A.8 SIP for Chlorinated Solvents in Groundwater for Remediation (AZ)

Adapted with permission from: Chiang, S. D., R. Mora, W. H. Diguiseppi, G. Davis, K. Sublette, P. Gedalanga, and S. Mahendra. 2012. “Characterizing the intrinsic bioremediationThe treatment of environmental contamination through the use of techniques that rely on biodegradation. Bioremediation has two essential components: biostimulation and bioaugmentation. potential of 1,4-dioxane and trichloroethene using innovative environmental diagnostic tools.” Journal of Environmental Monitoring 14: 2317-2326. Reproduced by permission of The Royal Society of Chemistry (RSC). http://pubs.rsc.org/en/content/articlelanding/2012/em/c2em30358b.

EMD Technology

Primary: Stable Isotope Probing (SIP)
Complementary: Enzyme Activity Probes (EAPs), qPCR

Contacts

Rebecca Mora

AECOM

(714) 689-7254

[email protected]

Air Force Civil Engineer Center (AFCEC)

Environmental Center of Excellence (ECoE)

Environmental Restoration Technical Support Branch (CZTE)

Adria Bodour, Ph.D.

(210) 395-8426

[email protected]

A.8.1 Site Background and Knowledge from Traditional Methods

Air Force Plant 44 is a missile assembly plant that historically used trichloroethene (TCE) and 1,1,1-trichloroethane (1,1,1-TCA) as solvents. 1,4-dioxane was a stabilizer in 1,1,1-TCA and consequently, was also released to the environment. Currently, primary contaminants at the site are TCE, 1,4-dioxane, and 1,1-dichloroethene (1,1-DCE).

A groundwater extraction, treatment (air stripping), and reinjection system has been operating since 1987. Treatment was upgraded to advanced oxidation in 2009 to treat 1,4-dioxane, in addition to volatile organic compounds (VOCs). Monitored natural attenuation (MNA) is being considered as part of the final remedy to reduce the operational timeframe of the pump and treat system. TCE and 1,4-dioxane contaminant trend analysis indicates concentrations are declining steadily over time. Examination of groundwater geochemical parameters (dissolved oxygen, nitrate, ferrous iron, sulfate, methane, and oxidation reduction potential) indicated conditions were aerobic.

It has been established that TCE can be biodegraded to carbon dioxide under aerobic conditions through co-metabolism without accumulation of toxic intermediate products. Biodegradation of 1,4-dioxane, which historically was thought to be insignificant, has been confirmed in recent years and can occur through co-metabolism as well as where 1,4-dioxane is used as a growth-supporting substrateAny substance that is acted upon by an enzyme. (Zenker et al. 2000; Fam 2005; and Mahendra and Alvarez-Cohen 2006). The 1,4-dioxane biodegradationA process by which microorganisms transform or alter (through metabolic or enzymatic action) the structure of chemicals introduced into the environment (USEPA 2011). pathway, which also results in mineralization to carbon dioxide, was documented in Mahendra et al. (2007), and is the same for co-metabolic and growth-supporting processes.

A.8.2 EMD Objectives and Approach

The study was designed to evaluate intrinsic aerobic biodegradation (via co-metabolism and/or growth-supporting processes) of TCE and 1,4-dioxane to determine whether MNA could be considered as a component of the site remedial strategy. Four EMDs were used to evaluate site-specific biodegradation and confirm degradation mechanisms. The EMDs were applied using a stepwise approach which involved separate sequential sampling events. This approach allowed for optimization of sampling location selection for the more expensive analyses as they were based on results of previous steps.

The study involved answering the following questions using specific EMDs, which were applied in the order they are presented:

Are bacteria and enzymesAny of numerous proteins or conjugated proteins produced by living organisms and facilitating biochemical reactions (based on USEPA 2004a). capable of aerobically degrading TCE and/or 1,4-dioxane present at the site?
Are TCE and/or 1,4-dioxane being aerobically degraded at the site?
Are enzymes capable of degrading TCE and/or 1,4-dioxane metabolically active at the site?

A.8.3 Methods and Results

To address Question 1, Bio-Trap^® and groundwater samples were collected from wells throughout the TCE and 1,4-dioxane plume (source area, mid-plume, and downgradient) and analyzed by qPCR for available qPCR targets related to TCE and/or 1,4-dioxane aerobic degradation. Table A.8-1 includes the qPCR targets.

