Techn. Universiẗat, Diss., 2005--Berlin.

Chloroethenes such as tetrachloroethene (PCE), trichloroethene (TCE), dichloroethene (DCE) and vinyl chloride (VC), are major contaminants in subsurface systems threatening water quality and human health. Under anaerobic conditions, PCE and TCE can be reductively dechlorinated to ethene. Recent findings indicate that members of the Dehalococcoides group are responsible for ethene formation at chloroethene-contaminated sites. Dehalococcoides species exhibit diverse dechlorination activities, but share highly similar 16S rRNA genes. Hence, additional gene targets that go beyond the 16S rRNA gene are needed to reliably detect and quantify Dehalococcoides populations involved in high rate chloroethene detoxification at contaminated sites. Dehalococcoides sp. strain BAV1 couples growth to reductive dechlorination of VC to ethene. To shed light on the genes involved in reductive dechlorination in strain BAV1, degenerate primers targeting reductive dehalogenase (RDase) genes of Dehalococcoides were designed using available sequence information. PCR amplification with these primers yielded seven putative RDase genes with genomic DNA from strain BAV1 as template. Transcription analysis identified one RDase gene possibly involved in VC dechlorination, which was named bvcA. The bvcA gene was not present in Dehalococcoides strains that failed to couple growth with reductive dechlorination of VC (i.e., Dehalococcoides isolates CBDB1, FL2 and 195). Primers specific for bvcA detected this gene in several, but not all, Dehalococcoides-containing, ethene-producing mixed cultures. Apparently, the bvcA-targeted primers do not capture the diversity of VC RDase genes. Nevertheless, a relevant target was identified, and bvcA-targeted primers are commercially applied to monitor Dehalococcoides sp. strain BAV1 and related organisms at contaminated sites undergoing bioremediation treatment. Additional RDase genes were identified in Dehalococcoides sp. strain FL2, and expression analysis was performed when FL2 was grown with cis-DCE and TCE as electron acceptors. Multiple RDase genes were transcribed with each electron acceptor. This work identified novel process-specific target genes that are useful for site assessment and bioremediation monitoring at chloroethene-contaminated sites. In particular, bvcA emerged as a relevant target for monitoring the critical detoxification step from VC, to ethene. Additionally, the RDase genes retrieved in this work form a basis for further exploration of the specific functions and regulation mechanisms involved in reductive dechlorination processes.

Zugl.: Freiberg (Sachsen), Bergakad., Diss., 2008.

The findings of this study demonstrate that Dehalococcoides species are intimately involved in complete reductive detoxification of chlorinated ethenes and are widely distributed in anoxic sediments and aquifers, including non-contaminated (pristine) environments. Careful examination of enrichment culture dechlorination kinetics, 16S rRNA gene based analyses, and reductive dehalogenase gene targeted PCR approaches revealed that complete reductive dechlorination is carried out by multiple dechlorinators. Two new dechlorinating species were isolated from contaminated and non-contaminated site materials. The first new isolate, designated strain SZ, was isolated from PCE-to-ethene dechlorinating microcosms established with creek sediment. 16S rRNA gene sequence of the strain SZ indicates that the new isolate is affiliated with the genus Geobacter most closely related to G. thiogenes. Strain SZ is capable of stepwise dechlorination of PCE to cis-DCE, while the closest relatives were not able to dechlorinate PCE or TCE. Dechlorination of PCE or TCE by strain SZ was supported by acetate, hydrogen or pyruvate as electron donor. Chloroethene-dechlorinating populations have been shown to have distinct electron donor requirements. However, none of previously described chlorinated ethene degrading population can use both, acetate and hydrogen, as electron donors. PCE dechlorination by strain SZ uses both acetate and hydrogen as electron donors suggesting that the ability to versatile electron donor utilization may increase the efficiency of bioremediation approaches. Importantly, strain SZ reduced two environmental priority pollutants, PCE and U(VI) concomitantly and detected from both bio-stimulated chloroethene and uranium contaminated sites, strongly suggesting that strain SZ play a important roles in in-situ bioremediation of chloroethene and U(VI) contaminated sites. The second, a new Dehalococcoides species designated strain GT, was isolated from contaminated site materials. Strain GT uses trichloroethene (TCE), cis-DCE, 1,1-dichloroethene (1,1-DCE), and the human carcinogen vinyl chloride (VC) as growth supporting electron acceptors producing products ethene and inorganic chloride. The new isolate shares common traits of Dehalococcoides such as ampicillin resistance, strict hydrogen-dependent metabolism, and a low hydrogen consumption threshold concentration. Culture-dependent and independent, 16S rRNA gene and reductive dehalogenase gene targeted PCR approaches suggested culture purity.

