润滑油生物降解菌种选育及特性研究
发布时间：2018-10-10 17:29
【摘要】：润滑油在工农业生产中有着广泛的应用。由于润滑油回收利用渠道不畅通，回收技术不成熟，回收费用高等因素，导致润滑油的回收利用率低。同时由于生产工艺不合理以及操作不当等外界因素导致大量润滑油流入环境。这类不可再生润滑油中含有环烷烃，芳香烃及重金属等难降解组分，如果不加以有效处理，会造成环境污染。物化方法在处理废润滑油污染时，，虽然能够在短时间里取得一定的效果，但是二次污染严重。而生物法处理废润滑油是利用微生物的本质属性，即生物降解特性，将有毒害污染物作为底物进行彻底降解。作为生物处理技术的核心，开发高效降解菌种是目前润滑油污染治理的研究重心。 本实验旨在从土壤中筛选出具有自主知识产权的高效废润滑油菌种。课题组以废旧润滑油和油污染土壤为分离源，对后者首先进行预处理及梯度驯化，再以传统的微生物分离纯化技术为基础，分别从废润滑油和油污染土壤中共分离出51株单菌落；继而利用24孔板实验初筛得到15株润滑油降解菌，之后又通过摇瓶降解实验和脂肪酶的测定定量化优选出6株高效润滑油降解菌种；最后以这些菌种为物质基础对其润滑油生物降解能力进行了研究。研究证实这6株菌均具有较好的耐油特性，能够以润滑油为唯一碳源实现有效降解，并得到如下结论： 1）废润滑油及润滑油长期污染土壤中都含有高效石油烃降解菌种，是优良菌种的理想来源； 2）利用24孔板实验，以细胞培养液和静止细胞为接种体，能够定性的评价菌种对润滑油的生物降解能力，采取该方法筛选出15株目的菌种，其油膜变化情况与孔板培养液中细胞生长情况吻合; 3）利用摇瓶降解实验，并辅以细胞脂肪酶测定实验，筛选出6株降解润滑油优势菌种，尽管对6株菌的脂肪酶含量检测发现其中2株菌的脂肪酶含量较低，但在反复的摇瓶降解实验中证实了这2株菌确有较为突出的润滑油降解能力，因此，细胞脂肪酶测定只能作为评价菌种降解润滑油能力的一个参考依据，即有部分菌种代谢产生脂肪酶能力较弱，但不意味着其润滑油生物降解特性一定很差； 4）实验采用了国标和国际流行方法对脂肪酶活力进行了检测，结果表明氢氧化钠滴定法不适合于本实验体系，表现在对各株菌的检测结果均较低且酶活力基本都保持一致，而利用紫外分光光度法成功地检测了细胞的脂肪酶活力； 5）通过对6株菌的生物降解特性研究发现，6号菌种在600mg/L以上的润滑油浓度中不能生长，但在500mg/L的浓度下，能够实现较高的降解效果。4，5号菌种在700mg/L的润滑油中也无法生长，但在600mg/L浓度以下时，均具有高效的降解效果。除6号菌种外，其他5支菌种在144小时的降解条件下，能够实现对浓度为500mg/L的润滑油的完全降解。而6号菌种也能达到80%左右的降解效率。
[Abstract]:Lubricating oil is widely used in industrial and agricultural production. The recovery and utilization rate of lubricating oil is low due to the lack of smooth recovery channels, immature recovery technology and high recovery cost. At the same time, due to unreasonable production process and improper operation and other external factors leading to a large number of lubricating oil into the environment. This kind of non-renewable lubricating oil contains non-degradable components such as cycloalkanes, aromatics and heavy metals, which will cause environmental pollution if not treated effectively. Although the physicochemical method can obtain certain effect in a short time when dealing with the pollution of waste lubricating oil, the secondary pollution is serious. The biological treatment of waste lubricating oil is based on the essential properties of microorganisms, I. e., biodegradation, which uses toxic pollutants as the substrate for thorough degradation. As the core of biological treatment technology, the development of high-efficiency biodegradable bacteria is the focus of research on the treatment of lubricating oil pollution. The purpose of this study was to screen high efficient waste lubricating oil bacteria with independent intellectual property rights from soil. Using waste lubricating oil and oil-contaminated soil as the source of separation, the research group firstly pretreated and acclimated the latter, and then based on the traditional microbial separation and purification technology. A total of 51 single colonies were isolated from waste lubricating oil and oil-contaminated soil, and 15 strains of degrading bacteria were obtained by 24 hole plate test. After that, six strains of high-efficiency lube oil degrading bacteria were selected by shaking flask degradation experiment and lipase determination. Finally, the biodegradation ability of lubricating oil was studied on the basis of these strains. The results showed that all the 6 strains had good oil tolerance and could be effectively degraded with lubricating oil as the sole carbon source. The conclusions are as follows: 1) waste lubricating oil and lubricating oil contaminated soil for a long time contain high efficient petroleum hydrocarbon degrading bacteria, which is the ideal source of excellent bacteria, 2) using 24 hole plate experiment, The biodegradability of lubricating oil was evaluated qualitatively by using cell culture medium and stationary cells as inoculum, and 15 target strains were screened by this method. The variation of oil film was consistent with that of cell growth in the culture medium. 3) by using shaking flask degradation test and cell lipase assay, 6 strains of dominant bacteria were selected. Although the lipase content of 6 strains was lower than that of 2 strains, it was proved by repeated shaking flask degradation experiments that the two strains had outstanding lube oil degradation ability. The determination of cellular lipase can only be used as a reference for evaluating the ability of bacteria to degrade lubricating oil, that is, the ability of some strains to produce lipase is weak, but it does not mean that the biodegradability of lubricating oil must be very poor. 4) the activity of lipase was detected by national standard and international popular method. The results showed that sodium hydroxide titration was not suitable for the experiment system. The results showed that the detection results of each strain were low and the enzyme activity was basically the same, while the lipase activity of the cells was successfully detected by ultraviolet spectrophotometry. 5) the biodegradation characteristics of 6 strains of bacteria were studied. It was found that strain 6 could not grow in the concentration of lubricating oil above 600mg/L, but at the concentration of 500mg/L. Strain 4 and 5 could not grow in the lubricating oil of 700mg/L, but when the concentration of 600mg/L was below the concentration, they all had high degradation effect. With the exception of strain No. 6, the other five strains could completely degrade the lubricating oil with concentration of 500mg/L under the condition of 144-hour degradation. The degradation efficiency of strain 6 can reach about 80%.
