摘要
为满足“资源节约型、环境友好型”社会建设和绿色工业发展对绿色净水剂的需求，生物破乳剂作为一种高效、低毒、环保的绿色净水剂，成为破乳剂领域研究的新热点。在高含水原油乳状液以及其它工业乳状液副产品的处理和处置中，应用生物破乳剂替代大量使用的化学破乳剂，对提高乳状液脱水率，降低环境污染风险具有深远意义。但是，生物破乳剂的实际应用进程受到破乳剂产生菌代谢过程不稳定、破乳有效成分复杂及缺少大规模发酵生产经验等问题的制约。基于此，开展破乳菌筛选方法；菌种破乳效能强化；粗产品分离与鉴定；发酵工艺优化及产破乳剂相关蛋白质功能解析等方面的研究，将为生物破乳剂的大规模生产、推广和应用奠定良好基础。
将可用于间接表征生物破乳剂破乳效能及破乳菌初筛的6种生物表面活性剂检测方法与破乳试验相结合，提出破乳菌筛选原则，构建高效筛选模式。确定了高效破乳菌(24h排油率≥90%)的判断依据：发酵液表面张力≤40mN/m或细胞表面疏水性(MATH)≥50%。筛选得到7株高效破乳菌，经16S rDNA鉴定分属于芽孢杆菌属(Bacilllus sp.)和戈登氏菌属(Gordonia sp.)，实验室编号分别为LXH-1、LXH-2、LXH-3、LL1、LL-1、LL-2和LL-3。
以生物破乳剂产生菌XH1为研究对象，改进破乳菌复壮方法，其核心是通过烃类物质-液体石蜡为底物排除负变细胞，调节菌株的破乳活性；改进的复壮方法可成功将菌株XH1的表面活性、破乳功能等特性恢复至原始水平，效果优于常规复壮。
研究表明烃类物质-液体石蜡对菌株XH1合成破乳有效组分具有刺激和促进作用，利用液体石蜡预先刺激强化菌种，再经生产培养基扩大培养，获得的生物破乳剂可将破乳半衰期t1/2从16h缩短为2h。根据胞外蛋白破乳比活性、细胞表面疏水性等相关试验结果，初步分析液体石蜡强化促进菌株XH1破乳特性的作用机制。
菌株XH1经液体石蜡强化后，破乳有效组分主要分布于上清液和附着在菌体表面。分离得到生物破乳剂粗产品，产量为2.01g/L，4mg的排油率(24hR.D.)>93%。利用可见-紫外光谱、红外光谱和薄层层析(TLC)确定粗产品主要功能组分为蛋白质和脂肽类物质；平均分子量为2.59×106Da。并从中粗提得到脂肽类物质的量为0.08±0.01g/g (24h R.D.:65.3%)；破乳活性蛋白复合物可由饱和度25%，45%的(NH4)2SO4粗提得到，产量为0.36±0.02g/g (24h R.D.:67.4%)。利用SDS-PAGE电泳和质谱技术确定疏水蛋白质Oxalate Decarboxylase为一种破乳有效蛋白质。
为了提高生物破乳剂的产率，采用响应面法(RSM)确定最优培养基组成为：8.5g/L葡萄糖、3%(v/v)液体石蜡、15g/L磷酸盐(K2HPO4&KH2PO4)、1.5g/L酵母膏和3.36g/L氯化铵；与优化前相比排油率(24h)提高了35.5%，破乳剂粗产品产量提高了1倍。进一步确定最佳发酵条件为：培养温度29℃、摇床转速200r/min、培养时间21h和种子液菌龄24h。
为了确定破乳菌XH1的发酵方式，首先基于Logistic方程、Luedeking-Piret方程及Luedeking-Piret-Like方程建立描述菌株XH1分批发酵生产破乳剂的动力学模型，经检验该组模型能较好的拟合XH1发酵过程。进一步比较不同的发酵方式，结果表明分批补料半连续式发酵优于分批发酵，最佳补料参数：初始葡萄糖为2.0g/L；补料方式为间歇式，间隔时间为5h；补料量为：控制每次补料量比前一次增加0.02%~0.04%。破乳菌XH1在最佳补料方式下破乳剂粗产品产率提高55.9%，产品对糖得率提高44%，高效破乳剂(24h排油率>80%)连续生产周期延长了50h。
为了确定参与生物破乳剂合成的相关蛋白质，采用SDS-PAGE电泳研究液体石蜡刺激强化培养和单一营养物质缺失对XH1破乳效能和菌体总蛋白变化的影响，获得差异表达蛋白复合物Ⅰ、Ⅱ和Ⅲ。通过nanoESI-Q-TOF MS/MS共鉴定出66个差异表达蛋白质，分属于碳水化合物的转运和代谢、能量产生与转换、翻译/核糖体结构和生物合成等14个蛋白质功能。确定以上差异表达蛋白质中与糖酵解途径(EMP)、三羧酸循环(TCA)以及蛋白质合成相关的酶与菌株XH1合成蛋白质和脂肽类生物破乳剂关系密切，进一步从蛋白质组层面分析不同发酵条件影响菌株XH1产生物破乳剂能力的作用机制。
基于以上研究结果，建立发酵工艺条件、差异蛋白质功能以及生物破乳剂合成过程三者的关系，从代谢调节、菌种特性和发酵工艺三个层面综合分析强化破乳菌XH1产破乳剂能力的策略，并提出菌种高效筛选→菌种改进复壮→液体石蜡刺激强化→半连续发酵生产的破乳菌XH1发酵形式，对于今后生物破乳剂大规模生产具有重要的参考价值。
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.
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