本文選題：食烷菌 + 石蠟降解��； 參考：《河南師范大學(xué)》2016年碩士論文

【摘要】：油田含蠟量較高,且多數(shù)為長碳鏈蠟分子,造成十分嚴(yán)重的結(jié)蠟現(xiàn)象,給采油帶來嚴(yán)重影響,所以采取措施進(jìn)行清防蠟是非常有必要的。通常采用熱水、熱油進(jìn)行洗井清蠟,或使用化學(xué)清防蠟劑進(jìn)行清防蠟。但這些措施工作量都大、且費(fèi)用高,化學(xué)清防蠟劑會降低原油質(zhì)量、對環(huán)境造成破壞、以及威脅工人的健康;熱水洗井會引起地層粘土膨脹造成采油堵塞;而長期進(jìn)行熱油洗井,則會對油井的結(jié)構(gòu)造成損壞。本文由此立題,從青海油田表層取回油水混合物樣品,經(jīng)過分離、純化得到一株菌,通過16SrDNA基因的測序,確定其系統(tǒng)分類學(xué)地位;并了解該菌株的生理生化特性;由于該菌株生長速度慢,需要確定該菌株的最適生長條件,進(jìn)而對菌株的培養(yǎng)條件進(jìn)行優(yōu)化;接著在石蠟降解過程中,探究了該菌株的最佳降蠟條件,但降解石蠟的效果不高,所以為了獲得高降解能力的菌株,本實驗利用常壓室溫等離子體(atmospheric and room temperature plasma,ARTP)進(jìn)行誘變育種,獲得了高降蠟?zāi)芰透叻�(wěn)定性的突變菌株Y7;且在培養(yǎng)過程中該菌株表現(xiàn)出一定的乳化性,表明該菌株能夠產(chǎn)生表面活性物質(zhì)——具有重要活性的次級代謝產(chǎn)物,又對該菌株所產(chǎn)生的生物表面活性劑進(jìn)行了初步研究。主要研究結(jié)論包括以下幾個方面:(1)在樣品中分離、純化、并且經(jīng)過16SrDNA測序,確定了本實驗得到的菌株是印度洋深海食烷菌(Alcanivorax dieselolei),它屬于Proteobacteria變形桿菌門,Gammaproteobacteria變形桿菌綱,Oceanospirillales海洋螺菌目,Alcanivoracaceae烷烴降解菌科,Alcanivorax食烷菌屬;且為革蘭氏陰性菌,不產(chǎn)芽孢。(2)該菌株的最佳發(fā)酵條件為:裝液量為60 m L/250 m L,初始pH值7.0,溫度33℃,轉(zhuǎn)速150 rpm,發(fā)酵時長7 d;營養(yǎng)物質(zhì)配方NaCl濃度30.0 g/L,K_2HPO_4 2.0 g/L,MgSO_4濃度0.4 g/L,氮源為蛋白胨,碳源為植物油。優(yōu)化后的菌體數(shù)量最高達(dá)到3.688×1011個/m L,比優(yōu)化前數(shù)量上提高105倍。該實驗證明了菌株在優(yōu)化后的培養(yǎng)條件下生長,菌體數(shù)量顯著上升。(3)該菌株的最佳降蠟條件為:在石蠟液體培養(yǎng)基、初始石蠟濃度為20.00 g/L、處理時間9 d、搖床轉(zhuǎn)速180 rpm、MgSO_4、K_2HPO_4、Fe Cl3的濃度分別在0.80 g/L;3.00g/L;0.05 g/L、葡萄糖含量12.00 g/L、培養(yǎng)溫度33℃、培養(yǎng)基初始pH值為8.0、以及最佳NaCl濃度15.00 g/L時,降解石蠟的能力最好,可達(dá)到58.87%。(4)ARTP誘變處理該菌株,時間為30 s時更容易出現(xiàn)高降解石蠟的菌株。該操作在高純氦氣作為工作氣體的條件下,工作功率為120W,氣體流量10 SLM,待處理樣品與等離子體發(fā)生器射流出口間距設(shè)定為2 mm,減壓閥低壓輸出端壓力在0.1~0.15MPa。經(jīng)誘變以后,最終得到一株具有較高降蠟活性的誘變菌株Y7。在優(yōu)化后的降解石蠟條件下,該誘變菌株降解石蠟的能力較出發(fā)菌株提高了51.49%,達(dá)到89.05%。(5)突變菌株Y7具有很好的遺傳穩(wěn)定性,連續(xù)傳6代,每一代的降解石蠟?zāi)芰Χ挤�(wěn)定在80%以上。(6)該菌株產(chǎn)生的次級代謝產(chǎn)物——生物表面活性劑,研究表明:該生物表面活性劑存在于細(xì)胞外,乳化性和穩(wěn)定性良好,該表面活性劑是屬離子型,歸屬于脂肽類活性物質(zhì)。
[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.

【學(xué)位授予單位】：河南師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：TE357.9;Q939.97

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