表面活性剂产生菌及石油烃降解菌在石油烃降解中的作用
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摘要
随着社会和经济的发展,石油对环境的污染日趋严重,治理海洋石油污染已成为环境保护的重要任务之一。生物表面活性剂由于能促进疏水性化合物的溶解,且具有高效、无毒、无二次污染等优点,是极具发展潜力的促进石油烃生物降解的生物修复剂。因此,研究生物表面活性剂的产生条件和性能,探索生物表面活性剂对石油烃类生物降解的作用,从而为生物表面活性剂促进水体石油烃污染生物修复提供理论依据和应用基础。
本论文采集汕头海域的表层沉积物样品,以原油为唯一碳源和能源进行微生物的富集培养,并从中分离纯化出24株具有一定菌落形态的菌株,经称重法测定,其中的6株菌降解原油效率大于30%。进一步对原油降解效果较好的菌株S04、S07、S09、S017进行生理生化及分子鉴定,初步确定菌株S04和S07属于Acinetobacter sp.,菌株S09属于Xanthomonas sp.,而菌株S017属于Micrococcus sp.。不同的菌株对不同的石油烃化合物有不同的降解效果,对原油降解效果最好的是S04(78.87%),对柴油和石蜡降解效果最好的是S017(分别为36.5%4和38.65%)。此外,对影响菌株S04利用原油的外界环境因子进行分析发现,在0.75%原油起始浓度、pH 8.0、30℃等培养条件下,菌株降解原油效率最高;添加表面活性物质不利于S04降解原油,特别是离子表面活性剂Tween 80和Tween 20抑制S04降解原油的作用显著。
利用血平板法和表面张力法相结合,从汕头海域表层沉积物中分离到16株生物表面活性剂产生菌。选取生物表面活性剂高效产生菌SB12为进步的实验对象,经鉴定为假单胞菌(Pseudomonas sp.)。对菌株SB12产生物表面活性剂的发酵条件进行优化,以蛋白胨10 g/L、NaCL 1 g/L、酵母提取物5g/L为培养基,在接种量为1%,pH为9.5,25℃,培养6 d,最小表面张力可达到19.77 mN/m。分析发酵液中的生物表面活性剂的特性发现:SB12产生的表面活性剂的CMC为100mg/L,此时表面张力为22.9mN/m;理化性能的研究表明:30—100℃各温度,对表面活性剂几乎没有影响,121℃高温高压处理之后表面活性剂的表面性能仍能保持原始的90%以上;在pH4～12之间基本处于稳定;0～10%的盐浓度表面活性剂的影响不大。表面活性剂提取物都具有较好的乳化活性,乳化力可以达到91%。同时排油实验也显示排油性能良好。分离纯化得到的SB12发酵产物经用Bradford法和蒽酮硫酸法(0D620nm)进行主成分分析,SDS—PAGE,TLC得到的紫色斑点和FT-IR得到的吸收光谱,最终确认SB12分泌的生物表面活性剂为肽脂。
SB12及发酵液能加速原油的分散,加快原油在水体中的扩散速度,但是进一步的降解实验表面,SB12及其发酵液只对革兰氏阳性菌309降解原油有促进作用,且效果显著,可以分别提高15.2%,16.34%,但不利于促进革兰氏阴性菌S04、S07和S017对原油的降解。进一步的生物表面活性剂对降解菌的影响实验表面,SB12发酵液会明显抑制S04、S07和S017的生长,对S09作用不明显。
With the development of society and economy, oil pollution in environment had becoming more and more serious, remediation marine oil pollution become one of the most important tasks of environmental protection. Because of surfactant could promote the dissolution of hydrophobic compounds, and had a highly efficient, non-toxic quality, no secondary pollution, and other advantages, it had great potential for biodegradation petroleum hydrocarbons. Therefore, we study on biosurfactant production conditions and biosurfactant properties, exploring the effecs of biosurfactant on the biodegradation of petroleum hydrocarbon, for promoting petroleum hydrocarbons bioremediation in water and providing a theoretical and application basis.
