Cluster-Specific Biomarkers for Effective Assessment of 1,4-Dioxane Biodegradation Potentials for Natural Attenuation and Propane Biosparging

Natural attenuation (NA) and propane biostimulation (PB) are effective and cost-efficient in situ techniques for remediating 1,4-dioxane (dioxane)-contaminated groundwater. We designed and validated three primer/probe sets capable of distinguishing among three clusters of group-6 propane monooxygenases (PRMs) and evaluated their correlation with dioxane degradation in microcosms mimicking NA and PB treatments. These biomarkers demonstrated exclusive specificity and high sensitivity (500–1600 copies/mL groundwater). Microcosms prepared with groundwater at seven monitoring wells across two sites exhibited significant dioxane removal, particularly where active propane biosparging was implemented. Using Taqman-based qPCR assays, prmAI and prmAIII were most dominant, while prmAII and thmA were absent, indicating the pivotal roles of Cluster I and III PRMs in the observed dioxane biodegradation. Moreover, the average abundance of total prmA, as well as prmAI and prmAIII individually, correlated significantly with the dioxane degradation rates. Correlation and regression analyses highlighted a stronger association of prmAIII than prmAI, suggesting a greater influence of Cluster III PRMs under tested conditions. Samples with total prmA below 10^4.5copies/mL groundwater exhibited negligible dioxane removal, suggesting a practical threshold for assessing the bioremediation potential. These findings establish cluster-specific group-6 PRM biomarkers as effective tools for predicting and monitoring dioxane biodegradation in impacted aquifers.