Laboratory-scale microcosm study results show electron donor (yeast extract) amendment enhanced reductive dehalogenation of parent chlorinated aliphatic hydrocarbons (CAHs). Microcosm study samples were collected at a former wastewater treatment facility in southern New Hampshire where CAHs had historically been co-disposed with sludge and septage. Sampling locations for the study included upper and lower overburden hydrogeologic units. Groundwater conditions within the contaminant plume range from aerobic and chemically oxidizing along the plume periphery to anaerobic and chemically reducing within the core. Previous intrinsic bioremediation screening demonstrated reductive dehalogenation to be a significant natural attenuation mechanism for parent CAHs, but the process was becoming electron donor limited by depletion of dissolved organic carbon (DOC), initially provided by sludge and septage. A 54-day microcosm study was performed simulating intrinsic and enhanced bioremediation conditions, and controlling for abiotic transformation processes. Microcosm monitoring parameters included nitrate, sulfate, methane, chemical oxygen demand (COD), inorganic chloride, and CAHs. Results for enhanced bioremediation microcosms, relative to controls, showed greater parent CAH removal; depressed nitrate and sulfate; and elevated methane, COD, and inorganic chloride. Results suggest yeast extract stimulated parent CAH transformation by providing substrate, which exerted oxidant demand, scavenged available electron acceptors, and drove reductive dehalogenation. Preliminary results from an on-going pilot-scale yeast extract amendment study at the site are consistent with those from this microcosm study.
INTRODUCTION
CAHs in groundwater were initially detected in 1987 at concentrations on the order of 101 to 104 micrograms per liter (ug/l) at a southern New Hampshire site operated as a wastewater treatment facility from 1964 until closure in 1986. CAHs included the assumed parents tetrachloroethene (PCE), trichloroethene (TCE), and 1,1,1-trichloroethane (TCA), and the assumed daughters 1,1-dichloroethene (1,1-DCE), cis/trans-1,2-dichloroethenes (1,2-DCEs), 1,1/1,2-dichloroethanes (DCAs), and vinyl chloride (VC). Hydrogeological studies were performed at the site from 1987 to 1992 to identify the CAH source and evaluate the extent and magnitude of contamination. The source was identified as uncontrolled dumping of CAHs into on-site disposal pits where sludges from the wastewater treatment facility were disposed with septage from off-site sources. Significant quantities of dense, non-aqueous phase liquids are not believed to be present at the site based upon reductions in parent CAH concentrations with time. Two overburden groundwater flow systems separated by a silty clay aquitard exist at the site. The flow systems include an upper fine sand and silt unit of moderate hydraulic conductivity (10-3 to 10-4 cm/sec), and a lower sandy and silty till unit of lower hydraulic conductivity (10-3 to 10-5 cm/sec). Estimated average hydraulic gradients within the upper and lower units are about 10-3 and 10-4, respectively. Groundwater depth ranges from around 5 to 10 feet below ground surface seasonally due to variations in recharge. Groundwater recharge conditions exist at the site, with vertical hydraulic gradients of up to about 10-2.
Two distinct groundwater redox zones are present in the plume within which different CAH intrinsic bioremediation processes are important. Anaerobic, chemically reducing (methanogenic), conditions exist within the contaminant plume core in the vicinity of the disposal pits, with decreasing parent and increasing daughter CAH concentrations over time. Aerobic, chemically oxidizing, conditions exist along the plume periphery, with decreasing parent and daughter CAH concentrations over time, though parent CAHs are attenuating at slower rates than within the plume core.
Intrinsic bioremediation screening results suggest sludge/septage co-disposal with CAHs drove intrinsic bioremediation by exerting biochemical oxygen demand upon the groundwater system, which stimulated certain microbes to scavenge available electron acceptors and drove conditions methanogenic. Methanogenic conditions yielded hydrogen that stimulated CAH reductive dehalogenation within the core, and methane that stimulated CAH co-oxidation within the aerobic mixing zone along the plume periphery. Direct mineralization of daughter CAHs likely is also occurring along the periphery. Though intrinsic bioremediation was fueled by residual sludge/septage, breakthrough of electron acceptors (e.g., nitrate and sulfate) into the anaerobic core coupled with typically low DOC concentrations of £ 10 milligrams per liter (mg/L) suggest parent CAH reductive dehalogenation (and indirectly co-oxidation) is becoming increasingly substrate limited since sludge/septage disposal ceased in 1986. Though overburden site groundwater discharges into a river less than about 400 feet from the source, natural attenuation mechanisms have reduced CAH concentrations in groundwater such that there have been no exceedances of surface water quality standards at river sampling stations for three years.
