【作者】 杨丽；

【导师】 赵宇华；

【作者基本信息】 浙江大学 ， 微生物学， 2005， 硕士

【摘要】 本论文以浙江大学华家池校区农场的蔬菜大棚土为研究材料,综合运刚传统及现代分子生物学手段,采用室内培养、纯培养及统计分析相结合的方法,全面探讨了有机磷杀虫剂毒死蜱对土壤的微生物生态的毒性效应,并筛选分离到几株毒死蜱降解菌,进行了毒死蜱降解途径的初步探讨,为建立有效的有机磷杀虫剂污染预警指标体系、环境质量评价及毒死蜱降解菌的有效利用提供有益的参考。本研究所获主要结果如下: 1.以浙江大学华家池校区蔬菜大棚土为材料,采用室内培养方法,系统研究了用不同浓度的外源有机磷杀虫剂毒死蜱处理对蔬菜大棚土壤可培养微生物种群多样性、土壤生物活性以及酶活性动态变化过程的影响。研究结果表明:蔬菜大棚土壤各微生物类群和土壤酶对不同浓度的毒死蜱具有各自不同的反应。毒死蜱能刺激放线菌数量增加,但不利于好氧细菌和真菌的生长。毒死蜱刺激脱氢酶活性,抑制脲酶、磷酸酶和过氧化氢酶活性。 2.本研究从蔬菜大棚土壤中分离到三株毒死蜱降解细菌（DSP3、DSP6、DSPll）,能以毒死蜱为唯一碳源和能源生长。三株菌中,DSP3的生长和毒死蜱的降解性能最好。经鉴定,DSP3为产碱杆菌属（Alcaligenes）,在14d中对培养基中含量为100mg/L的毒死蜱的降解率可达94%。在土壤中,DSP3也具有良好的毒死蜱降解能力,并且推测DSP3和土著微生物间可以形成一种稳定的微生物群落结构。 3.DSP3菌株的最适生长温度为30℃,最适pH为8.5。培养基初始pH为8.5时,随着培养基中毒死蜱含量的增加,DSP3菌株的生长出现底物饱和现象。氮源NH₄NO₃、（NH₄）₂SO₄、NaNO₃和（NH₄）₂HPO₄对DSP3菌株生长的影响差异不人,但氮源为NH₄NO₃时毒死蜱的降解更为迅速。有机物酵母膏、蛋白胨及葡萄糖的加入在前三天,DSP3以这些有机物为碳源,延滞了毒死蜱的降解,这些碳源消耗后,再利用毒死蜱作为碳源,出现降解高峰,且都可不同程度地加快DSP3菌株对毒死蜱的降解。 4.毒死蜱降解菌株DSP3和菌株DSPll混合培养时,毒死蜱的降解速度显著加快。试验中观察到以毒死蜱为唯一碳源和磷源时DSP3菌株的降解率要高于含有无机磷源的删培养基;在MM培养基中添加适量的氮源,而不添加无机磷源也可以提高菌株DSP3的毒死蜱降解率。这反映出,在毒死蜱污染的生物治理中减少磷源,添加氮源与多种菌株混培可能是一种更为有效可行的方法。更多还原

【Abstract】 Chlorpyrifos （CP）, O, O-diethyl-O- （3, 5, 6-trichloro-2-pyridyl） phosphorothioate, a phosphorothioate insecticide, was commercially used for more than a decade, particularly to control foliar insects on cotton, paddy fields, pasture and vegetable crops. When it is applied to plants or mixed with soil, it will produce hazardous effects to the environment, especially to the ozone layer in the atmospheres. Leaching of the applied pesticide may pollute the surface /ground water, ultimately resulting in adverse effects on the biological systems. There is an increasing need to develop new methods to detect, isolate and characterize the strains playing a part in these degradation processes.The study was undertaken to look for some strains that were able to degrade chlorpyrifos. For the present work, the authors studied the effect of the bacterium DSP3 on chlorpyrifos in liquid culture and in soils, characterized the strain DSP3, and studied the ecological factors that govern chlorpyrifos degradation in soil. The mian results of the experiment are as fellows:1. It was found that influences of chlorpyrifos on the cultural microorgansisms in soil were studied based on the culture-dependent counting methods. It was found that the influences of chlorpyrifos varied on different microorganism populations in soil. The results showed that the numbers of actinomycetes were stimulated; however, aerobic bacteria and fungi were inhibited after application of chlorpyrifos. The activity of dehydrogenase was enhanced, however, urease, catalase and phosphorylase was strongly depressed.2. Three chlorpyrifos-degrading bacteria were isolated from soil samples from a vegetable plot in Huajiachi Campus of Zhejiang University, which are capable of using chlorpyrifos as sole carbon and energy sources. One of them named DSP3 was the best one on growth and chlorpyrifos degradation among the three strains. DSP3 was indentified to be belonged to Alcaligenes, with degradation rate of 94% within 14d at concentration of 100mg/kg chlorpyrifos in MM medium.3. The optimal temperature was 30°C, and the optimal pH was 8.5 for growth of DSP3 strain. As increasing chlorpyrifos content, substrate saturation appeared in medium at initial pH8.5. The effects of NH₄NO₃、（NH₄）₂SO₄、NaNO₃ and （NH4）₂HPO₄ were weaker on growth of DSP3 strain,but the degradation rate of chlorpyrifos was larger when utilized NH4NO3 as nitrogen source than utilized other nitrogen sources. Addition of yeast extraction, or peptone or glucose could promote the growth of DSP3 strain at different degree, but would arrearage chlorpyrifos degraded at first three days. When these organic compounds used up, there would be a degrading peak subsequently and all of these compounds promoted the degradation of DSP3 strain at different degree.4.Comparing to the pure culture of single DSP3 strain, the mixture of DSP3 strain and DSP11 strain had very little effective on total biomass, but could enhance the chlorpyrifos degradation rate remarkably. It could promote the degradation of DSP3 on chlorpyrifos, using chlorpyrifos as the sole source of carbon and phosphorus. DSP3 strain also could degrade chlorpyrifos more rapidly if added nitrogen source appropriately and none of phosphorus source into MM. The results showed that reducing the source of phosphorus, added the source of nitrogen and multi-strain mixture is probably more effective and feasible for bioremediation of chlorpyrifos contaminated environments.5. Some of the organophosphorus insecticides tested in the cross-feeding experiment were degraded by the isolate. All of those degraded organophosphorus insecticides have diethyl phosphorothionate side chains, which may explain the reason for their degradation. There was a phosphotriester bond in all compounds. It maybe concluded that the compounds were hydrolyzed at a phosphotriester bond. Due to its broad specificity against a range of organophosphorus compounds the isolated Alcaligenes faecalis strain possesses a great potential to be used for the remediation of highly toxic organophosphate nerve agents.更多还原