【作者】 谢慧；

【导师】 朱鲁生；

【作者基本信息】 山东农业大学 ， 土壤学， 2013， 博士

【摘要】 DDT是22种持久性污染物之一，其在土壤环境中的残留浓度依然居高不下，有关DDT残留及其风险一直是研究关注的热点之一，2008年我国环保部将DDT列入第一批“高污染、高环境风险”产品名录。如何快速消除农田土壤中的残留DDTs，是保障农产品安全和人体健康的重要问题。消除土壤中DDTs等有机污染物的有效方法通常是基于微生物降解作用的生物修复。本实验室采用富集培养法和直接培养法从农药厂的污水、污泥样品中获得数株降解细菌，通过对其降解产物的研究发现，菌株KK只能把p,p’-DDT降解为p,p’-DDE，而不能进一步降解；而部分菌株能将DDT降解为DDE，但是DDE仍然为具有毒性的环境内分泌干扰物，所以筛选到能降解DDE的菌株是本试验的目的，通过初步研究，确定DXZ9对培养液中p,p’-DDT和p,p’-DDE的降解能力分别为55.0%和39.88%。本论文在已有研究工作基础上，选用菌株DXZ9进行下一步研究，主要研究内容包括三个方面：菌株DXZ9在培养液中对p,p’-DDT和p,p’-DDE降解机理的研究；室内模拟研究了菌株DXZ9对土壤中p,p’-DDT和p,p’-DDE的降解能力；利用盆栽试验方法研究了菌株DXZ9与黑麦草对土壤中p,p’-DDT和p,p’-DDE的联合生物降解，主要研究结果如下：1.明确菌株对DDT的降解途径。本研究采用了两种有机溶剂提取方法提取降解产物，一种方法采用非极性溶剂苯进行提取，另外采用混合溶剂正己烷和乙酸乙酯提取。推断出菌株对p,p’-DDT的降解途径可能为：p,p’-DDT先降解为p,p’-DDE和p,p-DDD，然后生成中间产物9-Methylidenefluorene，最后可能分解为1,2-benzenedicarboxylic Acid,2,6-bis （1,1-dimethylethyl）-4-methyl-phenol,2-benzenedicarboxic Acid，Dodecanoic Acid和2,6-bis（1,1-dimethylethyl）-4-methyl-phenol。2.建立了适合本试验的有机氯农药残留测定方法。在该测定条件下：OV-1701（30m×0.25mm（ID）×0.25μm）；Col:150℃（保持1min）→以4℃/min升到240（保持5min）,以1.5℃/min升到270℃（保持5min），进样口：250℃；DET（ECD）：300℃；尾吹85KPa；载气80KPa。土壤样品中10种有机氯农药的色谱峰分离比较好，无明显的杂质干扰，说明选用的测定条件和样品的前处理方法比较适合土壤中这10种农药的残留测定，在该测定条件下10种有机氯农药的仪器最小检测量为10-12¹0-13g，方法的最小检测浓度在10-2¹0-3mg·kg^-1之间，灵敏度很高。六六六的四种异构体和DDT的四种异构体在土壤中的添加回收率范围在90.90%-104.4%之间，变异系数小于8.93%，完全符合农药残留分析方法要求。3.采用室内培养研究方法研究了菌株对土壤中DDT和DDE的降解作用，研究结果表明接种降解菌株能明显缩短DDTs在土壤环境中的降解半衰期。p,p’-DDT、p,p’-DDE和DDTs降解动态采用双室降解模型进行表示，由模型计算p,p’-DDT、p,p’-DDE和DDTs在自然土壤的T_1/2分别为420天、1203天和532天，添加降解菌株后T_1/2分别减少为42.5天、642天和221天，添加降解菌株能明显缩短p,p’-DDT、p,p’-DDE和DDTs在土壤环境中的降解半衰期。p,p’-DDT在第150天时，接种菌株处理和不接种菌株处理残留浓度分别为1.38mg.kg^-1和2.06mg.kg^-1，降解率分别为55.3%和33.3%；p,p’-DDE在第150天时，接种菌株处理和不接种菌株处理残留浓度分别降为1.19mg.kg^-1和1.43mg.kg^-1，降解率分别为37.0%和24.3%；到试验的第150天，DDTs土壤中残留浓度分别降低为2.67mg.kg^-1和3.60mg.kg^-1，降解率分别为45.2%和29.3%。4.在盆栽试验中，利用DGGE明确了菌株在土壤中的定殖状况。黑麦草和菌株联合修复过程中菌株的定殖状况采用DGGE电泳图谱的条带进行评价，DXZ9菌株在30天之内的处理土壤中优势较明显，条带比210天的目标条带清晰，说明菌株DXZ9在试验的初期能成为优势菌群的一种，随着时间的延长，菌株DXZ9优势度逐渐减弱。5.利用盆栽试验方法，得出联合修复处理效果最好，且菌株的作用大于黑麦草的作用，采用植物和微生物的联合修复能显著缩短DDTs在土壤环境中的降解半衰期。研究表明不同处理下土壤中p,p’-DDT和p,p’-DDE及DDTs的残留浓度变化差异很大，均表现为前期降解较快后期降解较慢的特点，根据降解率评价其降解效果看，S+G+D+B处理效果最好，对土壤中农药的降解率最高，p,p’-DDT和p,p’-DDE及DDTs降解率分别为80.7%、54.5%和69%；S+D+B处理也非常好，降解率分别为77.1%、52.4%和65.5%；S+G+D处理降解效果较好，降解率分别为72.2%、48.4%和60.8%；均比S+D处理即仅靠土著微生物降解效果要好，其降解率为30.0%、30.9%和28.8%。p,p’-DDT在S+G+D+B、S+D+B、S+G+D和S+D处理中的T_1/2分别为48.0、58.5、75.5和1055天，p,p’-DDE在四个不同处理中的T_1/2分别为162.5、229、235和745天，DDTs在四个不同处理中的T_1/2分别为71.8、86.5、114.8和1095天。通过比较不同处理土壤中农药的降解半衰期，由于接种菌株和种植黑麦草的强化降解作用，显著降低了土壤中的农药浓度。6.利用植物和微生物的联合修复能够显著减少DDTs污染土壤的毒性。土壤中p,p’-DDT和p,p’-DDE降解前后对生物的遗传毒性采用对赤子爱胜蚓体腔细胞DNA损伤程度这个指标进行评价，S+D处理对蚯蚓DNA损伤程度最大，S+G+D+B处理对蚯蚓DNA损伤程度最小，S+G+D处理对蚯蚓DNA损伤程度略大于S+D+B处理，说明接种降解菌株和植种黑麦草能显著降低土壤中污染物的毒性，结合前面的残留浓度发现，表现毒性小的处理土壤中p,p’-DDT和p,p’-DDE残留浓度亦小。210天时，S+G+D+B、S+D+B、S+G+D处理和S+D相比，土壤中污染物对生物的遗传毒性分别降低了58.1%、52%、36.9%，可见，利用植物和微生物的修复能够明显减少p,p’-DDT和p,p’-DDE污染土壤的毒性。7.明确了修复过程中对土壤微生物群落结构影响较小。黑麦草和菌株联合修复过程中，由于接种菌株和植种黑麦草及其农药的作用，土壤中微生物数量和土壤酶活性受到不同程度的影响，随着时间的延长，影响越来越小。在修复过程的这210天内，土壤中细菌的数量发生了比较大的变化，在第5天和第10天时接种菌株的处理和其他处理之前差异极其显著。利用DGGE研究表明采用植物和微生物的联合修复对土壤细菌群落结构存在一定的影响，但是随着时间的延长，影响逐渐变小。更多还原

