A.7 EAP for TCE in Groundwater for Remediation (KY)

A.7 EAP for TCE in Groundwater for Remediation (KY).

Adapted with permission from: Lee, Hope M., Looney, Brian B., Hampson, S.K. 2008. "Enzyme Activity Probe and Geochemical Assessment for Potential Aerobic Cometabolism of Trichloroethene in Groundwater of the Northwest Plume, Paducah Gaseous Diffusion Plant, Kentucky." WSRC-STI-2008-00309.

EMD Technology

Primary: Enzyme Activity Probes (EAP)
Complementary: Compound Specific Isotope Analysis (CSIA)

Contacts

Blaine Rowley

U.S. Department of Energy

(301) 903-2777

[email protected]

M. Hope Lee

Pacific Northwest National Laboratory

( 240) 818-2987

[email protected]

A.7.1 Site Background and Knowledge from Traditional Methods

Groundwater below a portion of Paducah Gaseous Diffusion Plant in Paducah Kentucky is contaminated with chlorinated solvents, primarily trichloroethene, TCE. TCE was first released to the subsurface as the result of site operations, which included disposal and burial of hazardous and radioactive materials across the site, from 1952 to the mid 90s (Lee et al. 2008b, p. 11). The dominant source of contamination at the site is associated with continued TCE and ⁹⁹Tc releases in the vicinity of building 400. The site is the location of an old river bed and as such groundwater moves quickly, approximately 3 feet per day; site operations, and the local geology have resulted in two defined groundwater contaminant plumes, northwest and northeast, which originate near building 400 and end in tributaries off site. Other defined contaminant plumes (not considered in this study) exist on site and are associated with waste and burial grounds; all of the mentioned groundwater plumes share similar geochemistry and thus potential for attenuation of the contaminants under intrinsic conditions.

In 1997 Clausen et al. documented biological attenuation of chlorinated solvents in groundwater at the Paducah site; however the observed half-life degradation rates were estimated to be between 9.4 and 26.7 years. Based on the chemical and geochemical sampling results, the relatively slow rates of degradation, aerobic co-metabolism of TCE was suspected as the dominant mechanism for attenuation but was not confirmed in the study. At that time, the estimated rates were considered much too slow to accomplish site goals and no further studies were proposed.

Unlike reductive dechlorination of TCE under anaerobic conditions, where biological break-down products, such as 1-2, cis-dichloroethene or vinyl chloride are produced, degradation of TCE under aerobic conditions can only be inferred by indirect measures of carbon dioxide, disappearance of the contaminants, geochemical conditions (elevated dissolved oxygen and an appropriate carbon substrateAny substance that is acted upon by an enzyme.), and EMDs.

A.7.2 EMD Objectives/Approach

Enzyme Activity Probes (EAPs) were used to directly measure enzyme activityRefers to when a microorganism performs a specific function (e.g., sulfate reduction, metabolism of benzene) of target enzymesAny of numerous proteins or conjugated proteins produced by living organisms and facilitating biochemical reactions (based on USEPA 2004a). known to be produced during co-metabolic degradation of chlorinated solvents such as TCE.

EAPs directly measure if methane and/or aromatic (substrates such as toluene, benzene, phenol) enzyme production is occurring. These enzymes are documented to be produced for the degradation of methane and aromatic compounds and also break down TCE in a process referred to as co-metabolism (Lee et al. 2005, 2008a; Keener et al. 1998, 2001; Clingenpeel et al. 2005; Wymore et al. 2007; Miller et al. 2002).

For this study, the EAP results were compared to the following additional lines of evidence: (1) quantitative and traditional PCR for the genes of interest (oxygenases), (2) carbon stable isotopic analysis, (3) geochemical conditions, (4) contaminants trends, and (5) conservative tracers (⁹⁹Tc).

Collectively, these additional lines of evidence were used to validate the EAPs as a useful tool for confirming the presence of co-metabolic processes and more importantly to confirm co-metabolic degradation of TCE was occurring in situ and at measurable rates at the Paducah site (Lee et al. 2008a).

A.7.3 Results

An assessment was conducted to determine if EAPs could be used to confirm co-metabolic destruction of TCE. In this study, 12 wells were selected along the centerline of the northwest plume as well as two control wells outside the contaminant plume at the site. The wells are shown in Figure A.7-1.

Figure A.7-1: Map displaying wells where either sMMO enzyme-activity probe, and/or one or more toluene enzyme-activity probes were used.

Source: PRS, 2007; reprinted in Lee et al. 2008b.

As shown in Table A.7-1, positive results were established for both toluene oxygenases (9 out of 12 wells) and the soluble methane monooxygenase (sMMO) enzymes (7 out of 12 wells). Inhibitor studies supported these findings (data not shown). Quantitative toluene probe results represent the percentage of the total biomass determined to be active; values greater than 3% are considered significant.