The Port of Houston and the Houston Ship Channel (HSC) are highly industrialized areas along Galveston Bay, Texas. The HSC is highly polluted with a host of persistent organic pollutants, including dioxins. The main objective of this study was to determine the potential for in situ bioremediation in the HSC sediments. Our study focused on the bacterial group Dehalococcoides, since it is the only known group to reductively dechlorinate dioxins. Culture independent methods were used to determine the presence or absence of Dehalococcoides in HSC sediments. Molecular methods including PCR, cloning, restriction enzyme digest, and sequencing were used to determine the diversity of Dehalococcoides as well as total bacterial diversity in HSC sediments. The metabolically active members of the microbial community in HSC sediments were also determined using the same molecular methods as described above. Dehalococcoides was detected in every sediment core and at various depths within each core. Depths ranged from 1cm (SG-6) to 30cm (11261). Dehalococcoides diversity was centered on Dehalococcoides ethenogenes strain 195 and Dehalococcoides sp. strain CBDB1. Overall bacterial diversity in HSC sediments was dominated by Proteobacteria, especially Deltaproteobacteria, and Chloroflexi, which include Dehalococcoides. Total bacterial diversity at a wetlands control site was dominated by Betaproteobacteria and Acidobacteria. Deltaproteobacteria and Chloroflexi were determined to be the major metabolically active groups within the HSC sediments. These findings indicate that the HSC sediments have great potential for successful in situ bioremediation. These results also support the use of Dehalococcoides as a biological proxy for dioxin contamination.

Perchloroethene and trichloroethene are among the most persistent groundwater pollutants, and Dehalococcoides is the only known species that can degrade these compounds completely to non-toxic ethene. Characterization of the reductive dehalogenase (RDase) enzymes responsible for dechlorination is important to understanding this process. A series of dechlorination assays were performed with whole cell suspensions and cell-free extracts of three Dehalococcoides-containing mixed microbial consortia to compare dechlorination kinetics and to characterize co-contaminant inhibition. Michaelis-Menten kinetic parameters Vmax and Km, as well as non-competitive inhibition coefficients for 1,1,1-trichloroethane and 1,1-dichloroethane inhibitors are reported. Secondly, blue native gel electrophoresis was developed as a method to isolate active protein complexes containing RDases. Thirdly, sources of variability in the isotopic fractionation of vinyl chloride to ethene reaction step were examined using cell-free extracts and whole-cell suspensions. Understanding the function and range of RDases are goals towards the successful application of Dehalococcoides-containing cultures to remediate contaminated sites.

Perchloroethene and trichloroethene are among the most persistent groundwater pollutants, and Dehalococcoides is the only known species that can degrade these compounds completely to non-toxic ethene. Characterization of the reductive dehalogenase (RDase) enzymes responsible for dechlorination is important to understanding this process. A series of dechlorination assays were performed with whole cell suspensions and cell-free extracts of three Dehalococcoides-containing mixed microbial consortia to compare dechlorination kinetics and to characterize co-contaminant inhibition. Michaelis-Menten kinetic parameters Vmax and Km, as well as non-competitive inhibition coefficients for 1,1,1-trichloroethane and 1,1-dichloroethane inhibitors are reported. Secondly, blue native gel electrophoresis was developed as a method to isolate active protein complexes containing RDases. Thirdly, sources of variability in the isotopic fractionation of vinyl chloride to ethene reaction step were examined using cell-free extracts and whole-cell suspensions. Understanding the function and range of RDases are goals towards the successful application of Dehalococcoides-containing cultures to remediate contaminated sites.

