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A.4 Application of qPCR for Chlorinated Solvents in Groundwater for Remediation (NY)

Adapted with permission from: Davis, G., B.R. Baldwin, A.D. Peacock, D. Ogles, G.M. White, S.L. Boyle, E. Raes, S.S. Koenigsberg, and K.L. Sublette. 2008. "Integrated approach to PCE-impacted site characterization, site management and enhanced bioremediationThe treatment of environmental contamination through the use of techniques that rely on biodegradation. Bioremediation has two essential components: biostimulation and bioaugmentation.." Remediation. 18(4):5-17.

EMD Technology

• Primary: Quantitative Polymerase Chain Reaction (qPCR)
• Complementary: EMD Sampling Methods

Contacts

Todd M. Caffoe, P.E.

NYSDEC - Region 8

6274 East Avon-Lima Road

Avon, New York 14414

(585) 226-5350

[email protected]

Aaron Peacock, Ph.D.

Haley and Aldrich

(865) 356-0498

[email protected]

A.4.1 Site Background and Knowledge from Traditional Methods

The site is located in upstate New York and industrial use contaminated the area with chlorinated solvents. Several remedial actions were implemented over a period of six years, including pump and treatment remediation followed by multi-phase high vacuum extraction. An estimated 9,600 pounds of volatile organic compounds were removed using these processes before deactivation once asymptotic conditions were achieved. Identification of an in situ remedial approach was sought to obtain site closure.

Additional details are as follows:

• The shallow aquifer was impacted by the chlorinated solvents tetrachloroethene (PCE) and trichloroethene (TCE).
• Examination of groundwater geochemical parameters (dissolved oxygen, nitrate, ferrous iron, sulfate and others) suggested mildly anaerobic conditions. Furthermore, elevated levels of sulfate were observed at the site.
• Under anaerobic conditions, PCE and TCE can undergo sequential reductive dechlorination through the degradation products cis-dichloroethene (DCE) and vinyl chloride to ethene.
• Detection of degradation products in groundwater samples suggested that reductive dechlorination was occurring at least to a limited degree under existing site conditions.
• High DCE concentrations combined with relatively low vinyl chloride and ethene concentrations suggested that DCE was accumulating (commonly referred to as “DCE stall”).

A.4.2 EMD Objectives and Approach

The objectives of this study were to 1) quantify the types of microorganisms present under baseline conditions, 2) confirm the presence/absence of microorganisms capable of complete or partial reductive dechlorination of PCE to ethene, and 3) quantify the changes induced in the indigenous microbial communityThe microorganisms present in a particular sample. due to injection of different electron donors (biostimulationA remedial technique which provides the electron donor, electron acceptor, and/or nutrients to an existing subsurface microbial community to promote degradation.). Design criteria for the study included the following:

• At the time of site characterization, DehalococcoidesDehalococcoides is a genus of organohalide-respiring bacteria (for example, bacteria that use chlorinated solvents as metabolic electron acceptors) within the phylum Chloroflexi, in the domain Bacteria, and currently represented by a single species, Dehalococcoides mccartyi (Dhc). This species is the only one known with strains that dechlorinate dichloroethenes (DCEs) and vinyl chloride (VC) to ethene and inorganic chloride. mccartyi (Dhc) populations were below the laboratory detection limit indicating that the complete reductive dechlorination of PCE to ethene was unlikely.
• qPCR results during the baited Bio-Trap^® study and subsequent pilot study demonstrated that addition of electron donorA chemical compound that donates electrons to another compound (based on USEPA 2011). B (two different electron donors were tested and are referred to here as A and B) would stimulate growth of Dhc and promote reductive dechlorination.
  
• During performance monitoring, qPCR data revealed that the observed lag prior to the onset of enhanced reductive dechlorination was due to a temporary increase in methanogens and decrease in Dhc abundance following electron donor addition.
  
• Continued qPCR monitoring demonstrated the rebound in the Dhc abundance and most importantly the increase in vinyl chloride reductase genes. Thus, continued reductive dechlorination of vinyl chloride to ethene could be expected.

Site characterization and remedy selection focused on answering the following questions:

• Under existing site conditions, are microorganisms (Dhc) present that are capable of complete reductive dechlorination of PCE to ethene?
• Will adding an electron donor (biostimulation) promote growth of Dhc and enhance reductive dechlorination of chlorinated ethenes?

