Isolation Of A Biosurfactant-Producing Bacterium, Cloning Of Key Genes And Construction Of A Genetically Engineered Microorganism

Biosurfactants were amphipathic molecules with a hydrophilic and hydrophobic group and had high surface-active features, which were usually synthesized by microorganisms. Compared with chemical surfactants, biosurfactants had many advantages such as good performance on oil-water interfacial tension reduction, emulsification, foaming and defoaming; easily biodegradable; non-toxic to the environment and so on. So biosurfactants had a wide range of potential application in bioremediation of various polluted environments, enhancing oil recovery and other fields. A biosurfactant-producing bacterial strain BSFD5 was isolated from the petroleum-polluted soil. It was identified as Pseudomonas aeruginosa according to its physiological-biochemical analysis and the similarity analysis of its 16S rRNA gene sequence.The crud biosurfactants were obtained from the fermentation liquid of BSFD5 by acid precipitation.The yield was 4.06 g/L and its CMC was 50 mg/L. The biosurfactant produced by strain BSFD5 was primally identified as rhamnolipid by the method of FT-IR and relative literature reports. The results of emulsification experiment revealed that the biosurfactant had high surface activity and emulsifying efficiency.Effect of different carbon and nitrogen sources, temperature, pH, aeration and metal ions on cell growth and rhamnolipid production were studied. The results showed that, when the optimal carbon sources were the 1:1 (w/w) mixture of glucose and rapeseed oil, the optimal nitrogen sources was NaNO3 and the optimal C/N ratio was 15, the highest rhamnolipid yield of 5.21 g/L was obtained. The optimal conditions for the growth of strain BSFD5 and the producing of rhamnolipid were 30℃, pH6～7 and 25～100 mL medium in 250 mL Erlenmeyer flask. Fe2+、Ca2+ showed the promotion effect on the growth of strain BSFD5 and the producing of rhamnolipid, whereas Zn2+ showed the obvious inhibitory effect.A GEM designated as KT2440-rhlABRI, capable of producing rhamnolipid was successfully constructed by homologous recombination. The rhamnosyltransferase gene rhlABRI was inserted into the chromosome of Pseudomonas putida KT2440 by using a homologous recombination vector with sacB gene as counter selectable marker. The GEM was relatively stable and without bringing any antibiotic marker. The homologous recombination events were confirmed by PCR detection.The effect of inoculating KT2440-rhlABRI on the degradation of pyrene in soil was studied under laboratory conditions. Various factors, including soil temperature, pH, inoculum size and initial pyrene concentration influenced its degradation efficiency. KT2440-rhlABRl could promote the degradation efficiency of pyrene by indigenous microorganisms under the condition of 20℃～40℃, pH6.0～8.0. The degradation efficiency was correlated positively to initial inoculum size. Pyrene could be degraded well at low concentration and the efficiency was promoted with the inoculating of GEMs whereas no obvious degradation efficiency was shown at high concentration. These results provide theoretic foundation for the application of GEMs in bioremediation.