Studies On Fermentation, Molecular Structure And The Physicochemical Properties Of Lipopeptide Biosurfactant

Biosurfactants synthesized by microorganisms are natural surface-active compounds. Basically there are six major classes of biosurfactants: glycolipids, lipopeptides/lipoproteins, phospholipids, lipopolysaccharides, substituted fatty acids and neutral lipids. Like synthetic surfactants, biosurfactants can reduce the surface and interfacial tensions of aqueous media. However,they have several outstanding advantages over the chemical surfactants, such as lower toxicity; higher biodegradability; better environmental compatibility; higher foaming; high selectivity and specific activity at extreme temperatures, pH, and salinity; and the ability to be synthesized from renewable feed stocks and so on. New structural types of biosurfactants can be produced and, thus, they expand the range of available surfactants. Biosurfactants are a unique class of compounds that have been show a variety of potential applications, including enhanced oil recovery, remediation of organics and metals, food additives, cosmetic additives, emulsifiers in agriculture and medicine and in biological control. This wide range of potential applications has increased interested in microbially produced surfactants. So biosurfactants will have a wide-range of applications for various industries in the future.The group of lipopeptide presents a heterogenous calss of biologically active peptides and most of them are known also to possess antibiotics activity. Among the many classes of biosurfactants, lipopeptides are particularly interesting because of their high surface activities and therapeutic potential, which is an inhibitor of fibrin clotting and cyclic AMP phosphodiesterase.(1) Microorganisms capable of producing biosurfactants could be isolated by a series of step including enrichment culture, hemolytic activity assay on blood agar plates and hydrolyzing oil activity assay on oil agar plates etc. Then the fermentation supernatant of first-isolated strains was secondly screened by a drop-collapsing method, oil displacement activity and surface tension method. Eight strains bacteria were isolated with higher surface activity from 220samples of oil-contaminated soil and water collected from Daqing Oil Field and Shengli Oil Field. The results of TLC and IR analysis showed that biosurfactants of four strains bacteria was lipopeptide and strain SD59 was the best one among them.By the experimental results of the morphological structure, physiological characteristics and by the comparison of its whole sequence of 16S rDNA with the type strains in Genebank, it was showed that 16S rDNA of the stain SD59 and that of Bacillus licheniformis was more than 98 percent of homogenous. Therefore bacterium strain SD59 was identified as Bacillus licheniformis SD59.(2) The conditions for shaking flask fermentation of strain SD59 which has the high productivity of lipopeptide biosurfcant have been studied. The influences of the nutrient and environmental factors on the fermentation production are investigated through the complex orthogonal experiments and one-factor experiments. The optimized composition of medium(g/L) : soluble starch 20.0, NH4NO3 6.0, Na2HPO4-12H2O 8.0, KH2PO4 2.0, MgSO4 7H2O 0.3, CaCl2 2H2O 0.08, yeast extract 0.6, initial pH 7.2.The optimal culture condition: 500 mL flask containing 200 mL medium,5.0% seed culture is used, temperature is 36~38C. On such a condition, the productivity of lipopeptide can reach above 1.2g/L.SD596, a mutant strain which has high productivity was obtained through mutation breeding by ultraviolet, microwave and diethyl sulfate, from the original strain SD59. Lipopeptide productivity of Bacillus licheniformis mutant SD596 was improved 2.8 times higher than that of initial strain after mutated by ultraviolet, microwave and diethyl sulfate. The biosurfactant of SD596 has higher surface activity and it is the same kind as that of SD59 by the FT-IR analysis. On the optimized conditions of strain SD59, the lipopeptide production of SD596 is as high as 3.445 g/L whic...