Screening Of Biosurfactant-producing Bacterial Strains And Optimization Of Culture Conditions

Biosurfactants are natural surface-active compounds mainly synthesized by microorganisms, which have distinct advantages like no secondly pollution and friendly to environment compared with chemical surfactants. With the development of modern biological technology, biosurfactants have been shown a variety of potential applications, including medicine, agriculture, oil production and environmental remediation, so it has already caused many researchers a strong interest in the production of biosurfactants making use of biological technology. The paper concentrates on the screening and identification of biosurfactant-producing bacterias, the identification and components analysis with HPLC-MS of the product, and the optimization of culture medium and cultivation conditions. The main results are as follows:Sampling in the specific oil contaminated environment, nine strains with great ability of emulsification were selected through enrichment method, plate isolation, direet oil-utilizing method and agar plate method. Then the strain SFH-6 with the most ability of surface activity had been selected from the nine bacterias by secondary screening. With the following identifications of colony morphology, cell morphology, physiological and biochemical characteristics and 16S rRNA gene sequence analysis, the strain SFH-6 was identified as Pseudomonas Aeruginosa, named Pseudomonas aeruginosa SFH-6. Its fermentation broth diluted 10 times could make the diameter at most 5.2 cm of clear zone obtained with the oil spreading, which showed high surface activity and stable property.The product after acid hydrolysis produced by strain SFH-6 with glycerol as carbon source had more reducing sugar in the measured by DNS colorimetry, which indicated that the surface-active compounds produced by strain SFH-6 was glycolipid outside the cell, then the reducing sugar was identified as rhamnose by paper chromatography analysis. Further more, the result of electric charge measurement showed the product the same as rhamnolipid was anionic biosurfactant. Those demonstrated the surface-active compound synthesized by strain SFH-6 was rhamnolipid. Nine rhamnolipid congeners, formed by linking one or two rhamnose molecules to one or twoβ-hydroxy fatty acids of saturated or unsaturated alkyl chain between C8 and C12, were separated and identified from by HPLC-MS analysis, and the highest content(2-O-α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxyldec -anoyl -β-hydroxydecanoate) of which was 59.79%.The culture medium and the fermentation conditions of rhamnolipid production by strain SFH-6 were optimized by single-factor experiment. The optimal culture medium for rhamnolipid production were as follows(g/L): glycerol 30, NH4NO3 2.5, KH2PO4 10, Na2HPO4 4. The optimal fermentation conditions for rhamnolipid production were as follows: seed age 20 h, temperature 30℃, the quantity of inoculation 5%, volume in shake flask 35 mL/250 mL, rotation speed of rocking bed 180 r/min, initial pH 7.0 and culture time 72 h. The rhamnolipid yield of strain SFH-6 was 8.59 g/L with the optimal culture medium and optimal fermentation conditions, and the output had increased by 85% compared with the initial.