油污土中降解柴油细菌的分离鉴定及降解能力的研究
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摘要
随着石油工业迅速发展,环境也面临污染问题。柴油是目前运输工具中使用最多的燃料之一,在其加工过程中,因其不合理的处置会导致土壤污染,使土壤环境严重破坏。在此形势下,修复工作也因此而开展。其中,微生物修复由于经济、有效并对环境破坏性小等优点,近年来发展尤为迅猛。本论文从北京通州区取土样,在实验室内制备长,宽,高均为1米的砂箱装置。之后模拟地下储油罐,人为进行历时一年的柴油污染,并从中均匀分布的各点取样,运用生物富集分离方法,得到4株降解柴油菌株。最后,以此土著菌为研究对象,研究柴油污染土壤的微生物修复。通过考虑菌株五个生长环境因素(温度、pH、氮、磷源营养元素、盐度),利用已筛选出的细菌对柴油污染土壤进行修复实验研究,主要成果如下:
(1)通过直接分离、富集培养和纯化的方法,分离纯化得到4株以柴油为碳源进行生长的细菌:B-1、B-2、B-3、B-4;对其进行生理生化试验,结合16S rDNA全序列分析, 4株细菌鉴定结果为B-1为微杆菌属Microbacterium sp. B-2为短波单胞菌属Brevundimonas sp. B-3为Tetrathiobacter kashmirensis,B-4为假单胞菌属Pseudomonas sp.。其中,Tetrathiobacter kashmirensis是一株新确定的菌种,在柴油降解方面的研究还未有文献报道。B-4是典型的石油烃降解菌,已广泛用于石油烃降解。
(2)室内摇床实验筛选得出B-3、B-4的最佳生长条件:温度25℃~30℃,pH值7~7.5,最适氮源NH4Cl,最适磷源K2HPO4或KH2PO4,盐浓度0.5%~5%。
(3)两株菌对柴油的降解能力研究表明,B-3的降解能力优于B-4,柴油浓度低于0.378mg/g时,B-3的降解效率较低,浓度超过0.378mg/g后,降解效率随柴油浓度增加,B-3降解效率渐增强,达到60.98%。B-4的最适柴油浓度为0.252mg/g,其降解率为48.97%。当浓度超过这一范围,两株菌的降解率均下降。说明细菌自身有耐受范围,高浓度污染物,对细菌产生毒害,抑制微生物生长。
(4)土体系的培养基情况,了解两株菌的降解性能。其整体降解率在30℃与35℃时降解效果好,B-3优于B-4。在低温范围20℃与25℃,实验进行15天左右时,B-3随温度升高,其降解率与其呈正相关,B-4同之。因此,在实际修复工程中,在15-20天左右时,为微生物补充营养物质,使其具充足营养发挥高效降解性能。
The rapid development in the oil industry today, the environment is also faced with serious pollution problems. Diesel is one of the most used fuel in transportation. And it causes soil contamination in the course of processing, unreasonable waste disposal as well as the accident occurred, particularly underground storage tank leakage. In this situation, the repair work carried out so many. Among them, microbiology-repaired was carried out in recent years., because of the economic, efficient and environment friendly advantages. In this paper, we took the soil sample from the Tongzhou district, Beijing. Simulated underground storage tank, preparde a length, width and height were one meter sand box device, then Man-made polluted the siol from diesel to last for one year. Take the soil sample from which top-down distribution of the points, use the bio-enrichment separation methods, the four diesel-degrading bacterial strain were taken. And then, study this bacterial strains, and the combination method of indoor shaking-flask in which five factors(temperature, pH, nitrogen, phosphorus source, salinity) were considered and the indoor Shaking- flasked experiment, and the main results are as follows:
(1) The microbiology experiment was carried out to enrich, separate, screen and identify by means of molecular biology to indigenous dominant bacteria in the sand box, four diesel-degrading strains:B-1, B-2, B-3, B-4. then through morphological observation and 16S rDNA sequence analysis, the results showed these strains belong to Microbacterium sp. Brevundimonas sp., Tetrathiobacter kashmirensis, Pseudomonas sp. .This is also the first report about biodegrading diesel contaminated soil using strain B-3, and B-4 is Pseudomonas sp., a very typical of diesel-degrading bacteria. So in this paper, the study key is B-3, the B-4 as a frame of reference.
(2) The indoor shaking-flask experiment showed that the two diesel-degrading bacteria B-3 and B-4 had the optimal growth and diesel degradation conditions: initial temperature is 25℃~30℃, pH is 7~7.5, optimal nitrogen source is NH4Cl, optimal phosphorus source is K2HPO4 or KH2PO4, the salinity is 0.5%~5%.
(3) Under the optimum culture conditions, we studied degradation characteristics of strain B-3 and strain B-4. The degradation rate of strain B-3was obviously higher than that of strain B-4 for different initial diesel concentration. When the diesel fuel concentrations below 0.378mg/g, the degradation efficiency of strain B-3 is relatively low. When it more than 0.378mg/g, as the concentration increases, strain B-3 degradation efficiency gradually increase, up to 60.98%. Then strain B-4 optimal concentration in diesel is 0.252mg/g, the degradation rate is 48.97%. When the concentration exceeds this range, it has made them can not be tolerated. This two bacteria degradation rate decreased. It showed that bacteria themselves have the tolerance range of pollutants. Because of pollutants in high concentration, it would produced a toxic effect on the strains, inhibit the growth of bacterial.