To address these questions, a preliminary study was conducted in which sets of three passive microbial sampling devices (specifically, Bio-Traps^®) were deployed in select monitoring wells located within the dissolved plume:

• The control Bio-Trap^® represented existing subsurface conditions and therefore contained no addition electron donors (Control).
• The second Bio-Trap^® contained a commercial electron donor A (BioStim A).
• The third Bio-Trap^® contained an alternative commercial electron donor B (BioStim B).

Following a 60 day in-well deployment period, the passive microbial sampling devices were recovered for Quantitative Polymerase Chain Reaction (qPCR) analysis to quantify:

• Dhc – the only known speciesThe lowest taxonomic rank, and the most basic unit or category of biological classification.(www.biology-online.org) of microorganisms capable of complete reductive dechlorination of PCE to ethene.
• Vinyl chloride reductase geneA segment of DNA containing the code for a protein, transfer RNA, or ribosomal RNA molecule (based on Madigan et al. 2010). (bvcA) – functional geneA segment of DNA that encodes an enzyme or other protein that performs a known biochemical reaction. For example, the functional gene tceA encodes the reductive dehalogenase enzyme that initiates reductive dechlorination of TCE. Other genes can code for RNA entities which can regulate the activity of other DNA target sequences. encoding the enzymesAny of numerous proteins or conjugated proteins produced by living organisms and facilitating biochemical reactions (based on USEPA 2004a). responsible for reductive dechlorination of vinyl chloride to ethene.

A.4.3 Results

The results of the studies are presented here. Figure A.4-1 includes the results of the qPCR analyses across the three wells where the Bio-Traps^® were deployed.

Figure A.4-1: Results of qPCR quantification of Dhc following recovery of Bio-Traps^® from select monitoring wells following a 60 day deployment period.

Source: Adapted from Davis et al. 2008. Used with permission.

Observations and implications for the qPCR results (Figure A.4-1):

• In the Control Bio-Traps^®, Dhc populations were below the laboratory detection limit indicating that complete reductive dechlorination of PCE to ethene was unlikely under existing site conditions.
• With BioStim A Bio-Traps^®, Dhc were only detected at Well 1 and at a low concentration suggesting that the addition of electron donor A did not promote growth of these key halorespiring bacteria, at least within the deployment period.
• Conversely, Dhc were detected in each of the BioStim B samplers and at concentrations up to 10⁴ cells/bead.

The results suggested the following for the site remedy selection:

• Monitored natural attenuation (MNA) was eliminated as a potential remedy based upon Dhc populations being below laboratory detection limits under existing site conditions and historical groundwater monitoring data suggesting DCE stall.
• Biostimulation with electron donor A was eliminated as a potential remedy based upon the fact that Dhc populations in the BioStim A samplers were not substantially greater than in the Control sampler.
• Biostimulation with electron donor B was selected for subsequent pilot and full scale corrective actions based in part upon qPCR evidence demonstrating growth of Dhc in the preliminary Bio-Trap^® study.

In the pilot scale test, electron donor B was injected in the vicinity of Well 12. Groundwater samples were obtained for VOC analysis. Standard, un-amended Bio-Traps^® deployed in the injection zone wells were recovered quarterly for qPCR analysis of:

• Dhc – the only known group of microorganisms capable of complete reductive dechlorination of PCE to ethene.
• Methanogens – methanogens can compete with Dhc and other reductive dechlorinating bacteria for available electron donors.
• Vinyl chloride reductase gene (bvcA) – functional gene encoding the enzymes responsible for reductive dechlorination of vinyl chloride to ethene.

• The results of the monitoring during the pilot scale testing are shown in Figure A.4-2 (Days 0 through 200):

  

  Figure A.4-2. Performance monitoring results through day 200.

  Source: Adapted from Davis et al. 2008. Used with permission.

• Prior to the electron donor injection, Dhc were detected on the order of 10³ cells/bead. However, methanogens, who can compete with Dhc for available electron donors were present on the order of 10⁴ cells/bead.
• After approximately 100 days, the DCE concentration had increased while production of vinyl chloride and ethene was not evident suggesting that electron donor addition had not enhanced continued reductive dechlorination of DCE.
• The qPCR results revealed that electron donor addition had initially promoted growth of methanogens while Dhc populations decreased substantially. A temporary increase in competing microorganisms combined with a decrease in Dhc is not uncommon following an electron donor addition.
• By day 200, the Dhc population had at least rebounded to levels detected prior to injection while the methanogen population decreased.  Substantial reductive dechlorination of DCE to vinyl chloride and ethene, however, was still not observed.