A laboratory-scale microcosm study was performed to evaluate enhanced bioremediation treatment of parent CAHs within the plume in consideration of a substrate-limited groundwater condition inhibiting further parent CAH transformation. In addition, the state regulatory agency requested an engineered groundwater treatment technology be selected that would destroy CAHs on site. The remedial concept assumes daughter CAHs, not destroyed by reductive dehalogenation in the anaerobic treatment zone, will continue to be attenuated at distal locations by co-oxidation, mineralization, adsorption, and dilution in the saturated zone and co-oxidation, mineralization, dilution, adsorption, and volatilization in the discharge zone. Microcosm study objectives included selection of a suitable electron donor, and an evaluation of substrate re-initiation of parent CAH reductive dehalogenation. Results of the microcosm study would be used as the technical basis of a Remedial Action Plan to reduce parent CAHs in groundwater.
METHODS
Electron Donor Selection
Yeast extract was selected as an electron donor for the microcosm study based upon literature review. Yeast extract is a complex, highly water soluble growth media that provides substrate and inorganic nutrients for stimulating microbial activity such as reductive dehalogenation. Reductive dehalogenation involves the microbial-mediated replacement of chlorine with hydrogen in the presence of suitable substrate, followed by subsequent CAH transformation to less oxidized CAH species. Reductive dehalogenation typically occurs sequentially from PCE to TCE to DCEs to VC to ethene. Rasmussen et al. (1994) showed a positive correlation between increased substrate complexity and reductive dehalogenation rates. Odom et al. (1995a) demonstrated yeast extract amendment stimulated reductive dehalogenation of PCE and TCE to 1,2-DCEs in laboratory-scale microcosms within about 180 days, and that yeast extract was more effective than other substrates such as lactate, methanol, and formate for stimulating reductive dehalogenation under a variety of biogeochemical conditions. Haston et al. (1996) demonstrated yeast extract amendment stimulated PCE transformation in laboratory-scale microcosms in about two months. Yeast extract enhances reductive dehalogenation by stimulating: 1) obligate aerobic and facultative aerobic heterotrophs to scavenge electron acceptors that may otherwise inhibit methanogenic conditions from developing; and 2) methanogenic bacteria to convert substrate to molecular hydrogen, the virtual "fuel" driving reductive dehalogenation.
Though yeast extract has greater unit cost than other potential substrates, subsurface site conditions were not anticipated to require significant electron donor loading to re-initiate reductive dehalogenation. For example, plume center mass is anoxic and chemically reducing, with groundwater screening results for one well indicating <0.2 mg/L dissolved oxygen (DO), <-30 millivolts (mV) oxidation-reduction potential (ORP), and >1,700 ug/l methane. Additionally, groundwater quality data suggest significant reductive dehalogenation occurred historically before substrate became depleted. For example, the ratio of parent to total CAH concentration has steadily declined from <50 percent (%) parents in 1988 to <12% in 1997 at all monitoring well locations within the plume, with the greatest observed reductions occurring within the core.
Groundwater Sampling
Groundwater samples were collected from three monitoring wells using an aseptic technique following DO, ORP, and pH stabilization. The aseptic technique involved washing sampling implements with non-phosphate, laboratory-grade detergent to remove particulate matter, then rinsing sequentially with tap water, distilled water, alcohol, chlorine bleach, and de-ionized water heated at 212oF for two hours. Samples were collected in sterile 500-milliliter amber glass containers without headspace.
Sampling locations depicted in Figure 1 included one upper hydrogeologic unit monitoring well (GZ-2) and two lower unit wells (GZ-3L and GZ-4L). Groundwater conditions at well locations GZ-4L and GZ-2 were aerobic (4.7 and 6.1 mg/l DO, respectively) and chemically oxidizing (+180 and +150 mV ORP, respectively), and generally yield parent and daughter CAH concentrations of <104 ug/l, whereas groundwater conditions at well location GZ-3L were anaerobic (0.2 mg/l DO) and chemically reducing (-55 mV ORP), and generally yield lower parent CAH concentrations of <102 ug/l, but higher daughter CAH concentrations of >105 ug/l. Sampling locations GZ-2 and GZ-4L were selected to reflect representative CAH concentration conditions. Sampling location GZ-3L reflects a worse-case condition due to significantly higher daughter CAH concentrations.
Microcosm Study
Microcosms were constructed to simulate intrinsic and enhanced bioremediation conditions, and included an abiotic control for microbial activity. Microcosm conditions are summarized in Table 1.
Table 1. Summary of Microcosm Conditions
| SAMPLING LOCATION | MICROCOSM NO. | [CAHtotal] (ug/l) |
HYDROGEOLOGIC UNIT | MICROCOSM CONDITION | |
| Upper | Lower | ||||
| GZ-4L | 1 | <104 | X | Intrinsic Bioremediation | |
| 2 | X | Inhibited Control | |||
| 3 | X | Enhanced Bioremediation | |||
| GZ-3L | 4 | >105 | X | Intrinsic Bioremediation | |
| 5 | X | Inhibited Control | |||
| 6 | X | Enhanced Bioremediation | |||
| GZ-2 | 7 | <104 | X | Intrinsic Bioremediation | |
| 8 | X | Inhibited Control | |||
| 9 | X | Enhanced Bioremediation | |||
NOTES:
- [CAHtotal] indicates total CAH concentration, including both parents and daughters.
