漆酶介导土壤PAHs降解体系下微生物生态过程研究

Benzo[a]pyrene, a high molecular weight polycyclic aromatic hydrocarbons (PAHs), has become a matter of great concern due to its low water solubility and high carcinogenicity. Laccases produced by white-rot fungi are capable of oxidizing benzo[a]pyrene rapidly, thus have a promising prospect in PAHs remediation. However, current studies mainly focus on the initial transformation step by laccases, but do not provide adequate information about subsequential degradation of the PAHs metabolites that were produced from the initial transformation. In this work, the subsequential degradation (from the perspective of PAHs mineralization and detoxification) on the metabolites of benzo[a]pyrene primarily oxidized by laccase, and the corresponding response of soil microorganism (from the perspective of microbial community composition using the technique of PCR-DGGE, clone libraries, and real-time quantitative PCR) was investigated in soil slurry and microcosm. The results of this study will be significant to elucidate the role of soil microorganism in the subsequential degradation of this laccase-dependent remediation approach, and further benefit for its practical application.

苯并[a]芘因其极低的水溶性和高致癌性，成为土壤多环芳烃(PAHs)修复的重点与难点。漆酶能够快速转化苯并[a]芘等高环PAHs，因此在PAHs修复中显现出广阔的应用前景，但目前针对土壤PAHs经漆酶转化之后的后续降解过程尚存诸多疑问。本项目拟针对该后续降解过程，选用苯并[a]芘为高环PAHs代表，设计泥浆法摇瓶实验和土壤微域实验研究苯并[a]芘经漆酶转化之后的矿化与解毒过程；并采用PCR-DGGE、克隆文库和定量PCR等分子生态学方法，探讨该后续降解对土壤微生物群落结构的影响。该项目有助于加深对漆酶介导下土壤苯并[a]芘微生物降解过程的了解，并对建立以漆酶为基础的微生物修复技术提供重要理论依据。

漆酶能够快速转化苯并[a]芘（BaP）等高环多环芳烃（PAHs），在PAHs修复中显现广阔应用前景。本项目重点研究了土壤体系下漆酶转化BaP对污染物归趋及土壤微生物生态效应的影响。研究结果表明，该转化方式一方面提高了污染物的矿化率（由0.1%提高至0.5%），另一方面促进了污染物与土壤腐殖质的稳固态结合（约增加10%的化学结合态）。研究采用荧光定量PCR（qPCR）和454高通量测序技术研究了该修复过程中土壤微生物的响应。结果发现细菌16s rRNA基因和双加氧酶基因拷贝数并未显著提高，但是微生物群落结构发生了显著分异，而且漆酶转化BaP降低了污染物对土壤微生物的生态干扰效应，暗示该修复方式主要形成脱毒效应。综上研究，证实了利用漆酶进行土壤PAHs修复是一种有效的生物学方法。本项目资助下，我们还扩展了细菌漆酶对PAHs的转化研究，结果表明枯草芽胞杆菌CotA蛋白能够高效的转化BaP，是一种潜在的修复PAHs的生物资源。相对真菌漆酶而言，细菌漆酶在土壤中的稳定性、分子改造及大规模生产等方面具有潜在应用价值。该项目研究为发展以漆酶为基础的生物修复技术提供了重要理论依据。