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.
A bench-scale study was performed to evaluate the enhancement of tetrachloroethene (PCE) dissolution from a dense nonaqueous phase liquid (DNAPL) source zone due to reductive dechlorination. The study was conducted in a pair of two-dimensional bench-scale aquifer systems using soil and groundwater from Dover Air Force Base, DE. After establishment of PCE source zones in each aquifer system, one was biostimulated (addition of electron donor) while the other was biostimulated and then bioaugmented with the KB1 dechlorinating culture. Biostimulation resulted in the growth of iron-reducing bacteria (Geobacter) in both systems as a result of the high iron content of the Dover soil. After prolonged electron donor addition methanogenesis dominated, but no dechlorination was observed. Following bioaugmentation of one system, dechlorination to ethene was achieved, coincident with growth of introduced Dehalococcoides and other microbes in the vicinity and downgradient of the PCE DNAPL (detected using DGGE and qPCR). Dechlorination was not detected in the nonbioaugmented system over the course of the study, indicating that the native microbial community, although containing a member of the Dehalococcoides group, was not able to dechlorinate PCE. Over 890 days, 65% of the initial emplaced PCE was removed in the bioaugmented, dechlorinating system, in comparison to 39% removal by dissolution from the nondechlorinating system. The maximum total ethenes concentration (3 mM) in the bioaugmented system occurred approximately 100 days after bioaugmentation, indicating that there was at least a 3-fold enhancement of PCE dissolution atthis time. Removal rates decreased substantially beyond this time, particularly during the last 200 days of the study, when the maximum concentrations of total ethenes were only about 0.5 mM. However, PCE removal rates in the dechlorinating system remained more than twice the removal rates of the nondechlorinating system. The reductions in removal rates over time are attributed to both a shrinking DNAPL source area, and reduced flow through the DNAPL source area due to bioclogging and pore blockage from methane gas generation.
The use of enhanced in situ anaerobic bioremediation (EISB) and bioaugmentation in fractured bedrock is limited compared to its use in granular media. We evaluated EISB for the treatment of trichloroethene (TCE)-impacted groundwater in fractured carbonate rock at a site in Southern Ontario, Canada, with cool average groundwater temperature (∼ 13 °C). Borehole-connectivity, contaminant concentrations, and groundwater properties were investigated. Changes in dechlorinating and nondechlorinating populations (fermenters, acetogens, methanogens, and sulfate reducers) were assessed via quantitative PCR (qPCR). During biostimulation with ethanol, concentrations of TCE daughter products cis-dichloroethene (cDCE) and vinyl chloride (VC) decreased in association with an enrichment of vcrA (VC reductive dehalogenase)-carrying Dehalococcoides, whereas ethene production was only moderate. Following bioaugmentation with the mixed dechlorinating culture KB-1, greater concentrations of chloride-a product of dechlorination-was observed in most wells; in addition, ethene production increased significantly in monitoring well locations that had strong hydraulic connectivity to the groundwater recirculation system, while Dehalococcoides and vcrA concentrations did not appreciably vary. Interestingly, increases of 3-4 orders of magnitude of an ethanol-fermenting Bacteroidetes population also present in KB-1 were correlated to improved conversion to ethene, an observation which suggests there could be a causal relationship-for example, better syntrophy and/or synergy among bacterial populations.
Chlorinated ethenes are commonly found in contaminated groundwater. Remediation strategies focus on transformation processes that will ultimately lead to nontoxic products. A major concern with these strategies is the possibility of incomplete dechlorination and accumulation of toxic daughter products (cis-1,2-dichloroethene (cDCE), vinyl chloride (VC)). Ethene mass balance can be used as a direct indicator to assess the effectiveness of dechlorination. However, the microbial processes that affect ethene are not well characterized and poor mass balance may reflect biotransformation of ethene rather than incomplete dechlorination. Microbial degradation of ethene is commonly observed in aerobic systems but fewer cases have been reported in anaerobic systems. Limited information is available on the isotope enrichment factors associated with these processes. Using compound-specific isotope analysis (CSIA) we determined the enrichment factors associated with microbial degradation of ethene in anaerobic microcosms (ε = -6.7‰ ± 0.4‰, and -4.0‰ ± 0.8‰) from cultures collected from the Twin Lakes wetland area at the Savannah River site in Georgia (United States), and in aerobic microcosms (ε = -3.0‰ ± 0.3‰) from Mycobacterium sp. strain JS60. Under anaerobic and aerobic conditions, CSIA can be used to determine whether biotransformation of ethene is occurring in addition to biodegradation of the chlorinated ethenes. Using δ(13)C values determined for ethene and for chlorinated ethenes at a contaminated field site undergoing bioremediation, this study demonstrates how CSIA of ethene can be used to reduce uncertainty and risk at a site by distinguishing between actual mass balance deficits during reductive dechlorination and apparent lack of mass balance that is related to biotransformation of ethene.
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