【Abstract】 DDT （1,1,1-trichloro-2,2-bi （4-chlorophenyl） ethane） belongs to the one of the22persistent organic pollutes, high DDT levels in the soil were still being observed recently inChina in the soil, and its residue and the risk was still an issue of great contention, DDT wasclassed the first product directory with the high pollutes and high environment risk. It was theimportant thing that speedy removal the residue of DDTs in the soil for protection the safetyof agricultural products and health of the people. The effective way of removal of DDTs fromthe contaminated soils was bioremediation using the microorganism degrading. Many DDT-degrading bacteria were isolated from the sewage and sewage sludge from the pecticidefactory, it was discovered by the research of metabolite that the p,p’-DDT was degraded intop,p’-DDE by the bacterial KK and p,p’-DDE was not further degraded, but p,p’-DDE wasdegraded furthermore by the other bacteria named with DXZ9, by the preliminary research,the degrading rates of p,p’-DDT and p,p’-DDE by the DXZ9were55.0%and39.88%respectively in the medium. Basing on the research foundation, the DXZ9was chosen andfurther researched. The contents of the thesis included three aspects, the degradingmechanism of p, p’-DDT and p, p’-DDE in the medium was researched firstly; and secondly,the biodegradation of p, p’-DDT and p, p’-DDE in the soil was researched by the DXZ9in theexperiment culture; in the end, the combined biodegradation of p, p’-DDT and p,p’-DDE inthe soil was researched by the DXZ9and the ryegrass in the pot.1. The step of a proposed pathway for the biodegradation of DDT by DXZ9was confirmed.The degradation products were extracted by the different polar organic solvents, the twodifferent polar organic solvents were used, one was benzene, the other was hexane and ethylacetate, from this study we can reasonably infer that p,p’-DDT firstly degraded into p,p’-DDEand p,p’-DDD, and then translated into9-Methylidenefluorene, and in the end degraded into1,2-benzenedicarboxylic Acid,2-benzenedicarboxic Acid， Dodecanoic Acid and2,6-bis（1,1-dimethylethyl）-4-methyl-phenol。2. The residual methods of the ten organochlorine pesticides in the soil were established.The column was OV-1701（Australia） capillary column （30m length,0.25mm i.d., and0.25μm film thicknesses）. The oven temperature was programmed from an initial temperature of150℃（1.0min hold）, and to240℃at a rate of4C/min, maintained at240℃for5min, and then to270℃at a rate of1.5℃/min, maintained at270℃for5min; Injector temperatureswere maintained at250℃, Detector temperatures （ECD） were maintained at300℃; the flowpressure of make up and the carrier gas was both80kpa. The chromatograms of tenorganochlorine pesticides in the soil were separated very well without the interference fromthe impurity peak. The chromatograms of the soil samples were separated very well not onlyin the high addition level but also in the low