芘降解菌群驯化过程中的演变  

Population shift and degrading characteristics of a pyrene-degrading bacterial consortium during incubation process

作　　者：李晓明[1] 张明江[1] 金京华 刘双江[1] 姜成英[1] 

机构地区：[1]中国科学院微生物研究所,微生物资源前期开发国家重点实验室,北京100101 [2]轻工业环境保护研究所,北京100089

出　　处：《微生物学报》2012年第10期1260-1267,共8页Acta Microbiologica Sinica

基　　金：国家"863计划"(2007AA061101)~~

摘　　要：【目的】筛选、驯化获得芘的高效降解菌群,为利用其对多环芳烃污染土壤进行生物修复奠定理论基础。【方法】利用芘作为底物在矿物盐培养基(MSM)中富集驯化,得到了一个混合菌群PYR,利用分光光度计和HPLC测定混合菌群的生长与降解的关系,并对混合菌群降解多环芳烃底物的广谱性和降解芘性能的稳定性进行了测定;通过冷冻干燥的方法对菌群进行了保藏,通过HPLC的方法对保藏后复壮的菌群的降解性能进行了测定;通过培养和非培养方法对菌群的多样性进行了调查,通过构建16S rRNA基因文库的方法,分析焦化厂原始土壤中菌群组成和混合菌群转接3次(PYR-3)、6次(PYR-6)和9次(PYR-9)后的组成变化。【结果】该混合菌群可以利用芘作为唯一碳源和能源生长,12 d对芘的降解率为89%,对于菲(86%)及荧蒽(49%)也具有较高的降解率,但不能降解萘和茚并芘,并且该菌群的降解活性经过多次转接和冷冻干燥保藏保持稳定,从混合菌群中分离得到了9株菌,这9株菌分布在无色杆菌属(Achromobacter),芽胞杆菌属(Bacillus),节杆菌属(Arthrobacter),微小杆菌属(Exiguobacterium)和类土地杆菌属(Parapedobacter)。系统进化分析表明变形菌门是土壤原样(100%)及以芘为底物富集的混合菌群中的主要类群(PYR-3,83%),与原土壤样品相似,PYR-3中γ-Proteobacteria(占变形菌门的77%)中假单胞菌属的菌依然占主导地位,但由于菌群的多样性增加,假单胞菌属所占比例减少。随着富集代数的增加,菌群的多样性进一步增加,γ-Proteobacteria的比例在混合菌群中的比例下降(PYR-6中占变形菌门的33%,PYR-9中占变形菌门的18%),而β-Proteobacteria在混合菌群中的比例上升(PYR-3中占变形菌门的13%,PYR-6中占变形菌门的36%,PYR-9中占变形菌门的55%)。【结论】混合菌群具有很强的芘的降解性能,并且随着传代次数的增加,菌群的组成趋于稳定。[Objective] The work aimed to obtain pyrene-degrading bacterial consortium and use it for bioremediation of polycyclic aromatic hydrocarbons-contaminated soil.[Methods] We enriched and incubated a bacterial consortium utilizing pyrene as the sole carbon source from the contaminated soil of Beijing Coking Chemical Plant.We analyzed the degrading ability and growth of the consortium by high performance liquid chromatography（HPLC） and spectrophotometer.We investigated the degradation activities of the consortium after several times transfer and freeze-drying deposit.We also investigated the shift of bacterial consortium composition after several times transfer by combining of culture-dependent and culture-independent methods.We constructed and analyzed the 16S rRNA gene clone libraries at different transfer times（3 times PYR-3,6 times PYR-6 and 9 times PYR-9） for monitoring the bacterial population changes.[Results]The degradation rate of pyrene,phenanthrene and fluoranthene reached 89%,86% and 49% respectively after incubation of the consortium for 12 days.The degradation activities of the consortium were stable after several times transfer and freeze-drying deposit.We isolated nine strains from the consortium,affiliated to genus of Achromobacter,Bacillus,Arthrobacter,Exiguobacterium and Parapedobacter.Phylogenetic analyses showed that Proteobacteria were the main group at contaminated soil sample（100%） and bacterial consortium incubated in pyrene（PYR-3,83%）.Bacterial community structure was shifted and biodiversity was increased during the transfer process,the proportions of γ-Proteobacteria was decreased from 77%（PYR-3） to 33%（PYR-6） and 18%（PYR-9）,while β-Proteobacteria increased from 13%（PYR-3） to 36%（PYR-6） and 55%（PYR-9）.[Conclusion] The bacterial consortium could utilize pyrene as sole carbon and energy source for growth and the degradation ability for pyrene was stable.

关 键 词：芘 细菌群落 菌群结构 16S rRNA基因文库 生物降解 

分 类 号：X172[环境科学与工程—环境科学]