• 关键词：菲  反硝化降解  富集菌群  定量PCR  Illumina MiSeq测序

• 基金项目：国家自然科学基金（No.41371477）；中央高校基本科研业务费专项（No.XDJK2014B047）；西南大学光炯创新实验项目（No.2016012）

• 黄星云
• 西南大学资源环境学院, 重庆 400715

• 张泽宇
• 西南大学资源环境学院, 重庆 400715

• 熊苏雅
• 西南大学资源环境学院, 重庆 400715

• 王镱洁
• 西南大学资源环境学院, 重庆 400715

• 王明霞
• 西南大学资源环境学院, 重庆 400715

• 周志峰
• 西南大学资源环境学院, 重庆 400715

• 摘要：从潜在多环芳烃（Polycyclic Aromatic Hydrocarbons，PAHs）污染的油田区域采集土壤样品，以菲为唯一碳源且添加硝酸根的培养基来富集土壤中的菲反硝化降解菌群.随后，通过定量PCR（Polymerase Chain Reaction）测定了获取的富集菌群中反硝化相关功能基因（硝酸还原酶基因narG、亚硝酸还原酶基因nirS）的丰度，并通过Illumina MiSeq测序对其中的细菌群落结构进行解析.结果表明，获取到的3个菌群（PDN-1、PDN-2和PDN-3）12 d内对菲的降解率分别为45.18%、34.04%和25.92%.各富集培养菌群中narG的丰度均高于nirS，且菲降解率最高的PDN-1中的反硝化相关基因丰度较低.Illumina MiSeq测序结果表明，菲降解率最高的富集菌群PDN-1同时也具有较高的细菌多样性指数，变形菌门（Proteobacteria）、疣微菌门（Verrucomicrobia）和拟杆菌门（Bacteroidetes）是各富集菌群中的优势菌门，且Proteobacteria在3个富集菌群PDN-1（97.78%）、PDN-2（96.57%）、PDN-3（93.90%）中的比例均最高.变形菌门的Pseudomonas（γ-Proteobacteria）和Methylophilus（β-Proteobacteria）则是各富集菌群中最大的优势菌属，前者为公认的PAHs降解菌，而后者则为能够利用还原型"一碳化合物"的特殊菌属.细菌多样性与菲的降解率呈正相关，表明菲的反硝化降解可能是多种细菌参与的共同结果.上述结果可为揭示典型PAHs反硝化降解的微生物机制提供理论依据，同时为深入研究反硝化与菲代谢的偶联机理打下基础.

• Abstract：In this study, soil sample with the potential risk of PAHs contamination was collected from the agricultural field of Jianghan oil field. Using a selective media containing nitrate and phenanthrene (sole carbon source), the soil bacterial consortium harboring the ability to degrade phenanthrene under denitrification condition was enriched. Subsequently, the abundances of denitrification concerning genes (narG: nitrate reductase gene; nirS: nitrite reductase gene) were analyzed by quantitative PCR (polymerase chain reaction), and the bacterial community structures in the enriched consortiums were analyzed by Illumina MiSeq Sequencing. The result indicates that the phenanthrene degradation rates of these three consortiums (represented as PDN-1, PDN-2, and PDN-3) were 45.18%, 34.04% and 25.92%, respectively, within 12 days. The abundance of narG was higher than that of nirS in each enriched consortium, but the abundances of narG and nirS in the consortium PDN-1 with higher phenanthrene degradation rate were not higher than those of the other two consortiums. Moreover, the result of Illumina MiSeq Sequencing shows that the consortium (PDN-1) with higher phenanthrene degradation rate also had higher bacterial diversity, and Proteobacteria, Verrucomicrobia, and Bacteroidetes were the dominant phyla in the enriched consortiums. Among those phyla, Proteobacteria was the most abundant, occupied almost 97.78%, 96.57%, and 93.90% (ratios) of total bacterial phyla in PDN-1, PDN-2, and PDN-3, respectively. Moreover, the bacterial genus Pseudomonas (γ-Proteobacteria) and Methylophilus (β-Proteobacteria) were dominating in those enriched consortiums. Pseudomonas had been confirmed as important degrader of varied kinds of organic pollutants, and Methylophilus was a representative bacterial genu with the ability to consume reduced one-carbon compounds. Taken together, the positive relationship between phenanthrene degradation rate and bacterial diversity might illustrate that the anaerobic phenanthrene degradation under denitrification might be a co-operation of varied bacterial genus. Those results could be helpful for deeper understanding the microbial mechanism of phenanthrene degradation under denitrification, and provide some basic information for further investigation of the coupling mechanism between denitrification and PAHs degradation.

• Key words：phenanthrene  degradation under denitrification  enriched bacterial consortium  quantitative PCR  Illumina MiSeq Sequencing

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