Dehalococcoides

Coastal wetlands, such as mangroves, seagrass beds, and salt marshes, are highly threatened by increasing anthropic pressures, including chemical pollution. Persistent organic pollutants (POPs) have attracted attention in these particularly vulnerable ecosystems, due to their bioaccumulative, pervasive, and ecotoxic behavior. This article reviews and summarizes available information regarding current levels, biogeochemical cycling, and effects of POPs on coastal wetlands. Sediment POP levels were compared with international quality guidelines, revealing many areas where compounds could cause damage to biota. Despite this, toxicological studies on some coastal wetland plants and microorganisms showed a high tolerance to those levels. These taxonomic groups are likely to play a key role in the cycling of the POPs, with an active role in their accumulation, immobilization, and degradation. Toxicity and biogeochemical processes varied markedly along three main axes; namely species, environmental conditions, and type of pollutant. While more focused research on newly and unintentionally produced POPs is needed, mainly in salt marshes and seagrass beds, with the information available so far, the environmental behavior, spatial distribution, and toxicity level of the studied POPs showed similar patterns across the three studied ecosystems.

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-1 sediment), was able to efficiently sustain the biodegradation of PCBs when controlled anaerobic conditions were imposed.

The toxicity of polychlorinated biphenyls (PCB) can be efficiently reduced in contaminated marine sediments through the reductive dechlorination (RD) process lead by anaerobic organohalide bacteria. Although the process has been extensively investigated on PCB-spiked sediments, the knowledge on the identity and metabolic potential of PCB-dechlorinating microorganisms in real contaminated matrix is still limited. Aim of this study was to explore the composition and the dynamics of the microbial communities of the marine sediment collected from one of the largest Sites of National Interest (SIN) in Italy (Mar Piccolo, Taranto) under conditions promoting the PCBs RD. A long-term microcosm study revealed that autochthonous bacteria were able to sustain the PCB dechlorination at a high extent and the successive addition of an external fermentable organic substrate (lactate) caused the further depletion of the high-chlorinated PCBs (up to 70%). Next Generation Sequencing was used to describe the core microbiome of the marine sediment and to follow the changes caused by the treatments. OTUs affiliated to sulfur-oxidizing ε-proteobacteria, Sulfurovum, and Sulfurimonas, were predominant in the original sediment and increased up to 60% of total OTUs after lactate addition. Other OTUs detected in the sediment were affiliated to sulfate reducing (δ-proteobacteria) and to organohalide respiring bacteria within Chloroflexi phylum mainly belonging to Dehalococcoidia class. Among others, Dehalococcoides mccartyi was enriched during the treatments even though the screening of the specific reductive dehalogenase genes revealed the occurrence of undescribed strains, which deserve further investigations. Overall, this study highlighted the potential of members of Dehalococcoidia class in reducing the contamination level of the marine sediment from Mar Piccolo with relevant implications on the selection of sustainable bioremediation strategies to clean-up the site.

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.

Chlorinated compounds pose environmental concerns due to their toxicity and wide distribution in several matrices. Microorganisms specialized in leading anaerobic reductive dechlorination (RD) processes, including Dehalococcoides mccartyi (Dhc), are able to reduce chlorinated compounds to harmless products or to less toxic forms. Here we report the first detailed study dealing with the RD potential of heavy polluted marine sediment by evaluating the biodegradation kinetics together with the composition, dynamics and activity of indigenous microbial population. A microcosm study was conducted under strictly anaerobic conditions on marine sediment collected near the marine coast of Sarno river mouth, one of the most polluted river in Europe. Tetrachloroethene (PCE), used as model pollutant, was completely converted to ethene within 150 days at reductive dechlorination rate equal to 0.016 meq L −1 d −1. Consecutive spikes of PCE allowed increasing the degradation kinetics up to 0.1 meq L −1 d −1 within 20 days. Strictly anaerobiosis and repeated spikes of PCE stimulated the growth of indigenous Dhc cells (growth yield of ~7.0E + 07 Dhc cells per μM Cl −1 released). Dhc strains carrying the reductive dehalogenase genes tceA and vcrA were detected in the original marine sediment and their number increased during the treatment as demonstrated by the high level of tceA expression at the end of the microcosm study (2.41E + 05 tceA gene transcripts g −1). Notably, the structure of the microbial communities was fully described by Catalysed Reporter Deposition Fluorescence In Situ Hybridization (CARD-FISH) as wells as the dynamics of the dechlorinating bacteria during the microcosms operation. Interestingly, a direct role of Dhc cells was ascertained

