中度嗜盐菌生物强化修复盐碱土壤多环芳烃污染的机制研究

Polycyclic aromatic hydrocarbons (PAHs) are widespread in oil field contaminated soil, with some exhibiting carcinogenic, teratogenic, and mutagenic properties. This oil field contaminated soil usually has a high salt content, high pH, as well as a high concentration of PAHs. The efficiency for bioremediation of PAHs in saline-alkali soil is very low using conventional techniques. This bioremediation effect is often unsatisfactory to remove PAHs by bioaugmentation using nonhalophilic bacteria in saline-alkali soil. In order to enhance the efficiency of bioremediation of PAHs, methods that add moderately halophilic bacteria Martelella sp. AD-3 are suggested as a feasible strategy. The focus of this research is to assess the survival of degrading bacteria, the bioavailability of PAHs, metabolic activity, in addition to other systematic studies of key processes. There are a number of goals of the research, with the first being to resolve the main factors that affect the degradation of bacterial survival ability. Additionally, this work clarifies measures and mechanisms of the pollutant bioavailability, while helping to reveal the migration and transformation of the PAHs in the saline-alkali environment. Finally, the research has demonstrated the degradation mechanisms of PAHs pollutants using moderately halophilic bacteria in a saline-alkali environment, which in turn provides a scientific basis for microbial remediation of contaminated soil in this environment. The results of the research have successfully achieved efficient, stable and safe bioremediation and provide the theoretical basis for future real-world implementation.

多环芳烃（Polycyclic aromatic hydrocarbons，PAHs）是油田污染土壤中广泛存在的"三致"化合物，油田PAHs污染土壤通常具有高盐，高pH和PAHs浓度高等特点，将非嗜盐的PAHs高效降解菌引入到盐碱环境中去除PAHs的效果往往不理想。针对盐碱土壤PAHs污染生物修复效率较低的现状，采用向污染土壤中添加中度嗜盐PAHs高效降解菌 Martelella sp. AD-3的生物强化方法，以该菌在生物修复过程中的存活能力，对多环芳烃的生物可利用性、代谢活性等关键过程为主线进行系统研究，期望解析影响降解菌存活能力的主要因素，阐明促进污染物生物可利用性的措施和机制，有助于揭示PAHs在高盐污染环境中的迁移转化规律和中度嗜盐菌在盐碱环境中降解PAHs污染物的机制，为盐碱环境中PAHs污染的微生物修复提供科学依据，实现污染土壤的高效、稳定和安全生物修复奠定理论基础.

针对非嗜盐的PAHs高效降解菌在高盐，高pH和多环芳烃（Polycyclic aromatic hydrocarbons，PAHs）浓度高的油田PAHs污染土壤中不能够适应盐碱环境而存活能力较低，往往去除PAHs的效果不理想的问题，本研究在探索中度嗜盐PAHs高效降解菌Martelella sp. AD-3降解PAHs降解特性、降解途径以及阐明分子机制基础上，采用向污染土壤中添加该中度嗜盐PAHs高效降解菌的生物强化方法，实现提高污染土壤的修复效率。研究结果显示，AD-3菌在盐碱土壤中具有较强的存活能力和降解活性，其中在3%盐度，pH9.0条件下，能够完全降解蒽、芴等多种PAHs，并首次分离到3-羟基-2-萘甲酸等关键代谢产物，该菌且能够进一步降解中间代谢产物，实现对PAHs的完全彻底代谢。利用Illumina Miseq和Pacbio RS II技术完成了对AD-3菌基因组测序的完成图（DDBJ/EMBL/GenBank 序列号为 no. AYGY02000000），通过对全基因组的生物信息学分析，初步阐明了AD-3菌有关降解PAHs和嗜盐的分子特征。在基因组测序基础上，利用特异性引物克隆了龙胆酸1,2-双加氧酶（Gentisate1,2-dioxygenase, GDO）基因，并实现其在大肠杆菌E.coli21(DE3)中成功诱导表达，经亲和层析纯化重组蛋白GDO，并对其进行了酶学性质的分析，首次发现GDO开环龙胆酸时两个氧原子分别来自于H2O和O2这一加氧机制。为盐碱环境中PAHs污染的微生物修复提供科学依据，实现污染土壤的高效、稳定和安全生物修复奠定理论基础。