A.5 qPCR for Remediation (CA)

Adapted with permission from: ESTCP, 2010, “A Low Cost Passive Approach for Bacterial Growth and Distribution for Large‐Scale Implementation of Bioaugmentation,” Project ER-200513, Washington, DC. www.serdp.org.

EMD Technology

• Primary: Quantitative Polymerase Chain Reaction (qPCR)
• Complementary: Compound Specific Isotope Analysis (CSIA)

Contacts

Joey Trotsky

Naval Facilities Engineering and Expeditionary Warfare Center

1100 23rd Ave

Port Hueneme, CA  93043

805-982-1258

[email protected]

 

CDM Smith

Ryan A. Wymore

720-264-1110

[email protected]

A.5.1 Background and Knowledge from Traditional Methods

Naval Weapons Station Site 70 is the former National Aeronautics and Space Administration (NASA) Research Testing and Evaluation Area, which was a rocket engine test facility in Seal Beach, California. Past operations at the facilities reportedly included the use of trichloroethene (TCE) along with other contaminants. Currently groundwater is contaminated with TCE in the area of interest.

Sequential reductive dechlorination of TCE under anaerobic conditions is a well documented pathway to remediate TCE in groundwater. However, certain conditions are required for complete dechlorination to ethene. These conditions include groundwater geochemistry, presence of dechlorinating bacteria 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. (Dhc), and pH. Because very little dechlorination of TCE to degradation products cis-1,2-DCE (DCE), vinyl chloride, and ethene had occurred, it was apparent that the conditions required for complete dechlorination were not present. Geochemical data suggest that high sulfate concentrations may be limiting full anaerobic dechlorination of TCE to ethene, but there were also concerns that the Dhc presence was not strong enough for complete dechlorination.

Figure A.5-1. Site location showing TCE concentration contours and passive/active treatment cells.

Source: Adapted from ESTCP 2010.

A.5.2 EMD Objectives and Approach

The Environmental Security Technology Certification Program (ESTCP) recently funded a study to evaluate passive and active approaches to bioaugmentationThe introduction of cultured microorganisms into the subsurface environment for the purpose of enhancing bioremediation of organic contaminants (USEPA 2011) to clean up the TCE contaminated groundwater. Analytical techniques were used to assist in the evaluation of the study included quantitative polymerase chain reaction (qPCR)A laboratory analytical technique for quantification of a target gene based on DNA. and carbon specific isotopeTwo atoms with the same number of protons but a different number of neutrons. analysis (CSIA). More details are available in ESTCP 2010.

The overall objective of this study was to compare bioaugmentation strategies using passive and active distribution approaches for chlorinated solvent contaminated groundwater. To support this objective, the following objectives required qPCR and CSIA to evaluate performance:

• Use qPCR methods to demonstrate that at least one commercially available bioaugmentation culture can carry out complete dechlorination in the presence of high sulfate concentrations.
• Use qPCR to determine if Dhc are present on site; if so, use qPCR to select a culture that contains a Dhc strain or 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. not present naturally at site.
• Use qPCR to determine bacterial growth and distribution throughout the treatment cells using both bioaugmentation approaches.
• Use CSIA as a supplemental tool to help determine extent of dechlorination in both treatment cells during the test period.

A.5.3 Results

qPCR was used during three key phases of the demonstration: baseline monitoring, pre-conditioning, and post-bioaugmentation performance assessment. The use of qPCR during each phase is discussed further below. 

A.5.3.1 Baseline monitoring

During baseline monitoring activities, TCE was detected at concentrations up to 60,000 micrograms per liter (µg/L). However, intermediate products DCE, vinyl chloride, and ethene were either not detected or were less than 5% of the TCE concentrations. Additionally, although the groundwater geochemistry appeared relatively anaerobic, sulfate was detected between 1,000 and 8,000 milligrams per liter (mg/L). Lastly, qPCR analysis indicated that Dhc was not present in the study area in the majority of the samples collected. In the few areas where Dhc was present, only tceA was present at levels above detection but below reporting limits, while bvcA and vcrA were not observed.

These results indicated that high sulfate concentrations were likely limiting complete anaerobic dechlorination. Additionally, because the Dhc only contained tceA (which encodes enzymesAny of numerous proteins or conjugated proteins produced by living organisms and facilitating biochemical reactions (based on USEPA 2004a). to degrade TCE to vinyl chloride) and not bvcA or vcrA (which both encode enzymes to degrade vinyl chloride to ethene), the naturally occurring Dhc was not likely to perform complete dechlorination.

A.5.3.2 Pre-conditioning

Based on these data, a “pre-conditioning” step was performed by adding sodium lactate to the study area to decrease sulfate concentrations and to create reducing conditions suitable for bioaugmentation. Figures A.5-2a-c show results of the pre-conditioning phase (prior to the bioaugmentation event shown by the vertical orange line). Although the pre-conditioning step was successful at creating highly reducing conditions and reducing sulfate concentrations (Figure A.5.2a), complete dechlorination was still not occurring (Figure A.5.2b). This result was expected, as qPCR results indicated that even after pre-conditioning, Dhc populations remained undetected or below 10⁴ geneA segment of DNA containing the code for a protein, transfer RNA, or ribosomal RNA molecule (based on Madigan et al. 2010). copies per liter (Figure A.5-2c). Additionally, functional gene analysis indicated that even with the slight increase in Dhc populations after pre-conditioning, the functional gene vcrA was still not detected throughout the study area.

