克拉玛依油田石油污染土壤细菌群落结构与环境因子的关系

Effect of environmental factors on bacterial community structure in petroleum contaminated soil of Karamay oil field

摘要:

[目的] 以16S rRNA为分子标记，探讨克拉玛依油田石油污染土壤中细菌群落多样性和系统发育，并分析环境因子对群落分布的影响，为生物降解石油污染物提供理论基础。[方法] 在克拉玛依油田分别采集深度为5、20、50 cm的石油污染土壤样品，测定环境参数；提取石油污染土壤细菌群落基因组DNA，分别构建3个土层细菌16S rRNA基因文库，利用限制性片段长度多态性分析（Restrictionfragment length polymorphisms RFLP）技术初步分群，确定各文库中的代表菌株并测定16S rRNA基因序列；利用软件Biodap计算各群落多样性和丰富度指数，以Neighbor-Joining法构建3个土层细菌的系统发育树；运用软件CANOCO 4.5结合不同样品环境因子的差异进行典型对应分析（CCA），并探讨了环境因子对细菌多样性的影响。[结果] 环境参数结果表明20 cm土层总磷（TP）、总氮（TN）含量最低，50 cm含量最高；5 cm土层中有机碳（TOC）含量最高，50 cm含量最低。基于16S rRNA序列的生物多样性和物种丰富度指数表明20 cm土层生物多样性和丰富度指数较高，而50 cm土层各项指数均较低。各土层供试序列RFLP聚类分析表明，克拉玛依油田石油污染土壤细菌种群具有丰富的多样性。Neighbor-Joining构建的系统发育分析表明，石油污染土壤被分为5个类群（I–V），分别为变形菌门（Proteobacteria）、放线菌门（Actinobacteria）、厚壁菌门（Firmicute）、拟杆菌门（Bacteroidetes）、浮霉状菌门（Planctomycetes），其中群Ⅰ占78.57%，广泛分布于不同的生态环境；其中来自5 cm土层代表菌的69.23%分布于群Ⅰ。CCA分析结果显示TN、TP和TOC对大部分细菌影响较大；TOC含量对Pseudomonas影响明显。[结论] 克拉玛依油田石油污染土壤细菌群落具有丰富的多样性；环境因子是影响石油污染土壤细菌群落空间分布的重要因素。

Abstract:

[Objective] This study aimed to study the phylogenetic diversity and community structure of bacteria in petroleum contaminated soils from Karamay oil field, and to analyze the relationship between the community variation and the environment parameters, to provide a reference for bioremediation of petroleum contaminated soils.[Methods] We collected samples from petroleum contaminated soils in 5 cm, 20 cm and 50 cm depth layers, and measured the environment parameters subsequently. We constructed three 16S rRNA gene clone libraries of these soil samples, and then determined the operation taxonomy units (OTUs) restriction fragment length polymorphism method, and finally sequenced the representative clones of every OUT. The diversity, richness and evenness index of the bacteria communities were calculated by using Biodap software. Neighbor-Joining phylogenetic tree was constructed based on 16S rRNA gene sequences of bacteria from Karamay oil field and the references from related environments. Canonial correspondence analysis (CCA) was used to analyze the relationship between environment parameters and species by using CANOCO 4.5 software.[Results] Environment parameters showed that 50 cm dee_p soil contained the highest amount of total nitrogen (TN) and total phosphorus (TP), whereas the 20 cm depth soil contained the lowest amount. The 5 cm depth soil contained the highest amount of total organic carbon (TOC), whereas the 50 cm depth soil contained the lowest amount. Among the 3 layers, 20 cm depth had the highest diversity and richness of bacteria, whereas the bacteria in 50 cm depth was the lowest. Phylogenic analyses suggested that the bacteria in Karamay oil field could be distributed into five groups at the level of phylum, Cluster I to V, respectively belong to Proteobacteria, Actinobacteria, Firmicute, Bacteroidetes, Planctomycetes. Cluster I accounts for 78.57% of all tested communities. CCA results showed that TN, TP, TOC significantly affected the bacteria community structure. Especially, TOC content is significantly related to the distribution of Pseudomonas.[Conclusion] The petroleum-contaminated soil inhabited abundant of bacteria. The diversity index and spatial distribution of these communities were affected by the environment parameters in the soil.