(4) The indoor simulation experiments of the degrading diesel oil contaminated soil. Especially in the soil medium, understanding the degrading performance of strain B-3 and strain B-4. The overall degradation rate of bacteria at the temperature of 30℃and 35℃, the degradation effect is good. B-3 was superior to B-4. When the low-temperature range is 20℃and 25℃, the experiment lasted for 15 days, as the temperature increased, the degradation rate of strain B-3 was positively correlated with temperature. B-4 was also the same with this. Therefore, in the actual restoration project, when it lasted for 15-20 days, to provide a micro-nutrient for bacterias. So that, with adequate nutrition, micro-organisms will play a high degradation.
(1)通过直接分离、富集培养和纯化的方法,分离纯化得到4株以柴油为碳源进行生长的细菌:B-1、B-2、B-3、B-4;对其进行生理生化试验,结合16S rDNA全序列分析, 4株细菌鉴定结果为B-1为微杆菌属Microbacterium sp. B-2为短波单胞菌属Brevundimonas sp. B-3为Tetrathiobacter kashmirensis,B-4为假单胞菌属Pseudomonas sp.。其中,Tetrathiobacter kashmirensis是一株新确定的菌种,在柴油降解方面的研究还未有文献报道。B-4是典型的石油烃降解菌,已广泛用于石油烃降解。
(2)室内摇床实验筛选得出B-3、B-4的最佳生长条件:温度25℃~30℃,pH值7~7.5,最适氮源NH4Cl,最适磷源K2HPO4或KH2PO4,盐浓度0.5%~5%。
(3)两株菌对柴油的降解能力研究表明,B-3的降解能力优于B-4,柴油浓度低于0.378mg/g时,B-3的降解效率较低,浓度超过0.378mg/g后,降解效率随柴油浓度增加,B-3降解效率渐增强,达到60.98%。B-4的最适柴油浓度为0.252mg/g,其降解率为48.97%。当浓度超过这一范围,两株菌的降解率均下降。说明细菌自身有耐受范围,高浓度污染物,对细菌产生毒害,抑制微生物生长。
(4)土体系的培养基情况,了解两株菌的降解性能。其整体降解率在30℃与35℃时降解效果好,B-3优于B-4。在低温范围20℃与25℃,实验进行15天左右时,B-3随温度升高,其降解率与其呈正相关,B-4同之。因此,在实际修复工程中,在15-20天左右时,为微生物补充营养物质,使其具充足营养发挥高效降解性能。
The rapid development in the oil industry today, the environment is also faced with serious pollution problems. Diesel is one of the most used fuel in transportation. And it causes soil contamination in the course of processing, unreasonable waste disposal as well as the accident occurred, particularly underground storage tank leakage. In this situation, the repair work carried out so many. Among them, microbiology-repaired was carried out in recent years., because of the economic, efficient and environment friendly advantages. In this paper, we took the soil sample from the Tongzhou district, Beijing. Simulated underground storage tank, preparde a length, width and height were one meter sand box device, then Man-made polluted the siol from diesel to last for one year. Take the soil sample from which top-down distribution of the points, use the bio-enrichment separation methods, the four diesel-degrading bacterial strain were taken. And then, study this bacterial strains, and the combination method of indoor shaking-flask in which five factors(temperature, pH, nitrogen, phosphorus source, salinity) were considered and the indoor Shaking- flasked experiment, and the main results are as follows:
(1) The microbiology experiment was carried out to enrich, separate, screen and identify by means of molecular biology to indigenous dominant bacteria in the sand box, four diesel-degrading strains:B-1, B-2, B-3, B-4. then through morphological observation and 16S rDNA sequence analysis, the results showed these strains belong to Microbacterium sp. Brevundimonas sp., Tetrathiobacter kashmirensis, Pseudomonas sp. .This is also the first report about biodegrading diesel contaminated soil using strain B-3, and B-4 is Pseudomonas sp., a very typical of diesel-degrading bacteria. So in this paper, the study key is B-3, the B-4 as a frame of reference.
(2) The indoor shaking-flask experiment showed that the two diesel-degrading bacteria B-3 and B-4 had the optimal growth and diesel degradation conditions: initial temperature is 25℃~30℃, pH is 7~7.5, optimal nitrogen source is NH4Cl, optimal phosphorus source is K2HPO4 or KH2PO4, the salinity is 0.5%~5%.
(3) Under the optimum culture conditions, we studied degradation characteristics of strain B-3 and strain B-4. The degradation rate of strain B-3was obviously higher than that of strain B-4 for different initial diesel concentration. When the diesel fuel concentrations below 0.378mg/g, the degradation efficiency of strain B-3 is relatively low. When it more than 0.378mg/g, as the concentration increases, strain B-3 degradation efficiency gradually increase, up to 60.98%. Then strain B-4 optimal concentration in diesel is 0.252mg/g, the degradation rate is 48.97%. When the concentration exceeds this range, it has made them can not be tolerated. This two bacteria degradation rate decreased. It showed that bacteria themselves have the tolerance range of pollutants. Because of pollutants in high concentration, it would produced a toxic effect on the strains, inhibit the growth of bacterial.
(4) The indoor simulation experiments of the degrading diesel oil contaminated soil. Especially in the soil medium, understanding the degrading performance of strain B-3 and strain B-4. The overall degradation rate of bacteria at the temperature of 30℃and 35℃, the degradation effect is good. B-3 was superior to B-4. When the low-temperature range is 20℃and 25℃, the experiment lasted for 15 days, as the temperature increased, the degradation rate of strain B-3 was positively correlated with temperature. B-4 was also the same with this. Therefore, in the actual restoration project, when it lasted for 15-20 days, to provide a micro-nutrient for bacterias. So that, with adequate nutrition, micro-organisms will play a high degradation.
引文
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