食细菌线虫调控细菌群落影响污染土壤石油降解的机理研究

The remediation of soil petroleum contamination is becoming increasingly important. Bacteria are the key organisms for petroleum degradation in contaminated soil and bacterial-feeding nematodes are the main functional group of soil nematodes. In addition to their roles in metabolic activity, soil bacterial-feeding nematodes are involved in soil ecological processes through grazing on soil bacteria, resulting in changes in the number of microbes, microbial activity and microbial community structure. However, the potential impact of bacterial-feeding nematodes on the petroleum degradation through grazing on soil bacteria is currently unknown. In this study, various species of bacterial-feeding nematodes in various densities will be inoculated into petroleum-contaminated soils with the following variable conditions: soil temperature, moisture and organic matrix. The impact of bacterial-feeding nematodes on the dynamic of bacterial community structure will be investigated using methods called denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP). Further, the effect of bacterial-feeding nematodes on the number of petroleum-degrading bacteria in contaminated soil will be investigated using a method called fluorescence in situ hybridization (FISH) using specific oligonucleotide probe markers. The goal of this project is to reveal the role of bacterial-feeding nematodes in degrading petroleum in contaminated soils, to identify the environmental factors that are favorable for bacterial-feeding nematodes and petroleum-degrading bacteria enhancing the petroleum degradation, to explore the biological mechanisms of bacterial-feeding nematodes' influence on petroleum degradation, and to clarify the mechanisms of molecular biology for "grazing and enhancing" relationship between bacterial-feeding nematodes and petroleum-degrading bacteria. It is expected that all the research into this program will pave the way for discovery and rational use of beneficial soil organisms that can regulate the degradation of petroleum in soils.

石油污染土壤的修复日益重要，细菌是污染土壤中降解石油的主要生物。食细菌线虫是土壤线虫的重要功能类群，可通过取食细菌影响微生物的数量、活性和群落结构，参与土壤的多种生态过程，但食细菌线虫如何调控细菌群落影响污染土壤石油降解的机理尚不清晰。本研究拟通过在不同土壤温度、湿度和有机质条件下,接种不同种类和密度的食细菌线虫，采用变性梯度凝胶电泳及末端限制性片段长度多态性技术，研究食细菌线虫作用下石油污染土壤细菌群落结构的变化；然后利用荧光原位杂交技术，探究食细菌线虫对石油降解菌数量的影响；在此基础上分析污染土壤石油降解与食细菌线虫及石油降解菌群落组成和数量的相关关系，确认在食细菌线虫与石油降解菌互作条件下影响石油降解的环境因子及生物学机理，明确食细菌线虫在石油污染土壤中的生态功能，为揭示食细菌线虫与石油降解菌之间的"牧食与促进作用"的分子生物学机制及发掘和利用调控石油降解的有益土壤生物提供理论基础。

石油污染所造成的土壤生态的破坏已成为阻碍社会经济可持续发展的重大环境问题，土壤石油污染修复尤为迫切。土壤线虫广泛存在于石油污染土壤中，其中食细菌线虫是优势类群。但有关石油污染土壤中食细菌线虫的生态功能及食细菌线虫与细菌之间的相互作用对土壤石油降解的影响还不明确。本项目通过在石油污染土壤中接种不同种类和密度的食细菌线虫，利用分子生物学技术，研究石油的降解及细菌数量和群落结构的变化；研究在不同土壤温度、水分和有机质条件下，食细菌线虫与细菌相互作用对石油降解的影响及细菌数量和群落结构的变化。.结果表明：⑴ 接种不同密度食细菌线虫对石油的降解影响不同，其中处理SPN5的石油残留量最低，即1克干土添加5条食细菌线虫降解石油效果最优。石油污染土壤中的食细菌线虫可以增强土壤微生物活性，促进石油降解。⑵ 在石油污染土壤中分别接种3种食细菌线虫，处理SPN2的石油降解效果最佳，即头叶属线虫对石油降解的促进作用最好。接种食细菌线虫可增加石油污染土壤的基础呼吸、脱氢酶及脲酶活性，其中SPN2的脱氢酶、脲酶活性最高；食细菌线虫降低了土壤细菌/真菌的比值，改变了微生物群落结构。⑶ 添加有机质可促进食细菌线虫与微生物互作对污染土壤石油的降解作用。有机质添加可改变石油污染土壤微生物活性，促进土壤微生物的生长。添加有机质能显著增加土壤微生物总量和细菌总量，增加土壤中革兰氏阴性菌和阳性菌数量。⑷ 不同温度培养时，处理SPT3的石油降解效果最佳，即30℃培养条件下石油降解速度最快。随着温度升高，微生物总数下降，真菌与细菌群落的相对丰度降低。不同水分条件下，处理SPM3的石油降解效果最佳，即土壤含水量为饱和含水量的60%时，石油降解最快。研究结果表明食细菌线虫和细菌之间的相互作用可有效提高土壤的酶活性，改变微生物群落结构，促进污染土壤石油降解，即在石油污染土壤中食细菌线虫与细菌之间存在“牧食作用”，初步揭示了土壤食细菌线虫促进石油降解的生物学机制。