Screening And Fermentation Optimization Of Biosurfactant Producing Bacteria

Biosurfactant is a kind of amphiphilic compound formed by microorganisms.Compared with chemically synthesized surfactants,biosurfactants have the advantages of variety,low toxicity,environmental friendliness and high biodegradability,etc.,and have wide application prospects in the fields of environmental remediation,food and microbial oil recovery in recent years.But biosurfactant producing bacteria are not abundant and the biosurfactant yield is low.Therefore,it is necessary to screen different kinds of biosurfactant producing bacteria as much as possible,and optimize fermentation to improve the yield.This study used 96 microplate method to screen potential strains producing surfactants from the laboratory simulation of petroleum contaminated soil samples and the Inner Mongolia xilingol league area 51 blocks in oil well produced fluid samples.Then,the functional identification of the strains was carried out through the oil drain ring and surface tension measurement.One strain was selected as the target strain for subsequent experiments,and optimized for fermentation.The main research results of this thesis are as follows:（1）225 strains of potential biosurfactant producing bacteria were screened by the96 microplate method from simulation of petroleum contaminated soil and oil well produced liquid samples,which belonged to 3 phyla,5 classes,14 orders,20 families,26 genera,46 species,and 59 strains by calculating 16S r DNA gene sequence distance.Then,the 59 strains with different sequences were screened by oil ring and surface tension test.Three strains could produce oil ring and 23 strains could reduce surface tension were screened out respectively.The strain named L01 was selected for subsequent experiments,and identified by 16S r DNA as Pseudomonas Aeruginosa JCM5962.（2）The product of strain L01 was charged.The biosurfactant was anionic,and the type detected by thin Layer chromatography（TLC）was glycolipids.In the TLC process,the extraction agent and the developing solution were compared,and ethyl acetate was selected as the extraction agent,n-hexane:ammonia:isopropanol=2:1:4（v/v/v）as the developing solution.The product detected by Fourier infrared spectroscopy（FTIR）is rhamnosin.The emulsification performance,critical micelle concentration（CMC）,pH and temperature influence for the product of strain L01 were studied.It was found that the product could emulsify soybean oil,and the CMC was 1.00 mg/m L.It is stable at different pH and temperatures.（3）Single factor method,orthogonal method and response surface method were used to optimize fermentation,and the results were as follows:when the concentration of glucose was 30 g/L,NaNO₃ was 2.4 g/L,phosphate was 23.3 g/L（Na₂HPO₄·12H₂O was 11.65 g/L,KH₂PO₄ was 11.65 g/L）,pH was 6.8,and culture time was 132 h,the maximum yield was 8.43 g/L.Compared with the initial yield,the increase is 1.8 times.Fifty-nine different strains were screened out,which enriched the resources of biosurfactant producing bacteria.A stable and adaptable strain L01 was optimized for fermentation,and the biosurfactant,yield was increased.