【作者】 王霞；

【导师】 薛林贵；

【作者基本信息】 兰州交通大学 ， 微生物学， 2019， 硕士

【摘要】 近年来,温室在世界各地广泛应用,温室既带来经济效益,但也存在环境污染问题。农药的不合理使用不仅会破坏土壤质量,还会污染空气、水体,甚至通过食物链进入人体,危害人体健康。噻虫胺是日光温室中经常使用的、高水溶性和低挥发性的小分子物质,且在土壤中的半衰期很长,容易浸入地表径流或地下水。此外,噻虫胺对一些非目标生物如蜜蜂,鸟类和水生无脊椎动物产生影响,因此,噻虫胺农药污染土壤的生物修复技术的研究,具有十分重要的意义。本研究以10年连作温室蔬菜大棚土壤为研究对象,以噻虫胺为目标农药,在室内土壤生态模拟条件下,研究了噻虫胺残留对土壤pH、土壤酶活性以及土壤可培养微生物数量的影响,采用富集驯化法筛选并构建复合菌系,并采用单一变量法和液相色谱串联质谱法研究了复合菌系对噻虫胺的最佳降解条件和降解途径,通过高效液相色谱法研究了噻虫胺在不同处理土壤中的残留变化及复合菌系对噻虫胺污染土壤的生物修复效果,其主要研究结果如下:不同浓度噻虫胺均对土壤pH产生抑制作用;5 mg kg^–1和20 mg kg^–1噻虫胺对蔗糖酶活性表现为促进作用,但50 mg kg^–1噻虫胺对蔗糖酶活性产生抑制作用;不同浓度噻虫胺对脲酶活性作用表现为“促进–抑制–恢复”变化趋势;低浓度的噻虫胺对过氧化氢酶活性作用表现为“抑制–促进–抑制”变化趋势,但高浓度噻虫胺对过氧化氢酶活性表现为抑制作用;对于碱性磷酸酶,不同浓度噻虫胺均表现为抑制作用;不同浓度噻虫胺对土壤细菌和真菌数量表现为先抑制后促进的作用,但对放线菌表现为强烈的抑制作用,且浓度越大,抑制作用越强。从甘肃天水蔬菜大棚土壤中分离筛选到5株噻虫胺高效降解细菌,分别鉴定为苍白杆菌（Ochrobactrum anthropic）、肠杆菌属（Enterobacter sp.）、不动杆菌属（Acinetobacter johnsonii）、单胞菌属（Pseudomonas putida）和寡养单胞菌属（Stenotrophomonas maltophilia）,为了提高菌株对噻虫胺的降解效率,本研究构建并筛选了复合菌系SCAH,该复合菌系在以噻虫胺(500 mg L^–1)为唯一碳源的无机盐培养基中,培养15 d后,其降解率达到79.3%。考察了复合菌系SCAH降解噻虫胺的特性,发现其在pH值为6.0、温度为35℃、接种量为3%时对噻虫胺的降解率最高,培养10 d后,其降解率达69.8%。通过LC-MS图谱对复合菌系SCAH降解噻虫胺的途径作出了推论,发现噻虫胺的主要代谢反应如下:（1）硝基亚氨基部分转化为尿素化合物N-（2-氯-1,3-噻唑-5-基甲基）-N-甲基脲（TZMU）;（2）部分硝基胍中N–N键的断裂和噻唑基中C–Cl键的断裂形成N-（1,3-噻唑-5-基甲基）-N’甲基弧（TZMG）,并通过噻唑基甲基部分与硝基胍部分之间的C–N键的断裂进一步代谢为5-氨基甲基噻唑（MTZ）。在土壤模拟研究中,发现生物强化与生物刺激联合处理组（NBA）修复噻虫胺污染土壤的效果最好,修复45 d后,该处理组对噻虫胺的降解率达到95.7%。采用高效液相色谱法测定噻虫胺在土壤中的残留,其最低检测浓度为0.01 mg kg^–1,平均添加回收率为84.2%⁸6.4%,相对标准偏差为5.5%⁷.4%,结果可靠,符合农残分析标准。噻虫胺在四种不同处理土壤中残留量随着时间的推移逐渐减少,符合一级动力学方程,在土壤中观察到的降解速率常数（k）为0.008 mg kg^–1 d^–10.066 mg kg^–1d^–1,半衰期为10.6⁹2.4d。更多还原