Table A.7-1. Comparison of qualitative and quantitative enzyme-activity probe results to contaminant concentrations in groundwater from monitoring wells at the Northwest Plume at PGDP

Table A.7-1. Comparison of qualitative and quantitative enzyme-activity probe results to contaminant concentrations in groundwater from monitoring wells at the Northwest Plume at PGDP. Source: Table 3, Lee et al. 2008b.
Well	Aquifer	Screened interval depth (ft)	TCE * (µg/L)	Tc-99 (pCi/L)	Qualitative preliminary data (6/4/7)		Toluene probes quantitative data (% of Total DAPI)			Total –DAPI cells/mL
					sMMO probe Coumarin	Toluene probes	3HPA	PA	Cinnamo- nitrile
1MW168	URGA	63 - 68	10,000 to 100,000	3260	-	-	0.00	1.27	0.00	1.90E+05
MW66		55 - 60	1,000 to 10,000	3670	+	+++	3.90	5.73	2.49	3.67E+05
MW194		47 - 52	<MCL	17	+	+++	1.78	5.41	6.80	1.76E+05
MW197		58 - 63	<MCL	283	-	+	1.09	3.95	0.14	1.59E+06
MW185	MRGA	68 - 73	1,000 to 10,000	1260	-	++	1.84	1.41	0.20	9.75E+05
MW242		65 - 75	100 to 1,000	341	-	-	0.46	0.16	1.14	7.76E+05
MW243		65 - 75	100 to 1,000	3860	-	-	0.77	1.08	0.31	4.27E+05
MW381		66 - 76	100 to 1,000	329	-	++	6.36	3.64	0.57	9.66E+05
MW262	LRGA	90 - 95	1,000 to 10,000	4178	+	+++	3.83	3.86	7.92	3.52E+05
MW340		85.5 - 95.3	1,000 to 10,000	747	+	+	0.05	1.32	0.00	7.25E+05
MW236		69.5 - 79.5	100 to 1,000	936	+	+++	3.66	5.95	1.05	8.84E+05
MW125		78 - 88	100 to 1,000	273	+	++	1.74	7.97	2.54	7.99E+05
URGA: Upper Regional Gravel Aquifer MRGA: Middle Regional Gravel Aquifer LRGA: Lower Regional Gravel Aquifer TCE: Trichloroethene Tc-99: Technicium 99 3HPA: 3-hydroxy-phenylacetylene PA: Phenylacetylene DAPI: 4',6-Diamidino-2-Phenylindole (double stranded DNA staining) MCL: Maximum Contaminant Level									ft bgs– feet below ground surface µg/L – micrograms per liter pCi/L – picocuries per liter cells/mL – per milliliter

DNA Control Study: A DNA control study was performed to determine if key genes of interest were present in the groundwater samples collected from the northwest plume, corresponding to groundwater samples taken for EAP analyses. Positive responses were observed for the PCR and qPCR analyses, providing a secondary line of evidence that the oxygenaseAn enzyme that catalyzes the incorporation of molecular oxygen into a compound (based on Madigan et al. 2010). genes of interest, various oxygenases, were present in situ and correlated well with the positive results observed with EAPs.
Geochemical Data: Although the groundwater geochemistry varied spatially within the plume, the results were generally conducive to aerobic co-metabolism (neutral pH, dissolved oxygen from 0.6 to 5.8 mg/L).
CSIA Study: The CSIA data showed that isotopic fractionationSome processes (for example, those which involve breaking chemical bonds) have slightly different rates for different isotopes, leading to a more rapid consumption of one isotope over the other. This characteristic is manifested in a change in the isotopic ratio of the residual compound. from paired groundwater wells within the northwest plume was indicative of aerobic degradation of the contaminant TCE. In this case, differences between δ¹³C values (even small, > -0.9), in groundwater monitoring wells and ‘fresh solvent’ or current source zone values, is indicative of slow aerobic degradation. The δ ¹³C values obtained in this evaluation are similar to other published data (-0.8 to -1.3), but differ from those focused on reductive dechlorination, which typically result in a decreasing trend over the length of the contaminant plume. These data support the conclusion that degradation of the TCE occurs along the centerline of this plume. These data serve as another line of evidence for natural attenuation of chlorinated solvents at the Paducah site and validate the EAP results which confirmed the presence and activity of key enzymes involved in the co-metabolic degradation of TCE.

Table A.7-2: Summary of CSIA Results (Source: Table 7, Lee et al, 2008b)
Sample ID	δ¹³C (per mil)	Comments
Northwest plume wells along the flow path
MW-168	-24.8	Near source
MW-262	-25.8
MW-340	-25.9
MW-185	-25.9
MW-242	-24.6
MW-243	-25.3
MW-125	-25.6
MW-381	-25.4
MW-236	-25.3	Distal portion of plume
MW-66	-25.3	Near downgradient source
MW-197	-23.1	Control well, outside of plume

A.7.4 Conclusions

Through EAP, the key enzymes produced in the aerobic co-metabolic degradation of TCE were confirmed present and active in situ.
CSIA determined that aerobic biodegradationA process by which microorganisms transform or alter (through metabolic or enzymatic action) the structure of chemicals introduced into the environment (USEPA 2011). of TCE was likely occurring within the northwest plume, based on isotopic fractionation; these data correlated well with the EAP data.
DNA analyses also supported the EAP data, providing evidence that the genes of interest, oxygenases, were present throughout the groundwater locations sampled.
EAPs provided key data in support of natural attenuation processes occurring within the northwest plume at the Paducah site.

