Formatted Title
Substrate-Mediated Biodefluorination of Fluorotelomer-Based PFAS by Rhodococcus jostii RHA1
Background/Objectives
Fluorotelomer carboxylic acids (FTCAs) are industrial chemicals commonly detected in landfill leachates and environmental matrices. Unlike their perfluorinated analogues, FTCAs contain unfluorinated carbons which may facilitate microbial enzymatic activity. Rhodococcus jostii RHA1 (RHA1) is of interest for the biodegradation of FTCAs because of its robust enzymatic capabilities. The genome of RHA1 is known to contain many oxygenase and ligase enzymes that allow RHA1 to utilize a broad substrate range as carbon and energy sources. This promiscuity in substrate utilization makes RHA1 an ideal organism for understanding co-metabolism of toxins during metabolization of growth substrates, and RHA1 has been implicated in the degradation of organic pollutants such as polychlorinated biphenyls. This research will therefore involve the characterization of RHA1 enzymatic capabilities as well as investigating how carbon co-substrates affect the biodegradation kinetics of FTCAs and mediate biotransformation pathways and biodefluorination degree.
Approach/Activities
RHA1 is grown with a variety carbon substrates (Luria-Bertani broth, glucose, pyruvate, or 1-butanol) to determine when the exponential growth phase of each substrate occurs. The cells are grown and harvested within their exponential phase of growth, washed with phosphate-buffered saline, and suspended in ammonium mineral salt medium with 6:2 FTCA (40 µM). Aliquots are taken at 0, 1, 4, 8, and 24 hours and measured for fluoride release via ion selective electrode to confirm defluorination is occurring. Targeted analysis is performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify the removal of FTCAs and the generation of transformation products when different substrates are supplemented. Non-targeted analysis combining fluorine-19 nuclear magentic resonance spectroscopy (19F NMR) and high resolution mass spectrometry (HRMS) will be performed for detection of any novel transformation products. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) will be used to measure the expression levels of target enzymes to deduce any changes from the start of the experiment.
Results/Lessons Learned
Exponential phase was reached within 48 hours for all treatment groups. It is expected that the carbon substrates will have measurable effects on the defluorination of 6:2 FTCA. When grown with different carbon sources RHA1 is expected to present various fluoride release capacities and velocities. Upon quantification of transformation products, we can determine if 6:2 FTCA biodegradation is occurring co-metabolically and highlight the difference in the composition of products based on the co-substrate used. The results of RT-qPCR will highlight key enzymes in the transformation of 6:2 FTCA by RHA1. Using these data we can propose a removal pathway for 6:2 FTCA and identify key enzymes in the degradation of 6:2 FTCA.