本文關(guān)鍵詞：五氯硝基苯降解細(xì)菌的分離鑒定及菌株DH19的降解特性、代謝途徑和SufB基因功能研究　出處：《吉林農(nóng)業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 五氯硝基苯 降解菌 Arthrobacter nicotianae DH19 氣相色譜-串聯(lián)質(zhì)譜 降解機(jī)制 代謝途徑

【摘要】：五氯硝基苯是一種典型的有機(jī)氯類保護(hù)性殺菌劑,因其殺菌譜廣、價(jià)格低廉且藥效持久等特點(diǎn),在生產(chǎn)中應(yīng)用廣泛。但其殘留期長(zhǎng),難降解且在環(huán)境中能代謝產(chǎn)生多種有毒類物質(zhì),對(duì)農(nóng)產(chǎn)品質(zhì)量及環(huán)境安全造成嚴(yán)重威脅,制約了我國(guó)農(nóng)產(chǎn)品的出口貿(mào)易。因此,發(fā)展生物修復(fù)對(duì)降低和消除其環(huán)境污染意義重大。本研究從常年施用五氯硝基苯的人參地塊采集土壤樣品,從中分離并篩選出能夠以五氯硝基苯為唯一碳源進(jìn)行生長(zhǎng)的細(xì)菌菌株,并對(duì)其進(jìn)行純化、鑒定,明確其降解特性,優(yōu)化其發(fā)酵條件,研究菌株的降解機(jī)制及其在降解五氯硝基苯的過(guò)程中產(chǎn)生的代謝產(chǎn)物,對(duì)降解五氯硝基苯的代謝途徑進(jìn)行分析推斷,并通過(guò)基因重組的方法對(duì)降解基因的功能進(jìn)行研究。本研究為提高五氯硝基苯污染的生物修復(fù)效率提供理論支持和合理依據(jù),為降解菌的產(chǎn)業(yè)化菌劑開發(fā)利用奠定基礎(chǔ),提高我國(guó)人參等多種植物產(chǎn)品的質(zhì)量,解除我國(guó)的作物出口貿(mào)易受到嚴(yán)重制約的尷尬局面,對(duì)于有效保障農(nóng)產(chǎn)品質(zhì)量安全,擴(kuò)大對(duì)外出口貿(mào)易,保護(hù)生態(tài)環(huán)境具有十分重要的意義。本研究主要取得以下結(jié)果:1.通過(guò)富集馴化再分離法對(duì)人參種植地區(qū)多年被五氯硝基苯污染的土壤樣本進(jìn)行分離,共獲得403株降解細(xì)菌。采用透明圈法初篩獲得64株能以五氯硝基苯為唯一碳源生長(zhǎng)的降解菌株。采用紫外分光光度計(jì)法通過(guò)不同菌株對(duì)五氯硝基苯的降解率進(jìn)行分析,最終獲得10株降解效果顯著的菌株,當(dāng)接種7 d時(shí),菌株QTH3,CB8,DH19,JA30,BS34,HL37,SJH42,CB44,FS49和CB56對(duì)五氯硝基苯的降解率分別為88.89%、57.13%、90.86%、83.30%、86.68%、83.46%、84.36%、86.97%、78.06%和70.99%。2.通過(guò)形態(tài)特征、生理生化反應(yīng)、分子鑒定及BIOLOG微生物自動(dòng)鑒定系統(tǒng)對(duì)五氯硝基苯降解菌株QTH3,CB8,DH19,JA30,BS34,HL37,SJH42,CB44,FS49和CB56進(jìn)行鑒定,鑒定菌株QTH3為惡臭假單胞菌(Pseudomonas putida),CB8為短小黃桿菌(Brevibacterium sanguinis),DH19為煙草節(jié)桿菌(Arthrobacter nicotianae),JA30為紅平紅球菌(Rhodococcus erythropolis),BS34為煙草節(jié)桿菌(Arthrobacter nicotianae),HL37為短小橘桿菌(Brevibacterium antarcticum),SJH42為赤紅球菌(Rhodococcus ruber),CB44為施氏假單孢菌(Pseudomonas stutzeri),FS49為紅平紅球菌(Rhodococcus erythropolis),CB56為大頭茶菌屬(Gordonia amicalis)。3.采用GC-MS/MS測(cè)定了五氯硝基苯在降解菌株DH19作用下的降解動(dòng)態(tài),結(jié)果表明菌株DH19對(duì)五氯硝基苯,五氯苯胺及甲基五氯苯硫醚均有明顯降解效果,接種14 d后對(duì)五氯硝基苯的降解率為98.64%,五氯苯胺和甲基五氯苯硫醚的殘留量分別為未檢出和0.30 mg/kg。4.為明確不同條件下降解菌株dh19對(duì)五氯硝基苯的降解特性,本研究在單因素實(shí)驗(yàn)的基礎(chǔ)上,采用響應(yīng)曲面法對(duì)dh19的最適降解條件進(jìn)行三因素三水平的響應(yīng)面分析,得出最佳條件為溫度30℃,ph6.846和接種量1.45g/l。