【作者】 刘击；

【导师】 闵航；

【作者基本信息】 浙江大学 ， 微生物学， 2007， 博士

【摘要】 作为一种废弃物资源化技术,堆肥及其产品广泛应用于农、林业生产,而且日益显示出在污染土壤的修复中具有巨大潜力。本文主要对利用堆肥进行污染环境的修复等相关问题进行了较为系统的研究。文中详细分析了城市生活垃圾堆肥化过程,对比研究了城市生活垃圾堆肥（CMSW）和蘑菇堆肥（SMC）对多环芳烃菲和三环唑污染土壤的修复效果,初步研究了堆肥修复污染土壤的动力学过程、污染物相互作用等实际问题,并应用修复样品中GST和C23O酶活性及其基因变化情况评价了修复过程的演化。主要结果如下:1.对好氧堆肥过程中各种物理化学参数、指标酶活性以及微生物数量和群落结构的研究表明,堆肥过程中生物酶活性和微生物群落结构、数量随堆肥温度变化而改变。进入稳定期后,堆肥中物质组成趋于稳定、总体代谢活性及各种酶活性达到极大值,微生物种类、数量显著高于土壤。此时堆肥可以用于污染土壤的生物修复。2.利用CMSW和SMC对不同浓度菲及三环唑污染土壤进行修复。结果表明,2种堆肥均能消除菲降解的延滞期、提高菲降解的速度,促进菲在土壤中的降解过程。对于极性较强的污染物三环唑（pKa=1.4）,其在土壤以及土壤/CMSW混合物中没有明显降解。而土壤/SMC体系中,不同初始浓度的三环唑降解率平均提高15%,且其降解曲线均为直线。相比于其它堆肥,SMC更适合于各种有机污染物污染土壤的修复。另外,三环唑降解速率随其初始浓度提高而减小,推测高浓度的三环唑可抑制相应降解微生物的生长和活性。3.三环唑在较低浓度(30 mg L^-1)时可对菲降解菌株ZX4的生长产生抑制。随着三环唑浓度上升,菲降解微生物生长延滞期增长,生长速率和生长量显著降低。当其浓度增至60 mg L^-1时,能显著抑制各种菲降解微生物的生长。对ZX4和M1中菲降解关键酶分析表明,三环唑能对菲降解初始关键酶（菲双加氧酶）活性产生显著的抑制,这种抑制作用极有可能是由结构类似分子之间的相互竞争所引起。因而加入60 mg kg^-1三环唑后,菲污染士壤中PAH降解微生物生长延滞期增加了7d,且生长速度显著降低。而土壤/SMC体系中,三环唑并未对降解微生物生长产生明显的影响。三环唑能显著抑制土壤中菲的降解过程。含有60 mg kg^-1三环唑的土壤中,菲降解延滞期增至14d,随后菲浓度呈直线下降。而对照土壤中,菲浓度在前7d内无显著改变,随后呈指数降低。土壤/SMC体系中,菲在对照和复合污染样品中均呈指数降解的趋势,其降解曲线基本吻合。且与培养基利土壤中不同,土壤/SMC体系中三环唑对菲降解未产生显著影响。三环唑对菲降解的抑制作用随环境中的微生物多样性增加而减小,同时各污染物的降解速度与微生物多样性之间表现为一定的正相关性。表明微生物多样性的增加能降低污染物之间的相互作用,提高污染物的降解速度。4.对比过氧化氢酶、多酚氧化酶、呼吸强度以及PAH降解微生物数量可见,SMC能显著增加土壤中各种生物活性,而对PAH降解微生物数量没有显著影响。另外,土壤及土壤/SMC体系中PAH降解微生物数量受环境中污染物种类和浓度的显著影响。过氧化氢酶、多酚氧化酶和呼吸强度随污染物不同浓度而有所差异,但并不显著。对菲和三环唑吸附量的研究表明,SMC仅在一定程度上增加土壤中的菲和三环唑的吸附比例,并未产生显著的影响。5.通过MPN-PCR对C23O基因的研究表明,菲污染样品中C23O变化趋势与PAH降解微生物数量具有显著的正相关性（R²=0.871,p＜0.01）,其能很好地反映样品中菲整体的降解情况。单独添加三环唑未对样品中C23O基因数量产生显著影响。对细菌群落结构的DGGE分析表明,细菌种群主要受菲浓度影响。三环唑对细菌没有明显毒性,并不会对环境中的细菌群落结构、多样性产生明显的负面效应。真菌群落多样性分析表明,菲和三环唑加入后,体系中真菌群落结构未发生迅速改变。随时间的延长,真菌群落结构随污染物种类及其降解情况而有所差异。加入SMC后,土壤中具有菲降解能力的真菌种类有所增加,但真菌种群多样性并未随SMC加入而有显著改变。6.谷胱甘肽S转移酶（GST）广泛存在于各种真核、原核生物之中,参与细胞内各种异源物质的代谢过程。在添加菲、三环唑的土壤及土壤/SMC中,污染物种类、浓度并未对GST含量产生显著影响,样品中GST基因种类以及GST含量主要随各污染物降解过程、趋势的不同发生相应变化。结合污染物在样品中的降解过程可见,污染物的降解能刺激GST基因种类及其表达量的增加。因此,GST含量变化可以看作是对环境中污染物整体代谢状况的一个反映。更多还原

