石油烃降解菌的驯化分离及降解条件研究
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摘要
从天津海滨潮间带取被石油烃严重污染的沉积物样品(含油量0.2 g/g),用柴油为唯一碳源,对其中的石油烃降解菌群进行逐级驯化筛选,以得到高降解能力的石油烃降解菌群。之后,采用选择培养基平板涂布法,对石油烃降解菌群进行分离纯化,得到单株细菌,并对其中两株长势好的石油烃降解菌用16s rDNA测序法进行菌种鉴定,结果表明,这两株菌分别为假单胞菌属和海旋菌属;重点研究了有机N源营养对优势菌群降解柴油的影响,试验确定了能促进生物降解的酵母膏的最适浓度;最后,分别以有机N源酵母膏和无机N源(NH4)2SO4为N源,在30d降解时间内,通过GC-MS测定和分析,对降解菌群降解柴油的情况进行了对比,并对其降解机理进行了初步分析和探讨。
实验结果如下:
(1)逐级驯化后得到的石油烃降解菌群具有广盐性,适盐浓度在1%~7%的范围内,最适盐度为3.5%,此盐度与海水盐度近似。
(2)以酵母膏作为N源,能促进生物降解的降解条件为:酵母膏8.0 g/L,柴油1%,FePO4·4H2O 6.0 mg/L,接种量2%,温度26℃,摇床转速150 rpm,自然pH。
(3)通过30d的降解对比实验,结果表明,分别以有机N源酵母膏和无机N源(NH4)2SO4为N源,对柴油的最终降解效果是相同的,但前者的降解时间比后者短10倍多。原因是酵母膏除提供氮源外,还能为细菌提供易利用碳和各种维生素及氨基酸等物质,使细菌在生物降解初期迅速生长和大量繁殖,由于生物量的增加,促使了柴油的快速降解。另外,在生物降解过程中,酵母膏代谢所产生的NH3,成为整个生物降解体系中的缓冲物,稳定降解过程中的pH,将pH维持在微生物喜好的偏碱性环境,使微生物很好地生长和繁殖。因此,与传统的无机氮源相比,酵母膏被证明是生物降解试验中的优势氮源。
以酵母膏为N源的降解实验表明,对于浓度≤1%的柴油,3d内可完全降解。
Using diesel oil as the only carbon source, and domesticated gradually, a bacterial consortium with high hydrocarbon degrading ability were obtained from oil-polluted sediments(oil content 0.2 g/g) in intertidal zone of Tianjin seashore. Several bacterial strains were isolated by viable culturing. Two of them strain H1 and strain H2 were identified by 16s rDNA sequence analysis, belonging to Pseudomonas sp. and Thalassospira sp., respectively. Through degradation experiments, we studied the effect of organic nitrogen source on biodegradation and found the optimal yeast extract concentration. By contrasting the biodegradation and GC-MS determination during a period of 30 days, using organic and inorganic nitrogen source, yeast extract and (NH4)2SO4 , respectively, we found the biodegradation was accelerated under the condition of yeast extract, simultaneously, their degrading mechanisms were also analysed and discussed.
The results of experiments as follows:
(1)The isolated hydrocarbon degrading bacteria consortium can adapt a wide salinity range from 1% to 7%. The optimal salinity is 3.5%, which is analogous to the salinity of sewater.
(2)Using yeast extract as the nitrogen source , the condition which can accelerate the biodegradation is yeast extract 8.0 g/L, diesel oil 1%(V/V), FePO4·4H2O 6.0 mg/L without adjusting the pH, inoculum quantity 2%(V/V), 26℃and 150 rpm.
