Distribution of organohalide-respiring bacteria between solid and aqueous phases.

Contemporary microbial monitoring of aquifers relies on groundwater samples to enumerate nonattached cells of interest. One-dimensional column studies quantified the distribution of bacterial cells in solid and the aqueous phases as a function of microbial species, growth substrate availability and porous medium (i.e., Appling soil versus Federal Fine Ottawa sand with 0.75% and 0.01% [w/w] organic carbon, respectively). Without supplied growth substrates, effluent from columns inoculated with the tetrachloroethene- (PCE-) to-ethene-dechlorinating bacterial consortium BDI-SZ containing Dehalococcoides mccartyi (Dhc) strains and Geobacter lovleyi strain SZ (GeoSZ), or inoculated with Anaeromyxobacter dehalogenans strain W (AdehalW), captured 94-96, 81-99, and 73-84% of the Dhc, GeoSZ, and AdehalW cells, respectively. Cell retention was organism-specific and increased in the order Dhc < GeoSZ < AdehalW. When amended with 10 mM lactate and 0.11 mM PCE, aqueous samples accounted for 1.3-27 and 0.02-22% of the total Dhc and GeoSZ biomass, respectively. In Appling soil, up to three orders-of-magnitude more cells were associated with the solid phase, and attachment rate coefficients (katt) were consistently greater compared to Federal Fine sand. Cell-solid interaction energies ranged from -2.5 to 787 kT and were consistent with organism-specific deposition behavior, where GeoSZ and AdehalW exhibited greater attachment than Dhc cells. The observed disparities in microbial cell distributions between the aqueous and solid phases imply that groundwater analysis can underestimate the total cell abundance in the aquifer by orders-of-magnitude under conditions of growth and in porous media with elevated organic carbon content. The implications of these findings for monitoring chlorinated solvent sites are discussed.

Design and application of an internal amplification control to improve Dehalococcoides mccartyi 16S rRNA gene enumeration by qPCR.

Dehalococcoides mccartyi (Dhc) strains are keystone bacteria for reductive dechlorination of chlorinated ethenes to nontoxic ethene in contaminated aquifers. Enumeration of Dhc biomarker genes using quantitative real-time PCR (qPCR) in groundwater is a key component of site assessment and bioremediation monitoring. Unfortunately, standardized qPCR procedures that recognize impaired measurements due to PCR inhibition, low template DNA concentrations, or analytical error are not available, thus limiting confidence in qPCR data. To improve contemporary approaches for enumerating Dhc in environmental samples, multiplex qPCR assays were designed to quantify the Dhc 16S rRNA gene and one of two different internal amplification controls (IACs): a modified Dhc 16S rRNA gene fragment (Dhc*) and the firefly luciferase gene luc. The Dhc* IAC exhibited competitive inhibition in qPCR with the Dhc 16S rRNA gene template when the ratio of either target was 100-fold greater than the other target. A multiplex qPCR assay with the luc IAC avoided competitive inhibition and accurately quantified Dhc abundances ranging from ∼10 to 10(7) 16S rRNA gene copies per reaction. The addition of ∼10(6) E. coli luc IAC to simulated groundwater amended with the Dhc-containing consortium KB-1 yielded reproducible luc counts after DNA extraction and multiplex qPCR enumeration. The application of the luc IAC assay improved Dhc biomarker gene quantification from simulated groundwater samples and is a valuable approach for "ground truthing" qPCR data obtained in different laboratories, thus reducing ambiguity associated with qPCR enumeration and reproducibility.

Chlorinated ethene source remediation: lessons learned.

Chlorinated solvents such as trichloroethene (TCE) and tetrachloroethene (PCE) are widespread groundwater contaminants often released as dense nonaqueous phase liquids (DNAPLs). These contaminants are difficult to remediate, particularly their source zones. This review summarizes the progress made in improving DNAPL source zone remediation over the past decade, and is structured to highlight the important practical lessons learned for improving DNAPL source zone remediation. Experience has shown that complete restoration is rare, and alternative metrics such as mass discharge are often useful for assessing the performance of partial restoration efforts. Experience also has shown that different technologies are needed for different times and locations, and that deliberately combining technologies may improve overall remedy performance. Several injection-based technologies are capable of removing a large fraction of the total contaminant mass, and reducing groundwater concentrations and mass discharge by 1 to 2 orders of magnitude. Thermal treatment can remove even more mass, but even these technologies generally leave some contamination in place. Research on better delivery techniques and characterization technologies will likely improve treatment, but managers should anticipate that source treatment will leave some contamination in place that will require future management.

Liquid-liquid mass transfer of partitioning electron donors in chlorinated solvent source zones.

