不同地域土壤微生物对多环芳烃污染的响应及其分子机制

In this research, we will collect soils that were polluted by polycyclic aromatic hydrocarbons (PAHs) from different provinces/geographical regions in China. The 16 PAHs under priority control will be detected by utilizing GC-MS, and the information will be used to assess soil PAHs pollution status and their ecological and health risk. Using the high-throughput sequencing technologies, multivariate statistics and topological analysis, soil microbial community structures and the interactions between soil microbial community and PAHs pollution will be evaluated. Meanwhile, the PAHs pollution bioindicators and PAHs-degrading microorganisms will be identified from the different PAHs polluted soils. The real-time quantitative PCR, PCR-DGGE and molecular cloning techniques will be used to investigate the abundance and diversity of PAHs-degrading functional genes, such as polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase (PAH-RHD) gene, and their responses to PAHs pollution in the test soils. To facilitate revealing the biotransformation of PAHs in soil, the functional genes of the isolated PAHs-degrading microorganisms will be further testified by utilizing transposon mutagenesis method. Results from this research will provide critical information to understand the response and molecule mechanism of soil microbial community and PAHs-degrading functional genes to PAHs pollution in different regions of China, obtain the genetic and genome resources of PAHs-degrading microorganisms, promote bioremediation of PAHs polluted soil, and finally ensure soil security in China.

本项目拟采集我国不同省份地域的PAHs污染土壤，GC-MS测定土壤中16种优先控制的PAHs，分析土壤PAHs污染状况及其生态、健康风险；运用第二代高通量测序技术及多元统计、网络拓扑学等方法，探究土壤微生物群落和PAHs污染间的交互关联作用，揭示不同地域土壤微生物群落对PAHs污染的响应及差异性，获得不同地域土壤PAHs污染指示性和降解功能性微生物；采用实时荧光定量PCR、PCR-DGGE和分子克隆等方法，解析不同地域PAHs污染土壤中PAH-RHD等降解功能基因丰度和多样性的分布差异及其对PAHs污染响应的分子机理，并运用转座子突变技术，验证功能菌中相关PAHs降解功能基因，分析其对PAHs的转化作用。研究成果将为深入诠释我国不同地域土壤微生物对PAHs污染的响应差异及其分子机制、获得降解功能微生物及其功能基因等种质资源、推进PAHs污染土壤高效修复以及保障我国土壤安全等提供科学支撑。

我国12个不同省份钢铁厂、焦化厂等高能耗工厂周边土壤中多环芳烃（PAHs）总浓度的变化范围为4.09-51.56 mg/kg，土壤有机碳是PAHs含量的主控因子，两者间呈极显著正相关关系；PAHs所致的苯并[a]芘总毒性当量生态风险为0.015-75.107 mg/kg，已构成潜在的生态风险。高通量测序结果表明，不同地域土壤细菌、真菌群落结构呈现出明显的分布差异，且与PAHs污染水平有关。网络与功能预测分析结果表明，丰富类群更多地占据网络核心，处于生态地位的中心，且在细菌-真菌互作网络中丰富真菌更多作为“关键物种”维持网络的稳定性；稀有物种则是更多地体现代谢功能的多样性，许多关键物种都具有对多种重金属的抗性与高效降解PAHs的能力；土壤细菌、丰富细菌群落以确定性过程为主，而真菌、稀有细菌以随机性过程为主。首次构建了三域微生物高通量绝对定量法并揭示了PAHs污染土壤中古菌呈不连续分布，细菌、真菌单域以及三域微生物整体的属水平均服从偏对数正态分布；长期PAHs污染下土壤细菌-细菌、细菌-真菌相互作用关系紧密，且互利关系多于竞争关系，并趋向以降解功能菌群聚集。从不同地域PAHs污染土壤中筛选到20余株菲芘降解功能菌，从属于分枝杆菌属、红球菌属等12个菌属；获得的降解菌株含有RHDα-GP、nidA、nidB、narAa、ndo、pdo等多种功能基因，全基因组更全面揭示了菌株WY10、WB9的功能基因以及对菲芘的高效降解途径与交互代谢协同机制；钢铁厂周边土壤中含有RHDα-GP、nidA、nidB等功能基因，携带这些功能基因的微生物对土壤PAHs降解起着主导作用。菌株WY10和WB9对PAHs污染土壤有良好的修复效果但修复机制不同；群落结构多样性分析表明接种降解菌WY10和WB9对于土壤细菌群落结构并无显著影响，说明它们是土壤PAHs污染修复高效且次生污染小的环境友好型生物材料。