Study On The Ability Of Rhamnolipid Production And Hydrocarbon Pollutants Biodegradation By Pseudomonas Aeruginosa

Hydrocarbon contaminants is consist of two kinds of carbon and hydrogen elements, such as fluoranthene, pyrene, benzo [a] pyrene, naphthalene, phenanthrene, which is widely distributed in the environment and has a harm effect on environment and the human being. These toxic substances are not only caused serious pollution of soil, air, water, etc, but also lead organisms to cancer, malformation, mutation. There are physical, chemical and biological method for removal of hydrocarbon pollutants. Biological method is a kind of removing the hydrocarbon pollutants thoroughly by microorganism, plants and enzyme, one group among these promising methods is microbial degradation. However, the strong hydrophobicity of the hydrocarbon pollutants that are difficult to be degraded by microorganism, becomes the bottleneck of bioremediation by the microbiological method. Biosurfactant produced by different microorganisms are biodegradable, high efficiency, low toxicity, and no secondary pollution of environment. Based on these properties, bio surfactants was gained more and more attention since they have applications in many fields including environmental remediation, and also they play an important role in enhancing degradation efficiency of many organic compounds.A rhamnolipid bio surfactant-producing and hydrocarbon degrading bacterium Pseudomonas aeruginosa strain DN1 was isolated from petroleum-contaminated soil samples in our laboratory. On this basis, the ingredient and culture conditions of the BPLM inorganic salt culture medium were screened and optimized for high rhamnolipid production of strain DN1 using the method of rapid high-throughput screening according to the luxCDABE luminous genetic characteristics of pMS402 plasmid. The results showed that the best carbon source was palm oil 63.34ml/L?V/V? and the best nitrogen source was NaNO₃5 g/L?W/V? of BPLM, the C/N ratio was 20, the optimum temperature was 32?-36?, the optimum pH was 5-7, respectively. Though high-throughput screening spent 20 hour and saved almost 80% culture period compared to flask fermentation, those results were consistent with ingredient and culture conditions. The rhamnolipid yields of strain DNl with the optimized BPLM medium in shaking flask was 25.91g/L, was more 72.98% than the production of the initial BPLM optimization ?14.978 g/L?. After fermentation of strain DN1 in 500 ml optimized BPLM medium, the rhamnolipid products were extracted by methanol-chloroform method, and were analyzed by the HPLC and ESI-MS. The results showed that there were 13 different structures of rhamnolipid, including six kinds of monorhamnolipids and seven kinds of structure of dirhamnolipids produced by strain DN1. The dominant monorhamnolipids were Rha-C₈-C₈, Rha-C₁₀:1-C₈, Rha-C₁₄-C₁₆, Rha-C₁₆-C₁₆, Rha-C_12?2 and Rha-C₁₂, the relative molecular mass were 448,474,644,672,358 and 362, the chemical formula were C₂₂H₄₀O₉, C₂₄H₄₂O₉, C₃₆H₆₈O₉, C₃₈H₇₂O₉, C₁₈H₃₀O₇, C₁₈H₃₄O₇. The dominant dirhamnolipids were Rha-Rha-C₁₀, Rha-Rha-C₁₂, Rha-Rha-C₁₄, Rha-Rha-C₁₀-C₁₀, Rha-Rha-C₁₀-C₁₂, Rha-Rha-C₈-C₁₀ and Rha-Rha-C₁₀-C_8:1, the relative molecular mass were 480,508,536,650,678,622 and 620, the chemical formula were C₂₂H₄₀O₁₁, C₂₄H₄₄O₁₁, C₂₆H₃₈O₁₁, C32H58O13, C₃₄H₆₂O₁₃, C30H54O13 and C₃₀H₅₂O₁₃. Other structures of rhamnolipids had only minor concentrations. Furthermore, the rhamnolipid from the fermentation broth of strain DN1 showed the critical micelle concentration ?CMC? of 90 mg/L by using the SVT-20 spinning drop interfacial tension machine, and the surface activity stability results showed that these rhamnolipids were effective in different temperature, pH and salt ionic concentration. In addition, the degradation of hydrocarbons by the strain DN1 was monitored by ultraviolet spectrophotometer and oil content analyzer, and the results showed that the degradable efficiency were 91%,96%, 95.2%,94.4% and 95% on 5%?V/V? of crude oil,200 mg/L ?W/V? of fluoranthene,200 mg/L ?W/V? of pyrene,500 mg/L ?W/V? of naphthalene and 500 mg/L ?W/V? phenanthrene, respectively, at the same time the OD values of the living bacterium at 600nm were 4.16,2.91, 3.18,4.16 and 3.0, respectively. Moreover, the results of gas chromatography showed that the crude oil and other hydrocarbons had been almostly biodegraded by strain DN1 after 14d cultivation. Therefore, the feature of rhamnolipid production of strain DN1 was beneficial to the emulsibility of hydrocarbon pollutants and biodegradation effectively.