红树林沉积物中多环芳烃降解菌Aquabacter sediminisP-9T的分离、鉴定及降解机制的研究

Isolation, identification, and mechanism of the polycyclic aromatic hydrocarbons degrader Aquabacter sediminis P-9T in mangrove sediment

摘要:

多环芳烃(polycyclic aromatic hydrocarbons, PAHs)是一类具有致畸、致癌和致突变特性的高风险有毒污染物，近年来，关于红树林生态系统受到PAHs污染的报道引起广泛关注，微生物降解被认为是处理PAHs污染的有效、经济和多功能替代方法。目前，研究者发现了大量细菌利用PAHs污染作为碳源和能源，然而其普遍存在降解效率偏低、降解谱系窄、对高盐环境适应性差等问题，红树林来源的PAHs降解菌的降解机理尚待充分挖掘。【目的】在红树林沉积物中定向筛选针对PAHs的高效广谱降解菌，并深入探讨其降解效能与作用机制，以期为红树林生态系统中微生物污染修复技术的创新研发提供坚实的科学基础。【方法】从深圳福田红树林沉积物筛选出一株降解PAHs的潜在新种菌株P-9^T，通过形态学观察、生理生化特性检测和16S rRNA基因序列分析，对该菌株进行鉴定；基于菌株基因组测序与分析预测该菌株的PAHs代谢潜能；在不同温度(25-40 ℃)、不同pH (5.0-9.0)和不同底物条件下，对菌株P-9^T的降解能力进行测定；利用高效液相色谱-质谱联用(HPLC-ESI-MS/MS)技术检测菌株降解PAHs的中间代谢产物，初步揭示P-9^T降解PAHs的代谢机制。【结果】菌株P-9^T为黄色杆菌科(Xanthobacteraceae)水杆菌属(Aquabacter)的潜在新种，暂命名为Aquabacter sediminis P-9^T，也是Aquabacter属中首次发现的PAHs降解菌种；在菌株A. sediminis P-9^T的基因组中则发现了一条完整的苯甲酸盐降解通路，以及参与萘和其他PAHs降解的脱氢酶、水杨酸羟化酶和细胞色素P450等关键基因。菌株P-9^T可以在25-40 ℃和pH 5.0-8.0的条件下降解菲，并且可分别以萘、菲、芘为唯一碳源生长繁殖。在菲初始浓度为50 mg/L下培养5 d降解率达100%。利用HPLC-ESI-MS/MS技术检测发现，该菌株对萘、菲、芘降解的中间代谢产物包括1-羟基-2-萘甲酸、1,2-萘二酚和儿茶酚和1-羟基-2-萘醛。推断菌株P-9^T是采用水杨酸途径降解PAHs。【结论】A. sediminis P-9^T是Aquabacter属的新种，而且是该属中首次发现的PAHs降解菌，最佳降解条件为37 ℃和pH 7.0，可通过水杨酸途径高效降解萘、菲及芘。

Abstract:

Polycyclic aromatic hydrocarbons (PAHs) are a kind of teratogenic, carcinogenic, and mutagenic organic pollutants. In recent years, the pollution of PAHs in mangrove ecosystems has attracted widespread attention, for which microbial degradation has been recognized as an effective, economical, and multifunctional treatment approach. Researchers have identified a large number of bacteria that utilize PAHs as carbon and energy sources, whereas these bacteria generally suffer from low degradation efficiency, narrow degradation spectra, and poor adaptability to high-salinity environments. In addition, the degradation mechanisms of PAH-degrading bacteria from mangroves remain to be fully explored. [Objective] The present study screened efficient and broad-spectrum PAH-degrading strains in mangrove sediments and explored their degradation efficiency and mechanism, with a view to providing a solid scientific foundation for the innovative research and development of microbial remediation technologies in mangrove ecosystems in the future.[Methods] A new PAH-degrading strain P-9^T was isolated from mangrove sediments collected from Futian, Shenzhen and identified based on the phenotypic and biochemical characteristics and phylogenetic relationship. The genomic DNA of this strain was extracted and sequenced, and the potential of P-9^T in degrading PAHs was investigated by genomic analysis. The degradation ability of P-9^T was measured at different temperatures (25–40 ℃), pH (5.0–9.0), and substrate conditions. Finally, according to the intermediate metabolites detected by high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS), we preliminarily revealed the mechanism of P-9^T in degrading PAHs. [Results] Strain P-9^T was proposed to represent a potential novel species belonging to Aquabacter of Xanthobacteraceae and named as Aquabacter sediminis P-9^T, which was also the first PAH-degrader identified within this genus. A complete benzoate degradation pathway and key genes encoding dehydrogenase, salicylate hydroxylase, and cytochrome P450 involved in PAHs degradation were found identified in the genome of P-9^T. Strain P-9^T could use naphthalene, phenanthrene, or pyrene as the sole carbon and energy source and grow at 25–40 ℃ and pH 5.0–8.0. In a mineral salt medium (MSM) with phenanthrene (50 mg/L) as the substrate, the degradation efficiency reached 100% after five days. Several metabolites, such as 1-hydroxy-2-naphthoic acid, 1,2-naphthalenediol, catechol, and 1-hydroxy-2-naphthaldehyde were detected, which indicated that strain P-9^T might degrade PAHs via the salicylate pathway. [Conclusion] A. sediminis P-9^T, the first strain of Aquabacter identified to be capable of degrading PAHs could efficiently degrade naphthalene, phenanthrene, and pyrene via the salicylate pathway, with the optimum degradation performance at 37 ℃ and pH 7.0.