A combination of batch and column experiments evaluated the mass transfer of two candidate partitioning electron donors (PEDs), n-hexanol (nHex) and n-butyl acetate (nBA), for enhanced bioremediation of trichloroethene (TCE)-dense nonaqueous phase liquid (DNAPL). Completely mixed batch reactor experiments yielded equilibrium TCE-DNAPL and water partition coefficients (KNW) for nHex and nBA of 21.7 ± 0.27 and 330.43 ± 6.7, respectively, over a range of initial PED concentrations up to the aqueous solubility limit of ca. 5000 mg/L. First-order liquid-liquid mass transfer rates determined in batch reactors with nBA or nHex concentrations near the aqueous solubility were 0.22 min(-1) and 0.11 min(-1), respectively. Liquid-liquid mass transfer under dynamic flow conditions was assessed in one-dimensional (1-D) abiotic columns packed with Federal Fine Ottawa sand containing a uniform distribution of residual TCE-DNAPL. Following pulse injection of PED solutions at pore-water velocities (vp) ranging from 1.2 to 6.0 m/day, effluent concentration measurements demonstrated that both nHex and nBA partitioned strongly into residual TCE-DNAPL with maximum effluent levels not exceeding 35% and 7%, respectively, of the applied concentrations of 4000 to 5000 mg/L. PEDs persisted at effluent concentrations above 5 mg/L for up to 16 and 80 pore volumes for nHex and nBA, respectively. Mathematical simulations yielded KNW values ranging from 44.7 to 48.2 and 247 to 291 and liquid-liquid mass transfer rates of 0.01 to 0.03 min(-1) and 0.001 to 0.006 min(-1) for nHex and nBA, respectively. The observed TCE-DNAPL and water mass transfer behavior suggests that a single PED injection can persist in a treated source zone for prolonged time periods, thereby reducing the need for, or frequency of, repeated electron donor injections to support bacteria that derive reducing equivalents for TCE reductive dechlorination from PED fermentation.

Hydraulics of recirculating well pairs for ground water remediation.

Recirculating well pairs are a proven means of implementing bioremediation and may also be useful for applying other in situ ground water remediation technologies. A bromide tracer test was performed to characterize the hydraulic performance of a recirculating well pair installed at Moffett Field, California. In particular, we estimate two important properties of the recirculating well pair: (1) the fraction of captured water that is recycled between the wells, and (2) the travel-time distribution of ground water in the induced zone of recirculation. We also develop theoretical estimates of these two properties and demonstrate they depend upon a dimensionless pumping rate, denoted xi. The bromide breakthrough curve predicted from theory agrees well with that determined experimentally at Moffett Field. The minimum travel time between the wells is denoted t(min). In theory, t(min) depends inversely on Q, the pumping rate in the recirculating wells, and is proportional to a2, the square of the distance between the wells. Both the experimental and theoretical travel-time distributions indicate that at least half the recirculating water travels between the wells along fast flowpaths (travel time < 2*t(min)). Therefore, when designing recirculating well pairs, engineers should ensure that t(min) will be sufficiently high to allow biologically mediated reactions (or other in situ remediation processes) sufficient time to proceed.

Comparing on-site to off-site biomass collection for Dehalococcoides biomarker gene quantification to predict in situ chlorinated ethene detoxification potential.

Biostimulation and bioaugmentation have emerged as constructive remedies for chlorinated ethene-contaminated aquifers, and a link between Dehalococcoides (Dhc) bacteria and chlorinated ethene detoxification has been established. To quantify Dhc biomarker genes, groundwater samples are shipped to analytical laboratories where biomass is collected on membrane filters by vacuum filtration for DNA extraction and quantitative real-time PCR analysis. This common practice was compared with a straightforward, on-site filtration approach to Sterivex cartridges. In initial laboratory studies with groundwater amended with known amounts of Dhc target cells, Sterivex cartridges yielded one-third of the total DNA and 9-18% of the Dhc biomarker gene copies compared with vacuum filtration. Upon optimization, DNA yields increased to 94 +/- 38% (+/-SD, n = 10), and quantification of Dhc biomarker genes exceeded the values obtained with the vacuum filtration procedure up to 5-fold. Both methods generated reproducible results when volumes containing >10(4) total Dhc target gene copies were collected. Analysis of on-site and off-site biomass collection procedures corroborated the applicability of the Sterivex cartridge for Dhc biomarker quantification in groundwater. Ethene formation coincided with Dhc cell titers of >2 x 10(6) L(-1) and high (i.e., >10(5)) abundance of the vinyl chloride reductive dehalogenase genes vcrA and/or bvcA; however, high Dhc cell titers alone were insufficient to predict ethene formation. Further, ethene formation occurred at sites with high Dhc cell titers but low or no detectable vcrA or bvcA genes, suggesting that other, not yet identified vinyl chloride reductive dehalogenases contribute to ethene formation. On-site biomass collection with Sterivex cartridges avoids problems associated with shipping groundwater and has broad applicability for biomarker monitoring in aqueous samples.

Implementing heterogeneous catalytic dechlorination technology for remediating TCE-contaminated groundwater.

To transition catalytic reductive dechlorination (CRD) into practice, it is necessary to demonstrate the effectiveness, robustness, and economic competitiveness of CRD-based treatment systems. A CRD system scaled up from previous laboratory studies was tested for remediating groundwater contaminated with 500-1200 microg L(-1) trichloroethylene (TCE) at Edwards Air Force Base (AFB), California. Groundwater was pumped from a treatment well at 2 gal min(-1), amended with hydrogen to 0.35 mg L(-1) and contacted for 2.3 min with 20 kg eggshell-coated Pd on alumina beads (2% Pd by wt) packed in a fixed-bed reactor, and then returned to the aquifer. Operation was continuous for 23 h followed a 1 h regeneration cycle. After regeneration, TCE removal was 99.8% for 4 to 9 h and then declined to 98.3% due to catalyst deactivation. The observed catalyst deactivation was tentatively attributed to formation of sulfidic compounds; modeling of catalyst deactivation kinetics suggests the presence of sulfidic species equivalent to 2-4 mg L(-1) hydrogen sulfide in the reactor water. Over the more than 100 day demonstration period, TCE concentrations in the treated groundwater were reduced by >99% to an average concentration of 4.1 microg L(-1). The results demonstrate CRD as a viable treatment alternative technically and economically competitive with activated carbon adsorption and other conventional physicochemical treatmenttechnologies.