Remediation of aquifers impacted with 1,4-dioxane can be complex because dioxane is highly mobile in groundwater, recalcitrant to biodegradation, and is not easily removed by volatilization or adsorption. Monitored natural attenuation (MNA), which relies primarily on removal of the chemical of concern by biodegradation, is often the most cost-effective approach to manage large and dilute groundwater plumes such as those encountered in sites impacted by 1,4-dioxane. However, the burden of proof that MNA is an appropriate solution lies on the proponent, which requires site-specific demonstration of removal of the chemical of concern with strong evidence to support the presence of expression of biodegradation capabilities. 

This project was conducted in two phases. In Phase I, the team investigated the catabolic genes coding for bacterial enzymes that are responsible for 1,4-dioxane metabolism in archetype 1,4-dioxane degraders and designed accordant genetic probes for these biomarkers that enable rapid quantification of 1,4-dioxane biodegradation activities in complex environmental assemblages (i.e., groundwater and aquifer materials) collected at dioxane impacted sites. 

During Phase II, catabolic gene probe(s) were developed and tested to quantify the presence and expression of 1,4-dioxane biodegradation genes to support decisions to select or reject MNA and assess its performance at 1,4-dioxane-impacted sites. Specific tasks included: 

1. Identification of genes responsible for initiating 1,4-dioxane biodegradation, development of a primer/probe biomarker to target the identified genes, and assessment of reliability (selectivity/sensitivity) of gene biomarker to evaluate the presence and activity of 1,4-dioxane degraders at a broad range of impacted sites;
2. Statistical analysis to determine how the abundance of this catabolic biomarker (and associated degradation activity) is influenced by geochemical factors and impact scenarios;
3. Identification (and perhaps isolation) of 1,4-dioxane degraders from these sites, using enrichment and dilution to extinction or fluorescence activated flow cytometry with cell sorting techniques; and
4. Characterization of catabolic gene sequences involved in 1,4-dioxane metabolism in isolated or sorted cells using DNA and messenger RNA (mRNA) sequencing approaches to obtain more sequences for possible adjustment/optimization of our previous biomarker design.
5. 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;
6. 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;
7. Statistically analyze how the abundance of this catabolic biomarker (and associated degradation activity) is influenced by geochemical factors and contamination scenarios;
8. 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
9. 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.

In Phase I, biomarkers for 1,4-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 1,4-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 1,4-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. 

During Phase II, the developed thmA/dxmA biomarker and quantitative polymerase chain reaction (qPCR) assays were used to conduct a broad survey of the presence of 1,4-dioxane degraders and assess their activity at numerous, diverse 1,4-dioxane-impacted sites. This was critical to determine if the biomarker was selective, sensitive as well as reliable as a tool to assess dioxane biodegradation potential with potential to infer on site-specific biodegradation rates. The data obtained during this task was statistically evaluated (multivariate analysis of variance) in conjunction with site-specific geochemical characteristics and 1,4-dioxane concentrations to heuristically discern general conditions that are amenable for MNA, which served to identify factors that may broadly affect biodegradation activity. Furthermore, a method was developed to isolate and identify the phylogenetic and metabolic diversity of indigenous 1,4-dioxane degraders using gene probes to target mRNA from catabolic genes (i.e., actively degrading bacteria) and fluorescence-activated flow cytometry with cell sorting. The catabolic genes in isolated bacteria were sequenced and annotated to obtain additional information about 1,4-dioxane metabolic pathways, and to optimize the design of catabolic biomarkers for further evaluation of the performance of dioxane MNA and bioremediation.

Through multiple alignment analyses, it was found that critical nucleotide sequences of key catabolic genes coding for the active site of the enzymes are highly conserved. These sequences were used to develop the thmA/dxmA probe capable of targeting putative 1,4-dioxane degrading monooxygenase. This primer-probe set was both highly selective (no false positives) and sensitive (7,000 ~ 8,000 copies/g soil) when tested against a 1,4-dioxane-degrading reference strain (e.g., CB1190). A significant correlation between biodegradation rates and the abundance of thmA/dxmA genes were observed. In contrast, a nonspecific 16S rRNA gene copy numbers were neither sensitive nor reliable as an indicator of 1,4-dioxane biodegradation activity. 

While many geochemical factors (pH, temperature, nutrients) and the abundance of specific degraders may be important for 1,4-dioxane biodegradation, these factors may not exert as strong of an influence on the potential biodegradation activity as the concentration of 1,4-dioxane. Unequivocally, in situ 1,4-dioxane concentrations significantly influenced the observed 1,4-dioxane biodegradation rates with higher 1,4-dioxane concentrations. Whereas auxiliary carbon sources may temporarily enhance 1,4-dioxane biodegradation, they may also exert counterproductive long-term consequences. Non-inducing growth substrates can boost the overall microbial biomass but inhibit the indigenous 1,4-dioxane degraders. 

Fluorescently-labeled oligonucleotide probes and flow cytometry is a cultivation-free strategy to separate and concentrate genus-specific subpopulations of interest. This will expedite discovering novel Pseudonocardia strains and discerning the molecular basis for specific metabolic traits of interest. In addition, using dilution to extinction method, two 1,4-dioxane degrading consortia were enriched from unimpacted environments. These have comparable 1,4-dioxane degradation capabilities with two archetype 1,4-dioxane degraders. Mycobacterium was the dominant genus and group 5 and group 6 soluble diiron monooxygenase (SDIMOs) was prevalent after enrichment. 

Through whole-genome sequencing, RNA sequencing and RT-qPCR, a group 6 propane monooxygenase (prmABCD) was identified in Mycobacterium dioxanotrophicus PH-06. Accordingly, a primer/probe set was developed and validated through four 1,4-dioxane-degrading consortia and strong correlation was found between 1,4-dioxane degradation rates and the abundance of biomarkers. This new biomarker is highly selective and sensitive and would minimize the false negatives of the previous biomarker (thmA/dxmA). Furthermore, the preliminary results of construction of a reporter strain to monitor 1,4-dioxane degradation activities would facilitate future development.

This project supports the rational selection of MNA over other remediation approaches that are usually more complex and costly (e.g., pump-and-treat with advanced chemical oxidation treatment). It also provides a scientific basis for the interruption of remediation strategies when these are no longer removing 1,4-dioxane faster than MNA. Together, these findings could aid decision-makers to more appropriately select for cost-effective clean up strategies saving the industry, state and federal government from multi-billion-dollar risk penalties. This project provides more reliable risk assessment and management approaches for hazardous wastes that have come from environmental advocacy groups, industry and the regulatory community. (Project Completion - 2020)

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. doi.org/10.1016/j.jenvman.2017.10.031.

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. doi.org/10.1021/acs.estlett.7b00456.

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. doi.org/10.1128/genomea.00625-17.

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. doi.org/10.1111/1751-7915.12850.

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. doi.org/10.1016/j.watres.2010.02.007.

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. doi.org/10.1111/j.1745-6592.2011.01350.x.

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. https://doi.org/10.1021/ez400176h.

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. doi.org/10.1021/es402228x.

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. doi.org/10.1007/s10532-014-9714-1.

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. doi.org/10.1016/j.watres.2017.01.047.

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. doi.org/10.1007/s00253-018-8801-3.

Patents

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. 3^rd Place at Geosyntec^TM 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.