Formatted Title

A Newly Designed QPCR Assay Implicates a Rhodococcus sp. RR1 prmA-like Gene in 1,4-Dioxane Metabolism

Background/Objectives

Background/Objectives. The groundwater contaminant, 1,4-dioxane, is commonly found at chlorinated solvent sites and has chemical characteristics that result in migration and persistence. Unfortunately, traditional remediation methods such as air stripping or activated carbon are largely ineffective. Ex situ oxidation methods, including ozone and hydrogen peroxide or hydrogen peroxide and ultraviolet light, can be costly at high concentrations. As a result, much attention has focused on approaches that result in the aerobic 1,4-dioxane biodegradation. Although many functional genes have been associated with the breakdown of this chemical, the occurrence of these genes at contaminated sites is largely unknown. To address this, the current study targeted these genes using both amplicon sequencing and quantitative PCR (qPCR).

Approach/Activities

Approach/Activities. The biodegradation of 1,4-dioxane was examined in sediment from an impacted site in California. Microcosms were set up in triplicate with the following treatments: 1) with both 1,4-dioxane and propanol, 2) with 1,4-dioxane and without propanol, 3) without 1,4-dioxane and without propanol, and 4) without 1,4-dioxane and with propanol. Also, three abiotic controls were included. DNA was extracted in triplicate from the sediment before and after the incubation. One DNA extract was submitted in triplicate for functional gene sequencing (targeting the conserved regions in the soluble di-iron monooxygenases alpha subunit gene) and the other extracts were used for qPCR. The amplicon sequences obtained were compared to 12 genes previously associated with 1,4-dioxane metabolism and co-metabolism. Based on the results of this comparison, two new qPCR assays were designed (using Primer-BLAST and the PrimerQuest™ Tool from Integrated DNA Technologies) targeting Rhodococcus jostii RH1 prmA and Rhodococcus sp. RR1 prmA. The abundances of these two genes, as well as the 16S rRNA gene, were then determined in the sediment DNA extracts.

Results/Lessons Learned

Results/Lessons Learned. 1,4-Dioxane biodegradation occurred after approximately 40 days in the microcosms both with and without propanol. Quantitative PCR indicated Rhodococcus sp. RR1 prm, but not Rhodococcus jostii RH1 prmA, was present in the extracted DNA. When the qPCR prmA data were normalized by 16S rRNA gene copies, several interesting trends were noted. This included a statistically significant increase in the ratio of gene copies (prmA/16S rRNA) in the microcosms amended with 1,4-dioxane (without propanol) compared to the ratio before any treatment, indicative of growth of the microorganisms harboring this gene. Also, the microcosms amended with 1,4-dioxane (without propanol) exhibited a larger ratio compared to the same treatment without 1,4-dioxane, also indicative of a positive influence on the target gene due to the presence of 1,4-dioxane. Further, the microcosms amended with 1,4-dioxane (with propanol) also exhibited a larger ratio compared to the same treatment without 1,4-dioxane. Overall, the data suggest 1,4-dioxane biodegradation resulted in increasing copy numbers of the target gene (Rhodococcus sp. RR1 prm). The newly designed assay should be of interest to those responsible for the bioremediation of 1,4-dioxane contaminated sites.