Isolation and Characterization of 4-tert-Butylphenol-Utilizing Sphingobium fuliginis Strains from Phragmites australis Rhizosphere Sediment

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.4-tert-Butylphenol is an alkylphenol with a tertiary branched side chain of four carbon atoms at the para position of phenol. It is an industrially important chemical and is abundantly and widely used for the production of phenolic, polycarbonate, and epoxy resins. Production of 4-tert-butylphenol in the European Union in 2001 was 25,251 tons (t) (9). In Japan, according to the National Institute of Technology and Evaluation (http://www.safe.nite.go.jp/english/sougou/view/ComprehensiveInfoDisplay_en.faces), production of 4-tert-butylphenol amounted to 27,761 t in 2007. 4-tert-Butylphenol is widely distributed in aquatic environments, including river waters (20), seawaters (17), river sediments (17), marine sediments (23), and effluent samples from sewage treatment plants and wastewater treatment plants (22). Furthermore, 4-tert-butylphenol interacts with estrogen receptors (29, 30, 34, 35, 39) and exhibits other toxic effects on aquatic organisms and humans (4, 15, 16, 25, 26, 42, 43). Therefore, it is necessary to study the biodegradation of 4-tert-butylphenol to understand its fate in the aquatic environment, to establish technologies to treat the waters polluted by it, and to remove it from contaminated environments.Studies of the biodegradation of alkylphenols have focused mainly on branched 4-nonylphenol. Several strains of sphingomonad bacteria, including Sphingomonas sp. strain TTNP3 (38), Sphingobium xenophagum Bayram (11), and Sphingomonas cloacae S-3^T (10), have recently been isolated from activated sludge. These strains can degrade branched 4-nonylphenol and utilize it as a sole carbon source. In addition, several Pseudomonas strains that can degrade medium-chain 4-n-alkylphenols (e.g., 4-n-butylphenol) and utilize them as sole carbon sources have been isolated from activated sludge or contaminated soil; they include Pseudomonas veronii INA06 (1), Pseudomonas sp. strain KL28 (21), and Pseudomonas putida MT4 (36). Biodegradation of branched 4-nonylphenol and 4-n-butylphenol has been well studied, but little is known about the biodegradation of 4-tert-butylphenol, although this compound has a structure similar to those of branched 4-nonylphenol and 4-n-butylphenol. There is only one report on the biotransformation of 4-tert-butylphenol—by resting cells of S. xenophagum strain Bayram grown on technical nonylphenol—but this strain cannot grow on 4-tert-butylphenol (11, 14). To our knowledge, there are no reports of bacteria that utilize 4-tert-butylphenol as the sole carbon source, and the biochemical pathway of 4-tert-butylphenol utilization has not been described.Here we characterize three Sphingobium fuliginis strains—TIK-1, TIK-2, and TIK-3—isolated from rhizosphere sediment of the reed Phragmites australis. These strains could use 4-tert-butylphenol as a sole carbon source. On the basis of additional tests of strain TIK-1, we propose that it degrades 4-tert-butylphenol through 4-tert-butylcatechol along a meta-cleavage pathway. We also show that strain TIK-1 cells grown on 4-tert-butylphenol can degrade a wide range of 4-alkylphenols via a meta-cleavage pathway.