【Abstract】 This thesis mainly focuses on the application of the produced compost to remediate the phenanthrene- and/or tricyclazole- contaminated soils as well as variations of microbial community structure, some enzyme activities during the process of remediation, and the kinetics based on the experimental data. The results obtained are shown as in follows:1. In this study, the process parameters, activities of characterized enzymes, shift in microbial population and community structure were investigated during composting the stimulated municipal solid waste （MSW）. The results showed that the temperature was the dominant factor to lead the composting during the whole compost process, and and it showed a noticeable influenced on the enzymatic activity and microbial communities. At the stable stage of compost process, the catabolic activity, enzyme activity and microbial population and diversity was got a prominent improvement, as which was high enough to perform remediation of contaminated soil.2. Composted Municipal Solid Waste （CMSW） and Spent Mushroom Compost （SMC） were taken as additives for remediating phenanthrene- and tricyclazole- polluted soil. The HPLC analysis on concentration of pollutants demonstrated that the CMSW and SMC both could improve the degradation process of phenanthrene in soil by shortening the lag phase, increasing the degrading rate, while SMC resulted in a more rapid biodegradation of phenanthrene with different initial concentrations, compared with CMSW.A less than 18% of the added tricyclazole was removed within 56 days in soil and soil/CMSW mixture, whereas an increased removal rate to 33% in soil/SMC was recorded, and the removal rate was proved to be negatively related with the initial concentration of tricyclazole. It might be due to the enhanced toxicity of added pollutant, which inhibited the growth and activity of corresponding degrader.3. As one of phenanthrene analogues, tricyclazole was found to inhibit the growth of phenanthrene-degrading strains ZX4 even in lower dose (30 mg L^-1) with an obviously prolonged lag phase. Further studies revealed that the influence from tricyclazole was positively related with its concentration and the growth of most of phenanthrene degraders was prominently delayed at 60 mg L" of tricyclazole. With 60 mg kg^-1 of tricyclazole, the population of PAH degrader rose slowly following a 2 weeks lag. However, tricyclazole generated no negative effect on PAH degrader in soil/SMC mixture, where the population of degrader rose quickly in similar trend as the control, without any obvious delay.Based on HPLC detection, tricyclazole was proved with a prominent inhibition effect on degradation of phenanthrene in soil with a prolonged lag phase of degradation, and phenanthrene was removed linearly but exponentially without tricyclazole.The concentration of phenanthrene in both soil/SMC remediation systems was diminished at exponential rate. It was deduced that SMC could not only get ride of the inhibition of tricyclazole on growth of PAH degrader, but dispel the influence on degradation of phenanthrene generated by tricyclazole likewise.In other way, a negative influence was observed on the degradation of tricyclazole at the presence of phenanthrene. It might due to the effect of firstly utilizing phenanthrene by microbial degrader or the enhanced toxicity of total pollutants.4. The investigation on variation of Catalase, polyphenol Oxidase, respiration activity and population of PAH degrader revealed that SMC improved the activities of many microbes and enzymes which were not significantly affected by contaminants, but produced no impact on succession of PAH degrader. Instead, the population of PAH degraders was mainly affected by the properties and concentration of the pollutants.Despite the rich humus in SMC, extraction analysis of phenanthrene and tricyclazole in SMC indicated that SMC only resulted in slight adsorption rate of both pollutants.5. As an indicator of the degradation potential of PAHs, C23O activity, determined with MPN-PCR showed a significant correlationship with PAH degraders （R²=0.871, p<0.01） and could reflect the variation of phenanthrene in both environments. On the other hand, no influence was observed via the added tricyclazole.An analysis on variation of microbial communities with DGGE implied that the microorganisms were also under control of phenanthrene, due to its enormous toxicity. In phenanthrene contaminated samples, microbial diversity was negatively correlated with the concentration of pollutants.On the contrary, fungal communities in contaminated samples did not response to the pollutants rapidly, but differed with each other according with the type and concentration of pollutants in the end of the remediation. In both soil/SMC mixtures, the populations and diversity of xenobiotic-degrading fungi were increased comparing to the soil samples, due to the addition of SMC containing abundant catabolic fungi.6. Glutathione S-transferases （GSTs） were found in most of microorganisms, with a variety of evidences about their ability on catabolism of many xenobiotics in cell. Two types of GSTs were identified by DGGE analysis, one was the related phenanthrene degradation and the other was not. Therefore, the ELISA on concentration of GST in environments might reflect the total amount of GST.The content of GSTs was not significantly affected by added pollutants either phenanthrene or tricyclazole contaminated samples, but varied with the degradation of each contaminants. In general, the content of GST could indicate the catabolism of pollutants, rather than the residue.更多还原