Degradation of 4-fluorophenol by Arthrobacter sp. strain IF1

Abstract

A Gram-positive bacterial strain capable of aerobic biodegradation of 4-fluorophenol (4-FP) as the sole source of carbon and energy was isolated by selective enrichment from soil samples collected near an industrial site. The organism, designated strain IF1, was identified as a member of the genus Arthrobacter on the basis of 16S ribosomal RNA gene sequence analysis. Arthrobacter strain IF1 was able to mineralize 4-FP up to concentrations of 5 mM in batch culture. Stoichiometric release of fluoride ions was observed, suggesting that there is no formation of halogenated dead-end products during 4-FP metabolism. The degradative pathway of 4-FP was investigated using enzyme assays and identification of intermediates by gas chromatography (GC), GC-mass spectrometry (MS), high-performance liquid chromatography, and liquid chromatography-MS. Cell-free extracts of 4-FP-grown cells contained no activity for catechol 1,2-dioxygenase or catechol 2,3-dioxygenase, which indicates that the pathway does not proceed through a catechol intermediate. Cells grown on 4-FP oxidized 4-FP, hydroquinone, and hydroxyquinol but not 4-fluorocatechol. During 4-FP metabolism, hydroquinone accumulated as a product. Hydroquinone could be converted to hydroxyquinol, which was further transformed into maleylacetic acid and beta-ketoadipic acid. These results indicate that the biodegradation of 4-FP starts with a 4-FP monooxygenase reaction that yields benzoquinone, which is reduced to hydroquinone and further metabolized via the beta-ketoadipic acid pathway.

Fig. 1

Phylogenetic tree of the 16S rRNA gene sequence of strain IF1. The scale bar represents 0.1 fixed mutation per site. Bootstrap values were derived from 1,000 analyses. The DNA sequences were aligned using ClustalX, and the tree was constructed by the neighbor-joining program from a similarity matrix of pairwise comparisons made by using the Kimura-2-parameter algorithm. Tree a shows the global position of the strain IF1 while tree b shows the Bayesian tree and the more precise relationship of strain IF1 to other members of the genus Arthrobacter. The values in parenthesis in this tree are the posterior probabilities for the nodes that show where IF1 resides

Fig. 2

Fluoride release and biomass formation by strain IF1 with different concentrations of 4-FP. a Stoichiometric fluoride release. Symbols: squares, optical density at 600 nm; circles, fluoride concentration. b Growth in the presence of different levels of 4-FP. The initial concentrations used were: triangles, 1 mM 4-FP; circles, 4 mM 4-FP; diamonds, 5 mM 4-FP; squares, 7 mM 4-FP

Fig. 3

Biodegradation of 4-FP and fluoride liberation in a batch culture of strain IF1. The carbon source used is 1 mM 4-FP. Symbols: triangles, 4-FP concentration; diamonds, optical density; squares, fluoride concentration

Fig. 4

Accumulation of hydroquinone during growth of strain IF1 on 4 mM 4-FP. The optical density (diamonds), 4-FP concentration (squares), and hydroquinone concentration (x marks) are indicated. Hydroquinone was identified by GC-MS and quantified by GC-FID

Fig. 5

Proposed pathway for 4-fluorophenol degradation by Arthrobacter sp. strain IF1. Hydroquinone and hydroquinole were identified as intermediates by GC-MS