U.S. EPA Contaminated Site Cleanup Information (CLU-IN)

U.S. Environmental Protection Agency
U.S. EPA Technology Innovation and Field Services Division

Dense Nonaqueous Phase Liquids (DNAPLs)

Detection and Site Characterization

Microcosm Delineation for Biodegradation

Abstracts of Journal Articles

Application of Bio-Trap Samplers in Conjunction with Real-Time PCR (CENSUS) Analysis to Directly Assess Petroleum Hydrocarbon Degradation
Davis, G.A., D. Ogles, D. McElroy, and J. Streufert (Microbial Insights, Rockford, TN); A. Peacock (Univ. of Tennessee, Knoxville); K. Sublette (Univ. of Tulsa, Tulsa, OK). The 11th Annual International Petroleum Environmental Conference, 11-15 October 2004, Albuquerque, NM. The Integrated Petroleum Environmental Consortium (IPEC), Univ. of Tulsa, OK.

Bio-Trap samplers are small devices that can provide an integrated picture of a microbial community over time, thus compensating for inherent variability associated with other subsurface sampling approaches. The samplers were combined with molecular-based diagnostic tools to evaluate microbial communities from three different BTEX-contaminated aquifers as a basis for developing remediation strategies. Molecular diagnostic tools based upon real-time PCR (CENSUS) were used to quantify the abundance of specific microbial functional genes associated with BTEX degradation. This combined approach led to an increased understanding of site dynamics and provided a rapid, direct method to obtain accurate information on the effectiveness of monitored natural attenuation and enhanced bioremediation strategies.

A New Tool for Characterization of Microbial Activity in Contaminated Soils During Remediation and Restoration
Sublette, K.L., C. Mehta, A. Moralwar, and L. Ford (Univ. of Tulsa, Tulsa, OK); K. Duncan (Univ. of Oklahoma, Norman); G. Thoma (Univ. of Arkansas, Fayetteville); A. Peacock and D. White (Univ. of Tennessee, Knoxville); G. Davis (Microbial Insights, Inc., Rockford, TN). The 10th Annual International Petroleum Environmental Conference, 11-14 November 2003, Houston, TX. The Integrated Petroleum Environmental Consortium (IPEC), Univ. of Tulsa, OK. [abstract only]

Bio-traps containing Bio-Sep® beads have been used to monitor subsurface microbial communities in soil where crude oil was remediated with and without fertilizer addition and in associated tilled and undisturbed prairie controls. Beads were deployed at 1, 3, and 5 inches in each plot in triplicate in mesh bags for five weeks in May and June of 2003. The soil surrounding the beads also was retrieved. Triplicates (soil and beads) were then composited and analyzed for biomarkers, including phospholipids fatty acids and 16S rDNA. The microbial communities in the beads were found to be enriched in C16 fatty acids relative to the soil communities, were more aerobic in character, and exhibited faster growth rates. Bead communities were less diverse than soil communities, and dominant bacteria in the soil were not necessarily dominant in the beads. Differences could be seen in bead communities within the same plot at different depths, which was not the case for soils. Bio-Sep® beads appear to offer potential advantages over soil analysis in the sampling of soil microbial communities in terms of selection of the more active community members and detection of subtle differences with depth.

Quantitative Detection of and Bioaugmentation with Reductive Dechlorinators
Koenigsberg, S.S. and E. Rasch, Regenesis Bioremediation Products, San Clemente, CA. The 20th Annual International Conference on Soils, Sediments and Water, 18-21 October 2004, University of Massachusetts at Amherst.

