Electrokinetic-enhanced bioaugmentation for remediation of chlorinated solvents contaminated clay.

Successful bioremediation of contaminated soils is controlled by the ability to deliver bioremediation additives, such as bacteria and/or nutrients, to the contaminated zone. Because hydraulic advection is not practical for delivery in clays, electrokinetic (EK) injection is an alternative for efficient and uniform delivery of bioremediation additive into low-permeability soil and heterogeneous deposits. EK-enhanced bioaugmentation for remediation of clays contaminated with chlorinated solvents is evaluated. Dehalococcoides (Dhc) bacterial strain and lactate ions are uniformly injected in contaminated clay and complete dechlorination of chlorinated ethene is observed in laboratory experiments. The injected bacteria can survive, grow, and promote effective dechlorination under EK conditions and after EK application. The distribution of Dhc within the clay suggests that electrokinetic transport of Dhc is primarily driven by electroosmosis. In addition to biodegradation due to bioaugmentation of Dhc, an EK-driven transport of chlorinated ethenes is observed in the clay, which accelerates cleanup of chlorinated ethenes from the anode side. Compared with conventional advection-based delivery, EK injection is significantly more effective for establishing microbial reductive dechlorination capacity in low-permeability soils.

Field evaluation of biological enhanced reductive dechlorination of chloroethenes in clayey till.

The performance of enhanced reductive dechlorination (ERD) for in situ remediation of cis-1,2-dichloroethene (cDCE) and vinyl chloride in clayey till was investigated in a pilot test. A dilute groundwater solution containing emulsified soybean oil and Dehalococcoides bacteria was injected into a sand-filled hydraulic fracture. Fermentation of the ERD solution caused the establishment of a dechlorinating bioactive zone in the fracture within 1 month of injection. By 148 days, all the cDCE in the fracture was dechlorinated to ethene. Analysis of a clay core from Day 150 indicated that electron donor and fermentation products diffused from the fracture at least 10 cm into clay and that stimulated dechlorination occurred in the clay in the presence of Dehalococcoides (7.9.10(4) cells g(-1)). Comparison of chloroethene profiles in the Day 150 core to modeled diffusion profiles indicated degradation occurred in a bioactive zone extending approximately 5 to 6 cm into the clay matrix. These data suggest that a bioactive zone established in a sand-filled fracture can expand into the adjacent clayey till matrix and facilitate mass transfer from the matrix to the bioactive zone. These findings offer promise for ERD and support further development of methods for deploying ERD in clayey till and other low-permeability deposits.

Concurrent ethene generation and growth of Dehalococcoides containing vinyl chloride reductive dehalogenase genes during an enhanced reductive dechlorination field demonstration.

Dehalococcoides bacteria that produce catabolic vinyl chloride (VC) reductive dehalogenase enzymes have been implicated as a requirement for successful biological dechlorination of VC to ethene in groundwater systems. Therefore, the functional genes in Dehalococcoides that produce VC reductase (e.g., vcrA) may be important biomarkers for predicting and monitoring the performance of bioremediation systems treating chloroethenes via enhanced reductive dechlorination (ERD). As part of an ERD field demonstration, 45 groundwater samples were analyzed for vcrA using quantitative PCR. The demonstration delivered lactate continuously via groundwater recirculation over 201 days to an aquifer contaminated with cis-1,2-dichloroethene (cDCE, approximately 150 microM) and VC (approximately 80 microM). Ethene (approximately 4 microM) and Dehalococcoides containing vcrA (average concentration of 4 x 10(3) gene copies L(-1)) were detected a priori in the demonstration plot; however, aquifer materials in a bench treatability test were able to dechlorinate cDCE with only a 4-month lag period. Given the short (7-month) schedule for the field demonstration, the field plot was bioaugmented on Day 69 with a mixed culture (KB-1) that included Dehalococcoides containing vcrA. Stimulated ethene generation commenced within four weeks of donor addition. Ethene concentrations increased until Day 145, and reached maximum concentrations of 10-25 microM. Concentrations of vcrA increased concurrently with ethene production until Day 145, and plateaued thereafter at 10(7) to 10(8) gene copies L(-1). These results indicate simultaneous growth of Dehalococcoides containing vcrA and ethene generation in an ERD field application. The quantitative increase in concentrations of Dehalococcoides containing vcrA at this site provides further evidence that the vcrA gene is an effective biomarker for field-scale ERD systems.

Perchlorate-reducing microorganisms isolated from contaminated sites.

