The 16S rRNA gene provides insufficient information to infer the range of chloroorganic electron acceptors used by different Dehalococcoides organisms. To overcome this limitation and provide enhanced diagnostic tools for growth measurements, site assessment, and bioremediation monitoring, a quantitative real-time PCR (qPCR) approach targeting 16S rRNA genes and three Dehalococcoides reductive dehalogenase (RDase) genes with assigned function (i.e., tceA, bvcA, and vcrA) was designed and evaluated. qPCR standard curves generated for the RDase genes by use of genomic DNA from Dehalococcoides pure cultures correlated with standard curves obtained for both Bacteria-and Dehalococcoides-targeted 16S rRNA genes, suggesting that the RDase genes are useful targets for quantitative assessment of Dehalococcoides organisms. RDase gene probe/primer pairs were specific for the Dehalococcoides strains known to carry the diagnostic RDase gene sequences, and the qPCR method allowed the detection of as few as 1 to 20 and quantification of as few as 50 to 100 tceA, bvcA, or vcrA gene targets per PCR volume. The qPCR approach was applied to dechlorinating enrichment cultures, microcosms, and samples from a contaminated site. In characterized enrichment cultures where known Dehalococcoides strains were enumerated, the sum of the three RDase genes equaled the total Dehalococcoides cell numbers. In site samples and chloroethane-dechlorinating microcosms, the sum of the three RDase genes was much less than that predicted by Dehalococcoides-targeted qPCR, totaling 10 to 30% of the total Dehalococcoides cell numbers. Hence, a large number of Dehalococcoides spp. contain as-yet-unidentified RDase genes, indicating that our current understanding of the dechlorinating Dehalococcoides community is incomplete.Chlorinated solvents are a well-recognized class of groundwater (GW) contaminants (1,7,41,53). Substantial knowledge regarding diverse groups of bacteria that partially dechlorinate tetrachloroethene (PCE) or trichloroethene (TCE) to cis-1,2-dichloroethene (cis-DCE) (5,12,19,30,50) or trans-DCE (13) has been accrued over the past decade. Partial reductive dechlorination contributes to the formation of DCEs and, in some cases, vinyl chloride (VC) (1,26,45). Environmental accumulation of VC is particularly troublesome because this compound is a proven human carcinogen; therefore, incomplete dechlorination of PCE and TCE does not result in detoxification.Recently, Dehalococcoides organisms were discovered as a deeply branching group within the green nonsulfur bacteria (Chloroflexi) (44). This physiologically and phylogenetically distinct bacterial group requires hydrogen as an electron donor and specific chloroorganic compounds as electron acceptors to support their energy metabolism. Dehalococcoides ethenogenes strain 195 was the first isolate described to dechlorinate PCE to VC and ethene (35); however, the last transformation step from VC to ethene occurred slowly in a cometabolic process (34). Similarly, metabolic dechlorination of TCE...

A strictly anaerobic bacterium was isolated from tetrachloroethene (PCE)-to-ethene dechlorinating microcosms established with river sediment without prior exposure to chlorinated solvents. The isolation procedure included the addition of 2-bromoethanesulfonate to select against methanogenic archaea, >50 consecutive 1-2% (v/v) transfers to reduced mineral salts medium amended with trichloroethene (TCE), acetate, and hydrogen, the addition of ampicillin, and the dilution-to-extinction principle. Culture-dependent and 16S rRNA gene-targeted approaches suggested culture purity. Microscopic examination revealed a homogeneous culture of an organism with a distinct, disc-shaped morphology. The isolate shared >99% 16S rRNA gene sequence similarity with members of the Pinellas group of the Dehalococcoides cluster, and was designated Dehalococcoides sp. strain FL2. Strain FL2 could be propagated with TCE, cis-1,2-dichloroethene (cis-DCE), or trans-DCE as the electron acceptors, acetate as the carbon source, and hydrogen as the electron donor in defined, completely synthetic medium. No other growth-supporting redox couples were identified. Trichloroethene, cis-DCE and trans-DCE were dechlorinated at rates of 27.5, 30.4 and 18.8 micromol l-1 day-1 respectively. Quantitative real-time polymerase chain reaction (PCR) with a fluorescently labelled linear hybridization probe confirmed growth with these electron acceptors, and suggested that strain FL2 captures energy from both the TCE-to-cis-DCE and 1,2-DCE-to-VC dechlorination steps. Tetrachloroethene and vinyl chloride (VC) were slowly and cometabolically dechlorinated in the presence of a growth-supporting chloroethene, but ethene formation was incomplete, even after prolonged incubation. At room temperature, strain FL2 grew with a doubling time of 2.4 days, and yielded 166.1+/-10.2 mg of protein per mole of chloride released. In the presence of excess electron acceptor, strain FL2 consumed hydrogen to a concentration of 0.061+/-0.016 nM. Dechlorination ceased following the addition of 0.5 mM sulfite, whereas sulfate (10 mM) and nitrate (5 mM) had no inhibitory effects.

