Developing and Field-Testing Genetic Catabolic Probes for Monitored Natural Attenuation of 1,4-Dioxane with a One-Year Timeframe

ER-2301

Objective

Remediation of aquifers contaminated with 1,4-dioxane (dioxane) is a difficult task because dioxane can be recalcitrant to biodegradation, is not easily removed by volatilization or adsorption, and is highly mobile in groundwater. Monitored natural attenuation (MNA), which relies primarily on biodegradation, is often the most cost-effective approach to manage large and dilute groundwater plumes of priority pollutants, such as those formed by dioxane. However, the burden of proof that MNA is an appropriate solution lies on the proponent, which requires demonstration of the presence and expression of relevant biodegradation capabilities.

This project seeks to develop catabolic gene probe(s) to quantify the presence and expression of dioxane biodegradation capacity to support decisions to select or reject MNA at dioxane-impacted sites. Specific tasks for this one-year limited- scope project are to:

  1. Discern dioxane degradation genes in Pseudonocardia dioxanivorans CB1190 (CB1190)
  2. Design probes to target conserved regions of these genes (focusing on the active sites of soluble di-iron monooxygenases (SDIMOs), which should enable quantification of similar genetic biomarkers in other dioxane-degrading bacteria
  3. Determine the selectivity and sensitivity of these degenerate functional gene probes by testing them with a wide variety of reference strains
  4. Validate these probes with samples from various dioxane-impacted sites.

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Technical Approach

CB1190 is the only annotated strain capable of mineralizing dioxane. The researchers found several monooxygenase genes in CB1190 that were up-regulated by dioxane and their role in dioxane catabolism will be discerned in this project using a combinatorial approach consisting of expression cloning and gene knock-outs. Sequence alignments of the relevant monooxygenase genes and other putative dioxane-degrading SDIMO genes will be used to design degenerate primers targeting DNA sequences for both the highly conserved binuclear iron center and active residues of the α-subunits. The iCODEHOP or GeneFisher2 programs will be used as a starting point for probe design. The reliability, selectivity, and sensitivity of these probes will be determined using pure cultures capable of metabolizing or co-metabolizing dioxane, using real-time quantitative PCR to quantify the presence and expression of the genetic biomarker(s). Probe validation would involve determining whether degradation activity (in microcosms) correlates with biomarker copy numbers, if copy numbers increase when dioxane is consumed, and if the relative abundance of these copy numbers (determined as % of the total population) also increases relative to background samples due to the selective pressure exerted by dioxane.

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Benefits

Since many dioxane plumes could be effectively managed by MNA at a small fraction of alternative treatment costs, it is important to develop simple and reliable forensic tools that enable site-specific decisions to select or reject MNA and to assess performance. This project will generate novel insight into the molecular basis of dioxane biodegradation, as well as a gene probe set to unequivocally characterize both dioxane biodegradation potential and activity for enhanced MNA forensics. (Anticipated Project Completion - 2013)

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Points of Contact

Principal Investigator

Dr. Pedro Alvarez

Rice University

Phone: 713-348-5903

Fax: 703-348-5203

Program Manager

Environmental Restoration

SERDP and ESTCP

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.