藻—菌体系降解原油性能及其体系生物多态性的研究
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摘要
石油污染导致了世界性的严重的生态问题,对环境以及人类健康造成了巨大的危害,石油污染生物修复技术已经成为人们研究的热点。针对目前水体石油污染生物修复技术中存在实际问题,如降解菌剂在修复环境中存活率低、溶解氧限制等。本研究利用藻-菌体系降解石油污染物,拟进一步提高水体石油污染生物修复技术的修复效率和实际可应用性。
用平板分离法从石油污染的港口水体中得到两株带菌单种藻,经鉴定为颤藻(Oscillatoriales GH1)和斜生栅藻(Scenedesmus obliqnuus GH2)。两株微藻均有较好的原油耐受性能,能在0.1-1%体积浓度原油培养基中生长良好。
紫外分光光度法测定单种藻原油降解性能结果表明:单种颤藻具备高效降解原油的性能,而单种栅藻对原油降解效果远不及单种颤藻。因而将栅藻GH2再与三种石油组分降解菌进行人工藻-菌体系的构建:单种栅藻的附生菌与石油组分降解菌不能很好的相互适应,使得降解率反而降低;纯栅藻能与三种石油组分降解菌构建一个高效降解原油的人工藻-菌体系。
柱层析法-GC/MS对藻-菌体系降解原油的过程进行分析:单种颤藻(颤藻GH1-附生菌体系)4天能完全去除直链烷烃,5天能将烷基环己烷系列同系物全部降解,7天可去除所有的烷基苯系列同系物。7天对多环芳烃物质中萘系列、芴系列、菲系列物质总体降解率分别达98%、85%和80%。多环芳烃的降解基本遵循低环的和取代基少的物质先被降解,高环的和取代基多的物质后降解的规律。人工藻-菌体系4天能基本去除所有直链烷烃,10天则能将支链烷烃也基本除去;烷基环己烷和烷基苯系列同系物也在7天内被完全去除;对多环芳烃物质中萘系列、芴系列、菲系列物质总体降解率分别达90%、76%和70%。菲等高环芳烃能与低环物质同时被降解,降解过程中检测到2-甲基-1丙基萘、1-丙基萘、1-苄基-3甲基苯、二苯甲烷等与已知降解途径相异的中间产物。
用分子生物学方法对藻-菌体系降解原油的作用机理进行研究:颤藻-附生菌体系中有7种附生菌,包括2种鞘氨醇单胞菌Sphingomonas、1种根瘤菌Rhizobium、1种水单胞菌Aquimonas、1种黄杆菌Flavobacteriaceae以及2种不可培养细菌。根瘤菌、水单胞菌和C5不可培养细菌在烷烃、烷基环己烷、烷基苯的降解过程中一直呈优势菌;当鞘氨醇单胞菌和黄杆菌大量生长时,多环芳烃中高分子物质大量被降解。人工藻-菌体系中,DGGE显示三种石油组份降解菌的生长与原油降解存在密切相关性。GS3C和GP3A在培养初期即大量生长,使得长链烷基类物质从第2天就开始被大量降解;芳烃物质的降解使得GY2B在第4天条带陡然增亮。同时,GY2B与GS3C和GP3的共代谢作用,使得长链烷基苯物质迅速降解。
Oil pollution can lead to serious world-wide ecological problems, and make great threat to the environment and human health. There are considerable interests in microbial degradation and detoxification of these pollutants. Currently, there are some problems in traditional bioremediation technology, such as the degrading-bacteria couldn’t survival in environment, insufficiency of dissolved oxygen, and so on. In this study, microalgal-bacterial consortia were used for crude oil degradation, with the aim to improve the feasibility of bioremediation technology for oil polluted water.
Two non-axenic microalgae (unialgal culture) were isolated from oil-polluted port water, including one filamentous cyanobacteria named GH1 and one green algae named GH2. GH1 was identified as Oscillatoriales and GH2 was indentified as Scenedesmus obliqnus. Both two algae tolerance for oil, and grew well in medium contain 0.1-1% crude oil. Degradation properties of unialgal culture were tested by UV spectrophotometry and results showed the unialgal GH1 showed high oil degradation efficiency, but unialgal GH2 inferior to GH1. Thus, three oil component degrading bacteria were used for artificial consortia construction with GH2. Unialgal GH2 was not suitable for the consortium construction, axenic Scenedesmus obliquus GH2 combined with the bacteria formed an optimal algal-bacterial consortium.
