Quantitative PCR (qPCR) was coupled with reverse transcription (RT) to analyze both gene copy numbers and transcripts of the 16S rRNA gene and three reductive dehalogenase (RDase) genes (tceA, vcrA, and bvcA) as biomarkers of "Dehalococcoides" spp. in the groundwater of a trichloroethene-dense nonaqueous-phase liquid site at Fort Lewis, WA, that was sequentially subjected to biostimulation and bioaugmentation. Dehalococcoides cells carrying the tceA, vcrA, and bvcA genes were indigenous to the site. The sum of the three identified RDase gene copy numbers closely correlated to 16S rRNA gene copy numbers throughout the biostimulation and bioaugmentation activity, suggesting that these RDase genes represented the major Dehalococcoides metabolic functions at this site. Biomarker quantification revealed an overall increase of more than 3 orders of magnitude in the total Dehalococcoides population through the 1-year monitoring period (spanning biostimulation and

Sodium lactate additions to a trichloroethene (TCE) residual source area in deep, fractured basalt at a U.S. Department of Energy site have resulted in the enrichment of the indigenous microbial community, the complete dechlorination of nearly all aqueous-phase TCE to ethene, and the continued depletion of the residual source since 1999. The bacterial and archaeal consortia in groundwater obtained from the residual source were assessed by using PCR-amplified 16S rRNA genes. A clone library of bacterial amplicons was predominated by those from members of the class Clostridia (57 of 93 clones), of which a phylotype most similar to that of the homoacetogen Acetobacterium sp. strain HAAP-1 was most abundant (32 of 93 clones). The remaining Bacteria consisted of phylotypes affiliated with Sphingobacteria, Bacteroides, Spirochaetes, Mollicutes, and Proteobacteria and candidate divisions OP11 and OP3. The two proteobacterial phylotypes were most similar to those of the known dechlorinators Trichlorobacter thiogenes and Sulfurospirillum multivorans. Although not represented by the bacterial clones generated with broad-specificity bacterial primers, a Dehalococcoides-like phylotype was identified with genus-specific primers. Only four distinct phylotypes were detected in the groundwater archaeal library, including predominantly a clone affiliated with the strictly acetoclastic methanogen Methanosaeta concilii (24 of 43 clones). A mixed culture that completely dechlorinates TCE to ethene was enriched from this groundwater, and both communities were characterized by terminal restriction fragment length polymorphism (T-RFLP). According to T-RFLP, the laboratory enrichment community was less diverse overall than the groundwater community, with 22 unique phylotypes as opposed to 43 and a higher percentage of Clostridia, including the Acetobacterium population. Bioreactor archaeal structure was very similar to that of the groundwater community, suggesting that methane is generated primarily via the acetoclastic pathway, using acetate generated by lactate fermentation and acetogenesis in both systems.Inadequate disposal practices of chlorinated solvents have resulted in the widespread contamination of groundwater with trichloroethene (TCE), a known toxin and suspected carcinogen. In situ biostimulation via anaerobic microbial reductive dechlorination is an attractive remediation strategy for TCE due to low operation and maintenance costs and minimal secondary-waste production relative to traditional extraction methods. Direct addition of nutrients such as electron donors to the contaminated subsurface can stimulate the activity of indigenous microbial communities. After oxygen is consumed, anaerobic populations reduce alternative electron acceptors such as nitrate, ferric iron, sulfate, and carbon dioxide (10). Chloroethenes can also be used as terminal electron acceptors (37,41,48,49), a process known as dehalorespiration, whereby hydrogen atoms sequentially replace chlorine atoms, leading to detoxification of the chloroethene...

Approximately 190 kg of 2 μm‐diameter zero‐valent iron (ZVI) particles were injected into a test zone in the top 2 m of an unconfined aquifer within a trichloroethene (TCE) source area. A shear‐thinning fluid was used to enhance ZVI delivery in the subsurface to a radial distance of up to 4 m from a single injection well. The ZVI particles were mixed in‐line with the injection water, shear‐thinning fluid, and a low concentration of surfactant. ZVI was observed at each of the seven monitoring wells within the targeted radius of influence during injection. Additionally, all wells within the targeted zone showed low TCE concentrations and primarily dechlorination products present 44 d after injection. These results suggest that ZVI can be directly injected into an aquifer with shear‐thinning fluids to induce dechlorination and extends the applicability of ZVI to situations where other emplacement methods may not be viable.

