Correlations between maximum reductive dechlorination rates and specific biomass parameters in Dehalococcoides mccartyi consortia enriched on chloroethenes PCE, TCE and cis-1,2-DCE.

One of the challenges to implementing the modeling of the biological reductive dechlorination (RD) process is the evaluation of biological parameters that represent the abundance/activity levels of the microorganisms involved in the biodegradation of chloroethenes. Here we report a combined analysis of kinetic and specific biomass parameters conducted on three dechlorinating consortia enriched on PCE, TCE and cis-1,2-DCE. In these consortia, Dehalococcoides mccartyi (Dhc) represented ≥70% of the bacterial population identified via 16S rRNA gene amplicon sequencing. Quantitative biomolecular methods were used to generate specific biomass parameters targeting either the Dhc population (16S rRNA genes or cells) or specific genes encoding RD process-involved reductive dehalogenases. The correlation factor between the abundance of active Dhc cells or tceA gene copies and maximum RD rates allowed to predict an increment of 7E+09 of active Dhc cells or 5E+09 tceA gene copies/L under controlled conditions. Diversely, the utilization of gene transcripts as biomass parameters for RD modeling did not provide reliable correlations with kinetic performances. This study provides valuable insights for further modeling of the RD process through the utilization of specific biomass parameters.

Marine hydrocarbon-degrading bacteria breakdown poly(ethylene terephthalate) (PET).

Pollution of aquatic ecosystems by plastic wastes poses severe environmental and health problems and has prompted scientific investigations on the fate and factors contributing to the modification of plastics in the marine environment. Here, we investigated, by means of microcosm studies, the role of hydrocarbon-degrading bacteria in the degradation of poly(ethylene terephthalate) (PET), the main constituents of plastic bottles, in the marine environment. To this aim, different bacterial consortia, previously acclimated to representative hydrocarbons fractions namely, tetradecane (aliphatic fraction), diesel (mixture of hydrocarbons), and naphthalene/phenantrene (aromatic fraction), were used as inocula of microcosm experiments, in order to identify peculiar specialization in poly(ethylene terephthalate) degradation. Upon formation of a mature biofilm on the surface of poly(ethylene terephthalate) films, the bacterial biodiversity and degradation efficiency of each selected consortium was analyzed. Notably, significant differences on biofilm biodiversity were observed with distinctive hydrocarbons-degraders being enriched on poly(ethylene terephthalate) surface, such as Alcanivorax, Hyphomonas, and Cycloclasticus species. Interestingly, ATR-FTIR analyses, supported by SEM and water contact angle measurements, revealed major alterations of the surface chemistry and morphology of PET films, mainly driven by the bacterial consortia enriched on tetradecane and diesel. Distinctive signatures of microbial activity were the alteration of the FTIR spectra as a consequence of PET chain scission through the hydrolysis of the ester bond, the increased sample hydrophobicity as well as the formation of small cracks and cavities on the surface of the film. In conclusion, our study demonstrates for the first time that hydrocarbons-degrading marine bacteria have the potential to degrade poly(ethylene terephthalate), although their degradative activity could potentially trigger the formation of harmful microplastics in the marine environment.

Combining electrokinetic transport and bioremediation for enhanced removal of crude oil from contaminated marine sediments: Results of a long-term, mesocosm-scale experiment.

Marine sediments represent an important sink of harmful petroleum hydrocarbons after an accidental oil spill. Electrobioremediation techniques, which combine electrokinetic transport and biodegradation processes, represent an emerging technological platform for a sustainable remediation of contaminated sediments. Here, we describe the results of a long-term mesocosm-scale electrobioremediation experiment for the treatment of marine sediments contaminated by crude oil. A dimensionally stable anode and a stainless-steel mesh cathode were employed to drive seawater electrolysis at a fixed current density of 11 A/m2. This approach allowed establishing conditions conducive to contaminants biodegradation, as confirmed by the enrichment of Alcanivorax borkumensis cells harboring the alkB-gene and other aerobic hydrocarbonoclastic bacteria. Oil chemistry analyses indicated that aromatic hydrocarbons were primarily removed from the sediment via electroosmosis and low molecular weight alkanes (nC6 to nC10) via biodegradation.

First evidence on the occurrence and dynamics of Dehalococcoides mccartyi PCB-dechlorinase genes in marine sediment during Aroclor1254 reductive dechlorination.

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 µmolCl-1 and 7.30E+05 Dehalococcoides per µmolCl-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.

Reductive dechlorination of tetrachloroethene in marine sediments: Biodiversity and dehalorespiring capabilities of the indigenous microbes.

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 ~7.0 E + 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 (2.41 E + 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 suggesting the existence of strains adapted at salinity conditions. Additionally, non-Dhc Chloroflexi were retrieved in the original sediment and were kept stable over time suggesting their likely flanking role of the RD process.

GeneCARD-FISH: detection of tceA and vcrA reductive dehalogenase genes in Dehalococcoides mccartyi by fluorescence in situ hybridization.

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.

Quantitative estimation of Dehalococcoides mccartyi at laboratory and field scale: comparative study between CARD-FISH and Real Time PCR.

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 (10-10(8) cells mL(-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 10(3) gene copies mL(-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.

Field distribution and activity of chlorinated solvents degrading bacteria by combining CARD-FISH and real time PCR.

Nowadays several advanced molecular techniques are applied for quantifying bacteria involved in contaminant degradation processes. However, despite the fact that significant efforts have been taken to make these tools more reliable and specific, their application for the analysis of field samples is hardly ever applied. In this study, a combination of three methods (CARD-FISH, qPCR and RT-qPCR) was successfully applied to evaluate the distribution and the activity of known chlorinated solvent dechlorinating bacteria in a contaminated site where no remedial actions have been undertaken. CAtalysed Reporter Deposition Fluorescence In Situ Hybridization (CARD-FISH) specifically provided the cell densities of known dechlorinating bacteria and was found to be more sensitive than quantitative PCR (qPCR) for the quantification of 'Dehalococcoides' cell numbers in the aquifer. Among the screened dechlorinators, 'Dehalococcoides' spp. were mainly found and nearly homogenously distributed in the aquifers at concentrations ranging from 8.1×10(5)±1.2×10(5) to 2.5×10(7)±5.6×10(6)cells per liter of groundwater (with a relative abundance out of the total Bacteria of 0.7-15%). Further, the dechlorination potentialities of 'Dehalococcoides' species living in the aquifer were evaluated by analyzing the abundance and the expression of 16S rRNA genes and reductive dehalogenase (RDase) encoding functional genes by qPCR and Reverse Transcription qPCR (RT-qPCR). 'Dehalococcoides'tceA gene, known to be associated to strains capable of reducing chlorinated solvents beyond cis-DCE, was found and expressed in the field. Overall, this study proved the existence of a well-established dechlorinating microbial community able to use contaminants as substrates for their metabolic activity and indicated the occurrence of reductive dechlorination at the site.