施氏假单胞菌YC-YH1的萘降解特性及产物分析

【目的】萘是一种重要的环境污染物，它在环境中的积累会对人类健康造成危害，生物降解是解决这一问题的有效方法。本实验室保存的施氏假单胞菌YC-YH1对萘具有较强的降解能力，在此基础上，研究和分析菌株对萘的降解特性、环境因素对萘降解率的影响以及代谢产物。【方法】本文首先采用单因素实验法研究pH、温度、接种量、萘初始浓度对萘降解率的影响；并在单因素实验结果的基础上，利用Design-Expert 8.0.5软件和Box-Behnken设计对pH、温度、接种量3个影响因素进行响应面优化分析，建立环境因素对萘降解率影响的优化模型。利用LC-MS检测萘降解过程中产生的重要代谢产物，从而推测菌株对萘的代谢途径。【结果】响应面分析结果表明，优化模型极显著(P<0.001)，拟合度良好，预测结果可信度高。降解实验证明，在培养温度为32.4 °C、pH为7.10、接种量5.74% (体积比)的优化条件下培养3 d即可将浓度为100 mg/L的萘100%降解。LC-MS分析表明，菌株降解萘的过程中，能够被检测到的主要代谢产物有1,2-二羟基萘、水杨酸、邻苯二酚等。【结论】施氏假单胞菌YC-YH1对萘有高的降解效率，pH、温度、接种量3个因素对菌株的降解率有较大影响。利用响应面法优化菌株对萘的降解条件，能够提高YC-YH1菌株对萘的生物降解性能。初步推测菌株YC-YH1对萘的降解是通过水杨酸途径，萘首先被其代谢为1,2-二羟基萘，而后被转化为水杨酸和邻苯二酚，最后进入三羧酸循环被彻底降解。

Characteristics and product analysis of naphthalene degradation by Pseudomonas stutzeri YC-YH1

Objective] Naphthalene is the simplest of polycyclic aromatic hydrocarbons and is considered as an environmental pollutant that has gained a lot of public concern owing to its carcinogenicity and persistent organic properties. Biodegradation is generally recognized as the mass balance-wise most important route of naphthalene degradation. A previous study has revealed that Pseudomonas stutzeri YC-YH1 could degrade naphthalene effectively. This study aimed to characterize degradation, optimize the degradation rate of Pseudomonas stutzeri YC-YH1 for naphthalene and investigate naphthalene degradation pathways. Methods] Firstly, effects of pH, temperature, inoculum amount and initial concentration of naphthalene for the naphthalene-degrading rate of Pseudomonas stutzeri YC-YH1 were determined by single factor experiments. Then based on the results of the single factor experiments, the software Design Expert 8.0.5 and Box-Behnken design was used to analyze and optimize the model of naphthalene degradation rate by three key factors (pH, temperature, and inoculums amount) through response surface methodology. LC-MS was used to analyze the degradation pathway and products. Results] An optimization model was then constructed, and its validity was verified. The model was significant (P<0.001) and fitting well. In confirming experiments, under the optimization condition of temperature 32.4 °C, pH 7.10, inoculation 5.74% (V/V), the degradation rate of 100 mg/L naphthalene was 100% after incubation for 3 days. LC-MS data indicate that it is possible to find three major metabolites of naphthalene, which include 1,2- dihydroxynaphthalene, salicylic acid and catechol. Conclusion] The results suggested that three key factors (pH, temperature, and inoculums amount) could affect the degradation rate of naphthalene, and optimization of the response surface methodology could improve the biodegradation efficiency of naphthalene by Pseudomonas stutzeri YC-YH1. The possible degradation pathway of strain YC-YH1 was catechol pathway. Naphthalene was first turned into1,2-dihydroxynaphthalene, and then salicylic acid and pyrocatechol, finally entered the tricarboxylic acid cycle.