- Microcosms consisted of pre-sterilized 500-mL amber glass jars topped with screw-down lids equipped with Teflon® septa.
- Intrinsic bioremediation microcosms contained groundwater, CAHs, and indigenous microbial seed. Inhibited control microcosms contained groundwater, CAHs, and 0.5 grams mercuric chloride (biocide) for inhibiting microbial activity. Enhanced bioremediation microcosms contained groundwater, CAHs, indigenous microbial seed, and yeast extract (0.3% m/v DIFCO Laboratories BACTO® Yeast Extract, Product Code 0127). Biocide/yeast extract amendments were performed during sample collection to minimize volatile loss due to handling.
- Microcosms were incubated without headspace at 20oC for 54 days.
- Bioremediation monitoring consisted of pre-incubation (time initial, ti) and post-incubation (time final, tf) analytical laboratory testing. Differences between ti and tf results are assumed to reflect biochemical processes occurring as a result of simulation conditions. Refer to the Analytical Program section for additional information.
- The pH within each microcosm ranged from 6.5 to 6.7 standard units during the incubation period, which is within the 4 to 10 standard unit range of microbial viability described by Cookson (1995).
Seed Cultures/Contaminant Microbial Ecology
Microcosms contained site groundwater without formation matrix material. Because a majority of microbial abundance is attached to formation matrices, and thus not recoverable during groundwater sampling, microbial seed for the microcosm study were cultured from aliquots of each groundwater sample. Indigenous seed included obligate/facultative aerobes, methanotrophs, and obligate anaerobes. Ambient microbial population densities within each microcosm were increased one order of magnitude to conservatively compensate for that fraction not recovered during groundwater sampling. Bacterial population density data for each microcosm, reflecting a one-order magnitude increase of the ambient population density, are summarized in Table 2.
Table 2. Summary of Bacterial Population Densities in Microcosms, CFU/ml
| Microcosm Nos. | POPULATION DENSITIES (CFU/ml) | |||||
| Obligate/Facultative Aerobes | Methanotrophs | Obligate Anaerobes | ||||
| Ambient | Microcosm | Ambient | Microcosm | Ambient | Microcosm | |
| 1 & 3 | 1E4 | 1E5 | 1E3 | 1E4 | 1E2 | 1E3 |
| 4 & 6 | 1E4 | 1E5 | 1E3 | 1E4 | 1E1 | 1E2 |
| 7 & 9 | 1E5 | 1E6 | 1E2 | 1E3 | 1E1 | 1E2 |
Notes:
- CFU/ml indicates colony forming units per milliliter.
- Enumerations were performed for obligate/facultative aerobes, methanotrophs, and obligate anaerobes using sterile nutrient broth, methane, and Bushnell-Haas broth/acetic acid, respectively.
Obligate/facultative aerobic heterotrophs use DOC, including some daughter CAHs, as electron donors and a variety of electron acceptors including molecular oxygen, nitrate, sulfate, and certain oxidized metals. Methanotrophs use methane as electron donor and molecular oxygen as an electron acceptor. Soluble methane monooxygenase (sMMO), an enzyme methanotrophs use to metabolize methane, can affect co-oxidation of certain parent and daughter CAHs under certain conditions. Equation 1 illustrates methane metabolism to carbon dioxide and water and Equation 2 illustrates TCE co-oxidation to carbon dioxide, water, and chloride:
 |
Equation 1 |
 |
Equation 2 |
Obligate anaerobes use carbon dioxide as an electron acceptor and molecular hydrogen as an electron donor, or simple organic compounds as both electron donors and acceptors (fermentation). Certain obligate anaerobes effect co-metabolic (serendipitous) reductive dehalogenation of certain parent and daughter CAHs while a smaller fraction use certain parents and daughters as electron acceptors in the process of metabolic reductive dehalogenation (dehalorespiration). Equation 3 illustrates oxidation of molecular hydrogen and co-metabolism of PCE to TCE and hydrochloric acid, and Equation 4 illustrates oxidation of a potential substrate (methanol) and dehalorespiration of PCE to ethene, hydrochloric acid, and carbon dioxide:
 |
Equation 3 |
 |
Equation 4 |
Though Equations 3 and 4 consider but one electron donor (i.e., hydrogen and methanol, respectively) and Equation 4 does not show intermediate products, both equations illustrate the overall process of PCE reductive dehalogenation.
