五氯硝基苯降解细菌的分离鉴定及菌株DH19的降解特性、代谢途径和SufB基因功能研究

发布时间：2017-12-28 02:31

本文关键词：五氯硝基苯降解细菌的分离鉴定及菌株DH19的降解特性、代谢途径和SufB基因功能研究　出处：《吉林农业大学》2016年博士论文　论文类型：学位论文

【摘要】：五氯硝基苯是一种典型的有机氯类保护性杀菌剂,因其杀菌谱广、价格低廉且药效持久等特点,在生产中应用广泛。但其残留期长,难降解且在环境中能代谢产生多种有毒类物质,对农产品质量及环境安全造成严重威胁,制约了我国农产品的出口贸易。因此,发展生物修复对降低和消除其环境污染意义重大。本研究从常年施用五氯硝基苯的人参地块采集土壤样品,从中分离并筛选出能够以五氯硝基苯为唯一碳源进行生长的细菌菌株,并对其进行纯化、鉴定,明确其降解特性,优化其发酵条件,研究菌株的降解机制及其在降解五氯硝基苯的过程中产生的代谢产物,对降解五氯硝基苯的代谢途径进行分析推断,并通过基因重组的方法对降解基因的功能进行研究。本研究为提高五氯硝基苯污染的生物修复效率提供理论支持和合理依据,为降解菌的产业化菌剂开发利用奠定基础,提高我国人参等多种植物产品的质量,解除我国的作物出口贸易受到严重制约的尴尬局面,对于有效保障农产品质量安全,扩大对外出口贸易,保护生态环境具有十分重要的意义。本研究主要取得以下结果:1.通过富集驯化再分离法对人参种植地区多年被五氯硝基苯污染的土壤样本进行分离,共获得403株降解细菌。采用透明圈法初筛获得64株能以五氯硝基苯为唯一碳源生长的降解菌株。采用紫外分光光度计法通过不同菌株对五氯硝基苯的降解率进行分析,最终获得10株降解效果显著的菌株,当接种7 d时,菌株QTH3,CB8,DH19,JA30,BS34,HL37,SJH42,CB44,FS49和CB56对五氯硝基苯的降解率分别为88.89%、57.13%、90.86%、83.30%、86.68%、83.46%、84.36%、86.97%、78.06%和70.99%。2.通过形态特征、生理生化反应、分子鉴定及BIOLOG微生物自动鉴定系统对五氯硝基苯降解菌株QTH3,CB8,DH19,JA30,BS34,HL37,SJH42,CB44,FS49和CB56进行鉴定,鉴定菌株QTH3为恶臭假单胞菌(Pseudomonas putida),CB8为短小黄杆菌(Brevibacterium sanguinis),DH19为烟草节杆菌(Arthrobacter nicotianae),JA30为红平红球菌(Rhodococcus erythropolis),BS34为烟草节杆菌(Arthrobacter nicotianae),HL37为短小橘杆菌(Brevibacterium antarcticum),SJH42为赤红球菌(Rhodococcus ruber),CB44为施氏假单孢菌(Pseudomonas stutzeri),FS49为红平红球菌(Rhodococcus erythropolis),CB56为大头茶菌属(Gordonia amicalis)。3.采用GC-MS/MS测定了五氯硝基苯在降解菌株DH19作用下的降解动态,结果表明菌株DH19对五氯硝基苯,五氯苯胺及甲基五氯苯硫醚均有明显降解效果,接种14 d后对五氯硝基苯的降解率为98.64%,五氯苯胺和甲基五氯苯硫醚的残留量分别为未检出和0.30 mg/kg。4.为明确不同条件下降解菌株dh19对五氯硝基苯的降解特性,本研究在单因素实验的基础上,采用响应曲面法对dh19的最适降解条件进行三因素三水平的响应面分析,得出最佳条件为温度30℃,ph6.846和接种量1.45g/l。在最佳条件下,对菌株dh19的生长量和五氯硝基苯的降解曲线进行了研究。接种7d后,五氯硝基苯的降解率在90%以上,且菌体生长量随着时间的延长逐渐增加。由此表明,菌株dh19可以利用五氯硝基苯作为生长和降解的唯一碳源和能量来源。5.采用gc-ms/ms测定了菌株dh19对六六六、ddt、氯氰菊酯和氟氯氰菊酯的降解,接种5d后,降解菌株dh19对六六六、ddt、氯氰菊酯和氟氯氰菊酯的降解率均在80%以上,接种7d后,降解率均在95%以上,降解菌株dh19对六六六、ddt、氯氰菊酯和氟氯氰菊酯均有较好降解效果。6.建立了五氯硝基苯降解酶促反应体系,结果表明菌株dh19的降解作用以胞内降解酶的粗酶液为主,对五氯硝基苯的降解率为73.47%,而胞外蛋白粗酶液对五氯硝基苯的降解率仅为4.7%。对菌株dh19产酶培养基的优化,研究表明:菌株dh19产酶培养基的最佳碳源及浓度为8g/l的葡萄糖,最佳氮源及浓度为1.6g/l的牛肉浸膏,种子液最佳的培养时间为16h,发酵液培养的最佳时间为18h,发酵培养基初始ph为8,接种量为2%时,降解菌株dh19的胞内降解酶的相对比活力最高,降解效果最佳。7.采用气相色谱-串联质谱法检测五氯硝基苯在菌株dh19作用下的代谢过程中产生的离子碎片,与nist数据库进行比对分析,推断出2,3,4,5,6-五氯苯胺,3,5-二氯苯胺,3,4-二氯苯胺,苯胺和邻苯二酚这5种可能的代谢产物。基于此,本研究推断了五氯硝基苯在菌株dh19的作用下的降解过程,推断了一条从五氯硝基苯最终完全代谢成h2o和co2的降解代谢途径。8.通过对菌株dh19基因组中的部分基因进行pcr扩增,成功扩增到了铁硫簇中sufb,sufc,sufd基因,并对sufb基因进行了克隆与分析,预测了该基因的启动子和终止子。此后,成功构建了sufb基因缺失突变体△sufb-dh19(kmr和gmr)和sufb互补菌株▲sufb-dh19(spr和smr),并对dh19野生菌株、sufb基因缺失突变体△sufb-dh19(kmr和gmr)和sufb互补菌株▲sufb-dh19(spr和smr)的降解效果进行了比较。摇培7d后,野生菌株降解五氯硝基苯后的残留量为9.88mg/kg,降解率为89.71%,互补菌株▲sufb-dh19降解五氯硝基苯的残留量为25.77mg/kg,降解率为73.16%,突变菌株△sufb-dh19在7d后降解五氯硝基苯后的残留量为34.75mg/kg,降解率为63.80%。因此,铁硫簇中的sufb基因对a.nicotianaedh19降解五氯硝基苯具有一定的正调控作用。9.通过利福平抗性标记对菌株dh19的定殖动态进行研究,结果表明菌株dh19在含有五氯硝基苯的土壤中可稳定定殖。通过对菌株dh19对五氯硝基苯及其代谢产物五氯苯胺和甲基五氯苯硫醚的田间降解效果评价试验表明,无论是未灭菌土和灭菌土,菌株dh19对五氯硝基苯及五氯苯胺和甲基五氯苯硫醚的降解效果明显。因此,该菌株可在生产中用于降解五氯硝基苯、五氯苯胺和甲基五氯苯硫醚,具有一定的开发潜力和应用前景。
