利用根表功能细菌生物膜减低植物PAHs污染的机理及效能优化

Anthropogenic soil contamination has become a worldwide environmental problem in recent decades. Organic pollutants present in soil may be taken up by plants and translocated into shoots, which is the major pathway for toxic organic substances to reach the food chain. Because plants form the basis of animal and human food chains, potentially harmful organic contaminants could find their way into animal and human populations via this route. Clearly, increased understanding of how to reduce the uptake and accumulation of organic contaminants by plants from the soil could have considerable benefit for risk assessments..　Rhizobacteria usually form biofilms on root surfaces, protecting plants from external harsh environments or promoting plant growth. As known, polycyclic aromatic hydrocarbons (PAHs) are a group of persistent organic contaminants commonly found in the soil environment. Due to their highly mutagenic and carcinogenic properties, considerable interest in the control of uptake and accumulation of PAHs by plants has emerged in recent decades. In the previous project, several species of PAH-degrading bacteria (PAHDB) with the ability of biofilm formation were isolated and re-colonized on the root surfaces of target plants. These strains could form bacterial biofilms on plant root surfaces and reduce the PAH concentration and accumulation inner plant, as well as restrain the PAH translocation from plant root to shoot. Based on these results, we proposed a biofilm-aided technique to eliminate plant PAH contamination in polluted soils. However, few literatures are available heretofore on the mechanisms involved in the process mentioned above..　To this end, this project seeks to (1) investigate the sorption, distribution and degradation of PAHs on PAHDB-based biofilms; (2) clarify the molecular mechanisms involved in PAH degradation by PAHDB and investigate the transfer of PAH-degrading genes on PAHDB-based biofilms; (3) analyze the responses of resident bacterial populations on root surfaces to PAHDB-based biofilms and eludate their cooperation on PAH degradation; (4) clarify the impacts of PAHDB-based biofilms on PAH metabolism and enzyme activities inner plants; (5) optimize the conditions of biofilm-aided technique to eliminate plant PAH contamination in polluted soils. Basing on these results, the mechanisms of the biofilm-reduced uptake and accumulation of PAHs by plants will be systematically elucidated and the performance of biofilm-aided technique will be optimized. Results of this work would benefit agriculture production, food safety, and human and ecological health in PAH-contaminated sites.

植物可以吸收土壤中的有机污染物进而导致作物污染风险。如何消除植物有机污染、在污染区生产安全的农产品成为当前急需解决的问题。多环芳烃（PAHs）是土壤环境中常见的一类持久性有机污染物，在前期研究中，我们提出并证实利用具有PAHs降解功能的成膜细菌在植物根表形成的细菌生物膜（即根表功能细菌生物膜）可以减低植物PAHs污染。然而，其中涉及的机理尚不明确。基于此，本项目拟综合利用微生物学和分子生物学方法、温室盆栽实验以及稳定同位素核酸探针技术，阐明根表功能细菌生物膜对根际PAHs的吸持和降解作用，明确根表细菌生物膜内功能细菌和土著细菌种群的协同代谢作用以及基因水平转移情况，探讨根表功能细菌生物膜对植物体内PAHs代谢的影响。在此基础上，系统地阐明利用根表功能细菌生物膜减低植物PAHs污染的机理，并优化这一效能，为防治土壤有机污染、减低作物污染风险、保障农产品安全以及合理利用污染土壤资源等提供依据。

利用具有多环芳烃（PAHs）降解功能的根表细菌生物膜可减低植物PAHs污染；但其作用机制尚不清楚。基于此，本项目以前期分离的根表菲降解细菌Sphingobium sp. RS2和芘降解细菌Mycolicibacterium sp. Pyr9为材料，系统开展了相关研究，并取得了以下研究结果：（1）利用基因组和转录组测序分析，结合qPCR技术，揭示了菌株RS2和Pyr9中负责PAHs降解的关键基因，并研究了PAHs诱导下各基因的表达情况；（2）利用温室盆栽实验，分别将RS2和Pyr9定殖于空心菜根表，结合绿色荧光蛋白基因（gfp）标记或抗生素标记，追踪了RS2和Pyr9在空心菜根表和体内的定殖和分布，明确了由其形成的根表生物膜对空心菜吸收积累PAHs的影响；（3）利用基于16S rRNA基因序列的高通量测序分析，解析了不同PAHs污染强度下RS2和Pyr9的定殖对空心菜中土著细菌群落结构的影响，并研究了RS2和Pyr9与土著菌群的互作关系；（4）利用qPCR技术，解析了在空心菜根表定殖细菌形成生物膜后，其根表和体内PAHs降解关键基因的丰度和表达量变化；（5）利用酶学分析方法，研究了空心菜体内PAHs代谢相关酶系对功能细菌在空心菜根表定殖成膜的响应；（6）优化了利用根表PAHs降解细菌生物膜减低植物PAHs污染的环境条件、定殖方式和功能菌群。.基于此，系统解析了利用根表PAHs降解细菌生物膜减低植物PAHs污染的机理，并优化了其作用条件；研究成果可为发展作物PAHs污染防护技术、有效规避植物有机污染风险等提供理论依据。依托本项目，共发表论文14篇，其中在《Environmental Micobiology》、《Environment International》等国际知名刊物上发表论文12篇；培养博士研究生3名，硕士研究生4名；授权国家发明专利2项；项目组成员在国际学术会议口头报告3人次。