Analytical Program
Time initial (ti) and time final (tf) microcosm samples were submitted to GZA GeoEnvironmental, Inc.'s Environmental Chemistry Laboratory (ECL) in Newton Upper Falls, Massachusetts and Matrix Analytical, Inc. (MAI) in Framingham, Massachusetts for the analytical program summarized in Table 3.
Table 3. Summary of Microcosm Analytical Program
| PARAMETER AND ANALYTICAL METHOD | SELECTION RATIONALE |
| Nitrate, Environmental Protection Agency (EPA) Method 353.2 (MAI) | Alternative terminal electron acceptor |
| Sulfate, EPA Method 45-OOE-SO4 (MAI) | Alternative terminal electron acceptor |
| Methane, gas chromatographic (GC) headspace screening (ECL) | Product of methanogenesis |
| COD, EPA Method 410.4 (MAI) | Substrate surrogate |
| Inorganic chloride, EPA Method 325.2 (MAI) | Reductive dehalogenation product |
| CAHs, EPA Method 8010 | Contaminants of concern |
Notes:
- GC headspace screening was performed using the Walsh and Pickering (1995) method.
- Note tf DO and ORP data were not collected due to equipment problems.
Laboratory analyses did not include soluble iron (II) or manganese (III) because microcosms did not contain formation matrix material. Moreover, previous groundwater monitoring results demonstrated insoluble iron (III) and manganese (IV) did not generally limit development of methanogenic conditions.
RESULTS AND DISCUSSION
Nitrate, sulfate, methane, COD, and inorganic chloride results for the microcosm study are summarized in Table 4, and CAH results are summarized in Table 5. Results are generally discussed in the order presented in those tables.
Nitrate and Sulfate
Intrinsic bioremediation microcosms contained detectable ti nitrate and sulfate, suggesting there was insufficient ambient DOC to stimulate facultative aerobes to scavenge these electron acceptors via nitrate and sulfate reduction, respectively. Significantly, the ti sulfate concentration for each microcosm exceeded the upper limit threshold for reductive dehalogenation (i.e., 25 parts per million [ppm], Wiedemeier et al., 1996). Nitrate and sulfate results were generally consistent with historical data, suggesting groundwater is becoming DOC limited. The tf nitrate and sulfate results for intrinsic bioremediation microcosms and inhibited controls were generally similar, further suggesting natural conditions were not conducive for scavenging electron acceptors.
Yeast extract amendment stimulated certain facultative aerobic bacteria to scavenge nitrate below method detection limits (MDLs) within each enhanced bioremediation microcosm, and sulfate below MDLs within Microcosm Nos. 3 and 6, and from 35 ppm to 23 ppm within Microcosm No. 9. These results suggest yeast extract provided sufficient DOC to scavenge nitrate and sulfate, such that the methanogenic conditions required for reductive dehalogenation could become established. Odom et al. (1995a and 1995b) report reductive dehalogenation may proceed in the presence of sulfate providing nitrate has been depleted. Though sulfate was not depleted below the MDL in Microcosm No. 9, the tf concentration was below the 25-ppm inhibitory threshold.
For certain intrinsic and enhanced bioremediation microcosms in which nitrate and sulfate reduction were indicated (e.g., Microcosm Nos. 3, 4, 6, and 9), the data suggest ambient DO was scavenged during the study because these respiratory processes are typically inhibited by the presence of about >0.2 mg/l DO.
Methane
The ti methane concentrations ranged from not detected above MDLs (Microcosm Nos. 7 through 9) to 2,600 ug/l (Microcosm Nos. 4 through 6). Lack of detectable methane for Microcosm Nos. 7 through 9 reflects shallow groundwater conditions at upper unit well GZ-2, because groundwater is more oxic and chemically oxidizing than lower overburden groundwater due to atmospheric contact and shorter residence time. The highest ti methane concentration was detected in microcosms containing groundwater from monitoring well GZ-3L. Elevated methane at this well reflects the influence of residual sludge/septage upon groundwater quality. Consistent with that conclusion, historical COD concentrations at that well have consistently been higher than those for other site wells. Notwithstanding, yeast extract amendment to Microcosm No. 6 stimulated a two-fold increase in methane production. The tf methane concentrations for each enhanced bioremediation microcosm were higher than for the other microcosms, indicating yeast extract amendment stimulated methanogenic activity.
COD
The ti COD concentrations ranged from <5 mg/l (Microcosm Nos. 1 through 3) to 180 mg/l (Microcosm Nos. 4 through 6). Elevated ti COD concentrations for Microcosm Nos. 4 through 6 are consistent with methane data, reflecting the continued presence of residual sludge/septage at well GZ-3L, though the ti concentration for these microcosms was about six-fold greater than the arithmetic mean for three other sampling rounds at that well. Well GZ-3L is located about 200 feet downgradient of the former sludge disposal pit, and is the closest site well to that pit. With the exception of Microcosm No. 4, the ti COD concentration for intrinsic bioremediation microcosms was insufficient to completely scavenge nitrate and sulfate. The tf COD concentrations for enhanced bioremediation microcosms were higher than for other microcosms, reflecting supplemental substrate provided by yeast extract.