[Abstract]:Fungiclor is a kind of typical organochlorine protective fungicide, due to its characteristics of wide bactericidal spectrum, low price and lasting effect, widely used in production. But its residue period is long, difficult to degrade, and can produce various toxic substances in the environment, which poses a serious threat to the quality and environmental safety of agricultural products, and restricts the export trade of China's agricultural products. Therefore, the development of bioremediation is of great significance to reduce and eliminate environmental pollution. This study plots from ginseng soil samples collected from the annual application of PCNB, isolated and screened to PCNB as the sole carbon source for growth of bacterial strain, and to purify and identify the specific degradation characteristics, optimization of the fermentation conditions, the degradation mechanism of metabolites of strains and produced in the degradation process of pentachloronitrobenzene the metabolic pathway on degradation of PCNB inferred that study and function by gene recombination method of degrading gene. This study provides theoretical support and reasonable basis for improving the efficiency of bioremediation of PCNB pollution, lay the foundation for the industrialization of microbial degradation bacteria development and utilization, improve the quality of Chinese ginseng and other plant products, lifting China crop export trade seriously restrict the embarrassing situation, to effectively guarantee the quality and safety of agricultural products, the expansion of foreign the export trade is of great significance to protect the ecological environment. The research results are as follows: 1. through the enrichment separation method of ginseng planting area has been polluted soil samples of PCNB were isolated, there were 403 strains of degrading bacteria. By using the method of transparent circle screening to get 64 strains of PCNB degradation strains for the sole carbon source for growth. Using UV spectrophotometer method through degradation of PCNB rate of different strains were analyzed, obtained 10 strains of significant degradation strains, when inoculated with 7 d strains, QTH3, CB8, DH19, JA30, BS34, HL37, SJH42, CB44, FS49 and CB56 of PCNB degradation rate were 88.89% 57.13%, 90.86%, 83.30%, 86.68%, 83.46%, 84.36%, 86.97%, 78.06% and 70.99%. 2. the morphological characteristics, physiological and biochemical reaction, molecular identification and BIOLOG microbial identification system of PCNB degradation strains QTH3, CB8, DH19, JA30, BS34, HL37, SJH42, CB44, FS49 and CB56 were identified, strain QTH3 was identified as Pseudomonas putida (Pseudomonas putida), CB8 sanguinis (Brevibacterium for short Flavobacterium DH19), tobacco (Arthrobacter nicotianae), Arthrobacter JA30 erythropolis (Rhodococcus erythropolis), BS34 (Arthrobacter nicotianae) tobacco Arthrobacter, Bacillus HL37 for short (Brevibacterium antarcticum), orange SJH42 (Rhodococcus ruber), Rhodococcus ruber CB44 Pseudomonas stutzeri (Pseudomonas stutzeri). FS49 erythropolis (Rhodococcus erythropolis), CB56 sp. (Gordonia Gordonia amicalis). 