An extensive microcosm survey of perchlorate-contaminated sites was undertaken to assess the ability of indigenous microorganisms to degrade perchlorate. Samples from 12 contaminated sites and from one pristine location were analysed. Perchlorate was degraded to below detection limit in all electron donor-amended microcosms. Perchlorate-reducing microorganisms (PRMs) were numerous at most of these sites. Sixteen distinct PRMs were isolated that were phylogenetically related to either Dechloromonas in the Beta Proteobacteria (9/16 isolates) or to Azospirillum in the Alpha Proteobacteria (7/16 isolates). The majority of previously isolated PRMs are in the Beta Proteobacteria related to Dechloromonas or Dechlorosoma. This study indicates that PRMs of the genus Azospirillum may be more prevalent at contaminated sites than the current record of isolates suggests. Cell yields, electron donor to perchlorate ratios and maximum specific growth rates were similar among the isolates and similar to the few previously published values. However, the Monod half-saturation constants for perchlorate for the two Azospirillum isolates characterized were lower than those measured for other genera, suggesting that they may be more effective at low concentrations of perchlorate. These results extend the current understanding of PRMs from diverse environments and provide added confidence that microbial perchlorate reduction is ubiquitous, even at highly contaminated sites, and can be harnessed effectively for bioremediation.

Comparison of anaerobic dechlorinating enrichment cultures maintained on tetrachloroethene, trichloroethene, cis-dichloroethene and vinyl chloride.

An anaerobic mixed microbial culture was enriched from soil and groundwater taken from a site contaminated with trichloroethene (TCE). This enrichment culture was divided into four subcultures amended separately with either perchloroethene (PCE), TCE, cis-dichloroethene (cDCE) or vinyl chloride (VC). In each of the four subcultures, the chlorinated ethenes were rapidly, consistently, and completely converted to ethene at rates of 30-50 micromol/l of culture per day, or an average 160 micro-electron equivalents/l of culture per day. These cultures were capable of sustained and rapid dechlorination of VC, and could not dechlorinate 1,2-dichloroethane, differentiating them from Dehalococcoides ethenogenes, the only known isolate capable of complete dechlorination of PCE to ethene. Chloroform (CF) and 1,1,1-trichloroethane, frequent groundwater co-contaminants with TCE and PCE, inhibited chlorinated ethene dechlorination. Most strongly inhibited was the final conversion of VC to ethene, with complete inhibition occurring at an aqueous CF concentration of 2.5 microM. Differences in rates and community composition developed between the different subcultures, including the loss of the VC enrichment culture's ability to dechlorinate PCE. Denaturing gradient gel electrophoresis of amplified bacterial 16S rRNA gene fragments identified three different DNA sequences in the enrichment cultures, all phylogenetically related to D. ethenogenes. Based on the PCR-DGGE results and substrate utilization patterns, it is apparent that significant mechanistic differences exist between each step of dechlorination from TCE to ethene, especially for the last important dechlorination step from VC to ethene.

Field demonstration of successful bioaugmentation to achieve dechlorination of tetrachloroethene to ethene.

A laboratory microcosm study and a pilot scale field test were conducted to evaluate biostimulation and bioaugmentation to dechlorinate tetrachloroethene (PCE) to ethene at Kelly Air Force Base. The site groundwater contained about 1 mg/L of PCE and lower amounts of trichloroethene (TCE) and cis-1,2-dichloroethene (cDCE). Laboratory microcosms inoculated with soil and groundwater from the site exhibited partial dechlorination of TCE to cDCE when amended with lactate or methanol. Following the addition of a dechlorinating enrichment culture, KB-1, the chlorinated ethenes in the microcosms were completely converted to ethene. The KB-1 culture is a natural dechlorinating microbial consortium that contains phylogenetic relatives of Dehalococcoides ethenogenes. The ability of KB-1 to stimulate biodegradation of chlorinated ethenes in situ was explored using a closed loop recirculation cell with a pore volume of approximately 64,000 L The pilot test area (PTA) groundwater was first amended with methanol and acetate to establish reducing conditions. Under these conditions, dechlorination of PCE to cDCE was observed. Thirteen liters of the KB-1 culture were then injected into the subsurface. Within 200 days, the concentrations of PCE, TCE, and cis-1,2-DCE within the PTA were all below 5 microg/L, and ethene production accounted for the observed mass loss. The maximum rates of dechlorination estimated from field date were rapid (half-lives of a few hours). Throughout the pilot test period, groundwater samples were assayed for the presence of Dehalococcoides using both a Dehalococcoides-specific PCR assay and 16S rDNA sequence information. The sequences detected in the PTA after bioaugmentation were specific to the Dehalococcoides species in the KB-1 culture. These sequences were observed to progressively increase in abundance and spread downgradient within the PTA. These results confirm that organisms in the KB-1 culture populated the PTA aquifer and contributed to the stimulation of dechlorination beyond cDCE to ethene.