""""Three microbial cultures were enriched on PCE (hereafter named RM3), TCE (B2), or cis-DCE (B3) (as electron acceptors) and lactate (as electron donor). All cultures, which were found to completely transform the chlorinated contaminants to ethene, were kinetically characterised and analysed to define the dynamics and activity of dechlorinating microbial populations. Specific biodegradation rates were estimated by means of conventional biomass parameter (i.e. VSS, volatile suspended solids) and by cell numbers of dechlorinating population specifically discriminated and quantified by biomolecular tools.
Kinetic batch experiments, carried out after a pseudo-steady state performance had been established, revealed that the observed dechlorination rates followed the order RM3 (PCE-enriched)>B2 (TCE-enriched)>B3 (cis-DCE-enriched).
Dechlorinating bacteria were quantified by combining in situ hybridization techniques and PCR-based approaches. Fluorescence in situ hybridization (FISH and CARD-FISH) allowed the cell numbers estimation of active and not active i) dechlorinating bacteria involved in the partial degradation of PCE or TCE to cis-DCE (Desulfitobacterium spp., Dehalobacter spp., spp., Geobacter spp., Sulfurospirillum spp.) as well as ii) “Dehalococcoides” spp. known to be able to completely dechlorinate such compounds to harmless ethene. Gene expression profile of reductive dehalogenase genes (tceA, bvcA, vcrA) were also estimated and correlated to kinetic performances and dechlorinating bacteria abundances. The potential for field application of the outputs of this study will be discussed.""""

""""The extent of chlorinated solvents biodegradation in contaminated aquifers is critically dependent upon several factors, namely, the presence of contaminant degrading bacteria and the creation of optimal environmental conditions to stimulate biodegradative activity. The early estimation of the occurrence and abundance of contaminant key-degrading bacteria at field scale can therefore strongly assist the definition of the bioremediation potential as well as the decision making process prior to initiating bioremediation activities and the detailed site characterization. In this study, a wide screening was made on groundwater samples taken from several Italian chlorinated solvents contaminated sites to estimate the dechlorinators cell densities and activity. In particular, the analysis was made by means of a combination of biomolecular tools including in situ detection methods (CARD-FISH) for the accurate estimation of dechlorinators abundances and RT-qPCR for the expression analysis of reductive dehalogenase.  Here, the outputs obtained by the application of the biomolecular tools were further compared with the results obtained by conventional treatability approach (i.e. microcosm study).
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Background Vinyl chloride is a widespread groundwater pollutant and Group 1 carcinogen. A previous comparative genomic analysis revealed that the vinyl chloride reductase operon, vcrABC, of Dehalococcoides sp. strain VS is embedded in a horizontally-acquired genomic island that integrated at the single-copy tmRNA gene, ssrA. Results We targeted conserved positions in available genomic islands to amplify and sequence four additional vcrABC -containing genomic islands from previously-unsequenced vinyl chloride respiring Dehalococcoides enrichments. We identified a total of 31 ssrA-specific genomic islands from Dehalococcoides genomic data, accounting for 47 reductive dehalogenase homologous genes and many other non-core genes. Sixteen of these genomic islands contain a syntenic module of integration-associated genes located adjacent to the predicted site of integration, and among these islands, eight contain vcrABC as genetic 'cargo'. These eight vcrABC -containing genomic islands are syntenic across their ~12 kbp length, but have two phylogenetically discordant segments that unambiguously differentiate the integration module from the vcrABC cargo. Using available Dehalococcoides phylogenomic data we estimate that these ssrA-specific genomic islands are at least as old as the Dehalococcoides group itself, which in turn is much older than human civilization. Conclusions The vcrABC -containing genomic islands are a recently-acquired subset of a diverse collection of ssrA-specific mobile elements that are a major contributor to strain-level diversity in Dehalococcoides, and may have been throughout its evolution. The high similarity between vcrABC sequences is quantitatively consistent with recent horizontal acquisition driven by ~100 years of industrial pollution with chlorinated ethenes.