Using qPCR Assays to Predict Rates of Cometabolism of TCE in Aerobic Groundwater

Abstract

Monitored natural attenuation (MNA) is commonly used as a remedy for trichloroethene (TCE) in anaerobic groundwater; however, MNA has not been applied to TCE contamination in aerobic groundwater. Under aerobic conditions, bacteria initiate the degradation of many organic substances with oxygenase enzymes. Several of these enzymes are known to degrade TCE through a fortuitous reaction known as cometabolism. There are commercially available qPCR assays that can determine the number of gene copies of these enzymes. If the qPCR assay could be used to predict the first‐order rate constant for cometabolism of TCE, the qPCR assay could be used to screen sites to determine whether MNA was a plausible remedy for TCE contamination. This study reevaluated data from water samples that were collected from 19 wells on five sites in Minnesota, New York, and Utah. Data had previously been published on the rate constant for cometabolism of TCE in the water samples as determined by a 14C‐assay and the abundance of gene copies for five enzymes that cometabolize TCE as determined using a qPCR assay. The Michaelis‐Menten (Haldane) kinetic parameters for cometabolism of TCE and the abundance of DNA for the five oxygenase enzymes were used to predict the rate constant for cometabolism of TCE. The predicted rate constants were evaluated and validated by comparing them to the rate constants derived from the 14C‐assay. For predicted rate constants greater than 0.003 per year, the predicted rate constants agreed with the measured rate constants within a factor of three. The qPCR assay serves as a convenient screening tool to determine whether MNA is a plausible remedy for an aerobic plume of TCE.

Distinct Catalytic Behaviors between Two 1,4-Dioxane-Degrading Monooxygenases: Kinetics, Inhibition, and Substrate Range

“…On the basis of our RT-qPCR analysis (Figure S1), V max of PRM and THM can be converted to (9.52 ± 0.71) × 10 –12 and (1.13 ± 0.14) × 10 –11 mg dioxane/h/transcript copy, respectively. These values may be of significant value to evaluate real-time dioxane degradation activities in the field when total RNA is recovered from environmental samples …”

“…These values may be of significant value to evaluate real-time dioxane degradation activities in the field when total RNA is recovered from environmental samples. 32 V max of PRM is significantly smaller than THM (p < 0.05), indicating that PRM has a relatively lower maximum catalytic capacity for dioxane transformation. However, when dioxane concentration is lower than 430 mg/L, PRM surpasses THM in the dioxane degradation rate, primarily due to its greater affinity to dioxane.…”

“…An additional important impediment is a practice of normalizing the rate of compound removal to the amount of protein associated with the active cells (e.g., V max values in Table S1). Wilson et al suggested that the lab-derived kinetic parameters could be used along with data on the abundance of catabolic biomarkers to screen for intrinsic degradation activity . Thus, normalization of degradation rates to the abundance of gene or transcript copies measured by quantitative polymerase chain reaction (qPCR) or reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses can be extrapolated to the field system within some useful level of agreement.…”

Framework for field-scale application of molecular biological tools to support natural and enhanced bioremediation

“…Subsequently generated contaminant, geochemical, and MBT data can be interpreted through spatial and/or temporal comparative analysis to support determination of 10.3389/fmicb.2022.958742 bioremediation potential. MBT data interpretation should be aligned with previously developed MLOE approaches and/or MNA guidance, and should consist of assessing contaminant trends for direct or indirect evidence of biodegradation, geochemical conditions to identify biodegradation mechanisms and potential electron acceptor/donor limitations of biological activity, presence and abundance of contaminant-degrading microorganisms or genes, and the inferred extent of intrinsic biodegradation (USEPA, 1999;Wilson et al, 2019;Taggart and Clark, 2021;Adamson et al, 2022). This approach is demonstrated by Baldwin et al (2008) where benzene, toluene, ethylbenzene and xylenes (BTEX) concentrations were correlated with aromatic oxygenase genes to assess bioremediation potential.…”

“…During the past 20 years, molecular biological tools (MBTs) have been increasingly utilized due to scientific advancements and decreased analytical costs, to directly assess microbiological processes at environmental sites ( Wilson et al, 1999 , 2019 ; Madsen, 2000 ; Beller et al, 2002 ; Winderl et al, 2007 ; Cupples, 2008 ; Gedalanga et al, 2016 ; Taggart and Clark, 2021 ; Adamson et al, 2022 ). MBTs, such as quantitative polymerase chain reaction (qPCR), can be used to directly assess abundance of contaminant-degrading microorganisms or functional genes which encode for contaminant-degrading enzymes present in the environment.…”