Inorganic Chloride
The ti chloride concentration ranged from 14 mg/l (Microcosm Nos. 7 through 9) to 85 mg/l (Microcosm Nos. 1 through 3), with the highest chloride concentrations detected at lower unit wells. Elevated chloride in samples from the lower unit reflect a longer residence time for solute accumulation.
Enhanced bioremediation microcosms had slightly higher tf inorganic chloride concentrations than intrinsic bioremediation microcosms. While this increase may reflect CAH dehalogenation, given ti total CAH concentrations of about 5 to 20 mg/l, it is likely a majority of the increase reflects amended substrate given yeast extract is about 0.5%mass chloride. The highest tf chloride concentrations were within inhibited microcosms, reflecting mercuric chloride amendment.
CAHs
Parent CAHs
Parent CAH concentration data for intrinsic bioremediation microcosms are generally similar to inhibited controls, suggesting parent CAH intrinsic bioremediation was limited over the study duration. Enhanced bioremediation microcosms generally had a lower tf parent CAH concentration than intrinsic bioremediation microcosms, indicating yeast extract amendment stimulated parent CAH dehalogenation. Certain ti parent CAH results for Microcosm Nos. 4 through 9 are either the same or lower than tf results; however, this is attributed to high dilution factors used by the analytical laboratory to meet EPA Method 8010 quality assurance acceptance limits. Dilution factors reflected high daughter CAH concentrations, and imposed high MDLs on CAH results. Note that CAH values reported in Table 5 are one half MDL values for those instances in which CAHs were not detected. For example, the reported tf PCE value for Microcosm No. 4 is 100 ug/l because EPA Method 8010 did not detect PCE above 200 ug/l for that microcosm.
Parent CAH reductive dehalogenation appeared most important for enhanced bioremediation Microcosm No. 3, in which the TCE concentration was reduced from 430 ug/l (ti) to 2 ug/l (tf). The significant decrease in TCE concentration for this microcosm is attributed to the relatively high ti TCE concentration available for dehalorespiration. Reductive dehalogenation was likely co-metabolic in the other microcosms due to lower ti parent concentrations that may not have been sufficient to support a respiratory pathway. This is consistent with the methane data for Microcosm No. 3 indicating there was no increase in methane concentration in response to yeast extract amendment, because methane is not a chemical product of dehalorespiration. Yeast extract amendment stimulated greater parent CAH removal within lower overburden Microcosm Nos. 3 and 6 than within upper overburden Microcosm No. 9, consistent with groundwater conditions at those monitoring well locations (i.e., upper overburden groundwater is generally more oxic and chemically oxidizing than lower overburden groundwater). Decreased removal efficiency for upper unit groundwater likely reflects a lag time associated with microbial acclimation to perturbed geochemical conditions.
Daughter CAHs
As with parent data, the general similarity of tf daughter CAH results between intrinsic bioremediation microcosms and inhibited controls suggests daughter CAH intrinsic bioremediation was limited over the study duration. Among intrinsic bioremediation microcosms, the greatest daughter removal, 8,041 ug/l (ti) to 4,989 ug/l (tf), occurred in Microcosm No. 1. Increased intrinsic bioremediation daughter removal for this microcosm is attributed to ti conditions at this location that are conducive for CAH methane co-oxidation (i.e., >5 mg/l DO, >+150 mV ORP, 610 ug/l methane, and 1E4 CFU/ml methanotrophs). Given suitable ti conditions and the fact no methane was detected above the MDL at tf, the data suggest methane was utilized and daughters destroyed. We note Microcosm No. 7 also had aerobic, chemically oxidizing ti conditions and high daughter CAHs; however, methane was not detected in that microcosm above the MDL. Therefore, co-oxidation may have been methane limited in Microcosm No. 7.
Enhanced bioremediation microcosms generally had lower tf 1,1,-DCE and 1,2-DCEs concentrations than intrinsic bioremediation microcosms, suggesting yeast extract amendment may also have stimulated dehalogenation of these daughters. Based on EPA Method 8010 results, enhanced bioremediation microcosms generally had a tf 1,1-DCA and VC results similar to intrinsic bioremediation microcosms; however, the samples were diluted due to high daughter CAH concentrations such that results reflected high MDLs, as discussed previously. GC headspace screening of sample aliquots from each microcosm using the Walsh and Pickering (1995) method showed that tf DCA and VC concentrations for enhanced bioremediation microcosms were higher than for intrinsic bioremediation microcosms, indicating yeast extract amendment drove reductive dehalogenation and yielded these daughters. For example, GC screening results indicated a >18% to >50% increase in DCA and VC concentration, respectively, for enhanced bioremediation microcosms relative to controls.
