石化污染土壤中原位降解功能微生物磁纳米识别及强化修复技术研究

Petrochemical contamination has caused serious damage to soil environment. There are three key restrictive factors in the bioremediation of petrochemical contaminated sites: pollutants biological availability, functional microbial activity in situ degradation and soil microcosms. The evaluation of the biological availability of existing pollutants and the technical bottleneck problem in the research of functional microbe in situ degradation are investigated in this research. Some core technology such as magnetic-nanoparticles mediated isolation and the analysis of molecular ecology in microbial community will be combined. The interaction model of ‘contaminant-microorganism-soil microcosms’ in bioremediation process is also established. As an emerging molecular research techniques, the surface modification of magnetic nanoparticles are used to magnetize, adsorb and label the environmental microbial cells and then the microbial cells are cultivated under specific conditions in situ. The living functional microbes gradually proliferate and demagnetize and then use magnetic fields to separate living and inert bacterial cells effectively. Thus it realizes the enrichment and isolation of living functional microbe in situ. Meanwhile, the whole-cell biosensors, Illumina Genome Analyzer, real-time PCR and Biolog are used in the research to evaluate the ecotoxicity and bio-available potential of contaminated sites. Those techniques are also used to identify and analyze the structure of soil microbial community. The research reveals the core ecological function of functional genes and microorganism in situ degradation. The activity and efficiency of functional microbe are intensified in situ degradation and the soil bioremediation capability is also enhanced. The ecological function and bioremediation mechanism of soil ecosystem are improved under the condition of environmental pollution.

石化污染对我国土壤环境造成严重破坏。石化污染场地生物修复存在三大核心限制性因素，既污染物生物可利用性、原位降解功能微生物活性和土壤微环境。本研究将结合磁性纳米颗粒分离和微生物群落分子生态分析等核心技术，建立生物修复过程中“污染物-微生物-土壤微环境”相互作用模型。作为新兴分子研究手段，开发表面改性磁纳米颗粒，对环境微生物细胞进行磁化吸附标记，于特定原位条件下培养后使活性功能微生物逐渐增殖、去磁，进而利用磁场分离实现活体功能微生物的原位富集。同时借助全细胞生物传感器、Illumina高通量测序、定量PCR及BIOLOG 等综合技术，进行污染场地生态毒性和生物可利用潜力评价、土壤微生物群落结构识别，揭示降解功能基因和原位降解功能菌在污染场地生物修复过程中的核心生态作用。最终优化强化原位降解功能微生物的活性和降解效率，提高土壤生物修复能力。完善环境污染条件下土壤生态系统的生态功能和生物修复机制。

随着石油化工产业不断发展，在石化生产、加工区，受污染的土壤及水体明显增加，生物修复由于在技术、经济上的优势，逐渐在石化污染环境修复领域受到广泛关注。本研究以石化污染场地中常见的污染物乙腈作为目标基质，结合磁纳米颗粒富集技术（magnetic-nanoparticle mediated isolation，以下简称MMI）和同位素标记技术（Stable isotope probing，以下简称SIP），同时借助Illumina高通量测序技术、荧光定量PCR技术以及宏基因组测序，建立了一种简单便捷地识别污染原位修复过程中功能微生物的方法，强化原位降解功能微生物活性和降解效率，为复杂的污染场地原位修复功能菌的鉴别提供更多方法。.本研究采用SIP和MMI两种方法研究了石化污染生物修复中短期和长期两个阶段污染物降解功能微生物，通过高通量测序技术，分析了活性乙腈降解菌的群落结构，同时研究了不同分离方法及处理方式对功能微生物群落结构的影响。研究表明短期处理阶段两种方法筛选出的降解菌差异较大，MMI法可以弥补SIP法不适用于低同化率污染物的缺陷，实现乙腈降解菌的准确鉴别和原位分离；在长期处理阶段，由于同化率的增大使得SIP法与MMI法筛选出了相同的功能微生物，一些生长缓慢但也具有乙腈降解能力的微生物被筛选出来，丰富了乙腈降解的菌株资料。利用宏基因组测序技术研究乙腈代谢过程，结果表明，SIP与MMI两反应体系的氮代谢路径相同，腈化物都是在腈水解酶（EC 3.5.5.1）的作用下一步水解为氨；而对于腈水解酶（EC 3.5.5.1）在不同反应体系中的丰度分析结果显示，其在MMI体系中的丰度显著高于SIP体系，说明MMI法能够实现对功能降解菌的富集，提高乙腈的降解率，是在复杂环境介质中鉴别分离功能微生物的一种有效的技术。本研究对石化污染中特定污染物乙腈的降解机理和代谢途径的研究提供了新的方法和思路，并为石化污染原位修复过程中乙腈的去除提供了数据资料。