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A.9 SIP for Fuel Oil in Groundwater for Remediation (NJ)

Adapted with permission from:  K. Key, K.L. Sublette, T. Johnnes, E. Raes, E. Sullivan, D. Ogles, B.R. Baldwin, and A, Biernacki. 2013. An in situ bioreactor for the treatment of petroleum hydrocarbons in groundwater. Remediation. Spring. (Publication Pending).

EMD Technology

• Primary: Stable Isotope Probing (SIP)
• Complementary: Quantitative Polymerase Chain Reaction (qPCR)

Contacts

Mr. Jon Malkin

New Jersey Department of Environmental Protection

(609) 633-1201

[email protected]

Mr. Eric J. Raes, PE, PP

Engineering and Land Planning Associates, Inc.

(908) 238-0544, ext. 10

[email protected]

A.9.1 Site Background and Knowledge from Traditional Methods

In 1994, a release of No. 2 fuel oil occurred beneath a historic house constructed in 1839. Fuel oil compounds persist beneath the structure. To date, several remedial efforts have been completed including soil removal, oxygen release compound injections, and a small-scale chemical oxidation remediation. Biostimulation was observed after the chemical oxidation, but was not sustained. Chemical testing indicated the following:

• Low levels of fuel oil related compounds persist in groundwater above regulatory levels.
• Two oxygen release product injections failed to achieve and sustain acceptable levels.
• Chemical oxidation injection failed to achieve and sustain acceptable levels due to low permeability soils and the source location.

A.9.2 EMD Objectives and Approach

SIP was used for this site to evaluate the impacts of in situ chemical oxidation on the indigenous microbial organisms. Once biostimulationA remedial technique which provides the electron donor, electron acceptor, and/or nutrients to an existing subsurface microbial community to promote degradation. was observed, SIP and qPCR were used to confirm that biostimulation processes could be sustained through the use of an in situ bio-reactor (ISBR).

The initial testing included the following activities.

• SIP was used prior to, during, and subsequent to the chemical oxidation (persulfate-based chemical oxidation) program to monitor the effects of the remedial action on the indigenous microbial communityThe microorganisms present in a particular sample..
• Each EMD sampling device (Bio-Trap^®) was pre-loaded (baited) with a known, small quantity (97+/- ug/bead) of ¹³C labeled naphthalene. Mass loss by chemical oxidation and mass loss and mineralization of naphthalene through microbial degradation was monitored. Mineralization was quantified through dissolved inorganic carbon readings and geneA segment of DNA containing the code for a protein, transfer RNA, or ribosomal RNA molecule (based on Madigan et al. 2010). expression by mRNA/qPCR analysis (Geyer et al. 2005).
• Background groundwater samples collected away from the release revealed almost no microbial activityRefers to when a microorganism performs a specific function (e.g., sulfate reduction, metabolism of benzene). This result explained the failure of the two previous, oxygen release compound remedial efforts at the site.

The use of EMDs during chemical oxidation revealed that the site was suitable for biostimulation of the petroleum compounds. The full-scale effort included sustainable aerobic biostimulation and microbial analyses

Sustainable aerobic biostimulation was achieved through the installation of a novel ISBR (see Figure A.9-1). The central portion of the ISBR is filled with Bio-Sep beads; the sparge stone resides on the bottom of bead bed and serves two functions:

• Dissolved oxygen is provided to hydrocarbon-degrading microbes that populated the Bio-Sep medium. 
• The tiny air bubbles create a circulation element within the ISBR, where contaminated groundwater is pulled in from the bottom of the ISBR, passes through the Bio-Sep medium and exits the top, as illustrated in the dye test study below. This configuration allows for healthy microbes from within the ISBR to migrate into the well column and into the formation beyond the well to further promote the biodegradationA process by which microorganisms transform or alter (through metabolic or enzymatic action) the structure of chemicals introduced into the environment (USEPA 2011). of the residual petroleum hydrocarbons in the aquifer.

Figure A.9-1. Photographs of the ISBR; photographs show dye released at base of ISBR is “uplifted” through the beads and exits the top into the well.

Source: K. Sublette 2012. Used with permission.

Microbial analyses using qPCR of the EMD sampling device (Bio-Trap^® Sampler) three months after the ISBR was installed in the well confirmed that microbial gene NAH expression was occurring, supporting the conclusion that biodegradation of the petroleum hydrocarbons was occurring.

