| In order to meet the ‘resourceconserving’ and ‘environmentally friendly’ socialconstruction and green industrial development on demand for green purifying agent,biodemulsifier as a high efficient, low toxicity and environmental green purifyingagent, has been the key point of demulsifier research area. Biodemulsifier was usedin the treatment and disposal of high water-cut crude oil emulsions and many otherindustial emulsions by-products to substitute for chemical demulsifier. That wasprofound significance to increase dewatering rate of emulsions and decreaseenvironmental risk. However, some key problems restrict the practical application ofbiodemulsifier such as unstable metabolic process of demulsifying bacteria, difficultanalysis on complicated effective ingredients, major research at laboratory level andabsence of large scale fermentation experience. So, research on isolation methods tohigh efficiency strains, enhancement of demulsifying ability, separation andidentification of crude products, fermentation process optimization and analysis onprotein related to demulsifying activity will be great foundation of lagre scaleproduction, promotion and application.A complementary advantage of highly-efficient isolation mode was put forwardby demulsifying test combining with six detecting methods to biosurfactants whichcould be used in indirect characterization of demulsifying efficiency ofbio-demulsifier and primary isolation of demulsifying strains. Judgement standardof high efficiency demulsifying strains (24h demulsifying ratio R.D.≥90%) wasdetermined as surface tensions≤40mN/m or microbial adhesion to the hydrocarbon(MATH)≥50%. Seven strains with high demulsifying ability were identified asBacilllus sp. and Gordonia sp.by16S rDNA analysis,respectively, named as LXH-1,LXH-2, LXH-3, LL1, LL-1, LL-2and LL-3.High efficiency bio-demulsifier producing bacterium XH1selected as thisstudy object, was rejuvenated by modified rejuvenation method. The key of thismethod was exclusion negative variation cells by hydrocarbon-liquid paraffin assubstrate and adjusting strain demulsifying activity.Modified mothod could makethe surface activity and demulsifying function of XH1rejuvenate to original level,which was superior to the routine method.It shows that hydrocarbon-liquid paraffin possessed the stimulation andfacilitation to demulsifying effective components production by XH1. Based on this,using paraffin stimulated and enhanced the demulsifying characteristics of the strainfirst. It was found that the bio-demulsifierafter enlarge cultivation in productionmedium could decrease the half-life period (t1/2) of demulsification from16h to2h. According to the related measured results of demulsifying specific activity ofextracellular protein, microbial cell surface hydrophobic property, and so on, theeffect mechanism of paraffin stimulation and enhancement was preliminaryanalysed.The effective components from the whole culture of XH1distributed into thesupernatants and adhersion to cell surfaces, due to XH1stimulated and enhanced byliquid paraffin. The yield of bio-demulsifier crude product was2.01g/L and4mgdosage of24h R.D. was>93%. The major effective components were determined asprotein and lipopeptid substances by UV-vis and FT-IR spectrum, average molecularweight was2.59×106Da. Lipopeptid crude extract could be obtained0.08±0.01g/gfrom crude products of biodemulsifiers, possessing of65.3%R.D.(24h).Demulsifying active protein complexs could be precipitated by25%~45%(NH4)2SO4, and demulsifying ratio and yield were67.4%and0.36±0.02g/g,respectively. Using SDS-PAGE and mass spectrogram technology, one hydrophobicprotein was determined as effective ingredient, identified as Oxalate Decarboxylase.In order to improve the bio-demulsifier productivity, using Response SurfaceMethodology (RSM) to optimize bio-demulsifier producing medium, the optimalcompositions were as follows:8.5g/L glucose,3%(v/v) liquid paraffin,15g/Lphosphate（K2HPO4&KH2PO4）,1.5g/L yeast extract, and3.3.6g/L ammoniumchloride. Compared with unoptimization, the demulsifying ratio (24h) and crudeproduct yield were increased by35.5%and100%, respectively. Further optimizingfermentation conditions, it obtained that the optimum incubation temperature,shacking table speed, incubation time, and inoculum age were29℃,200r/min,21h,and24h, respectively.In order to determine strain XH1fermentation mode, based on the equations of“Logistic”,“Luedeking-Piret”,“Luedeking-Piret-Like”, the kinetic models ofbio-demulsifier batch fermentation of XH1and their parameters were obtained byOrigin7.5software. This group of models had a good fit with fermentation processof XH1and indicated that feeding carbon souces in fermentation was help tobiosynthesis of biodemulsifier. Comparing diverse fermentation approaches, theresults show that semicontinuous fermentation with fed-batch was superior to batchfermentation. The optimal feeding parameters as follows:2.0g/L initial glucoseconcentration, fed-batch type:5h fed-time interval, fed glucose mass:0.02%~0.04%increase of per feeding glucose compared with last time. The yieldsof crude products and from glucose utilization under optimal fed types wereincreased by55.9%and44%, respectively. Moreover, continuous production periodof highly-efficient bio-demulsifier (24h R.D.>80%) were prolonged by50h.In order to determine the protein participating in bio-demulsifier biosynthesis,using SDS-PAGE investigated the effects of liquid paraffin stimulation and enhancement, and medium absence of sole nutrient substance on demulsifyingefficieny and total protein differential expression of XH1. And it was obtained thatthe differential expression protein complexes Ⅰ, Ⅱ, and Ⅲ related todemulsifying ability of XH1. The above complexes were identified and analyzedwith the help of nanoESI-Q-TOF MS/MS and bioinformatics tools, showing thatsixty-six unique proteins belong to fourteen function classifications includingcarbohydrate transfer and metabolism, energy generation and transformation, andtranslation/ribosome structure and biosynthesis. Above the differential expressionproteins, the enzyme related to glycolytic pathway (EMP), tricarboxylic acid cycle(TCA) or protein synthesis had close relations with the biosynthesis of protein andlipopeptid type bio-demulsifier from strain XH1. Furthermore, on the proteomicslevel, the fermentation conditions effect mechanism on ability of producingbio-demulsifier was analysed.Through above research results established the relationships of fermentationprocess, differential expression protein and bio-demulsifier synthesis process.Enhancement strategy of the bio-demulsifier production ability of strain XH1wasanalyzed by synthesis from metabolic regulation, strain characteristic andfermentation process levels. Sequentially, a new fermentation approach includingdemulsifying strain selection'modified rejuvenation'liquid paraffinbio-stimulation and enhancement'semicontinuous fermentation was proposed,which had a significant reference value of the large scale bio-demulsifierproduction. |
PDF Full Text Request