With the discovery that the microorganisms responsible for the complete biodegradation of chlorinated solvents may not be present at all sites, bioaugmentation with mixed cultures capable of degrading a wide range of contaminants is now being successfully used in the field to increase the rate of reductive dechlorination and reduce the time that it takes to obtain site closure. By using a "differential diagnosis" approach, bioaugmentation can be used to close sites that may otherwise not have achieved complete reductive dechlorination within a reasonable time frame. Real-time polymerase chain reaction (RT-PCR) is a technique in which the number of organisms in a sample can be determined by measuring the amount fluorescence of produced during the PCR reaction. Available commercially as Bio-Dechlor CENSUS(SM), this technique is now being used not only to determine if the necessary microorganisms are present within the aquifer in the appropriate numbers and whether the addition of external organisms is required, but also to track the changes induced in the microbial community by the addition of a carbon source or the addition of organisms. Recent advances in this technology have allowed functional genes to be targeted, or gene sequences that are specific to organisms able to perform the desired reductive dechlorination tasks, as opposed to only genes on the 16S rRNA sequence that are common to all organisms with this phylogenic group. DNA-based methods such as denaturing gradient gel electrophoresis (DGGE) and lipid analyses such as phospholipid fatty acid analysis (PLFA) can also be used to observe the effects of bioaugmentation or biostimulation on the microbial community as a whole, often more quickly than the effects of the application can be observed through VOC analysis only. The integration of these techniques and others into existing monitoring programs provides a greater understanding of what is occurring within the subsurface.

Case Studies

Advancement of Nucleic Acid-Based Tools for Monitoring In Situ Reductive Dechlorination
Vangelas, K., E. Edwards, F. Loeffler, B.B. Looney. Paper No: WSRC-STI-2006-00332, 23 pp, Nov 2006

Regulatory protocols generally recognize that destructive processes are the most effective mechanisms that support natural attenuation of chlorinated solvents. In many cases, these destructive processes will be biological processes and, for chlorinated compounds, will often be reductive processes that occur under anaerobic conditions. The existing EPA guidance (EPA, 1998) provides a list of parameters that provide indirect evidence of reductive dechlorination processes. In an effort to gather direct evidence of these processes, scientists have identified key microorganisms and are currently developing tools to measure the abundance and activity of these organisms in subsurface systems. The research presented in this paper continues the development efforts to provide a suite of tools to enable direct measures of biological processes related to the reductive dechlorination of TCE and PCE. The study investigated the strengths and weaknesses of the 16S rRNA gene-based approach to characterizing natural attenuation capabilities in samples. The results suggest that an approach based solely on 16S rRNA may not provide sufficient information to document the natural attenuation capabilities in a system because it does not distinguish between strains of organisms that have different biodegradation capabilities. The results of the investigations provided evidence that tools focusing on relevant enzymes for functionally desired characteristics may be useful adjuncts to the 16SrRNA methods.

Metabolic Biomarkers for Monitoring in Situ Anaerobic Hydrocarbon Degradation
Young, Lily Y. and Craig D. Phelps, Rutgers, New Brunswick, NJ. Environmental Health Perspectives, Vol 113 No 1, p 62-67, Jan 2005

Researchers have made great progress in understanding the metabolism of hydrocarbons by anaerobic bacteria. Organisms capable of utilizing benzene, toluene, ethylbenzene, xylenes, alkanes, and polycyclic aromatic hydrocarbons have been isolated and described, and the mechanisms of degradation for these compounds have been elucidated. This basic research has led to the development of methods for detecting in situ biodegradation of petroleum-related pollutants in anoxic groundwater. Knowledge of the metabolic pathways used by anaerobic bacteria to break down hydrocarbons has allowed the identification of unique intermediate compounds that can be used as biomarkers for in situ activity. One of these unique intermediates is 2-methylbenzylsuccinate, the product of fumarate addition to o-xylene by the enzyme responsible for toluene utilization. Laboratory studies show that this compound can be used as a reliable indicator of anaerobic toluene degradation. Field studies confirmed that the biomarker is detectable in field samples and its distribution corresponds to areas where active biodegradation is predicted. Three biomarkers [2-naphthoic acid (2-NA), tetrahydro-2-NA, and hexahydro-2-NA] were identified for naphthalene that can be used in the field to identify areas of active in situ degradation.

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