Bioremediation of Trichloroethylene-Contaminated Sediments
Augmented with a Dehalococcoides Consortia

P. C. McKinsey, R. L. Brigmon, M. A. Heitkamp, C. J. Berry,
J. Rossabi, T. E. Poppy, and D. B. Martin
Westinghouse Savannah River Company
Aiken, South Carolina 29808

F. E. Loeffler
Georgia Institute of Technology
Atlanta, Georgia


At the Department of Energy’s (DOE) Savannah River Site (SRS) in Aiken, SC there are a number of sites contaminated with Chlorinated Ethenes (CE) due to past disposal practices. Sediments from two CE contaminated SRS locations were evaluated for trichloroethylene (TCE) biodegradation through anaerobic laboratory microcosms. The testing included addition of amendments and bioaugmentation of sediments. The anaerobic microcosms were first amended with substrates including acetate, lactate, molasses, soybean oil, methanol, sulfate, yeast extract, Regenesis HRC®, and MEAL (methanol, ethanol, acetate, lactate mixture). Microcosms were analyzed after biostimulation for 9 months and no significant TCE biodegradation was observed.

At 10 months, additional TCE, fresh amendments, and a mixed culture containing Dehalococcoides ethenogenes were added to active microcosms. A significant decrease in TCE concentrations and an increase in biodegradation products cis-dichloroethylene (cDCE) and vinyl chloride (VC) were noted within 2 weeks of bioaugmentation. Microcosms amended with lactate and sulfate showed complete transformation of TCE (3 ppm) to ethene within 40 days after bioaugmentation. Microcosms amended with other substrates - soybean oil, acetate, yeast extract, and methanol - also show enhanced biodegradation of TCE to ethene. Microcosms amended with molasses and Regenesis HRC showed limited TCE transformation. No TCE transformation was seen in killed control microcosms. On the basis of these successful results, plans are underway for field-scale in-situ deployment of biostimulation/bioaugmentation at SRS.


While there has been significant progress in aggressive source area treatments of VOC contamination at SRS, residual CE contamination is still a problem at several locations (Kastner et al., 1999). Sediments from two CE contaminated Savannah River Site (SRS) locations were evaluated for TCE degradation through anaerobic laboratory microcosms.

The purpose of this project was to evaluate the potential of Monitored Natural Attenuation (MNA), biostimulation, and/or bioaugmentation for accelerated remediation of trichloroethylene and perchloroethylene (PCE) contaminated aquifers at C-Area and D-Area of the Savannah River Site (SRS). Characterization work has shown both sites to be oligotrophic with only minimal natural microbial degradation of Volatile Organic Compounds (VOCs). In addition, both sites have potential plume outcrop areas in seeplines. A microcosm study designed to estimate the bioremediation potential of SRS seepline soils demonstrated that sorption removed as much as 90% TCE (Brigmon, et al. 1998). A limited amount of aerobic TCE biodegradation and anaerobic reductive dechlorination was observed, including the formation of byproduct cis-1, 2-dichloroethylene (c-DCE). These plume outcrop areas may not support sufficient aerobic microbial activity to mineralize PCE and TCE degradation products as cDCE or vinyl chloride (VC), as detected in SRS groundwater and surface waters. The detection of biodegradation products cDCE and VC in these plumes indicates favorable in situ microbial activity. An opportunity exists for nutrient addition combined with bioaugmentation for in situ microbial degradation of VOCs in the two SRS sites. Molecular microbiological examinations of reductive dechlorination of chlorinated ethenes (CE) have shown that key CE degrading genera, including Dehalococcoides (PCE/TCE ® ethene), Desulfuromonas and Dehalobacter (both convert PCE ® cis-DCE) species must optimally be present for complete degradation (Löffler et al. 2000). Molecular detection of these species has been demonstrated in PCE/TCE contaminated aquifers, soils and sediments, and batch reactors having dechlorination activities (Fennell et al., 2001; Hendrickson et al., 2002; Löffler et al., 2000). The experimental approach and methods described here determined the optimal enhancements (i.e., nutrients) and bioaugmentation (addition of microorganisms) for potential SRS field-scale bioremediation applications.

Methods and Materials

In April 2001, two sediment cores were collected in Shelby tubes by rotosonic drilling from SRS Site D-Area in the vicinity of a VOC plume. The two cores were from depths of 38-39.9 ft and 27-29.3 ft. below ground surface. In August 2001, a sediment core was collected in Shelby tube by rotosonic drilling at depth 58-62' below ground surface from SRS C-Area (Figure 1). All cores were wax sealed in the field and immediately transferred to the laboratory where they were placed in an anaerobic chamber (90% N2, 5% CO2, and 5% H2). Ten liters of groundwater for the SRS D-Area study was collected from a groundwater monitoring well located 10 meters from the core location.

