Identification of metabolites from degradation of pyrene and fluoranthene via co-metabolism with phenanthrene by Sphingomonas sp. strain P2 and preliminary study on genes involved in phenanthrene degradation

Abstract

Metabolites from pyrene and fluoranthene degradation via co-metabolism with phenanthrene by Sphingomonas sp. P2. were investigated. After 4-day cultivation of Sphingomonas sp. P2 in carbon free mineral medium (CFMM) supplemented with phenanthrene and pyrene or fluoranthene in 30 l-fermenters, no metabolite from pyrene and fluoranthene was found, whilst five metabolites in phenanthrene degradation pathway in the presence of pyrene were detected and purified by silica gel open column, thin-layer and high performance liquid chromatography. Based on gas chromatography-mass spectral, 1H and 13C nuclear magnetic resonance spectral analyses, two novel metabolites in phenanthrene degradation were characterized. One was identified as 5,6-benzocoumarin deriving from catabolism initiated by dioxygenation at the 1 and 2 positions of phenanthrene ring and the other one as 1,5-dihydroxy-2-naphthoic acid. Other metabolites from phenanthrene degradation, including 7,8-benzocoumarin, 1-hydroxy-2-naphthoic acid, and coumarin were also identified. The detection of coumarin, 5,6-benzocoumarin and 7,8-benzocoumarin suggested that these three compounds were formed from nonenzymatic conversion of certain metabolites in phenanthrene degradation pathway. Therefore, the results obtained suggested that phenanthrene could be degraded by Sphingomonas sp. P2 via dioxygenation at both 1,2 and 3,4 positions and subsequently undergone meta-cleavage. Pathway for phenanthrene degradation is proposed. Prelominary study of gene encoding dioxygenase revealed that this gene could not be amplified by PCR using nahAc, phnAc, modified pPAH and Rieske type primers while Escherichia coli clones containing dioxygenase gene from shot gun cloning have not yer obtained.