Degradation process was analyzed by GC/MS. Consortium of Oscillatoriales GH1 and associated bacteria could eliminate n-alkanes, alkylcycloalkanes and alkylbenzens in 4 days, 5 days and 7 days, respectively. The degradation rates for alkylated naphthalenes, alkylated fluorenes and alkylated phenanthrenes were achieved 98%, 85% and 80% in 7 days, respectively. The orders of degradation were basically follow the regular pattern that the multimethyl tricyclic PAHs are more biorefractory. The artificial consortium (axenic Scenedesmus obliquus GH2- oil component-degrading bacteria) could eliminate n-alkanes and branch- alkanes in 4days and 10days, respectively. Alkylcycloalkanes and alkylbenzens also completely removed in 7days. The degradation rates for alkylated naphthalenes, alkylated fluorenes and alkylated phenanthrenes were achieved 90%, 76% and 70%, respectively. Higher melocular PAHs were degraded with low melocular materials simultaneously by the consortium, and some intermediates were distinct with the known biodegradation pathway.
Molecular biology method was used to analyze the mechanism of oil degradation by miocroalgal-bacterial consortium. There were seven kinds of associated bacteria in unialgal GH1, including two Sphingomonas, one Rhizobium, one Aquimonas, one Flavobacteriaceae and two unculturable bacteria. Rhizobium, Aquimonas, and C5 unculturable bacteria were dominating through the degradation process of alkanes, alkylcycloalkanes and alkylbenzens. The massive growth of Sphingomonas and Flavobacteriaceae leads to abundant decrease of high melocular PAHs. In the artificial consortium, microorganisms’population dynamics showed the degradation process closely correlated with the growth of bacterial strains and their metabolic activity: GS3C and GP3A were predominated and responsible for the aliphatic hydrocarbons degradation; the extensive decrease of PAHs leaded to sharp increase of GY2B; the cooperation of GS3C and GY2B cause co-oxidation of alkylbenzenes.
用平板分离法从石油污染的港口水体中得到两株带菌单种藻,经鉴定为颤藻(Oscillatoriales GH1)和斜生栅藻(Scenedesmus obliqnuus GH2)。两株微藻均有较好的原油耐受性能,能在0.1-1%体积浓度原油培养基中生长良好。
紫外分光光度法测定单种藻原油降解性能结果表明:单种颤藻具备高效降解原油的性能,而单种栅藻对原油降解效果远不及单种颤藻。因而将栅藻GH2再与三种石油组分降解菌进行人工藻-菌体系的构建:单种栅藻的附生菌与石油组分降解菌不能很好的相互适应,使得降解率反而降低;纯栅藻能与三种石油组分降解菌构建一个高效降解原油的人工藻-菌体系。
柱层析法-GC/MS对藻-菌体系降解原油的过程进行分析:单种颤藻(颤藻GH1-附生菌体系)4天能完全去除直链烷烃,5天能将烷基环己烷系列同系物全部降解,7天可去除所有的烷基苯系列同系物。7天对多环芳烃物质中萘系列、芴系列、菲系列物质总体降解率分别达98%、85%和80%。多环芳烃的降解基本遵循低环的和取代基少的物质先被降解,高环的和取代基多的物质后降解的规律。人工藻-菌体系4天能基本去除所有直链烷烃,10天则能将支链烷烃也基本除去;烷基环己烷和烷基苯系列同系物也在7天内被完全去除;对多环芳烃物质中萘系列、芴系列、菲系列物质总体降解率分别达90%、76%和70%。菲等高环芳烃能与低环物质同时被降解,降解过程中检测到2-甲基-1丙基萘、1-丙基萘、1-苄基-3甲基苯、二苯甲烷等与已知降解途径相异的中间产物。
用分子生物学方法对藻-菌体系降解原油的作用机理进行研究:颤藻-附生菌体系中有7种附生菌,包括2种鞘氨醇单胞菌Sphingomonas、1种根瘤菌Rhizobium、1种水单胞菌Aquimonas、1种黄杆菌Flavobacteriaceae以及2种不可培养细菌。根瘤菌、水单胞菌和C5不可培养细菌在烷烃、烷基环己烷、烷基苯的降解过程中一直呈优势菌;当鞘氨醇单胞菌和黄杆菌大量生长时,多环芳烃中高分子物质大量被降解。人工藻-菌体系中,DGGE显示三种石油组份降解菌的生长与原油降解存在密切相关性。GS3C和GP3A在培养初期即大量生长,使得长链烷基类物质从第2天就开始被大量降解;芳烃物质的降解使得GY2B在第4天条带陡然增亮。同时,GY2B与GS3C和GP3的共代谢作用,使得长链烷基苯物质迅速降解。