addition level, it was indicated that the detectioncondition and pretreatment method were suitable for the ten organochlorine pesticides, at thiscondition, minimum detection quantity of the ten organochlorine pesticides was10-12¹0-13g,minimal detectable concentration of the methed was10-2¹0-3mg·kg^-1, the method is sensitiveenough to detect the presence of the low level residue of the pesticides in the soil. Recoveriesrate of the four DDT isomers and六六六isomers pesticides in the soil tested were90.90%-104.4%, and the coefficient variation （CV） was less8.93%, and it could fulfill therequirements of the high standard of the pesticides residue.3. The biodegradation of p, p’-DDT and p, p’-DDE in the soil was researched by the DXZ9in the indoor experiment, it was explicit that DDT was degraded by the DXZ9in the soil; thedegradation half-life of DDTs was reduced through inoculating strains. Double chamberdegradation model was used in the degradation dynamics of DDTs, by the model, thedegradation half-life of p,p’-DDT, p,p’-DDE and DDTs was420d、1203d and532d in thesoil without the inoculation DXZ9treatment respectively, the degradation half-life of DDTswas reduced in the soil with the inoculation DXZ9treatment, its was42.5d,642d and221drespectively. Comparing the two modes, it was obvious that the treatment with the DXZ9could reduce the half-lives of pesticides in the soil. The residual concentrations of p, p’-DDTwere1.38mg·kg^-1and2.06mg·kg^-1in the soil with the two different treatments, the degradedrate were55.3%and33.3%. The residual concentrations of p,p’-DDE were1.19mg·kg^-1and1.43mg·kg^-1in the soil with the two different treatments, the degraded rate were37.0%and24.3%. The residual concentrations of DDTs were2.67mg·kg^-1and3.60mg·kg^-1in the soilwith the two different treatments, the degraded rate were45.2%and29.3%.4. The colonization of DXZ9was confirmed by denaturing gradient gel electrophoresis（DGGE） in the pot. On the basis of the monitoring predominant bacterium using denaturinggradient gel electrophoresis （DGGE）, the colonization of DXZ9was evaluated by the band ofDGGE during the combination with ryegrass and the microorganism, DXZ9was predominantwithin the30d in the soil, the band was more distinct than the band of the210d, it is evident that DXZ9was predominant bacterium on the preliminary period of experiment, as time wenton, the dominance of DXZ9began to wear off gradually.5. It was explicit that the combined remediation was the best method by the pot experiment,and the contribution of the DXZ9was more than the ryegrass, the degradation half-life ofDDTs in the soil was reduced significantly through the combined remediation with theryegrass and the microorganism. The result showed that the residual concentration of p, p’-DDT p, p’-DDE and DDTs was significant difference in the soils with different treatments.There was the same trend that the degraded speed was very quickly in the earlier stage slowlyin the later stage, evaluating the degrading