Total CAHs
Consistent with the tf parent and daughter CAH data, the tf total CAH data for intrinsic bioremediation microcosms are generally similar to inhibited control microcosms, again suggesting intrinsic bioremediation was limited over the study duration. Further consistency with the parent and daughter CAH data is indicated by enhanced bioremediation microcosm data that show lower tf total CAH concentrations than intrinsic bioremediation microcosms, indicating yeast extract amendment enhanced CAH biotransformation.
Parent CAH Ratio
As discussed previously, sample dilution was performed due to relatively high ti and tf daughter CAH concentrations within each microcosm. Because dilution imposed relatively high MDLs on the CAH results summarized in Table 5, which tended to mask parent CAH concentrations (e.g., tf data for Microcosm Nos. 4, 6, 7, 8, and 9), normalized parent CAH data (parent CAH ratios) are also included in that table. Parent CAH ratio refers to the ratio, in %, of total parent CAH concentrations divided by total CAH concentrations.
Parent CAH tf ratios were lower for enhanced bioremediation microcosms than for intrinsic bioremediation microcosms. Because reductive dehalogenation is generally more important for parent than daughter CAHs, the CAH signature is consistent with enhanced reductive dehalogenation due to yeast extract amendment. Consistent with the parent CAH data discussed above, there was greater reduction in the parent CAH ratio for enhanced bioremediation Microcosm No. 3 (82%Reduction) than for enhanced bioremediation Microcosm Nos. 6 (58%Reduction) and 9 (30%Reduction). The significant reduction in parent CAH ratio for this microcosm is likely related to metabolic transformation (dehalorespiration) rather than co-metabolic transformation, as discussed previously.
Microbial Data
As shown in Table 2, ti microbial populations for intrinsic and enhanced bioremediation microcosms included obligate/facultative aerobic heterotrophs, methanotrophs, and obligate anaerobes. Given their respective metabolic pathways and the microcosm results summarized herein, certain generalizations may be made about their roles in CAH transformation during the study. Obligate/facultative aerobes metabolized certain daughter CAHs in intrinsic bioremediation microcosms. These same aerobes metabolized yeast extract and scavenged major electron acceptors (i.e., molecular oxygen, nitrate, and sulfate) in enhanced bioremediation microcosms, thereby driving conditions anaerobic and chemically reducing. Methanotrophs participated in electron acceptor depletion in certain intrinsic bioremediation microcosms by metabolizing methane, and likely effected CAH co-oxidation in Microcosm No. 1. Once major alternative electron acceptors were depleted, obligate anaerobes fermented yeast extract, producing hydrogen, which drove reductive dehalogenation in enhanced bioremediation microcosms. Certain obligate anaerobes (and possibly some facultative aerobes) used parent CAHs as terminal electron acceptors in the process of dehalorespiration (e.g., Microcosm No. 3).
CONCLUSIONS
Prior to yeast extract amendment, parent CAH intrinsic bioremediation was limited by ambient DOC concentrations insufficient to stimulate indigenous bacteria to scavenge alternate electron acceptors and drive reductive dehalogenation. Yeast extract amendment provided supplemental substrate, which stimulated re-establishment of anaerobic, chemically reducing conditions by stimulating certain microbes to scavenge electron acceptors such as molecular oxygen, nitrate, and sulfate and providing substrate for anaerobic metabolism. Hydrogen, yielded during yeast extract fermentation, drove parent and select daughter CAH reductive dehalogenation over the 54-day study duration. Microcosm study results suggest biostimulation via yeast extract amendment may be an effective treatment technology for parent CAHs in site groundwater.
ONGOING WORK
Based on microcosm study results as well as ongoing groundwater monitoring, a yeast extract amendment pilot study began in July 1997 with injection of a 1,750-pound yeast extract load into a test zone at the former wastewater treatment facility site. Yeast extract solution was prepared using formation make-up water to minimize dilution. Results from seven post-injection groundwater sampling rounds within the test zone are consistent with microcosm study results. For example, yeast extract amendment resulted in significant depression of DO, ORP, nitrate, and sulfate values within two weeks of injection, relative to baseline conditions, and induced a 40% to 70% reduction in parent CAH concentrations. Subsequent yeast extract amendment was performed at another location at the site in November 1997, with similar results to date. The pilot study is ongoing, and plans include scaling-up the bioremediation program in the spring of 1999.
REFERENCES CITED
Cookson, J.T., 1995, Bioremediation Engineering: Design and Application, McGraw Hill, Inc., 524 p.
Haston, Z.C., Sharma, P.K., James, N.P., and McCarty, P.L., 1996, Enhanced dehalorespiration of chlorinated ethenes, in proceedings, Conference on the Natural Attenuation of Chlorinated Solvents, United States Air Force and Battelle Memorial Institute, p. 11-13.
