石油污染土壤的生态风险评价和生物修复 Ⅰ.一株具有乳化石油能力的细菌分离鉴定

ECO-RISK & BIOREMEDIATION OF PETROLEUM CONTAMINATED SOIL Ⅰ.ISOLATION AND IDENTIFICATION OF PETROLEUM-EMULSIFYING BACTERIA

以江汉原油为唯一碳源,经过BH培养基富集培养,以血平板从不同石油污染土壤中分离纯化获得多株细菌。通过测定这些细菌对柴油的乳化程度,发现菌株X13-1具有较强乳化能力,其1 h、24 h的乳化率分别为80%和75%,明显高于文献报道。此外,该菌在以石蜡为唯一碳源的BH培养基中生长较好,说明其具有较强的分解石油的能力。经Biolog细菌自动鉴定仪鉴定,同时进一步通过PCR扩增获得该菌的16SrDNA,并测序。对其16S rDNA分析表明,该菌株与GenBank中的醋酸钙不动杆菌同源性为97%,确定该菌可能为醋酸钙不动杆菌(Acinetobacter calcoaceticus)。

Crude oil is a kind of organic matter, relatively insoluble in water, which seriously affects its bioavailability. Some bacteria are found to be able to produce biosurfacant, which can emulsify the crude oil and enable the bacteria to adhere to the hydrophobic surface of the oil very strongly, thus enhancing bioavailability of the crude oil in the environment. It is reported that bacteria with hemolytic circle in the blood plate show emulsifying activity. Therefore strains of the bacteria were isolated from the petroleum contaminated soil through enrichment culture in BH medium with crude oil from Jianghan as the sole carbon source and hemolytic activity assay on blood agar plates. Nine strains with high degrading and emulsifying ability were isolated. Strain X13- 1 was found at the top of the list of the bacteria with emulsifying ability reaching 80% and 75% at lh and 24 h respectively. OD600 nm of the culture fluid with paraffin as the sole carbon source was used to indirectly measure petroleum-degrading abilities of bacteria. The OD600nm of strain X13-1 was up to 0.566. Besides its emulsifying ability and petroleum-degrading ability, strain X13-1 was chosen for ulterior research. Cell wall composition, morphology, metabolism Biolog Micro-station Identification System and the sequence analysis of 16S rDNA fragment amplified from total DNA of bacterium X13-1 were used to identify the strain of bacterium. The results show that the bacterium is G^- and bacilliform. SIM （Similarity） in Biolog to typical Acinetobacter calcoaceticus is 0.826 and 16S rDNA sequence homology with typical A. calcoaceticus in the genebank is 97%. To sum up the above-described characters, it can be concluded that strain X13-1 is likely to be an A. calcoaceticus.