Theme: 07. Per- and Polyfluorinated Alkyl Substances (PFAS)

Topic Area: 7g. PFAS and Bugs: The Search Continues

Formatted Title:

Precursor Biotransformation Leads to PFAS Assimilation

Background/Objectives:

Per- and polyfluoroalkyl substances (PFAS) are anthropogenic chemicals demonstrated to impact human health.^1,2A major group of PFAS are per- and polyfluoroalkyl carboxylic acids and per- and polyfluoroalkyl sulfonic acids. A few studies have demonstrated the microbial transformation of sulfur containing PFAS (S-PFAS), such as 6:2-FTS.^3–5 Details of S-PFAS transformation and product characterization are lacking. Pseudomonas sp. strain 273 utilizes S-PFAS, 6:2-FTS and 8:2 FTS as S sources under S-limiting growth conditions. Mass balance approaches determined that a fraction of the S-PFAS could not be accounted for in the soluble fraction. Elaborate lipidomics analyses determined that fluorotelomer sulfonated catabolites were covalently incorporated into phospholipids, suggesting the bacterial lipid bilayer is a sink of organofluorine.

Approach/Activities:

Our prior study demonstrated that during fluoroalkane utilization by Pseudomonas sp. strain 273, inorganic fluoride is released to the surrounding environment (~97% of total) and concomitantly organofluorine catabolites are directed towards biosynthesis of the lipid bilayer.⁶ Here we use our high-throughput cultivation system to screen various microbes capable of utilizing S-PFAS as a S source in S-limiting conditions. Microbial strains that showed positive responses (i.e., growth) to S-PFAS amendment are used for characterization incorporation of S-PFAS transformation product. The soil bacterium Pseudomonas sp. strain 273 was grown in 160 mL vessels containing defined sulfur-free basal salt medium with acetate in the presence of 100 µm 8:2 FTS or 6:2 FTS. Biomass was collected by centrifugation and subjected to an established lipid extraction protocol. Untargeted lipidome analysis was performed with an Ultimate 3000 liquid chromatograph coupled to an LTQ Velos Pro Orbitrap mass spectrometer at Oak Ridge National Laboratory. To facilitate the analysis of S-PFAS transformation product incorporation data, a customized database of PFAS incorporated into lipids was constructed to efficiently analyze datasets and recognize fluorinated phospholipids.

Results/Lessons Learned:

Pseudomonas sp. strain 273 grew in medium containing 6:2-FTS or 8:2 FTS as the sole source of S, but no growth occurred in the same medium without the S-PFAS. During growth experiments, 6:2-FTS and 8:2 FTS was consumed, whereas in sterilized control cultures no significant loss of the S-PFAS was observed, indicating the transformation of the S-PFAS required live cells. The analysis of lipid extracts from cells grown with 6:2 FTS and 8:2-FTS as the source of S, organofluorine, specifically 5:3 FTUA and 7:3 FTUA, was detected in the lipid bilayer of Pseudomonas sp. strain 273. In contrast, no organofluorine was detected in lipid fractions extracted from biomass grown in the same medium amended with sulfate as the S source. The results suggest that Pseudomonas sp. strain 273 incorporates 5:3 FTUA and 7:3 FTUA via yet to be identified mechanisms into phospholipids. This finding explains why prior efforts have failed to account for the transformation products of S-PFAS utilization in the soluble fraction. Apparently, Pseudomonas sp. strain 273 incorporates FTCA intermediates into its phospholipid fraction during growth with S-PFAS as S sources. The covalent incorporation of FTCA precursors into membrane structures delays, or possibly prevents, the conversion to perfluorinated carboxylic acids. The assimilation of precursors into bacterial phospholipids impacts the fate and transport of PFAS in groundwater aquifers, and the conversion of precursors to more recalcitrant perfluorinated carboxylic acids.

Robert Hettich

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Precursor Biotransformation Leads to PFAS Assimilation