This study was performed to screen microorganisms with high productivity of bioemulsifier, to maximize its yield and to investigate the surface active properties of the bioemulsifier. More than 100 bacterial strains capable of utilizing soybean oil or n-paraffin as carbon source were isolated from various kinds of soil and municipal samples. Among them, a strain with the highest emulsifying activity was selected. The strain was identified as Pseudomonas aeruginosa YPJ-80 from morphological, biochemical, and chemotaxonomical characteristics. It was shown that Pseudomonas aeruginosa YPJ-80 produced RL(rhamnolipid) 1, 3 by deoxyhexose detection and thin layer chromatography(TLC).
Quantitative methods for evaluating bioemulsifier and biosurfactant productivity were investigated. Standard emulsification dilution(SED) was proposed to evaluate bioemulsifier yield quantitatively. SED was defined as the dilution factor which makes emulsifying activity of culture broth to one.
Cultural conditions of Pseudomonas aeruginosa YPJ-80 were investigated to maximize bioemulsifier production and optimum condition was at temperature at 32.5℃, initial pH at 8.0, and inoculum size of 8 %(v/v). Of various carbon sources tested for the bioemulsifier production, vegetable oils such as olive oil or soybean oil yielded most. The bioemulsifier production with soluble carbon sources such as glycerol or glucose, yielded an comparable but less amount of bioemulsifier. Filtrated yeast extract was slightly better nutrient than autoclaved yeast extract and filtrated peptone. Ammonium sulfate and ammonium nitrate were better inorganic nitrogen sources for bioemulsifier production than sodium nitrate but sodium nitrate was the best nitrogen source for the biosurfactant production among those tested, evaluated by surface tension of culture broth. On a medium containing glucose as carbon source, bioemulsifier production changed little by C/N ratio in 10~20 range, and carbon concentration 1~3 %. Bioemulsifier production was increased with phosphate concentration up to 0.225 %.
Several potentially important cultivation variables involved in bioemulsifier production by Pseudomonas aeruginosa YPJ-80 in the flask cultures were investigated by employing a Plackett-Burman design. The cultivation variables investigated were as followed; the addition of citric acid, EDTA, and inorganic metal salt, and aeration and the use of dual carbon source. Among them, only aeration showed a strong positive effect on the bioemulsifier production. On the other hand, each variables showed positive effect on the utilization of glucose, except the addition of soybean oil.
Bioemulsifier, biosurfactant and rhamnolipid production started when the nitrogen level was very low. Phosphate consumption was negligible and only a small amount of extracellular protein was produced. SED/rhamnose ratio decreased with culture time.
Production of emulsifier was studied in a fermentor. Fermentor conditions were maintained at pH 8.0, aeration 1vvm, and agitation speed 500 rpm. After 24 hours of fermentation, surface tension, SED and rhamnose concentration were reached 30.7 mn/m, 200, 590 mg/l, respectively and held steady thereafter. SED/rhamnose decreased with culture time, as was on the flask culutre.
Crude bioemulsifier consisted of protein 31.5%, rhamnose 25%, volatile substance 10.5% in weight base. Therefore, neglecting the other impurity, 58% of weight was RL, which corresponded to 2.32 times of rhamnose weight. This implies that RL3 was produced as main RL.
HLB of crude RL was investigated for its applicability. HLB by dispersibility in water was 7-9 in acidic condition, 13 or higher in basic condition.
Minimum surface tension was shown to be 30.1 mN/m, and changed with pH condition. CMC increased as acidic condition changes to basic condition. CMC increased from 100 ppm in pH 4.0 to 800 ppm in pH 7.0. Surface tension increased with increasing pH and in pH range 5.0 to 9.0 shows most drastic changes. Emulsifying activity against an equal mixture of 2-methylnaphthalene and hexadecane increased linearly with the concentration of bioemulsifier. Emulsifying activity was largely unaffected by pH, except in pH 6, emulsifying activity was a lot higher. Emulsifying activity of RL was compared with that of emulsan against hexadecane, olive oil, soybean oil, and an equal mixture of 2-methynaphthalene and hexadecane. RL showed higher emulsifying activity than emulsan in each cases. Against vegetable oil, emulsifying activity was higher than those against hydrocarbon or crude oil. Optimal pH for emulsification vary with types of oil. This is thoght to have caused by a change of HLB with pH.