Table A.8-1. Biomarkers related to biodegradation of TCE and 1,4-dioxane
Biomarker Code	Bacteria or Enzymes	TCE	1,4-Dioxane
MOB	Methane oxidizing bacteria (Methanotrophs)	Yes	Yes
sMMO	Soluble methane monooxygenase	Yes	Yes
PHE	Phenol hydroxylase/ Toluene 2-,3-,4-monooxygenase	Yes	Yes
RMO	Toluene 3-,4-monooxygenase	Yes	Yes
TOD	Toluene 2,3-dioxygenase	Yes	No

Figure A.8-1 shows the results for qPCR quantification of bacteria and enzymes capable of degrading TCE and 1,4-dioxane from the Bio-Trap^® samplers.

Figure A.8-1: Results for qPCR quantification of bacteria and enzymes capable of degrading TCE and 1,4-dioxane in Bio-Trap^® samples from select monitoring wells.

Source: Adapted from Chiang , S.D., R. Mora, W. H. Diguiseppi, G. Davis, K. Sublette, P. Gedalanga, and S. Mahendra. 2012. “Characterizing the intrinsic bioremediation potential of 1,4-dioxane and trichloroethene using innovative environmental diagnostic tools.” Journal of Environmental Monitoring 14: 2317-2326. Reproduced by permission of The Royal Society of Chemistry (RSC). http://pubs.rsc.org/en/content/articlelanding/2012/em/c2em30358b.

Figure A.8-2 shows the results for qPCR quantification for groundwater samples.

Figure A.8-2: Results for qPCR quantification of bacteria and enzymes capable of degrading TCE and 1,4-dioxane in groundwater samples from select monitoring wells.

Source: Adapted from Chiang, S.D., R. Mora, W. H. Diguiseppi, G. Davis, K. Sublette, P. Gedalanga, and S. Mahendra. 2012. “Characterizing the intrinsic bioremediation potential of 1,4-dioxane and trichloroethene using innovative environmental diagnostic tools.” Journal of Environmental Monitoring 14: 2317-2326. Reproduced by permission of The Royal Society of Chemistry (RSC). http://pubs.rsc.org/en/content/articlelanding/2012/em/c2em30358b.

The observations based on qPCR results include:

Bacteria and enzymes capable of degrading TCE and 1,4-dioxane were present and abundant at the site.
No correlation exists between the abundance of each target and contaminant concentrations (that is, targets were not more abundant at locations with high contaminant concentrations).
Some differences were noted between Bio-Trap^® and groundwater samples, especially with regard to the RMO and PHE biomarkers.

To address Question 2, SIP was performed. While the qPCR step revealed the potential for biodegradation of TCE and 1,4-dioxane, SIP provides direct proof of contaminant biodegradation. Bio-Traps^® baited with specially- synthesized TCE and 1,4-dioxane that were approximately 15% ¹³C (as compared to the typical 1% ¹³C present in organic compounds) were deployed in select wells. Once SIP Bio-Traps^® were retrieved after approximately 60 days of incubation, the BioSep beads were analyzed for:

¹³C incorporation into phospholipidA type of biomolecule that is a primary structural component of the membranes of almost all cells. fatty acids (PLFAPhospholipid fatty acids derived from the two hydrocarbon tails of phospholipids.), an essential component of cell membranes, indicating the contaminant supports bacterial growth;
Dissolved inorganic carbon (DIC) (equivalent to carbon dioxide) indicating complete mineralization of the contaminant; and
Percent loss of the ¹³C baited compound off of the Bio-Trap^® during deployment.

Figure A.8-3 includes the results for ¹³C incorporation into PLFA for ¹³C TCE and ¹³C 1,4-dioxane.

Figure A.8-3: SIP results for ¹³C incorporation into PLFA for ¹³C TCE and ¹³C 1,4-dioxane baited Bio-Traps^® from select monitoring wells.

