高效石油降解菌群的构建及其固定化研究
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摘要
随着石油污染的日益加重,高效无污染的生物修复技术逐渐成为治理石油污染的重要方法之一,受到广泛关注。石油是由饱和烃、芳香烃类化合物和少量的沥青质、树脂类等组成的复杂混合物,而单菌株所能代谢的石油组分通常十分有限,难以将石油从环境中彻底去除。混合菌群之间的协同作用有助于提高微生物对石油的降解能力。因此如何利用不同菌种间的相互作用,优化组合出合理、高效的降解菌群是目前的研究热点。固定化微生物技术可以使微生物高度密集并保持其生物活性,强化对石油的降解效果。
本研究把实验室已筛选得到的烷烃降解菌GS3C、菲降解菌GY2B和芘降解菌(GP3A和GP3B)进行驯化,结果表明这些菌能以原油为唯一碳源进行良好的生长繁殖,并对原油有一定的降解效果,去除率都在30%以上。经过GC-MS分析,GS3C能基本去除原油中的直链烷烃化合物, GY2B对芳烃类化合物有很好的降解效果,GP3A对C<20的中短链烷烃化合物和芳烃类化合物有一定的降解作用,GP3B对烷烃类和芳烃类化合物的降解效果一般。
利用这四种菌构建高效混合菌群,筛选出最优混合菌G8,对石油的去除率达到68.76%,比单菌株提高了近30%。经GC-MS分析, G8保留了单菌株GS3C和GY2B的降解特性,能基本去除原油中的烷烃类化合物,同时对芳烃类化合物表现出较强的降解能力,通过三种菌的共同作用增强了对原油的完全去除能力。
对混合菌G8的降解性能进行研究发现混合菌G8的最佳混合比例为1:1:1,其最适生长条件为:温度为30℃,初始pH值为7.0,接种量为4%,石油浓度为2000 mg/L,在最佳环境条件下降解5d后使石油去除率达到69.20%。
采用稻草秸秆和玉米秸秆2种载体对混合菌G8进行表面吸附固定,结果表明固定化菌对原油的去除效果比游离菌或者载体与菌液的混合物好,不灭菌的秸秆对原油的去除效果都比灭菌的好。其中稻草秸秆固定化菌的去除率达到90%～95%,玉米秸秆固定化菌的去除率为95%～98%,都比单纯的游离菌G8提高了近40%。选择不灭菌的玉米秸秆为最佳载体固定混合菌G8,对原油的去除率达到98.19%,效果最佳。
With the oil pollution becoming more and more serious, biodegradation has become an important method for controlling the oil pollution, because it is efficient and pollution-free. Oil is a complex mixture which composed of saturated hydrocarbon, aromatic compounds, some asphaltene and resin. Single strains have limited ability of metabolizing oil components, so it is difficult to completely remove the oil from the environment. The synergies of mixed strains help to improve the ability of degradation. Therefore, the research focus is how to use the interaction between different strains to build a reasonable and efficient degradation flora. Immobilized microorganism technology can enhance oil degradation by keeping high density micro-organisms and biological activity.
In this study, the alkane degrading bacteria GS3C, phenanthrene degrading bacteria GY2B and pyrene degrading bacteria (GP3A and GP3B) were domesticated by crude oil. The results show that these strains can grow and metabolize by using crude oil as sole carbon source. So they can degrade crude oil and the removal ratio reaches 30%. GC-MS analysis shows that GS3C can basically remove the straight-chain alkane compounds in crude oil. GY2B has a good degradation of the aromatic compounds. GP3A can remove the short-chain alkanes and aromatic compounds and GP3B have less degradation of alkanes and aromatic compounds.
Efficient mixed strains were constructed by using these four strains. The optimal mixed strains G8 was selected and its oil removal ratio is 68.76%, nearly 30% higher than the single strains. The GC-MS analysis indicated that G8 retained the degradation characteristics of GS3C and GY2B. It can basically remove the alkane compounds in crude oil and has a good degradation on the aromatic compounds. Through the joint of three kinds of strains, mixed strains G8 have more capacity of degrading the crude oil.
The results show the best mixing ratio of mixed bacteria G8 is GS3C:GY2B:GP3B= 1:1:1. The removal efficiency of combination G8 reached 69.20% in 5 days when the initial crude oil concentration was 2000 mg/L, which was 30% higher than that of individual bacterial strain. The optimum conditions for G8 growth were 30℃,initial pH 7, and 4% inoculation volume.
