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This work investigated the effect of physiochemical conditions of fermentation on the de-emulsification capability of biological de-emulsifier production by a demulsifying strain(a Bacillus sp. isolated by our laboratory from oil contaminated soil). A quadratic orthogonal regressive rotation design was used to establish an effective statistical model for evaluation the combined effects of the incubation time(A), culturing temperature(B), glucose concentration(C) in substrate, in oculum age(D) and shaker agitation(E). The results of analysis of variance (ANOVA) of face-centered central composite design (FCCCD) showed that the adequately high R2 value of 0.976 and F-value of 30.69 indicated the statistical significance of the quadratic model which there were seven significant model terms (Prob>F

Bio-demulsifier as a biological agent will become a substitute of chemical demulsifier for its high efficiency, low cost, high adoptability and environment friendly. In this paper, a set of high efficiency, rapid and simple method was established to isolate deemulsification strains. Blue-gel plates test, oil agar plates test, oil-spreading test and demulsifying test were combined used in this method. Using this method, ten strains with high de-emulsification capability which the demulsifying ratios in O/W Kerosene emulsion (in 24h)were more than 50% were primary screened from activated sludge samples. Two highly efficient demulsifying strains which the demulsifying ratio in O/W Kerosene emulsion (in 24h) were more than 90%were secondary isolated and identified as Gordonia sp. through 16S rDNA gene and physiological test. The whole culture of the strain appeared very high deemulsification activity with low biomass.