Isolation and Identification of Lipase-Producing Bacteria

The oil-contaminated soil sample was collected from the Southwest University of Science and Technology, Mianyang, Sichuan Province (N 31.53791, E 104.69388). For the isolation and screening of lipase-producing strains, 5 g of the soil sample was weighed in 45 mL of physiological saline, incubated for 1 h at 30 °C while being shaken at 200 r/min, and then rested. A quantity of 1 mL of supernatant was added to 20 mL of enrichment medium (yeast paste 0.2 g/L, NaCl 0.5 g/L, Na₂HPO₄ 3.5 g/L, KH₂PO₄ 1.5 g/L, MgSO₄-7H₂O 0.5 g/L), and this was incubated for 24 h at 30 °C while being shaken at 200 r/min. After gradient dilution of the enrichment medium, 100 μL of 10⁻⁶~10⁻⁸ dilution was coated onto the oil assimilation plate containing rhodamine B (25 mL olive oil emulsion was mixed well with 175 mL LB medium, 200 μL 10% rhodamine B solution was added, and the mixture was shaken). After incubation at 30 °C for 2 d, a pure culture capable of producing degradation circles was obtained, named WCO-9. The single colony of strain WCO-9 was selected and inoculated in an LB liquid medium. Cells in the logarithmic growth phase were obtained by shaking at 200 r/min and incubating at 30 °C for 12 h and were then stored in glycerol tubes. Finally, the WCO-9 strain was identified and registered by the Guangdong Microbial Culture Collection Center with the serial number GDMCC No: 61851. The scanning electron microscopy observation, 16S rDNA analysis and whole genome sequencing were performed using cells from the logarithmic growth stage.