A.7.5 Costs

Analytical costs associated with EMDs are included in Table A.7-3 below.

Table A.7-3. Summary of analytical costs associated with the EMDs during the study.
EMD	No. of Samples	Cost per Sample	Total Cost
EAP and PCR	12	2700	$32,400
CSIA	12	500	$ 6,000
Total			$38,400

A.7.6 Outcomes and Challenges

The technologies, CSIA, enzyme activity probes, and microcosmA sample that is regarded as a small but representative portion of something larger. In environmental studies microcosm are typically small samples of soil, sediment, or water incubated in enclosed containers under laboratory conditions. studies, were still in demonstration phase when applied at the Paducah site. A collaborative effort was undertaken to demonstrate the appropriate knowledge and understanding was in place prior to applying the technology which proved very successful for this site. Specific criteria were defined before a groundwater sample was taken and included the definition for success for any one of the technologies. As a result, the contractor, technical staff, and regulators all agreed prior to data collection, what would be deemed sufficient evidence for aerobic co-metabolism to be a significant process within the sampled groundwater plume.

While there was a large amount of historical and current groundwater monitoring data including contaminant concentrations and a range of geochemical parameters, many of the aerobic biological indicators were not known or previously measured. A large effort was undertaken by the site in collaboration with the state and federal regulators to determine the appropriate geochemical parameters to be measured in support of aerobic degradation of chlorinated solvents. During the investigation it was also determined that the method for determining oxygen concentrations in groundwater had been changed several times over the past decade, and more importantly the regulators were not confident in the methods used. Working with the site contractor, a more robust method (time and expense) was used to monitor oxygen concentrations throughout several groundwater plumes at the site. It was also concluded that there were insufficient developed wells within the predominant groundwater plume and the DOE along with the site, installed 75 new MW locations across the site in order to better monitor groundwater and in order to develop a more complete conceptual site model.

A.7.7 References

Clausen, J.L. N.C. Sturchio, L.J. Heraty, L. Huang, T. Abrajano. 1997. "Evaluation of Natural attenuation processes for trichloroethylene and technetium-99 in the northeast and northwest plumes at the Paducah Gaseous Diffusion Plant, Paducah, Kentucky." Lockheed Martin, for the Department of Energy. KY/EM-113.

Clingenpeel, S.R., Keener, W.K., Keller, C.R., De Jesus, K., Howard, M.H., and Watwood, M.E. 2005. "Activity-dependent fluorescent labeling of bacterial cells expressing the TOL pathway." Journal of Microbiological Methods 60:41-46. PMID 15567223.

Keener, W. K., M. E. Watwood, and W. A. Apel. 1998. "Activity-dependent fluorescent labeling of bacteria that degrade toluene 2,3-dioxygenase." Applied and Environmental Microbiology 49:455-462.

Keener, W. K., M. E. Watwood, K. D. Schaller, M. R. Walton, J. K. Partin, W. A. Smith, and S. R. Clingenpeel. 2001. "Use of selective inhibitors and chromogenic substrates to differentiate bacteria based on toluene oxygenase activity." J. Microb. Meth. 46:171-185.

Lee, M.H., S.C. Clingenpeel, O.P. Leiser, and M.E. Watwood. 2005. Molecular and Physiological Characterization of Aerobic TCE Degradation Potential. Eighth International In Situ and On-Site Bioremediation Symposium. Battelle Press, Columbus, OH.

Lee, M.H., S.C. Clingenpeel, O.P. Leiser, R.A. Wymore, K.S.Sorenson, Jr., and M.E. Watwood 2008a. "Activity-Dependent Labeling of Oxygenase Enzymes in a Trichloroethene-Contaminated Groundwater Site." Environmental Pollution 153:238-246.

Lee, Hope M., Looney, Brian B., Hampson, S.K. 2008b. "Enzyme Activity Probe and Geochemical Assessment for Potential Aerobic Cometabolism of Trichloroethene in Groundwater of the Northwest Plume, Paducah Gaseous Diffusion Plant, Kentucky." WSRC-STI-2008-00309.

Miller, A.R., W.K. Keener, M.E. Watwood, and , F. Roberto. 2002. "A rapid fluorescence-based assay for detecting soluble methane monooxygenase." Applied Microbiology and Biotechnology 58:183-188. PMID 11876411.

PRS, 2007. Tricholorethene and Technetium-99 Contamination in the Regional Gravel Aquifer for the Calendar Year 2005, Paducah Gaseous Diffusion Plant, Paducah, Kentucky, PRS/Project/0019.

Wymore, R.A., M.H. Lee, A.R. Miller, W.K. Keener, F.S. Colwell, M.E. Watwood, and K.S. Sorenson, Jr. 2007. "Field Evidence for Intrinsic Aerobic Chlorinated Ethene Cometabolism by Methanotrophs Expressing sMMO." Bioremediation Journal 11(3):125-139

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