Microwell Fluoride Screen for Chemical, Enzymatic, and Cellular Reactions Reveals Latent Microbial Defluorination Capacity for -CF3 Groups

Abstract

The capacity to defluorinate polyfluorinated organic compounds is a rare phenotype in microbes but is increasingly considered important for maintaining the environment. New discoveries will be greatly facilitated by the ability to screen many natural and engineered microbes in a combinatorial manner against large numbers of fluorinated compounds simultaneously. Here, we describe a low-volume, high-throughput screening method to determine defluorination capacity of microbes and their enzymes. The method is based on selective binding of fluoride to a lanthanum chelate complex that gives a purple-colored product. It was miniaturized to determine biodefluorination in 96-well microtiter plates by visual inspection or robotic handling and spectrophotometry. Chemicals commonly used in microbiological studies were examined to define usable buffers and reagents. Base-catalyzed, purified enzyme and whole-cell defluorination reactions were demonstrated with fluoroatrazine and showed correspondence between the microtiter assay and a fluoride electrode. For discovering new defluorination reactions and mechanisms, a chemical library of 63 fluorinated compounds was screened in vivo with Pseudomonas putida F1 in microtiter well plates. These data were also calibrated against a fluoride electrode. Our new method revealed 21 new compounds undergoing defluorination. A compound with four fluorine substituents, 4-fluorobenzotrifluoride, was shown to undergo defluorination to the greatest extent. The mechanism of its defluorination was studied to reveal a latent microbial propensity to defluorinate trifluoromethylphenyl groups, a moiety that is commonly incorporated into numerous pharmaceutical and agricultural chemicals. IMPORTANCE Thousands of organofluorine chemicals are known, and a number are considered to be persistent and toxic environmental pollutants. Environmental bioremediation methods are avidly being sought, but few bacteria biodegrade fluorinated chemicals. To find new organofluoride biodegradation, a rapid screening method was developed. The method is versatile, monitoring chemical, enzymatic, and whole-cell biodegradation. Biodegradation of organofluorine compounds invariably releases fluoride anions, which was sensitively detected. Our method uncovered 21 new microbial defluorination reactions. A general mechanism was delineated for the biodegradation of trifluoromethylphenyl groups that are increasingly being used in drugs and pesticides.

Keywords: PFAS; Pseudomonas putida F1; bacteria; defluorination; fluoride; high throughput; organofluorine; screening; trifluoromethyl.

FIG 1

UV-Vis spectra of the lanthanum-alizarin complex bound to phosphate (orange), water (red), and fluoride (purple). The cuvettes show how the orange, red, and purple species can be differentiated visually. The upper right diagram shows the putative structure of the lanthanum-alizarin complex with bound fluoride as previously reported in several studies (34–36).

FIG 2

Plotted is the ratio of the lambda maximum of fluoride-bound complex (620 nm) over the lambda maximum of the unbound complex (530 nm). The 530-nm absorbance does not change during the increasing 620-nm absorbance, so the 530-nm absorbance is used as a fixed point to correct for minor baseline fluctuations. This trend of 620-nm/530-nm absorbance is linear from 0 to 100 μM fluoride, which also can be seen by eye in the assay. We define a dark purple well as having a 620/530 ratio of >0.7 and a light purple well as having a ratio of <0.7. (Inset) Comparison of fluoride detection in water versus 20 mM HEPES buffer down to a 0.62 nmol concentration; 20 mM HEPES does not inhibit the ability to detect fluoride compared to water.

FIG 3

Chemical and enzymatic defluorination of fluoroatrazine catalyzed by base or triazine hydrolase (TrzN). The assay wells correspond to fluoride detection over time. Wells shown are a 1:4 dilution of the original sample.

FIG 4

Gas chromatogram and mass spectra showing the initial fluorinated products from the oxidation of 4-fluorobenzotrifluoride by P. putida F1 and following a short-time derivatization with bis-trimethylsilane. Both derivatized and underivatized products are shown. The GC abundance for products eluting after 15.5 min is 20-fold higher, indicating that the derivatized catechol of 4-fluorobenzotrifluoride is the major product. The key masses of the MS are highlighted. Full MS data are available in the supplemental material (Fig. S8). ¹⁹F- and ¹H-NMR data are provided in Materials and Methods.

FIG 5

Schematic showing the oxygenation and defluorination of 4-fluorobenzotrifluoride. Arrows show pathway differences between P. putida F1 and P. putida F39/D, with the size of the arrows illustrating the relative magnitude of the products. In the top row, the products II and V are formed by toluene dioxygenase and cis-dihydrodiol dehydrogenase, respectively. Compounds III and IV derive from spontaneous dehydration of compound II. Compounds VI and VII arise from V and IV, respectively, via the relatively low pK_a phenolic groups undergoing deprotonation and fluoride elimination to form the quinone methide intermediates shown in brackets, following established chemistry (51, 55). The pK_as of the hydroxyl groups are indicated in the inset at the upper right, predicted using SciFinder (54). The pK_as for dihydroxy compounds are for the hydroxyl group ortho- to the trifluoromethyl group.