Simultaneous Biodegradation of Polyaromatic Hydrocarbons by a Stenotrophomonas sp: Characterization of nid Genes and Effect of Surfactants on Degradation

Abstract

A polyaromatic hydrocarbon degrading bacterium was isolated from a petroleum contaminated site and designated as Stenotrophomonas sp. strain IITR87. It was found to utilize pyrene, phenanthrene and benzo(a)pyrene as sole carbon source, but not anthracene, chrysene and fluoranthene. Gas chromatography and mass spectroscopy analysis resulted in identification of pyrene metabolites namely monohydroxypyrene, 4-oxa-pyrene-5-one, dimethoxypyrene and monohydroxyphenanthrene. Southern hybridization using naphthalene dioxygenase gene (nidA) as probe against the DNA of strain IITR87 revealed the presence of nidA gene. PCR analysis suggests dispersed occurrence of nid genes in the genome instead of a cluster as reported in a PAH-degrading Mycobacterium vanbaalenii PYR-1. The nid genes in strain IITR87, dioxygenase large subunit (nidA), naphthalene dioxygenase small subunit (nidB) and aldehyde dehydrogenase gene (nidD) showed more than 97 % identity to the reported nid genes from Mycobacterium vanbaalenii PYR-1. Most significantly, the biodegradation of PAHs was enhanced 25-60 % in the presence of surfactants rhamnolipid and Triton X-100 due to increased solubilization and bioavailability. These results could be useful for the improved biodegradation of high-molecular-weight PAHs in contaminated habitats.

Keywords: Biodegradation; Dioxygenase; Stenotrophomonas; Surfactants.

Fig. 1

Growth analysis of Stenotrophomonas sp. strain IITR87 on different PAHs. Strain was cultured in presence of using 100 mg L⁻¹ of each PAH as sole source of carbon in mineral medium for 25 days. Control: Pyrene (open square), Phenanthrene (open triangle), Benzo(a) pyrene (open diamond). Experimental:- Pyrene (filled square), Phenanthrene (filled triangle), Benzo(a)pyrene (filled diamond)

Fig. 2

GC mass spectra of pyrene metabolites obtained from Stenotrophomonas sp. strain IITR87 grown in minimal media at 30 °C in presence of pyrene (100 mg L⁻¹) along with their retention time. a neutral and b acidic condition pyrene (13.04), monohydroxypyrene (13.91), 4-oxa-pyrene-5-one (14.22), 1,2-dimethoxypyrene (15.28), monohydroxyphenanthrene (12.30)

Fig. 3

Effect of surfactants concentration on the dissolutions of mixed PAHs by Rhamnolipid and Triton X-100 (a). Degradation of mixed PAHs by Stenotrophomonas sp. strain IITR87 in presence of rhamnolipid (b) and Triton X-100 (c) at a concentration of 1.23 and 0.92 mM respectively. Phenanthrene (phe), pyrene (pyr), benz(a)pyrene (bap). Values are represented as the mean and standard error of duplicate measurement (n = 2). C Control, rhl Rhamnolipid, tx Triton X-100