Study On The Characteristics And Demulsification Efficiency Of Bio-Demulsifier Producing Bacteria

For its high demulsification efficiency, non-toxicity, non-secondary pollution, easily biodegradable and environmentally safe, the bio-demulsifier is one of the most potential research and development orientations for demulsifiers. Instead of chemical demulsifier, application of the high efficiency and environmental friendly bio-demulsifier has important significance in improving the dewatering efficiency of the crude oil emulsion, reducing environment pollution and realizing sustainable development.For the bio-demulsifier, most of the recent researches mainly focused on the selection of demulsifying bacteria and the optimization for their growth and demulsification conditions. Researches on fermentation kinetics, effective component analysis and demulsification mechanisms were rare. Based on isolating and selecting demulsifying bacteria, their growth characteristics were investigated and the fermentation kinetics were analyzed. The characteristics of the demulsifier producing bacteria were studied,and its influencing factors were investigated. Demulsifying effective substances of the bio-demulsifier were confirmed and its physiochemical properties were analyzed. Analysis of demulsifying process was carried out and the demulsifying mechanisms were discussed.A set of high efficiency and convenient method was established to isolate de-emulsification strain, by which, one strain with high and steady demulsification activity was isolated. And for O/W model emulsions, more than 85% efficiency of demulsification was obtained for the strain within 24h and 100% efficiency was acquired within 48h. The physiological-biochemical characteristics of the strain were analyzed and the strain was identified by 16S rDNA. The results indicated that the strain belonged to Bacillus mojavensis, named as XH-1. The serial generation transfer test showed that strain XH-1 had stable hereditary demulsification.Effects of cultivation conditions and nutritional factors on bio-demulsifier producting capability and growth of the strain XH-1 were investigated through demulsifying test and biomass estimation. The experimental results indicated that the optimal cultivation conditions were determined as follows: pH 6.5, temperature 30℃, revolution speed 140 rpm/min, cultivation time 18~20h. It indicated that strain XH-1 could utilized extensive substances as its carbon and nitrogen source. The optimal carbon source for XH-1was the mixture of glucose (10g/L) and liquid paraffin [2 ~ 4 %( v/v)], and the optimal nitrogen source was NH4NO3 (4.0 g/L) and yeast (1.0g/L). Upon studying the influential environmental factors of de-emulsification, the demulsification rate with the changes of contacting time was approximate first order reaction, and the best proportion of the complete culture bacterial solution and O/W model emulsions for de-emulsifying were 1:5. The most favorable environmental conditions for demulsification occurred when temperature was 30℃and pH was 5~9.The fermentation process of demulsifying bacteria was studied in batch system. Based on the equations of"Logistic equation"and"Luedeking-Piret equation", the kinetic models reflecting its fermentation features of strain growth, substrate consumption and product generation were constructed respectively. The experiment data was processed with the Origin 7.5 software and the batch fermentation kinetics model parameter was obtained. The good agreement of predicted values with the experimental results showed that the kinetic models were applicable for predicting the actual fermentation process.The microscope observation and physiochemical analysis showed that demulsifying effective components of XH-1 were proteins. These proteins existed in the bacterial extracellular polymer substances, which were attached around the bacterial surface or suspended in the fermentation solution. The bacterium itself did not have demulsification capability.The demulsifying proteins were subjected to separation by SDS-PAGE. It showed that the proteins appeared in a variant band between 50kD~80kD molecular weight. By shotgun proteomic analysis, a total of 20 proteins were identified with high-confidence. Among them, 6 proteins were unknown or unnamed proteins and the demulsifying efficiencies of other proteins were also unreported.The demulsifying behavior of bio-demulsifier was investigated, and combined with the physiochemical properties of the demulsifying effective components, the primary demulsifying mechanism was discussed. On this basis, combined with related theories, the hypothesis of synergistic effect demulsification by protein was proposed. The zeta potential of the model emulsion and its effect on demulsification of model emulsion in the presence of demulsifiers were analyzed. It indicated that there was no obviously electric neutralization phenomenon. Water-oil interfacial tension and its effects on demulsification of model emulsion in the presence of demulsifiers were studied. The results showed that by utilizing its amphiphilicity and macromolecular structure, the protein demusifiers display favorable extensibility and adsorptive ability, which were crucial for excellent demulsification ability. The demulsifying process included solubilization effect and displacement effect. When the dosage of bio-demulsifier was small, the displacement effect dominated; the interfacial tension decreased and the demulsification efficiency enhanced with the increase of bio-demulsifier amount. When the dosage of bio-demulsifier was high, the solubilization effect dominated; the interfacial tension rose and the demulsification efficiency reduced with the increase of bio-demulsifier amount.