Study On The Characterization Of A Marine Microbial Biosurfactant Produced By Vibrio Sp.3B-2

Microbial biosurfactants (BS) and biopolymers are key players for microbial enhanced oil recovery (MEOR) and bioremediation of crude oil. Recently, BS are becoming increasingly popular due to their advantage, such as non-toxic, selective and environmentally friendly. Besides, BS can quickly be degraded by microrganisms and will not cause secondary pollution to the environment when used. BS are metabolites of surface activity which are produced by microorganisms and simultaneously have hydrophilic groups and hydrophobic groups. Thus, this thesis studies the screening of the BS producing strains, the identification of the strain, BS production characteristics of the strain, purification and characterization of BS.1. In this research, we used plates screening method and rescreening method to screen a high-yield BS strain 3B-2 from crude oil contaminated sediment of Bohai bay. The strain was identified as Vibrio sp. according to the morphological characteristics and 16S rDNA sequence analysis. It is currently preserved in China Center for Type Culture Collection with a serial number CCTCC No. CJ11052.2. The fermentation conditions for BS were optimized using single factor experiments and response surface methodology (RSM) design. As a result, the optimum cultivation conditions:temperature, medium volume, inoculum size, were 28℃,100 mL/250 mL, and 8%, respectively. The optimized media:lactose 0.5%, yeast extract 1.1%, NaCl 2%. The maximum yield of BS was that protein concentration was 6.5 g/L, surface tension was 41.36 mN/m, oil displacing diameter was 6.5cm after 48 h under these conditions. The strain fermentation curve showed the highest yield 6g/L at 32 h.3. The BS produced by Vibrio sp.3B-2 was successively purified by ultracentri-fugation, ultrafiltration, DEAE-FF ion-exchange column chromatography and gel filtration, SDS-PAGE. The results showed that BS was purified as two type of BS named as B11 and B12, respectively. The B11 and B12 were purified to homogeneity level with 9.1-fold and 10.4-fold. The surface activity of purified B11 and B12 were 12.67 cm/mg and 14.50 cm/mg, respectively. The molecular weight of B11 and B12 was approximately 40 kDa and 22 kDa. respectively, by SDS-PAGE. The amino acid sequence of B11 and B12 were determined, and it showed no homology to any known BS sequence. And the amino acid sequence of B11 was G-Y-N-G-L-A-E-V-G-K, V-T-V-A-H-P-D-G-L-Q-D-K, D-L-V-P-N-P-P-K, S-G-V-N-Y-G-V-T-T-L-P-K, the amino acid sequence of B12 was A-F-E-L-P-A-L-P-Y-A-K, D-A-L-E-P-H-I-S-A-E-T-L-D-Y-H-H-G-K, L-N-G-L-I-P-G-T-E-F-E-G-K, T-L-E-E-I-I-K, F-T-D-S-A-I-N-N-F-G-S-S-W-T-W-L-V-K;B11 and B12 showed novel surfactants by BLAST ratio of results.4. The characteristics of the BS was studied by using three strains of oil degrading bacteria which were isolated by laboratory. In terms of oil degradation the composite strain (HC8-3, CN-3) possed good degradation ability for linear alkanes, strain 3B-2 has strong degradation for branched alkanes, Composite strain 3B-2 and CN-3 have very good synergy for linear alkanes and branched alkanes, The crude oil can achieve best degradation effect when the strain HC8-3, CN-3,3B-2 were mixed. In terms of oil-dispersing ability, We can judge by gas chromatogram that the biosurfactants of strains 3B-2 can better degradate property than the Tween 80 of dispersion effect, and can be well dispersed into small particles of crude oil, It can accelerate the degradation efficiency for oil-degrading bacteria.There are synergies between biosurfactants of Vibrio sp. strain 3B-2 and other oil degrading strains, which has great important theory significance and the strong application value on the remediation of oil-contaminated ocean. Compared with chemical method and physical method, biological method (for example, adding oil degrading strains or microbial biosurfactants), is of great advantages, such as high selectivity, environmentally friendly, and without secondary pollution. Therefore, the research of a high-yield strain and the characteristics of biosurfactant have important theoretical and practical significance.