Degradation of phenanthrene by different bacteria: evidence for novel transformation sequences involving the formation of 1-naphthol

Abstract

Four polycyclic aromatic hydrocarbon (PAH)-degrading bacteria, namely Arthrobacter sulphureus RKJ₄, Acidovorax delafieldii P_4-1, Brevibacterium sp. HL₄ and Pseudomonas sp. DLC-P₁₁, capable of utilizing phenanthrene as the sole source of carbon and energy, were tested for its degradation using radiolabelled phenanthrene. [9-¹⁴C]Phenanthrene was incubated with microorganisms containing 100 mg/l unlabelled phenanthrene and the evolution of ¹⁴CO₂ was monitored: within 18 h of incubation, 30.1, 35.6, 26.5 and 2.1% of the recovered radiolabelled carbon was degraded to ¹⁴CO₂ by RKJ₄, P_4-1, HL₄ and DLC-P₁₁, respectively. When mixtures of other PAHs such as fluorene, fluoranthene and pyrene, in addition to phenanthrene, were added as additional carbon sources, there was a 36.1 and 20.6% increase in ¹⁴CO₂ production from [9-¹⁴C]phenanthrene in the cases of RKJ₄ and HL₄, respectively, whereas P_4-1 and DLC-P₁₁ did not show any enhancement in ¹⁴CO₂ production. Although, a combination of many bacteria enhances the degradation of organic compounds, no enhancement in the degradation of [9-¹⁴C]phenanthrene was observed in mixed culture involving all four microorganisms together. However, when different PAHs, as indicated above, were used in mixed culture, there was a 68.2% increase in ¹⁴CO₂ production. In another experiment, the overall growth rate of P_4-1 on phenanthrene could be enhanced by adding the non-ionic surfactant Triton X-100, whereas RKJ₄, HL₄ and DLC-P₁₁ did not show any enhancement in growth. Pathways for phenanthrene degradation were also analysed by thin-layer chromatography, gas chromatography and gas chromatography-mass spectrometry. Common intermediates such as o-phthalic acid and protocatechuic acid were detected in the case of RKJ₄ and o-phthalic acid was detected in the case of P_4-1. A new intermediate, 1-naphthol, was detected in the cases of HL₄ and DLC-P₁₁. HL₄ degrades phenanthrene via 1-hydroxy-2-naphthoic acid, 1-naphthol and salicylic acid, whereas DLC-P₁₁ degrades phenanthrene via the formation of 1-hydroxy-2-naphthoic acid, 1-naphthol and o-phthalic acid. Both transformation sequences are novel and have not been previously reported in the literature. Mega plasmids were found to be present in RKJ₄, HL₄ and DLC-P₁₁, but their involvement in phenanthrene degradation could not be established.