Development of Biomarkers for Assessing In Situ RDX Biodegradation Potential
ER-1606
Objective
Explosives have contaminated many military sites in North America and Europe. The explosive RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) is a particularly problematic groundwater contaminant because of its relatively high solubility, recalcitrance, toxicity, and frequency of use. The most cost-effective remediation approach for RDX is likely to be bioremediation; however, because little is known about the microorganisms involved, it is not yet possible to predict the effectiveness and reliability of this cleanup strategy.
This project was conducted in two phases. The objective of Phase I was to identify the microorganisms responsible for the biodegradation of RDX in complex, mixed culture samples through the application of stable isotope probing. This effort was completed in 2010. The objective of Phase II is to build on the research conducted under Phase I, by applying the techniques developed in Phase I to a wider range of samples, including those from RDX contaminated sites. The Phase II efforts are planned to be completed in 2013.
Technical Approach
Phase I efforts involved the screening of ten soils for RDX degradation and the development and application of DNA stable isotope probing to identify RDX degraders. This approach had two distinct advantages: i) RDX degradation can be investigated in mixed cultures and environmental samples (more typical of contaminated site conditions) and ii) only active organisms are targeted.
Phase II efforts will involve the application of stable isotope probing to the other five soils previously identified as having RDX degradation activity. In addition, soils will be obtained from RDX contaminated sites for stable isotope probing analysis. Following this, enrichments and isolations will be performed to confirm and further examine the RDX degrading species. The second task will involve the development of primer pairs based on the stable isotope probing and enrichment studies. Finally, the effectiveness of using these biomarkers for predicting RDX degradation in a large number of environmental samples will be determined.
Benefits
Most data on RDX biodegradation has originated from pure cultures or enrichments, with little information of how these organisms act in environmental samples or in situ. This lack of knowledge makes it difficult to determine if natural attenuation is an appropriate remediation approach for RDX. The innovative molecular methods applied in this project could provide this much-needed information. Further, once RDX-degrading organisms are identified and methods have been developed to enumerate them, this information then can be used to investigate their presence at contaminated sites so that potential for natural attenuation can be determined. Such information will facilitate the establishment of more cost-effective and efficient RDX remedial plans, expediting the cleanup of RDX-contaminated DoD sites.
Interim Results
In Phase I, RDX degradation activity was noted in six of ten soils and degradation occurred only under anaerobic conditions. The stable isotope probing experiments involved exposing soil microcosms to labeled RDX, DNA extraction, DNA ultracentrifugation (to separate the labeled nucleic acid from the unlabeled background nucleic acid), terminal restriction fragment length polymorphism (TRFLP) and 16S rRNA gene sequencing to identify the organisms responsible for label uptake from RDX. Partial 16S rRNA gene sequencing indicated the organisms responsible belonged to either the Sphingobacteria or the Acidobacteria. These bacteria have had no previous links to RDX degradation, indicating the discovery of novel RDX degraders. In summary, the proof-of-concept that stable isotope probing could be used to identify in situ RDX degraders in complex, mixed culture samples was proven successful. The results of this study can be found in the Phase I Final Report.
Project Documents
Points of Contact
Principal Investigator
Dr. Alison Cupples
Michigan State University
Phone: 517-432-3370
Fax: 571-355-0250
Project Documents
Document Types
- Fact Sheet - Brief project summary with links to related documents and points of contact.
- Final Report - Comprehensive report for every completed SERDP and ESTCP project that contains all technical results.
- Cost & Performance Report - Overview of ESTCP demonstration activities, results, and conclusions, standardized to facilitate implementation decisions.
- Technical Report - Additional interim reports, laboratory reports, demonstration reports, and technology survey reports.
- Guidance - Instructional information on technical topics such as protocols and user’s guides.
- Workshop Report - Summary of workshop discussion and findings.
- Multimedia - On demand videos, animations, and webcasts highlighting featured initiatives or technologies.
- Model/Software - Computer programs and applications available for download.
- Database - Digitally organized collection of data available to search and access.