This study compares three molecular techniques, including terminal restriction fragment length polymorphism (T-RFLP), RFLP analysis with clone sequencing, and quantitative PCR (Q-PCR) for surveying differences in microbial communities at two contaminated field sites that exhibit dissimilar chlorinated solvent degradation activities. At the Idaho National Engineering and Environmental Laboratory (INEEL), trichloroethene (TCE) was completely converted to ethene during biostimulation with lactate. At Seal Beach, California, perchloroethene (PCE) was degraded only to cis-dichloroethene (cDCE) during biostimulation but was degraded to ethene after bioaugmentation with a dechlorinating culture containing Dehalococcoides strains. T-RFLP analysis showed that microbial community composition differed significantly between the two sites, but was similar within each site among wells that had low or no electron donor exposure. Analysis of INEEL clone libraries by RFLP with clone sequencing revealed a complex microbial population but did not identify any Dehalococcoides strains. Q-PCR targeting the 16S rRNA gene of Dehalococcoides strains - known for their unique capability to dechlorinate solvents completely to ethene - revealed a significant population at INEEL, but no detectable population at Seal Beach prior to bioaugmentation. Detection of Dehalococcoides by Q-PCR correlated with observed dechlorination activity and ethene production at both sites. Q-PCR showed that Dehalococcoides was present in even the pristine well at INEEL, suggesting that the difference in dechlorination ability at the two sites was due to the initial absence of this genus at Seal Beach. Of the techniques tested, Q-PCR quantification of specific dechlorinating species provided the most effective and direct prediction of community dechlorinating potential.

The impact of fecal contamination from human and agricultural animal waste on water quality is a major public health concern. Identification of the dominant source(s) of fecal pollution in a watershed is necessary for assessing the safety of recreational water and protecting water resources. A field study was conducted using quantitative PCR (qPCR) for the 16S rRNA gene of Brevibacterium sp. LA35 to track feces-contaminated poultry litter in environmental samples. Based on sensitivity and specificity characteristics of the qPCR method, the Bayesian conditional probability that detection of the LA35 marker gene in a water sample represented a true-positive result was 93%. The marker's covariance with fecal indicator bacteria (FIB) and metals associated with poultry litter was also assessed in litter, runoff, surface water, and groundwater samples. LA35 was detected in water and soil samples collected throughout the watershed, and its concentration covaried with concentrations of Escherichia coli, enterococci, As, Cu, P, and Zn. Significantly greater concentrations of FIB, As, Cu, P, and Zn were observed in edge-of-field runoff samples in which LA35 was detected, compared to samples in which it was not detected. Furthermore, As, Cu, P, and Zn concentrations covaried in environmental samples in which LA35 was detected and typically did not in samples in which the marker gene was not detected. The covariance of the poultry-specific LA35 marker gene with these known contaminants from poultry feces provides further evidence that it is a useful tool for assessing the impact of poultry-derived fecal pollution in environmental waters.

A high-density phylogenetic microarray (PhyloChip) was applied to track bacterial and archaeal populations through different phases of remediation at Ft. Lewis, WA, a trichloroethene (TCE)-contaminated groundwater site. Biostimulation with whey, and bioaugmentation with a Dehalococcoides-containing enrichment culture were strategies implemented to enhance dechlorination. As a measure of species richness, over 1300 operational taxonomic units (OTUs) were detected in DNA from groundwater samples extracted during different stages of treatment and in the bioaugmentation culture. In order to determine active members within the community, 16S rRNA from samples were analyzed by microarray and ~600 OTUs identified. A cDNA clone library of the expressed 16S rRNA corroborated the observed diversity and activity of some of the phyla. Principle component analysis of the treatment plot samples revealed that the microbial populations were constantly changing during the course of the study. Dynamic analysis of the archaeal population showed significant increases in methanogens at the later stages of treatment that correlated with increases in methane concentrations of over two orders of magnitude. Overall, the PhyloChip analyses in this study have provided insights into the microbial ecology and population dynamics at the TCE-contaminated field site useful for understanding the in situ reductive dechlorination processes.

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The effectiveness of whey as an electron donor that stimulates bioremediation and enhances dissolution of trichloroethene (TCE) dense nonaqueous phase liquid (DNAPL) was investigated. Laboratory experiments were conducted to evaluate increased mass transfer of TCE from the DNAPL to the aqueous phase in abiotic batch microcosms amended with several concentrations of whey, and in abiotic columns using high-and low-concentration whey mixtures. The effective solubility of TCE was a factor of 6 higher in microcosms amended with 10% w/w whey compared to 1% w/w whey or nanopure water. Increased aqueous-phase concentrations of TCE were a function of both the concentration of whey and time. In the columns, a factor of 5 increase in TCE concentrations was observed in the effluent during amendment with 10% w/w whey compared to potable water and 1% w/w whey. A field study involving three whey injections was performed at a site that had been actively undergoing bioremediation in a residual source area using lactate for 5 years. Results of the field test show a factor of 3 increase in total molar concentrations of chloroethenes and ethene following injection of 10% w/w whey compared to 5% lactate. In addition, complete dechlorination of TCE to ethene continued.