Development of a colorimetric colony-screening assay for detection of defluorination by micro-organisms

Abstract

Aims: To develop a colorimetric colony‐screening assay to facilitate the isolation of micro‐organisms capable of defluorination. Methods and Results: A metal‐dye chelate, zirconium‐xylenol orange was used to detect fluoride ions released from a fluorinated substrate through microbial metabolism. Depolymerised zirconium reagent gave the greatest visual contrast for the presence of fluoride compared to more polymerised forms of zirconium reagent. The sensitivity of the assay was greatest when the molar ratio of depolymerised zirconium to xylenol orange was 1 : 2. Using depolymerised zirconium and xylenol orange (150 and 300 nmol l−1 respectively), the assay could detect a fluoride application spot (5 mmol l−1) containing 50 nmoles of fluoride ions. Most media constituents were well tolerated by the assay, although phosphate ions needed to be restricted to 0·1 g l−1 and some proteins digest to between 1 and 5 g l−1. A microbial enrichment culture growing on solidified medium containing 20 mmol l−1 fluoroacetate was screened using the assay, and defluorinating bacteria belonging to the genus Burkholderia isolated. Conclusions: A method was developed that is sensitive, rapid and reliable for detecting defluorination by micro‐organisms growing on solidified medium. Significance and Impact of the Study: This method can be used to facilitate the isolation of micro‐organisms capable of defluorination.

Defluorination of Sodium Fluoroacetate by Bacteria from Soil and Plants in Brazil

“…These results were similar to those reported by Davis and colleagues [26] with Burkholderia sp., which also degraded SF in 32 hours, releasing 20 mmol L −1  F − with 20 mmol L −1 of SF.…”

Isolation and Identification of Sodium Fluoroacetate Degrading Bacteria from Caprine Rumen in Brazil

“…These bacteria displayed SF degradation activity, releasing 20 mmol L −1 of fluoride ion after 32 hours of incubation in Brunner medium with 20 mmol L −1 of SF ( Figure 2 ). These results are similar to those reported by Davis et al [ 34 ] for Burkholderia sp. The Pseudomonas fluorescens control strain (DSM 8341) reached the same level of defluorination, and there was no release of fluoride ions when the Brunner medium was incubated without bacteria, due to the stability of the strong carbon-fluorine bond in the fluoroacetate [ 17 ].…”

Isolation and survey of novel fluoroacetate-degrading bacteria belonging to the phylum Synergistetes

“…Following several transfers, dilutions of enrichment cultures were plated onto solidified PA10 media (1.5% agarose) containing 1 g L À1 complete amino acids supplement (Q bio gene, Vista, CA) and fluoroacetate (20 mM). Colonies were screened for their ability to degrade fluoroacetate using a colorimetric plate assay (Davis et al, 2011). Colonies were streaked onto new agarose plates and transferred to fresh media until single phenotypes were obtained.…”