Development of a microbial dewaxing agent using three spore forming bacteria.

Microbial enhanced oil recovery (MEOR) is a cost effective and efficient method for recovering residual oil. However, the presence of wax (paraffin) in residual oil can substantially reduce the efficiency of MEOR. Therefore, microbial dewaxing is a critical process in MEOR. In this study, a bacterial dewaxing agent of three spore-forming bacteria was developed. Among these bacteria, Bacillus subtilis GZ6 produced the biosurfactant surfactin. Replacing the promoter of the surfactin synthase gene cluster (srfA), increased the titer of surfactin in this strain from 0.33 g/L to 2.32 g/L. The genetically modified strain produced oil spreading rings with diameters increasing from 3.5 ± 0.1 to 4.1 ± 0.2 cm. The LadA F10L/N133R mutant was created by engineering an alkane monooxygenase (LadA) using site-directed mutagenesis in the Escherichia coli host. Compared to the wild-type enzyme, the resulting mutant exhibited an 11.7-fold increase in catalytic efficiency toward the substrate octadecane. When the mutant (pIMPpladA2mu) was expressed in Geobacillus stearothermophilus GZ178 cells, it exhibited a 2.0-fold increase in octadecane-degrading activity. Cultures of the two modified strains (B. subtilis GZ6 (pg3srfA) and G. stearothermophilus GZ178 (pIMPpladA2mu)) were mixed with the culture of Geobacillus thermodenitrificans GZ156 at a ratio of 5:80:15. The resulting composition increased the rate of wax removal by 35% compared to the composition composed of three native strains. This study successfully developed a multi-strain bacterial agent with enhanced oil wax removal capabilities by genetically engineering two bacterial strains.