The Synthesis Of Biosurfactant And The Application Research In Degradation Of Anthracene

Efficient biosurfactant (BS) producing bacteria were isolated by plate screening method and repulsion oil method from Liaohe oil field soil, and were named as A3, As and Y, respectively. The surface tension values of their fermented liquid were 35.7 mN/m,37.5 mN/m and 34.7 mN/m, respectively with the repulsion oil diameter up to five centimeters, which presented good surface activity.The molecular biological identification for the strains A3, As and Y were conducted by the analysis of the 16S rRNA gene sequences. The biosurfactant was extracted from the fermented liquid for thin layer chromatographic analysis to determine the chemical properties of biosurfactant. The results showed that the strain A3, As, Y belong to Pseudomonas sp.According to the surface tension value of the fermented liquid, L16(45) orthogonal experiments were conducted to optimize fermentation conditions of strain A3, As, Y, and the mixture of three strains, and temperature, pH, concentration of NaCl, carbon source concentration and nitrogen source concentration were chosed as impact factors. The best fermentation conditions for strain A3 were as follows:30 ℃, pH6.0,4 g/L NaCl,35 g/L glucose and 4 g/L sodium nitrate.The biosurfactants in the fermented liquid extraction were extracted using five extraction methods. Significant increases of the extraction amount of biosurfactants were observed after the optimization of fermentation conditions for strain A3(8.4 times), strain As(6.4 times), strain Y(5.6 times) and A3 As(5.7 times). Among these, the highest surfactant yield of 20.55 g/L was obtained in strain As. For three pure bacteria and mixed bacteria of A3-As, CHCl3 extraction method was the best of all, which extract the maximum amount of biosurfactant. However, for the other mixed strains, the difference among these five methods was not significant.Critical Micelle Concentration of the biosurfactant from strain A3 is 200 mg/L. The stability experiments of pH, temperature and degree of mineralization were studied because the stability of the biosurfactant is an important index to characterize its excellent properties. It was found that the change of surface tension value was small and all the surface tension value were about 47 mN/m with the temperature ranging from 15.2℃ to 150℃. Sodium chloride concentration from 0 g/L to saturation had a small effect on the surface tension value which was about 42 mN/m. With pH value changing from 2.0 to 6.0, the surface tension value increased from 31 mN/m to 50 mN/m. However, the surface tension value remained 50 mN/m when pH changed from 6.0 to 13.0. The experiments results showed that the biosurfactant from strain A3 could keep excellent biological activity in extreme circumstances, which was beneficial to its application in real environmemt.The biodegradation experiments of anthracene were conducted in following aspects:the biodegradation of anthracene by Irpex lacteus F17, the effects of concentration of BS, adding time of BS, temperature and pH value on the anthracene biodegradation. Besides, the influence of wood chips on anthracene biodegradation and the activity changes of extracellular enzymes secreted by Irpex lacteus F17 were invested. The experiments results showed that the biodegradation rate of anthracene could reach 59.0% after 15 days without adding the biosurfactant, while the addition of biosurfactant could significantly increase the degradation rate of anthracene. The biodegradation rate increased obviously with the concentration of BS increasing from100 mg/L to 1000 mg/L, and the biodegradation rate of anthracene reached 93.3% after 15 days. Adding time of BS, temperature, pH and wood chips had important influences on the biodegradation of anthracene. When BS was added to the medium one day earlier than Irpex lacteus F17, which promoted the dissolution of anthracene, could lead to the increase of biodegradation rate due to the better use of anthracene by Irpex lacteus F17. Both pH value and temperature had important effects on the growth of microorganisms. The highest biodegradation rate of anthracene was obtained at pH5.0, and 28℃～30℃ was optimal temperature range for Irpex lacteus F17 in the biodegradation of anthracene. The adding of wood chips in culture medium could induce the synthesis of degradation enzymes secreted by Irpex lacteus F17, which contributed to improving the biodegradation rate of anthracene. MnP, LiP and Lac secreted by Irpex lacteus F17 could be detected in the process of biodegradation, however, LiP and Lac might play important roles during the biodegradation of anthracene.The results of this study could provide a new source of bacteria to the synthesis of biosurfactants. And also, an appropriate method were developed to improve the yield of biosurfactant. The good properties of biosurfactant broaden the application in environments, which lay foundations for the development and application of new surfactants.