The mixed strains G8 was adsorbed and immobilized by rice straw and corn stalks. The results showed that the immobilized strains have better removal of oil than free strain or the mixture of carrier and strains and the no-sterilized straw have better removal of the oil than sterilization one. The mixed strains G8 which immobilized by rice straw was removed 90%～95% of oil. The one immobilized by corn stalk was removed 95%～98% of oil, nearly 40% higher than the one by non-immobilized G8 . The non-sterile corn stalks as the best carrier, the removal of crude oil reached 98.19 %.
本研究把实验室已筛选得到的烷烃降解菌GS3C、菲降解菌GY2B和芘降解菌(GP3A和GP3B)进行驯化,结果表明这些菌能以原油为唯一碳源进行良好的生长繁殖,并对原油有一定的降解效果,去除率都在30%以上。经过GC-MS分析,GS3C能基本去除原油中的直链烷烃化合物, GY2B对芳烃类化合物有很好的降解效果,GP3A对C<20的中短链烷烃化合物和芳烃类化合物有一定的降解作用,GP3B对烷烃类和芳烃类化合物的降解效果一般。
利用这四种菌构建高效混合菌群,筛选出最优混合菌G8,对石油的去除率达到68.76%,比单菌株提高了近30%。经GC-MS分析, G8保留了单菌株GS3C和GY2B的降解特性,能基本去除原油中的烷烃类化合物,同时对芳烃类化合物表现出较强的降解能力,通过三种菌的共同作用增强了对原油的完全去除能力。
对混合菌G8的降解性能进行研究发现混合菌G8的最佳混合比例为1:1:1,其最适生长条件为:温度为30℃,初始pH值为7.0,接种量为4%,石油浓度为2000 mg/L,在最佳环境条件下降解5d后使石油去除率达到69.20%。
采用稻草秸秆和玉米秸秆2种载体对混合菌G8进行表面吸附固定,结果表明固定化菌对原油的去除效果比游离菌或者载体与菌液的混合物好,不灭菌的秸秆对原油的去除效果都比灭菌的好。其中稻草秸秆固定化菌的去除率达到90%～95%,玉米秸秆固定化菌的去除率为95%～98%,都比单纯的游离菌G8提高了近40%。选择不灭菌的玉米秸秆为最佳载体固定混合菌G8,对原油的去除率达到98.19%,效果最佳。
With the oil pollution becoming more and more serious, biodegradation has become an important method for controlling the oil pollution, because it is efficient and pollution-free. Oil is a complex mixture which composed of saturated hydrocarbon, aromatic compounds, some asphaltene and resin. Single strains have limited ability of metabolizing oil components, so it is difficult to completely remove the oil from the environment. The synergies of mixed strains help to improve the ability of degradation. Therefore, the research focus is how to use the interaction between different strains to build a reasonable and efficient degradation flora. Immobilized microorganism technology can enhance oil degradation by keeping high density micro-organisms and biological activity.
In this study, the alkane degrading bacteria GS3C, phenanthrene degrading bacteria GY2B and pyrene degrading bacteria (GP3A and GP3B) were domesticated by crude oil. The results show that these strains can grow and metabolize by using crude oil as sole carbon source. So they can degrade crude oil and the removal ratio reaches 30%. GC-MS analysis shows that GS3C can basically remove the straight-chain alkane compounds in crude oil. GY2B has a good degradation of the aromatic compounds. GP3A can remove the short-chain alkanes and aromatic compounds and GP3B have less degradation of alkanes and aromatic compounds.
Efficient mixed strains were constructed by using these four strains. The optimal mixed strains G8 was selected and its oil removal ratio is 68.76%, nearly 30% higher than the single strains. The GC-MS analysis indicated that G8 retained the degradation characteristics of GS3C and GY2B. It can basically remove the alkane compounds in crude oil and has a good degradation on the aromatic compounds. Through the joint of three kinds of strains, mixed strains G8 have more capacity of degrading the crude oil.
The results show the best mixing ratio of mixed bacteria G8 is GS3C:GY2B:GP3B= 1:1:1. The removal efficiency of combination G8 reached 69.20% in 5 days when the initial crude oil concentration was 2000 mg/L, which was 30% higher than that of individual bacterial strain. The optimum conditions for G8 growth were 30℃,initial pH 7, and 4% inoculation volume.
The mixed strains G8 was adsorbed and immobilized by rice straw and corn stalks. The results showed that the immobilized strains have better removal of oil than free strain or the mixture of carrier and strains and the no-sterilized straw have better removal of the oil than sterilization one. The mixed strains G8 which immobilized by rice straw was removed 90%～95% of oil. The one immobilized by corn stalk was removed 95%～98% of oil, nearly 40% higher than the one by non-immobilized G8 . The non-sterile corn stalks as the best carrier, the removal of crude oil reached 98.19 %.
引文
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