A novel Dehalococcoides isolate capable of metabolic trichloroethene (TCE)-to-ethene reductive dechlorination was obtained from contaminated aquifer material. Growth studies and 16S rRNA gene-targeted analyses suggested culture purity; however, the careful quantitative analysis of Dehalococcoides 16S rRNA gene and chloroethene reductive dehalogenase gene (i.e., vcrA, tceA, and bvcA) copy numbers revealed that the culture consisted of multiple, distinct Dehalococcoides organisms. Subsequent transfers, along with quantitative PCR monitoring, yielded isolate GT, possessing only vcrA. These findings suggest that commonly used qualitative 16S rRNA gene-based procedures are insufficient to verify purity of Dehalococcoides cultures. Phylogenetic analysis revealed that strain GT is affiliated with the Pinellas group of the Dehalococcoides cluster and shares 100% 16S rRNA gene sequence identity with two other Dehalococcoides isolates, strain FL2 and strain CBDB1. The new isolate is distinct, as it respires the priority pollutants TCE, cis-1,2-dichloroethene (cis-DCE), 1,1-dichloroethene (1,1-DCE), and vinyl chloride (VC), thereby producing innocuous ethene and inorganic chloride. Strain GT dechlorinated TCE, cis-DCE, 1,1-DCE, and VC to ethene at rates up to 40, 41, 62, and 127 mol liter ؊1 day ؊1 , respectively, but failed to dechlorinate PCE. Hydrogen was the required electron donor, which was depleted to a consumption threshold concentration of 0.76 ؎ 0.13 nM with VC as the electron acceptor. In contrast to the known TCE dechlorinating isolates, strain GT dechlorinated TCE to ethene with very little formation of chlorinated intermediates, suggesting that this type of organism avoids the commonly observed accumulation of cis-DCE and VC during TCE-to-ethene dechlorination.Chlorinated ethenes are pervasive groundwater contaminants resulting from extensive usage, improper disposal, and accidental spills, and the incomplete microbial dechlorination of tetrachloroethene (PCE) and trichloroethene (TCE) leads to dichloroethene and vinyl chloride (VG) accumulation. A breakthrough in the anaerobic treatment of chloroethene-contaminated sites was the discovery of bacteria that use chloroorganic compounds as electron acceptors to drive their energy metabolism. This metabolic reductive dechlorination process, also known as (de)chlororespiration, is a focus of current bioremediation approaches to contain or remediate chloroethene plumes.Numerous bacterial isolates that reductively dechlorinate chloroethenes have been described previously (26); however, no single organism has the ability to couple energy generation with each reductive dechlorination step leading from PCE to ethene. The majority of isolates dechlorinate PCE to cis-1,2-dichloroethene (cis-DCE), and the ability to respire PCE to cis-DCE is distributed among phylogenetic groups and includes members of the classes Deltaproteobacteria, Gammaproteobacteria, and Firmicutes (14). Bacteria with the ability for incomplete dechlorination of PCE to cis-DCE are often present at co...

