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

Biosurfactants are amphipathic molecules with both hydrophilic and hydrophobic moieties that partition preferentially at the interface between fluid phases with different degrees of polarity and hydrogen bonding such as oil/water or air/water interfaces. However, interest in biosurfactants has been steadily increasing in recent years, which due to their diversity, environmentally friendly nature, and higher biodegrade ability compared with chemical surfactants, and their potential applications in medicine, agriculture, food-processing industries, environmental remediation and crude oil recovery. However, biosurfactant production technology is still in the stage of laboratory research currently. The research includes the following aspects:the screening and identification of biosurfactant -producing bacteria, the components analysis and the physicochemical properties of the metabolites, the optimization of culture mediums and fermentation conditions for shaking flask fermentation of the strain. The main results are as follows:Sampling in the oil contaminated environment,13 strains with great ability of emulsifying crude oil oil were selected through enrichment method, agar plate isolation, and shake-flask fermentation. The strain RDY7-1 had been selected from the 13 bacterias by shake-flask fermentation screening. Its fermentation broth had the most powerful surface activity, and could make an approximate 6.70cm diameter clear oil spreading zone. According to the identifications of colony morphology, cell morphology, physiological and biochemical properties of bacteria and 16S rDNA gene sequence analysis, the strain RDY7-1 was closely related to the genus Bacillus licheniformis, then named as Bacillus licheniformis RDY7-1.The fermentation broth of strain RDY7-1 was characterized by electric charge, sugar composition analysis. The surface-active compounds produced by strain RDY7-1 were secreted outside the cell and were not glycolipid. The result of electric charge measurement showed that the surface-active compounds were lipopetide and anionic biosurfactant. The main metabolites of strain RDY7-1 were extracted and purified. The amino acid compositions of the hydrolytic metabolites were determined on an amino acid analyzer. The analysis indicated the composition was determined to be aspartic acid, glutamic acid, valine, isoleucine and leucine in the ratios 1.4:1.9:1.5:1:4. Purified samples were characterized by TLC and FTIR as cyclic lipopeptide (CLPs).Through a series of laboratory experiments about the physicochemical property of lipopeptide produced by stain RDY7-1, and it was shown good thermal stability and chemical stability. The fermentation broth can maintain highly degreasing activity in wide pH value range (2.0-10.0). In the condition of 120℃with 2 h, the degreasing activity maintained at about 84.0% contrast to the control group. When the NaCl concentration of lipopeptide solution was 9.0%, degreasing activity reduced to about 95.2%. After 8 week 4℃Conservation, degreasing activity of the lipopeptide solution was maintain oil discharge activity of 82.6%.The optimal culture mediums and fermentation conditions of lipopetide production by strain RDY7-1 were optimized by single-factor experiment. The optimal culture mediums of lipopetide production were as follows (g/L):glucose 25, NH4NO32.5, KH2PO4 3, Na2HPO4 2, MgSO4 0.3, Fe2(SO4)3 0.02. The optimal fermentation conditions for lipopetide production in shaking flask were as follows:culture time 96 h, inoculum age 14 h, the inoculation amount 5 %, temperature 37℃, volume in shake flask 35 mL/250 mL, initial pH 8.0, and rotation speed of rocking bed 180 r/min. The Oil—exclusion diameter of strain RDY7-1 was 10.80 cm with the optimal culture medium and optimal fermentation conditions, and the Oil—exclusion. diameter had increased by 61.2% compared with the initial.