By enriching with crude oil as a sole carbon source, 24 bacteria were isolated from residual soil from the bay of Shantou. With preliminary degradation test, 6 of them had degradation rate over 30%; and 4 of them S04, S07, S09 and S017 were further studied on their physiological and biochemical characteristics and 16S rRNA sequences, strain S04 and S07 were identified as Acinetobacter sp., S09 was identified as Xanthomonas sp., S017 was identified as Micrococcus sp.. Degradation ability of different components of the residual oil, the results showed that different component had different degree of degradation, different bacteria have different degradation for the different component. S04 had highest rate of crude oil degradation, and the degradation rate was 78. 87%, S017 had highest degradation rates for diesel fuel and paraffine, and the degradation rates were 36. 54 and 38. 65. The biodegradation of crude oil were influenced by many exterior environmental factors. The initial concentration of crude oil at 0. 75%、pH 8.0、30℃had highest degradation. Add surfactants inhibit S04 to degraded crude oil, especially Tween 80 and Tween 20.
The main physical and chemical characteristics of SB12 were studied. The critical micelle concentration (CMC) of this biosurfactant was 50mg/L, while its surface tension was 22. 9mN/m. The biosurfactant produced by SB12 had wide rang of temperature adaptation. Surface tension did not change practically after thermally treated for 30 mins at 30-100℃, even at the higher temperature up to 121℃, and the activity can keep over 90%; the biosurfactant also had a wide adaptable pH range of 4. 0～12. 0 and 0～10% salinity had little effect to its activity. The biosurfactant produced by SB12 had good emulsify efficiency, its emulsifcaiton index was up to 91%. Oil displacement activity also was very well. After all, the biosurfactants were extracted from SB12 and purified. Determination of the mainly carbohydrate content of this biosurfactant was performed at 620 nm using the anthrone reagent method, and protein was assayed by the Bradford method. And with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), amethyst blotting of TLC and IR, it was identified as lipopeptide.
SB12 and its fermentation medium could speed up the crude oil spreading in the water phase, but the further degradation experiment indicated that SB12 and SB12 fermentation medium were only good for gram-positive bacteria S09 to degradate crude oil, significant effects can be increased by 15.2%, 16.34%, but SB12 and its fermentation medium could not promote gram-negative bacteria, S04, S07 and S017, to degradate crude oil, further study of biosurfactant on the effects of bacterias with degrading ability, show that SB12 would inhibit the growth of S04, S07 and S017, but had little effect on S09.
本论文采集汕头海域的表层沉积物样品,以原油为唯一碳源和能源进行微生物的富集培养,并从中分离纯化出24株具有一定菌落形态的菌株,经称重法测定,其中的6株菌降解原油效率大于30%。进一步对原油降解效果较好的菌株S04、S07、S09、S017进行生理生化及分子鉴定,初步确定菌株S04和S07属于Acinetobacter sp.,菌株S09属于Xanthomonas sp.,而菌株S017属于Micrococcus sp.。不同的菌株对不同的石油烃化合物有不同的降解效果,对原油降解效果最好的是S04(78.87%),对柴油和石蜡降解效果最好的是S017(分别为36.5%4和38.65%)。此外,对影响菌株S04利用原油的外界环境因子进行分析发现,在0.75%原油起始浓度、pH 8.0、30℃等培养条件下,菌株降解原油效率最高;添加表面活性物质不利于S04降解原油,特别是离子表面活性剂Tween 80和Tween 20抑制S04降解原油的作用显著。
利用血平板法和表面张力法相结合,从汕头海域表层沉积物中分离到16株生物表面活性剂产生菌。选取生物表面活性剂高效产生菌SB12为进步的实验对象,经鉴定为假单胞菌(Pseudomonas sp.)。对菌株SB12产生物表面活性剂的发酵条件进行优化,以蛋白胨10 g/L、NaCL 1 g/L、酵母提取物5g/L为培养基,在接种量为1%,pH为9.5,25℃,培养6 d,最小表面张力可达到19.77 mN/m。分析发酵液中的生物表面活性剂的特性发现:SB12产生的表面活性剂的CMC为100mg/L,此时表面张力为22.9mN/m;理化性能的研究表明:30—100℃各温度,对表面活性剂几乎没有影响,121℃高温高压处理之后表面活性剂的表面性能仍能保持原始的90%以上;在pH4～12之间基本处于稳定;0～10%的盐浓度表面活性剂的影响不大。表面活性剂提取物都具有较好的乳化活性,乳化力可以达到91%。同时排油实验也显示排油性能良好。分离纯化得到的SB12发酵产物经用Bradford法和蒽酮硫酸法(0D620nm)进行主成分分析,SDS—PAGE,TLC得到的紫色斑点和FT-IR得到的吸收光谱,最终确认SB12分泌的生物表面活性剂为肽脂。
