老化油泥对土壤的动态污染机制与微生物多样性影响效应研究

This project aims to research the dynamic pollution mechanism and microbial diversity effect of aging oil sludge on soil. Sludge retention time (SRT) of aging oil sludge in oil field is estimated based on survey. Typical soils polluted by aging oil sludge of medium- and long-term （>2 years) are sampled both in plane and in profile. The compositions and contents of main pollutants such as total petroleum hydrocarbon (TPH) and heavy metals are analyzed. Then the soil structure, physical and chemical properties, such as soil porosity, moisture rate, pH value, organic carbon composition, salinity are integrated studied, revealing the spatial pollution characteristics of contaminated soil derived from aging sludge with different SRTs in different regions. Through genomic DNA extraction, PCR amplification and fluorescent quantitation, Miseq high-throughput gene sequencing technology is adopted to get microbial diversity information. The result could help to determine the effect on characteristics of microbial diversity of aging sludge with different SRTs. Dynamic simulated experiment is designed to study short-term (≤ 2 years) contamination effects of sludge aging process on soil. The soil structure and composition under different sludge pollution time, the spatial-temporal distribution and variation trend of main pollutants are researched to expound the dynamic pollution mechanism of sludge aging process on soil. The variable characteristic of soil microbial diversity is analyzed, so as to determine sludge aging effects on the variation tendency of soil microbial diversity. Based on the above research, degradation bacteria with special effects to petroleum pollution is screened, providing fundamental data for the bioremediation of aging oil sludge contaminated soil in oil producing region.

通过调研确定油田老化油泥泥龄，对典型老化油泥中长期污染土壤进行平面和剖面取样（2年以上），分析土壤样品中总石油烃、重金属等主要污染物组成和含量，结合土壤结构和孔隙率、持水率、pH值、有机碳等物理化学性质，揭示不同地区、不同泥龄老化油泥对土壤的空间污染特征与差异；通过基因组DNA提取、PCR扩增、荧光定量，采用Miseq高通量基因测序技术，获取生物多样性信息，确定不同泥龄老化油泥对土壤中微生物多样性特征的影响。开展油泥老化过程对土壤短期（≤2年）污染效应的动态模拟实验，研究油泥污染过程中不同时间段的土壤结构、成分及主要污染物的时空分布和变化趋势，阐述油泥老化过程对土壤的动态污染机制；分析土壤微生物多样性变化特征，明确油泥老化过程对土壤微生物多样性变化规律的影响。在此基础上，筛选石油污染优势特色降解菌，为采油区老化油泥污染土壤微生物修复提供基础数据。

以胜利油田采油区土壤作为研究对象，通过分析受老化油泥污染土壤的理化性质和微生物多样性变化特征，揭示了典型老化油泥对土壤的中长期污染特征及对土壤微生物多样性的影响效应；进一步通过对大庆油田典型老化油泥污染土壤进行研究，揭示了老化油泥对土壤的中长期污染与微生物多样性效应的区域特征和差异；通过油泥和盐渍化联合作用对土壤污染的动态模拟实验，揭示了油泥老化过程和盐渍化联合污染胁迫对土壤的动态污染机制及微生物多样性的影响，筛选和确定适应多种土壤类型，对油泥污染物具有耐受性的优势特色降解菌，为石油污染土壤的原位微生物修复提供理论依据和数据支持。. 实验结果表明：石油污染物的输入会导致土壤理化性质的显著改变，使土壤总有机碳、重金属（Cu、Zn、Cr）、总石油烃等的含量显著提升，pH值降低。Miseq高通量测序分析结果揭示：受老化油泥污染后，土壤微生物多样性显著改变，且老化油泥污染愈强，土壤中微生物多样性指数愈高。胜利油田和大庆油田两油田的细菌门类分布表明：老化油泥污染，明显有利于变形菌（Proteobacteria）、放线菌（Actinobacteria）、拟杆菌（Bacteroidetes）、和芽单胞菌（Gemmatimonadetes）等门类的发育，但对厚壁菌（Firmicutes）的发育具有强烈抑制效应；绿湾菌（Chloroflexi）和酸杆菌（Acidobacteria）的生长在胜利油田被促进，在大庆油田被抑制。. 细菌科、属变化特征揭示，被相同泥龄老化油泥污染的土壤中，石油污染物对厌氧绳菌科（Anaerolineaceae）、假单胞菌属（Pseudomonas）均表现出明显促进作用。这两种细菌作为特色耐油微生物，具备对老化油泥污染土壤进行原位修复的潜质。. 石油和盐渍化联合动态模拟实验揭示：在石油和盐渍联合污染胁迫下，芽单胞菌科（Gemmatimonadaceae）既能高效降解石油烃又对盐碱有较高耐受度，表现出良好增长趋势。而伴随油泥老化过程，乳杆菌科（Lactobacillaceae）和红螺菌科（Halorhodospiraceae）在加入原油后出现；其中，乳杆菌科（Lactobacillaceae）能够利用碳水化合物等进行发酵为其他微生物提供代谢底物。