一株降解石蜡菌株的分离鉴定及其表面活性剂的研究

发布时间：2018-05-06 13:22

本文选题：食烷菌 + 石蜡降解　；参考：《河南师范大学》2016年硕士论文

【摘要】：油田含蜡量较高,且多数为长碳链蜡分子,造成十分严重的结蜡现象,给采油带来严重影响,所以采取措施进行清防蜡是非常有必要的。通常采用热水、热油进行洗井清蜡,或使用化学清防蜡剂进行清防蜡。但这些措施工作量都大、且费用高,化学清防蜡剂会降低原油质量、对环境造成破坏、以及威胁工人的健康;热水洗井会引起地层粘土膨胀造成采油堵塞;而长期进行热油洗井,则会对油井的结构造成损坏。本文由此立题,从青海油田表层取回油水混合物样品,经过分离、纯化得到一株菌,通过16SrDNA基因的测序,确定其系统分类学地位;并了解该菌株的生理生化特性;由于该菌株生长速度慢,需要确定该菌株的最适生长条件,进而对菌株的培养条件进行优化;接着在石蜡降解过程中,探究了该菌株的最佳降蜡条件,但降解石蜡的效果不高,所以为了获得高降解能力的菌株,本实验利用常压室温等离子体(atmospheric and room temperature plasma,ARTP)进行诱变育种,获得了高降蜡能力和高稳定性的突变菌株Y7;且在培养过程中该菌株表现出一定的乳化性,表明该菌株能够产生表面活性物质——具有重要活性的次级代谢产物,又对该菌株所产生的生物表面活性剂进行了初步研究。主要研究结论包括以下几个方面:(1)在样品中分离、纯化、并且经过16SrDNA测序,确定了本实验得到的菌株是印度洋深海食烷菌(Alcanivorax dieselolei),它属于Proteobacteria变形杆菌门,Gammaproteobacteria变形杆菌纲,Oceanospirillales海洋螺菌目,Alcanivoracaceae烷烃降解菌科,Alcanivorax食烷菌属;且为革兰氏阴性菌,不产芽孢。(2)该菌株的最佳发酵条件为:装液量为60 m L/250 m L,初始pH值7.0,温度33℃,转速150 rpm,发酵时长7 d;营养物质配方NaCl浓度30.0 g/L,K_2HPO_4 2.0 g/L,MgSO_4浓度0.4 g/L,氮源为蛋白胨,碳源为植物油。优化后的菌体数量最高达到3.688×1011个/m L,比优化前数量上提高105倍。该实验证明了菌株在优化后的培养条件下生长,菌体数量显著上升。(3)该菌株的最佳降蜡条件为:在石蜡液体培养基、初始石蜡浓度为20.00 g/L、处理时间9 d、摇床转速180 rpm、MgSO_4、K_2HPO_4、Fe Cl3的浓度分别在0.80 g/L;3.00g/L;0.05 g/L、葡萄糖含量12.00 g/L、培养温度33℃、培养基初始pH值为8.0、以及最佳NaCl浓度15.00 g/L时,降解石蜡的能力最好,可达到58.87%。(4)ARTP诱变处理该菌株,时间为30 s时更容易出现高降解石蜡的菌株。该操作在高纯氦气作为工作气体的条件下,工作功率为120W,气体流量10 SLM,待处理样品与等离子体发生器射流出口间距设定为2 mm,减压阀低压输出端压力在0.1~0.15MPa。经诱变以后,最终得到一株具有较高降蜡活性的诱变菌株Y7。在优化后的降解石蜡条件下,该诱变菌株降解石蜡的能力较出发菌株提高了51.49%,达到89.05%。(5)突变菌株Y7具有很好的遗传稳定性,连续传6代,每一代的降解石蜡能力都稳定在80%以上。(6)该菌株产生的次级代谢产物——生物表面活性剂,研究表明:该生物表面活性剂存在于细胞外,乳化性和稳定性良好,该表面活性剂是属离子型,归属于脂肽类活性物质。
[Abstract]:The wax content of the oil field is high, and most of them are long carbon chain wax molecules, which cause very serious waxing phenomenon and bring serious influence on oil recovery. So it is necessary to take measures to clean and prevent wax. Usually, hot water, hot oil are used to wash well, or use chemical cleaning agent to prevent wax. High quality, chemical dewaxing agent can reduce the quality of crude oil, cause damage to the environment, and threaten the health of workers; hot water washing will cause clay expansion in the formation to cause oil recovery, and long-term hot oil washing will cause damage to the structure of the oil well. This paper, in this paper, retrieves the oil and water mixture samples from the surface of the Qinghai oil field and separated, The phylogenetic status of the strain was determined by the sequencing of the 16SrDNA gene, and the physiological and biochemical characteristics of the strain were determined. The optimum growth condition of the strain was determined because of the slow growth rate of the strain, and then the culture conditions of the strain were optimized. Then the optimum strain was explored in the process of paraffin degradation. The effect of paraffin degradation was not high, so in order to obtain high degradation ability, the strain of atmospheric and room temperature plasma (ARTP) was used in the experiment to induce mutagenesis, and the mutant strain Y7 with high wax lowering ability and high stability was obtained, and the strain showed certain milk