Analysis Of Effective De-emulsification Components Produced By A Strain And Optimization Of Its Intensified Culture Condition

Microbial de-emulsifier is a complex system containing many kinds of effective ingredients. At the present time, the limit understanding of de-emulsification substances and uncontrolled productivity result in unstable de-emulsifying effectiveness and low de-emulsification activity. For this reason, the use of microbial de-emulsifier is limited. Profound investigation on effective de-emulsification substances would make it possible to adjust and control the performance of the microbial de-emulsifier. At the same time, seeking for new ways and methods to develop de-emulsification performance would also help to make big progress in bio-demulsifier research. Experiments were carried out to optimized fermentation factors for more de-emulsifer production based on superior demulsifying bacteria isolation. Environmental factors effected de-emulsification activity was clarified. More researches focused on analysis of effective de-emulsification substances. These effective ingredients were extracted, purified then characterized by modern analysis techniques. Basic studies using intensified culture were carried out to explore a simple and available way improving de-emulsification activity.Referring to methods of isolation bio-surfactant producing bacteria combining with de-emulsifying test, a superior strain was isolated. The strain was identified by 16SrDNA as Bacillus mojavensis, named as XH-1. The fermentation conditions which affected its growth characteristics and de-emulsification ability of cell culture were investigated by testing biomass and oil drain rate of the culture. The experimental results indicated that the optimal cultivation conditions using MMSM medium were: medium pH 6.5, cultivation temperature 30℃, rotary speed 140 rpm/min, and cultivation time 24h. Under this fermentation condition, the cell culture exhibited best de-emulsifying ability, with 40 %( v/v) input amount, the oil drain rate on O/W model emulsions was: 100% under room temperature in 48h; 80% at 46℃in 10h and 100% at 55℃in 10h.Distribution of de-emulsification activity in the culture and the stability of the active substances against high temperature and pH were studied with the help of UV, IR spectrum. The results revealed that protein was one of the active ingredients of the de-emulsifier. The active substances which were attached around the bacterial surface or suspended in the culture existed outside of bacterial cells. The bacterial cells itself did not have de-emulsification capability. Repeated freezing and thawing method and denaturant treating were used to evaluate the de-emulsification activity of the supernatant. At least 30% of its activity remained which indicated the existence of non-protein ingredients.De-emulsification substances were extracted from the supernatant. Optimized method was chosen by comparing the de-emulsification activity and productivity of the extracted products. The results showed that polysaccharides, lipoid, lipopeptide extracted from XH-1 supernatant could not break any O/W model emulsion. Effective substances could be precipitated by 25%~45% (NH4)2SO4 and organic solvents. Extracted products settled by ethanol showed high productivity and high de-emulsification activity. The extracted method using ethanol also showed good repeatability. With the optimizing extraction techniques, extracted productivity would reach the maximum point of 91%. The oil drain rate of the crude products solution to model emulsions was 100% under room temperature in 48h. The results of ingredients measuring and molecular weight distribution analysis showed that the extracted product was a complex system containing macromolecule substances as protein, polysaccharides and micromolecule substances which molecular weight was under 1000D.HIC and SEC purifying methods were conducted to separate and purify the activity ingredients. Only one elution peak was detected when eluted the purifying product solution through HLPC. Mass spectrograpHic analysis showed micromolecule substances with PEG structure played important role in de-emulsifying process. The molecular weight of these substances which existed as micelle in solution varied from 200-1000D. SDS-PAGE revealed the differences between fractions with varied de-emulsification activity. The differential band was digested by enzyme and characterized by mass spectrogram. Comparing the results with data base, four reliable proteins were identified. Only one of them was hydropHobic protein which was confirmed as activity ingredient, identified as pHospHate-binding protein.To intensify the de-emulsification activity of the microbial de-emulsifier, two superior strains (Bacillus mojavensis, Bacillus subtilis) were cultured in a mineral salt medium to obtain a microbial compound de-emulsifier with high capability of de-emulsification. The cell culture could demulsify the O/W model emulsion completely in 48 hours under room temperature. Comparing with that of the single strain culture, the fermentation process of the mixed culture was shortened more than 6 hours. With less addition volume, the mixed culture showed higher de-emulsification activity and stability. The mixed culture kept high de-emulsification activity in the emulsion with different pH value ranged from 3~7. Thermo stability was detected. When the emulsion temperature varied from 20~120℃, changing rate of the oil drain rate was under 15%. After the strains were mixed cultured, the metabolites produced during the fermentation process still took the main function of de-emulsification. The supernatant remained 87% de-emulsification ability of the cell culture while the bacterial cells could hardly break the emulsion. Intensified and mixed culturing is an effective way to enhance their de-emulsification abilities.