高产生物表面活性剂菌株的筛选、生产条件优化及其在石油烃降解中的应用
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摘要
石油类污染因具有较高的生物毒性、在环境中持留时间长等原因,而成为环境污染修复的研究热点。环境污染的生物修复具有成本低、可原位处理、能够最大限度地降低污染物浓度和环境负面影响小等多重优势,因而非常适合我国目前的污染修复。生物表面活性剂因具有化学合成表面活性剂所无法比拟的环境兼容性而日益受到国内外的关注,同时生物表面活性剂还能促进微生物对石油烃等难溶有机污染物的利用和降解。因此,筛选高效生物表面活性剂产生菌及其用于石油烃污染的生物修复研究具有广泛的生物学和经济意义。
本论文从油污染土壤中筛选高产生物表面活性剂的菌株,对其产生物表面活性剂发酵工艺进行优化,并考察其对柴油污染土壤的生物修复能力,得到如下实验结果:
⑴从油污染环境中,经富集培养、血平板分离、摇瓶培养和排油活性测定等方法筛选到高产生物表面活性剂菌株72,依据其生长形态及理化性质特征初步鉴定为假单胞菌属,命名为Pseudomonas sp. 72。并以此为出发菌株(排油直径为8.62cm),对其进行紫外诱变,筛选到一株排油直径达11.82cm,排油直径提高了37.1%的Y72-8。
⑵Y72-8的对数生长期为产生物表面活性剂的高峰期,产生物表面活性剂机理为生长相关型,所合成的生物表面活性剂为糖脂类物质。Y72-8同时具有较好的耐酸性、耐盐性和耐温性,因而也具有了较广的应用范围。
⑶通过对Y72-8产生物表面活性剂工艺的优化,结果显示在蔗糖20g/L、硫酸铵1.6g/L、酵母膏6.4g/L、磷酸二氢钾1g/L、微量元素液2mL/L、种子液菌龄16h、接种量5%、培养温度30℃、pH7.5的条件下产生物表面活性剂最佳,产量为2.5397g/L,与优化前的1.5415g/L相比,提高了64.8%,且整个发酵周期缩短至24h。
⑷Y72-8产生物表面活性剂与其对柴油的降解能力密切相关,对柴油的摄取机制是三种模式的组合,即“单一扩散-溶解”模式、“与大油滴的直接接触”模式和“与细微油滴接触”模式。
⑸Y72-8对土壤油污环境中有良好的适应性和降解能力。在200g柴油/Kg土壤污染浓度下,接种Y72-8的新鲜土壤中,培养30d,其柴油降解率可达81.07%,接种Y72-8的灭菌土壤中柴油降解率为62.71%,混合菌群的降解效果明显高于单株培养菌。可以推测Y72-8与土著微生物间可以形成一种稳定的微生物群落结构,这一结构对土壤中柴油的生物降解具显著的优势,为进一步研究柴油降解菌群的构建提供了基础。
Because petroleum hydrocarbon contaminant has relatively high biological toxicity and withholding in environment is relatively permanent, remediation of petroleum hydrocarbon contaminated environment is becoming a research hotspot. Bioremediation of petroleum hydrocarbon contaminated environment, which exploits the ability of organisms to degrade and/or detoxify contamination, has been established as an efficient, economic, versatile, and environmentally sound treatment. Decontamination of polluted sites in our country with bioremediation has received increasing interest recently.
Compared with chemical surfactant, biosurfactant has obtained international attention because of its environment friendly and broad application potential. Meanwhile, biosurfactant had the potential for enhancing the bioavailability and biodegradation of insoluble hydrocarbons. Therefore, the works of screening of strains with high-producing biosurfactant and its application to the degradation of petroleum hydrocarbon contaminant have extensive biological and economic significance.