during the process of culture. It shows that the strain can produce surfactant, a secondary metabolite with important activity, and a preliminary study on the bioactive agent produced by the strain. The main conclusions are as follows: (1) separation, purification, and 16SrDNA sequencing in the sample, and determine the strain obtained in this experiment. It is the India ocean deep-sea food alkanes (Alcanivorax dieselolei), which belongs to the Proteus Proteobacteria, Gammaproteobacteria Proteus, Oceanospirillales marine stud, Alcanivoracaceae alkanes degradation bacteria, Alcanivorax alkanes, and Gram-negative bacteria, and no spore. (2) the optimum fermentation condition of the strain is: liquid The initial pH value was 60 m L/250 m L, the initial pH value was 7, the temperature was 33, the speed was 150 rpm, and the fermentation length was 7 d; the nutrient formula NaCl concentration was 30 g/L, K_2HPO_4 2 g/L, the MgSO_4 concentration 0.4, the nitrogen source was peptone, and the carbon source was vegetable oil. The maximum number of bacteria was 3.688 * 1011 than the optimization before the optimization, which was proved to be twice as high as that before the optimization. The experiment proved that the number was 105 times higher than that before the optimization. The strain increased significantly under the optimized culture conditions. (3) the best wax reduction conditions of the strain were: in the paraffin liquid medium, the initial paraffin concentration was 20 g/L, the treatment time was 9 D, the rotational speed of the rocking bed was 180 rpm, the concentration of MgSO_4, K_2HPO_4, Fe Cl3 was 0.80 g/L, 3.00g/L; 0.05 g/L, 12 g/L glucose, culture temperature. At 33 degrees centigrade, the initial pH value of the medium is 8, and the best NaCl concentration is 15 g/L, the ability to degrade paraffin is best. The strain can be treated with 58.87%. (4) ARTP mutagenesis. When the time is 30 s, the strain of high degradation paraffin is easier to appear. The operation power is 120W, and the gas flow rate is 10 SLM under the condition of high pure helium gas as working gas. The outlet space between the treated sample and the plasma generator is set to 2 mm, and the pressure of the low pressure output end of the pressure reducing valve after 0.1~0.15MPa. is mutagenic, and a mutagenic strain Y7. with higher paraffin activity is finally obtained. Under the optimized condition of paraffin degradation, the ability of the mutagenesis strain to degrade paraffin is increased by 51.49% than that of the starting strain. The 89.05%. (5) mutant strain Y7 has a good genetic stability and a continuous transmission of 6 generations. The degrading paraffin ability of each generation is more than 80%. (6) the secondary metabolite produced by the strain, biological surfactants, shows that the biosurfactant exists outside the cell, the emulsification and stability are good, and the surfactant is isolated. The subtype belongs to the lipopeptide active substance.

【学位授予单位】：河南师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：TE357.9;Q939.97

【相似文献】