Odom, J.M., Nagel, E., and Tabinowski, J., 1995b, Chemical-biological catalysis for in situ anaerobic dehalogenation of chlorinated solvents, Bioremediation of Chlorinated Solvents, ed. Hinchee, R.E., Leeson, A., and Semprini, L., Battelle Press, Inc., 338 p.
Odom, J.M., Tabinowski, J., Lee, M.D., and Fathepure, B.Z., 1995a, Anaerobic biodegradation of chlorinated solvents: Comparative laboratory study of aquifer microcosms, Bioremediation of Chlorinated Solvents, ed. Hinchee, R.E., Leeson, A., and Semprini, L., Battelle Press, Inc., 338 p.
Rasmussen, G., Komisar, S.J., and Ferguson, J.F., 1994, Transformation of tetrachloroethene to ethene in mixed methanogenic cultures: Effect of electron donor, biomass levels, and inhibitors, Bioremediation of Chlorinated and Polycyclic Aromatic Hydrocarbon Compounds, ed. Hinchee, R.E., Leeson, A., Semprini, L., and Ong, S.K., Lewis Publishers, 525 p.
Walsh, K.M. and Pickering, E.W., 1995, The rapid analysis of VOCs in environmental samples by static headspace/gas chromatography, in press, International Symposium on Volatile Organic Compounds in the Environment, American Society for Testing and Materials.
Wiedemeier T. H., Swanson, M.A., Moutoux, D.E., Wilson, J.T., Kampbell, D.H., Hansen, J.E., and Haas, P., 1996, Overview of the technical protocol for natural attenuation of chlorinated aliphatic hydrocarbons in ground water under development for the U.S. Air Force Center for Environmental Excellence, in proceedings, Natural Attenuation of Chlorinated Organics in Ground Water Symposium, p. 35-59
TABLE 4
SUMMARY OF INDICATOR PARAMETER RESULTS
Former Wastewater Treatment Facility Site
Southern New Hampshire
| Groundwater Sampling Location |
Microcosm No. |
Microcosm Simulation |
NO3- (mg/l) |
SO4-2 (mg/l) |
CH4 (mg/l) |
COD (mg/l) |
Cl- (mg/l) |
|
| GZ-4L (Lower Unit) |
1 | Intrinsic | ti | 0.08 | 47 | 610 | <5 | 85 |
| tf | 0.16 | 51 | <10 | 20 | 68 | |||
| 2 | Control | ti | 0.08 | 47 | 610 | <5 | 85 | |
| tf | 0.10 | 53 | 430 | 20 | 75 | |||
| 3 | Enhanced | ti | 0.08 | 47 | 610 | <5 | 85 | |
| tf | <0.05 | <10 | 610 | 1,580 | 70 | |||
| GZ-3L (Lower Unit) |
4 | Intrinsic | ti | 1.1 | 35 | 2,600 | 180 | 56 |
| tf | <0.05 | <10 | 2,200 | 63 | 54 | |||
| 5 | Control | ti | 1.1 | 35 | 2,600 | 180 | 56 | |
| tf | <0.05 | 32 | 1,300 | 98 | 68 | |||
| 6 | Enhanced | ti | 1.1 | 35 | 2,600 | 180 | 56 | |
| tf | <0.05 | <10 | 5,800 | 1,520 | 57 | |||
| GZ-2 (Upper Unit) |
7 | Intrinsic | ti | 0.53 | 35 | <10 | 30 | 14 |
| tf | 0.85 | 32 | <10 | 15 | 13 | |||
| 8 | Control | ti | 0.53 | 35 | <10 | 30 | 14 | |
| tf | 0.96 | <10 | <10 | 20 | 33 | |||
| 9 | Enhanced | ti | 0.53 | 35 | <10 | 30 | 14 | |
| tf | <0.05 | 23 | 690 | 1,400 | 20 | |||
NOTES:
1. ti refers to time zero.
2. tf refers to time final.
3. Abbreviations: mg/l = milligrams per liter; mg/l = micrograms per liter; Cl- = Chloride; NO3-= Nitrate; SO42- = Sulfate; CH4 = Methane; and COD = Chemical Oxygen Demand.
