Isolation and characterization of 4-tert-butylphenol-utilizing Sphingobium fuliginis strains from Phragmites australis rhizosphere sediment

Abstract

We isolated three Sphingobium fuliginis strains from Phragmites australis rhizosphere sediment that were capable of utilizing 4-tert-butylphenol as a sole carbon and energy source. These strains are the first 4-tert-butylphenol-utilizing bacteria. The strain designated TIK-1 completely degraded 1.0 mM 4-tert-butylphenol in basal salts medium within 12 h, with concomitant cell growth. We identified 4-tert-butylcatechol and 3,3-dimethyl-2-butanone as internal metabolites by gas chromatography-mass spectrometry. When 3-fluorocatechol was used as an inactivator of meta-cleavage enzymes, strain TIK-1 could not degrade 4-tert-butylcatechol and 3,3-dimethyl-2-butanone was not detected. We concluded that metabolism of 4-tert-butylphenol by strain TIK-1 is initiated by hydroxylation to 4-tert-butylcatechol, followed by a meta-cleavage pathway. Growth experiments with 20 other alkylphenols showed that 4-isopropylphenol, 4-sec-butylphenol, and 4-tert-pentylphenol, which have alkyl side chains of three to five carbon atoms with α-quaternary or α-tertiary carbons, supported cell growth but that 4-n-alkylphenols, 4-tert-octylphenol, technical nonylphenol, 2-alkylphenols, and 3-alkylphenols did not. The rate of growth on 4-tert-butylphenol was much higher than that of growth on the other alkylphenols. Degradation experiments with various alkylphenols showed that strain TIK-1 cells grown on 4-tert-butylphenol could degrade 4-alkylphenols with variously sized and branched side chains (ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, tert-pentyl, n-hexyl, n-heptyl, n-octyl, tert-octyl, n-nonyl, and branched nonyl) via a meta-cleavage pathway but not 2- or 3-alkylphenols. Along with the degradation of these alkylphenols, we detected methyl alkyl ketones that retained the structure of the original alkyl side chains. Strain TIK-1 may be useful in the bioremediation of environments polluted by 4-tert-butylphenol and various other 4-alkylphenols.

FIG. 1.

Phylogenetic relationships between strains TIK-1, TIK-2, and TIK-3, type strains of Sphingomonadaceae and Pseudomonas sp., and previously isolated 4-nonylphenol-degrading and 4-octylphenol-degrading (*) and medium-chain 4-n-alkylphenol-degrading (**) bacterial strains, established by the neighbor-joining method on the basis of 16S rRNA gene sequences. Numbers on branches indicate bootstrap confidence estimates obtained with 1,000 replicates. The scale bar represents an evolutionary distance (K_nuc) of 0.02.

FIG. 2.

Degradation of 4-tert-butylphenol and cell growth of S. fuliginis strain TIK-1 in basal salts medium (BSM) containing 1.0 mM 4-tert-butylphenol (A) and degradation of 4-tert-butylcatechol and cell growth of strain TIK-1 in BSM containing 1.0 mM 4-tert-butylcatechol (B). The concentrations of 4-tert-butylphenol (closed squares), 4-tert-butylcatechol (closed diamonds), and 3,3-dimethyl-2-butanone (open triangles) and the cell densities (optical density at 600 nm [OD₆₀₀]; open circles) were monitored over 24 h. Data points represent the means of results from triplicate experiments, and error bars indicate 95% confidence intervals.

FIG. 3.

Influence of 3-fluorocatechol on degradation of 4-tert-butylphenol and 4-tert-butylcatechol by whole cells of S. fuliginis strain TIK-1 grown on 4-tert-butylphenol. Whole cells without 0.2 mM 3-fluorocatechol treatment were incubated in 50 mM potassium phosphate buffer (pH 7.5) containing 0.5 mM 4-tert-butylphenol (A) or 0.5 mM 4-tert-butylcatechol (B). In addition, whole cells subjected to 0.2 mM 3-fluorocatechol treatment for 30 min before the degradation tests were incubated in 50 mM potassium phosphate buffer (pH 7.5) containing 0.5 mM 4-tert-butylphenol (C) or 0.5 mM 4-tert-butylcatechol (D). Concentrations of 4-tert-butylphenol (closed squares) and 4-tert-butylcatechol (open diamonds) were monitored over 360 min. Data points represent the means results from of triplicate experiments, and error bars indicate 95% confidence intervals.

FIG. 4.

Proposed pathway for the metabolism of 4-tert-butylphenol by S. fuliginis strain TIK-1. (I) 4-tert-butylphenol; (II) 4-tert-butylcatechol; (III) 3,3-dimethyl-2-butanone; (IV) pyruvic acid.