Enhancement Of Demulsifying Properties And Environmental Adaptability Of Demulsifying Bacteria Mutant

Development and application of green biological agents in environmentalprotection fields has been taken more seriously. Bio-demulsifier has graduallybecome a hot research, because of its environmentally friendly property includinglow toxicity, non-secondary pollution and biodegradation. Bio-demulsifier can beused to treat the petroleum field emulsions and other industrial emulsionsin stead ofchemical demulsifier widely used, which has profound meaning for enhancing thedehydration rate of the emulsions and decreasing the environmental risks. However,with deep bio-demulsifier research, some significant problems have become theobstructions of large scale production and promotion. For example, bio-demulsifieryields and performance of wild strains cannot meet the requirements of industrialproduction, application of bio-demulsifier is restricted by environmental conditionsand incubation way is sole.This paper conducts correlative studies according to above problems forfilling the gaps of this fields and improving application. That is mainly includingusing the mutation breeding approaches to improve the demulsifying ability andextreme environment adaptation of bio-demulsifier producing bacteria; based onproteomics analysis of environmental adaptation mutation mechanism ofthermotolerant, acid resistance and alkali resistance mutants; selection of highefficiency demulsifying new strains used to study complex culture and compoundbio-demulsifier development.In order to enhance the demulsifying ability of wild strains, wild demulsifyingstrain XH1(Bacillus mojavensis) was proceed mutation breeding. Comparing themutation effect of the different mutation factors on demulsifying strain XH1, it isdetermined that the optimal mutation approach was UV+nitrosoguanidine(NTG) ofcompound mutation. The mutant with the highest demulsifying ability was strainXN5, which exhibited a15.12%increase of demulsifying ratios compared withoriginal strain XH1. Through study on the effect of cultivation condition on growthand demulsifying ability of mutant XN5, it obtained the optimal cultivationconditions were as follows:35℃cultivation temperature,140r/min shaking tablespeed,7.0initial pH-value,21h incubation time.The correlative techniques of proteomics including SDS-PAGE,2-DE andnanoESI-Q-TOF MS/MS were used to analysis of environmental adaptationmutation mechanism of temperature-fast, acid-fast and alkali-fast mutants. Thedifferential expression protein was separated, identified and function analyzedwhich was from the original strain XH1grown on routine conditions, the mutant isolated by temperature grown on50℃incubation temperature, the mutant isolatedby acid environment grown on medium with pH3, the mutant isolated by alkalienvironment grown on medium with pH11, respectively. The results show thatsignificant variation of the numbers of the differential expression protein spots fromthermotolerant, acid resistance and alkali resistance mutants were14,8and9.Through identification and function analysis of differential expression protein ofdemulsifying strain XH1produced by mutation under the different environmentalconditions, the mutation mechanism of demulsifying bacteria was preliminaryanalyzed on the proteomics levels.Combination the detection methods ofbiosurfactants and demulsifying test was used to isolate the demulsifying bacteriafrom the activated sludge in aeration tank of Taiping waste water treatment plant.One strain with high and steady demulsifying characteristics (98.5%of24hdemulsifying ratios), named as L1, identified as Gordonia sihwensis. Thedemulsifying activity of bio-demulsifier produced by L1could not be influenced bythe temperature treatment, autoclaved sterilization and freeze thawing. Throughresearch on the effects of the cultivation conditions on demulsifying efficiency ofstrain L1grown on mineral salt medium(MSM), it obtained that the optimalcultivation conditions were as follows:30℃cultivation temperature,140minshaking table speed,7.0initial pH-value,60h incubation time.In order to study on the optimal cultivation conditions of mixed cultureproducing compound bio-demulsifier, strain L1and XH1selected were incubated inmodified mineral salt medium(MMSM) without glucose. Then the response surfacemethodology (RSM) was used to optimize mixed culture conditions. According tothe results of the model analysis and verification, it was determined that the optimalconditions were follows:3:2inoculum proportion (strain L1:XH1),3.6%(v/v) liquidparaffin contents, the fourth day fed glucose, continue incubating21h after feedingglucose. Compared with single strain culture, the compound bio-demulsifierproduced by complex culture of L1and XH1exhibited a decrease of dose, anincrease of stability and a shortened demulsifying time. And the complex cultureincreased the utilization ratio of main nutrients in the medium, and the per tonbio-demulsifier cost decreased by72.6%.