TABLE 5
SUMMARY OF CAH RESULTS
Former Wastewater Treatment Facility Site
Southern New Hampshire
| Groundwater Sampling Location |
Microcosm No. |
Microcosm Simulation |
Parent CAHs (mg/l) | Daughter CAHs (mg/l) | Total CAHs (mg/l) | Parent CAH Ratio (%) |
Parent % Reduction |
|||||||||||
| TCA | PCE | TCE | DCE | DCEs | DCAs | VC | Parents | Daughter | Total | |||||||||
| GZ-4L (Lower Unit) |
1 | Intrinsic | ti | 140 | 9 | 430 | ti | 19 | 7,910 | 42 | 70 | ti | 579 | 8,041 | 8,620 | ti | 12.6 | -10 |
| tf | 120 | 8 | 370 | tf | 19 | 4,909 | 42 | 19 | tf | 498 | 4,989 | 5,487 | tf | 13.9 | ||||
| 2 | Control | ti | 140 | 9 | 430 | ti | 19 | 7,910 | 42 | 70 | ti | 579 | 8,041 | 8,620 | ti | 12.6 | -12 | |
| tf | 130 | 7 | 330 | tf | 18 | 4,709 | 39 | 75 | tf | 467 | 4,841 | 5,308 | tf | 12.8 | ||||
| 3 | Enhanced | ti | 140 | 9 | 430 | ti | 19 | 7,910 | 42 | 70 | ti | 579 | 8,041 | 8,620 | ti | 12.6 | 82 | |
| tf | 85 | 1 | 2 | tf | 17 | 4,709 | 53 | 60 | tf | 88 | 4,839 | 4,927 | tf | 2.3 | ||||
| GZ-3L (Lower unit) |
4 | Intrinsic | ti | 100 | 100 | 100 | ti | 100 | 17,100 | 100 | 2,300 | ti | 300 | 19,600 | 19,900 | ti | 2.4 | 46 |
| tf | 140 | 100 | 20 | tf | 39 | 17,053 | 150 | 2,400 | tf | 260 | 19,642 | 19,902 | tf | 1.3 | ||||
| 5 | Control | ti | 100 | 100 | 100 | ti | 100 | 17,100 | 100 | 2,300 | ti | 300 | 19,600 | 19,900 | ti | 2.4 | 13 | |
| tf | 130 | 1 | 92 | tf | 35 | 17,047 | 140 | 2,000 | tf | 223 | 19,222 | 19,445 | tf | 2.1 | ||||
| 6 | Enhanced | ti | 100 | 100 | 100 | ti | 100 | 17,100 | 100 | 2,300 | ti | 300 | 19,600 | 19,900 | ti | 2.4 | 58 | |
| tf | 88 | 100 | 12 | tf | 28 | 16,049 | 160 | 2,000 | tf | 200 | 18,237 | 18,437 | tf | 1.0 | ||||
| GZ-2 (Upper Unit) |
7 | Intrinsic | ti | 230 | 1 | 42 | ti | 19 | 5,928 | 110 | 1 | ti | 273 | 6,058 | 6,331 | ti | 6.6 | 20 |
| tf | 140 | 100 | 32 | tf | 16 | 6,918 | 94 | 100 | tf | 272 | 7,128 | 7,400 | tf | 5.3 | ||||
| 8 | Control | ti | 230 | 1 | 42 | ti | 19 | 5,928 | 110 | 1 | ti | 273 | 6,058 | 6,331 | ti | 6.6 | 14 | |
| tf | 100 | 100 | 100 | tf | 100 | 5,500 | 100 | 100 | tf | 300 | 5,800 | 6,100 | tf | 5.7 | ||||
| 9 | Enhanced | ti | 230 | 1 | 42 | ti | 19 | 5,928 | 110 | 1 | ti | 273 | 6,058 | 6,331 | ti | 6.6 | 30 | |
| tf | 100 | 100 | 100 | tf | 100 | 5,400 | 100 | 100 | tf | 300 | 5,700 | 6,000 | tf | 4.6 | ||||
NOTES:
1. ti refers to time zero.
2. tf refers to time final.
3. Abbreviations: CAHs = Chlorinated aliphatic hydrocarbons; mg/l = micrograms per liter; TCA = 1,1,1-Trichlorethane; PCE = Tetrachloroethene; TCE = Trichloroethene; DCE = 1,1- Dichloroethene; DCEs = total cis- and trans-1,2 Dichloroethenes; DCAs = 1,1- and 1,2-Dichloroethanes; and VC = Vinyl chloride.
4. Parents refers to the assumed chemical reactants, 1,1,1-TCA, PCE, and TCE; Daughters refer to assumed chemical products DCE, DCEs, DCAs, and VC. Total refers to parent plus daughter CAH concentrations.
5. Results consider one half the method detection limit (MDL) for those instances when a particular parent/daughter CAH was reported as non-detect. Note that values in bold represent non-detects.
6. "Parent CAH Ratio" refers to the ratio, in percent (%), of the total parent CAH concentration divided by the total CAH concentration. Due to relatively high EPA Method 8010 MDLs, owing to high dilution factors associated with high daughter CAH concentrations, the parent CAH ratio is based upon gas chromatographic (GC) screening results (Walsh and Pickering, 1995) because: 1) dilutions were not required, and 2) GC screening MDLs were significantly lower than EPA Method 8010 MDLs.
7. "Parent % Reduction" refers to percent reduction of the parent CAH ratio for the respective microcosm.
