【作者】 王亮；

【导师】 李风亭； 王磊；

【作者基本信息】 同济大学 ， 环境科学， 2007， 博士

【摘要】 近年来，由石油及其制品外泄造成的水体与土壤污染日益严重，已引起人们广泛的重视。目前，石油类污染物已成为地表水中超标的主要污染物之一，也是土壤污染的一大罪魁，并可以通过食物链进行累积，从而危及人类的生存环境。在众多治理技术中，生物技术以其高效、经济和无二次污染等诸多优点越来越受到人们的关注。机油是应用最为广泛的石油制品之一，它的主要成分是一系列长链烷烃，属于难降解的有机物，目前对于机油这一重要石油制品生物降解的研究鲜见报道。基于此，本文以一种新的筛选思路，从多处被石油的污染的土壤中筛选驯化得到对机油有较高降解能力的混合菌群，并针对此混合菌群进行了降解条件、固定化、生物表面活性物质以及群落结构的一系列研究。在对菌群进行常规分离效果不佳的情况下，运用分子生物学的手段对菌群的内部结构进行了分析对比研究。上述研究结果表明：1．混合菌群通过驯化后，对含高浓度机油废水具有较强的降解能力。在温度32℃，pH 7.2，转速190 r／min的条件下，对初始浓度约2g／L的人工含油废水，接种量为0.1％（菌体湿重g／培养液体积L），经过7d的降解，可降至403mg／L，去除率达到81.4％；对降解液溶解性有机碳（STOC）的检测结果表明，在降解过程中没有中间产物的积累，菌群对机油的降解较为彻底；2．菌群对氯离子、铜离子、SDS等限制性因素均有一定抵抗能力，但当碳源发生变化时，选择性不强的葡萄糖、甘油等会使菌群内部结构发生变化，导致对机油的降解能力下降；3．机油浓度分别为489、1075、2088 mg／L时，菌群对机油的生物降解符合Quiroga-Sales二级反应动力学模型，动力学方程为-dS／dt=k₂S²+k₁S+k₀，r²均达到0.98以上；4．经过包埋固定化后，菌群对外界环境条件变化的敏感性降低，同时对机油的去除能力也降低。吸附固定化除油效果不明显；5．菌群分泌的表面活性物质包含糖脂类和脂肽类两类，它能够将培养基表面张力从68.5 mN／m降低到35.1 mN／m，且对温度、氯离子、钙离子、pH值等具有一定的耐受能力；6．对不同培养条件下菌群样品采用PCR-DGGE手段分析发现，碳源对菌群中优势菌种更替影响较大。高盐度会刺激菌群中出现新的优势菌种。对菌群经过一段时间的连续曝气培养后，一类与摇床培养时菌群中的优势菌种除油机理不同的细菌逐渐成为优势菌种，这类细菌对烷烃的摄取机制为直接接触机制。7．从优势条带测序分析结果来看，多数匹配度较高的比对结果为不能单独培养的菌种（Uncultured），这也是进行常规平板涂布分离无法得到这些菌种的重要原因。更多还原

【Abstract】 In recent years, the pollution caused by the leakage and spill of crude oil and itsrefined products has become so serious that it has aroused the attention of people.Many oil products have become the main contaminants of surface water and soil. Assome of them can be accumulated through food chain, thus it endangershuman-beings as well. Therefore, as a more effective, economical and green method,bio-treatment technology is now gaining more concern than ever.Machine oil is one of the most widely used petroleum products, which aremainly composed of a series of long-chain alkanes. There is few report about thebiodegradation of machine oil. For this reason, the compound bacteria was screenedfrom several samples of oil-contaminated soil. The biodegradation conditions,immobilization and biosurfactant were studied. Because of the failure of theconventional separation method for the compounded-bacteria, the molecular biologymethod was used to analyze and compare the microbial community structure of thecompounded-bacteria. The results of the study are as follows:1. The machine oil removal efficiency achieved 81.4% by 7-day-biodegradationusing the compound bacteria while the initial machine oil concentration was about2000 mg/L, the biodegradation conditions were 32℃, pH 7.2, 190 r/min（rotationspeed） and inoculum-concentration was 0.1%;2. The compound bacteria have the ability to resist the chloride ions, copper ions,SDS and other restrictive matters. The machine oil removal efficiency will decrease ifthe compound bacteria has been culturing several days in the culture medium whosecarbon resource is non-selective, such as glucose, glycerin etc.3. The second-order equation-dS/dt=k₂S²+k₁S+k₀, proposed by Quirogaand Sales was applied to the experimental data obtained from the biodegradationtest（machine oil concentration of 489, 1075, 2088 mg/L）, and the correlationcoefficient values r² are all above 0.98.4. Embeding immobilization can help to resist the change of the externalcondition. The machine oil removal effectiveness of adsorption immobilization was not better than the non-adsorption unit.5. The biosurfactant secreted by the bacteria contains two kinds of matter, one isglycolipid and another is lipopeptide, which can decrease the surface tension of theculture medium from 68.5 mN/m to 35.1 mN/m and can resist the high concentrationof chloride and calcium ion and change of pH.6. The type of carbon resource of the culture medium determined the dominantstrain of the compound bacteria by using the PCR-DGGE technology. Some newstrains would be dominant under the condition of high salinity. A new kind of strainbecame dominant after a period of contious aeration culture. The machine oilbiodegradation mechanism of this kind of strain which is different from that culturedin the shake table, is direct contact mechanism.7. The sequencing results showed that most of the dominant strains were thestrains that could not be sperate cultured. This is the reason why these strains couldnot be isolated by using the spread plate method.更多还原