Developing and Field-Testing Genetic Catabolic Probes for Monitored Natural Attenuation of 1,4-Dioxane

Dr. Pedro Alvarez | Rice University



Remediation of aquifers contaminated with 1,4-dioxane is a difficult task because dioxane is highly mobile in groundwater, can be recalcitrant to biodegradation, and is not easily removed by volatilization or adsorption. 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 and requires site-specific demonstration of the presence and expression of relevant biodegradation capabilities.

The overall objective of this project is 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. In Phase I, the team uncovered the catabolic genes coding for bacterial enzymes that are responsible for dioxane metabolism in archetype dioxane degraders and designed accordant genetic probes for these biomarkers that enable rapid quantification of dioxane biodegradation activities in complex environmental assemblages (i.e., groundwater and aquifer materials) collected at dioxane impacted sites. This effort was completed in 2013. Specific tasks in Phase II include:

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    Assess the reliability (selectivity/sensitivity) of the recently identified thmA/dxmA gene biomarker to evaluate the presence and activity of dioxane degraders at a broad range of contaminated sites;
  2. Statistically analyze how the abundance of this catabolic biomarker (and associated degradation activity) is influenced by geochemical factors and contamination scenarios;
  3. Identify (and perhaps isolate) dioxane degraders from these sites, using enrichment and dilution to extinction or fluorescence activated flow cytometry with cell sorting techniques; and
  4. Characterize catabolic gene sequences involved in dioxane metabolism in isolated or sorted cells using DNA and mRNA sequencing approaches, and obtain more sequences for possible adjustment/optimization of the previous biomarker design.

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Technical Approach

In Phase I, biomarkers for dioxane-degrading genes were discerned at the molecular scale and probes were developed to quantify them and assess MNA at the field scale. The first step was to identify genes responsible for initiating dioxane biodegradation. dxmADBC operon was identified in the archetype dioxane metabolizer, Pseudonocardia sp. CB1190, using reverse transcription PCR and bioinformatics analyses. Then, a primer/probe set to target a putative dioxane monooxygenase gene and measure its concentration using quantitative PCR with Taqman chemistry was developed. This process involved multiple sequence alignment of the four thmA/dxmA genes available on the NCBI database. The reliability, selectivity, and sensitivity of these probes were determined using pure cultures capable of metabolizing or co-metabolizing dioxane, using real-time quantitative PCR (qPCR) to quantify the presence and expression of the genetic biomarker(s). Probe validation involved 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.

In Phase II, the team will use the developed thmA/dxmA biomarker and qPCR assays to conduct a broad survey of the presence of dioxane degraders and assess their activity at numerous, diverse dioxane-impacted sites (Task 1). This is critical to determine if the biomarker is a selective, sensitive, and reliable tool to assess dioxane biodegradation potential and infer site-specific biodegradation rates. The data obtained during this effort will be statistically evaluated (multivariate ANOVA) in conjunction with site-specific geochemical and contamination information to heuristically discern general conditions that are amenable for MNA and identify factors that may broadly affect biodegradation activity (Task 2). Furthermore, new indigenous bacteria with the potential to biodegrade dioxane will be identified (and some will perhaps be isolated) from these sites, using enrichment and dilution to extinction, or gene probes that target mRNA from catabolic genes (i.e., actively degrading bacteria) with the help of fluorescence activated flow cytometry and cell sorting techniques (Task 3). This will advance the understanding of the phylogenetic and metabolic diversity of indigenous dioxane degraders. Catabolic genes in isolated or sorted cells will then be sequenced and annotated to obtain additional information about dioxane metabolic pathways, and to optimize the design of catabolic biomarkers for evaluation of the performance of dioxane MNA and bioremediation (Task 4).

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Interim Results

In Phase I, the serendipitous discovery that the critical nucleotide sequence of the key catabolic genes (coding for the active site of the enzymes) is highly conserved enables detection of multiple dioxane degraders. Real-time PCR using reference strain genomic DNA demonstrated the high selectivity (no false positives) and sensitivity of this probe (7,000 - 8,000 copies/g soil). Microcosm tests prepared with groundwater samples from 16 monitoring wells at five different dioxane-impacted sites showed that enrichment of this catabolic gene (up to 114-fold) was significantly correlated to the amount of dioxane degraded. Notably, a significant correlation was also found between biodegradation rates and the abundance of thmA/dxmA genes. In contrast, 16S rRNA gene copy numbers (a measure of total bacteria) were neither sensitive nor reliable indicators of dioxane biodegradation activity. Overall, these results suggest that this novel catabolic biomarker (thmA/dxmA) has great potential to rapidly assess the performance of natural attenuation or bioremediation of dioxane plumes. The results can be found in the Phase I Final Report.

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Since many large, dilute dioxane plumes could be effectively managed by MNA at a small fraction of alternative treatment costs, it is important to develop simple and reliable approaches 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 gene probe(s) set to unequivocally characterize dioxane biodegradation potential and assess activity for enhanced MNA forensic analysis. This will advance the scientific basis to characterize uncultivable dioxane degraders and to synthesize conjugative plasmids that deliver dioxane degradation capabilities to non-competent indigenous bacteria, which would significantly enhance site cleanup activities. (Anticipated Project Completion - 2018)

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Da Silva, M. L. B., Woroszylo, C., Castillo, N. F., Adamson, D. T., and P.J. Alvarez. 2018. Associating Potential 1,4-Dioxane Biodegradation Activity with Groundwater Geochemical Parameters at Four Different Contaminated Sites. Journal of Environmental Management, 206:60-64.