effect according to the degrading rate, thetreatment of S+G+D+B was the best method, the degrading rate of DDTs was the highest, thedegrading rate of p, p’-DDT, p, p’-DDE and DDTs were80.7%,54.5%and69%respectively.The treatment of S+D+B was better method, the degrading rate of p, p’-DDT, p, p’-DDE andDDTs were77.1%,52.4%and65.5%respectively. The treatment of S+G+D was bettermethod, the degrading rate of p, p’-DDT, p, p’-DDE and DDTs were72.2%,48.4%and60.8%respectively. The last one was the treatment with S+D, which was degraded by the indigenousmicroorganism, the concentration of p,p’-DDT, p,p’-DDE and DDTs varied very little in thetreatment with S+D during the210d experimental period, the degrading rate of p,p’-DDT,p,p’-DDE and DDTs were30.0%,30.9%and28.8%respectively. The degradation half-life ofp,p’-DDT was48.0d,58.5d,75.5d and1055d in the soil with S+G+D+B, S+D+B, S+G+Dand S+D treatment; The degradation half-life of p,p’-DDE was162.5d,229d,235d and745din the soil with the same four treatments, and the degradation half-life of DDTs was71.8d,86.5d,114.8d and1095d in the soil. Comparing the degradation half-lives of pesticides in thesoil with different treatments, we could conclude that the residual concentration of pesticidewas reduced significantly by the combination of bioremediation and phytoremediation.6. It is thus obvious that the gene toxicity of the polluted soil was reduced significantly bythe phytoremediation and microbial remediation. The gene toxicity of the metabolites of thep,p’-DDT and p,p’-DDE in the soil, which was evaluated by the coelomocytes comet olivetails moment of the earthworm （Eisenia foetida）, the gene toxicity was the maximum in thesoil with S+D treatment, the gene toxicity was minimum in the soil with S+G+D+Btreatment, the gene toxicity of treatment with S+G+D was more than treatment with S+D+B,so it could be concluded that the gene toxicity was reduced significantly by the inculcationDXZ9and cultivating ryegrass. On the210d, the gene toxicity of pollutes in the soil wasreduced58.1%、52%and36.9%comparing the treatments S+G+D+B, S+D+B, S+G+D withS+D. 7. It was explicit that the effect of the microbial community structure diversity of the soilwas less during the combination with ryegrass and the microorganism. During thecombination with ryegrass and the microorganism, under the action of the bacterial strain,ryegrass and pesticide, the microorganism amount and the activity of soil enzyme wereaffected with different degrees in the soil, and as time went on, the effect was tapering off. Inthe period of210d experiment, the bacteria amount in the soil changed very much, there wassignificant difference between the treatment with inoculated strains and treatment withoutinoculated strains, there was significance at1%level on the5d and10d. The bacteriacommunity structure was different in the different treatments during the combination withryegrass and the microorganism by DGGE, but as time went on, the effect was tapering off.更多还原