滨海湿地芦苇根际PAHs降解功能古菌群落组成与多样性研究

Microbial degradation plays an important role in organic pollutant degradation in rhizosphere. However, studies on microbial degradation of polycyclic aromatic hydrocarbons (PAHs) in rhizosphere have mostly focused on eubacteria, relatively little is known on the identity, contribution and ecological mechanisms of archaea in PAH degradation in rhizosphere. The coastal wetlands in our country are generally polluted with PAHs severely. The microbial degradation in the rhizosphere of common reed (Phragmites australis), which is usually a dominant plant species in coastal wetland, represents a very important ecosystem function and service of coastal wetlands. This project focuses on archaea in the rhizosphere of the common reed originally collected from the Yellow River Delta, Shandong. By constructing spatial gradient in rhizosphere in the lab and applying selective inhibitors to bacteria and archaea, the relative contribution of archaea to the degradation of phenathrene will be quantified. By using stable isotope probing and high throughput sequencing, the identity, composition and relative abundances, and spatiotemperal dynamics of archaea that function in the degradation processes will be characterized. Based on 16S rDNA data of Archaea, archaea-related virtual metagenomes will be constructed and, aiming to explore the correlation between archaeal genome and PAH degradation in the rhizosphere of common reed. The outcome of this project will further our understanding of community composition and diversity of archaea participating PAH degradation in coastal wetlands, and will provide essential data towards biodegradation of organic pollutants with archaea in coastal wetland soils.

微生物降解在植物根际有机污染物降解过程中起到重要作用。但目前根际多环芳烃微生物降解的研究常以细菌为主，对参与降解过程的古菌及其作用与贡献还需进一步明确。我国滨海湿地多环芳烃污染严重，湿地芦苇根际生物降解可能是区域性污染修复的重要途径。本项目拟以黄河三角洲滨海湿地的芦苇为取材来源,在室内构建根际梯度微域，通过添加选择性古菌及细菌抑制剂，探明芦苇根际古菌对多环芳烃降解的贡献率；利用稳定同位素探针与高通量测序技术，解析污染物添加后根际多环芳烃降解功能古菌的群落组成、相对丰度及其时空异质性；基于古菌群落16S rDNA序列信息构建根际古菌的虚拟宏基因组，探索古菌功能基因组与多环芳烃降解过程的联系。本项目旨在揭示滨海湿地芦苇根际参与多环芳烃降解的古菌群落组成与多样性，为植物根际古菌参与有机污染物降解的内在机理提供重要参考。

生物修复技术是目前最具潜力的土壤有机污染修复技术。植物根际微生物在有机污染物降解过程中起到重要作用，但目前古菌微生物群落在根际有机污染物降解过程的作用与贡献尚不清楚。阐明古菌在根际有机污染物降解过程中的作用机制，对优化根际有机污染物降解，强化生物修复技术具有重要作用。本项目利用选择性微生物抑制剂抑制古菌活性，定量古菌群落在根际多环芳烃降解中的贡献。根际微域中，抑制古菌活性显著增高生物有效性多环芳烃组分含量，且增高土壤有机质锁定的多环芳烃组分含量，表明古菌群落能够降低土壤多环芳烃被腐殖质锁定。基于稳定同位素探针技术(SIP) 标记多环芳烃降解微生物类群，结果表明多环芳烃降解古菌类群主要包括Halobiforma, Haloferax, Halonasta, Halorubulius, Methanolobus, Methanocaldococcus, Methanosarcina, Thermofilum, Vulcanisaeta和Woesearchaeota。 距离根系较近的根际梯度中的菲降解古菌主要以产甲烷菌为主，而在距离根系较远的根际微域中菲降解古菌则是以嗜盐古菌类群为主。近根际微域的多环芳烃降解微生物群落的多样性显著低于未被标记微生物群落，而远根际微域的近根际微域的多环芳烃降解微生物与未被标记微生物群落的多样性则较接近。多环芳烃降解菌和未被标记微生物类群的群落结构差异也随距离根系表面的矩阵增加而增加。基于 Spearman 相关性矩阵构建的相关性网络具有明显的非标度网络特征，说明构建的相关性网络为非随机结构 。网络节点的连接度与被标记序列比例成显著正相关，表明微生物类群的降解能力越高，则在群落中与其他类群微生物有更高的相互作用。基于古菌16s rRNA基因序列利用 PICRUSt 构建根际梯度中古菌宏基因组，表明多环芳烃降解多环芳烃降解古菌群落具有较高的降解多环芳烃的潜力，多环芳烃降解古菌宏基因组中菲降解通路的基因具有明显的根际梯度。菲代谢通路中，双加氧酶和单加氧酶的相对丰度均随到根系距离增加而升高，但醛缩酶和水杨酸羧化酶相对丰度则随到根系距离增加而降低。本项目定量了根际微域古菌群落对多环芳烃降解的贡献，探明了根际微域参与多环芳烃降解的古菌群落的组成，阐明了古菌群古菌群落在根际参与多环芳烃降解的内在机制，为优化土壤有机污染物生物修复技术提供了理论支持。