A.9.3 Results

The results from sampling during and after chemical oxidation events demonstrated:

• The persulfate-based injection, while removing mass from the baited EMD sampling device (54%), had little impact on the microbial community, which was predominantly dormant for aerobic processes prior to and during the chemical oxidation remedial action.
• Subsequent to the injection, biostimulation of petroleum-degrading microorganisms was inadvertently promoted. Significant ¹³C was incorporated in the dissolved inorganic carbon (DIC) indicating that naphthalene losses were the result of biodegradation with mineralization of the hydrocarbon. In addition, mRNA analyses of naphthalene dioxygenase (NAH) and phenol hydroxylase (PHE) genes were conducted to investigate the potential for and the microbial gene expression of metabolic pathways responsible for aerobic biodegradation of naphthalene (Baldwin et al. 2003).  NAH expression, which had not been noted previously, was detected after chemical oxidation  treatment was completed indicating activity of aerobic naphthalene-utilizing bacteria (Baldwin et al. 2010). The biostimulation could have been predicted, as the chemical oxidation process generates partially-oxidized materials that are more readily consumed by bacteria. In addition, the chemical oxidation included ozone (O₃) injection, which left behind dissolved oxygen in the groundwater. This was used by petroleum-degrading bacteria, which were previously detected but were not functional under background conditions.
• The biostimulation was not sustainable, as the partially-oxidized materials and dissolved oxygen were not maintained, as observed in the microbial analysis (lack of NAH expression) collected four months after the chemical oxidation events. Figure A.9-2 includes the data from the SIP results.
• The use of SIP defined a transition point in the remedial strategy, whereas a less costly and more effective sustainable biostimulation remedy was pursued in lieu of the continuation of the more costly chemical oxidation strategy.

Figure A.9-2. SIP results before, during, and after ISCO.

Source: E. Raes 2012. Used with permission.

After six months of operations, a groundwater sample was collected from well WP-1R; the results were below the NJDEP GWQS for the first time in 17 years (see Figure A.9-3). In fact, the results were reported as nondetect for all targeted volatile organic and base neutral compounds, and nondetect for VOCs TICs. Base neutral TICs were reported as 135 mg/l.

Figure A.9-3. Comparison of SIP results after ISCO and ISBR operations.

Source: E. Raes 2012. Used with permission.

A.9.4 Conclusions

• Through SIP, aerobic biodegradation processes under background conditions were confirmed to be insufficient.
• A more effective and less costly remedial strategy was determined through the use of EMDs, specifically the combination of SIP and qPCR analyses (Suzuki et al. 2000).
• Using EMD sampling devices coupled with EMD laboratory techniques, sustainable biostimulation was confirmed (Johnson et al. 2005).

A.9.5 Costs

Table A.9-1 summarizes the analytical costs associated with the EMDs used in this study.

A.9.6 Outcomes and Challenges

The most significant challenges were as follows:

• The ISBR was installed in the compliance well. The regulatory agency required the ISBR be removed, and for groundwater levels to remain below their regulatory standard for 90 days after biostimulation ceased at the site.
• Determining what, if any, permits were required, was a challenge because the SIP used EMD sampling devices that were a relatively new technology to the regulatory agency. In the end, the agency decided no permits were required.
• Sample handling became a significant issue, when the field technician failed to properly store and ship the Bio-Traps^®. While mass loss and DIC analysis were still possible, the mRNA data was compromised and invalidated since the holding times for microbial analyses were exceeded.

A.9.7 References

Baldwin, B. R., Nakatsu, C. H., and Nies, L. 2003. "Detection and enumeration of aromatic oxygenaseAn enzyme that catalyzes the incorporation of molecular oxygen into a compound (based on Madigan et al. 2010). genes by multiplex and real-time PCR. [Evaluation Studies Research Support, Non-U.S. Gov't]." Applied and Environmental Microbiology 69(6):3350-3358.

Baldwin, B. R., Biernacki, A., Blair, J., Purchase, M. P., Baker, J. M., Sublette, K., Ogles, D. 2010. "Monitoring gene expression to evaluate oxygen infusion at a gasoline-contaminated site." Environmental Science & Technology 44(17):6829-6834. doi: 10.1021/es101356t.

Geyer, R., Peacock, A. D., Miltner, A., Richnow, H. H., White, D. C., Sublette, K. L., & Kastner, M. 2005. "In situ assessment of biodegradation potential using biotraps amended with ¹³C-labeled benzene or toluene. [Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S.]." Environmental Science & Technology 39(13):4983-4989.

Johnson D.R., P.K. Lee, V.F. Holmes, L. Alvarez-Cohen. 2005.  "An internal reference technique for accurately quantifying specific mRNAs by real-time PCR with application to the tceA reductive dehalogenaseAn enzyme that catalyzes the removal of a halogen atom from an organic compound. gene." Applied and Environmental Microbiology 71(7):3866-3871.

Key, K.L. Sublette, D. Ogles, B.R. Baldwin, and Raes, E.J. 2013. "An in situ bioreactor for the treatment of petroleum hydrocarbons in groundwater." Remediation. Spring.

Suzuki, M. T., Taylor, L. T., & DeLong, E. F. 2000. "Quantitative Analysis of Small-Subunit rRNA Genes in Mixed Microbial Populations via 5′-Nuclease Assays." Applied and Environmental Microbiology, 66(11):4605-4614. doi: 10.1128/aem.66.11.4605-4614.

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