The study consisted of four sets of anaerobic microcosms per site – (1) live, active microcosms with varying nutrient amendments and addition of TCE, (2) live control microcosms with TCE but without nutrient amendments, (3) live control microcosms with no TCE addition, and (4) killed control microcosms (with nutrient and TCE amendments). Each microcosm treatment was prepared in triplicate. Nutrient amendments tested were methanol, acetate, lactate, MEAL (methanol, ethanol, acetate, and lactate in combination), soybean oil, and molasses. Additionally, the affects of added SO4-2 as an electron acceptor and yeast extract were tested. Microcosms prepared from SRS C-Area sediments also tested Regenesis HRC® compound as a nutrient addition (Odencrantz et al., 1995). Regenesis HRC® is a commercial polylactate ester formulated for slow release of lactate upon hydration (Regenesis Bioremediation Products, San Clemente, CA). A list of all amendments and concentrations is in Table 1.

Cores were sectioned aseptically in the anaerobic chamber with autoclaved and ethanol-rinsed tools. The sediments were then removed from the inner section of the cores and mixed in sterile pans. Microcosms were assembled in the anaerobic chamber in sterile glass 240 ml serum bottles sealed with screw cap mininert valves (Supelco).

Table 1. A list of all microcosm amendments and concentrations.

Table 1. Microcosm Amendments


Concentration in microcosm (aqueous)

Sodium acetate (trihydrate)


Sodium lactate
(60% syrup)



10 mM



Yeast Extract

20 mg/l



Soybean Oil

1 gm/liter


1 gm/liter


1 gm/liter


1 mg/liter

Included in each SRS D-Area microcosm were 50 grams of mixed core sediment, 100 ml groundwater with nutrient amendment(s), and TCE as the electron acceptor (chlorinated compound). TCE was added with a starting concentration of 2500 ug/kg sediment in each microcosm. The groundwater in the D-Area study was unfiltered and unsterilized (except in killed controls) and was "degassed" by storage in a hood prior to adding it to the microcosms. Sterile resazurin (1mg/liter) was added to the groundwater as an indicator of anaerobiosis.

Microcosms for the SRS C-Area study were prepared in the same way except that deionized water (DI) was used in place of groundwater. The DI was sterile filtered (0.2m m), autoclaved, and degassed by storage in the anaerobic hood prior to use in the microcosms. TCE was added to C-Area microcosms with a starting concentration of 2000 ug/kg in each microcosm.

Killed control microcosms were prepared for both site studies to evaluate abiotic TCE degradation. Sodium azide (0.1%) was added to filter sterilized and autoclaved water used in the killed control microcosms. Before microcosm preparation, sediments used in killed controls were autoclaved three times over a 5-day period. All microcosms were incubated shielded from light at 25°C.

Due to lack of biodegradation activity, it was decided to bioaugment the microcosms ten months after initial preparation. Fresh TCE and nutrient amendments were added to all microcosms. TCE was spiked so that the starting total concentration of TCE in the SRS D-Area microcosms was 3000 ug/kg, and the initial TCE concentration in the C-Area microcosms was 5500 ug/kg. Nutrients were added at the same concentrations as initial concentrations (Table 1). Five days after nutrient and TCE additions, 1.5ml of a fresh bacteria consortium containing halorespirers including Dehalococcoides ethenogenes was added to each active microcosm. No bacterial culture was added to the killed controls. All additions were performed in the anaerobic chamber.

When initial preparation of microcosms was complete, microcosms were shaken vigorously to ensure proper mixing and a 1.0 ml water sample was taken with a Hamilton gas-tight syringe. The sample was injected into a pre-labeled 22 ml Agilent Gas Chromatography (GC) vial filled with 9ml of DI water. The vial was immediately capped with Teflon-lined butyl rubber seals, crimped, and inverted in a vial tray. The sample vials were subsequently run on the gas chromatographic headspace analysis system for analysis of targeted compounds (VC, c-DCE, TCE and PCE) (Enzien et al., 1994). The 1-mL water sample taken immediately after microcosm preparation was called the zero time. The sample for GC analysis taken immediately after bioaugmentation was called the bioaugmentation zero time. This same sampling process was used for subsequent samples taken during microcosm incubation.