【Abstract】 Recent years,greenhouses are widely used around the world.Greenhouse has economic benefits and also has environmental pollution problem.The abuse of pesticides not only has damaged the quality of the soil,but also polluted the air,water bodies,and even entered the human body through the food chain,endangering human health.Clothianidin is a relatively small molecule with a high water solubility and low volatility,and has a long half-life in soil,which was used in greenhouses frequently.In terms of chemical properties and environmental persistence,the residue of clothianidin could leach and move into surface water and groundwater.In addition,the residue of clothianidin also can be toxic to nervous system of non-target species such as honeybee,birds,and aquatic invertebrates.Therefore,the research on bioremediation technology of clothianidin pesticide contaminated soil is of great significance.This study took 10 years of vegetable greenhouse soil as the experiment object and determinated clothianidin as a target pesticide by sampling survey.The effects of clothianidin residues on soil pH,soil enzyme activity and soil microbial quantity were studied by soil ecological simulation test in the laboratory.The research constructed and screened bacterial consortia SCAH by enrichment culture and directed domestication.The degradation characteristics and degradation mechanism of the bacterial consortia SCAH were studied.The bioremediation effect of the bacterial consortia SCAH on the clothianidin contaminated soil was studied by comprehensive analysis of the residual changes of clothianidin in different treated soils.The main test results are as follows:Different concentrations of clothianidin showed inhibition effect on soil pH.5 mg kg^–1and 20 mg kg^–1 clothianidin showed a promoting effect on invertase activity,but 50 mg kg^–1clothianidin showed inhibition effect.Different concentrations of clothianidin showed a trend of"promoting–inhibition–recovery"on urease activity.Low concentration of clothianidin showed a trend of"inhibition–promotion–inhibition"on catalase activity,but high concentration clothianidin showed inhibition on catalase activity.For alkaline phosphatase,different concentrations of clothianidin showed inhibition.Different concentrations of clothianidin showed the effect of"inhibition–promoting"on soil bacteria and fungi,but showed strong inhibition on actinomycetes,and the greater the concentration of clothianidin,the stronger the inhibition.Five high-efficiency degrading bacteria were isolated and screened from the soil of Tianshui vegetable greenhouse in Gansu Province and identified as Ochrobactrum anthropi,Enterobacter sp,Acinetobacter johnsonii,Pseudomonas putida and Stenotrophomonas maltophilia,respectively.In order to improve the degradation efficiency of clothianidin,the bacterial consortia SCAH was constructed and screened.In the mineral salt medium,79.3%of clothianidin(500 mg L^–1)was degraded by bacterial consortia SCAH after 15 days.The characteristics of the bacterial consortia SCAH degradation of clothianidin were investigated.The results showed that the degradation rate of clothianidin was the highest at pH 6.0,temperature of 35°C and inoculum of 3%.After 10 days of culture,the degradation rate reached 69.8%.Based on the results of LC-MS,the proposed metabolic pathway of clothianidin by the consortia SCAH was speculated.The major metabolic reactions of clothianidin were concluded to be as follows:（1）transformation of the nitroimino moiety to the urea compound N-（2-chloro-1,3-thiazole-5-ylmethyl）-N￠-methylurea（TZMU）.（2）The cleavage of the N–N bond in a part of the nitroguanidine and the cleavage of the C–Cl bond in the thiazole formed the N-（1,3-thiazole-5-ylmethyl）-N′-methylguanidine（TZMG）,and was further metabolized to 5-amino-methlthiazol（MTZ）via cleavage of the C–N bond between the thiazolylmethyl moiety and the nitroguanidine moiety.In the soil simulation study,it was found that higher degradation rates were shown in the combination of bioaugmentation and biostimulation compared with other treatment groups.After 45 days,the degradation rate of clothianidin achieved 95.7%.The residue of clothianidin in soil was determined by high performance liquid chromatography.The minimum detection concentration was 0.01 mg kg^–1,the recovery was84.2%⁸6.4%,and the relative standard deviation was 5.5%⁷.4%.The results were reliable and met the requirements of pesticide residue analysis.The residual amount of clothianidin in four different treatment soils gradually decreased with time,which accorded with the first-order kinetic equation.The degradation rate constant（k）observed in soil were0.008 mg kg^–1 d^–10.066 mg kg^–1 d^–1,and the half-life is 10.6⁹2.4 days.更多还原