Source: Chiang, S.D., R. Mora, W. H. Diguiseppi, G. Davis, K. Sublette, P. Gedalanga, and S. Mahendra. 2012. “Characterizing the intrinsic bioremediation potential of 1,4-dioxane and trichloroethene using innovative environmental diagnostic tools.” Journal of Environmental Monitoring 14: 2317-2326. Reproduced by permission of The Royal Society of Chemistry (RSC). http://pubs.rsc.org/en/content/articlelanding/2012/em/c2em30358b.

Figure A.8-4 shows the results for ¹³C incorporation into DIC (carbon dioxide) for ¹³C TCE and ¹³C 1,4-dioxane.

Figure A.8-4: SIP results for ¹³C incorporation into DIC (carbon dioxide) for ¹³C TCE and ¹³C 1,4-dioxane baited Bio-Traps^® from select monitoring wells.

Figure A.8-5 shows the results for percent loss of ¹³C TCE and ¹³C 1,4-dioxane.

Figure A.8-5: SIP results for percent loss of ¹³C TCE and ¹³C 1,4-dioxane from baited Bio-Traps^® from select monitoring wells.

The observations based on SIP results include:

¹³C incorporation into PLFA for the ¹³C TCE baited Bio-Traps^® was observed in two wells (E-15M and M-69). Because TCE is not directly metabolized under aerobic conditions (only co-metabolically metabolized), the enrichment is likely due to direct metabolism of co-metabolic TCE intermediate products such as formic and glyoxylic acids.
¹³C incorporation into PLFA for the ¹³C 1,4-dioxane baited Bio-Traps^® was observed in three out of four wells with significant incorporation in Well E-15M. These data indicate that 1,4-dioxane is supporting bacterial growth at the site.
¹³C incorporation into DIC was detected in all of the ¹³C TCE and 1,4-dioxane baited Bio-Traps^® indicating at least some conversion of both contaminants to carbon dioxide.
¹³C TCE loss ranged from variable ranging from 0% to 43% while ¹³C 1,4-dioxane loss was consistent and significant ranging from 82% to 90%. The loss of ¹³C 1,4-dioxane was likely due to physical leaching of 1,4-dioxane off the BioSep beads into the aquifer.

To address question number 3, groundwater samples were collected and analyzed using enzyme activity probes (EAPs)Transformation of surrogate compounds (probes) resembling contaminants produces a fluorescent (or other distinct) signal in cells which is then detected using a microscope.. Four probes, phenylacetylene (PA), 3-hydroxyphenylacetylene (3-HPA), trans-cinnamonitrile (CINN), and 3-ethylnyl benzoate (3EB), were used to measure the activityRefers to when a microorganism performs a specific function (e.g., sulfate reduction, metabolism of benzene) of toluene monooxygenase and/or dioxygenase enzymes (PHE, RMO, TOL, and TOD). One probe, coumarin, was used to measure the activity of sMMO. Table A.8-2 includes the EAP results for toluene oxygenases and soluble methane monooxygenase enzymes.

Table A.8-2. EAP results for toluene oxygenases and soluble methane monooxygenase enzymes from selected monitoring wells in cells per milliliter.

	Probes for toluene oxygenases (PHE, RMO, TOL, TOD)				Probe for sMMO
Well	PA	3-HPA	CINN	3EB	Coumarin
M-69	-	1.05x10⁴	-	-	15.22
M-69	8.21x10³	1.25x10⁴	-	-	-
M-01A	2.54x10⁴	-	2.14x10⁴	8.12x10³	-
M-81	2.15x10⁴	2.04x10⁴	-	-	42.11
M-105	2.68x10⁴	2.21x10⁴	1.12x10⁴	-	-
M-101	3.54x10⁴	-	-	-	-
M-95	2.45x10⁴	-	1.42x10⁴	-	-

The observations based on EAP results include:

There is widespread enzyme activity in the wells that were sampled, with each well showing at least one positive result with one EAP and five out of six wells showing activity for more than one EAP.
These results are evidence that intrinsic aerobic biodegradation is occurring at the site.

