Composition Analyses Of Biosurfactants Produced By Marine Alkane-degrading Microorganisms And Preliminary Investigation On The Genes Involved In Biosurfactant Synthesis

Biosurfactant is one kind of amphipathic molecule that not only has the hydrophilic groups but also has the hydrophobic ones.It is noticed in many fundamental and applied research fields because of its numerous merits,such as excellent emulsification,aidant dissolvability,degradable and nontoxic.When microorganisms are supplied with insoluble components in water as the sole carbon and energy source,biosurfactant will be present in the metabolizable productions of the culture broth.In this thesis,some bacteria were incubated,which not only could degrade hydrocarbon well but also could lower the surface tension of its culture broth.The components of biosurfactant that each bacteria produced were separated,purified and identified.Moreover,the metabolization related regulation factors were analyzed. Three strains that contained two marine bacteria(Alcanivorax dieselolei B-5 and Dietzia sp.N3ZF-1) and one yeast strain(Pichia guilliermondii 510-6jm) were choosed as laboratorial objects.When the n-hexadecane was supplied as the sole carbon and energy source,B-5 can grow well and the surface tension of its culture broth could be reduced to 31 mN m^-1.After centrifugation,biosurfactant was detected present in hydrophobic layer floating on the culture.Hydrophobic layer was first extracted with chloroform/methanol(1:1,v/v),then further purified step by step with normal phase silica gel column,Sephadex LH20 gel column and preparative thin layer plate. Finally,the main component was determined to be lipopeptide.Through the chemically characterized with NMR,LCQ-MS,AAS and GC-MS,it was found to be a mixture of proline lipids.The monomer of proline lipids was composed of a proline residue and a fatty acid(C_14:0,C_16:0 or C_18:0).The critical micelle concentration(CMC) of the proline lipids was determined to be 40mg 1^-1.Moreover, the activity variations in large ranges of pH,temperature and salinity were also tested,the results showed reasonable stability.So these proline lipids have great potential in application.Dietzia sp.N3ZF-1 was isolated from the sediments of South China Sea as an alkane degrading bacterium,which can lower the surface tension of its culture broth to 33.6mNm^-1.Through the same ways of extration and purification with strain B-5, the pure biosurfactantal compounds of the strain were obtained,which were finally identified as dirhamnolipids.Despite of the dirhamnolipids produced by N3ZF-1 are not novel in structure,Dietzia sp.N3ZF-1 is the first case in this genus that could produce biosurfactant. The yeast strain 510-6jm could degrad alkanes actively and lower the surface tension of its culture broth to 28mN m^-1.Its biosurfactant was identified as mannosylerythrutol lipids(MELs),which was a new category of biosurfactant that has great postential in application.Although there were some yeasts reproted producing MELs,but this is the first report in Pichia genus.In addition,with designing primers and progressing PCR,two key genes involved in MLEs synthesis were detected.In conclusion,through the componential analysis of biosurfactant that produced by three microorganisms and the investigation of metabolization related regulation factors about biosurfactant,a novel category of biosurfactant is produced and the theory foundation of its metabolizable regulation is enriched.