【作者】 林爱军；

【导师】 朱鲁生；

【作者基本信息】 山东农业大学 ， 农药学， 2002， 硕士

【摘要】 环境生物技术是目前环境科学和生物科学研究的热点和重点之一，利用生物技术治理环境污染是目前污染治理的主要手段。农药是目前环境中数量最多、影响范围最广的有毒环境污染物质，给人民的生命健康带来严重的威胁，所以应该加强农药生物降解的研究，为合理开发使用农药、治理农药污染和保护生态环境提供理论依据。 本文以二甲戊乐灵为研究对象，通过富集培养的方法筛选高效农药降解微生物，包括真菌和细菌；本文研究纯培养条件下农药的生物降解，培养条件对农药降解和微生物生长的影响，优化了农药降解的微生物培养条件；本文研究了一株二甲戊乐灵高效降解细菌的生理特性，初步研究降解酶的诱导性，提取降解质粒并采用多种方法进行质粒检测。研究结果如下： 1．本文建立了培养液中二甲戊乐灵的多种提取测定方法。二甲戊乐灵测定采用气相色谱分析的方法，GC-14C型气相色谱仪(带⁶³Ni-ECD)，玻璃填充柱，固定相为3％OV-17。真菌培养液中二甲戊乐灵的提取采用分液漏斗萃取，气相色谱检测，1.0、10.0、100.0mg·L^-1三个浓度的添加回收率分别为96.67±1.98％，97.62±1.83％、97.43±2.17％；细菌培养液中二甲戊乐灵采用试管振荡提取的方法，1.0、10.0、100.0mg·L^-1三个浓度的添加回收率分别为99.88±0.55％，98.70±0.41％、98.13±0.42％，均符合农药残留分析的要求。 2．采用富集培养的方法筛选二甲戊乐灵的高效降解真菌，富集液分离得到23株真菌，降解试验表明有16株真菌在5d内对100mg·L^-1二甲戊乐灵的降解率在50％以上，10株在60％以上。根据降解能力和生理耐受能力，确定编号为YF-209、YF-212、YF-216的三株真菌为高效降解菌，经鉴定此三株真菌分别属于土生曲霉组（Aspergillus terreus）、毛连孢属（Monilochaetes）和烟色曲霉组（Aspergillusfurnigatus）。 3．采用两种不同成分的培养液富集二甲戊乐灵的降解细菌，含少量外加碳源的富集液分离获得12株细菌，但经降解试验未发现高效降解细菌，以二甲戊乐灵为唯一碳源的富集液中分离得到18株细菌，有5株细菌在3天内的降解率在60％以上，其中编号为HB-1、HB-7、WB-1的三株细菌在80％以上。将此三株细菌确定为二甲戊乐灵的高效降解细菌。HB-1、HB-7、WB-1三株细菌经鉴定分别属于假单胞菌属（Pseudomonas）、芽孢杆菌属（Bacillus）和玫瑰色微球菌属（Mirococcus luteus）。在此三株细菌中，以HB-7的降解能力最强。 4．二甲戊乐灵真菌降解特性研究表明，培养液外加碳源浓度、初始pH值、二甲戊乐灵浓度、培养温度等培养条件可以影响真菌的生长和农药降解率。研究表明在中性培养液外加碳源浓度为0.5～1.0％、二甲戊乐灵浓度100mg·L^-1、在20～30℃培养条件下此三株真菌的降解作用和生长量均最大。 5．二甲戊乐灵细菌降解特性研究表明，细菌生长量和二甲戊乐灵的降解也受培养条件的影响。通过研究各个培养条件对细菌生长和二甲戊乐灵降解的影响，表明在 林爱军：二甲戊乐灵降解微生物筛选及降解特性研究中性培养液外加碳源浓度为0．IAI．2％、H甲戊乐灵浓度100mg·L”’、在30C左右培养时此三株细菌的生长量和对二甲戊乐灵的降解作用均最大。 6．微生物对农药的生理耐受能力和降解能力有一定的关系，但并没有直接的联系，生理耐受能力高的微生物并不一定降解能力大。 7．环境中农药的降解模型有多种，在纯培养条件下微生物对农药的降解可以采用指数模型表示。 8．采用生物方法处理环境污染时，所应用的微生物要安全高效，不会引起二次污染。通过小白鼠腹腔注射和烟草过敏试验表明所获得的高效降解菌株HB－7不会引起植物和哺乳动物病变，不产生外毒素，可以应用于环境污染的治理或生物修复。 9．采用不同数量的底物诱导菌株HB－7，然后测定其降解能力，表明HB－7降解能力大小和底物性质有一定关系。分别测定了菌体和除去菌体的培养液的降解能力，发现去除去菌体后的培养液不具有降解二甲戊乐灵的能力，表明 HB刁产生的二甲 午戊乐灵降解酶是胞内酶。 10．采用琼脂糖凝胶电泳、质粒消除和转化等方法检测菌株HB－7的质粒。研究结果表明，HB－7的质粒与其降解H甲戊乐灵的能力有直接关系，该质粒易于消除，很不稳定，可以利用质粒转导的方法合成其它可以降解二甲戊乐灵的微生物，使原来没有降解能力的细菌获得降解二甲戊乐灵的能力。更多还原