The results of the monitoring during the pilot scale testing are shown in Figure A.4-3 (Days 300 through 400):

• By day 300, the Dhc population had increased by three orders of magnitude to over 10⁶ cells/bead.

• By day 400, the DCE concentration had decreased from 30 to 5.8 mg/L with corresponding production of vinyl chloride.

  

Figure A.4-3. Performance monitoring results through day 400.

Source: Adapted from Davis et al. 2008. Used with permission.

• Production of vinyl chloride, which is documented to be more hazardous than DCE, TCE or PCE, occurred.
• The qPCR quantification of the bvcA vinyl chloride reductase gene indicated the presence of microorganisms capable of reductive dechlorination of vinyl chloride to ethene. Thus, at day 400, the qPCR results provided stakeholders with reassurance that the increase in vinyl chloride concentration would be temporary, and complete reductive dechlorination to ethene could be expected.

The results of the monitoring during the pilot scale testing are shown in Figure A.4-4 (Days 400 through 500):

• The Dhc population and abundance of vinyl chloride reductase genes were even greater at day 500 than at day 400.

• Consistent with the qPCR results, the vinyl chloride concentration decreased, with a corresponding increase in ethene production.

  

Figure A.4-4. Performance monitoring results through day 500.

Source: Adapted from Davis et al. 2008. Used with permission.

A full scale biostimulation project was implemented at the site with similar results. More information about the project is reported in Davis et al. 2008.

A.4.4 Conclusions

Site characterization and remedy selection resulted in the following conclusions:

• qPCR results demonstrated that the observed DCE stall was due to the lack of microorganisms capable of continued reductive dechlorination of DCE to vinyl chloride and ethene.
• MNA was eliminated as a potential remedy. Dhc and the bvcA vinyl chloride reductase gene were not detected under baseline conditions, indicating that complete reductive dechlorination of PCE to ethene was unlikely.
• Biostimulation with electron donor B was selected as the site remedy. qPCR results demonstrated that electron donor B, but not electron donor A, stimulated growth of Dhc species capable of complete reductive dechlorination of PCE to ethene.

Performance monitoring resulted in the following conclusions:

• qPCR analysis revealed that the initial lag in reductive dechlorination (through day 200) was due to stimulation of competing microorganisms (methanogens) and a decrease in chlorinated ethene degrading bacteria (Dhc) following electron donor injection.
• During the pilot study, qPCR results conclusively demonstrated that electron donor B stimulated growth of bacteria capable of complete reductive dechlorination of PCE.
• Increases in Dhc and the vinyl chloride reductase gene bvcAcorresponded to decreases in DCE concentration and production of the degradation products vinyl chloride and ethene, linking the changes in contaminant concentrations to growth of known halorespiring bacteria.
• When vinyl chloride concentrations increased, qPCR quantification of the vinyl chloride reductase gene provided stakeholders with reassurance that the increase in vinyl chloride concentration would be temporary and that complete reductive dechlorination to ethene could be expected.
• Maintenance of elevated populations of Dhc and bvcA vinyl chloride reductase gene strongly suggested that enhanced reductive dechlorination would continue and that a second electron donor injection was not necessary.

A.4.5 Costs

The cost for a study as described above is around $4,500 per well. This cost includes using a control and two different biostimulations per monitoring well, along with monitoring for qPCR analysis (Dhc, vinyl chloride reductase, iron/sulfate reducing bacteria, methanogens), geochemistry, and contaminant concentrations.

Continuing with quarterly qPCR monitoring of Dhc, vinyl chloride reductase (bvcA), iron/sulfate reducing bacteria, and methanogens was approximately $500 per sample, per event.

A.4.6 Outcomes and Challenges

• The qPCR results were accepted as a valuable line of evidence and the site was reclassified to “No Further Action Required” status.
• Integrating qPCR results with traditional chemical and geochemical analyses provided the converging lines of evidence required to direct site management activities.
• In addition to qPCR, quantification of contaminant degrading microorganisms and quantification of competing microorganisms (such as methanogens and sulfate reducing bacteria) can provide valuable insight when biostimulation is not performing as well as anticipated.

A.4.7 References

Davis, G., B.R. Baldwin, A.D. Peacock, D. Ogles, G.M. White, S.L. Boyle, E. Raes, S.S. Koenigsberg, and K.L. Sublette. 2008. "Integrated approach to PCE-impacted site characterization, site management and enhanced bioremediation." Remediation 18(4): 5-17.

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