Study On The Fermentation And Separation Of Biosurfactants By A Newly Isolated Pseudomonas Aeruginosa S1

As one of the most extensive studied and powerful biosurfactants,rhamnolipids（RLs）have of great prospects in applications in the fields of environmental protection,home&personal care industry,agriculture,medicine,etc.However,the industrial production of rhamnolipids is greatly inhibited by limited over-producing strain and the low level of fermentation production.Furthermore,the high-cost resulted by inefficient and complex separation processes presents as the major obstacle for the real production of RLs at large scale.Therefore,it is of great significance to improve the fermentation level of RLs as well as development of a green and efficient separation and purification process for the industrial application.In this study,the fermentation parameters during production of biosurfactant by a newly screened strain P.aeruginosa S1 was systematically optimized and an efficient advanced fed-batch fermentation process was developed to further enhance the fermentation level of P.aeruginosa S1.Moreover,an aqueous two-phase extraction process based on alcohol/inorganic salts has been developed for recovery of RLs from fermentation broth.The main findings in this thesis are as follows:（1）The newly strain P.aeruginosa S1 which was isolated from crude oil contaminated soil,exhibited a high degradation capacity of crude oil and the surface tension of fermentation broth was readily reduced to 34.1 m N/m.Interesting,it was for the first reported that P.aeruginosa is able to co-produce RLs and surfactin,two of the most powerful biosurfactants,when corn oil was used as the sole carbon source.In addition,the biosurfactants products showed higher surface activities than rhamnolipids,and presented of great potential in application in enhance oil recovery as a result of p H-regulated surface activity.（2）The fermentation parameters of S1 were systematically optimized in terms of the addition of Na NO₃ and Fe²⁺/Fe³⁺,and the level of fermentation was significantly improved.The results showed that high concentration（12-16 g/L）of Na NO₃ was more conducive to the production of biosurfactants.When Na NO₃ concentration reach 12 g/L,both Fe SO₄·7H₂O and Fe Cl₃·6H₂O can further enhance the RLs production.At the dosage of 1-2 mg/L,the rhamnolipids yield reached 30.1-32.4 g/L which have an increase of about 120%compared with that obtained by Fe-free medium（14.7 g/L）.In addition,by optimizing the types and concentration of Fe²⁺/Fe³⁺,the co-production of RLs and surfactin was achieved and a mixture of RLs:surfactin=28.4:9.89（w/w）was obtained.Moreover,the advanced sequential fed-batch fermentation with high cell density further improved the fermentation level of S1.The RLs productivity as improved to 0.5-1.05 g/L/h and was well maintained for a fermentation period of 90 h,resulting in a total produced RLs of 64.4 g/L.The obtained surfactin productivity by P.aeruginosa S1 in this study was 1.03 g/L/h which was much higher than that reported in most of literatures.Finally,22.0 g/L RLs and 11.5 g/L surfactin were achieved after 5 days of fermentation under the optimizations of mass transfer and Fe³⁺concentration.（3）An efficient separation process of RLs was developed based on isopropanol/sodium sulfate aqueous two-phase system,and the factors determining separation efficiency were investigated.The results showed that more than 95%of RLs with>90%purity was recovered from fermentation broth via this newly developed aqueous two-phase system which is comprise of 30%（v/v）isopropanol and 20%（w/v）sodium sulfate combined with a following desalination process.In addition,the strategy of recycling isopropanol and sodium sulfate were also developed in consideration of cost and avoiding pollution at the following downstream process.This process was also valid for separation of rhamnolipids from fermentation broth produced by P.aeruginosa PAO1 and ATCC 9027,can deal with the unpredictable non-precipitable of rhamnolipids triggered by addition of acid to p H of below 3.