qPCR has been indicated as the MBT of choice, the use of CARD-FISH recently demonstrated to provide a more accurate quantification of Dhc cells in a wide concentration range, overcoming the drawbacks of loosing nucleic acids during the preparation of the sample associated with qPCR. CARD-FISH assays usually target 16S rRNA and up to date no protocol able to discriminate different Dhc strains by detecting RDase genes has been developed. This study reports the first evidence of in situ detection of tceA and vcrA genes into Dhc cells by applying a new procedure named geneCARD-FISH. Dhc strains carrying tceA and vcrA genes were identified and quantified in a PCE-to-ethene dechlorinating microbial enrichment and overall they represented 58.63% ± 2.45% and 40.46% ± 1.86% of the total Dhc cells, respectively. These values were markedly higher than those obtained by qPCR, which strongly underestimated the actual concentration of vcrA gene (0.08% ± 0.01% of Dhc 16S rRNA gene copies). The assay was successfully applied also for the analysis of environmental samples and remarkably strengthens the biomonitoring activities at field scale by providing the specific in situ discrimination of Dhc cells carrying the key-RDase genes. [ABSTRACT FROM AUTHOR]

In situ bioreactive capping is a promising technology for mitigation of surface water contamination by discharging polluted groundwater. Organohalide respiration (OHR) of chlorinated ethenes in bioreactive caps can be stimulated through incorporation of solid polymeric organic materials (SPOMs) that provide a sustainable electron source for organohalide respiring bacteria. In this study, wood chips, hay, straw, tree bark and shrimp waste, were assessed for their long term applicability as an electron donor for OHR of cis-dichloroethene (cDCE) and vinyl chloride (VC) in sediment microcosms. The initial release of fermentation products, such as acetate, propionate and butyrate led to the onset of extensive methane production especially in microcosms amended with shrimp waste, straw and hay, while no considerable stimulation of VC dechlorination was obtained in any of the SPOM amended microcosms. However, in the longer term, short chain fatty acids accumulation decreased as well as methanogenesis, whereas high dechlorination rates of VC and cDCE were established with concomitant increase of Dehalococcoides mccartyi and vcrA and bvcA gene numbers both in the sediment and on the SPOMs. A numeric simulation indicated that a capping layer of 40 cm with hay, straw, tree bark or shrimp waste is suffice to reduce the groundwater VC concentration below the threshold level of 5 μg/l before discharging into the Zenne River, Belgium. Of all SPOMs, the persistent colonization of tree bark by D. mccartyi combined with the lowest stimulation of methanogenesis singled out tree bark as a long-term electron donor for OHR of cDCE/VC in bioreactive caps. [ABSTRACT FROM AUTHOR]

We have developed a novel approach to identifying and quantifying closely related organohalide-respiring bacteria. Our approach made use of the unique genomic associations of specific reductive dehalogenase subunit A encoding genes ( rdhA) that exist in known strains of Dehalococcoides mccartyi and Desulfitobacterium and the distinguishing covariance pattern of observed rdhA genes to assign genes to unknown strains. To test this approach, we operated five anaerobic reductively dechlorinating chemostats for 3-4 years with tetrachloroethene and trichloroethene as terminal electron acceptors and lactate/formate as electron donors. The presence and abundance of rdhA genes were determined comprehensively at the community level using a custom-developed Reductive Dehalogenase Chip ( RDH Chip) DNA microarray and used to define putative strains of Dehalococcoides mccartyi and Desulfitobacterium sp. This monitoring revealed that stable chemical performance of chemostats was reflected by a stable community of reductively dechlorinating bacteria. However, perturbations introduced by, for example, electron donor limitation or addition of the competing electron acceptor sulfate led to overall changes in the chemostat performance, including incomplete reduction in the chloroethene substrates, and in the population composition of reductively dehalogenating bacteria. Interestingly, there was a high diversity of operationally defined D. mccartyi strains between the chemostats with almost all strains unique to their specific chemostats in spite of similar selective pressure and similar inocula shared between chemostats. [ABSTRACT FROM AUTHOR]

Abstract: Stimulated anaerobic dechlorination is generally considered a valuable step for the remediation of aquifers polluted with chlorinated ethenes (CEs). Correct simulation and prediction of this process in situ, however, require good knowledge of the associated biological reactions. The aim of this study was to evaluate the dechlorination reaction in an aquifer contaminated with trichloroethene (TCE) and its daughter products, discharging into the Zenne River. Different carbon sources were used in batch cultures and these were related to the dechlorination reaction, together with the monitored biomarkers. Appropriate kinetic formulations were assessed. Reductive dechlorination of TCE took place only when external carbon sources were added to microcosms, and occurred concomitant with a pronounced increase in the Dehalococcoides mccartyi cell count as determined by 16S rRNA gene-targeted qPCR. This indicates that native dechlorinating bacteria are present in the aquifer of the Zenne site and that the oligotrophic nature of the aquifer prevents a complete degradation to ethene. The type of carbon source, the cell number of D. mccartyi or the reductive dehalogenase genes, however, did not unequivocally explain the observed differences in degradation rates or the extent of dechlorination. Neither first-order, Michaelis–Menten nor Monod kinetics could perfectly simulate the dechlorination reactions in TCE spiked microcosms. A sensitivity analysis indicated that the inclusion of donor limitation would not significantly enhance the simulations without a clear process understanding. Results point to the role of the supporting microbial community but it remains to be verified how the complexity of the microbial (inter)actions should be represented in a model framework. [Copyright &y& Elsevier]

Abstract: Dehalococcoides mccartyi detectability in the field is a fundamental tool to assess the efficiency of natural attenuation or engineered bioremediation in chlorinated solvent-contaminated sites. This study reports on the direct comparison of quantitative data obtained by Real Time PCR (qPCR) and CAtalyzed Reporter Deposition-Fluorescence In situ Hybridization (CARD-FISH) over a wide range of Dehalococcoides concentrations (10–108 cellsmL−1) both in three independent 10-fold serial dilutions of a laboratory dechlorinating enrichment and in 49 groundwater samples from 6 different contaminated sites. Dehalococcoides enumeration by CARD-FISH yielded a linear curve in the analyzed concentration range which was consistent with the expected concentrations and showed good reproducibility in triplicate assays. Alternatively, qPCR did not allow for the discrimination of 16S rRNA gene concentrations lower than 103 gene copiesmL−1 either in the dechlorinating mixed culture or in field samples. Overall this study highlights the limits of qPCR quantification, especially in samples where low concentrations of this microorganism may be expected, and suggests the use of a confirmatory methodology under these particular conditions. [Copyright &y& Elsevier]