Team:NWU-CHINA/Preparation
Preparation
Acquire sample
We got crude oil from Yanchang oil field production of Xiasiwan (36°27′36″ N, 108°55′12″ E), a terrestrial oil production located in Northern Shaanxi, China for decades. Petroleum hydrocarbons con- taminated soils were also collected from Yanchang oil field production of Xiasiwan. The soils were taken from 2 to10 cm depth layer and sieved through a 2-mm screen, then sample was stored at 5 °C in the dark until required.
Sample separation
We separate a strain capable of efficiently degrading alkanes from samples.
There are many bacterial strains isolated from petroleum-contaminated soil which had the ability to either produce visual rhamnolipids as indicated by the presence of blue halos, or utilize crude oil as a sole source of carbon and energy. Among these isolates, one strain code as DN1 was not only capable of highly producing the biosurfactants, but it was also able to degrade crude oil effectively. Then the complete 16SrRNA gene sequence (1475bp) of strain DN1 was obtained and phylogenetic analysis was conducted to determine that it belonged to Gamma Proteobacteria, showing the highest 16SrRNA gene sequence similarity of 98% with Pseudomonas aeruginosa(Figure 1). This strain was oval to rod-shaped (0.5–0.8 mm_1.5–3.0 mm), gram-negative and motile with a single polar flagellum (Figure 2). Colonies(2mm–3 mm)growing on LB agar for 24h at 37℃ were rough, circular convex, wet and yellowish-brown in color (Figure 3). The strain was tolerant to a wide variety of physical conditions, including temperature and pH, with the optimum growth occurring at a pH6.5–7.5 and 30–37°C. It was positive for citrate, catalase, oxidase, aerobic nitrite reduction, anaerobic nitrate reduction and denitrification, as well as for the hydrolysis of gelatin. According to API-test results, the biochemical profiles seemed to ferment some carbohydrates by the strain. Morphological, physiological and phylogenetic properties indicated that strainDN1(CCTCCNO:M2011287)was a member of the genus Pseudomonas, and the accession number in the GenBank is KP119458.
Reference
[1] Kuang-Yi Ma, Meng-Yan Sun, Wen Dong, Chun-Qiu He, Fu-Lin Chen, Yan-Ling Ma .Effects of nutrition optimization strategy on rhamnolipid production in a Pseudomonas aeruginosa strain DN1 for bioremediation of crude oil[J].Biocatalysis and Agricultural Biotechnology, 2016, 6(1):144-151.
[2] Wen Dong, Chunqiu He, Yanpeng Li, Chao Huang, Fulin Chen, Yanling Ma⁎.Complete genome sequence of a versatile hydrocarbon degrader, Pseudomonas aeruginosa DN1 isolated from petroleum-contaminated soil[J].Gene Reports, 2017, 7(1):123-126.