A.8.4 Conclusions

qPCR results showed that bacteria and enzymes capable of degrading TCE and 1,4-dioxane under aerobic conditions are present and abundant at the site.
SIP results showed that ¹³C from 1,4-dioxane was incorporated into bacterial PLFAs, indicating the contaminant may serve as a growth-supporting substrate at the site.
SIP results demonstrated TCE and 1,4-dioxane mineralization to carbon dioxide is occurring at the site.
EAP results confirmed that enzymes capable of degrading TCE and 1,4-dioxane under aerobic conditions are not just present, but metabolically active at the site.
EMD results confirmed that aerobic degradation of TCE and 1,4-dioxane is occurring and may be responsible for decreasing contaminant trends at the site.
MNA can be considered as a component of the site remedy.
This was the first study confirming intrinsic biodegradation of 1,4-dioxane under field conditions.

A.8.5 Costs

Table A.8-3 summarizes the analytical costs associated with the EMDs used in this study.

Table A.8-3: Summary of analytical costs associated with the EMDs during the study.

EMD	No. of Samples	Cost per Sample	Total Cost
qPCR (5 biomarkers)	25	$425	$10,625
CSIA (TCE)	5	$350	$1,750
SIP (TCE)	6	$1,650	$9,900
SIP (1,4-Dioxane)	4	$2,070	$8,280
EAP (5 probes)	7	$2,375	$16,625
Total			$47,180

A.8.6 Outcomes and challenges

The significant outcomes and challenges were as follows:

A significant physical loss of ¹³C 1,4-dioxane from the SIP Bio-Traps^® likely occurred during deployment. However, valuable data regarding aerobic biodegradation of 1,4-dioxane at the site was still obtained.
SIP and EAP are expensive EMDs which limited their broad application throughout the plume.
Using multiple EMDs provided multiple lines of evidence to support the conclusion that intrinsic aerobic biodegradation of both TCE and 1,4-dioxane is occurring at the site.
qPCR and EAP confirmed the presence and activity of desired enzymes but SIP provided unambiguous results confirming TCE and 1,4-dioxane biodegradation at the site.
SIP can be used to prove that degradation is occurring but it cannot provide information on the rate at which it is occurring. A 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. study using groundwater and soil from the site is currently being conducted by Dr. Shaily Mahendra, at UCLA, to evaluate the intrinsic aerobic biodegradation rate for TCE and 1,4-dioxane at the site as well as any increases in the rate through biostimulationA remedial technique which provides the electron donor, electron acceptor, and/or nutrients to an existing subsurface microbial community to promote degradation. (e.g., addition of a primary substrate such as methane or propane) or bioaugmentationThe introduction of cultured microorganisms into the subsurface environment for the purpose of enhancing bioremediation of organic contaminants (USEPA 2011) with Pseudonocardia dioxanivorans, a bacteria known to grow on and degrade 1,4-dioxane.
Using a stepwise approach allowed evaluation of the results of each EMD before selecting sampling locations for the next EMD. This resulted in a more optimized and cost-effective approach.

A.8.7 References

Chiang, S.D., R. Mora, W. H. Diguiseppi, G. Davis, K. Sublette, P. Gedalanga, and S. Mahendra. 2012. “Characterizing the intrinsic bioremediation potential of 1,4-dioxane and trichloroethene using innovative environmental diagnostic tools.” Journal of Environmental Monitoring 14: 2317-2326. Reproduced by permission of The Royal Society of Chemistry (RSC). http://pubs.rsc.org/en/content/articlelanding/2012/em/c2em30358b.

Fam, S.A., Fogel, S., and M. Findlay, 2005, “Rapid Degradation of 1,4-Dioxane using a Cultured Propanotroph,” Proceedings of the International In Situ and On-Site Bioremediation Symposium, Baltimore, Maryland, June 6-9, 2005.

Mahendra, S. and L. Alvarez-Cohen, 2006, “Kinetics of 1,4-Dioxane Biodegradation by Monooxygenase-expressing Bacteria,” Environmental Science &Technology, 40 (17):5435-5442.

Mahendra, S., C. J. Petzold, E. E. Baidoo, J. D. Keasling, and L. Alvarez-Cohen, 2007, “Identification of the Intermediates and End-products of 1,4-Dioxane Biodegradation by Monooxygenase-expressing Bacteria,” Environmental Science & Technology, 41 (21): 7330 -7336.

Zenker, M.J., R.C. Borden, and M.A. Barlaz, 2000, “Mineralization of 1,4-dioxane in the presence of a structural analog,” Biodegradation. Volume 11, Number 4, 239-246.

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