三氯乙烷污染地地下水中Dehalobacter属细菌定量与多样性分析

目的]对某公司6个以1,1,1-三氯乙烷(1,1,1-Trichloroethane,1,1,1-TCA)为主要污染物的地下水样品中的降解微生物Dehalobacter spp.(Dhb)进行相对定量和多样性分析.方法]采用气相色谱法测定6个样品中1,1,1-TCA、1,1-二氯乙烷(1,1-DCA)和氯乙烷(CA)的浓度；通过定量PCR法分别测定6个样品中Dhb占总菌的百分比；以16S rRNA基因通用引物和Dhb特异性引物扩增获得的PCR产物构建了6个样品的Dhb特异性克隆文库,所得序列与GenBank中的最相似序列构建系统发育树.结果]6个样品中均有1,1-DCA和(或)CA的检出,推测此6处地下水中1,1,1-TCA可能存在生物降解.定量PCR结果表明,6个样品中Dhb丰度差异较大.6个Dhb特异性克隆文库获得41条序列,序列比对结果表明,与它们最相似的已知分类地位的序列全部属于Dhb属.这些序列按99％的相似性被划分成7个可操作性分类单元(OTU).其中24条序列属于OTU1,该OTU的序列与已知能阵解1,1,1-TCA的Dehalobacter sp.str.TCA1的16S rRNA基因序列相似性达98％;文库中的3个OTU与GenBank中16S rRNA基因序列同源性最高仅为95％-96％.结论]该污染场地地下水中存在多样性较丰富的降解微生物Dehalobacter属细菌,它们可能与现场的1,1,1-TCA生物降解有关.

Diversity of 1,1,1-trichloroethane degrading Dehalobacter spp. in contaminated groundwater

Objective] In order to study the diversity of Dehalobacter spp. from six 1,1,1-trichloroethane (TCA) contaminated groundwater samples. Methods] The concentration of 1,1,1-TCA, 1,1-dichloroethane (DCA) and chloroethane (CA) of each sample was determined using gas chromatography. Real-time qPCR was performed on each sample to estimate the ratio of Dehalobacter spp. (Dhb) to the total bacteria. PCR products amplified by combining 16S rRNA gene universal primer and Dehalobacter group-specific primer were used to construct six Dhb group-specific clone libraries, respectively. Phylogenetic tree was constructed based on the sequences from the clone libraries and their nearest neighbors from GenBank. Results] The existence of 1,1-DCA and (or) CA in the 6 samples may associate with 1,1,1-TCA biodegradation. Real-time qPCR results demonstrated that the ratio of Dhb to the total bacteria varied in each sample. In total 41 sequences were obtained from these 6 clone libraries and BLAST showed all their nearest sequences were affiliated to Dhb. These sequences were clustered into 7 operational taxonomic units (OTUs) at a threshold of 99% similarity. OTU1 (24 sequences) has a similarity of 98% with the 1,1,1-TCA reductively dechlorinating strain Dehalobacter sp. str. TCA1. Three other OTUs only has 95%?96% similarity with their nearest 16S rRNA gene sequences in GenBank. Conclusion] This study demonstrated the existence of Dhb in all 6 samples with a relatively high diversity and these Dhb bacteria might be responsible for the biodegradation of 1,1,1-TCA in the groundwater of sampling sites.