【学位授予单位】：重庆工商大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：X172
本文编号：2262657
[Abstract]:Lubricating oil is widely used in industrial and agricultural production. The recovery and utilization rate of lubricating oil is low due to the lack of smooth recovery channels, immature recovery technology and high recovery cost. At the same time, due to unreasonable production process and improper operation and other external factors leading to a large number of lubricating oil into the environment. This kind of non-renewable lubricating oil contains non-degradable components such as cycloalkanes, aromatics and heavy metals, which will cause environmental pollution if not treated effectively. Although the physicochemical method can obtain certain effect in a short time when dealing with the pollution of waste lubricating oil, the secondary pollution is serious. The biological treatment of waste lubricating oil is based on the essential properties of microorganisms, I. e., biodegradation, which uses toxic pollutants as the substrate for thorough degradation. As the core of biological treatment technology, the development of high-efficiency biodegradable bacteria is the focus of research on the treatment of lubricating oil pollution. The purpose of this study was to screen high efficient waste lubricating oil bacteria with independent intellectual property rights from soil. Using waste lubricating oil and oil-contaminated soil as the source of separation, the research group firstly pretreated and acclimated the latter, and then based on the traditional microbial separation and purification technology. A total of 51 single colonies were isolated from waste lubricating oil and oil-contaminated soil, and 15 strains of degrading bacteria were obtained by 24 hole plate test. After that, six strains of high-efficiency lube oil degrading bacteria were selected by shaking flask degradation experiment and lipase determination. Finally, the biodegradation ability of lubricating oil was studied on the basis of these strains. The results showed that all the 6 strains had good oil tolerance and could be effectively degraded with lubricating oil as the sole carbon source. The conclusions are as follows: 1) waste lubricating oil and lubricating oil contaminated soil for a long time contain high efficient petroleum hydrocarbon degrading bacteria, which is the ideal source of excellent bacteria, 2) using 24 hole plate experiment, The biodegradability of lubricating oil was evaluated qualitatively by using cell culture medium and stationary cells as inoculum, and 15 target strains were screened by this method. The variation of oil film was consistent with that of cell growth in the culture medium. 3) by using shaking flask degradation test and cell lipase assay, 6 strains of dominant bacteria were selected. Although the lipase content of 6 strains was lower than that of 2 strains, it was proved by repeated shaking flask degradation experiments that the two strains had outstanding lube oil degradation ability. The determination of cellular lipase can only be used as a reference for evaluating the ability of bacteria to degrade lubricating oil, that is, the ability of some strains to produce lipase is weak, but it does not mean that the biodegradability of lubricating oil must be very poor. 4) the activity of lipase was detected by national standard and international popular method. The results showed that sodium hydroxide titration was not suitable for the experiment system. The results showed that the detection results of each strain were low and the enzyme activity was basically the same, while the lipase activity of the cells was successfully detected by ultraviolet spectrophotometry. 5) the biodegradation characteristics of 6 strains of bacteria were studied. It was found that strain 6 could not grow in the concentration of lubricating oil above 600mg/L, but at the concentration of 500mg/L. Strain 4 and 5 could not grow in the lubricating oil of 700mg/L, but when the concentration of 600mg/L was below the concentration, they all had high degradation effect. With the exception of strain No. 6, the other five strains could completely degrade the lubricating oil with concentration of 500mg/L under the condition of 144-hour degradation. The degradation efficiency of strain 6 can reach about 80%.
【学位授予单位】：重庆工商大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：X172
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