白腐真菌-土著菌群协同矿化苯并[a]蒽机制研究

Remediation of high molecular weight polycyclic aromatic hydrocarbons (PAHs) contaminated soil always attracts the interests of environmental scientists. White rot fungi (WRF) can effectively transform PAHs to intermediate products with higher water-solubility, which can be further degraded by indigenous microflora. As a consequence, the mineralization of high molecular weight PAHs is enhanced. Therefore, WRF-mediated remedial method might develop to be an environmental friendly and effective technology; however, the mineralization mechanism was still unclear. In this proposal, benz[a]anthracene was employed as the model high molecular weight PAHs. The transformation rate, the mineralization rate, the water-soluble metabolites concentration and the transformation products structure of benz[a]anthracene by different enzymes systems from WRF are analyzed base on the isotope tracer technique, to determine the key enzymes in the initial transformation of benz[a]anthracene. Following, the shifts of the community structure and abundances of the indigenous bacteria and fungi, as well as particular guilds are monitored by PCR-DGGE and real-time quantitative PCR, to recognize the microbial processes in the successive benz[a]anthracene biodegradation. The aims of the proposal are to unravel the mechanisms of high molecular weight PAHs mineralization by synergism of WRF and indigenous microflora, and furthermore, to lay the scientific foundation for its practical application.

高分子量多环芳烃(polycyclic aromatic hydrocarbons, PAHs)污染土壤修复是环境领域的研究热点和难点。白腐真菌可高效转化高分子量PAHs，其形成的代谢产物由于水溶性增强，可被土著菌群后续降解，最终导致矿化，因此白腐真菌介导的PAHs修复方法有望成为安全高效的土壤修复技术，但是目前对其矿化机制还缺乏深入认识。本项目以苯并[a]蒽为模式高分子量PAHs，采用同位素示踪技术，分析白腐真菌不同酶系对苯并[a]蒽的转化率、矿化率、水溶性产物含量和产物结构，明确苯并[a]蒽初始转化阶段起关键作用的白腐真菌酶系；采用PCR-DGGE和定量PCR技术，分析苯并[a]蒽后续降解过程中土壤细菌、真菌、特定功能微生物群落结构和数量变化，阐明苯并[a]蒽后续降解阶段的微生物过程。本项目旨在深入认识白腐真菌协同土著菌群矿化高分子量PAHs的机制，为修复技术的深度应用奠定理论基础。

多环芳烃是一类重要持久性有机污染物，其污染问题日益严重。白腐真菌具有较强的降解能力，有望用于多环芳烃污染土壤的修复。但是，目前对白腐真菌代谢多环芳烃的机制尚不清楚，对白腐真菌与土著菌协同矿化多环芳烃的过程并不明确。本项目以血红密孔菌为研究对象，研究其对几种典型多环芳烃的降解和矿化机制。摇瓶实验表明，血红密孔菌具有较高的漆酶产量以及对菲和苯并[a]蒽的降解率。血红密孔菌菌丝体和胞外液均可以转化菲和苯并[a]蒽。通过气质联用技术鉴定了白腐真菌及其菌丝体和胞外液对菲和苯并[a]蒽的代谢产物，并总结了代谢途径，发现血红密孔菌可以通过至少两条途径代谢菲：（1）先胞外漆酶初始转化菲成为1,4-dihydrophenanthrene-9,10-dione，然后转化为 phenylmethanol；（2）P450以及其它胞内酶把菲先转为为2-dibenzofuranol，然后转化为2-methylphenol。而血红密孔菌对苯并[a]蒽只有一条代谢途径，即先通过胞外漆酶转化为benz[a]anthracene-7,12-dione，然后转化为2,3-dihydroxy-3-phenylpropanoic acid 和citramalic acid。. 通过摇瓶实验发现，血红密孔菌及其菌丝体和胞外液可转化蒽；血红密孔菌及其菌丝体也可以转化芘。对于蒽，血红密孔菌主要通过胞外漆酶、P450和胞内漆酶三条途径进行初始转化；但对于芘，血红密孔菌可以通过P450一条途径进行初始转化。. 通过微宇宙实验结合同位素示踪技术发现，蒽在土壤中可以迅速发生矿化，土著细菌可能在蒽矿化中发挥了重要作用，真菌也起到了一定作用。木质素抑制了土壤中蒽的矿化，这有可能是由于木质素是比蒽更容易利用碳源。稳定性同位素探针结合高通量测序技术结果表明，土壤中Acidobacteria_Gp1是参与蒽代谢的主要菌种。. 这些结果丰富了对白腐真菌及土著菌降解多环芳烃机制的认识，为完善白腐真菌修复多环芳烃污染土壤技术奠定了基础。