【作者】 张曦；

【导师】 陈英旭； 施积炎；

【作者基本信息】 浙江大学 ， 环境工程， 2013， 硕士

【摘要】 近年来,随着我国工业化进程的迅速发展,土壤污染问题日趋严重。人们为了恢复污染土壤的生态功能,采取了一系列方法对土壤进行修复,但在评价土壤污染状况、确定修复方法和评估修复效果的过程中,如何选择合适的土壤污染评价指标成为关键。本论文主要针对浙江省台州市电子垃圾拆解区多氯联苯污染农田土壤和富阳地区铜矿冶炼厂周边重金属污染农田土壤,首先比较了两种常用土壤蛋白质提取方法,考察其用于后续研究的可行性；然后结合传统生物学手段(酶活性和微生物生物量碳)和现代分子生物学分析技术(基因组学和蛋白质组学)等方法,研究上述污染土壤中生物指标的变化。通过和常规生物评价指标相比,考察土壤蛋白质在污染土壤评价中应用的可行性,初步揭示污染物胁迫下微生物群落多样性和土壤蛋白质的变化规律,以期为蛋白质在实际污染土壤评价中的应用提供理论依据。主要结论如下：(1)柠檬酸-SDS分步提取法相对于NaOH提取法能够有效去除土壤中有机质同时获得大量蛋白质样品,是相对高效的土壤蛋白质提取方法。(2)酸性磷酸酶、蛋白酶和多酚氧化酶在PCBs污染土壤中的活性显著升高(p<0.05),且多酚氧化酶活性与PCBs浓度之间呈显著正相关(p<0.05)；土壤蛋白质在PCBs胁迫下则显著减少(p<0.05),与PCBs浓度之间呈显著负相关(p<0.01),且随着PCBs浓度升高,土壤中大分子蛋白质逐渐减少,出现大量小分子蛋白质。与多酚氧化酶相比,蛋白质与PCBs浓度之间关系更紧密,更适合作为土壤PCBs污染的指示物。(3)土壤酸性磷酸酶和脱氢酶活性在重金属胁迫下受到抑制而其中脱氢酶对重金属更加敏感,且脱氢酶活性与Cu、Zn和Pb含量之间呈显著负相关(p<0.05)；土壤微生物生物量碳和蛋白质含量与Cu、Zn、Pb和Cd含量之间呈显著负相关(p<0.05),且蛋白质在重金属轻微污染时便大幅减少。后续通过使用PCR-DGGE和SDS-PAGE等分子生物学手段分析发现,重金属胁迫下土壤细菌和放线菌群落结构变化明显,随着重金属含量增加,细菌和放线菌的Shannon指数逐渐降低,且土壤中低分子量蛋白质大量表达。通过比较酸性磷酸酶、脱氢酶、微生物量和蛋白质对重金属的响应,蛋白质更适合作为土壤重金属污染的指示物。更多还原

【Abstract】 With the development of industries in China, the issue of soil pollution is becoming increasingly serious and some measurements have been taken to recover the ecological functions of contaminated soil. There is an urgent need to select an appropriate indicator in the process of assessing soil pollution, choosing remediation methods, and evaluating the effect of remediation. Consequently, we chose soil contaminated by polychlorinated biphenyls (PCBs) and heavy metals as study objects to explore the changes of soil biological indicators under pollutants stress. Firstly we compared two soil protein extract methods that were used most to investigate their extract efficiency for following researches. Comparing with some conventional biological indicators by combining traditional biological technology (e.g. enzymes activity, microbial biomass carbon, etc.) and modern molecular biotechnology (e.g. genomics, proteomics, etc.), the potential of soil protein as an indicator for soil pollution was discussed. In order to provide theoretical basis for using protein as an indicator for natural soil contamination, we did some preliminary work about the variations of microbial community structure and soil protein under contaminant stress. The main conclusions are as follows:(1) Compared with NaOH extraction, Citrate-SDS extraction can remove soil organic matter effectively and obtain more soil protein. The Citrate-SDS extraction is a relative high efficiency extract method for soil protein.(2) Acid phosphatase, protease, and polyphenol oxidase activity increased significantly (p<0.05) in soil contaminated by PCBs. Furthermore, the polyphenol oxidase activity was significantly positively correlated (p<0.05) with PCBs concentration. On the contrast, the content of soil protein was significantly negatively correlated (p<0.01) with PCBs concentration. The protein was separated by SDS-PAGE showed that with PCBs concentration increasing, there were less large molecular weight proteins and more low molecular weight proteins. Compared with polyphenol oxidase, the relationship between protein and PCBs was more closely. Therefore, it is more reliable to be an assessment indicator for soil contaminated by PCBs.(3) Soil acid phosphatase and dehydrogenase were inhibited by heavy metals, the dehydrogenase was more sensitive to heavy metals. Dehydrogenase was significantly negatively correlated (p<0.05) with Cu, Zn and Pb. Similarly, soil microbial biomass carbon (MBC) and soil protein were significantly negatively correlated (p<0.05) with Cu, Zn, Pb and Cd. Moreover, soil protein decreased sharply once the soil slightly contaminated by heavy metals. Subsequently, the variations of soil bacteria and actinomycete community structures were studied by modern molecular biological methods (e.g. SDS-PAGE, PCR-DGGE, etc.). The microbial community structures changed significantly under heavy metals stress. With the increase of heavy metals concentration, the Shannon index decreased and there were more low molecular weight proteins in soil. Compared with the responses of acid phosphatase, dehydrogenase, MBC and protein to heavy metals, protein is more suitable to be an indicator for soil polluted by heavy metals.更多还原