Biodegradation of di-n-butyl phthalate by Rhodococcus sp. JDC-11 and molecular detection of 3, 4-phthalate dioxygenase gene.

Rhodococcus sp. JDC-11, capable of utilizing di-n-butyl phthalate (DBP) as the sole source of carbon and energy, was isolated from sewage sludge and confirmed mainly based on 16S rRNA gene sequence analysis. The optimum pH, temperature, and agitation rate for DBP degradation by Rhodococcus sp. JDC-11 was 8.0, 30 degrees C, and 175 rpm, respectively. In addition, the effect of glucose concentration on DBP degradation indicated that low concentration of glucose inhibited the degradation of DBP while high concentrations of glucose increased its degradation. Meanwhile, the substrates utilization test showed that JDC-11 could also utilize other phthalates. Furthermore, the major metabolites of DBP degradation were identified as mono-butyl phthalate and phthalic acid by gas chromatography-mass spectrometry and the metabolic pathway of DBP degradation by Rhodococcus sp. JDC-11 was tentatively speculated. Using a set of new degenerate primer, partial sequence of the 3, 4-phthalate dioxygenase gene was obtained from the strain. Sequence analysis revealed that the phthalate dioxygenase gene of JDC-11 was highly homologous to the large subunit of phthalate dioxygenase from Rhodococcus coprophilus strain G9.

Degradation of di-n-butyl phthalate by newly isolated Ochrobactrum sp.

A gram negative isolate designated JDC-41 was obtained from river sludge using mixtures of phthalate esters as the sole source and energy. The isolate was identified as Ochrobactrum sp. based on its 16S rRNA gene sequence. Over 87% of supplied di-n-butyl phthalate (DBP) was degraded by JDC-41 in a pH neutral mineral salts medium at 30 degrees C within 48 h. Increased DBP (50-500 mg/L) in the culture correspondingly increased degradation half-life from 3.83 to 18.12 h. DBP induced cells more rapidly degraded DBP.

[Isolation and identification of four DBP-degrading strains and molecular cloning of the degradation genes].

Four di-butyl-phthalate(DBP)-degrading bacterial strains, JDC-1, JDC-8, JDC-9 and JDC-12, were isolated from soil. The strains were gram positive. The 16S rRNA sequence analysis revealed that the four strains had similarities of 99% with Arthrobacter sp.. According to the morphologic, physiobiochemical characteristics and the analysis of their 16S rRNA, all the four strains were identified as Arthrobacter sp.. A 900 bp DNA fragment was obtained from the four strains by PCR amplified and clone. When compared with the large subunit of phthalate dioxygenase gene (phtA) of Arthrobacter keyseri, more than 96% similarities were evident in the nucleotide sequences. The optimal growth conditions and degradation rates of DBP were tested and the result indicated that the optimal growth conditions of the four bacteria strains were pH 7.0-8.5 and 30-35 degrees C. All the four bacteria strains performed efficiently for DBP degrading capabilities under optimal conditions. The most efficient strain JDC-1 degraded 500 mg/L DBP completely within 28 h whereas the least efficient strain JDC-8 degraded 500 mg/L DBP completely within 40 h. This study is helpful to the investigation of DBP-degrading mechanisms and the development of microbial resources.