3. by GC-MS/MS degradation of PCNB in degrading strain DH19 under the action of the determination, the results show that the PCNB strain DH19, five chloro aniline and five methyl chlorobenzene sulfide has obvious degradation effect, 14 d after inoculation of PCNB degradation rate was 98.64%, the residues of five chloro aniline and five methyl chlorobenzene sulfide was not detected and 0.30 mg/kg respectively. 4. clearly different degrading strain dh19 degradation characteristics of pentachloronitrobenzene, this study on the basis of single factor experiment, the response surface of dh19 optimal degradation conditions of three factors and three levels of response surface analysis, the optimum conditions for the temperature of 30 DEG C, ph6.846 and 1.45g/l inoculation. Under the optimum conditions, the degradation curve of strain dh19 growth and PCNB were studied. 7d after inoculation, the PCNB degradation rate of more than 90%, and the biomass was increased. This shows that the strain dh19 can utilize PCNB as sole carbon source and energy source for growth and degradation. 5. by gc-ms/ms, 666 DDT, the degradation of cypermethrin and cyfluthrin in strain dh19 were measured 5D after inoculation, degrading strain dh19 degradation of 666, DDT, cypermethrin and cyfluthrin was above 80% in 7d after inoculation, the degradation rate was above 95%, degradation dh19, DDT, 666 strains of cypermethrin and cyfluthrin had better degradation effect. 6. established PCNB degradation enzymatic reaction system. The results show that the degradation effect of strain dh19 in crude enzyme intracellular degradation enzyme, the PCNB degradation rate was 73.47%, while the cellular protein degradation enzyme of PCNB was only 4.7%. According to the research on Optimization of fermentation medium of strain dh19: the yield of dh19 was the best carbon source and concentration of medium was 8g/l glucose, the best nitrogen source and concentration of 1.6g/l beef extract, seed liquid is the best culture time is 16h, the best time of fermentation liquid culture was 18h, the initial fermentation medium pH is 8, the inoculation amount was 2%, the degradation of strain dh19 intracellular degradation enzyme compared to the highest activity and the best effect of degradation. 7. the ionic fragments produced by gas chromatography tandem mass spectrometry for the determination of pentachloronitrobenzene in strain dh19 under the action of the metabolic process, compared with the NIST database, deduce 2,3,4,5,6- five 3,5- two chloro aniline, chloro aniline, 3,4- two chloro aniline, aniline and catechol 5 possible metabolites. Based on this, the study concluded that the degradation of PCNB in strain under the action of dh19, inferred from a completely PCNB metabolized into H2O and CO2 degradation pathway. 8., by amplifying the PCR gene in the genome of dh19 strain, the sufb, sufc and sufd genes in iron sulfur cluster were successfully amplified, and the sufb gene was cloned and analyzed, and the promoter and terminator of the gene were predicted. After that, successful construction
【学位授予单位】：吉林农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：X172

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