Functional characterization of reductive dehalogenases by using blue native polyacrylamide gel electrophoresis.

Dehalococcoides mccartyi strains are obligate organohalide-respiring bacteria harboring multiple distinct reductive dehalogenase (RDase) genes within their genomes. A major challenge is to identify substrates for the enzymes encoded by these RDase genes. We demonstrate an approach that involves blue native polyacrylamide gel electrophoresis (BN-PAGE) followed by enzyme activity assays with gel slices and subsequent identification of proteins in gel slices using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). RDase expression was investigated in cultures of Dehalococcoides mccartyi strain BAV1 and in the KB-1 consortium growing on chlorinated ethenes and 1,2-dichloroethane. In cultures of strain BAV1, BvcA was the only RDase detected, revealing that this enzyme catalyzes the dechlorination not only of vinyl chloride, but also of all dichloroethene isomers and 1,2-dichloroethane. In cultures of consortium KB-1, five distinct Dehalococcoides RDases and one Geobacter RDase were expressed under the conditions tested. Three of the five RDases included orthologs to the previously identified chlorinated ethene-dechlorinating enzymes VcrA, BvcA, and TceA. This study revealed substrate promiscuity for these three enzymes and provides a path forward to further explore the largely unknown RDase protein family.

Stable carbon isotope enrichment factors for cis-1,2-dichloroethene and vinyl chloride reductive dechlorination by Dehalococcoides.

Compound-specific stable isotope analysis (CSIA) is a promising tool for monitoring in situ microbial activity, and enrichment factors (ε values) determined using CSIA can be employed to estimate compound transformation. Although ε values for some dechlorination reactions catalyzed by Dehalococcoides (Dhc) have been reported, reproducibility between independent experiments, variability between different Dhc strains, and congruency between pure and mixed cultures are unknown. In experiments conducted with pure cultures of Dhc sp. strain BAV1, ε values for 1,1-DCE, cis-DCE, trans-DCE, and VC were -5.1, -14.9, -20.8, and -23.2‰, respectively. The ε value for 1,1-DCE dechlorination was 48.9% higher than the value reported in a previous study, but ε values for other chlorinated ethenes were equal between independent experiments. For the dechlorination of cis-DCE and VC by Dhc strains BAV1, FL2, GT, and VS, average ε values were -18.4 and -23.2‰, respectively. cis-DCE and VC ε values determined in pure Dhc cultures with different reductive dehalogenase genes (e.g., vcrA vs bvcA) varied by less than 36.8 and 8.3%, respectively. In the BDI consortium, ε values for cis-DCE and VC dechlorination were -25.3‰ and -19.9‰, 31.6% higher and 15.3% lower, respectively, compared to the average ε value for Dhc pure cultures. As cis-DCE and VC ε values are all within the same order-of-magnitude and fractionation is always measured during Dhc dechlorination, CSIA may be a valuable approach for monitoring in situ cis-DCE and VC reductive dechlorination.

Effects of elevated temperature on Dehalococcoides dechlorination performance and DNA and RNA biomarker abundance.

Coupling thermal treatment with microbial reductive dechlorination is a promising remedy for tetrachloroethene (PCE) and trichloroethene (TCE) contaminated source zones. Laboratory experiments evaluated Dehalococcoides (Dhc) dechlorination performance, viability, and biomarker gene (DNA) and transcript (mRNA) abundances during exposure to elevated temperatures. The PCE-dechlorinating consortia BDI and OW produced ethene when incubated at temperatures of 30 °C, but vinyl chloride (VC) accumulated when cultures were incubated at 35 or 40 °C. Cultures incubated at 40 °C for less than 49 days resumed VC dechlorination following cooling; however, incubation at 45 °C resulted in complete loss of dechlorination activity. Dhc 16S rRNA, bvcA, and vcrA gene abundances in cultures showing complete dechlorination to ethene at 30 °C exceeded those measured in cultures incubated at higher temperatures, consistent with observed dechlorination activities. Conversely, biomarker gene transcript abundances per cell in cultures incubated at 35 and 40 °C were generally at least one order-of-magnitude greater than those measured in ethene-producing cultures incubated at 30 °C. Even in cultures accumulating VC, transcription of the vcrA gene, which is implicated in VC-to-ethene dechlorination, was up-regulated. Temperature stress caused the up-regulation of Dhc reductive dehalogenase gene expression indicating that Dhc gene expression measurements should be interpreted cautiously as Dhc biomarker gene transcript abundances may not correlate with dechlorination activity.

Solution and microbial controls on the formation of reduced U(IV) species.