在最佳條件下,對(duì)菌株dh19的生長(zhǎng)量和五氯硝基苯的降解曲線進(jìn)行了研究。接種7d后,五氯硝基苯的降解率在90%以上,且菌體生長(zhǎng)量隨著時(shí)間的延長(zhǎng)逐漸增加。由此表明,菌株dh19可以利用五氯硝基苯作為生長(zhǎng)和降解的唯一碳源和能量來(lái)源。5.采用gc-ms/ms測(cè)定了菌株dh19對(duì)六六六、ddt、氯氰菊酯和氟氯氰菊酯的降解,接種5d后,降解菌株dh19對(duì)六六六、ddt、氯氰菊酯和氟氯氰菊酯的降解率均在80%以上,接種7d后,降解率均在95%以上,降解菌株dh19對(duì)六六六、ddt、氯氰菊酯和氟氯氰菊酯均有較好降解效果。6.建立了五氯硝基苯降解酶促反應(yīng)體系,結(jié)果表明菌株dh19的降解作用以胞內(nèi)降解酶的粗酶液為主,對(duì)五氯硝基苯的降解率為73.47%,而胞外蛋白粗酶液對(duì)五氯硝基苯的降解率僅為4.7%。對(duì)菌株dh19產(chǎn)酶培養(yǎng)基的優(yōu)化,研究表明:菌株dh19產(chǎn)酶培養(yǎng)基的最佳碳源及濃度為8g/l的葡萄糖,最佳氮源及濃度為1.6g/l的牛肉浸膏,種子液最佳的培養(yǎng)時(shí)間為16h,發(fā)酵液培養(yǎng)的最佳時(shí)間為18h,發(fā)酵培養(yǎng)基初始ph為8,接種量為2%時(shí),降解菌株dh19的胞內(nèi)降解酶的相對(duì)比活力最高,降解效果最佳。7.采用氣相色譜-串聯(lián)質(zhì)譜法檢測(cè)五氯硝基苯在菌株dh19作用下的代謝過(guò)程中產(chǎn)生的離子碎片,與nist數(shù)據(jù)庫(kù)進(jìn)行比對(duì)分析,推斷出2,3,4,5,6-五氯苯胺,3,5-二氯苯胺,3,4-二氯苯胺,苯胺和鄰苯二酚這5種可能的代謝產(chǎn)物�；诖�,本研究推斷了五氯硝基苯在菌株dh19的作用下的降解過(guò)程,推斷了一條從五氯硝基苯最終完全代謝成h2o和co2的降解代謝途徑。8.通過(guò)對(duì)菌株dh19基因組中的部分基因進(jìn)行pcr擴(kuò)增,成功擴(kuò)增到了鐵硫簇中sufb,sufc,sufd基因,并對(duì)sufb基因進(jìn)行了克隆與分析,預(yù)測(cè)了該基因的啟動(dòng)子和終止子。此后,成功構(gòu)建了sufb基因缺失突變體△sufb-dh19(kmr和gmr)和sufb互補(bǔ)菌株▲sufb-dh19(spr和smr),并對(duì)dh19野生菌株、sufb基因缺失突變體△sufb-dh19(kmr和gmr)和sufb互補(bǔ)菌株▲sufb-dh19(spr和smr)的降解效果進(jìn)行了比較。搖培7d后,野生菌株降解五氯硝基苯后的殘留量為9.88mg/kg,降解率為89.71%,互補(bǔ)菌株▲sufb-dh19降解五氯硝基苯的殘留量為25.77mg/kg,降解率為73.16%,突變菌株△sufb-dh19在7d后降解五氯硝基苯后的殘留量為34.75mg/kg,降解率為63.80%。因此,鐵硫簇中的sufb基因?qū).nicotianaedh19降解五氯硝基苯具有一定的正調(diào)控作用。9.通過(guò)利福平抗性標(biāo)記對(duì)菌株dh19的定殖動(dòng)態(tài)進(jìn)行研究,結(jié)果表明菌株dh19在含有五氯硝基苯的土壤中可穩(wěn)定定殖。通過(guò)對(duì)菌株dh19對(duì)五氯硝基苯及其代謝產(chǎn)物五氯苯胺和甲基五氯苯硫醚的田間降解效果評(píng)價(jià)試驗(yàn)表明,無(wú)論是未滅菌土和滅菌土,菌株dh19對(duì)五氯硝基苯及五氯苯胺和甲基五氯苯硫醚的降解效果明顯。因此,該菌株可在生產(chǎn)中用于降解五氯硝基苯、五氯苯胺和甲基五氯苯硫醚,具有一定的開發(fā)潛力和應(yīng)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
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：X172

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1 王燕;五氯硝基苯降解細(xì)菌的分離鑒定及菌株DH19的降解特性、代謝途徑和SufB基因功能研究[D];吉林農(nóng)業(yè)大學(xué);2016年

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本文編號(hào)：1344235