【作者】 董亮；

【导师】 谢冰； 徐亚同；

【作者基本信息】 华东师范大学 ， 环境科学， 2006， 硕士

【摘要】 人工湿地中有着非常复杂的化学、物理和生物的反应和循环，这些反应是由湿地中的动植物和微生物共同参与完成的。微生物是人工湿地净化废水的主要作用者，它们把有机质作为营养源转化为组成物质和能量。本研究以上海梦清园内的芦苇人工湿地为研究对象，在水力负荷60-70cm／d条件下，芦苇湿地对苏州河水氨氮、BOD和SS的去除率分别达到30％，50％和60％左右。为了揭示微生物在湿地污染物降解中的作用，我们采用分子生物学方法，分别是ERIC-PCR、PCR-SSCP和PCR-DGGE等研究了湿地底泥中微生物的群落结构并对其中的氨氧化细菌菌群进行了初探，结合水质变化指标，分析了芦苇人工湿地污水处理系统中微生物的变化规律。主要研究结论如下： 1、在底泥DNA提取方法探索中，运用超声波、SDS、苯酚、蛋白酶K结合裂解细胞的方法，成功地将湿地底泥中的总DNA提取出来。并通过控制超声波的作用频率、时间，采用间歇式振打的方式，有效地减少DNA的破碎；大多数纯化DNA的方法都会造成纯化后DNA的严重损失，与其他方法相比，低熔点的琼脂糖方法纯化DNA的损失相对较小。 2、在用ERIC-PCR技术研究复合微生物制剂中微生物群落构成时，当扩增体系中模板DNA量在45-75ng获得了很好的扩增效果，条带清晰，且重现性好，说明菌剂十分稳定。由于环境中可培养的细菌是有限的，使得直接从菌剂中提取的DNA和纯培养后的菌剂DNA扩增图谱存在差异。 3、实验发现不同的凝胶组成、PCR产物的稀释倍数和上样量(所加PCR产物的体积)、电泳温度、电压等因素都会对SSCP电泳的结果产生影响。经过对各种凝胶和电泳条件的比较，得出了针对本实验样品的最佳优化条件：凝胶浓度10％，交联度49：1，5％的尿素，PCR产物稀释4倍，上样量为4μl，凝胶中不加甘油，电压为150V。 4、芦苇人工湿地中优势微生物主要是一些芽孢杆菌，这些菌对环境的适应能力很强，它们在污染物的处理过程发挥着一定的作用，但这些细菌的具体功能有待进一步研究。 5、PCR-SSCP和ERIC-PCR方法对芦苇人工湿地的微生物群落结构研究后得到的结果基本相同，主要结果是(1)芦苇湿地微生物DNA多样性呈现季节性和地带性变化，在夏季温度高时最高，进水口附近的多样性高于出水口，实验围隔高于对照围隔；(2)芦苇湿地各采样点的相似性都很低，各样点微生物在不同采样季节有较大的不同，相比较春夏两季，秋冬季有较高的相似性，说明芦苇人工湿地中微生物在生长繁殖时期更容易受到外界更多还原

【Abstract】 Many experiments proved that the constructed wetlands are effective in degradation of pollutants, particularly nitrogen treatment. Microorganisms played very important role in constructed wetlands. The species and quantity of the microorganism in the constructed wetland are extremely abundant, which offered enough decomposer for sewage disposal system of constructed wetland. Mengqing park bulrush wetland located beside the downstream Suzhou Creek, Shanghai, China was studied in this research. Under hydraulic loading of 60-70 cm/d, the mean removal rate of ammonia, BOD and SS could reach to about 30 %, 50% and 60% respectively in wetland. To elucidate the roles of microorganisms on pollutants degradation in bulrush wetland, molecular methods, such as ERIC-PCR, PCR-SSCP and PCR-DGGE were used to explore the microbial communities strucutrue and funcational microbacteria(ammonia-oxidizing bacteria). The conclusions were drawn as follows:1, Among DNA extracted methods, it is found that using the method combined ultrasonication, SDS, phenol with Proteinase K to lyses cells could successfully extract DNA from sediments. Moreover, the fragments of DNA could be decreased effectively by controlling ultrasonic frequency and using shaking with intermission. The purification methods all cause DNA losing. However, low melting temperature agrose was better than others.2, The ERIC-PCR fingerprints of exogenous microorganism were legibility and good reproducibility, which showed that the exogenous microorganism products were changeless. Meanwhile, we compared the fingerprints of the addition to the pure culture and DNA extracted directly from the microorganism products and found that they were quite different. The reason was that the bacteria cultivated were limited.3, Many factors, such as the gel-concentration, the dilution of the PCR products, the sample quantity of the PCR products, the electrophoresis temperature, voltage etc, can influence the results of SSCP. The optimal conditions were as followed : the gel-concentration is 10 %( acrylamide-bisacrylamide[49:1], 5% urea, no glycerol), the dilution of the PCR products is 1 : 4(total vol. 20 μl), the sample quantity of the PCR products applied in electrophoresis is 4 μl , during electrophoresis, the voltage is 150V, with constant temperature.更多还原