Oil pollution can lead to serious world-wide ecological problems, and make great threat to the environment and human health. There are considerable interests in microbial degradation and detoxification of these pollutants. Currently, there are some problems in traditional bioremediation technology, such as the degrading-bacteria couldn’t survival in environment, insufficiency of dissolved oxygen, and so on. In this study, microalgal-bacterial consortia were used for crude oil degradation, with the aim to improve the feasibility of bioremediation technology for oil polluted water.
Two non-axenic microalgae (unialgal culture) were isolated from oil-polluted port water, including one filamentous cyanobacteria named GH1 and one green algae named GH2. GH1 was identified as Oscillatoriales and GH2 was indentified as Scenedesmus obliqnus. Both two algae tolerance for oil, and grew well in medium contain 0.1-1% crude oil. Degradation properties of unialgal culture were tested by UV spectrophotometry and results showed the unialgal GH1 showed high oil degradation efficiency, but unialgal GH2 inferior to GH1. Thus, three oil component degrading bacteria were used for artificial consortia construction with GH2. Unialgal GH2 was not suitable for the consortium construction, axenic Scenedesmus obliquus GH2 combined with the bacteria formed an optimal algal-bacterial consortium.
Degradation process was analyzed by GC/MS. Consortium of Oscillatoriales GH1 and associated bacteria could eliminate n-alkanes, alkylcycloalkanes and alkylbenzens in 4 days, 5 days and 7 days, respectively. The degradation rates for alkylated naphthalenes, alkylated fluorenes and alkylated phenanthrenes were achieved 98%, 85% and 80% in 7 days, respectively. The orders of degradation were basically follow the regular pattern that the multimethyl tricyclic PAHs are more biorefractory. The artificial consortium (axenic Scenedesmus obliquus GH2- oil component-degrading bacteria) could eliminate n-alkanes and branch- alkanes in 4days and 10days, respectively. Alkylcycloalkanes and alkylbenzens also completely removed in 7days. The degradation rates for alkylated naphthalenes, alkylated fluorenes and alkylated phenanthrenes were achieved 90%, 76% and 70%, respectively. Higher melocular PAHs were degraded with low melocular materials simultaneously by the consortium, and some intermediates were distinct with the known biodegradation pathway.
Molecular biology method was used to analyze the mechanism of oil degradation by miocroalgal-bacterial consortium. There were seven kinds of associated bacteria in unialgal GH1, including two Sphingomonas, one Rhizobium, one Aquimonas, one Flavobacteriaceae and two unculturable bacteria. Rhizobium, Aquimonas, and C5 unculturable bacteria were dominating through the degradation process of alkanes, alkylcycloalkanes and alkylbenzens. The massive growth of Sphingomonas and Flavobacteriaceae leads to abundant decrease of high melocular PAHs. In the artificial consortium, microorganisms’population dynamics showed the degradation process closely correlated with the growth of bacterial strains and their metabolic activity: GS3C and GP3A were predominated and responsible for the aliphatic hydrocarbons degradation; the extensive decrease of PAHs leaded to sharp increase of GY2B; the cooperation of GS3C and GY2B cause co-oxidation of alkylbenzenes.
引文
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