SB12及发酵液能加速原油的分散,加快原油在水体中的扩散速度,但是进一步的降解实验表面,SB12及其发酵液只对革兰氏阳性菌309降解原油有促进作用,且效果显著,可以分别提高15.2%,16.34%,但不利于促进革兰氏阴性菌S04、S07和S017对原油的降解。进一步的生物表面活性剂对降解菌的影响实验表面,SB12发酵液会明显抑制S04、S07和S017的生长,对S09作用不明显。
With the development of society and economy, oil pollution in environment had becoming more and more serious, remediation marine oil pollution become one of the most important tasks of environmental protection. Because of surfactant could promote the dissolution of hydrophobic compounds, and had a highly efficient, non-toxic quality, no secondary pollution, and other advantages, it had great potential for biodegradation petroleum hydrocarbons. Therefore, we study on biosurfactant production conditions and biosurfactant properties, exploring the effecs of biosurfactant on the biodegradation of petroleum hydrocarbon, for promoting petroleum hydrocarbons bioremediation in water and providing a theoretical and application basis.
By enriching with crude oil as a sole carbon source, 24 bacteria were isolated from residual soil from the bay of Shantou. With preliminary degradation test, 6 of them had degradation rate over 30%; and 4 of them S04, S07, S09 and S017 were further studied on their physiological and biochemical characteristics and 16S rRNA sequences, strain S04 and S07 were identified as Acinetobacter sp., S09 was identified as Xanthomonas sp., S017 was identified as Micrococcus sp.. Degradation ability of different components of the residual oil, the results showed that different component had different degree of degradation, different bacteria have different degradation for the different component. S04 had highest rate of crude oil degradation, and the degradation rate was 78. 87%, S017 had highest degradation rates for diesel fuel and paraffine, and the degradation rates were 36. 54 and 38. 65. The biodegradation of crude oil were influenced by many exterior environmental factors. The initial concentration of crude oil at 0. 75%、pH 8.0、30℃had highest degradation. Add surfactants inhibit S04 to degraded crude oil, especially Tween 80 and Tween 20.
The main physical and chemical characteristics of SB12 were studied. The critical micelle concentration (CMC) of this biosurfactant was 50mg/L, while its surface tension was 22. 9mN/m. The biosurfactant produced by SB12 had wide rang of temperature adaptation. Surface tension did not change practically after thermally treated for 30 mins at 30-100℃, even at the higher temperature up to 121℃, and the activity can keep over 90%; the biosurfactant also had a wide adaptable pH range of 4. 0～12. 0 and 0～10% salinity had little effect to its activity. The biosurfactant produced by SB12 had good emulsify efficiency, its emulsifcaiton index was up to 91%. Oil displacement activity also was very well. After all, the biosurfactants were extracted from SB12 and purified. Determination of the mainly carbohydrate content of this biosurfactant was performed at 620 nm using the anthrone reagent method, and protein was assayed by the Bradford method. And with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), amethyst blotting of TLC and IR, it was identified as lipopeptide.
SB12 and its fermentation medium could speed up the crude oil spreading in the water phase, but the further degradation experiment indicated that SB12 and SB12 fermentation medium were only good for gram-positive bacteria S09 to degradate crude oil, significant effects can be increased by 15.2%, 16.34%, but SB12 and its fermentation medium could not promote gram-negative bacteria, S04, S07 and S017, to degradate crude oil, further study of biosurfactant on the effects of bacterias with degrading ability, show that SB12 would inhibit the growth of S04, S07 and S017, but had little effect on S09.
引文
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