 

Figures A.5-2a-c from active cell well AMW-2 show decreased sulfate following initiation of pre-conditioning in April 2008. However, “DCE-stall” was observed until bioaugmentation in January 2009. qPCR results show that although Dhc started to appear by November 2008, populations remained low and vcrA was not present.

Source: ESTCP 2010.

A.5.3.3 Bioaugmentation

Following pre-conditioning, injection wells were inoculated in January 2009 with commercially available Dhc culture SDC-9^™. The use of qPCR and standard analytical techniques were used to evaluate the function of the bioaugmented culture in dechlorination.

qPCR was used to evaluate distribution of the bioaugmentation culture, including “first arrival,” as well as growth of Dhc over time.  Results showed that the bioaugmentation culture had transport times similar to that of a conservative tracer, with the first detection of Dhc at monitoring wells two weeks following bioaugmentation. As shown in Figures A.7.2b-c (active cell) above and A.7.3 a, b below (passive cell), enhanced dechlorination and sustained elevated Dhc (with vcrA) populations were observed almost immediately following bioaugmentation. In both the active and passive cells, Dhc and functional gene populations increased 4-7 orders of magnitude, indicating that bioaugmentation using both approaches was successful to introduce a more effective culture with increased abilities to fully dechlorinate TCE.

 

Figures A.5-3 a, b from passive cell well PMW-6 show minimal dechlorination until bioaugmentation in January 2009. PMW-6 was 8 feet downgradient from injection wells and showed almost immediate Dhc presence.  Additionally, since culture used had functional genes tceA and vcrA but no bvcA, these functional genes increased with Dhc but bvcA was no longer detected.

Source: ESTCP 2010.

A.5.3.4 CSIA to Verify Dechlorination

CSIA was used along with the qPCR and dechlorination data to verify that dechlorination was occurring during the study and the plume was not being diluted or displaced by injection activities. CSIA data were consistent with the dechlorination data, in that they suggested degradation to vinyl chloride and ethene was occurring where VOC data suggest active dechlorination was occurring.  An example CSIA chart is included as A.5.4 for PMW-6.  This chart shows that TCE, c-DCE, and VC become enriched in the heavier isotope (¹³C) during the course of the demonstration, indicating degradation is occurring. 

Figure A.5-4 CSIA Data

Source: ESTCP 2010.

A.5.4 Conclusions

The conclusions from this part of the site work include:

• qPCR showed that dechlorinating bacteria were not present at adequate levels prior to addition of electron donorA chemical compound that donates electrons to another compound (based on USEPA 2011)..
• The functional gene analysis using qPCR showed that even after electron donor addition, vinyl chloride reductase gene vcrA was not present in the indigenous community, and thus could be used as a “biomarkerA distinctive (unique) characteristic of a biomolecule that can be measured and used as an indicator of a target microorganism or biological process. For example, a specific DNA sequence (used as a probe on a microarray) could be a biomarker for a particular microorganism (e.g., Desulfotomaculum).” for the bioaugmentation culture.
• qPCR was used to track bacterial distribution following bioaugmentation, with results indicating that Dhc transport occurred nearly as fast as groundwater velocity.
• qPCR was used to assess growth of Dhc over time in response to bioaugmentation and repeated electron donor injections.  Results showed a strong correlation to presence of Dhc (and specifically vcrA) at or above 10⁶ gene copies/L to complete dechlorination.
• Overall, similar electron donor distribution and dechlorination performance was achieved in both passive and active cells; however, more donor was required and more operational issues were encountered with active approach.
• CSIA was used as a secondary line of evidence to demonstrate that complete dechlorination was occurring at the site.

A.5.5 Costs

Costs for analysis of qPCR samples for Dhc and the three functional genes bvcA, vcrA, and tceA were $300 per sample for this demonstration project. However, this cost was based on the large number of samples required for this project. Costs for this analysis for a nonresearch-based project may range from $350-$400/sample.

A.5.6 Outcomes and Challenges

• The regulatory agencies understood that the purpose of the work at this facility was for an applied research project. Therefore, no issues were encountered with regulatory acceptance.
• In addition, bioremediationThe treatment of environmental contamination through the use of techniques that rely on biodegradation. Bioremediation has two essential components: biostimulation and bioaugmentation. with bioaugmentation is the final CERCLA remedy for this site. qPCR is being used to track growth and distribution of Dhc as a part of the performance monitoring program for the final remedy.
• Integrating qPCR results with traditional chemical and geochemical analyses provided the converging lines of evidence required to optimize the demonstration activities.
• One challenge at the site was the high sulfate concentrations (up to 9,000 mg/L), which created uncertainty regarding whether complete dechlorination could be stimulated at the site, even with bioaugmentation. The qPCR results provided the earliest indication that Dhc could be distributed and could grow at this site, well before the chemistry data indicated that dechlorination was occurring.

A.5.7 References

ESTCP, 2010, “A Low Cost Passive Approach for Bacterial Growth and Distribution for Large‐Scale Implementation of Bioaugmentation,” Project ER-200513, Washington, DC.

 

Publication Date: April 2013