Reduction of U(VI) to U(IV) as the result of direct or indirect microbial activity is currently being explored for in situ remediation of subsurface U plumes, under the assumption that U(IV) solubility is controlled by the low-solubility mineral uraninite (U(IV)-dioxide). However, recent characterizations of U in sediments from biostimulated field sites, as well as laboratory U(VI) bioreduction studies, report on the formation of U(IV) species that lack the U═O(2)═U coordination of uraninite, suggesting that phases other than uraninite may be controlling U(IV) solubility in environments with complexing surfaces and ligands. To determine the controls on the formation of such nonuraninite U(IV) species, the current work studied the reduction of carbonate-complexed U(VI) by (1) five Gram-positive Desulfitobacterium strains, (2) the Gram-negative bacteria Anaeromyxobacter dehalogenans 2CP-C and Shewanella putrefaciens CN32, and (3) chemically reduced 9,10-anthrahydroquinone-2,6-disulfonate (AH(2)QDS, a soluble reductant). Further, the effects of 0.3 mM dissolved phosphate on U(IV) species formation were explored. Extended X-ray absorption fine structure (EXAFS) spectroscopy analysis demonstrated that the addition of phosphate causes the formation of a nonuraninite, phosphate-complexed U(IV) species, independent of the biological or abiotic mode of U(VI) reduction. In phosphate-free medium, U(VI) reduction by Desulfitobacterium spp. and by AH(2)QDS resulted in nonuraninite, carbonate-complexed U(IV) species, whereas reduction by Anaeromyxobacter or Shewanella yielded nanoparticulate uraninite. These findings suggest that the Gram-positive Desulfitobacterium strains and the Gram-negative Anaeromyxobacter and Shewanella species use distinct mechanisms to reduce U(VI).

Resolution of culture Clostridium bifermentans DPH-1 into two populations, a Clostridium sp. and tetrachloroethene-dechlorinating Desulfitobacterium hafniense strain JH1.

Clostridium bifermentans strain DPH-1 reportedly dechlorinates tetrachloroethene (PCE) to cis-1,2-dichloroethene. Cultivation-based approaches resolved the DPH-1 culture into two populations: a nondechlorinating Clostridium sp. and PCE-dechlorinating Desulfitobacterium hafniense strain JH1. Strain JH1 carries pceA, encoding a PCE reductive dehalogenase, and shares other characteristics with Desulfitobacterium hafniense strain Y51.

Electron donor availability for microbial reductive processes following thermal treatment.

Thermal treatment is capable of removing significant free-phase chlorinated solvent mass while potentially enhancing bioremediation effectiveness by establishing temperature gradients in the perimeter of the source zone and by increasing electron donor availability. The objectives of this study were to determine the potential for enhanced reductive dechlorination activity at the intermediate temperatures that establish in the perimeter of the heated source zone, and to evaluate the effect of electron donor competition on the performance of the microbial reductive dechlorination process. Microcosms, constructed with tetrachloroethene- (PCE-) and trichloroethene- (TCE-) impacted soils from the Great Lakes, IL, and Ft. Lewis, WA, sites were incubated at temperatures of 24, 35, 50, 70, and 95 °C for 4 months. Reductive dechlorination did not occur in microcosms incubated at temperatures above 24 °C even though mesophilic PCE-to-cis-1,2-dichloroethene dechlorinators were present in Ft. Lewis soil suggesting electron donor limitations. Five days after cooling the microcosms to 24 °C and bioaugmentation with the methanogenic, PCE-to-ethene-dechlorinating consortium OW, at least 85% of the initial PCE and TCE were dechlorinated, but dechlorination ceased prior to complete conversion to ethene. Subsequent biostimulation with hydrogen gas mitigated the dechlorination stall, and conversion to ethene resumed. The results of this study demonstrated that temperatures >35 °C inhibit reductive dechlorination activity at the Great Lakes and Ft. Lewis sites, and that the majority of reducing equivalents released from the soil matrix during heat treatment are consumed in methanogenesis rather than reductive dechlorination. These observations suggest that bioaugmenting thermal treatment sites with cultures that do not contain methanogens may allow practitioners to realize enhanced dechlorination activity, a potential benefit of coupling thermal treatment with bioremediation.

U(VI) reduction to mononuclear U(IV) by Desulfitobacterium species.

The bioreduction of U(VI) to U(IV) affects uranium mobility and fate in contaminated subsurface environments and is best understood in Gram-negative model organisms such as Geobacter and Shewanella spp. This study demonstrates that U(VI) reduction is a common trait of Gram-positive Desulfitobacterium spp. Five different Desulfitobacterium isolates reduced 100 microM U(VI) to U(IV) in <10 days, whereas U(VI) remained soluble in abiotic and heat-killed controls. U(VI) reduction in live cultures was confirmed using X-ray absorption near-edge structure (XANES) analysis. Interestingly, although bioreduction of U(VI) is almost always reported to yield the uraninite mineral (UO(2)), extended X-ray absorption fine structure (EXAFS) analysis demonstrated that the U(IV) produced in the Desulfitobacterium cultures was not UO(2). The EXAFS data indicated that the U(IV) product was a phase or mineral composed of mononuclear U(IV) atoms closely surrounded by light element shells. This atomic arrangement likely results from inner-sphere bonds between U(IV) and C/N/O- or P/S-containing ligands, such as carbonate or phosphate. The formation of a distinct U(IV) phase warrants further study because the characteristics of the reduced material affect uranium stability and fate in the contaminated subsurface.

