Microelectrode Arrays Electrofusion Technology For Breeding Of High-yield Biosurfactant Strains

Breeding high-yield biosurfactant strains is the premise and foundation of biosurfactant production.Protoplast electrofusion is used widely in cells and plants. However,there are many disadvantages of the present fusion technology for microorganism.For example,the size of microorganism is smaller than that of electrode and the action sites of electrical pulse are not easy to control.To solve these problems,a electrofusion system,based on an interdigital and pectinate microelectrode arrays,was developed independently and the breeding of high-yield biosurfactant strains was studied with this system.After primarily screened by using oil agar plates and secondarily screened by oil displacement activity from oil slurry,24 strains capable of producing biosurfactant were obtained,and the surface tension of their fermentation broth was measured.Finally,a high-yield biosurfactant strain BS-34 was screened,with the surface tension of 51.47mN/m and the emulsifying ability of 62%.The influence factors of protoplast preparation were studied.The results showed that under the conditions of using 1.5%snailase,at 33℃and pH 6.0,stirring slowly for 3h and 0.8mol/L D-sorbitol as osmotic stabilizer,the formation rate of protoplasts is 95.2%and the generation rate is 8.9%.The influence factors of electrofusion were studied and a new strain was obtained. The string ratio of cells was more than 90%under conditions that queueing voltage was 2 V,frequency was 1 MHz,and the ion concentration of the mixture was less than 0.1 mmol/LCa²⁺ and 0.1 mmol/LMg²⁺.The fusion rate was 8%when the intensity of voltage was 50V,the number of electrofusion pluse was eight,the interval was 80μs, and the buffer solution consisted of 0.1 mmol/LCa²⁺ and 0.1 mmol/LMg²⁺ and 0.8mol/L D-sorbitol.A new strain was obtained with the electro fusion system.Compared with the surface tension of the parents's fermentation broth,that of the new one's decresed to 37.59mN/m.While the emulsibility increased from 62%to 85%.This new electrofusion system succeeds in breeding high-yield biosurfactant strains by protoplast electro fusion,which promotes the progress of microbial electro fusion.