(3)Through contrast experiment, we found the ultimate biodegradation efficiency are same during 30 days, but under the condition of using yeast extract the biodegradation were accelerated almost decuple than that of (NH4)2SO4. The reason is that yeast extract not only supply nitrogen source, but also offer easily accessible carbon and other nutrients to increase the biomass in the initial phase, so enhancing the microbial degradation of diesel oil. In addition, NH3 produced by metabolism of yeast extract is the buffer in the microbial degradation system, which can stabilize pH in the low alkaline environment for the full growth of bacteria. Consequently, compared with the conventional inorganic nitrogen source, yeast extract is superior.
In the study, the hydrocarbon degrading bacteria consortium can degrade diesel oil (≤1%) completely in 3 days using yeast extract as the nitrogen source.
实验结果如下:
(1)逐级驯化后得到的石油烃降解菌群具有广盐性,适盐浓度在1%~7%的范围内,最适盐度为3.5%,此盐度与海水盐度近似。
(2)以酵母膏作为N源,能促进生物降解的降解条件为:酵母膏8.0 g/L,柴油1%,FePO4·4H2O 6.0 mg/L,接种量2%,温度26℃,摇床转速150 rpm,自然pH。
(3)通过30d的降解对比实验,结果表明,分别以有机N源酵母膏和无机N源(NH4)2SO4为N源,对柴油的最终降解效果是相同的,但前者的降解时间比后者短10倍多。原因是酵母膏除提供氮源外,还能为细菌提供易利用碳和各种维生素及氨基酸等物质,使细菌在生物降解初期迅速生长和大量繁殖,由于生物量的增加,促使了柴油的快速降解。另外,在生物降解过程中,酵母膏代谢所产生的NH3,成为整个生物降解体系中的缓冲物,稳定降解过程中的pH,将pH维持在微生物喜好的偏碱性环境,使微生物很好地生长和繁殖。因此,与传统的无机氮源相比,酵母膏被证明是生物降解试验中的优势氮源。
以酵母膏为N源的降解实验表明,对于浓度≤1%的柴油,3d内可完全降解。
Using diesel oil as the only carbon source, and domesticated gradually, a bacterial consortium with high hydrocarbon degrading ability were obtained from oil-polluted sediments(oil content 0.2 g/g) in intertidal zone of Tianjin seashore. Several bacterial strains were isolated by viable culturing. Two of them strain H1 and strain H2 were identified by 16s rDNA sequence analysis, belonging to Pseudomonas sp. and Thalassospira sp., respectively. Through degradation experiments, we studied the effect of organic nitrogen source on biodegradation and found the optimal yeast extract concentration. By contrasting the biodegradation and GC-MS determination during a period of 30 days, using organic and inorganic nitrogen source, yeast extract and (NH4)2SO4 , respectively, we found the biodegradation was accelerated under the condition of yeast extract, simultaneously, their degrading mechanisms were also analysed and discussed.
The results of experiments as follows:
(1)The isolated hydrocarbon degrading bacteria consortium can adapt a wide salinity range from 1% to 7%. The optimal salinity is 3.5%, which is analogous to the salinity of sewater.
(2)Using yeast extract as the nitrogen source , the condition which can accelerate the biodegradation is yeast extract 8.0 g/L, diesel oil 1%(V/V), FePO4·4H2O 6.0 mg/L without adjusting the pH, inoculum quantity 2%(V/V), 26℃and 150 rpm.
(3)Through contrast experiment, we found the ultimate biodegradation efficiency are same during 30 days, but under the condition of using yeast extract the biodegradation were accelerated almost decuple than that of (NH4)2SO4. The reason is that yeast extract not only supply nitrogen source, but also offer easily accessible carbon and other nutrients to increase the biomass in the initial phase, so enhancing the microbial degradation of diesel oil. In addition, NH3 produced by metabolism of yeast extract is the buffer in the microbial degradation system, which can stabilize pH in the low alkaline environment for the full growth of bacteria. Consequently, compared with the conventional inorganic nitrogen source, yeast extract is superior.
In the study, the hydrocarbon degrading bacteria consortium can degrade diesel oil (≤1%) completely in 3 days using yeast extract as the nitrogen source.
引文
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