In this study, a strain which could highly produce biosurfactant was screened, optimized condition of biosurfactant production and investigated for their capability in bioremediation of diesel fuel contaminated soil. Finally, we obtain the following results:
(1)A strain capable of high-producing biosurfactants was isolated from petroleum hydrocarbon contaminated environment by a series of steps including hydrocarbon enrichment culture, hemolytic activity assay on blood agar plates and oil displacement activity assay etc. Identified as Pseudomonas sp. based on its morphological and physiological characteristics,named as Pseudomonas sp.72. Breeding of high-yielding strains by UV mutagenesis were based on this. A high-yield strain Y72-8 was obtained, the oil-discharge diameter of which is 11.82cm, almost 37.1% higher than that of the original strain.
(2)The strain Y72-8 released biosurfactant in its logarithmic growth period. Hence, the model of growth-correlativity of the strain Y72-8 releasing biosurfactant was proposed. The biosurfactant produced by Y72-8 was glycolipid type. Meanwhile, Y72-8 showed great stability against temperature, pH and mineralization, therefore, has a wide field of application and a good developmental prospect.
(3)The optimal medium components and growth conditions for biosurfactant production by Y72-8 were studied. The results showed that the optimal medium for biosurfactant production by this strain was 20g/L sucrose, 1.6g/L(NH4)2SO4, 6.4g/L yeast extract, 1g/L KH2PO4, 2mL/L trace element solution, culture temperature 30℃, pH 7.5, seed age 16hrs, inoculation volume 5% and 120 r/min. Under such conditions, the yield of biosurfactant was as high as 2.5397g/L within 24h, almost 64.8% higher than before which is 1.5415g/L, decreased the fermentation period conspicuously.
(4) Y72-8 releasing biosurfactant is closely related to their capability in degradation of diesel oil. Degradation of diesel oil by Pseudomonas sp.Y72-8 is combination of three models. First,single solution-diffusion model. Second, direct contact with large oil-drop model. Third, contact with tiny oil-drop model.
(5)Using Y72-8 to treat petroleum hydrocarbon contaminated soil, the results showed that Y72-8 can better adapt to environment, in 200g diesel oil/Kg soil concentration, the degradation effect was obviously, more than 81.07% of the diesel oil was degradation after 30 days in inoculated fresh soil, which the degradation rate was 62.71% in inoculated sterile soil. Mixed bacteria is more beneficial to the biodegradation of diesel oil than single strain, we can make some conclusions that: the inoculated strains coexisted possibly with indigenous microorganism, and became high effective complex microbial community. This microbial community structures has obvious advantage on the bioremediation of diesel oil contaminated soil, and to provide a basis for further study on the construction of diesel degradable flora.
本论文从油污染土壤中筛选高产生物表面活性剂的菌株,对其产生物表面活性剂发酵工艺进行优化,并考察其对柴油污染土壤的生物修复能力,得到如下实验结果:
⑴从油污染环境中,经富集培养、血平板分离、摇瓶培养和排油活性测定等方法筛选到高产生物表面活性剂菌株72,依据其生长形态及理化性质特征初步鉴定为假单胞菌属,命名为Pseudomonas sp. 72。并以此为出发菌株(排油直径为8.62cm),对其进行紫外诱变,筛选到一株排油直径达11.82cm,排油直径提高了37.1%的Y72-8。
⑵Y72-8的对数生长期为产生物表面活性剂的高峰期,产生物表面活性剂机理为生长相关型,所合成的生物表面活性剂为糖脂类物质。Y72-8同时具有较好的耐酸性、耐盐性和耐温性,因而也具有了较广的应用范围。
⑶通过对Y72-8产生物表面活性剂工艺的优化,结果显示在蔗糖20g/L、硫酸铵1.6g/L、酵母膏6.4g/L、磷酸二氢钾1g/L、微量元素液2mL/L、种子液菌龄16h、接种量5%、培养温度30℃、pH7.5的条件下产生物表面活性剂最佳,产量为2.5397g/L,与优化前的1.5415g/L相比,提高了64.8%,且整个发酵周期缩短至24h。
⑷Y72-8产生物表面活性剂与其对柴油的降解能力密切相关,对柴油的摄取机制是三种模式的组合,即“单一扩散-溶解”模式、“与大油滴的直接接触”模式和“与细微油滴接触”模式。
⑸Y72-8对土壤油污环境中有良好的适应性和降解能力。在200g柴油/Kg土壤污染浓度下,接种Y72-8的新鲜土壤中,培养30d,其柴油降解率可达81.07%,接种Y72-8的灭菌土壤中柴油降解率为62.71%,混合菌群的降解效果明显高于单株培养菌。可以推测Y72-8与土著微生物间可以形成一种稳定的微生物群落结构,这一结构对土壤中柴油的生物降解具显著的优势,为进一步研究柴油降解菌群的构建提供了基础。
Because petroleum hydrocarbon contaminant has relatively high biological toxicity and withholding in environment is relatively permanent, remediation of petroleum hydrocarbon contaminated environment is becoming a research hotspot. Bioremediation of petroleum hydrocarbon contaminated environment, which exploits the ability of organisms to degrade and/or detoxify contamination, has been established as an efficient, economic, versatile, and environmentally sound treatment. Decontamination of polluted sites in our country with bioremediation has received increasing interest recently.
