高效柴油降解菌Acinetobacter sp.W3分离鉴定及降解酶基因扩增分析

Isolation,Identification of Alkane-degrading Bacteria Strain Acinetobacter sp.W3 and Alkane Hydroxylase Genes Analysis

从柴油污染的海水样品中分离高效柴油降解细菌,分析菌株对柴油的降解能力及降解酶基因,为海洋柴油污染的生物修复奠定基础。选取浙江定海港柴油污染的海水样品,进行降解菌的富集培养;采用常规方法分离筛选高效柴油降解菌。利用革兰氏染色、形态学观察、生理生化鉴定及16S rDNA分析等方法对降解菌株进行种属鉴定。采用紫外吸收法测定菌株对柴油的降解率。采用PCR方法、核酸序列测定和比对,对其降解酶基因进行扩增分析。筛选出一株高效降解菌,形态学观察及生理生化鉴定初步确定为不动杆菌。16S rDNA序列分析及比对结果表明,其16S rDNA序列与威尼斯不动杆菌(Acinetobacter venetianus)属的序列同源性达到99.7%,命名为不动杆菌W3(Acinetobactersp.W3),该菌对柴油的7 d降解率达到84.7%。PCR方法从Acinetobactersp.W3菌株中的基因组DNA和质粒DNA上扩增到了大小为540 bp的烷烃羟化酶基因alkB和864 bp的CYP153A部分DNA片段,分别与Acinetobacter venetianus1-D-2的alkB和Acinetobactersp.OC4、Acinetobactersp.EB104的CYP153具有99%和98%的同源性。从定海港口柴油污染海水分离得到一株高效柴油降解菌Acinetobactersp.W3,该菌属于不动杆菌属,含有烷烃降解酶基因,能高效降解柴油污染物,有望应用于海水柴油污染的生物修复。

It was to isolate and identify high efficiency diesel-degrading bacteria from diesel polluted sea water, and analyze the degrading ability and alkane hydroxylase genes of the diesel-degrading bacteria. Enrichment and isolation of diesel-degrading bacteria from sea water sample from Dinghai port were carried out. One bacteria strain with high efficiency degradability was separated and selected to be further study. The general characteristics were decided by the physiological and biochemical tests and 16S rDNA sequence. PCR method was used to analyze the alkane hydroxylase genes in separated bacteria. Results showed that the bacterium was tentatively classified as Acinetobacter sp. according to its physiological and biochemical characteristics. 16S rDNA sequencing result confirmed that it belonging to Acinetobacter sp, thus it was named as Acinetobacter sp. W3. The degrading ratio of Acinetobacter sp. W3 reached 84.7% at 7^th day. PCR results showed that Acinetobacter sp. W3 harbored both alkB and CYP153A genes in genomic DNA and plasmid DNA. The partical of these two genes, 540 bp of alkB and 864 bp of CYP153A, showed 99% identification to the alkB of Acinetobacter venetianus l-D-2, and 98% identification to the CYP153 of Acinetobacter sp. OC4 and Acinetobacter sp. EBI04, respectively. It proved that Acinetobater sp. W3 has the potential to be used in bioremediation of diesel polluted sea water.