A bacterial isolate, designated strain SZ, was obtained from noncontaminated creek sediment microcosms based on its ability to derive energy from acetate oxidation coupled to tetrachloroethene (PCE)-to-cis-1,2-dichloroethene (cis-DCE) dechlorination (i.e., chlororespiration). Hydrogen and pyruvate served as alternate electron donors for strain SZ, and the range of electron acceptors included (reduced products are given in brackets) PCE , respectively, with acetate as the electron donor. Alternate electron acceptors, such as U(VI) and nitrate, did not inhibit PCE dechlorination and were consumed concomitantly. With PCE, Fe(III) (as ferric citrate), and nitrate as electron acceptors, H 2 was consumed to threshold concentrations of 0.08 ؎ 0.03 nM, 0.16 ؎ 0.07 nM, and 0.5 ؎ 0.06 nM, respectively, and acetate was consumed to 3.0 ؎ 2.1 nM, 1.2 ؎ 0.5 nM, and 3.6 ؎ 0.25 nM, respectively. Apparently, electron acceptor-specific acetate consumption threshold concentrations exist, suggesting that similar to the hydrogen threshold model, the measurement of acetate threshold concentrations offers an additional diagnostic tool to delineate terminal electronaccepting processes in anaerobic subsurface environments. Genetic and phenotypic analyses classify strain SZ as the type strain of the new species, Geobacter lovleyi sp. nov., with Geobacter (formerly Trichlorobacter) thiogenes as the closest relative. Furthermore, the analysis of 16S rRNA gene sequences recovered from PCE-dechlorinating consortia and chloroethene-contaminated subsurface environments suggests that Geobacter lovleyi belongs to a distinct, dechlorinating clade within the metal-reducing Geobacter group. Substrate versatility, consumption of electron donors to low threshold concentrations, and simultaneous reduction of electron acceptors suggest that strain SZ-type organisms have desirable characteristics for bioremediation applications.Environmental pollutants, such as chlorinated solvents, toxic metals, and radionuclides (e.g., uranium), are strictly regulated due to their negative effects on ecosystem function and human health. Tetrachloroethene (PCE) and trichloroethene (TCE) are frequently used as solvents and degreasing agents. As a result of extensive usage, improper disposal, and accidental spills, PCE and TCE became widely distributed, pervasive groundwater contaminants. Uranium was released in significant amounts from nuclear weapon complexes managed by the U.S. Department of Energy (DOE) during the Cold War arms race. It was estimated that more than 80% of DOE sites have radionuclide contamination and at least 27% have volatile organic hydrocarbons as cocontaminants (47).The considerable knowledge that has accrued on the fate of specific contaminants and the bacteria involved in the transformation and degradation pathways has led to successful bioremediation field studies. For instance, acetate additions to stimulate dissimilatory metal-reducing organisms promoted reduction of soluble U(VI) to insoluble U(IV) and contributed to plume containment (2,...

Two tetrachlorethene (PCE)-dechlorinating populations, designated strains BB1 and BRS1, were isolated from pristine river sediment and chloroethene-contaminated aquifer material, respectively. PCE-to-cis-1,2-dichloroethene-dechlorinating activity could be transferred in defined basal salts medium with acetate as the electron donor and PCE as the electron acceptor. Taxonomic analysis based on 16S rRNA gene sequencing placed both isolates within the Desulfuromonas cluster in the ␦ subdivision of the Proteobacteria. PCE was dechlorinated at rates of at least 139 nmol min ؊1 mg of protein ؊1 at pH values between 7.0 and 7.5 and temperatures between 25 and 30°C. Dechlorination also occurred at 10°C. The electron donors that supported dechlorination included acetate, lactate, pyruvate, succinate, malate, and fumarate but not hydrogen, formate, ethanol, propionate, or sulfide. Growth occurred with malate or fumarate alone, whereas oxidation of the other electron donors depended strictly on the presence of fumarate, malate, ferric iron, sulfur, PCE, or TCE as an electron acceptor. Nitrate, sulfate, sulfite, thiosulfate, and other chlorinated compounds were not used as electron acceptors. Sulfite had a strong inhibitory effect on growth and dechlorination. Alternate electron acceptors (e.g., fumarate or ferric iron) did not inhibit PCE dechlorination and were consumed concomitantly. The putative fumarate, PCE, and ferric iron reductases were induced by their respective substrates and were not constitutively present. Sulfide was required for growth. Both strains tolerated high concentrations of PCE, and dechlorination occurred in the presence of free-phase PCE (dense non-aqueous-phase liquids). Repeated growth with acetate and fumarate as substrates yielded a BB1 variant that had lost the ability to dechlorinate PCE. Due to the 16S rRNA gene sequence differences with the closest relatives and the unique phenotypic characteristics, we propose that the new isolates are members of a new species, Desulfuromonas michiganensis, within the Desulfuromonas cluster of the Geobacteraceae.The first reported anthropogenic production of tetrachloroethene (PCE) dates back to 1821, when Faraday produced PCE by thermal decomposition of hexachloroethane (15). Starting at the beginning of the 20th century, increasing amounts of PCE and trichloroethene (TCE) were manufactured due to the extensive use of these compounds in industry, in the military, and in private households, mainly as nonflammable solvents (summarized in reference 9). This widespread use, along with careless handling and storage, made chlorinated ethenes abundant groundwater pollutants. Often, PCE and TCE contamination coincided with spills of other organic compounds, such as crude oil constituents, other solvents (often seen at industrial and military sites), or starch (at dry cleaning operations). Aerobic microorganisms rapidly degrade the nonchlorinated contaminants, thereby depleting oxygen. Under anaerobic conditions PCE and TCE can be used as alternate growth-supportin...