生物表面活性剂产生菌的优化培养及在稠油降粘中的应用研究
发布时间：2019-02-19 12:10
【摘要】：本实验首先利用蓝色凝胶平板培养法进行菌种筛选,从华北某油田的受污染土壤中分离筛选得到一株性能较好的生物表面活性剂产生菌H1。对这株菌进行生理生化试验和16s rRNA基因序列相似性分析以确定其种属,并对其所合成的生物表面活性剂进行提取以进行产物的薄层色谱和红外光谱分析,进而确定其该生物表面活性的种类。结果表明,该菌株经鉴定为肺炎克雷伯氏菌(Klebsiella pneumoniae)。薄层色谱和红外光谱分析表明,所提取的物质遇磷脂类指示剂有显色反应,红外光谱数据表明存在碳氧双键(C=O)和磷脂键(P-O-C),由此可推断菌株H1所产生物表面活性剂为磷脂类生物表面活性剂,其产量可达到0.7 g/L。后期实验中,采用油平板筛选法代替蓝色凝胶平板筛选法,从辽河油田某处土样中分离筛选出一株性能更为优良的生物表面活性剂产生菌,并将其命名为A3。对其进行生理生化实验和16s rRNA基因序列相似性分析初步鉴定。结果表明,菌株A3为铜绿色假单胞菌。对该菌所合成的生物表面活性剂进行提取纯化,再通过薄层层析和红外光谱分析确定该生物表面活性剂的种类。对菌株A3的发酵培养条件进行正交实验,选取的因子为碳源、氮源、温度、pH值和NaCl浓度。评价生物表面活性剂性能的一个重要指标就是它的稳定性,对A3产生物表面活性剂进行矿化度、温度和酸碱度的稳定性探究,并确定其临界胶束浓度值(critical micelle concentration, CMC)以便进行原油降粘实验。原油降粘实验包括：原油的粘度-温度探究,生物表面活性剂添加状态对降粘率的影响,对温度、矿化度的稳定性实验,生物表面活性剂添加量-降粘率实验,生物表面活性剂、化学表面活性剂降粘性能的对比实验。结果表明,合成生物表面活性剂的最适条件为：30 g/L葡萄糖,4g/L硝酸钠,4g/L氯化钠,初始pH值为6.0,最适培养温度为30℃。在上述条件下,产量可达到5.5 g/L,是优化前产量的5.4倍。菌株A3所产生物表面活性剂具有良好的矿化度稳定性及温度稳定性。该生物表面活性剂对稠油的降粘率最大可达到96.7%。本研究结果为生物表面活性剂的合成提供了新的菌源,也为提高生物表面活性剂的合成能力提供了科学依据。其良好的稳定性有利于拓宽生物表面活性剂的应用领域,从而为新型表面活性剂的开发和应用奠定基础。
[Abstract]:In this experiment, the blue gel plate culture method was first used to screen the bacteria, and a biosurfactant producing strain H1 was isolated and screened from contaminated soil in a North China oil field. Physiological and biochemical tests and 16s rRNA gene sequence similarity analysis were carried out to determine the species of the strain, and biosurfactants were extracted for TLC and IR analysis. Then the species of the biological surface activity is determined. The results showed that the strain was identified as Klebsiella pneumoniae (Klebsiella pneumoniae). TLC and FTIR analysis showed that the extracted substance had color reaction in the presence of phospholipid indicator, and the infrared spectrum data showed that there were carbon-oxygen double bond (CIO) and phospholipid bond (P-O-C). It can be concluded that the surfactant produced by strain H1 is phospholipid biosurfactant, and its yield can reach 0.7 g / L. In the later experiment, a biosurfactant producing strain with better performance was isolated from a soil sample of Liaohe Oilfield by oil plate screening instead of blue gel plate screening method, and it was named A3. Physiological and biochemical experiments and similarity analysis of 16s rRNA gene sequence were carried out. The results showed that strain A 3 was Pseudomonas copper-green. The biosurfactants synthesized by this bacterium were extracted and purified, and the kinds of biosurfactants were determined by TLC and FTIR. The fermentation conditions of strain A3 were studied by orthogonal experiment. The factors selected were carbon source, nitrogen source, temperature, pH value and NaCl concentration. An important index to evaluate the performance of biosurfactant is its stability. The stability of the surfactant produced by A3 is studied in terms of salinity, temperature and pH, and the critical micelle concentration (critical micelle concentration,) is determined. CMC) for crude oil viscosity reduction experiments. The viscosity reduction experiment of crude oil includes: the viscosity and temperature of crude oil, the effect of the addition state of biosurfactant on viscosity reduction, the stability experiment of temperature and salinity, the experiment of adding amount of biosurfactant to reduce viscosity, Comparative experiment on viscosity reduction performance of biosurfactants and chemical surfactants. The results showed that the optimum conditions for biosurfactant synthesis were as follows: 30 g / L glucose, 4g/L sodium nitrate, 4g/L sodium chloride, initial pH value was 6.0, and the optimum culture temperature was 30 鈩，
本文编号：2426487
[Abstract]:In this experiment, the blue gel plate culture method was first used to screen the bacteria, and a biosurfactant producing strain H1 was isolated and screened from contaminated soil in a North China oil field. Physiological and biochemical tests and 16s rRNA gene sequence similarity analysis were carried out to determine the species of the strain, and biosurfactants were extracted for TLC and IR analysis. Then the species of the biological surface activity is determined. The results showed that the strain was identified as Klebsiella pneumoniae (Klebsiella pneumoniae). TLC and FTIR analysis showed that the extracted substance had color reaction in the presence of phospholipid indicator, and the infrared spectrum data showed that there were carbon-oxygen double bond (CIO) and phospholipid bond (P-O-C). It can be concluded that the surfactant produced by strain H1 is phospholipid biosurfactant, and its yield can reach 0.7 g / L. In the later experiment, a biosurfactant producing strain with better performance was isolated from a soil sample of Liaohe Oilfield by oil plate screening instead of blue gel plate screening method, and it was named A3. Physiological and biochemical experiments and similarity analysis of 16s rRNA gene sequence were carried out. The results showed that strain A 3 was Pseudomonas copper-green. The biosurfactants synthesized by this bacterium were extracted and purified, and the kinds of biosurfactants were determined by TLC and FTIR. The fermentation conditions of strain A3 were studied by orthogonal experiment. The factors selected were carbon source, nitrogen source, temperature, pH value and NaCl concentration. An important index to evaluate the performance of biosurfactant is its stability. The stability of the surfactant produced by A3 is studied in terms of salinity, temperature and pH, and the critical micelle concentration (critical micelle concentration,) is determined. CMC) for crude oil viscosity reduction experiments. The viscosity reduction experiment of crude oil includes: the viscosity and temperature of crude oil, the effect of the addition state of biosurfactant on viscosity reduction, the stability experiment of temperature and salinity, the experiment of adding amount of biosurfactant to reduce viscosity, Comparative experiment on viscosity reduction performance of biosurfactants and chemical surfactants. The results showed that the optimum conditions for biosurfactant synthesis were as follows: 30 g / L glucose, 4g/L sodium nitrate, 4g/L sodium chloride, initial pH value was 6.0, and the optimum culture temperature was 30 鈩，
本文编号：2426487
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