The aim of this study was to access the efficacy of an enhanced in situ bioremediation technology at a 1,2-dichloroethane (1,2-DCA) polluted site in southern Taiwan. A water-soluble substrate was injected into the groundwater to provide carbon sources for microbial growth. After substrate injection, increased total organic carbon (TOC) concentrations and microbial populations including Dehalococcoides spp. and Desulfitobacterium spp. were observed in the groundwater. Microbial diversity was analyzed using denaturing gradient gel electrophoresis (DGGE) and 16S rDNA sequencing to identify the bacterial strains. The results showed that after 4.5 months of substrate injection, the reduction-oxidation potential (ORP) changed from aerobic to anaerobic conditions. The less oxygen-tolerable 1,2-DCA degrading bacteria Dehalococcoides spp. started to accumulate in groundwater. However, the more oxygen-tolerable Desulfitobacterium spp. didn¡¦t show a prominent change, although the ORP was suitable for Desulfitobacterium spp. to carry out reductive dechlorination. The DGGE results indicate that with the injected carbon sources and mineral nutrients, both the groundwater microbial diversity and the amount of dominant bacteria were increased. The 16S rDNA sequencing demonstrated that the amount and diversity of 1,2-DCA degradation-related bacteria also increased with the injection of substrate. Six groups of 1,2-DCA degradation related reactions were found: dechlorination, chlorinated-compound degradation, denitrification, iron-reduction, sulfate-reduction and methane-utilizing. Four species that can directly degrade 1,2-DCA were found: Dehalobacter sp., Dehalococcoides sp., Nitrosospira sp. and Pseudomonas sp. Moreover, 11 methane-utilizing bacterial species were also discovered. The presence of these methane-utilizing bacteria not only might assist the process of denitrification and sulfate-reduction, but also could diminish the emission of the greenhouse gas. The results of this study confirmed that the addition of substrates could affect the groundwater oxidation-reduction state and enhance the bioremediation at the 1,2-DCA-contaminated site. Thus, enhanced in situ bioremediation is a feasible technology for site remediation.

The WBC-2 consortium was characterized using quantitative PCR and analytical techniques to associate growth of dechlorinating bacteria to each step of the 1,1,2,2-Tetrachloroethane (TeCA) degradation pathway. The consortium was found to degrade TeCA through dichloroelimination to trans-1,2-dichloroethene (tDCE), and reductive dehalogenation to Vinyl Chloride (VC) and ethene. Thus the pathway was hypothesized to provide three distinct niches for three genera of dechlorinating bacteria, Dehalobacter, Dehalogenimonas and Dehalococcoides. Using qPCR to track growth over two time course experiments at different inoculum dilutions, the Dehalobacter species showed significant growth on the first step of TeCA dihaloelimination to tDCE Dehalococcoides and Dehalogenimonas species grew on the dechlorination products. The Dehalogenimonas species, a novel non-Dehalococcoides, was found to grow only on tDCE. The Dehalococcoides species also grew on cDCE, less well on tDCE, and on VC.

The WBC-2 consortium was characterized using quantitative PCR and analytical techniques to associate growth of dechlorinating bacteria to each step of the 1,1,2,2-Tetrachloroethane (TeCA) degradation pathway. The consortium was found to degrade TeCA through dichloroelimination to trans-1,2-dichloroethene (tDCE), and reductive dehalogenation to Vinyl Chloride (VC) and ethene. Thus the pathway was hypothesized to provide three distinct niches for three genera of dechlorinating bacteria, Dehalobacter, Dehalogenimonas and Dehalococcoides. Using qPCR to track growth over two time course experiments at different inoculum dilutions, the Dehalobacter species showed significant growth on the first step of TeCA dihaloelimination to tDCE Dehalococcoides and Dehalogenimonas species grew on the dechlorination products. The Dehalogenimonas species, a novel non-Dehalococcoides, was found to grow only on tDCE. The Dehalococcoides species also grew on cDCE, less well on tDCE, and on VC.