Compared with chemical surfactant, biosurfactant has obtained international attention because of its environment friendly and broad application potential. Meanwhile, biosurfactant had the potential for enhancing the bioavailability and biodegradation of insoluble hydrocarbons. Therefore, the works of screening of strains with high-producing biosurfactant and its application to the degradation of petroleum hydrocarbon contaminant have extensive biological and economic significance.
In this study, a strain which could highly produce biosurfactant was screened, optimized condition of biosurfactant production and investigated for their capability in bioremediation of diesel fuel contaminated soil. Finally, we obtain the following results:
(1)A strain capable of high-producing biosurfactants was isolated from petroleum hydrocarbon contaminated environment by a series of steps including hydrocarbon enrichment culture, hemolytic activity assay on blood agar plates and oil displacement activity assay etc. Identified as Pseudomonas sp. based on its morphological and physiological characteristics,named as Pseudomonas sp.72. Breeding of high-yielding strains by UV mutagenesis were based on this. A high-yield strain Y72-8 was obtained, the oil-discharge diameter of which is 11.82cm, almost 37.1% higher than that of the original strain.
(2)The strain Y72-8 released biosurfactant in its logarithmic growth period. Hence, the model of growth-correlativity of the strain Y72-8 releasing biosurfactant was proposed. The biosurfactant produced by Y72-8 was glycolipid type. Meanwhile, Y72-8 showed great stability against temperature, pH and mineralization, therefore, has a wide field of application and a good developmental prospect.
(3)The optimal medium components and growth conditions for biosurfactant production by Y72-8 were studied. The results showed that the optimal medium for biosurfactant production by this strain was 20g/L sucrose, 1.6g/L(NH4)2SO4, 6.4g/L yeast extract, 1g/L KH2PO4, 2mL/L trace element solution, culture temperature 30℃, pH 7.5, seed age 16hrs, inoculation volume 5% and 120 r/min. Under such conditions, the yield of biosurfactant was as high as 2.5397g/L within 24h, almost 64.8% higher than before which is 1.5415g/L, decreased the fermentation period conspicuously.
(4) Y72-8 releasing biosurfactant is closely related to their capability in degradation of diesel oil. Degradation of diesel oil by Pseudomonas sp.Y72-8 is combination of three models. First,single solution-diffusion model. Second, direct contact with large oil-drop model. Third, contact with tiny oil-drop model.
(5)Using Y72-8 to treat petroleum hydrocarbon contaminated soil, the results showed that Y72-8 can better adapt to environment, in 200g diesel oil/Kg soil concentration, the degradation effect was obviously, more than 81.07% of the diesel oil was degradation after 30 days in inoculated fresh soil, which the degradation rate was 62.71% in inoculated sterile soil. Mixed bacteria is more beneficial to the biodegradation of diesel oil than single strain, we can make some conclusions that: the inoculated strains coexisted possibly with indigenous microorganism, and became high effective complex microbial community. This microbial community structures has obvious advantage on the bioremediation of diesel oil contaminated soil, and to provide a basis for further study on the construction of diesel degradable flora.
引文
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