【Abstract】 Now environmental biotechnology has become a popular field of environment science and biology. The biological waste treatment is the most important means of pollution control. Pesticides are the poisonous pollutants more than others and have more influences than other pollutants. So the study of pesticides degradation ought to be enhanced. Pesticide degradation in environment mainly is biodegradation by microbe. In this research paper what are mainly studied was the biodegradation of pendimethalin. Some strains of microbe which could effectively degrade pendimethalin in culture were isolated. Properties of pendimethalin biodegradation were studied and biological properties of strain HB-7 were researched. These researches can offer more extract and valid bases and evidences for the biodegradation of pesticides and bioremediation of pesticides pollution. The results could be summarized as followings:1. In this paper the extraction and analysis methods were studied. The study of extraction rate of different methods in culture solution demonstrated that pendimethalin was strongly sorbed by microorganism especially by fungi. The pendimethalin was absorbed by microbe in short time and was not degradated. So the high extraction rate and simple operation should both considered in choosing the extraction methods. The pendimethalin was detected by GC-14C gas chromatography with 63Ni-ECD. The recovery rates in fungi culture of l.O.lO.O.lOO.Omg-L’1 was 96.67?.98%, 97.62?1.83%, 97.43+2.17% respectively and 99.88 + 0.55%, 98.70+0.41%, 98.13 + 0.42% in bacterial culture. The results could meet the requirements for analysis of pendimethalin residue.2. Fungi degrading pendimethalin were isolated by enrichment from soil and sludge. 23 fungi were got. 16 fungi could degrade more than 50% pendimethalin in 5 days in culture fluid and 10 fungi could degrade more than 60%. Three fungi YF-102, YF-212, YF-216 were considered as efficiently degrading strains identified as Aspergillus terreus, Monilochaetes and Aspergillus fivnigatus.3. The pendimethalin degrading bacteria were isolated by two methods. 12 bacteria strains were got by selective enrichment from soil and active sludge with cane sugar as carbon source in addition to pendimethalin. But no efficiently degrading strain was found.18 bacteria strains were got by selective enrichment from soil and active sludge with pendimethalin as sole carbon source. 5 strains could degrade more than 60% pendimethalin in 3 days in culture fluid and 3 strains HB-1, HB-7, WB-1 could degrade more than 80% considered as efficiently degrading strains. These 3 strains were preliminarily identified as Pseudomonas^ Bacillus and Mirococcus luteus.4. The effects of culture conditions on pendimethalin by fungi were studied.The efficiency of degradation was affected by culture conditions such as pH, temperature, the concentration of cane sugar and pendimethanlin. The optimal conditions of fungi and bacteria culture were proposed.5. The properties of biodegradation of bacteria were studied. The culture conditions such as pH , temperature, the concentration of cane sugar and pendimethanlin had effects on the degradation and bacterial growth. The optimal conditions were pH7.0, 30C, 0.1-0.2% cane sugar and no more than lOOmg-L"1 pendimethanlin.6.Microbial physiological tolerance of pendimethalin had something to do with the degrading capacity. But they had no directly relation and not all of the tolerant species were efficiently degrading strains.T.Many models could be used for describing pesticide -substrate bioavailability and rates of biodegradation in environment. Among these models C=C0e~Kt was used extensively. The results showed that C=Coe"Kt could be used for describing biodegradation of pendimethalin by bacteria.8. The study of environmental safety of strain HB-7 showed that it could not contaminate environment doing no harm to tobacco and fat. So the strain could be used in bioremediation of soils contaminated with pesticides and disposal of sewage.9.The study of in更多还原