He, Y., J. Mathieu, Y. Yang, P. Yu, M.L. da Silva, and P.J. Alvarez. 2017. 1,4-Dioxane Biodegradation by Mycobacterium dioxanotrophicus PH-06 is Associated with a Group-6 Soluble Di-iron Monooxygenase. Environmental Science & Technology Letters, 4(11):494-499.

He, Y., K. Wei, K. Si, J. Mathieu, M. Li, and P.J. Alvarez. 2017. Whole-genome Sequence of the 1,4-Dioxane-degrading Bacterium Mycobacterium dioxanotrophicus PH-06. Genome Announcements, 5(35):e00625-17.

He, Y., J. Mathieu, M.L. da Silva, M. Li, and P.J. Alvarez. 2018. 1,4‐Dioxane‐degrading Consortia can be Enriched from Uncontaminated Soils: Prevalence of Mycobacterium and Soluble Di‐iron Monooxygenase Genes. Microbial Biotechnology, 11(1):189-198.

Li, M., S. Fiorenza, J.R. Chatham, S. Mahendra, and P.J. Alvarez. 2010. 1,4-Dioxane Biodegradation at Low Temperatures in Arctic Groundwater Samples. Water Research, 44(9):2894-2900.

Li, M., P. Conlon, S. Fiorenza, R.J. Vitale, and P.J. Alvarez. 2011. Rapid Analysis of 1,4‐Dioxane in Groundwater by Frozen Micro‐extraction with Gas Chromatography/Mass Spectrometry. Groundwater Monitoring & Remediation, 31(4):70-76.

Li, M., J. Mathieu, Y. Liu, E.T. Van Orden, Y. Yang, S. Fiorenza, and P.J. Alvarez. 2013. The Abundance of Tetrahydrofuran/Dioxane Monooxygenase Genes (thmA/dxmA) and 1,4-Dioxane Degradation Activity are Significantly Correlated at Various Impacted Aquifers. Environmental Science & Technology Letters, 1(1):122-127.

Li, M., J. Mathieu, Y. Yang, S. Fiorenza, Y. Deng, Z. He, J. Zhou, and P.J. Alvarez. 2013. Widespread Distribution of Soluble Di-iron Monooxygenase (SDIMO) Genes in Arctic Groundwater Impacted by 1,4-dioxane. Environmental Science & Technology, 47(17):9950-9958.

Li, M., E.T. Van Orden, D.J. DeVries, Z. Xiong, R. Hinchee, and P.J. Alvarez. 2015. Bench-scale Biodegradation Tests to Assess Natural Attenuation Potential of 1,4-Dioxane at Three Sites in California. Biodegradation, 26(1):39-50.

Li, M., Y. Liu, Y. He, J. Mathieu, J. Hatton, W. DiGuiseppi, and P.J. Alvarez. 2017. Hindrance of 1,4-Dioxane Diodegradation in Microcosms Biostimulated with Inducing or Non-inducing Auxiliary Substrates. Water Research, 112:217-225.

Li, M., Y. Yang, Y. He, J. Mathieu, C. Yu, Q. Li, and P.J. Alvarez. 2018. Detection and Cell Sorting of Pseudonocardia Species by Fluorescence In Situ Hybridization and Flow Cytometry using 16S rRNA-targeted Oligonucleotide Probes. Applied Microbiology and Biotechnology, 102(7):3375-3386.


Li, M., J. Mathieu, and P.J. Alvarez. 2015. Monitoring of 1,4-Dioxane Biodegradation in Various Environments. U.S. Patent Application No. 14/562,517.

Dissertations and Theses

He, Y. 2019. Isolation of 1,4-Dioxane Degraders and Investigation of Responsible Catabolic Genes (PhD Dissertation). Rice University.

Li, M. 2010. 1,4-Dioxane Biodegradation at Low Temperatures in Arctic Groundwater Samples (Master’s Dissertation). Rice University.

Li, M. 2013. Genetic Catabolic Probes to Assess the Natural Attenuation of 1,4-Dioxane (PhD Dissertation). Rice University.

Van Orden, E.T. 2013. Microcosm Assessment of Aerobic Intrinsic Bioremediation and Mineralization Potential for Three 1,4-Dioxane Impacted Sites (Master’s Thesis). Rice University.

Awards and Honors

He, Y. 2017. Student Paper Competition Winner for the Fourth International Symposium on Bioremediation and Sustainable Environmental Technologies.

Li, M. 2013. 3rd Place at GeosyntecTM Student Paper Competition.

Li, M. 2014. Honor Award for the Excellence in Environmental Engineering and Science™ Competition in the category of University Research. American Academy of Environmental Engineering and Sciences (AAEES).

Li, M. 2014. Student Paper Winner for the Ninth International Conference on Remediation of Chlorinated and Recalcitrant Compounds.

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Points of Contact

Principal Investigator

Dr. Pedro Alvarez

Rice University

Phone: 713-348-5903

Program Manager

Environmental Restoration