【摘要】：本實(shí)驗(yàn)首先利用藍(lán)色凝膠平板培養(yǎng)法進(jìn)行菌種篩選,從華北某油田的受污染土壤中分離篩選得到一株性能較好的生物表面活性劑產(chǎn)生菌H1。對這株菌進(jìn)行生理生化試驗(yàn)和16s rRNA基因序列相似性分析以確定其種屬,并對其所合成的生物表面活性劑進(jìn)行提取以進(jìn)行產(chǎn)物的薄層色譜和紅外光譜分析,進(jìn)而確定其該生物表面活性的種類。結(jié)果表明,該菌株經(jīng)鑒定為肺炎克雷伯氏菌(Klebsiella pneumoniae)。薄層色譜和紅外光譜分析表明,所提取的物質(zhì)遇磷脂類指示劑有顯色反應(yīng),紅外光譜數(shù)據(jù)表明存在碳氧雙鍵(C=O)和磷脂鍵(P-O-C),由此可推斷菌株H1所產(chǎn)生物表面活性劑為磷脂類生物表面活性劑,其產(chǎn)量可達(dá)到0.7 g/L。后期實(shí)驗(yàn)中,采用油平板篩選法代替藍(lán)色凝膠平板篩選法,從遼河油田某處土樣中分離篩選出一株性能更為優(yōu)良的生物表面活性劑產(chǎn)生菌,并將其命名為A3。對其進(jìn)行生理生化實(shí)驗(yàn)和16s rRNA基因序列相似性分析初步鑒定。結(jié)果表明,菌株A3為銅綠色假單胞菌。對該菌所合成的生物表面活性劑進(jìn)行提取純化,再通過薄層層析和紅外光譜分析確定該生物表面活性劑的種類。對菌株A3的發(fā)酵培養(yǎng)條件進(jìn)行正交實(shí)驗(yàn),選取的因子為碳源、氮源、溫度、pH值和NaCl濃度。評價(jià)生物表面活性劑性能的一個(gè)重要指標(biāo)就是它的穩(wěn)定性,對A3產(chǎn)生物表面活性劑進(jìn)行礦化度、溫度和酸堿度的穩(wěn)定性探究,并確定其臨界膠束濃度值(critical micelle concentration, CMC)以便進(jìn)行原油降粘實(shí)驗(yàn)。原油降粘實(shí)驗(yàn)包括：原油的粘度-溫度探究,生物表面活性劑添加狀態(tài)對降粘率的影響,對溫度、礦化度的穩(wěn)定性實(shí)驗(yàn),生物表面活性劑添加量-降粘率實(shí)驗(yàn),生物表面活性劑、化學(xué)表面活性劑降粘性能的對比實(shí)驗(yàn)。結(jié)果表明,合成生物表面活性劑的最適條件為：30 g/L葡萄糖,4g/L硝酸鈉,4g/L氯化鈉,初始pH值為6.0,最適培養(yǎng)溫度為30℃。在上述條件下,產(chǎn)量可達(dá)到5.5 g/L,是優(yōu)化前產(chǎn)量的5.4倍。菌株A3所產(chǎn)生物表面活性劑具有良好的礦化度穩(wěn)定性及溫度穩(wěn)定性。該生物表面活性劑對稠油的降粘率最大可達(dá)到96.7%。本研究結(jié)果為生物表面活性劑的合成提供了新的菌源,也為提高生物表面活性劑的合成能力提供了科學(xué)依據(jù)。其良好的穩(wěn)定性有利于拓寬生物表面活性劑的應(yīng)用領(lǐng)域,從而為新型表面活性劑的開發(fā)和應(yīng)用奠定基礎(chǔ)。
[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，

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