【作者】 熊海铮；

【导师】 邓新平；

【作者基本信息】 西南大学 ， 农药学， 2011， 硕士

【摘要】 近年来,利用微生物降解农药、处理农药污染的研究,日益被人们所重视。筛选出高效降解菌,是此研究基础和重点,而筛选方法的合理性更是能否筛选出高效降解菌的关键。本文针对传统农药降解菌筛选方法中存在的一些弊端,对农药降解菌筛选方法进行了有创新和改良。以西南大学周边普通大田土壤为研究材料,通过使用高浓度NaCl筛选方法,分离得到多株耐盐株菌株,并以我国农田中广泛使用的几类农用化学物质为研究对象,对耐盐菌株的在一定浓度农药中生长及降解特性进行了初步探讨,为降解菌筛选方法的创新,提出了有益的参考。本研究主要结果如下：1耐盐菌株的筛选通过使用含有10%浓度NaCl的LB富集培养基对土壤中微生物进行筛选,分离出22株耐盐微生物,使用用含有15%浓度NaCl的LB培养基再次对22株耐盐菌株进行复筛得到6株极度耐盐菌株(el-e6),其它16株为中度耐盐菌株(m1-m16)。2耐盐菌株的耐药性检测分别使用四种农用化学物质对其抗药性进行检测,发现在200ppm药物浓度下,22株耐盐菌在48小时内生长被抑制,但效果并不明显,其最大生长量和所需时间均与不加药相差无儿,表现出较高的耐药性。说明耐盐菌对其他有毒害物质亦有耐受力。3耐盐菌株降解率研究通过对22株耐盐菌降解率的测定发现,在50mg/L药物浓度下,22株耐盐菌对四种农用化学物质均有一定的降解效果。在对广灭灵、高效氯氰菊酯、3,4-二氯苯胺、毒死蜱的处理2d后,中等耐盐菌株平均降解率分别为10.8%、39.5%、34.0%、28.6%。高耐盐菌株分别为25.1%、76.4%、83.9%、63.6%。可看出高耐盐居住的降解能力,远高于中等耐盐菌株,说明植株耐盐性与其降解效率有一定的相关关系。4对高耐盐菌株的鉴定及降解、生长研究通过对6株耐盐菌在50mg/L药物浓度下降解及生长对照曲线,可发现其降解效率可能受到菌株生长期和药物残留量两方面的影响：1)在菌株活性最强的对数生长期,菌株降解效率最高,而随着菌株生长进入衰亡期,其降解效率逐步降低。2)在药物残留量较高时,菌株降解效率较高,而随着药物残留量的减少,其降解效率也随之降低。符合一级动力学原理。通过16srRNA检测6株高耐盐农药降解菌株鉴定结果如下：el与蜡样芽孢杆菌Bacillus cereus strain TAUC5相似性最高(>99.9%)e2、e4与地衣芽孢杆菌Bacillus licheniformis strain BPRIST006相似性最高(97.8%)e3与短小杆菌Bacillus pumilus sp. GN-6目似性最高(99.1%),e5与Uncultured Bacillus sp.相似性最高(97.9%)。e6通过鉴定与放线菌链霉菌Streptomyces sp.702(2011)相似性最高(98.6%)。更多还原

【Abstract】 In recent years, research of degradation pesticide, processing pesticide-contaminated by using microbial was attended increasingly. Selected high efficiency degradation bacterium, is the foundation and focus of this research, and rationality of the method is the key of high efficiency degradation bacterium selection.Based on the disadvantage exist in traditional pesticide degradation bacterium screening methods, this article proved the innovation and improvement for the method. This research test if it is possible by using high salt as extreme condition to found farm chemical degradation bacteria. Four chemical: Beta-cypermethrin, Chlorpyrifos,clomazone,3,4-DCA,were used as contaminant in this experiment. So these series of researches have provided references to the further works on environal-microbe applying on farm chemical, the main results are as follows:1 The selection of salt-tolerant bacteriaA total of 22 bacteria isolates from the enrichment survived and grew on the CHS(10%NaCl)medium agar plate considered to be the moderately slat-tolerant bacteria (MSTB).All the colonies can be observed clearly in 4ds incubate time on CHS medium agar plate. In the experiment of isolating extremely salt-tolerant bacteria (ESTB),only 6 moderately slat-tolerant bacteria (MSTB) survived and grew on the EHS (15%NaCl) medium.2 Tolerant of farm chemicalAs the result shown, the growth of 6 ESTB strains are different by using normal LB medium.But the optical density value of all strains rise to more than 0.7 after 12h and 1.0 after 24h incubation. Although there is passive affection of growth in initial 24h by using 4 chemical adding medium. But the optical density also can rise to about 2.0 in 48h like grown in normal LB medium. Growth of almost another 16 strains are like the same.So we can observe difference of 22 strains growth and the affection of using normal LB medium and 4 chemical adding medium, but it is consider too slight to impacting the degradation.3 Degradation of 4 agricultural chemicals by STBsThe following can be concluded from above analysis on the bio-degradation efficiency (BE) after two days of culture of the isolated bacteria.1) The ESTBs had greater BE than the MSTBs, which is true identically for four agricultural chemicals. The improved BE between the ESTBs and NSTBs was the most for 3,4- dichloroaniline (49.9%), and the least for clomazone (14.2%) among the four chemicals.2) The bio-degradation efficiency was the highest for 3,4-dichloroaniline, and the least for clomazone.3) The most effective bacteria varied among the chemicals, e5 for clomazone (BE 32.0%) and beta-cypermethrin (BE 85.8%), e6 for 3,4- dichloroaniline (BE 94.9%) and e3 for chlorpyrifos (BE 79.3%).4 The identify and degradation research of ESTBsThrough the six in ESTBs 50mg/l concentration chemical solution growth and degradation curve, found its (DE) may be affected by growth time and drug residues:1) the most efficiency degradation was observed in grown period of logarithm, and on the contrary the lowest DE was shown in decay stage 2)the positive correlation was found between the DE and chemical residues The rate of degradation is vary fit first-order kinetics. Through the 16srRNA detection 6 ESTBs appraisal result is as follows:e1 is identified as Bacillus cereus strain TAUC5 (similarity>99.9%).e2,e4 is identified as Bacillus licheniformis (similarity≈97.8%).e3 is identified as Bacillus pumilus sp. GN-6(similarity≈99.1%). e5 is identified as Uncultured Bacillus sp. partial (similarity≈97.9%).e6 is identified as Streptomyces sp.702(2011) (similarity≈98.6%)更多还原