The amounts of VC, c-DCE, TCE and PCE components in the microcosm studies were determined by Gas Chromatography Mass Spectroscopy (GC/MS) analysis. Analyses were performed on an Agilent model 5890 Series II gas chromatograph interfaced with an Agilent Model 5972 mass selective detector. Samples were introduced to the GC using an Agilent 7694 Headspace Sampler, with approximately 10 ml liquid volume in 22 ml Agilent GC vials. Helium was used as the carrier gas with the pressure program as follows: initial pressure of 29.2 psi held for 10 minutes and then increased by 1.20 psi/minute up to a final pressure of 47.7 psi which was held for 0.58 minutes. Analytical separations were carried out on a HP-5 capillary column (50 m x 0.20 mm [inside diam] with a 0.33 m M film thickness). The temperature program was as follows: 40°C initial temperature held for 10 minutes followed by a linear increase of 10°C/minute to 200°C. Injector temperatures and mass spectropic detector temperatures were held at 250°C. The amounts of targeted compounds present were calculated by comparing peak area obtained by 1.0 ml headspace sampler injections (headspace sampler parameters: 30.0 minute oven time @ 90°C) with Supelco standards (EPA 524.2) containing all the targeted compounds using HP ChemStation software (HP G1034C MS ChemStation Software). The limit of detection for these components was set at 4 ug/L.

Figure 1. Chlorinated Ethene Plumes in C-Area

Figure 1. Chlorinated Ethene Plumes in C-Area


Table 2. Rates of Chlorinated Ethene Transformation

Table 2. Rates of Chlorinated Ethene Transformation

Results and Discussion

Resazurin indicated that all active microcosms went anaerobic within two weeks of initial preparation. The D-Area microcosms were analyzed for VOC concentrations at 0, 30, 90, 150, and 330 days. Most D-Area microcosms before bioaugmentation showed little evidence of biodegradation of TCE by biostimulation alone. The transient shifts in VOCs that were observed could have been due to sorption/desorption by the sediment. The lactate-sulfate treated D-Area microcosms did have a steady reduction and finally disappearance of TCE. Disappearance of TCE did not occur in any killed control microcosms. The C-Area microcosms before bioaugmentation were analyzed for VOC concentrations at 0, 60, and 210 days. The 210-day samples showed some evidence of biodegradation of TCE in the molasses and in the yeast extract amended microcosms. No killed control microcosms show significant changes in the TCE concentration when 0 time samples are compared to 210 day samples. No daughter products (c-DCE, VC, ethene) were noted in any C-Area microcosms.

Figure 2. C-Area Bioaugmented Microcosm Acetate Amended Ethene Detected at 20, 37, and 73 days

Figure 2. C-Area Bioaugmented Microcosm Acetate Amended
Ethene Detected at 20, 37, and 73 days

After bioaugmentation, rapid biodegradation of TCE was seen in all active C and D-Area microcosms except for those amended with molasses and HRC®. All TCE was rapidly transformed to c-DCE within 20 days in many microcosms. Numerous microcosms (C-Area Acetate, C-Area Soybean Oil + sulfate, D-Area Acetate, D-Area Lactate, D-Area Lactate+sulfate, D-Area MEAL+sulfate, D-Area MEAL+yeast ext, D-Area MEAL+yeast ext+sulfate, D-Area Yeast extract, and D-Area Methanol) showed VC production within 20 days of bioaugmentation. For C-Area sediment microcosms, only acetate (Figure 2) and methanol + sulfate showed complete breakdown of all chlorinated ethenes at 73 days after bioaugmentation. For D-Area sediment microcosms, acetate, lactate + sulfate (Figure 3), methanol, and MEAL (Methanol, ethanol, acetate, and lactate) + sulfate + yeast extract resulted in complete CE biodegradation at 73 days after bioaugmentation. Biodegradation rates were calculated for those treatments which gave complete CE biodegradation. Acetate treatment gave the best rate for D-Area and C-area sediments. Lactate+sulfate treatment additionally resulted in a high rate of transformation for D-area sediments (Table 2).

Figure 3. D-Area Bioaugmented Microcosm Lactate + Sulfate Amended Ethene Detected at 20, 37, and 74 days

Figure 3. D-Area Bioaugmented Microcosm Lactate + Sulfate
Amended Ethene Detected at 20, 37, and 74 days

These studies have shown rapid development of anaerobic conditions in aerobic sediments after nutrient addition. Although incubation with these nutrients (biostimulation) produced anaerobic conditions, minimal chlorinated solvent degradation was observed by indigenous microorganisms after up to 330 days of incubation. These results indicate that the natural microbial population, even with the addition of electron donors, is inefficient at removing the VOCs at the site. Since inoculation with halo-respiring bacteria (bioaugmentation) resulted in rapid and complete biodegradation of CE with select substrates, biostimulation coupled with bioaugmentation may provide the best alternative for in situ biodegradation of VOCs at SRS. These bench-scale studies have shown that biostimulation coupled with bioaugmentation is feasible for enhanced VOC biodegradation in SRS sediments collected from C-Area and D-Area.


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