Stable carbon isotope fractionation of 1,2-dichloropropane during dichloroelimination by Dehalococcoides populations.

The isotope fractionation of 1,2-dichloropropane (1,2-D) during dichloroelimination to propene by Dehalococcoides populations was explored in laboratory experiments in order to provide data for the characterization of the fate of 1,2-D in heterogeneous subsurface systems. Compound specific stable carbon isotope analysis (CSIA) was used to determine the bulk enrichment factors (epsilonbulk), reactive position specific enrichment factors (epsilonreactive), and apparent kinetic isotope effect (AKIE) values for 1,2-D dichloroelimination in two distinct Dehalococcoides-containing cultures. The epsilonbulk factors calculated in the two cultures were statistically identical, -10.8 +/- 0.9 and -11.3 +/- 0.8 per thousand, even though the cultures were derived from geographically distinct locations. AKIE values for 1,2-D dichloroelimination assuming stepwise and concerted reaction mechanisms were approximately 1.033 and 1.017, respectively. These values are within the range of previously reported values for dichloroelimination reactions and were equivalent to values reported for biotic 1,2-dichloroethane and abiotic 1,1,2,2,-tetrachloroethane and pentachloroethane dichloroelimination reactions.

Fate of TCE in heated Fort Lewis soil.

This study explores the transformation of trichloroethene (TCE) caused by heating contaminated soil and groundwater samples obtained from the East Gate Disposal Yard (EGDY) located in Fort Lewis, WA. After field samples transferring into glass ampules and introducing 1.5 micromol of TCE, the sealed ampules were incubated at temperatures of 25, 50, and 95 degrees C for periods of up to 95.5 days. Although TCE was completely transformed into cis-1,2-dichloroethene (cis-DCE) after 42 days at 25 degrees C by microbial activity, this transformation was not observed at 50 or 95 degrees C. Chloride levels increased after 42 days at 25 degrees C corresponding to the mass of TCE transformed to cis-DCE, were constant at 50 degrees C, and increased at 95 degrees C yielding a TCE degradation half-life of 1.6-1.9 years. These findings indicate that indigenous microbes contribute to the partial dechlorination of TCE to cis-DCE at temperatures of less than 50 degrees C, whereas interphase mass transfer and physical recovery of TCE will predominate over in situ degradation processes at temperatures of greater than 50 degrees C during thermal treatment at the EGDY site.

Detection and quantification of Geobacter lovleyi strain SZ: implications for bioremediation at tetrachloroethene- and uranium-impacted sites.

Geobacter lovleyi strain SZ reduces hexavalent uranium, U(VI), to U(IV) and is the first member of the metal-reducing Geobacter group capable of using tetrachloroethene (PCE) as a growth-supporting electron acceptor. Direct and nested PCR with specific 16S rRNA gene-targeted primer pairs distinguished strain SZ from other known chlorinated ethene-dechlorinating bacteria and closely related Geobacter isolates, including its closest cultured relative, G. thiogenes. Detection limits for direct and nested PCR were approximately 1 x 10(6) and 1 x 10(4) 16S rRNA gene copies per mul of template DNA, respectively. A quantitative real-time PCR (qPCR) approach increased the sensitivity to as few as 30 16S rRNA gene copies per mul of template DNA but was less specific. Melting curve analysis and comparison of the shapes of amplification plots identified false-positive signals and distinguished strain SZ from G. thiogenes when analyzed separately. These indicators were less reliable when target (strain SZ) DNA and nontarget (G. thiogenes) DNA with high sequence similarity were mixed, indicating that the development of qPCR protocols should not only evaluate specificity but also explore the effects of nontarget DNA on the accuracy of quantification. Application of specific tools detected strain SZ-like amplicons in PCE-dechlorinating consortia, including the bioaugmentation consortium KB-1, and two chlorinated ethene-impacted groundwater samples. Strain SZ-like amplicons were also detected in 13 of 22 groundwater samples following biostimulation at the uranium- and chlorinated solvent-contaminated Integrated Field-Scale Subsurface Research Challenge (IFC) site in Oak Ridge, TN. The numbers of strain SZ-like cells increased from below detection to 2.3 x 10(7) +/- 0.1 x 10(7) per liter groundwater, suggesting that strain SZ-like organisms contribute to contaminant transformation. The G. lovleyi strain SZ-specific tools will be useful for monitoring bioremediation efforts at uranium- and/or chlorinated solvent-impacted sites such as the Oak Ridge IFC site.

Geobacter lovleyi sp. nov. strain SZ, a novel metal-reducing and tetrachloroethene-dechlorinating bacterium.

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 and trichloroethene [cis-DCE], nitrate [ammonium], fumarate [succinate], Fe(III) [Fe(II)], malate [succinate], Mn(IV) [Mn(II)], U(VI) [U(IV)], and elemental sulfur [sulfide]. PCE and soluble Fe(III) (as ferric citrate) were reduced at rates of 56.5 and 164 nmol min(-1) mg of protein(-1), 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 electron-accepting 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.